ellis d.

avner
william e. harmon
patrick niaudet
norishige yoshikawa
francesco emma
stuart l. goldstein
Editors

Pediatric
Nephrology
Seventh Edition

OFFICIALLY
ENDORSED BY 1 3Reference
Pediatric Nephrology
Ellis D. Avner • William E. Harmon
Patrick Niaudet • Norishige Yoshikawa
Francesco Emma • Stuart L. Goldstein
Editors

Pediatric Nephrology
Seventh Edition

With 506 Figures and 310 Tables

Editors
Ellis D. Avner William E. Harmon
Department of Pediatrics Boston Children’s Hospital
Medical College of Wisconsin Harvard Medical School
Children’s Research Institute Boston, MA, USA
Children’s Hospital
Health System of Wisconsin
Milwaukee, WI, USA
Patrick Niaudet Norishige Yoshikawa
Service de Néphrologie Pédiatrique Department of Pediatrics
Hôpital Necker-Enfants Malades Wakayama Medical University
Université Paris-Descartes Wakayama City, Japan
Paris, France

Francesco Emma Stuart L. Goldstein

Division of Nephrology Division of Nephrology and
Bambino Gesù Children’s Hospital – IRCCS Hypertension
Rome, Italy The Heart Institute
Cincinnati Children’s Hospital
Medical Center, College of
Medicine
Cincinnati, OH, USA

ISBN 978-3-662-43595-3 ISBN 978-3-662-43596-0 (eBook)

ISBN 978-3-662-43597-7 (print and electronic bundle)
DOI 10.1007/978-3-662-43596-0
Library of Congress Control Number: 2015954467

Springer Heidelberg New York Dordrecht London

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Preface

Through its past six editions, Pediatric Nephrology has become the standard
medical reference for health care professionals treating children with kidney
disease. This new edition, published 6 years since the previous version,
reflects the tremendous increase in critical information required to translate
molecular and cellular pathophysiology into the prevention, diagnosis, and
therapy of childhood renal disorders. This text is particularly targeted to
pediatricians, pediatric nephrologists, pediatric urologists, and physicians in
training. It is also targeted to the increased number of health care professionals
comprising the multidisciplinary teams required to provide comprehensive
care for children with kidney disease and their families: geneticists, genetic
counselors, nurse specialists, dialysis personnel, nutritionists, social workers,
and mental health professionals. Finally, this reference is designed to serve the
needs of primary care physicians (internists and family practitioners) as well as
internist nephrologists who are increasingly involved in the initial evaluation
and/or the longitudinal care of children with renal disease under different
health care delivery systems evolving throughout the world.
To keep pace with the dramatic changes in pediatric renal medicine since
the publication of the previous edition, the content of the seventh edition of
Pediatric Nephrology has been completely revised, updated, and enlarged.
The seventh edition contains 83 chapters in 3 volumes, which are organized
into 12 main sections. The text begins with an overview of the basic develop-
mental anatomy, biology, and physiology of the kidney, which provides the
basic information necessary to understand the developmental nature of pedi-
atric renal diseases. This is followed by a comprehensive coverage of the
evaluation, diagnosis, and therapy of specific childhood kidney diseases,
including the extensive use of clinical algorithms. Of special note is a section
focused on rapidly evolving research methodologies, which are being trans-
lated into new clinical approaches and therapies for many childhood renal
diseases. The final sections focus on comprehensive, state-of-the-art reviews
of acute and chronic renal failure in childhood.
Many chapters of the seventh edition have been completely rewritten by
new authors, all recognized as global authorities in their respective areas. The
remainder of the text has been totally revised, with junior authors often joining
senior authors from the previous edition. The number of contributors has
increased by 20 %. In addition to the new section on Global Pediatric
Nephrology, which focuses on unique aspects of pediatric nephrology practice

v
vi Preface

and the epidemiology of pediatric renal disease in different regions of the

world, all of the chapters reflect a global perspective. This has been achieved
through a dynamic, evolving relationship between the Editors and the Inter-
national Pediatric Nephrology Association (IPNA). This has led to the con-
tinued endorsement of Pediatric Nephrology as the standard global
reference text in the field of childhood kidney disease. We are proud
that the IPNA logo adorns the cover of Pediatric Nephrology in recognition
of this endorsement. The Editors look forward to this dynamic interaction
with IPNA to take advantage of future opportunities that such collaboration
may provide in the areas of education and outreach.
Other major changes are also evidenced by the new publication of the
seventh edition of Pediatric Nephrology as a basic reference handbook in the
SpringerReference series. Representing advances in state-of-the-art electronic
publishing, there are regularly updated online versions of each chapter of this
text at SpringerLink.com. The new publishing format has led to a welcome
expansion of the published text to three volumes and the increased utilization
of high-resolution, color figures. Further, two new Editors, Professor
Francesco Emma of Ospedale Pediatrico Bambino Gesu in Rome, Italy, and
Professor Stuart L. Goldstein of the University of Cincinnati, USA, have
joined the Editorial Team. The addition of new Editors continues to provide
a dynamic mixture of continuity, new ideas, new perspectives, and globalism,
which has distinguished each new edition of the text. Professors Emma and
Goldstein join the four Editors from the sixth edition: Senior Editor
and Emeritus Professor Ellis D. Avner, from the Medical College of Wiscon-
sin, USA, and Professors William E. Harmon M.D. of Harvard University,
USA, Patrick Niaudet, from the Hôpital Necker-Enfants Malades in
Paris, France; and Norishige Yoshikawa from Wakayama, Japan. The current
Editors are internationally recognized leaders in complementary areas of
pediatric nephrology and along with the more than 150 contributors reflect
the global nature of the text and the subspecialty it serves.
The Editors wish to thank a number of individuals whose efforts were
critical in the success of this project. The book would never have reached
this seventh edition without the dedication of our professional colleagues at
Springer, Gabriele Schroder, Sandra Lesny, Gregory Sutorius, and particularly
Mr. Andrew Spencer, Senior Editor of Major Reference Works, who served as
our “guide for the perplexed” in all aspects of project management. We thank
our families, and particularly our wives (Jane, Diane, Claire, Hiro, and
Elizabeth) for their support and understanding. In particular, the Senior Editor
wishes to recognize his lifetime partner in all endeavors, Jane A. Avner, Ph.D.,
for her extraordinary editorial assistance. And finally, we thank our mentors,
our students, and most importantly, our patients and their families. Without
them, our work would lack purpose.
Preface vii

Finally, the Editors wish to dedicate this seventh edition of Pediatric Nephrol-
ogy to three former Editors who passed away in 2014. Professors Martin Barratt,
Malcolm A. “Mac” Holiday, and Robert Vernier were extraordinary physician-
scientists who served as mentors to a generation of pediatric nephrologists. This
text would not exist without their efforts, commitment, and selfless contributions.

Ellis D. Avner
William E. Harmon
Patrick Niaudet
Norishige Yoshikawa
Francesco Emma
Stuart L. Goldstein
Contents

Volume 1

Part I Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1 Embryonic Development of the Kidney . . . . . . . . . . . . . . . . . 3

Carlton Bates, Jacqueline Ho, and Sunder Sims-Lucas
2 Development of Glomerular Circulation and Function .... 37
Alda Tufro and Ashima Gulati
3 Renal Tubular Development . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Michel Baum
4 Clinical Perinatal Urology . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
David A. Diamond and Richard S. Lee
5 Renal Dysplasia/Hypoplasia . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Paul Goodyer and Indra R. Gupta
6 Developmental Syndromes and Malformations of the
Urinary Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Chanin Limwongse

Part II Homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

7 Physiology of the Developing Kidney: Sodium and Water

Homeostasis and Its Disorders . . . . . . . . . . . . . . . . . . . . . . . . 181
Nigel Madden and Howard Trachtman
8 Physiology of the Developing Kidney: Potassium
Homeostasis and Its Disorder . . . . . . . . . . . . . . . . . . . . . . . . . 219
Lisa M. Satlin and Detlef Bockenhauer
9 Physiology of the Developing Kidney: Acid-Base
Homeostasis and Its Disorders . . . . . . . . . . . . . . . . . . . . . . . . 247
Peter D. Yorgin, Elizabeth G. Ingulli, and Robert H. Mak
10 Bone Developmental Physiology . . . . . . . . . . . . . . . . . . . . . . . 279
MH Lafage-Proust

ix
x Contents

11 Physiology of the Developing Kidney: Disorders and

Therapy of Calcium and Phosphorous Homeostasis . . . . . . . 291
Amita Sharma, Rajesh V. Thakker, and Harald J€uppner

12 Nutrition Management in Childhood Kidney Disease:

An Integrative and Lifecourse Approach . . . . . . . . . . . . . . . . 341
Lauren Graf, Kimberly Reidy, and Frederick J. Kaskel

13 Physiology of the Developing Kidney: Fluid and

Electrolyte Homeostasis and Therapy of Basic
Disorders (Na/H2O/K/Acid Base) . . . . . . . . . . . . . . . . . . . . . . 361
Isa F. Ashoor and Michael J. G. Somers

Part III Translational Research Methods .................... 423

14 Translational Research Methods: Basics of Renal Molecular

Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425
Gian Marco Ghiggeri, Maurizio Bruschi, and
Simone Sanna-Cherchi

15 Translational Research Methods: The Value of Animal

Models in Renal Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447
Jordan Kreidberg

16 Basics of Clinical Investigation . . . . . . . . . . . . . . . . . . . . . . . . 473

Susan L. Furth and Jeffrey J. Fadrowski

17 Genomic Methods in the Diagnosis and Treatment of

Pediatric Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
Karen Maresso and Ulrich Broeckel

18 Translational Research Methods: Renal Stem Cells . . . . . . . 525

Kenji Osafune

19 Translational Research Methods: Tissue Engineering of the

Kidney and Urinary Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . 571
Austin G. Hester and Anthony Atala

Part IV Clinical Approach to the Child with Suspected Renal

Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593

20 Clinical Evaluation of the Child with Suspected

Renal Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595
Mohan A. Shenoy and Nicholas J. A. Webb

21 Laboratory Investigation of the Child with Suspected

Renal Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613
George van der Watt, Fierdoz Omar, Anita Brink, and
Mignon McCulloch
Contents xi

22 Growth and Development of the Child with

Renal Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 637
Bethany Foster

23 Diagnostic Imaging of the Child with Suspected

Renal Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
Jonathan Loewen and Larry A. Greenbaum

24 Pediatric Renal Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . 705

Agnes B. Fogo

Part V Glomerular Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751

25 Congenital Nephrotic Syndrome . . . . . . . . . . . . . . . . . . . . . . . 753

Hannu Jalanko and Christer Holmberg

26 Inherited Glomerular Diseases . . . . . . . . . . . . . . . . . . . . . . . . 777

Michelle N. Rheault and Clifford E. Kashtan

27 Idiopathic Nephrotic Syndrome in Children: Genetic

Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 805
Olivia Boyer, Kálmán Tory, Eduardo Machuca, and
Corinne Antignac

28 Idiopathic Nephrotic Syndrome in Children: Clinical

Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839
Patrick Niaudet and Olivia Boyer

29 Immune-Mediated Glomerular Injury in Children . . . . . . . . 883

Michio Nagata

30 Complement-Mediated Glomerular Injury in

Children . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 927
Zoltán Prohászka, Marina Vivarelli, and
George S. Reusz

31 Acute Postinfectious Glomerulonephritis in Children . . . . . . 959

Bernardo Rodríguez-Iturbe, Behzad Najafian,
Alfonso Silva, and Charles E. Alpers

32 Immunoglobulin A Nephropathies in Children

(Includes HSP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 983
Koichi Nakanishi and Norishige Yoshikawa

33 Membranoproliferative and C3-Mediated

GN in Children . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1035
Christoph Licht, Magdalena Riedl, Matthew C. Pickering, and
Michael Braun

34 Membranous Nephropathy in Children . . . . . . . . . . . . . . . . . 1055

Rudolph P. Valentini
xii Contents

Volume 2

Part VI Tubular Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1077

35 Nephronophthisis and Medullary Cystic Kidney Disease in

Children . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1079
Friedhelm Hildebrandt
36 Childhood Polycystic Kidney Disease . . . . . . . . . . . . . . . . . . . 1103
William E. Sweeney Jr., Meral Gunay-Aygun,
Ameya Patil, and Ellis D. Avner
37 Aminoaciduria and Glycosuria in Children . . . . . . . . . . . . . . 1155
Israel Zelikovic
38 Renal Tubular Disorders of Electrolyte Regulation
in Children . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1201
Olivier Devuyst, Hendrica Belge, Martin Konrad,
Xavier Jeunemaitre, and Maria-Christina Zennaro
39 Renal Tubular Acidosis in Children . . . . . . . . . . . . . . . . . . . . 1273
Raymond Quigley and Matthias T. F. Wolf
40 Nephrogenic Diabetes Insipidus in Children . . . . . . . . . . . . . 1307
Nine V. A. M. Knoers and Elena N. Levtchenko
41 Cystinosis and Its Renal Complications in Children . . . . . . . 1329
William A. Gahl and Galina Nesterova
42 Pediatric Fanconi Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . 1355
Takashi Igarashi
43 Primary Hyperoaxaluria in Children . . . . . . . . . . . . . . . . . . . 1389
Pierre Cochat, Neville Jamieson, and
Cecile Acquaviva-Bourdain
44 Pediatric Tubulointerstitial Nephritis . . . . . . . . . . . . . . . . . . . 1407
Uri S. Alon

Part VII Kidney Involvement in Systemic Diseases . . . . . . . . . . . 1429

45 Renal Involvement in Children with Vasculitis . . . . . . . . . . . 1431

Seza Ozen and Diclehan Orhan
46 Renal Involvement in Children with Systemic Lupus
Erythematosus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1449
Patrick Niaudet, Brigitte Bader-Meunier, and Rémi Salomon
47 Renal Involvement in Children with HUS . . . . . . . . . . . . . . . 1489
Carla M. Nester and Sharon P. Andreoli
Contents xiii

48 Sickle Cell Nephropathy in Children . . . . . . . . . . . . . . . . . . . 1523

Connie Piccone and Katherine MacRae Dell

49 Diabetic Nephropathy in Children . . . . . . . . . . . . . . . . . . . . . 1545

M. Loredana Marcovecchio and Francesco Chiarelli

50 Renal Manifestations of Metabolic Disorders

in Children . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1569
Francesco Emma, William G. van’t Hoff, and
Carlo Dionisi Vici

51 Infectious Diseases and the Kidney in Children . . . . . . . . . . 1609

Jennifer Stevens, Jethro A. Herberg, and Michael Levin

52 Nephrotoxins and Pediatric Kidney Injury . . . . . . . . . . . . . . 1655

Takashi Sekine

Part VIII Urinary Tract Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1693

53 Urinary Tract Infections in Children . . . . . . . . . . . . . . . . . . . 1695

Elisabeth M. Hodson and Jonathan C. Craig

54 Vesicoureteral Reflux and Renal Scarring in Children . . . . . 1715

Tej K. Mattoo, Ranjiv Mathews, and Indra R. Gupta

55 Pediatric Obstructive Uropathy . . . . . . . . . . . . . . . . . . . . . . . 1749

Bärbel Lange-Sperandio

56 Pediatric Bladder Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . 1779

Etienne Berard

57 Urolithiasis in Children . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1821

Vidar Edvardsson

58 Pediatric Renal Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1869

Elizabeth Mullen, Jordan Kreidberg, and
Christopher B. Weldon

Volume 3

Part IX Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1905

59 Epidemiology of Hypertension in Children . . . . . . . . . . . . . . 1907

Midori Awazu

60 Pathophysiology of Pediatric Hypertension . . . . . . . . . . . . . . 1951

Ikuyo Yamaguchi and Joseph T. Flynn

61 Evaluation of Hypertension in Childhood Diseases . . . . . . . . 1997

Eileen D. Brewer and Sarah J. Swartz
xiv Contents

62 Management of the Hypertensive Child . . . . . . . . . . . . . . . . . 2023

Demetrius Ellis and Yosuke Miyashita

Part X Acute Renal Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2099

63 Pathogenesis of Acute Kidney Injury . . . . . . . . . . . . . . . . . . . 2101

David P. Basile, Rajasree Sreedharan, and
Scott K. Van Why
64 Evaluation and Management of Acute Kidney Injury in
Children . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2139
Stuart L. Goldstein and Michael Zappitelli

Part XI Chronic Renal Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2169

65 Pathophysiology of Progressive Renal Disease

in Children . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2171
H. William Schnaper
66 Management of Chronic Kidney Disease in Children . . . . . . 2207
Rene G. VanDeVoorde, Craig S. Wong, and
Bradley A. Warady
67 Handling of Drugs in Children with Abnormal
Renal Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2267
Guido Filler, Amrit Kirpalani, and Bradley L. Urquhart
68 Endocrine and Growth Abnormalities in Chronic
Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2295
Franz Schaefer
69 Mineral and Bone Disorders in Children with Chronic
Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2349
Katherine Wesseling-Perry and Isidro B. Salusky
70 Peritoneal Dialysis in Children . . . . . . . . . . . . . . . . . . . . . . . . 2381
Enrico Verrina and Claus Peter Schmitt
71 Hemodialysis in Children . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2433
Lesley Rees
72 Immunology of Pediatric Renal Transplantation . . . . . . . . . 2457
Elizabeth G. Ingulli, Stephen I. Alexander, and David M. Briscoe
73 Pediatric Renal Transplantation . . . . . . . . . . . . . . . . . . . . . . . 2501
Nancy M. Rodig, Khashayar Vakili, and William E. Harmon
74 Immunosuppression for Pediatric Renal
Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2553
Jodi M. Smith, Thomas L. Nemeth, and Ruth A. McDonald
75 Complications of Pediatric Renal Transplantation . . . . . . . . 2573
Vikas R. Dharnidharka and Carlos E. Araya
Contents xv

Part XII IPNA: Global Pediatric Nephrology . . . . . . . . . . . . . . . . . 2605

76 IPNA: Global Pediatric Nephrology, Introduction and

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2607
Pierre Cochat and Isidro B. Salusky
77 AFPNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2613
Mignon McCulloch, Hesham Safouh, Amal Bourquia, and
Priya Gajjar
78 ALANEPE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2631
Vera Koch, Nelson Orta, and Ramon Exeni
79 AsPNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2639
Hui-Kim Yap, Man-Chun Chiu, Arvind Bagga, and
Hesham Safouh
80 Pediatric Nephrology in North America . . . . . . . . . . . . . . . . 2665
Victoria F. Norwood and Maury Pinsk
81 ANZPNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2673
Deborah Lewis
82 ESPN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2681
Rosanna Coppo
83 JSPN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2687
Kazumoto Iijima
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2697
Contributors

Cecile Acquaviva-Bourdain Centre de référence des maladies rénales rares

Néphrogones, Hôpital Femme Mère Enfant, Hospices Civils de Lyon &
Université de Lyon, Lyon, France
Service Maladies Héréditaires du Métabolisme et Dépistage Néonatal, Centre
de Biologie et Pathologie Est, Hospices Civils de Lyon, Lyon, France

Stephen I. Alexander Discipline of Paediatrics and Child Health, The

University of Sydney, Sydney, New South Wales, Australia
Division of Nephrology, Children’s Hospital at Westmead, Sydney, New
South Wales, Australia

Uri S. Alon Section of Pediatric Nephrology, The Children’s Mercy Hospital

and Clinics, University of Missouri at Kansas City, School of Medicine,
Kansas City, MO, USA

Charles E. Alpers Department of Pathology, University of Washington,

Seattle, WA, USA

Sharon P. Andreoli Division of Nephrology, Indianapolis, IN, USA

Corinne Antignac Laboratory of Hereditary Kidney Diseases, Inserm UMR

1163, Paris, France
Paris Descartes, Sorbonne Paris Cité University, Imagine Institute, Paris,
France
Department of Genetics, MARHEA reference center, Necker – Enfants
Malades Hospital, Paris, France

Carlos E. Araya University of Central Florida and Nemours Children’s

Hospital, Orlando, FL, USA

Isa F. Ashoor Division of Nephrology, Children’s Hospital, New Orleans,

LA, USA

Anthony Atala School of Medicine, Department of Urology, Wake Forest

Institute for Regenerative Medicine, Wake Forest University, Winston Salem,
NC, USA

xvii
xviii Contributors

Ellis D. Avner Department of Pediatrics, Medical College of Wisconsin,

Children’s Research Institute, Children’s Hospital Health System of Wiscon-
sin, Milwaukee, WI, USA

Midori Awazu Department of Pediatrics, Keio University School of Medi-

cine, Shinjuku-ku, Tokyo, Japan

Brigitte Bader-Meunier Service d’Immunologie et Rhumatologie

Pédiatrique, Hôpital Necker-Enfants Malades, Paris, France

Arvind Bagga Division of Nephrology, All India Institute of Medical

Sciences, New Delhi, India

David P. Basile Indiana University School of Medicine, Indianapolis, IN, USA

Carlton Bates Department of Pediatrics, Division of Pediatric Nephrology,

Children’s Hospital of Pittsburgh of UPMC, University of Pittsburgh School
of Medicine, Pittsburgh, PA, USA

Michel Baum Departments of Pediatrics and Internal Medicine, University

of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA

Hendrica Belge Zurich Institute for Human Physiology, University of

Zurich, Institute of Physiology, Zurich Center for Integrative Human Physiol-
ogy (ZIHP), Z€ urich, Switzerland

Etienne Berard Pediatric Nephrology Unit, Universitary Hospital of Nice

(France), Nice, France

Detlef Bockenhauer Great Ormond Street Hospital, Institute of Child

Health, University College London, London, UK

Amal Bourquia Paediatric Nephology, Red Cross War Memorial Children’s

Hospital, Dept. of Paediatric Medicine, University of Cape Town, Western
Cape, South Africa

Olivia Boyer Service de Néphrologie Pédiatrique, Hôpital Necker-Enfants

Malades, Université Paris-Descartes, Paris, France

Michael Braun Renal Section, Department of Pediatrics, Texas Children’s

Hospital, Balyor College of Medicine, Houston, TX, USA

Eileen D. Brewer Department of Pediatrics Renal Section, Baylor College of

Medicine and Texas Children’s Hospital, Houston, TX, USA

Anita Brink Department of Pediatrics and Child Health (Nuclear Medicine),

University of Cape Town, Red Cross War Memorial Children’s Hospital, Cape
Town, South Africa

David M. Briscoe Department of Pediatrics, Harvard Medical School,

Boston, MA, USA
Division of Nephrology, Transplant Research Program, Boston Children’s
Hospital, Boston, MA, USA
Contributors xix

Ulrich Broeckel Department of Pediatrics, Medical College of Wisconsin

and Children’s Hospital of Wisconsin, Milwaukee, WI, USA
Maurizio Bruschi Laboratory of Physiopathology of Uremia, Division of
Nephrology, Dialysis and Transplantation, Istituto Giannina Gaslini, Genoa,
Italy
Francesco Chiarelli University of Chieti, Chieti, Italy
Man-Chun Chiu Department of Pediatrics and Adolescent Medicine, Prin-
cess Margaret Hospital, Hong Kong University, Kowloon, Hong Kong
Pierre Cochat Centre de référence des maladies rénales rares Néphrogones,
Hôpital Femme Mère Enfant, Hospices Civils de Lyon & Université de Lyon,
Lyon, France
IBCP-UMR 5305 CNRS, Université Claude-Bernard Lyon 1, Lyon, France
Rosanna Coppo Nephrology Dialysis and Transplantation Unit, Azienda
Ospedaliera-Universitaria Città della Salute e della Scienza di Torino, Regina
Margherita Children’s University Hospital, Turin, Italy
Jonathan C. Craig Centre for Kidney Research, The Children’s Hospital at
Westmead, Westmead, Sydney, NSW, Australia
Sydney School of Public Health, University of Sydney, Sydney, NSW,
Australia
Katherine MacRae Dell Center for Pediatric Nephrology, Department of
Pediatrics, Cleveland Clinic Children’s and Case Western Reserve University,
Cleveland, OH, USA
Olivier Devuyst Zurich Institute for Human Physiology, University of
Zurich, Institute of Physiology, Zurich Center for Integrative Human Physiol-
ogy (ZIHP), Z€urich, Switzerland
Vikas R. Dharnidharka Pediatric Nephrology, Washington University
School of Medicine and St. Louis Children’s Hospital, St. Louis, MO, USA
David A. Diamond Department of Urology, Harvard Medical School,
Boston Children’s Hospital, Boston, MA, USA
Carlo Dionisi Vici Division of Metabolic Diseases, Bambino Gesù
Children’s Hospital – IRCCS, Rome, Italy
Vidar Edvardsson Landspitali – The National University Hospital of Ice-
land, Reykjavik, Iceland and Faculty of Medicine, School of Health Sciences,
University of Iceland, Reykjavik, Iceland
Demetrius Ellis Department of Pediatrics, Division of Pediatric Nephrology,
Children’s Hospital of Pittsburgh of UPMC, University of Pittsburgh School
of Medicine, Pittsburgh, PA, USA
Francesco Emma Division of Nephrology, Bambino Gesù Children’s
Hospital – IRCCS, Rome, Italy
xx Contributors

Ramon Exeni Department of Nephrology, Children’s Hospital “San Justo”,

Buenos Aires, Argentina

Jeffrey J. Fadrowski Department of Pediatrics, John Hopkins University

School of Medicine, Baltimore, MD, USA

Guido Filler Departments of Pediatrics, Medicine, and Pathology Laboratory

Medicine, Schulich School of Medicine and Dentistry, Western University,
London, ON, Canada
Children’s Hospital of Western Ontario, Children’s Health Research Institute
(CHRI), London, ON, Canada

Joseph T. Flynn Division of Nephrology, Seattle Children’s Hospital;

Department of Pediatrics, University of Washington School of Medicine,
Seattle, WA, USA

Agnes B. Fogo Department of Pathology, Microbiology and Immunology,

Vanderbilt University Medical Center, Nashville, TN, USA

Bethany Foster The Research Institute of the McGill University Health

Centre, Montreal, QC, Canada

Susan L. Furth Departments of Pediatrics and Epidemiology, Perelman

School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

William A. Gahl Section on Human Biochemical Genetics, Medical Genet-

ics Branch, National Human Genome Research Institute, National Institutes of
Health, Bethesda, MD, USA

Priya Gajjar Paediatric Nephology, Red Cross War Memorial Children’s

Hospital, Dept. of Paediatric Medicine, University of Cape Town, Western
Cape, South Africa

Gian Marco Ghiggeri Laboratory of Physiopathology of Uremia, Division

of Nephrology, Dialysis and Transplantation, Istituto Giannina Gaslini,
Genoa, Italy

Stuart L. Goldstein Division of Nephrology and Hypertension, The Heart

Institute, Cincinnati Children’s Hospital Medical Center, College of Medicine,
Cincinnati, OH, USA

Paul Goodyer Division of Pediatric Nephrology, Montreal Children’s

Hospital, McGill University, Montreal, QC, Canada

Lauren Graf Children’s Hospital at Montefiore, Albert Einstein College of

Medicine, Bronx, NY, USA

Larry A. Greenbaum Department of Pediatric Radiology, Emory University

School of Medicine, Atlanta, GA, USA

Ashima Gulati Department of Pediatrics, Nephrology Section, Yale School

of Medicine, New Haven, CT, USA
Contributors xxi

Meral Gunay-Aygun Medical Genetics Branch, The Intramural Program of

the Office of Rare Diseases, National Human Genome Research Institute,
Bethesda, MD, USA
Department of Pediatrics, McKusick-Nathans Institute of Genetic Medicine,
Johns Hopkins University School of Medicine, Baltimore, MD, USA
Indra R. Gupta Department of Pediatrics and Department of Human Genet-
ics, Division of Pediatric Nephrology, Montreal Children’s Hospital, McGill
University, Montréal, QC, Canada
William E. Harmon Boston Children’s Hospital, Harvard Medical School,
Boston, MA, USA
Jethro A. Herberg Imperial College London, London, UK
Austin G. Hester School of Medicine, Department of Urology, Wake Forest
Institute for Regenerative Medicine, Wake Forest University, Winston Salem,
NC, USA
Friedhelm Hildebrandt Harvard Medical School, Boston, MA, USA
Jacqueline Ho Department of Pediatrics, Division of Pediatric Nephrology,
Children’s Hospital of Pittsburgh of UPMC, University of Pittsburgh School
of Medicine, Pittsburgh, PA, USA
Elisabeth M. Hodson Centre for Kidney Research, The Children’s Hospital
at Westmead, Westmead, Sydney, NSW, Australia
Sydney School of Public Health, University of Sydney, Sydney, NSW,
Australia
Christer Holmberg Hospital for Children and Adolescents, University of
Helsinki, Helsinki, Finland
Takashi Igarashi National Center for Child Health and Development
(NCCHD), Tokyo, Japan
Kazumoto Iijima Department of Pediatrics, Kobe University Graduate
School of Medicine, Kobe, Japan
Elizabeth G. Ingulli Department of Pediatrics, University of California,
San Diego, CA, USA
Division of Nephrology, Rady Children’s Hospital San Diego, San Diego,
CA, USA
Kidney Transplant Program, Rady Children’s Hospital, San Diego, CA, USA
Hannu Jalanko Hospital for Children and Adolescents, University of
Helsinki, Helsinki, Finland
Neville Jamieson Department of Surgery, Addenbrookes Hospital,
Cambridge University Teaching Hospitals, Cambridge, UK
Xavier Jeunemaitre Department of Molecular Genetics, Hôpital Européen
George Pompidou, Paris, France
xxii Contributors

Harald J€uppner Departments of Medicine and Pediatrics, Endocrine Unit

and Pediatric Nephrology Unit, Massachusetts General Hospital and Harvard
Medical School, Boston, MA, USA
Clifford E. Kashtan Department of Pediatrics, Division of Pediatric
Nephrology, University of Minnesota Masonic Children’s Hospital, Minneap-
olis, MN, USA
Frederick J. Kaskel Division of Pediatric Nephrology, Children’s Hospital
at Montefiore, Albert Einstein College of Medicine, Bronx, NY, USA
Amrit Kirpalani Departments of Pediatrics, Schulich School of Medicine
and Dentistry, Western University, London, ON, Canada
Nine V. A. M. Knoers Departments of Medical Genetics, University Medical
Centre Utrecht, Utrecht, The Netherlands
Vera Koch Instituto da Criança- Pediatric Nephrology Unit, Department of
Pediatrics, University of Sao Paulo Medical School, Sao Paulo, Brazil
Martin Konrad Department of General Pediatrics, Pediatric Nephrology,
University Hospital, M€unster, Germany
Jordan Kreidberg Children’s Hospital Boston, Boston, MA, USA
MH Lafage-Proust INSERM U 1059, Université de Lyon, Saint-Etienne,
France
Bärbel Lange-Sperandio Dr. v. Hauner Children’s Hospital, Department of
Pediatric Nephrology, LMU, Munich, Germany
Richard S. Lee Department of Urology, Harvard Medical School, Boston
Children’s Hospital, Boston, MA, USA
Michael Levin Department of Medicine, Imperial College London, London,
UK
Elena N. Levtchenko Department of Pediatric Nephrology, Department of
Growth and Regeneration, University Hospitals Leuven, Katholieke
Universiteit Leuven, Leuven, Belgium
Deborah Lewis Sydney, NSW, Australia
Christoph Licht Division of Nephrology, The Hospital for Sick Children,
University of Toronto, Toronto, ON, Canada
Research Institute, Cell Biology Program, The Hospital for Sick Children,
Toronto, ON, Canada
Department of Paediatrics, University of Toronto, Toronto, ON, Canada
Chanin Limwongse Department of Medicine, Division of Medical Genetics,
Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkoknoi,
Bangkok, Thailand
Jonathan Loewen Department of Pediatric Radiology, Emory University
School of Medicine, Atlanta, GA, USA
Contributors xxiii

Eduardo Machuca Department of Nephrology, Medical School, Pontificia

Universidad Católica de Chile, Santiago, Chile
Nigel Madden NYU Langone Medical Center and NYU School of
Medicine, New York, NY, USA
Robert H. Mak Pediatric Nephrology, University of California, San Diego,
CA, USA
Pediatric Nephrology Division, Rady Children’s Hospital, San Diego, CA, USA
M. Loredana Marcovecchio University of Chieti, Chieti, Italy
Karen Maresso Section of Genomic Pediatrics, Medical College of
Wisconsin, Milwaukee, WI, USA
Ranjiv Mathews The Nevada School of Medicine, The Johns Hopkins
School of Medicine, Las Vegas, NV, USA
Tej K. Mattoo Pediatric Nephrology and Hypertension, Wayne State
University School of Medicine, Children’s Hospital of Michigan, Detroit,
MI, USA
Mignon McCulloch Department of Paediatric Intensive Care/Nephrology,
University of Cape Town, Red Cross War Memorial Children’s Hospital, Cape
Town, Western Cape, South Africa
Ruth A. McDonald Division of Nephrology, Seattle Children’s, University
of Washington, Seattle, WA, USA
Yosuke Miyashita Department of Pediatrics, Division of Pediatric Nephrol-
ogy, Children’s Hospital of Pittsburgh of UPMC, University of Pittsburgh
School of Medicine, Pittsburgh, PA, USA
Elizabeth Mullen Hematology Oncology, Dana-Farber/Boston Children’s
Blood Disorders and Cancer Center, Boston, MA, USA
Michio Nagata Graduate School of Comprehensive Human Sciences,
University of Tsukuba, Tsukuba, Japan
Behzad Najafian Department of Pathology, University of Washington,
Seattle, WA, USA
Koichi Nakanishi Department of Pediatrics, Wakayama Medical University,
Wakayama City, Japan
Thomas L. Nemeth Department of Pharmacy, Seattle Children’s, University
of Washington, Seattle, WA, USA
Carla M. Nester Stead Family Department of Pediatrics, Department of
Internal Medicine, Divisions of Nephrology, University of Iowa Children’s
Hospital, Carver College of Medicine, Iowa City, IA, USA
Galina Nesterova Section on Human Biochemical Genetics, Medical Genet-
ics Branch, National Human Genome Research Institute, National Institutes of
Health, Bethesda, MD, USA
xxiv Contributors

Patrick Niaudet Service de Néphrologie Pédiatrique, Hôpital Necker-

Enfants Malades, Université Paris-Descartes, Paris, France
Victoria F. Norwood University of Virginia, Charlottesville, VA, USA
Fierdoz Omar Chemical Pathology, University of Cape Town and National
Health Laboratory Service, Red Cross Children’s Hospital and Groote Schuur
Hospital, Cape Town, South Africa
Diclehan Orhan Department of Pediatric Pathology, Hacettepe University,
Sihhiye, Ankara, Turkey
Nelson Orta Service of Pediatric Nephrology, Children’s Hospital “Jorge
Lizarraga”, University of Carabobo, Valencia, Venezuela
Kenji Osafune Center for iPS Cell Research and Application (CiRA), Kyoto
University, Sakyo-ku, Kyoto, Japan
Seza Ozen Department of Pediatrics, Faculty of Medicine, Hacettepe
University, Sihhiye, Ankara, Turkey
Ameya Patil Department of Pediatrics, Medical College of Wisconsin, Chil-
dren’s Research Institute, Children’s Hospital Health System of Wisconsin,
Milwaukee, WI, USA
Connie Piccone Rainbow Babies and Children’s Hospital, Cleveland,
OH, USA
Matthew C. Pickering Centre for Complement and Inflammation Research,
Imperial College, London, UK
Maury Pinsk University of Alberta, Edmonton, AB, Canada
Zoltán Prohászka 3rd Department of Medicine, Faculty of Medicine,
Semmelweis University, Budapest, Hungary
Raymond Quigley Department of Pediatrics, University of Texas South-
western Medical Center at Dallas, Dallas, TX, USA
Lesley Rees Department of Nephrology, Great Ormond Street Hospital for
Children NHS Trust, London, UK
Kimberly Reidy Division of Pediatric Nephrology, Children’s Hospital at
Montefiore, Albert Einstein College of Medicine, Bronx, NY, USA
George S. Reusz 1st Department of Pediatrics, Faculty of Medicine,
Semmelweis University, Budapest, Hungary
Michelle N. Rheault Department of Pediatrics, Division of Pediatric
Nephrology, University of Minnesota Masonic Children’s Hospital, Minneap-
olis, MN, USA
Magdalena Riedl Research Institute, Cell Biology Program, The Hospital
for Sick Children, Toronto, ON, Canada
Department of Paediatrics, Innsbruck Medical University, Innsbruck, Tyrol,
Austria
Contributors xxv

Nancy M. Rodig Boston Children’s Hospital, Harvard Medical School,

Boston, MA, USA
Bernardo Rodríguez-Iturbe Nephrology Service, Hospital Universitario,
Maracaibo, Estado Zulia, Venezuela
Hesham Safouh Faculty of Medicine, Center for Pediatric Nephrology and
Transplantation (CPNT), Cairo University, Orman, Giza, Egypt
Rémi Salomon Service de Néphrologie Pédiatrique, Hôpital Necker-Enfants
Malades, Université Paris-Descartes, Paris, France
Isidro B. Salusky Division of Pediatric Nephrology, Clinical Translational
Research Center, David Geffen School of Medicine at UCLA, Los Angeles,
CA, USA
Simone Sanna-Cherchi Division of Nephrology, Columbia University,
College of Physicians and Surgeons, New York, NY, USA
Lisa M. Satlin Department of Pediatrics, Division of Pediatric Nephrology,
Icahn School of Medicine at Mount Sinai, New York, NY, USA
Franz Schaefer Division of Pediatric Nephrology, University Children’s
Hospital, Heidelberg, Germany
Claus Peter Schmitt Centre for Pediatric and Adolescent Medicine, Heidel-
berg, Germany
H. William Schnaper Division of Kidney Diseases, Ann and Robert H. Lurie
Children’s Hospital of Chicago, Department of Pediatrics, Northwestern
University Feinberg School of Medicine, Chicago, IL, USA
Takashi Sekine Department of Pediatrics, Toho University Faculty of
Medicine, Meguro-ku, Tokyo, Japan
Amita Sharma Department of Pediatrics, Pediatric Nephrology Unit, Mas-
sachusetts General Hospital and Harvard Medical School, Boston, MA, USA
Mohan A. Shenoy Department of Pediatric Nephrology, Royal Manchester
Children’s Hospital, Manchester, UK
Alfonso Silva Nephrology Service, Hospital Universitario, Maracaibo,
Estado Zulia, Venezuela
Sunder Sims-Lucas Department of Pediatrics, Division of Pediatric
Nephrology, Children’s Hospital of Pittsburgh of UPMC, University of Pitts-
burgh School of Medicine, Pittsburgh, PA, USA
Jodi M. Smith Division of Nephrology, Seattle Children’s, University of
Washington, Seattle, WA, USA
Michael J. G. Somers Division of Nephrology, Boston Children’s Hospital,
Harvard Medical School, Boston, MA, USA
Rajasree Sreedharan Medical College of Wisconsin, Milwaukee, WI, USA
Jennifer Stevens University Hospital Wales, Cardiff, S. Wales, UK
xxvi Contributors

Sarah J. Swartz Department of Pediatrics Renal Section, Baylor College of

Medicine and Texas Children’s Hospital, Houston, TX, USA
William E. Sweeney Jr. Department of Pediatrics, Medical College of Wis-
consin, Children’s Research Institute, Children’s Hospital Health System of
Wisconsin, Milwaukee, WI, USA
Rajesh V. Thakker Radcliffe Department of Medicine, Academic Endocrine
Unit, University of Oxford, OCDEM (Oxford Centre for Diabetes, Endocri-
nology and Metabolism), The Churchill Hospital Headington, Oxford, UK
Kálmán Tory Laboratory of Hereditary Kidney Diseases, Inserm UMR
1163, Paris, France
Department of Pediatrics, Semmelweis University, Budapest, Hungary
Howard Trachtman NYU Langone Medical Center and NYU School of
Medicine, New York, NY, USA
Alda Tufro Department of Pediatrics, Nephrology Section, Yale School of
Medicine, New Haven, CT, USA
Bradley L. Urquhart Departments of Pediatrics, Department of Medicine,
Physiology and Pharmacology, Western University, London, ON, Canada
Children’s Health Research Institute (CHRI), London, ON, Canada
Khashayar Vakili Boston Children’s Hospital, Harvard Medical School,
Boston, MA, USA
Rudolph P. Valentini Pediatric Nephrology, Children’s Hospital of Michi-
gan, Detroit, MI, USA
Wayne State University School of Medicine, Detroit, MI, USA
George van der Watt Chemical Pathology, University of Cape Town and
National Health Laboratory Service, Red Cross Children’s Hospital and
Groote Schuur Hospital, Cape Town, South Africa
Scott K. Van Why Medical College of Wisconsin, Milwaukee, WI, USA
William G. van’t Hoff Great Ormond Street Hospital, London, UK
Rene G. VanDeVoorde Cincinnati Children’s Hospital Medical Center,
Cincinnati, OH, USA
Enrico Verrina Nephrology, Dialysis and Transplantation Unit, Giannina
Gaslini Childrens Hospital, Genoa, Italy
Marina Vivarelli Division of Nephrology and Dialysis, Children’s Hospital
Bambino Gesù-IRCCS, Rome, Italy
Bradley A. Warady Pediatric Nephrology, Children’s Mercy Hospital,
Kansas City, MO, USA
Nicholas J. A. Webb Department of Pediatric Nephrology, Royal Manches-
ter Children’s Hospital, Manchester, UK
Contributors xxvii

Christopher B. Weldon Department of Surgery, Boston Children’s Hospital

and Harvard Medical School, Boston, MA, USA
Katherine Wesseling-Perry Division of Pediatric Nephrology, David
Geffen School of Medicine at UCLA, Los Angeles, CA, USA
Matthias T. F. Wolf Department of Pediatrics, University of Texas South-
western Medical Center at Dallas, Dallas, TX, USA
Craig S. Wong Pediatric Nephrology, University of New Mexico Health
Sciences Center, Albuquerque, NM, USA
Ikuyo Yamaguchi Division of Pediatric Nephrology, University of Texas
School of Medicine at San Antonio, University of Texas Health Science Center
at San Antonio, San Antonio, TX, USA
Hui-Kim Yap Yong Loo Lin School of Medicine, National University of
Singapore, Singapore, Singapore
Peter D. Yorgin Pediatric Nephrology, University of California, San Diego,
CA, USA
Pediatric Nephrology Division, Rady Children’s Hospital, San Diego,
CA, USA
Norishige Yoshikawa Department of Pediatrics, Wakayama Medical
University, Wakayama City, Japan
Michael Zappitelli Pediatrics, Division of Nephrology, Montreal Children’s
Hospital, McGill University Health Center, Montreal, QC, Canada
Israel Zelikovic Department of Physiology and Biophysics, Faculty of
Medicine, Technion – Israel Institute of Technology, Haifa, Israel
Division of Pediatric Nephrology, Rambam Medical Center, Haifa, Israel
Maria-Christina Zennaro Inserm U970, Paris Cardiovascular Research
Center, Paris, France
 Part I
Development
 Embryonic Development of the Kidney
1
Carlton Bates, Jacqueline Ho, and Sunder Sims-Lucas

Contents Vascular Development of the Kidney . . . . . . . . . . . . . . 14

Angiogenesis Versus Vasculogenesis . . . . . . . . . . . . . . . . . 15
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Origins of the Peritubular Capillary Endothelia . . . . . . 16
Studying the Kidney and Urinary Tract Molecular Control of Renal Vascular
Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Development of the Metanephros . . . . . . . . . . . . . . . . . . 7 Collecting System Development . . . . . . . . . . . . . . . . . . . . 17

Ureteric Bud Induction and Outgrowth . . . . . . . . . . . . . . . 17
Nephron Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Renal Branching Morphogenesis . . . . . . . . . . . . . . . . . . . . . 19
Specification of Nephron Progenitors/Cap Patterning of the Medullary and Cortical Collecting
Mesenchyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Ducts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Nephron Induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Nephron Segmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Lower Urinary Tract Development . . . . . . . . . . . . . . . . 21
Glomerulogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Anatomic and Functional Development . . . . . . . . . . . . . . 21
Molecular Control of Podocyte Terminal Molecular Control of Ureter and Bladder
Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Molecular Control of Glomerular Capillary Tuft Bladder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Ureter–Bladder Anastomosis . . . . . . . . . . . . . . . . . . . . . . . . . 25

Renal Stroma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

C. Bates (*) • J. Ho • S. Sims-Lucas

Department of Pediatrics, Division of Pediatric
Nephrology, Children’s Hospital of Pittsburgh of UPMC,
University of Pittsburgh School of Medicine, Pittsburgh,
PA, USA
e-mail: batescm@upmc.edu; jacqueline.ho2@chp.edu;
sunder.sims-lucas@chp.edu

# Springer-Verlag Berlin Heidelberg 2016 3

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_1
4 C. Bates et al.
Introduction
The mammalian kidney functions as a key regu-
lator of water balance, acid–base homeostasis,
maintenance of electrolytes, and waste excretion.
The performance of these activities depends on
the development of specific cell types in a precise
temporal and spatial pattern, to produce a suffi-
cient number of nephrons. Over the past several
decades, considerable advances have been made
in understanding the molecular basis for this
developmental program. Defects in this program
result in congenital anomalies of the kidney and
urinary tract, which are the leading causes of
chronic kidney disease and renal failure in chil-
dren. These developmental disorders range from
renal malformations, such as renal aplasia
(absence of the kidney), dysplasia (failure of nor-
mal renal differentiation), and hypoplasia (smaller
kidneys), to urinary tract abnormalities such as
vesicoureteral reflux and duplicated collecting
systems. This chapter describes the embryology
of the kidney and urinary tract, as a means to
understand the developmental origins of these
disorders.
Human kidney development starts in the fifth
week of gestation, and new nephrons are formed
until approximately 32–34 weeks gestation
[1–3]. Remarkably, nephron endowment is quite
variable ranging from 200,000 to 1.8 million
nephrons per person [4]. While the human kidney
continues to grow after 34 weeks gestation, this
occurs due to the growth and maturation of
existing nephrons, rather than the formation of
new nephrons. The mature mammalian kidney
cannot compensate for nephron loss due to renal
injury by the de novo generation of nephrons
[2, 5]. Therefore, the number of nephrons present
at birth in an individual is an important determi-
nant of long-term kidney health. Therefore,
reduced nephron number is associated with hyper-
tension and chronic kidney disease [6, 7].
Critical determinants of nephron endowment
are structural development and three-dimensional
nephron patterning. The formation of kidneys in
utero involves the coordinated regulation of
critical developmental processes: differentiation,
morphogenesis, and regulation of cell number.
Differentiation is the process by which precursor
cells or tissues mature into more specialized cells.
During kidney development, renal mesenchymal
cells have the potential to differentiate into
nephron epithelia or stromal cells and interstitial
fibroblasts. Morphogenesis describes the
process whereby cells and tissues acquire three-
dimensional patterns. This is particularly impor-
tant in the kidney, as the three-dimensional rela-
tionship between the nephrons, the vasculature,
and the collecting system is critical for
normal kidney structural function. Finally, the
regulation of cell number at different stages of
development is crucial. Such regulation maintains
a balance between cellular proliferation and
programmed cell death or apoptosis. All of these
processes are integrated tightly and regulated both
spatially and temporally in normal renal
development.
The molecular control of these developmental
processes has been the subject of several recent
comprehensive reviews [8–14]. Genetic, epige-
netic, and environmental factors all regulate dif-
ferentiation, morphogenesis, and cell number
within the developing kidney. Mutational ana-
lyses in animal models have provided significant
insights into the genetic control of renal develop-
ment. Genes critical for kidney development in
animal models include transcription factors that
act as master regulators of other genes, growth
factors that signal to other cells, and adhesion
molecules that regulate how cells interact with
each other and with the extracellular matrix.
Increasingly, analyses of humans with congenital
renal malformations (such as renal aplasia or
duplex kidneys) have identified gene mutations
originally described in animal models [15]. Recent
studies have also implicated epigenetic mecha-
nisms (defined as heritable changes in gene activ-
ity that are not caused by changes in DNA
sequence) in regulating nephron formation. Two
major classes of epigenetic molecules that appear
to regulate renal development include chromatin
remodeling proteins and regulatory RNAs such as
miRNAs [16–22]. Finally, environmental influ-
ences that may interact with genetic and
1 Embryonic Development of the Kidney
epigenetic factors are also important in determin-
ing nephron number and patterning. For example,
vitamin A deficiency leads to decreased nephron
number in rodents and has been implicated in
decreased renal size in humans [23, 24].
Studying the Kidney and Urinary Tract
Development
The methods for studying the molecular and
genetic control of kidney development have con-
tinued to evolve over the past several decades.
Visualization of tissue morphology and expres-
sion of individual genes and proteins in the devel-
oping kidney have traditionally been performed
on tissue sections by general staining (e.g., hema-
toxylin and eosin), detecting messenger RNA
(mRNA) via in situ hybridization or protein via
immunohistochemistry (Fig. 1a–c). The advent of
high-throughput technologies to detect gene
expression with microarrays and more recently
by high-throughput RNA sequencing has resulted
in the ability to assay the transcriptome of the
developing kidney and/or different kidney tissue
compartments in an unbiased fashion. These tech-
niques have led to large public databases that
describe the gene expression of the developing
kidney, including the GenitoUrinary Develop-
ment Molecular Anatomy Project (URL: www.
gudmap.org) and Eurexpress (URL: www.
eurexpress.org) [25–27].
A classical technique to analyze kidney devel-
opment is to culture rodent embryonic kidneys
in vitro as explants. Studies using these methods
were the first to show that reciprocal interactions
between the metanephric mesenchyme and the
ureteric bud are critical to induce the formation
of new nephrons and ureteric branching (Fig. 1d)
[28]. Moreover, kidney explants still allow one to
modulate the expression and function of specific
genes and proteins using reagents such as anti-
sense oligonucleotides or blocking antibodies
[29, 30]. While these experiments have been illu-
minating, the growth of embryonic kidney
explants differs from kidney development
in vivo in several key ways: lack of blood flow,
5
growth limitations from diffusion of the culture
media across the air–media interface, and distor-
tions in the three-dimensional kidney architecture
as explants flatten in culture.
Recently, several methods to generate more
physiological and quantifiable three-dimensional
reconstructions of developing kidneys and urinary
tracts have been developed. One method utilizes
exhaustive serial sectioning through developing
kidneys, histological staining, projection of each
serial image onto a monitor to identify each tissue
lineage, and rendering of the serial images into a
three-dimensional image (Fig. 1e) [31, 32]. This
technique allows for the quantification of both
developing nephron structures and the branching
ureteric tree. Another method uses optical projec-
tion tomography to image through the full thick-
ness of a developing kidney that has been whole
mount stained for a specific tissue and also per-
mits the quantification of these elements [33].
Physical, chemical, and genetic strategies can
be used to manipulate developing kidneys in vivo.
For example, ureteric obstruction in utero in sheep
and monkeys results in hydronephrotic kidneys
with renal dysplasia [34, 35]. In addition, dietary
manipulations including high doses of vitamin A or
dietary protein restriction result in kidney and uri-
nary tract defects [36, 37]. Transgenic approaches
have also been used to drive gene expression in
specific spatiotemporal patterns, usually in the
mouse. In these experiments, a transgenic construct
consisting of a tissue-specific promoter and the
gene of interest is randomly inserted into the
genome, leading to expression of that gene in a
tissue-specific pattern. The limitations of this
approach include: (1) that the random insertion
can result in unintended changes in gene expres-
sion (due to other nearby promoters/enhancers near
the site of integration), (2) that the insertion of the
transgene into the genome may lead to loss of
function of an endogenous gene, and that (3) epige-
netic factors may silence the construct. The
increased utilization of bacterial artificial chromo-
some (BAC) constructs, which contain more of
endogenous promoter elements than are found in
traditional plasmid constructs, has led to more
faithful and reliable transgene expression.
6 C. Bates et al.
Fig. 1 Experimental methods utilized to study kidney
development. (a) Hematoxylin and eosin (H&E)-stained
tissue section of a control postnatal day 0 mouse kidney.
The ureteric bud is outlined in yellow, and the arrow points
to the cap metanephric mesenchyme. (b) In situ hybridiza-
tion in a control mouse embryonic day 16.5 tissue section
for the transcription factor, Wt1, which stains the meta-
nephric mesenchyme and developing glomeruli. (c) Immu-
nofluorescent staining in a control embryonic day 14.5
mouse tissue section for the transcription factor, Wt1
(red), and lotus tetragonolobus lectin (LTL, green), which
stains the proximal tubule. (d) Embryonic culture of a
transgenic embryonic day 11.5 HoxB7GFP mouse kidney,
demonstrating branching ureteric structures (green) after
5 days of growth. (e) 3D reconstruction of an embryonic
day 13.5 mouse kidney with the ureteric epithelium
depicted in pink and developing nephron types including
renal vesicles (blue), comma-shaped bodies (red),
S-shaped bodies (purple), and glomeruli (green)
(Reproduced with kind permission from Springer Science
+Business Media: Sims-Lucas S. Kidney Development:
Methods and Protocols, Methods in Molecular Biology,
vol. 886, 2012, pp 81, Figure 3F) (f) H&E-stained tissue
section of a postnatal day 0 mouse kidney lacking
microRNAs in the ureteric lineage using a conditional
knockout approach (HoxB7Cre; Dicerflx/flx)
1 Embryonic Development of the Kidney
As opposed to transgenic approaches, homol-
ogous recombination is the method whereby a
gene is “knocked-out of” or “knocked-into” the
mouse genome. Using these methods, a gene of
interest is deleted so that it becomes
nonfunctional, or a gene (such as a green fluores-
cent reporter) is added to the genome at a specific
locus. A limitation to traditional knockout tech-
niques is that global loss of function of the gene
may result in extrarenal effects (such as early
embryonic lethality), which can impact or
severely limit the study of the gene’s function in
the kidney. Given these limitations, it has become
more common to perform conditional gene
targeting (e.g., with the Cre–loxP system)
(Fig. 1f) and/or inducible gene targeting (e.g.,
with tamoxifen), allowing for kidney- and/or uri-
nary tract-specific gene deletion (using a kidney
zebrafish specific Cre-) and/or at a particular time
(driving induction of gene targeting with a drug).
While most investigators using genetically
modified animals utilize mice, a growing number
of scientists study kidney development in other
model systems, such as avians, zebrafish, and
Xenopus. These simple systems are obviously
limited by their inability to recapitulate the com-
plex regulation of development previously
described in three-dimensional mammalian kid-
ney formation. However, their simplicity has
advantages in isolating possible molecular path-
ways involved in specific renal developmental
processes. These systems produce larger number
of embryos in a shorter period of time than in
mammals. Many of these models, such as
zebrafish and Xenopus, have only a
one-dimensional pronephric tubule as a kidney.
However, many of the genes which pattern such
simple structures have important roles in mamma-
lian metanephric kidney development. Increas-
ingly, investigators using have utilized more
sophisticated techniques such as transgenic fish
to examine the roles of genes or drugs in modify-
ing nephron progenitor populations [38]. Finally,
the advent of clustered regularly interspaced short
palindromic repeats (CRISPR)/CRISPR-
associated protein (Cas) techniques should even-
tually allow relatively quick and easy genetic
modifications of any animal model desired [39].
7
Development of the Metanephros
The mesoderm forms as one of the three embryonic
germ layers during gastrulation. The mammalian
kidney develops from the intermediate mesoderm,
lying between the somites and lateral plate meso-
derm, on the posterior abdominal wall of the devel-
oping embryo. In mammals, three pairs of
embryonic kidneys develop from the intermediate
mesoderm: the pronephros, the mesonephros, and
the metanephros (Fig. 2). At their maximal devel-
opment, the pronephros and mesonephros extend
from the cervical to the lumbar levels of the devel-
oping embryo. The pronephric and mesonephric
nephrons are induced to differentiate by signals
from the adjacent pronephric/mesonephric ducts,
paired epithelial tubules running in a longitudinal
course along the embryo on either side of the
midline. The mesonephric duct (also known as
the Wolffian duct) continues to grow caudally in
the embryos to eventually fuse with the cloaca,
which eventually gives rise to the bladder. The
pronephros is not functional in mammals, but is
the functional kidney in larval fish [40] and frogs
[41]. The mesonephros becomes the mature kidney
in these lower species and is functional in mam-
mals during embryogenesis. Ultimately, the pro-
nephros and mesonephros largely degenerate in
mammals. Portions of the mesonephros and meso-
nephric duct persist in mammals as the rete testis,
efferent ducts, epididymis, vas deferens, seminal
vesicle, and prostate in males [42]. In mammalian
females, remnants of the mesonephric tubules per-
sist as the epoophroron and paroophoron.
The mature mammalian kidney, the metaneph-
ros, is derived from two tissues, the ureteric bud
and metanephric mesenchyme (see a recent
review in Ref. [13]). Starting at approximately
embryonic day 10.5 in the mouse and the 5th
week of gestation in humans, reciprocal inductive
signals cause the ureteric bud to grow out from the
caudal portion of the mesonephric duct and the
metanephric mesenchyme to condense around the
ureteric bud to form nephron progenitors. The
ureteric bud ultimately gives rise to the collecting
system, including the collecting ducts, renal
calyces, renal pelvis, and ureters [2, 43]. In turn,
the nephrogenic metanephric mesenchyme
8 C. Bates et al.
Fig. 2 Schematic overview of kidney development.
Mammalian kidney development begins with the forma-
tion of the nephric duct, which is divided into three seg-
ments: pronephros, mesonephros, and metanephros. The
pronephros degenerates in mammals, whereas the meso-
nephros forms the male reproductive organs (rete testis,
efferent ducts, epididymis, vas deferens, seminal vesicles,
and prostate). The metanephros becomes the mature mam-
malian kidney and is derived from inductive interactions
between the metanephric mesenchyme and the ureteric bud
(Reproduced with kind permission from Springer Science
+Business Media: Moritz K, et al. Factors Influencing
Mammalian Kidney Development: Implications for Health
in Adult Life, Morphological Development of the Kidney,
Advances in Anatomy and Cell Biology, volume
196, 2008, pp 9–16, figure number 1)
differentiates into the epithelial cells that comprise
the mature nephron, including the parietal cells
and podocytes of the glomerulus, the proximal
convoluted tubule, the ascending and descending
limbs of the loops of Henle, and the distal convo-
luted tubule [2, 43]. Just after the condensation of
nephrogenic mesenchyme around the ureteric
bud, stromal metanephric mesenchyme (or renal
stroma) develops adjacent to the nephrogenic
mesenchyme. The renal stroma develops into
perivascular cells, vascular smooth muscle, fibro-
blasts, mesangial cells, renin-producing cells, and
even some peritubular endothelial cells (see
below).
The transcription factor, Odd-skipped related 1
(Osr1), is one of the several key molecules neces-
sary to specify portions of the posterior intermediate
mesoderm to become the mesonephric duct, ureteric
bud, and metanephric mesenchyme (nephrogenic
and stromal) [44]. Osr1-expressing cells in the inter-
mediate mesoderm have been shown to give rise to
most of the cellular components of the metanephric
kidney, including the nephron, vascular cells, inter-
stitial cells, and the mesonephric duct (including its
derivatives, viz., the ureteric bud/collecting system)
[45]. Other molecules critical for specification and
development of the mesonephric duct include the
transcription factors Paired box 2 (Pax2) [46], Pax8
[47], Lim homeobox 1 (Lhx1) [48], Gata binding
protein 3 (Gata3) [49], and the receptor tyrosine
kinase, Ret [50].
Many key signaling pathways have been shown
to drive reciprocal interactions between the ureteric
epithelium, the renal mesenchymal lineages, and
the renal vasculature. Ureteric bud outgrowth
depends on inductive signals from nephron pro-
genitors [51–53], stromal cells [54–58], and
angioblasts [59, 60], as well as from itself
[61]. The nephrogenic mesenchyme relies in part
on signaling from the ureteric bud and renal stroma
for self-renewal and for initiation of nephron for-
mation [8, 11, 62–64]. Subsequent nephrogenesis
(i.e., patterning and differentiation of nephron epi-
thelia) is highly dependent on factors from both
ureteric epithelial and stromal cells [57, 65, 66].
Nephron Formation
The metanephric mesenchyme gives rise to
nephrogenic mesenchyme or nephron progeni-
tors, which self-renew and have the capacity to
1 Embryonic Development of the Kidney
form the multiple epithelial cell types of the neph-
ron. Much research is geared toward understand-
ing this progenitor cell population, which is
critical for determining nephron endowment and
thus long-term kidney health.
Anatomically, there are several steps that take
place in nephron formation. After the ureteric bud
has penetrated the metanephric mesenchyme,
nephron progenitors condense around the first
ureteric ampulla, forming “cap mesenchyme.”
As the ureteric bud continues to branch and elon-
gate, the nephrogenic mesenchyme continues to
form new caps surrounding each ureteric tip. After
the initial few ureteric branches, the earliest cap
mesenchymal cells receive spatiotemporal cues to
begin the differentiation process to form
epithelialized renal vesicles [67]. Subsequent
growth and differentiation of the renal vesicle
results in formation of the comma-shaped body,
which then lengthens to form the S-shaped body.
The lower limb of the S-shaped body begins to
differentiate into glomerular podocytes. During
this time, endothelial cells migrate into the cleft
of the lower limb of the S-shaped body and will
ultimately form the glomerular capillary loops
[68, 69]. Simultaneously, nascent mesangial
cells, derived from renal stroma (see below), also
migrate into this cleft. Thus, the lower limb of the
S-shaped body forms the immature glomerulus.
Fig. 3 H&E-stained sections showing the four stages of
nephron formation in mice. (a) Image of a renal vesicle,
the first stage of nephron formation. (b) Image of a comma-
shaped body that has differentiated from a vesicle. (c)
Image of an S-shaped body, the third stage of nephron
formation. (d) Image of an immature glomerulus that
9
Concurrently, the middle and upper limbs of the
S-shaped body elongate and differentiate into
nephron tubules including proximal tubules,
loops of Henle (including descending and ascend-
ing limbs), and distal convoluted tubules. The
terminal ends of the distal convoluted tubules
eventually connect to ureteric epithelia, which
ultimately form the collecting system (collecting
ducts, renal pelvis, and ureters) (Fig. 3).
Nephrogenesis repeats in a radial fashion with
the first nephrons forming in the juxtamedullary
regions and last in the peripheral cortex, until the
full complement of nephrons is reached.
During prenatal and/or postnatal life, each
nephron increases in size and complexity as it
matures. Starting in the first month of life, matur-
ing proximal tubules transition from a columnar to
cuboidal epithelium, develop microvilli, and
increase their tubular dimensions [70, 71]. While
the earliest limbs of the Henle loop are located in
the renal cortex, subsequent maturation and elon-
gation of these limbs in utero results in the loops
pushing through the corticomedullary boundary
in term infants [72–74]. Postnatal maturation
results in the Henle loops eventually reaching
the inner renal medulla in the mature kidney.
Thus, the urinary concentrating capacity of new-
born infants is limited by a reduced medullary
tonicity gradient, due to the relatively shorter
differentiated from the lower limb of the S-shaped body
(Reproduced with kind permission from Springer Science
+Business Media: Sims-Lucas S. Analysis of 3D
Branching Pattern: Hematoxylin and Eosin Method,
Methods in Molecular Biology, volume 886, pp 73–86,
2012, Figure 3, panels A–D)
10
loops of Henle. Finally, as the distal convoluted
tubule matures, a portion of the cells are found in
close proximity to the future vascular pole of the
developing glomerulus, where they develop into
the macula densa [72].
Specification of Nephron Progenitors/
Cap Mesenchyme
Differentiation of the intermediate mesoderm and
metanephric mesenchyme into nephron progeni-
tors and their derivatives is genetically defined by
the sequential upregulation of several transcrip-
tion factors, cell adhesion molecules, and growth
factors. The intermediate mesoderm and early
metanephric mesenchyme express the transcrip-
tion factors Sal-like 1 (Sall1) [75], Sine oculis
homeobox homolog 1 (Six1) [76], Eyes absent
homolog 1 (Eya1) [77, 78], and the secreted pep-
tide growth factor, Glial-derived neurotrophic
factor (Gdnf) [79]. Induction of cap mesen-
chyme/nephron progenitors by the ureteric bud
tips is marked by expression of transcription fac-
tors such as Wilms tumor 1 (Wt1) [80]; Cbp/p300-
interacting transactivator, with Glu/Asp-rich
carboxy-terminal domain, 1 (Cited1) [81]; and
Sine oculis homeobox homolog 2 (Six2) [82], as
well as the transmembrane molecules cadherin-11
[83] and α8 integrin [84]. The earliest epithelial
derivative of the cap mesenchyme, the renal ves-
icle, is marked by the transcription factors Pax2
[85] and Lhx1 [86].
The use of tissue-specific gene knockouts gen-
erally via conditional transgenic techniques has
revealed the importance of several transcription
factors (including many mentioned above) in the
specification of the cap mesenchyme. Conditional
homozygous deletion of Eya1 [78], Six1 [76],
Pax2 [87, 88], Wt1 [80], Sall1 [75], Six2 [82], or
Lhx1 [86, 89] leads to bilateral renal aplasia or
severe renal dysgenesis from defects in cap mes-
enchyme specification and/or differentiation;
these mesenchymal defects are often accompa-
nied by ureteric induction and/or branching
abnormalities due in large part to loss of GDNF
signaling from the metanephric mesenchyme.
Pax2 mutant mice generate a metanephric
C. Bates et al.
mesenchyme that is unable to differentiate into
nephrons and fail to form the mesonephric duct,
which is required for ureteric bud induction
[88]. In Lhx1 [86, 89] and Sall1 [75] mutants,
the metanephric mesenchyme does induce ure-
teric bud formation, but it fails to elongate and
branch, and the mesenchyme is once again unable
to differentiate into nephrons. In Wt1 mutants, a
defective metanephric mesenchyme is formed,
but rapidly undergoes apoptosis [80]. Six2, a spe-
cific marker of nephron progenitors, is required
for the maintenance of progenitor cells, but not for
nephron differentiation; deletion of Six2 in mice
results in the formation of ectopic nephron tubules
and the rapid depletion of nephron progenitor
cells [82]. Similarly, the p53–E3 ubiquitin ligase,
murine double minute 2 (Mdm2), appears critical
for the maintenance of nephron progenitor
cells [91].
Recent studies have identified factors that gov-
ern the delicate balance of self-renewal and dif-
ferentiation of nephron progenitors, including
WNT genes. Studies from the mid-1990s showed
that lithium chloride, a potent inducer of Wnt
signaling, was able to drive tubulogenesis in iso-
lated rodent metanephric mesenchyme cultures
[92–94]. More recently Wnt9b, secreted from ure-
teric bud cells, was shown to be required for the
differentiation of nephron progenitor cells.
Genetic deletion of Wnt9b resulted in a failure of
nephron progenitors to undergo the mesenchymal
to epithelial transition that is required to form the
renal vesicle [95]. A subsequent study revealed
that Wnt9b, along with signals from the renal
stroma (see below), plays an essential role in
mediating the decision of nephron progenitors to
self-renew or differentiate [64, 96].
Another major signaling pathway that has been
shown to mediate nephron progenitor survival is
the fibroblast growth factor (FGF) signaling path-
way. FGF ligands are secreted peptides that bind
and signal through their receptor tyrosine kinases,
FGF receptors (FGFRs). Isolated nephrogenic
zone cell culture studies revealed that addition of
FGF1, 2, 9, and 20 ligands drives the expression
of nephron progenitor markers and progenitor
proliferation [97]. More recently, in vivo mouse
studies showed that Fgf9 and Fgf20 are critical for
1 Embryonic Development of the Kidney
maintaining nephron progenitor survival, prolif-
eration, and competence to respond to inductive
signals; furthermore, FGF20 mutations in humans
were shown to be associated with severe renal
dysplasia [98]. Other work in mice has identified
that the Fgfrs critical for metanephric mesen-
chyme development are Fgfr1 and Fgfr2. Condi-
tional deletion of Fgfr1 and Fgfr2 in the
metanephric mesenchyme leads to severe renal
dysgenesis [99–101].
Several studies have shown that a balance
between cell survival and apoptosis is necessary
for normal nephron progenitor function. Classic
in vitro studies using isolated metanephric mes-
enchyme have shown that nephron progenitors
undergo massive apoptosis when cultured without
an inducer [102]. Coculture with isolated ureteric
buds or heterologous inducers, such as embryonic
spinal cords, dampens apoptosis and drives pro-
genitor survival [43, 52]. Other studies have iden-
tified several factors that when added to
metanephric mesenchyme or nephrogenic zone
cell cultures drive progenitor survival, including
transforming growth factor-β2 (TGF-β2), TGFα,
leukemia inhibitory factor (LIF), epidermal
growth factor (EGF), FGF2, and bone morphoge-
netic protein 7 (BMP7) [62, 103–105]. Whether
some or all of these factors act as endogenous
nephron progenitor inducers remains to be deter-
mined. Counterbalancing cell survival, apoptosis
is required for normal nephron progenitor func-
tion. Moreover, suppression of apoptosis via phar-
maceutical or genetic means leads to kidney
malformations including abnormal ureteric
branching and defective nephrogenesis [106,
107]. Relative “overabundance” of nephron pro-
genitors also leads to epithelial and/or stromal cell
defects [108, 109].
Recent studies have revealed the importance of
epigenetic mechanisms that regulate nephron pro-
genitor specification, survival, and potential for
differentiation. Histone deacetylases (HDACs)
are enzymes that remove acetyl groups from his-
tones, which then modulate (usually stimulate)
gene transcription. Recent work has revealed
that Class I HDACs are highly expressed in neph-
ron progenitors and are required for the proper
expression of several key developmental genes
11
including Osr1, Eya1, Pax2, Wt1, and Wnt9b
(among others) [16]. MicroRNAs (miRNAs) are
small noncoding RNAs that bind to specific
mRNA targets to block translation and promote
mRNA degradation. Conditional targeting of
dicer, an enzyme required for the processing of
all miRNAs, in mouse nephron progenitors, led to
a loss of the progenitors due to excessive apopto-
sis, likely from upregulation of the proapoptotic
protein, Bim [22]. A more recent study revealed
that conditional deletion of a specific miRNA
cluster, the miR-17~92 complex, in nephron pro-
genitors led to decreases in progenitor prolifera-
tion, fewer numbers of nephrons, proteinuria, and
podocyte damage. Moreover, this report was the
first to identify a specific miRNA cluster essential
for kidney development [110].
Nephron Induction
The initial differentiation step of nephron progen-
itors is to undergo a mesenchymal to epithelial
transition to form the renal vesicle. In vitro exper-
iments utilizing isolated rodent metanephric mes-
enchymal rudiments (similar to and including
some of the survival studies noted above) have
identified exogenous factors that stimulate neph-
ron progenitors to undergo tubulo-epithelial dif-
ferentiation [52]. Some of these growth factors
can act alone or in concert with others and include
FGF2 [111], LIF [63, 105, 112], TGFβ2 [105,
113], growth/differentiation factor-11 (GDF-11)
[105, 114], and WNT1/4 [115, 116]. Sequestration
of Wnt ligands in intact rodent kidney explants by
addition of secreted frizzled-related proteins
(sFrps) leads to decreases in mesenchyme-derived
tubulogenesis [117]. More recent mouse genetic
experiments have identified at least some of the
critical endogenous pathways and growth factors
necessary for the induction of the mesenchymal to
epithelial transition. For example, global deletion
of Wnt4, normally expressed in renal vesicles, did
not perturb cap mesenchyme formation; however,
the mutant nephron progenitors were completely
unable to form renal vesicles [95]. Conditional
deletion of Fgf8 in the metanephric mesenchyme
leads to a block of nephrogenesis beyond the renal
12
vesicle stage. Fgf8 likely normally acts with Wnt4
to drive Lhx1 expression [118, 119]. Interestingly,
global deletion of Fgfr-like 1, a membrane-bound
Fgf receptor that lacks an intracellular tyrosine
kinase domain, also leads to a block in
nephrogenesis similar to Fgf8 conditional
mutants [120].
Nephron Segmentation
Establishment of a proper proximal–distal axis is
critical for normal nephron segmentation. Nega-
tive reciprocal interactions between Wt1 and
Pax2 at early stages of nephron development
appear vital to proximal–distal axis patterning
[121–123]. In the S-shaped body, Wt1 is localized
to the lower limb and inhibits Pax2 expression,
which together drives cells toward podocyte fates
[124]. Transgenic mice with overexpression of
Pax2 throughout the embryo including develop-
ing nephrons develop glomerular defects and
renal cystic dysplasia [125]. In contrast Pax2 is
expressed in the upper limb of the S-shaped body
and represses Wt1 expression, stimulating these
cells to become proximal and distal tubular neph-
ron segments [118, 126].
Two other transcription factors critical for
proximal–distal axis patterning of the nephron
include Lhx1 and Brain specific homeobox 1
(Brn1), both of which are expressed at the renal
vesicle stage. Conditional deletion of Lhx1
throughout the metanephric mesenchyme blocks
nephrogenesis at the renal vesicle stage and also
leads to a loss of Brn1 expression [86]. Condi-
tional targeting of Brn1 in the metanephric mes-
enchyme does not block proximal nephrogenesis;
however, the loop of Henle fails to form, and
distal convoluted tubules fail to terminally differ-
entiate [73]. These results suggest that Lhx1 acts
earlier in nephron patterning than Brn1, which is a
critical distal nephron patterning.
Notch receptor signaling, mediated largely by
Recombining binding protein suppressor of hair-
less (Rbpsuh), appears critical for proximal neph-
ron patterning [127–129]. Use of a Notch
inhibitor in mouse metanephric kidney explants
led to a loss of proximal cell fates, including
C. Bates et al.
glomeruli and proximal tubules [127]. In vivo,
conditional deletion of Notch2 or Rbpsuh in
mouse metanephric mesenchyme leads to an
absence of proximal tubules and glomerular epi-
thelium [128]. Finally, ectopic Notch expression
in nephron progenitor cells results in the prema-
ture differentiation of the progenitors into proxi-
mal nephron epithelia [130].
Glomerulogenesis
Glomerulogenesis is initiated when the comma-
shaped body differentiates into the S-shaped body
[2, 131]. During this time, immature podocytes
along the lower limb are highly proliferative and
have a columnar shape with apical cell attach-
ments and a single-layer basement membrane
[131]. Concurrently, endothelial and mesangial
cell progenitors are recruited into the lower cleft
of the S-shaped body, which will become the
vascular pole [132]. While mesangial cells origi-
nate from the renal stroma (see below), the devel-
opmental origin of the glomerular endothelium is
still unclear. Transplantation of avascular rodent
embryonic kidney rudiments under neonatal kid-
ney capsule led to the formation of endothelial
precursors or angioblasts originating from the
graft metanephric mesenchyme [132–134]. How-
ever, engraftment of embryonic rat kidney rudi-
ments onto avian chorioallantoic membrane led to
vascular ingrowth of avian vessels into the rat
glomeruli [135]. As will be expanded on in the
Vascular Development section below, it is likely
that both processes/sources contribute to the for-
mation of glomerular capillaries. As the S-shaped
body matures, the lower cleft transforms into a
cup shape configuration.
At this time the podocytes lose their prolifera-
tive ability [136] and differentiate, forming foot
processes and slit diaphragms, specialized intra-
cellular junctions critical for proper glomerular
filtration [137, 138]. Concurrently, the composi-
tion of the glomerular basement membrane
changes from laminin-1 to laminin-11 and from
α -1 and α -2 type IV collagen chains to α -3, α -4,
and α -5 type IV collagen chains [139]. Several
mouse knockout mice models have shown how
1 Embryonic Development of the Kidney
failure in these transitions leads to structural and
functional glomerular basement membrane
defects [140–142]. At this stage of development,
the nascent mesangial cells act as a scaffold for the
formation of the glomerular capillary loops and
ultimately form the supportive core for the entire
glomerulus via the deposition of extracellular
matrix [143, 144]. Developing glomerular endo-
thelial cells branch extensively during this time
and begin differentiating into fenestrated endothe-
lia [2] (see Vascular section below).
By 32–34 weeks gestation, glomerulogenesis/
nephrogenesis ceases in humans, whereas it per-
sists in mice and rats for 7–10 days following full
gestation [2]. In newborn humans, the superficial
glomeruli are the most immature and are smaller
than the deeper juxtamedullary glomeruli
[71]. While no new glomeruli are formed after
birth, they continue to grow and mature postna-
tally, reaching their adult size at approximately
three and a half years of age [71].
Molecular Control of Podocyte
Terminal Differentiation
Transcription factors and epigenetic factors,
including microRNAs, have been shown to be
critical for podocyte differentiation. Examples of
essential transcription factors include Wt1,
podocyte expressed 1 (Pod1), Lim homeobox 1b
(Lmx1b), and Mafb. Several studies utilizing
murine genetic knockout models of Wt1 have
demonstrated its critical roles in mediating
podocyte differentiation [145–148]. In humans,
WT1 mutations can lead to diffuse mesangial scle-
rosis, characterized by podocyte differentiation
defects resulting in varied glomerular lesions and
proteinuria, and can occur as an isolated disease or
in association with Denys–Drash or Frasier syn-
dromes [149–152]. Deletion of Pod1, expressed
in stromal cells, leads to nonautonomous
podocyte defects at the capillary loop stage in
mice [153]. Genetic deletion of Lmx1b or Mafb
leads to podocyte differentiation defects past the
capillary loop stage [154, 155]. Furthermore,
mutations in the LMX1b gene in humans lead to
nail–patella syndrome, which is often associated
13
with glomerular basement membrane thickening
and proteinuria that can progress to chronic kid-
ney disease [156, 157]. Three studies recently
showed the importance of microRNAs in
maintaining differentiated podocytes in the
mouse. Targeted ablation of dicer in murine
podocytes, resulting in a loss of all miRNAs, led
to podocyte injury, severe proteinuria, and tubular
damage starting 2 weeks after birth [20, 158, 159].
Molecular Control of Glomerular
Capillary Tuft Development
There are several signaling cascades that have
been implicated in the homing and maturation of
the endothelial and mesangial precursors to form
the glomerular capillary tuft. Vascular endothelial
growth factor (VEGF), which is secreted from the
podocytes at the S-shaped body stage, promotes
recruitment of endothelial precursors to the vas-
cular cleft [160, 161]. Angiopoietin-1 and -2,
growth factors expressed by podocytes and
mesangial cells, respectively, are also critical for
normal glomerular capillary development
[162]. Mesangial cell recruitment into the cleft is
largely mediated by the secretion of platelet-
derived growth factor (PDGF)-B by endothelial
cells, which binds PDGF receptor-β (PDGFRβ)
on the mesangial cell progenitors [163]. Mice that
lack either Pdgfβ or Pdgfrβ fail to form glomeru-
lar capillary tufts demonstrating the importance of
mesangial cell recruitment [164, 165]. Finally,
Notch2 and its ligand Jagged1 are critical for
glomerular endothelial and mesangial cell devel-
opment. Notch2 hypomorphic mice and Notch2/
Jagged1 compound heterozygous mice develop
glomerular aneurysms and possess no mesangial
cells [166].
Renal Stroma
The renal stroma, like the nephrogenic mesen-
chyme, is derived from Osr1-positive intermedi-
ate mesoderm and the metanephric mesenchyme
[167, 168]. A hallmark of the initial renal stroma
is the expression of the transcription factor,
14
Foxd1, which is seen as early as E11.5 in the
mouse. The renal stroma is initially located at
the periphery of the kidney and interdigitates
between the developing nephron units and ure-
teric tips. One function of the early renal stroma
is to support framework for the developing ves-
sels, nephron progenitors, and ureteric epithelia.
As embryonic kidney development progresses,
stromal cells are present in both the peripheral
renal cortex and the medulla surrounding devel-
oping collecting ducts. At this time, the cortical
stroma expresses Foxd1, Aldehyde dehydroge-
nase 1 family, member A2 (Raldh2), Retinoic
acid receptor α (Rarα), and Rarβ2, while the
medullary stroma expresses Fgf7, Pod1, and
Bmp4. Many of these stromally expressed genes
have been shown to be critical for nephrogenesis
and ureteric branching morphogenesis by virtue
of mouse knockout studies [54, 55, 57, 58, 153,
169]. At birth, many of the developmental stromal
cells have undergone apoptosis and are replaced
by nephron segments such as loops of Henle
[170]. Many stromal derivatives do survive giving
rise to fibroblasts, lymphocyte-like cells, glomer-
ular mesangial cells, renin-expressing cells, vas-
cular smooth muscle cells, pericytes, and a
subpopulation of peritubular endothelial cells
[168, 170, 171].
As noted, signaling from the renal stroma is
critical for ureteric morphogenesis. Three genes/
pathways expressed within the stroma, retinoic
acid, Foxd1, and Pod1, modulate ureteric
branching by regulating expression of Ret, a
receptor tyrosine kinase expressed in ureteric
tips and required for ureteric development (see
below). Vitamin A is converted to its active
form, retinoic acid, by the enzyme Raldh2 in the
renal stroma. Moreover, blockade of retinoic acid
signaling in mice, by deletion of Raldh2 or by
combined deletion of the retinoic acid receptors,
Rarα and Rarβ2, leads to hypoplastic kidneys
with a reduction in the number of ureteric
branches; the ureteric branching defects are linked
to the downregulation of Ret expression in mutant
embryos, which in the case of the retinoic acid
receptor mutants can be rescued by forced
re-expression of Ret in the ureteric tissues [55,
56, 172]. Foxd1 (expressed in cortical stroma
C. Bates et al.
and the renal capsule) or Pod1 (found in medul-
lary stroma) appears to appropriately restrict Ret
expression to ureteric tips; genetic deletion of
either gene leads to mis-expression of Ret
throughout the entire ureteric tree and subsequent
ureteric branching defects [54, 57, 153, 173].
Cross talk from the stroma is also critical for
nephron development. Mouse genetic studies
show that Foxd1 and Pod1 are necessary for nor-
mal nephron patterning (in addition to ureteric
morphogenesis) [54, 153]. Loss of Foxd1 in
mice leads to premature differentiation of stromal
cells, which inhibits Bmp7-mediated nephron
progenitor differentiation [174]. Two recent
studies have shown how complete ablation of
renal cortical stroma with diphtheria toxin leads
to abnormally thickened nephron progenitor
caps and a decreased ability of progenitors to
differentiate [64, 175]. Mechanistically, it
appears that loss of the protocadherin Fat4 in the
stroma perturbs the activity of the transcription
factors, Yap and Taz, which in turn disrupts
Wnt9b signaling and nephron differentiation
[64]. Thus, in addition to providing a “frame-
work” for the rest of the developing kidney, the
renal stroma actively signals to other renal line-
ages and differentiates into cells that populate the
mature kidney.
Vascular Development of the Kidney
The adult kidney receives approximately 25 % of
the cardiac output. Furthermore, the adult kidney
has a high complex vascular network with differ-
ent functions and therefore different specialized
endothelia depending on location [176]. Specifi-
cally, three major types of endothelial cells are
present within the kidney, including fenestrated
(in glomerular capillaries), fenestrated with dia-
phragms (in peritubular capillaries and ascending
vasa recta), and continuous capillaries
(in descending vasa recta) (Fig. 4). Not surpris-
ingly, these various endothelial cell types have
heterogeneous expression profiles and often
appear to have different developmental origins
[21, 177, 178].
1 Embryonic Development of the Kidney
Fig. 4 Electron microscopy demonstrating the varied
renal endothelium. (a, e) Glomerular capillaries contain
fenestrated endothelium without diaphragms (e, arrows)
and share a basement membrane (*) with podocyte foot
processes (large arrowhead) that are separated by slit
diaphragms (small arrowhead). (b, f, g) Peritubular capil-
laries have fenestrated endothelial cells that are covered
with diaphragms (f, g, arrows) and have a thick basement
membrane (*) separating them from the tubular cells.
(c, h, i) Ascending vasa recta (AVR) also have fenestrated
Angiogenesis Versus Vasculogenesis
Blood vessels can form by angiogenesis, in which
new vessels sprout from existing vessels, or by
vasculogenesis, in which de novo vessels form
from endothelial progenitors (Fig. 5). Extensive
linage tracing experiments and transplantation
studies have shown that both of likely occur in
renal vascular formation [176, 179–181].
The early renal artery and efferent arterioles
appear to be primary sites from which new
angiogenic vessels sprout within the developing
kidney. Simultaneously, renal endothelial
15
endothelium with diaphragms (c, h, i, arrow). (d, h, i)
Descending vasa recta (DVR) possess endothelium that is
non-fenestrated, thick, and continuous (d, h, i). RBC red
blood cell, EC endothelial cell. Panels (a–d) scanning
electron micrographs. Panels (e–i) transmission electron
micrographs (Reproduced with kind permission from
Springer Science+Business Media: Stolz DB and Sims-
Lucas S. Unwrapping the origins and roles of the renal
endothelium, Pediatr Nephrol. 2015;30(6):865–72,
Figure 2)
progenitors (marked by Flk1/Vegfr2) form primi-
tive vascular networks, particularly within the
renal stroma, that subsequently join with and are
pruned by the angiogenic vessels [182]. The
vasculogenic endothelial cell progenitors within
the kidney appear to arise from the
Osr1-expressing intermediate mesoderm, as is
the case with the rest of the metanephric
kidney [45]. Finally, specification of the
endothelium (whether angiogenic or
vasculogenic in origin), including arterial,
venous, capillary, or lymphatic fates, is driven
by growth factor signaling pathways and
16
Fig. 5 Schematic diagram of vascular formation in the
developing mouse kidney. Top panels. Angiogenic ves-
sels (red) grow out from the major branches of the renal
artery and track with the branching ureteric epithelium
(orange). Middle panels. Vasculogenic vessels form from
progenitor cells (yellow, middle panel) within the meta-
nephric mesenchyme (blue) and form a primitive vascular
plexus (yellow, right panel). Bottom panels. Schematic
transcription factors, including Vegf, Ephrin,
Notch, and Sox [183].
Origins of the Peritubular Capillary
Endothelia
While the formation of glomerular capillaries has
been extensively studied (see above), the origin of
C. Bates et al.
diagrams depicting how a combination of angiogenesis
and vasculogenesis likely leads to vessel formation in the
kidney. E10.5–12.5 = embryonic days 10.5–12.5
(Reproduced with kind permission from Springer Science
+Business Media: Stolz DB and Sims-Lucas
S. Unwrapping the origins and roles of the renal endothe-
lium, Pediatr Nephrol. 2015;30(6):865–72, Figure 1)
peritubular capillaries has been less well defined.
Recent studies, however, have found that
peritubular capillaries arise from a combination
of resident endothelial progenitors as well as
invading angiogenic vessels [182, 184,
185]. One intriguing study found that Foxd1-pos-
itive renal cortical stroma cells give rise to a subset
of the peritubular endothelia but not the glomeru-
lar endothelia [184].
1 Embryonic Development of the Kidney
Molecular Control of Renal Vascular
Development
A key signaling pathway mediating renal vascular
development is the VEGF pathway. VEGF
ligands are expressed early in the metanephric
mesenchyme and later in the developing glomer-
ular podocytes, distal tubules, and collecting
ducts and at low levels in the proximal tubules
[68, 69]. Developing endothelial cells, including
those that arise from existing vessels and those
forming de novo, express VEGF receptors; thus,
VEGF signaling appears to drive both angiogen-
esis and vasculogenesis within the kidney. Inter-
estingly, Vegfr2 is present on the apical surface of
ureteric epithelium, which likely accounts for
the stimulatory role of Vegf on ureteric growth
[132, 186].
Hypoxia-inducible factors (HIFs), a family of
transcriptions factors, are likely master regulators
of angiogenesis and vasculogenesis within the
developing kidney [187]. These molecules are
activated during periods of low oxygenation, as
occurs during embryogenesis, and are
downregulated postnatally. The HIF genes are
largely located in the nephrogenic zone, including
podocytes, developing collecting ducts, and
developing endothelial cells [187, 188]. HIF pro-
teins induce expression of VEGF ligands, Vegfr1,
and Vegfr2 during kidney development by directly
binding to hypoxia-responsive elements on those
genes [189–192].
Angiopoietin (Ang) growth factors that bind to
Tie receptors also appear to have critical roles in
renal vascular development and are at least in part
regulated by HIF and VEGF signaling [193,
194]. Ang1, which is expressed in the metaneph-
ric mesenchyme, maturing nephron tubules, and
podocytes, signals through Tie2, which is
expressed on endothelial cells [195]. Conditional
deletion of Ang1 or Tie2 in mice leads to glomer-
ular capillary defects including endothelial cells
that do not attach to the basement membrane [196,
197]. Ang2, expressed in vascular smooth muscle
cells and pericytes, binds to Tie1 that is expressed
by endothelial cells. Genetic deletion of Ang2
leads to upregulation of Tie2 signaling and signif-
icant defects in renal peritubular capillaries [198].
17
The Notch signaling pathway appears to regu-
late renal angiogenic vessel outgrowth [199].
Notch receptors induce sprouting by stimulating
the expression of Vegfr2 in vascular tip cells.
Simultaneously Notch inhibits Vegfr2 signaling
in adjacent vascular stalk cells, causing them to
remain dormant. Thus Notch regulates the pattern
of branching in angiogenic vessels.
Collecting System Development
Ureteric bud formation begins in the 5th week of
gestation in humans and at embryonic day 10.5 in
mice. As noted previously, signals from the meta-
nephric mesenchyme cause the ureteric bud to
form from the mesonephric duct and then invade
the mesenchyme. Overall, collecting duct system
development includes (i) ureteric bud outgrowth,
(ii) branching of the ureteric bud, and (iii) pattern-
ing of the collecting duct system, all of which is
discussed in more detail below.
Ureteric Bud Induction and Outgrowth
Failure of ureteric bud outgrowth results in renal
aplasia, which can occur unilaterally or bilaterally
[200]. The GDNF–RET signaling pathway is cru-
cial for bud outgrowth. The receptor tyrosine
kinase RET and its coreceptor GFRα1 are
expressed in the mesonephric duct, the initial ure-
teric bud, and later in the branching ureteric tips,
while its ligand, GDNF, is present in the meta-
nephric mesenchyme (Fig. 6) [201–203]. Targeted
deletion of Gdnf, Ret, or Gfrα1 in mice generally
results in bilateral renal aplasia due to a lack of
ureteric bud outgrowth [202, 204–209]. Heterozy-
gous mutations of RET have also been identified
in humans with bilateral renal aplasia, and a rare
RET polymorphism has been reported in individ-
uals with nonsyndromic vesicoureteral reflux
[210, 211]. Moreover, the renal aplastic pheno-
type is not fully penetrant in a subset of Gdnf /
or Ret/ mutant mice [202, 204], suggesting that
other molecular pathways play a role in ureteric
bud outgrowth. Some examples of other pathways
include signaling through integrins such as
18
Fig. 6 Schematic diagram of the molecular control of
ureteric bud induction. GDNF is secreted from the meta-
nephric mesenchyme and binds to its receptor, Ret (and its
coreceptor GFRα1) on the mesonephric duct, to induce
ureteric bud formation. Slit2/Robo2 and FoxC1 inhibit
the domain of GDNF expression and thus limit ureteric
α8 integrin [84] and enzymes involved in proteo-
glycan synthesis such as heparan sulfate
2-sulfotransferase (Hs2st) [212].
Many studies have focused on the molecular
mechanisms that regulate GDNF–RET expression
and/or signaling. Prior to kidney development,
Ret is expressed throughout the mesonephric
duct, and Gdnf is present throughout the interme-
diate mesoderm adjacent to the mesonephric duct
[50, 201]. At the time of ureteric bud induction,
Gdnf expression becomes restricted to the poste-
rior intermediate mesoderm next to the site of
ureteric bud outgrowth; once the ureteric bud has
invaded the mesenchyme, Ret expression
becomes restricted to ureteric bud tips [50]. In
vitro studies with Gdnf-soaked agarose beads
show that the entire length of the mesonephric
duct is competent to respond to Gdnf by initiating
ectopic ureteric bud formation [201, 213]. More-
over, mice that ectopically express Gdnf or Ret
in vivo develop renal malformations such as
duplex kidney and hydronephrosis [214,
215]. Together, these data show that GDNF–RET
signaling is highly spatially regulated for a single
ureteric bud to form in the correct location from
the mesonephric duct.
C. Bates et al.
bud induction to a single site from the mesonephric duct.
Sprouty1 (in the mesonephric duct) and Bmp4 (in tailbud-
derived mesenchyme around the mesonephric duct)
repress GDRF–Ret signaling and thus restrict ureteric
bud induction to its proper site
At least three genes, Foxc1, Slit2, and Robo2,
are thought to be crucial in restricting Gdnf to the
posterior intermediate mesoderm (Fig. 6). Homo-
zygous mutant mice for all three genes develop
ectopic ureteric buds, multiple ureters,
hydroureter, and anterior expansion of Gdnf
expression [216, 217]. Foxc1 encodes a transcrip-
tion factor co-expressed with Gdnf in the meta-
nephric mesenchyme [216]. In the central nervous
system, the secreted protein Slit2 functions as a
chemorepellent during migration of axons that
express its receptor Robo2 [218, 219]. In the
developing kidney, Slit2 is expressed in the meso-
nephric duct, and Robo2 is detected in the meta-
nephric mesenchyme [220]. ROBO2 missense
mutations in humans have been identified in fam-
ilies with vesicoureteral reflux and/or duplex
kidneys [221].
Two other genes, Sprouty1 (Spry1) and Bmp4,
act in a negative feedback loop with GDNF–RET
signaling (Fig. 6). Loss of Spry1, which is nor-
mally expressed in the mesonephric duct, results
in ectopic ureteric bud induction, multiple ureters,
multiplex kidneys, and hydroureter [222,
223]. Spry1 mutant embryonic kidneys have
increased expression of Gdnf and GDNF–RET
1 Embryonic Development of the Kidney
target genes and have increased sensitivity to
GDNF-induced ureteric induction in organ cul-
ture. Bmp4 is expressed in the tailbud-derived
mesenchyme (different than renal mesenchyme)
immediately next to the mesonephric duct and
ureteric bud [169, 224]. Mice heterozygous for
Bmp4 have ectopic or duplicated ureteric buds,
resulting in hypodysplastic kidneys, hydroureter-
onephrosis, and ureteral duplications [225,
226]. In vitro, Bmp4 has been shown to block
the ability of Gdnf to induce ureteric bud out-
growth from the mesonephric duct [85,
227]. BMP4 mutations have also been described
in humans with renal tract malformations [228].
The downstream effects of GDNF–RET sig-
naling, namely, ureteric bud proliferation, sur-
vival, and ureteric outgrowth and branching, are
mediated by the transcription factors, Etv4 and
Etv5. Combined deletion of Etv4 and Etv5 causes
bilateral renal aplasia in mice [229]. Etv4 and Etv5
drive expression of several critical genes in the
ureteric bud tip, including Wnt11, Cxcr4, Mmp14,
Myb, and Met [229]. Furthermore, genetic dele-
tion studies in mice have shown that Wnt11 is
necessary for normal Gdnf expression in the meta-
nephric mesenchyme [230].
Renal Branching Morphogenesis
After growing into the metanephric mesenchyme,
the ureteric bud bifurcates into a T-shaped struc-
ture. The ureteric bud then continues to branch,
ultimately generating about 15 generations of
branches, with the earliest branches remodeling
to form the calyces and renal pelvis [231]. The
process of ureteric branching includes (1) expan-
sion of the ureteric bud at its leading tip (termed
the ampulla), (2) division of the ampulla to form
new branches, and (3) elongation of newly formed
branches [232, 233]. In humans, during the first
9 generations of branching, ureteric bud tips
induce formation of new nephrons from the sur-
rounding cap mesenchyme at about a 1:1 ratio
[2]. Ureteric bud branching is completed by the
20th–22nd week of human gestation, and subse-
quent collecting duct maturation occurs by elon-
gation of peripheral (cortical) segments and
19
remodeling of central (medullary) segments
[2]. At this stage, four to seven new nephrons
are induced around each tip of a terminal
collecting duct branch [2, 43].
Localized cell proliferation contributes to ini-
tial ureteric bud outgrowth from the mesonephric
duct, formation and growth of ampullae, and elon-
gation of ureteric branches [61, 108, 234,
235]. Cell survival is also critical for normal
renal branching morphogenesis; defects in cell
survival are associated with renal cystic dysplasia
and urinary tract dilatation. Moreover, targeted
deletion of bcl2 [236] and AP-2 [237], genes
critical for cell survival, results in increased apo-
ptosis and collecting duct cysts in mice. In addi-
tion, experimental models of fetal and neonatal
urinary tract obstruction lead to apoptosis in
dilated collecting ducts [238, 239].
Several signaling pathways are necessary for
branching morphogenesis. In addition to its role in
ureteric bud induction, GDNF–RET signaling has
been shown to be critical for ureteric branching
in vivo and in vitro [213, 215]. As noted, Wnt11,
expressed in ureteric tips, is necessary for
maintaining normal Gdnf expression; conversely,
Wnt11 expression is reduced when Gdnf signaling
is absent. Furthermore, Wnt11 mutant mice
have defective ureteric branching morphogenesis
and thus develop renal hypoplasia
[230, 240]. Finally, conditional targeting of
β-catenin, a key mediator of canonical Wnt sig-
naling, in the ureteric lineage results in aberrant
branching, loss of ureteric bud tip gene expres-
sion, and premature expression of differentiated
collecting duct genes [241, 242].
Fibroblast growth factor signaling is also crit-
ical for ureteric branching. In vitro studies have
shown that exogenous Fgf ligands differentially
modulate ureteric bud growth and proliferation
[243]. FGF10 preferentially stimulates prolifera-
tion at ureteric bud tips, whereas FGF7 increases
cell proliferation throughout the developing
collecting ducts [243]. In vivo, global deletion of
Fgf7 or Fgf10 in mice results in ureteric branching
defects and hypoplastic kidneys [58, 244]. Condi-
tional targeting studies in mice have revealed that
Fgfr2 is likely the key Fgf receptor mediating
effects on ureteric branching [245–247]. Loss of
20
Fgfr2 in the ureteric bud results in hypoplastic
ureteric ampullae with reduced proliferation and
increased apoptosis, ultimately leading to a sig-
nificant reduction in ureteric branching and hypo-
plastic kidneys. In addition, an interesting study
revealed that while combined loss of Sprouty1 and
Gdnf in mice largely rescues ureteric defects com-
pared to when either locus is deleted alone, addi-
tional loss of Fgf10 in the combined mutants led
to complete loss of ureteric branching and renal
aplasia [248]; thus, FGF signals appear to be able
to largely substitute for GDNF in promoting ure-
teric morphogenesis in the absence of Sprouty1.
Finally, a combination of in vitro and in vivo
experiments revealed that Fgf signaling acts in a
coordinated fashion with Wnt11 and Gdnf to reg-
ulate ureteric morphogenesis, in concert with
Sprouty genes [249].
Patterning of the Medullary
and Cortical Collecting Ducts
From the 22nd–34th week of human gestation [2]
and embryonic day 15 birth in mice [43], the
cortical (peripheral) and medullary (central)
regions of the kidney become established. The
relatively compact, circumferential renal cortex
comprises approximately 70 % of the mature kid-
ney volume [250]. The renal medulla develops
modified a cone shape and occupies the remainder
of the mature kidney volume [250]. The apex of
the medullary cone consists of collecting ducts
converging in the inner medulla and is termed
the papilla. Ultimately, medullary collecting
ducts become morphologically distinct from cor-
tical collecting ducts. Medullary collecting ducts
become elongated and linear and remain relatively
unbranched in a region devoid of glomeruli. In
contrast, collecting ducts in the renal cortex
remain branched and induce nephrogenic cap
mesenchyme to form nephrons throughout
nephrogenesis.
These morphological differences are likely due
in part to distinct axes of growth in the developing
renal cortex and medulla. The renal cortex grows
circumferentially, which preserves the organiza-
tion of the peripheral tissues, including
C. Bates et al.
differentiating glomeruli, nephron tubules, and
collecting ducts [250]. In contrast, the developing
renal medulla expands longitudinally, perpendic-
ular to the axis of cortical growth, due to elonga-
tion of outer medullary collecting ducts
[250]. Stromal cells may be a source of stimula-
tory cues for the medullary growth [250]; studies
have shown that mice lacking the stromal tran-
scription factors Foxd1 and Pod1 have abnormal
medullary collecting duct patterning [54, 66,
251]. Finally, apoptosis appears to participate in
remodeling the branched medullary ureteric tis-
sues into elongated tubules, as programmed cell
death normally occurs prominently in developing
medullary ureteric epithelia that become the
papilla, calyces, and renal pelvis [108].
Multiple genes have been implicated in the
differentiation of cortical and medullary
collecting ducts, including those that encode for
soluble growth factors (Fgf7, Fgf10, Bmp4,
Bmp5, and Wnt7b), proteoglycans (Gpc3), cell
cycle regulatory proteins (p57KIP2), and compo-
nents of the renin–angiotensin axis (angiotensin
and angiotensin type 1 and 2 receptors). Fgf7
mutant mice have marked papillary underdevel-
opment, while Fgf10 null kidneys exhibit medul-
lary dysplasia with fewer loops of Henle and
medullary collecting ducts, increased medullary
stroma, and enlargement of the renal calyx [58,
244]. The ability of FGF ligands to bind properly
to their receptors requires interactions with cell
surface proteoglycans, including glypicans
[252]. Glypican-3 (GPC3) is required for normal
medullary patterning in humans and mice [253,
254]. Moreover, the medullary dysplasia observed
in Gpc3-deficient mice appears to result from
unrestrained proliferation and overgrowth of the
ureteric bud and collecting ducts, followed by
aberrant apoptosis [253, 254]. The Gpc3/ med-
ullary defects appear to be driven by altered
responses of mutant collecting duct cells to
growth factors including Fgfs [254–256]. Finally,
mice lacking the cell cycle protein p57KIP2 dem-
onstrate medullary dysplasia, with fewer inner
medullary collecting ducts [257]. Together, these
studies reveal the importance of balanced cell
proliferation and apoptosis in medullary
collecting duct patterning.
1 Embryonic Development of the Kidney 21

Proper elongation and growth of medullary
collecting ducts also appears to rely on oriented
cell divisions. Studies have shown that canonical
Wnt signaling in collecting ducts via Wnt7b leads
to proper oriented cell division and survival [258,
259]. Furthermore, α3β1 integrin and the receptor
tyrosine kinase c-Met act in concert to regulate
Wnt7b expression and signaling in medullary
collecting ducts [259].
Finally, angiotensin (Agt) and angiotensin
receptors (Agtrs) appear critical for the develop-
ment of the renal calyces, pelvis, and ureter. Mice
lacking Agt or Agtr1 genes demonstrate progres-
sive widening of the calyx and atrophy of the
papillae and underlying medulla [260,
261]. These defects appear to be caused by
decreased proliferation of the smooth muscle
cells that line the renal pelvis. Loss of Agtr2
causes a range of renal anomalies secondary to
ureteric mispatterning, including vesicoureteral
reflux, duplex kidney, renal ectopia, ureteropelvic
or ureterovesical junction stenoses, renal dyspla-
sia or hypoplasia, multicystic dysplastic kidney,
and renal aplasia [107].
Lower Urinary Tract Development
Anatomic and Functional Development
Concurrent with metanephric kidney formation,
the embryonic ureter and bladder develop the
former functioning to propel urine into the latter
which stores urine until an appropriate time to
expel it via the urethra. Similar to the kidney, the
ureter and bladder undergo maturation largely due
to reciprocal interactions between an epithelium
(i.e., urothelium) and surrounding mesenchyme
that forms the lamina propria, muscle, and adven-
titia (Fig. 7). For a recent detailed review on
anatomic and molecular control of lower urinary
tract development, see [262].

Fig. 7 H&E-stained
sections from E15.5 and
P1 mouse ureters and
bladders. E15.5 ureters and
bladders have early
urothelium (u), an inner
layer of mesenchyme
(future lamina propria,
arrows), and an outer layer
of condensing mesenchyme
(future muscle, m). P1
ureters and bladders have a
more stratified urothelium
(u) and well-developed
lamina propria (arrows) and
outer muscle (m) layers.
The adventitial layer of
fibroblasts that surrounds
the muscle in both tissues is
not labeled. Ureters =100
magnification; bladders
=40 magnification
22
Ureter development begins simultaneously
with metanephric kidney development around
the 5th week of gestation in humans and at
E10.5 in the mouse, when the ureteric bud arises
from the mesonephric duct [262]. Thus the
embryonic origin of the ureteral urothelium is
the intermediate mesoderm, the same as the meta-
nephric kidney. By E11.5 in the mouse, the ure-
teric bud has been segmented into a distal portion
that will develop into the ureter and a proximal
end that has invaded the metanephric mesen-
chyme to eventually branch and form the
collecting ducts and renal pelvis (see above).
The mesenchyme surrounding the early develop-
ing ureter consists largely of tailbud-derived mes-
enchyme that appears to be crucial for directing
the distal ureteric bud toward a ureter fate
[263]. Between E10.5 and E13.5, the nascent
ureter transitions from attaching to the nephric
duct to emptying directly into the early bladder
(see below). By E13.5, a thin outer ring of ureteral
mesenchyme condenses and expresses alpha
smooth muscle actin (αSMA) mRNA, the first
marker of differentiation toward a smooth muscle
fate [264]. αSMA protein expression is not noted
until E14.5 in the proximal portion of the ureter
(nearest the kidney) and then throughout the entire
length of ureter by E16.5; thus, ureter muscle
development progresses in a rostral to caudal
direction [265]. Concurrent with development of
the mesenchymal layers, the ureteral urothelium
gradually matures from a simple epithelium to a
stratified epithelium consisting of at least three
cell types: basal, intermediate, and superficial/
umbrella cells. Each of these cell types has distinct
structural features and molecular markers [266];
moreover, recent data strongly suggests that
urothelial basal cells, arising from the original
ureteric epithelium, serve as the progenitors for
other ureteral urothelial cell types [266]. A unique
feature of urothelium (compared with other epi-
thelia) is the apical expression of urothelial
plaques, consisting of uroplakins, which likely
have several functions, including providing a per-
meability barrier and acting as a binding site for
uropathogenic E. coli [266].
An important function of the ureter is to con-
tinuously propel urine from the renal pelvis to the
C. Bates et al.
bladder. The ability of the ureter to undergo peri-
staltic waves of contraction followed by relaxa-
tion appears to be intrinsic and not dependent on
urine flow; cultured explants of E13.5 mouse ure-
ters attached to kidneys begin to undergo sponta-
neous peristaltic contractions within a few days,
as do isolated and cultured E15.5 ureters
[268]. Elegant studies recently identified a popu-
lation of pacemaker cells at the junction of the
renal pelvis and the kidney that express
hyperpolarization-activated cation-3 (Hcn3)
channels (a family of channels that are also pre-
sent in cardiac pacemakers) [269]. Loss of Hcn3
activity in mice leads to abnormal coordination
and frequency of ureter contractions [269]. Fol-
lowing this study, another described a population
of secondary pacemakers that are located in the
muscle of the mouse proximal ureter (starting at
the ureteropelvic junction) and that have morpho-
logical and molecular features similar to intestinal
pacemakers including expression of c-kit
[268]. Thus, like the cardiac conduction system,
the ureter has primary and secondary pacemakers
that act to drive urinary propulsion in a coordi-
nated fashion.
The embryonic bladder initially forms around
the 5th gestational week in humans and at
E11.5–12.5 in the mouse [262, 270]. Unlike the
ureter, bladder urothelium is derived from the
endodermal urogenital sinus, formed from the
ventral region of the cloaca. At E11.5–12.5, the
urogenital sinus further subdivides into an ante-
rior portion, which will become the bladder, and a
posterior portion that forms the urethra and por-
tions of the female vagina. The mesenchyme that
surrounds the bladder urothelium is largely
thought to be from splanchnic mesoderm,
although fate-mapping studies reveal that tailbud
mesenchyme also contributes to bladder mesen-
chyme (similar to the ureter) [263]. By E13.5, the
bladder is recognized as a distinct structure that is
attached directly to the ureters. As is the case with
ureters, αSMA mRNA expression in the develop-
ing bladder muscle precedes protein expression;
mRNA expression appears as early as E11.5 in
mice [264], while protein expression begins at
E13.5 [270]. Unlike the ureter (and many other
organs with smooth muscle such as the intestine)
1 Embryonic Development of the Kidney
that has a thin ring of mesenchyme that begins to
differentiate into muscle, the entire outer half of
the bladder mesenchyme condenses simulta-
neously and strongly expresses αSMA by E15.5
[264, 271]. Similar to the ureter, the bladder
urothelium matures from a simple epithelium to
a stratified epithelium, consisting of cell types
similar to the ureter, most of which also express
uroplakins and urothelial plaques [266]. While
bladder urothelial basal cells were originally
thought to be the progenitor cell for the other
urothelial cell types, recent careful lineage tracing
studies revealed the presence of a previously
unidentified transient population of cells, known
as “P” cells that serve as the progenitors in
embryos [272]; moreover, there are dynamic
changes in relative composition of bladder
urothelial cell types throughout development
(Fig. 8).
P-cell Foxa2+ Upk+ P63+ Shh+ Krt5–
l-cell Foxa2– Upk+ P63+ Shh+ Krt5–
S-cell Foxa2– Upk+ P63– Shh– Krt5–
B-cell Foxa2– Upk– P63+ Shh+ Krt5+
100%
75%
50%
25%
0%
E11 E13 E14 E16 E18 Adult
Fig. 8 Graph demonstrating the proportion of differ-
ent murine bladder urothelial cell types during devel-
opment. “P” cells, an early transient progenitor population
appear first, followed dynamic changes in the proportion of
their derivatives [intermediate (I ), superficial (S), and basal
(B) cells] through adulthood. Immunohistochemical
markers of each cell type are listed (Foxa2 forkhead box
A2, Upk uroplakins, P63 tumor protein P63, Shh sonic
hedgehog, Krt5 keratin 5) (Reproduced with kind permis-
sion from Elsevier. Gandhi, D, et al. Retinoid signaling in
progenitors controls specification and regeneration of the
urothelium. Developmental Cell, volume 26, pp 469–482,
2013, adapted from Figure 2)
23
While the connection of the ureters with the
bladder is critical, how this occurs has been the
subject of some debate. What is clear is that
between E12.5 and E13.5, the common nephric
duct (caudal portion of the mesonephric duct
between the ureter base and future bladder)
moves adjacent to the bladder, allowing for the
ureters and rostral nephric ducts (future male
gonadal excretory ducts) to separate and empty
directly into the bladder. The triangular portion of
the bladder demarcated by the entry points of the
ureters and the bladder neck (where the remaining
embryonic nephric ducts empty) is known as the
trigone. Historically the common nephric duct
was thought to become incorporated into the blad-
der to form the trigone. However, elegant fate-
mapping studies reveal that the common nephric
duct undergoes complete apoptosis starting at
E12.5 in the mouse and that the urothelium of
the trigone originates entirely from the bladder
urothelium [273]. Moreover, a separate study
showed that the muscle present in the trigone
arises mostly from the bladder with only a few
fibers emanating from ureteral muscle
[274]. Thus, the trigone originates mostly from
bladder tissues and not the mesonephric duct.
Molecular Control of Ureter
and Bladder Development
Ureter
The pathways critical for early ureteric bud for-
mation were already covered in the section on
kidney development above. Thus, an overview
of the molecular control of ureter development
will be presented in this section (Fig. 9). Once
the ureteric bud has formed, Bmp4 is secreted by
tailbud-derived mesenchyme surrounding the dis-
tal ureteric bud, driving the epithelium toward a
urothelial fate; moreover, ectopic Bmp4 expres-
sion around proximal portions of the ureteric bud
directs it to become urothelium instead of
collecting duct epithelium [263]. The transcrip-
tion factor Tbx18 is also critical for normal
urothelial development and is expressed through-
out the mesenchyme investing the distal ureter.
The genetic ablation of Tbx18 in mice leads to
24 C. Bates et al.

Ptch

Hcn3
Smo
Shh
Muscle Gli3R

Adventitia UPKs Fzd Kit

β-catenin

Adventitia
Wnt
Tbx18
Ptch

s
ell
lc
Bmp4

lia
he
Shh Smo
s
ell Bmp4

ot
c Smad
Ur
al receptor
m lls
ro ce Tshz3
St le
Lamina propria c
Dlg1 us SMC genes
m

tia
(stromal cells) Urothelium th

nti
oo
Sm

ve
Ad
Agtr1

Cnb1 AngII

Fig. 9 Genetic pathways regulating ureter develop-
ment. The cell layers of the developing ureter include the
urothelium (green) and mesenchymal lamina propria/stro-
mal cells (pink), smooth muscle (yellow), and adventitia
(blue). Tbx18, expressed in mesenchyme, is critical for
smooth muscle and urothelial development. Uroplakins
(UPKs) are not only markers of urothelium but critical
for urothelial morphogenesis. Sonic hedgehog (Shh) is
secreted by urothelium, binding to patched (Ptch) receptors
to regulate morphogenesis of smooth muscle and Hcn3 and
c-Kit expressing pacemakers [via interactions with
Smoothened (Smo) and Gli3 repressor (Gli3R)]. Other
Shh targets including Bmp4 (acting through Smad
proteins) and Tshz3 regulate smooth muscle morphogene-
sis. Wnt ligands in urothelium bind to Fzd receptors and
stabilize β-catenin to stimulate smooth muscle develop-
ment and repress adventitial expansion. Angiotensin
2 (AngII) binds to type 1 receptors (Agtr1) that along
with calcineurin b1 subunits also pattern smooth muscle.
Dlg1 is critical for lamina propria/stromal cell morphogen-
esis (Reproduced with kind permission from Wiley Peri-
odicals, Inc. Rasouly, HM and Lu W. Lower urinary tract
development and disease. Wiley Interdisciplinary
Reviews: Systems Biology and Medicine, volume 5, pp
307–342, adapted from Figure 3)

severe mispatterning of the mesenchyme and then
secondary defects in urothelial development
[277]. Finally, not only are uroplakins markers
of urothelial maturation, but they are also critical
for normal urothelial morphogenesis. Loss of
either uroplakin II or uroplakin IIIa leads to severe
urothelial plaque defects, hypoplastic superficial
cells, urothelial leakiness, hydronephrosis, and
vesicoureteral reflux [276, 277]. Moreover, muta-
tions in UPKIIIa in humans have been associated
with severe renal dysplasia and reflux [279],
whereas the role of UPKII in humans is still
unclear [279].
Signaling from the ureteral urothelium has also
been shown to be critical for mesenchymal pat-
terning. The growth factor, Sonic hedgehog (Shh),
is secreted by the urothelium and binds to its
receptor, Patched 1, in surrounding mesenchyme.
Conditional ablation of Shh in developing ureteric
epithelium leads to loss of mesenchymal prolifer-
ation and smooth muscle differentiation
[265]. Furthermore, Shh signaling from
urothelium has also been shown to be critical for
the formation of Hcn3 and c-kit expressing pace-
makers within the ureteral muscle, via the hedge-
hog signaling mediators Smoothened and Gli13
1 Embryonic Development of the Kidney
repressor [268]. In addition, downstream targets
of Shh signaling have been identified as necessary
for ureteral muscle development. One target,
Bmp4, is essential for normal ureteral
muscle investment around the ureter, particularly
at the ureterovesical junction (in addition to
having its aforementioned role in urothelial dif-
ferentiation) [280]. The transcription factor,
Teashirt 3 (Tshz3), downstream of both Shh and
Bmp4, is critical for proximal ureteral
smooth muscle differentiation and for normal
ureteral peristaltic function; deletion of Tshz3 in
mice leads to severe muscle patterning defects and
congenital hydronephrosis [281]. A role for SHH
in human urinary tract development was
confirmed in patients with Pallister–Hall syn-
drome, who have mutations in GLI3 and urinary
tract anomalies such as hydroureter and
hydronephrosis [268, 282]. Finally, Wnt
ligands are also secreted by the urothelium, bind
to frizzled receptors in the mesenchyme, and sig-
nal primarily by stabilization of β-catenin
(Ctnnb1). Genetic ablation of Ctnnb1 leads to
failure of mesenchymal proliferation and differ-
entiation into smooth muscle cells, with concur-
rent expansion of the outer adventitial fibroblast
layer [283].
Other genes within the ureteral mesenchyme
have been shown to be critical for normal mesen-
chymal development. As alluded to, loss of the
transcription factor Tbx18 leads to decreased
proliferation and failure of smooth muscle differ-
entiation and ultimately severe hydrouretero-
nephrosis [275]. Similarly, conditional ablation
of the calcineurin b1 subunit in developing
ureteral mesenchyme leads to reduced smooth
muscle proliferation and hydronephrosis
[284]. Furthermore, genetic ablation of the angio-
tensin type 1 receptor (which is expressed in ure-
teral mesenchyme) leads to smooth muscle
hypoplasia, lack of peristalsis, and ultimately
hydronephrosis [285]. Finally, discs large
homolog 1 (Dlg1) is the only gene identified to
date that is necessary for lamina propria/stromal
cell development; genetic deletion of Dlg1 in
mice led to an absence of the entire ureteral lamina
propria layer and disorganized muscle
development [286].
25
Bladder
Compared with the ureter, much less is known
about the molecular control of bladder develop-
ment. Some of the pathways critical for formation
of the urogenital sinus (future bladder) have been
identified. Bidirectional signaling between
ephrin-B2 and EphB2, a ligand and its receptor
tyrosine kinase, is critical for septation of the
cloaca into the ventral urogenital sinus and the
dorsal anorectal canal [287]. Sonic hedgehog sig-
naling is also critical for the formation of the
urogenital sinus. In mice, the loss of Shh or com-
pound mutations in the downstream hedgehog
mediators, Gli2 and Gli3, leads to failure of cloa-
cal septation [288]. During later stages of bladder
formation, urothelial cell stratification is depen-
dent on retinoid signaling emanating from the
lamina propria surrounding the urothelium; forced
expression of a dominant negative retinoic acid
receptor in developing mouse urothelium leads to
a loss of S cells, I cells, and uroplakins [272].
There are also some limited data on genetic
pathways critical for bladder mesenchyme devel-
opment. As is true in the ureter, several studies
have shown that Shh signaling from the bladder
urothelium is necessary for bladder mesenchyme
morphogenesis; loss of Shh in mice leads to a
complete loss of smooth muscle formation
[289–292]. Another mouse line, termed the
megabladder mouse (mgb/), is a random trans-
gene insertional mutant that lacks outer mesen-
chymal condensation, has very limited αSMA
expression, and ultimately develops a massive
bladder with no functional detrusor [271]. While
the gene disrupted in mgb/ mice is still
unknown, Shh signaling appears perturbed, and
the bladders lack expression of myocardin, a tran-
scription factor critical for smooth muscle
development [293].
Ureter–Bladder Anastomosis
There are some data on genetic pathways neces-
sary for the proper connection between the ureter
and bladder, i.e., apoptosis of the common nephric
duct starting at E12.5 in mice. Retinoid signaling
26
from the bladder has been shown to be critical for
this process. Genetic ablation of retinaldehyde
dehydrogenase-2, an enzyme expressed in the
urogenital sinus and necessary for retinoic acid
synthesis, led to persistence of the common neph-
ric duct with ureters ending blindly in the meso-
nephric duct [273]. In addition, Ret has also been
shown to be essential for ureter–bladder anasto-
mosis. Mice with a point mutation in tyrosine
1015, the phospholipase Cγ binding site on Ret
(RetY1015), have a persistent common nephric
duct due to enhanced proliferation and decreased
apoptosis [294].
Lower urinary tract defects contribute substan-
tially to chronic kidney disease in children, neces-
sitating a better understanding about the genes
regulating lower urinary tract morphogenesis.
While our understanding of the molecular control
of ureter and bladder development trails that of the
kidney, more studies are emerging on how the
ureter and bladder form.
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 Development of Glomerular Circulation
and Function 2
Alda Tufro and Ashima Gulati

Contents Introduction
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
From the Malpighian corpuscle description and
Development of the Kidney Vasculature . . . . . . . . . . . 38
Vasculogenesis and Angiogenesis . . . . . . . . . . . . . . . . . . . . 38
Bowman’s sketch to defining its ultrastructure and
molecular function, the ways we look at the kid-
The Glomerular Filtration Barrier . . . . . . . . . . . . . . . . . 41
ney glomerulus have evolved tremendously. The
Components of the GFB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Glomerular Filtration Barrier (GFB) Selective first systematic exploration of the body with a
Permeability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 microscope led to the identification of “Malpi-
Glomerular Hemodynamics and Assessment of ghian corpuscles” as “glands” within the kidney
Renal Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 [1]. Two centuries later, a more sophisticated
Renal Blood Flow: Basic Concepts . . . . . . . . . . . . . . . . . . . 47 microscope enabled Sir William Bowman to iden-
Perinatal Considerations for Renal Blood Flow tify glomerular capillary tufts in animal and
and GFR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
The Concept of Clearance . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 human kidneys and demonstrate a relationship
between the capillary tuft and the renal tubule
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
[2]. Since then, the understanding of the human
glomerulus as a specialized structure uniquely
adapted for renal filtration at the proximal part of
the nephron has considerably advanced.
The human definitive kidney is a highly
complex organ system with an average number
of ~1 million functional units called nephrons.
Nephrogenesis involves the development of the
glomerulus (glomerulogenesis) and the renal
tubule (tubulogenesis) from mesenchymal pro-
genitors residing in the metanephric mesenchyme
and ureteric bud. The specification, maintenance,
and commitment of nephron progenitors and the
regulatory processes that transform nephron pro-
genitors into a functional nephron are
detailed elsewhere ([3]; see ▶ Chaps. 1, “Embry-
A. Tufro (*) • A. Gulati
onic Development of the Kidney,” and ▶ 18,
Department of Pediatrics, Nephrology Section, Yale
School of Medicine, New Haven, CT, USA “Translational Research Methods: Renal Stem
e-mail: alda.tufro@yale.edu Cells” in this text). The current chapter aims to
# Springer-Verlag Berlin Heidelberg 2016 37
E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_2
38
combine established knowledge and new findings
pertaining to the structural and functional aspects
of glomerular development, describe intricacies of
the glomerular filtration barrier at the molecular
level, and provide insight into future research
areas.
Three attributes make the human glomerulus a
fascinating focus of research and clinical signifi-
cance: first is the synchrony among
vasculogenesis, angiogenesis, and epithelial and
stromal differentiation; second, the glomerular
unique ultrastructure provides controlled regula-
tion of filtration while acting as a barrier; and
third, the glomerulus is the major controller of
renal hemodynamics and provides functional
advantages for effective filtration. Each of these
attributes and their relevance for clinical use will
be discussed in the following sections.
Development of the Kidney
Vasculature
The kidney vasculature develops in a patterned
fashion, allowing structural and functional devel-
opment of the glomerular circulation that eventu-
ally handles 20 % of the cardiac output for the
purpose of clearing metabolic waste products and
toxins and maintaining fluid and electrolyte bal-
ance [4]. The complex architecture of the renal
vasculature is essential for the organ function and
comprises an arterial tree and three capillary beds.
Single renal arteries branch and direct over 90 %
of the renal blood flow to glomeruli in the renal
cortex. The glomerular capillary bed length
amplifies the area for filtration, allowing the
highest fluid outflow rate in the body. Glomerular
capillaries are flanked by high-resistance afferent
and efferent arterioles, which regulate the rate of
blood flow through the capillary bed and thereby
glomerular filtration. Two postglomerular capil-
lary networks emerge in series from efferent arte-
rioles. Efferent arterioles from superficial
glomeruli give rise to cortical peritubular capil-
laries, while those from the juxtamedullary glo-
meruli give rise to vasa recta. Close alignment of
the postglomerular microvasculature with cortical
and medullary renal tubules is critical for oxygen
A. Tufro and A. Gulati
delivery to all nephron segments and for fluid and
solute reabsorption from them. As a result of fluid
removal by glomerular ultrafiltration, oncotic
pressure in postglomerular capillaries increases
above hydrostatic pressure. Thus, peritubular cap-
illaries are poised for fluid and solute
reabsorption.
Vasculogenesis and Angiogenesis
The formation of the kidney vasculature com-
prises in situ differentiation of endothelial cells
from hemangioblasts present in the metanephric
mesenchyme and capillary assembly, a process
called vasculogenesis, and angiogenesis wherein
capillary growth occurs by the sprouting and/or
splitting of existing capillaries within or surround-
ing the developing kidney [5–9]. In vivo,
vasculogenesis and angiogenesis might occur
sequentially or simultaneously [10].
Vasculogenesis and Angiogenesis
Contribute to the Kidney Vasculature
The origin of kidney endothelial cells and the
mechanisms involved in kidney vascularization
have been extensively examined in experimental
models and debated for quite some time. Genetic
fate mapping and the identification of molecular
guidance cues from angiogenic factors have
largely resolved these controversies. The hypoth-
esis of extrarenal angiogenic origin of kidney
endothelial cells is supported by elegant interspe-
cies transplantation experiments and by the
absence of vascular development in embryonic
kidneys cultured ex vivo [11–13]. As shown in
other embryonic vascular beds [14], Sariola and
colleagues showed evidence of angiogenesis in
undifferentiated embryonic mouse kidney rudi-
ments after the transplantation onto avian chorio-
allantoic membrane [15, 16]. Indeed, grafts were
well vascularized, and both the glomerular and the
vessel endothelium expressed an avian nuclear
marker, suggesting that the glomerular endothe-
lium is derived from extrinsic (avian) vasculature
rather than by the differentiation of endothelial
cells native to the metanephric mesenchyme
[15–17]. Abrahamson et al. demonstrated that
2 Development of Glomerular Circulation and Function
embryonic kidneys grafted under the renal cap-
sule of newborn mice were vascularized by host
endothelium, whereas adult hosts failed to
vascularize the grafts [18], suggesting that the
angiogenic activity of the host is crucial to this
process.
The hypothesis of the endogenous,
vasculogenic origin of glomerular endothelial
cells is supported by the identification of native
endothelial precursors in the metanephric mesen-
chyme, by the exposure of avascular metanephric
kidneys to hypoxia or angiogenic factors, and by
grafting experiments [18–21]. These findings
were facilitated by the landmark discovery of
vascular endothelial growth factor (VEGF) recep-
tors Flk1 and Flt1, as indispensable for endothe-
lial differentiation and vascular assembly and thus
genetic markers of endothelial precursors
[22–24]. Flk1+ and Flt1+ angioblasts were
detected within the avascular metanephric mesen-
chyme [19–21], demonstrating that endothelial
progenitors in situ enable vasculogenesis in the
developing kidney [25–28]. Flt-1 and Flk-1 are
expressed in isolated cells before any morpho-
logic evidence of vascular development in the
metanephric blastema [25]. Within 24 h in culture,
Flk-1-expressing cells align to form cord-like
structures, followed by the acquisition of lumen
and typical endothelial cell phenotype in the fol-
lowing 2 days. As renal vascularization proceeds,
Flt-1 and Flk-1 are expressed in contiguous endo-
thelial cells [25]. Exposure of avascular kidney
rudiments to hypoxia similar to that occurring in
embryonic tissues leads Flk1+ angioblasts present
within the metanephric blastema to form primitive
vascular networks via the upregulation of VEGF
[29]. Similarly, exposure to exogenous VEGF
enables avascular embryonic kidneys to develop
capillaries [25, 30]. Genetically tagged LacZ-
Flk1+ embryonic mouse kidneys grafted into the
anterior chamber of the rat eye develop glomeruli
vascularized by graft rather than host endothelial
cells [31]. Remarkably, the glomerular basement
membrane and mesangial matrix were noted to be
exclusively of graft origin, implying the self-
sufficient capability of the metanephric mesen-
chyme to form the glomerulus [19]. Furthermore,
Tie-1/LacZ metanephros transplanted into the
39
nephrogenic cortex of wild-type mice develop
transgene-expressing glomerular capillary loops
[31]. In contrast, glomerular Tie-1/LacZ + vessels
do not develop in rudiments placed in organ cul-
ture on 20 % O2 [31]. This capability of Flk1+
angioblasts to give rise to a primitive vasculature
has subsequently been confirmed in vitro and
in vivo explant assays [18, 19, 32, 33]. These
observations imply that endothelial progenitors
present at the onset of mouse nephrogenesis dif-
ferentiate and undergo morphogenesis to become
glomerular capillaries when experimental condi-
tions resemble those found in the metanephros
in vivo, such as moderate hypoxia. Together, a
large body of experimental data demonstrates that
both vasculogenesis and angiogenesis contribute
to the formation of kidney vasculature under the
influence of a variety of growth factors and guid-
ance proteins.
Blood vessels are comprised of endothelial
cells and associated vascular mural cells including
pericytes and vascular smooth muscle cells. While
the data outlined above provide evidence for the
presence of endothelial progenitors in the meta-
nephric blastema as well as ingrowing endothelial
cells, genetic fate mapping experiments demon-
strate that Foxd1+ cells present at the periphery of
the metanephric blastema give rise to most vascu-
lar mural cells, thus contributing to the overall
vascular structural development [34].
Angiogenic Factor Signaling Is Critical
for Patterning the Kidney Vasculature
The kidney endothelium undergoes specification
into distinct histological types in arteries, veins,
capillaries, and lymphatics, including continuous
endothelium or endothelium fenestrated with dia-
phragms. This specification is adapted to the tis-
sue permeability characteristics and is determined
by local microenvironmental cues, irrespectively
of the endothelial cell lineage [35]. For example,
the grafting of embryonic vessels with fenestrated
endothelium into the brain resulted in tight con-
tinuous endothelium typical of the blood-brain
barrier [36]. Though the progenitor lineage
might contribute to endothelial cell heterogeneity
within the kidney, endothelium specification by
local environmental cues involves dynamic cross
40
talk between various signaling pathways that
communicate by way of growth factors [37]. Sol-
uble angiogenic factors and guidance proteins
including VEGF-A, basic fibroblast growth factor
(bFGF), platelet-derived growth factor (PDGF),
and semaphorin 3A play a key role in kidney
vascularization.
VEGF-A is a pleiotropic glycoprotein origi-
nally described as a vascular permeability and
endothelial growth factor [38–40]. VEGF is a
direct-acting specific endothelial cell mitogen
that stimulates angiogenesis and regulates embry-
onic vessel development in a gene dosage-
dependent manner [41]. VEGF-A is required for
endothelial cell differentiation, survival, prolifer-
ation, and migration, as well as for vascular
assembly, maintenance, and remodeling; thus it
is critical for physiological angiogenesis [42, 43].
During kidney development, VEGF-A
expressed in the metanephric mesenchyme acts
as a chemoattractant for endothelial cells and
directs their migration toward developing neph-
rons [30]. Glomerular development starts when
nephrons are at the S-shaped body stage, at
which time podocytes differentiate and express
VEGF-A, leading endothelial progenitors to
migrate into and differentiate in the adjacent “vas-
cular cleft.” Podocytes synthesize three VEGF-A
isoforms (VEGF121-165-189) by alternative splicing
[44]. VEGF-A isoforms with differences in size,
membrane, and extracellular matrix-binding prop-
erties (VEGF121 and the most abundant VEGF165
are secreted) enable gradient formation and endo-
thelial cell chemoattraction [30]. The glomerular
endothelium forms a single capillary loop
initially that later forms the mature glomerular
tuft. VEGF-A is indispensable for normal devel-
opment of glomerular capillaries [45] and to
acquire their fenestrated phenotype [46–48]. The
principal source of VEGF-A in the renal glomer-
ulus is the podocyte [49]. Continuous expression
of VEGF-A in podocytes and tubular cells and
of Flk1 on the adjacent endothelia induces and
maintains the fenestrated endothelia and also reg-
ulates vascular permeability [26, 48, 50, 51].
Podocytes and tubular cells express VEGF-A
throughout life, unlike other tissues that cease
expressing VEGF-A at the completion of
A. Tufro and A. Gulati
development [25, 26]. Moreover, tightly regulated
VEGF-A is required to establish and maintain a
normal podocyte phenotype, i.e., foot processes
linked by slit diaphragms, as revealed by
both loss- and gain-of-function mouse models
[45, 52–54]. VEGF-A signals in autocrine and
paracrine fashion in podocytes, renal tubules,
and endothelial cells promoting the survival,
proliferation, and migration in vitro and in vivo
[44, 45, 55–62]. VEGF-A receptors are most
abundant in endothelial cells, but they are
expressed by multiple cells, including podocytes
and tubular cells [44, 57, 59, 60, 62–64].
Two VEGF-A tyrosine kinase receptors,
VEGFR-1 [previously known as fms-like tyrosine
kinase-1 (Flt-1)] and VEGFR-2 [formerly murine
fetal liver kinase 1 (flk-1)/human kinase insert
domain receptor (KDR)], and two co-receptors,
neuropilin-1 and neuropilin-2 (NRP1 and NRP2),
have been described. VEGF-A signals are trans-
duced through VEGFR2 and NRP1 and NRP2
amplify VEGFR2 signals, while VEGFR1 func-
tions mostly as a decoy [65]. The importance of
VEGF-A signaling for embryonic vascular devel-
opment is illustrated by gene deletion experi-
ments. Both heterozygous and homozygous
VEGF-A knockout mice die during embryogene-
sis due to major vascular defects [66, 67]. Flk-1-
deficient mice die in utero because of an early
defect in the development of hematopoietic cells
and endothelial cells [22]. Flt-1-deficient mice die
later in utero, due to the disorganization of the
early embryonic vasculature [23, 68].
Hypoxia leads to the stabilization of HIF-1α, a
transcriptional activator of hypoxia-inducible
genes, including VEGF-A, erythropoietin, and
other angiogenic factors expressed in the kidney,
such as angiopoietin-1, angiopoietin-2, PDGFBB,
and FGFβ [10, 69]. Although it is likely that
several signaling pathways contribute to the
angiogenic response to hypoxia in kidney devel-
opment resulting in glomerular vascularization,
undoubtedly VEGF-A plays a critical role
[10]. Accordingly, the deletion of podocyte
VEGF-A reduces the glomerular endothelial
cells [45]. In contrast, the ablation of semaphorin
3A, a guidance protein that functions as a negative
regulator of endothelial cell survival and
2 Development of Glomerular Circulation and Function
migration, which is also secreted by podocytes
[70, 71], results in glomerular capillary hyperpla-
sia [72]. Conversely, excess podocyte semaphorin
3A leads to glomerular endothelial apoptosis and
a low number of glomerular endothelial cells
[72]. Together, these studies indicate that glomer-
ular capillary development depends on pro- and
antiangiogenic signaling pathways shared with
other vascular beds, while it is clear that
podocytes play a key role controlling
glomerulogenesis.
Other hypoxia-regulated angiogenic factors
control the relationship between endothelial and
mesangial cells during glomerular development,
as revealed by targeted deletion experiments. The
ablation of PDGF B, or its receptor PDGFR beta,
expressed by endothelial and mesangial cells,
respectively, and involved in vessel maturation,
results in glomeruli with aneurysm-like glomeru-
lar capillaries and lacking mesangial cells
[73, 74]. Similarly, the targeted ablation of
CXCL12, its receptor CXCR4 or RBP-J, a trans-
ducer of notch signaling, and angiopoietin1
results in single-loop or ballooned glomerular
capillaries associated with mesangial cell deficit
in some mutants [75–79]. The studies summa-
rized here illustrate the critical role of hypoxia-
regulated angiogenic factors and their signaling in
the differentiation, proliferation, migration, and
mutual regulation among glomerular endothe-
lium, mesangial cells, and podocytes.
The development of postglomerular capillary
beds is thought to be driven and regulated by a
similar array of angiogenic factors secreted by
renal tubules and endothelial or mural cell pre-
cursors, including VEGF-A, angiopoietins, stro-
mal cell-derived factor-1, erythropoietin, and
angiotensin II. These factors are involved in the
remarkable alignment of postglomerular capil-
laries with the renal tubules. The deletion of
VEGF-A from developing renal tubules results
in a hypoplastic medulla with fewer peritubular
capillaries [79, 80]. Angiopoietin-2 regulates
peritubular capillary architecture by promoting
vascular mural cell differentiation in the presence
of VEGF [81]. SDF-1 signaling from vascular
mural cell progenitors to its receptor in endothelial
cells regulates the size and distribution of
41
peritubular capillaries [77]. Moreover, vasa recta
postnatal development is controlled by angioten-
sin II and mediated by increased tubular VEGF
secretion [82]. Notably, the deletion of Foxd1
progenitors, a transcription factor expressed in
the renal stroma, disrupts peritubular capillary
development, recruitment of vascular mural
cells, and nephron patterning [4]. This suggests
that regulators of the local environment, as
discussed above in regard to hypoxia, directly or
indirectly influence epithelial-endothelial-mural
cell cross talk and ultimately the patterning of
the renal vasculature.
The Glomerular Filtration Barrier
The renal glomerulus controls the filtration of
water and solutes while at the same time acting
as a barrier retaining vital molecules such as
plasma proteins. The glomerular filter functions
as a semipermeable, macromolecular sieve capa-
ble of excluding molecules larger than serum
albumin (MM 66,400) [83, 84]. The glomerular fil-
tration barrier (GFB) separating the vasculature
from the urinary space consists of a three-layered
in-series structural arrangement of highly special-
ized cells that interact with one another (Fig. 1).
The components of the GFB include three layers:
the fenestrated endothelial cells lined by a
glycocalyx, the glomerular basement membrane
(GBM), and the slit diaphragm, which links the
neighboring podocyte foot processes.
Components of the GFB
Table 1 and see also ▶ Chaps. 1, “Embryonic
Development of the Kidney,” and ▶ 18, “Transla-
tional Research Methods: Renal Stem Cells” of
this text.
The Podocyte
Podocytes, the glomerular visceral epithelial cells
that reside within the Bowman’s space and bathe
in the glomerular filtrate, are a specialized and
anatomically unique feature of the glomerulus.
The podocyte has microtubule-based cellular
42
Fig. 1 Ultrastructure of a
typical glomerular capillary
loop: podocyte foot
processes ( fp), slit-
diaphragm (thin arrow),
glomerular basement
membrane (GBM), capillary
(cap) endothelial cell (EC)
with fenestrations (thick
arrows)
extensions known as primary processes and
actin-based interdigitating secondary processes
known as foot processes, which rest on the GBM
[85, 86]. Mature podocyte foot processes are
connected by modified adherens junctions called
slit diaphragms (SD). During glomerulogenesis,
columnar podocytes are joined by tight junctions,
which migrate toward the basal side and acquire
SD features as differentiation proceeds. The major
molecular components of the glomerular filtration
barrier are described in Table 1.
The ultrastructure of the SD has been conven-
tionally studied using transmission electron
microscopy (TEM) (Fig. 1), EM tomography,
and scanning EM [87–90]. Rodewald and
Karnovsky described the SD as “zipper structure
arrangement” with regular pores with a mean
width of ~40A, which would restrict the passage
of most serum proteins [87]. Tryggvason
et al. [89] provided new insight into the three-
dimensional molecular structure of the podocyte
slit diaphragm by identifying the extracellular
domain of nephrin as the backbone of the SD
and irregular pores with similar average width.
Recent examination of the slit diaphragm using
scanning EM challenges the view of an ordered
“zipper-like” SD structure and suggests a
heteroporous structure with ellipsoidal pores of
~120 A radius measured by digital morphometry
[90]. Tracer studies followed by TEM show that
A. Tufro and A. Gulati
the filtration of large molecules such as ferritin
(MM ~500,000) is hindered at the GBM, whereas
smaller proteins such as horseradish peroxidase
(MM 40,000) are filtered by the GBM but retained
by the SD [91–93]. In addition to its structural
function as a filtration barrier and cell-cell junc-
tion, the SD is a signaling protein complex that
dynamically determines podocyte cell behavior.
Extracellular domains of nephrin, neph1,
P-cadherin, and FAT1 contribute to the SD protein
complex, which includes multiple scaffolding,
receptors, and actin-binding proteins, through
direct and indirect protein interactions. The list
of SD complex members is ever growing, and
their roles are documented in gene ablation exper-
iments, as well as human mutations leading to
proteinuria and glomerular disease ([94–111],
Table 1).
The Glomerular Basement Membrane
The GBM is the extracellular gel-like matrix com-
ponent that lies between podocytes and endothe-
lial cells, composed of four major
macromolecules: laminin, type IV collagen,
nidogen, and heparan sulfate proteoglycan [112]
(see Table 1). In the mature GBM, laminin is a
trimer consisting of α5, β2, and γ1 laminin chains
(α5β2γ1 or 521), and collagen IV is a trimer
consisting of α3, α4, and α5 chains [113]. In con-
trast, laminin α1β2γ1 and α1 and α2 collagen IV
2 Development of Glomerular Circulation and Function 43

Table 1 Known molecular components of the glomerular filtration barrier (Refs. [94–113])
Protein/
localization Gene Function Characteristic References
Slit diaphragm
Nephrin NPHS1 Finnish form of congenital Transmembrane protein [94–100,
nephrotic syndrome localized to SD 103, 104]
Infantile nephrotic syndrome
Autosomal recessive
inheritance
Neph-family Neph1 (Kirrel) Phenotype of the mice lacking Neph1–3 are components of [101, 102]
proteins Neph2 Neph1 resembles that of the SD
(Neph1–3) (Kirrel3) nephrin-deficient mice
Neph3
(Kirrel2)
Podocin NPHS2 Autosomal recessive steroid- Transmembrane protein [105, 106]
resistant nephrotic syndrome interacts with nephrin and
(infantile and childhood) neph1
CD2-associated CD2AP Autosomal recessive infantile Multidomain scaffolding [107–109]
protein steroid-resistant nephrotic protein at the SD
syndrome
Phospholipase C PLCE1 Autosomal recessive infantile Component of SD [110]
epsilon 1 and early childhood steroid-
resistant nephrotic syndrome;
major gene of DMS
Transient TRPC6 Autosomal dominant Component of SD [111]
receptor Juvenile and adult onset FSGS
potential cation
channel
subfamily C
member 6
FAT FAT Component of SD, co-localizes Adhesion molecule, large [195]
with ZO-1 protocadherin at SD contains
a larger extracellular domain
than traditional cadherins
Zonula ZO-1 Most commonly associated Present in cytoplasmic [195]
occludens-1 with tight junctions, sometimes component of SD
associated with adherens
junctions
Podocyte
WT-1 WT-1 First identified as a tumor Zinc finger transcription [193]
suppressor gene for Wilms factor and RNA-binding
tumor protein
Expression restricted to the
podocyte
Denys-Drash syndrome –
diffuse mesangial sclerosis
within the glomeruli
Frasier syndrome –
glomerulosclerosis
(continued)
44 A. Tufro and A. Gulati

Table 1 (continued)
Protein/
localization Gene Function Characteristic References
LMX1B LMX1B Regulates podocyte-specific LIM-homeodomain [193]
gene expression; KO mice transcription factor
show reduced expression of
foot processes
Unique role in renal
development and in the
patterning of the skeletal
system; mutation causes
nail-patella syndrome
Pod1/epicardin/ Pod1 KO mice glomerular Basic helix-loop-helix [193]
capsulin development arrests at single transcription factor
capillary loop stage
Integrins Alpha3beta1 integrin Adhesion protein in the [194]
Major integrin expressed by GBM
podocytes
Important for podocyte
differentiation
Receptor for some isoforms of
laminin
Major laminin-binding integrin
Alpha1beta1
Alpha2beta 2 are collagen IV
receptors
Podocalyxin Podxl Maintains podocyte cell Sulfated cell surface [195]
separation sialomucin-charged protein
GLEPP1 GLEPP1 Deficient mice are susceptible Cell surface protein [195]
to hypertension Receptor tyrosine
phosphatase
Synaptopodin Synaptopodin Foot process assembly Actin-associated [195]
cytoskeletal protein
Alpha-actinin 4 ACTN4 Familial FSGS has been Actin-binding protein [113]
described
VEGF-A VEGF Endothelial cell mitogen for Glycoprotein [25, 26]
endothelial cell differentiation,
survival, proliferation, and
migration
Angiopoietin-1 Angiopoietin-1 Remodeling and maturation of 70 kDa glycoprotein [31]
capillaries
GBM
Laminin LAMB2 Juvenile isoforms LM-111 and Large (~800 kd) heterotrimer [194]
(humans) LM-511 replaced by mature of alpha, beta, and gamma
isoform LM-521 in mature glycoprotein chains
glomeruli
No known human mutations in
LAMA1/B1/C1/A5
Mutations in human LAMB2
gene known (Pierson
syndrome)
(continued)
2 Development of Glomerular Circulation and Function 45

Table 1 (continued)
Protein/
localization Gene Function Characteristic References
Type-IV COL4A3, A4, Alpha-1,2 in early nephron, Triple helical heterotrimer of [194]
collagen A5 shift to 3,4,5 subunits in mature collagen IV alpha chains
GBM
Mutations in human COL4A3,
A4 (autosomal recessive), A5
(most common, X-linked)
genes – Alport syndrome
Nidogen One of the integral basement Two isoforms A and B, each [112, 113]
membrane protein consisting of a single
Function not yet determined polypeptide chain
Agrin (most Negatively charged GBM Heparan sulfate [112, 113]
abundant) and components originally thought proteoglycans with
perlecan to contribute to the charge negatively charged sulfated
selective barrier (however, glycosaminoglycan side
current evidence challenges chains
this REF)

are expressed in the developing GBM [114]. Spe-
cific basement membrane protein isoforms are
crucial for glomerular development, morphology,
and function, as mutations in laminin β2
(LAMB2) or collagen IV (Alport) alter the GBM
structure and lead to progressive glomerular dis-
ease. The mechanisms for transitions from imma-
ture to adult-type isoforms of the GBM proteins
are not completely understood, but these substitu-
tions are important for the structural and func-
tional integrity of the GFB [113, 115–117]. It
has long been accepted that the net negative
charge of the GBM is a crucial component of the
glomerular capillary wall’s filtration barrier to
plasma albumin, which is also negatively charged
and should therefore be repelled by the GBM
[91]. However, the concept of charge selectivity
has recently been called into question. Removal of
these negatively charged proteoglycans has no
effect on the glomerular filtration barrier perme-
ability to either albumin or to a negatively charged
tracer [118]. It has been suggested that GBM
charge plays a minor role in imparting the glomer-
ular filter with charge selectivity and argued that
the GBM functions as a gel where macromole-
cules traffic by diffusion depending on their
molecular size [119] or their size and anionic
charge [120].
The Glomerular Endothelial Cell Layer
and Its Contributions to the Filtration
Barrier
The endothelium functions as a barrier, regulates
vasomotor tone, and controls tissue inflammation
and thrombosis. Glomerular endothelial cells
form a fenestrated capillary bed and play a
role in the filtration function [121]. Endothelial
fenestrations are transcellular holes that allow
the plasma flowing through the capillaries to
reach the GBM, even though fenestrations
are plugged by a glycocalyx-like material
consisting of sulfated proteoglycans and
glycoproteins that impart barrier properties
[51, 55–57]. The glomerular endothelial
surface layer has a thickness similar to that of
the GBM consisting of two elements, the
glycocalyx and the cell coat [122]. It has been
proposed that the endothelial glycocalyx might
contribute significantly to the GFB
permselectivity [122–127]. The glycocalyx com-
ponents are covalently bound to the endothelial
cell membrane and are attached to the glycocalyx
by charge-charge interactions [128, 129]. Damage
to the glycocalyx and endothelial cell coat causes
proteinuria in the absence of detectable damage to
the GBM or the podocytes, but the evidence
remains indirect [130, 131].
46
As discussed above, the assembly and integrity
of endothelial cells are regulated by angiogenic
factor signaling. VEGF-A is necessary for the
survival of endothelial cells, to establish and
maintain the integrity of endothelial fenestrae, as
demonstrated by knockdown and loss of function
in vivo experiments, and endothelial cell damage
leading to TMA in humans treated with VEGF-A
receptor blockers [35, 46, 47, 61]. Angiopoietin-1
produced by mural cells supports endothelial cell
survival and decreases vascular permeability by
inhibiting VEGF-induced eNOS activation
[132]. Angiopoietin-2 is stored in Weibel-Palade
bodies and secreted by activated endothelial cells.
Angiopoietin-2 and angiopoietin-1 compete for
signaling via the Tie2 receptor; in the presence
of VEGF-A, angiopoietin-2 leads to angiogenesis,
whereas in the absence of VEGF-A, it causes
endothelial cell apoptosis [133]. Davis et al.
showed that podocyte-specific overexpression of
angiopoietin-2 leads to apoptosis of glomerular
endothelial cells, without affecting the podocytes
[134]. In addition, mesangial cells affect the glo-
merular endothelial cell properties and promote
glomerular endothelial cell survival by
inactivating TGFβ [78, 135–138].
Glomerular Filtration Barrier (GFB)
Selective Permeability
The glomerular filtration barrier has size-selective
properties and differentially handles molecules of
varying size. Small molecules up to the size of
inulin filter freely, whereas large molecules such
as plasma proteins are held back. The sieving
coefficient for a particular molecule is the ratio
of its concentration in the glomerular filtrate rela-
tive to the plasma concentration. The GFB pore
theory assumes that the capillary wall consists of
cylindrical pores of two or various sizes allowing
size-selective passage of molecules through them
[139–143]. The fiber matrix theory, an extension
of the pore theory, posits that the GFB consists of
pores filled with a fiber matrix [144]. Various
mathematical models have been proposed and
applied to the porous substructure of the barrier
to predict macromolecular sizes that will be
A. Tufro and A. Gulati
compatible for crossing the barrier. In general,
there has been a discrepancy between these calcu-
lations and the in vivo descriptions of molecular
sieving, suggesting that other mechanisms might
be involved [145, 146]. Kedem and Katchalsky
have proposed flux equations for defining the
physical properties of the GFB and require no
specification of its substructure [147]. Charge
selectivity has been an age-old phenomenon
assumed to operate at the level of the GBM due
to the presence of negatively charged proteogly-
cans, which were thought to repel the anionic
proteins. However, present-day emphasis is on
the endothelial layer glycocalyx property confer-
ring charge selectivity to the GFB. Smithies pro-
posed that the size selectivity of the glomerulus
resides solely in the GBM, which functions as a
concentrated gel and macromolecules permeate
mostly by diffusion and also by fluid flow (con-
vection). Hence, this gel permeation/ diffusion
model suggests that lower filtration rate causing
lower water flow and thus a higher concentration
of proteins in the proximal tubular fluid and the
tubular mechanisms of reabsorption saturate ear-
lier to increase proteinuria [119]. Data from chil-
dren with nephrotic syndrome fit this hypothesis
[148], provided the degree of podocyte efface-
ment is also taken into account, suggesting that
the latter influences the size selectivity of the GFB
beyond limitations of GFR. Recently, Moeller and
colleagues proposed an electrokinetic model,
whereby a local electric field “streaming poten-
tial” is established across the glomerular filter that
prevents plasma proteins from entering or cross-
ing the filter [120]. This hypothesis fits data from
several previous models but awaits further exper-
imental confirmation.
Glomerular Hemodynamics
and Assessment of Renal Function
The molecular basis of glomerular architecture
discussed previously argues that the glomerular
structure provides functional advantages leading
to controlled glomerular filtration. Mammalian
glomerular capillary pressure averages
50 mmHg, which is approximately 50 % of the
2 Development of Glomerular Circulation and Function
mean systemic arterial pressure, with waveform
characteristics similar to the central aorta
[149]. The glomerular capillaries form a high-
pressure system with about 4 times the pressure
of systemic capillaries and confer a functional
advantage due to higher blood flow rates enabling
higher clearance as they filter large volumes of
plasma (~180 l/day).
Renal Blood Flow: Basic Concepts
Renal blood flow is approximately 1 L/min (20 %
of cardiac output) Fig. 2. Renal plasma flow is the
portion of the renal blood flow available for ultra-
filtration: RPF = (1-Hct)*RBF, thus given a nor-
mal Hct of 40 %, RPF is approximately
600 ml/min. Renal blood flow (RBF) is deter-
mined systemically by the renal perfusion pres-
sure that depends on the systemic arterial blood
pressure (SBP) and locally by the renal vascular
resistance (RVR), so RBF/SBP/RVR.
The unique architecture of the renal vascula-
ture enables to control the RVR at two sites, the
afferent and efferent arterioles, where significant
pressure drops occur, leading to relatively high
glomerular capillary pressure and low peritubular
20 Fraction of cardiac output perfusing the kidneys
%
15
10
5
Age, days
0 Perinatal Considerations for Renal
15 20 30 40 50
Fig. 2 Renal blood flow as a percentage of cardiac output,
plotted versus age, in growing rats between 17 and 60 days
of age (From Ref. [196])
47
capillary pressure. Thus changes in arteriolar
resistance lead to changes in glomerular plasma
flow and glomerular capillary pressure, which can
influence GFR. For example, during sympathetic
stimulation or increased angiotensin II, both affer-
ent and efferent resistance increase; thus RPF
decreases, but GFR remains constant due to the
opposite effect of afferent and efferent resistance
on GFR. This intrinsic autoregulation occurring
via neurohumoral mechanisms is mainly a conse-
quence of local adjustment of RVR secondary to
changes in efferent arteriolar tone. The efferent
arteriole is normally in a state of only partial
constriction, and any increase or decrease in
blood flow is accompanied by a reciprocal change
in glomerular filtration pressure, with the result
that the filtration rate remains relatively
unchanged [150–152]. Though the efferent arteri-
ole seems to take precedence over the afferent as a
controller of renal hemodynamics, the contribu-
tions of pre-and postglomerular vasculature vary
owing to specific circumstances. The efferent arte-
riole seems to be the major regulator of renal
blood flow during conditions of stress or after
the administration of angiotensin-converting
enzyme inhibitors or receptor blockers
[150–152]. However, the large increase in RPF
to the remnant kidney after a nephrectomy leads to
a dramatic decrease in afferent arteriole resistance,
driving a rapid increase in GFR. Generally,
increased glomerular plasma flow leads to
increased GFR. The reduction in hematocrit in
euvolemic patients increases RPFs and is a mech-
anism contributing to hyperfiltration injury in ane-
mic states [153]. Under normal conditions, the
glomerular filtrate is a steady fraction of the
RPF, the so-called filtration fraction
(FF) [149]. FF = GFR/RPF. Because normal
GFR is 125 ml/min and RPF 600 ml/min, normal
FF is 0.2. FF is higher at low RPF and when
efferent arteriole resistance is increased.
60 Blood Flow and GFR
The fetal human kidney main function from ges-
tation week 16 onwards is to produce urine to
48
maintain normal amniotic fluid volume, as the
fetal fluid-electrolyte homeostasis and solute
clearance are achieved through the placenta and
maternal kidney function. Though nephrogenesis
is complete by 34–36 weeks of gestation, the
efficiency of the renal excretory system akin to
most other organ systems is age dependent even in
postnatal life related to structural maturation
[154]. It is of utmost importance to the clinician
to be aware of the functional capacity of the new-
born kidney and the extreme states of vulnerabil-
ity posed by prematurity and at the same time
understand the expected sequence of physiologic
adaptations of kidney function that shall occur
with time during a successful transition to
extrauterine life.
The proportion of cardiac output flowing
through the kidney increases with gestational
and postnatal age; the fetal kidney is receiving
only 2–3 % of the cardiac output in animals and
humans alike [155–157]. The low fetal renal
blood flow, which does not attain adult levels
until 2 years of age, forms the basis for reduced
capacity for glomerular filtration in the
newborn period and early childhood [158]
Fig. 3. The normally low GFR in neonates is
further decreased in premature infants due to
2
160 GFR, ml/ min/ 1.73m
150
100
50
Age, months
0 age (Fig. 6) [166]. However, this has been
0 5 10
Fig. 3 Glomerular filtration rate (GFR) during the first
year of life (From Ref. [197])
A. Tufro and A. Gulati
lower renal perfusion, incomplete nephrogenesis,
or reduced nephron number associated with pre-
maturity Figs. 4 and 5.
The most elaborate quantitative data on post-
natal RPF in neonates and small children has been
provided from the measurement of renal extrac-
tion ratios of PAH. PAH clearance in neonates
increases from about 65 % to attain 90 % of
adult value by 5 months of age. RPF increased
from about 140 ml/min/1.73 m2 at 8 days of age in
a full-term infant to 580 ml/min/1.73 m2 at
5 months of age. Comparatively, the RPF in nor-
mal adults is 625 ml/min [159].
In preterm neonates, the RPF (PAH clearance)
is presumably lower, though not measured. The
renal hemodynamic alterations between fetal and
adult life are mainly accompanied by a gradual
decrease in RVR and consequent increments in
RBF [160]. See comment in PubMed Commons
below. Inulin clearance of premature and term
neonates remains low despite the correction for
body size [161]. This has been attributed to factors
related to the low permeability of the glomerular
filtration barrier, small available surface area for
filtration, low systemic arterial pressure, and rela-
tively high resistance of the afferent glomerular
arteriole [154]. Measurements of filtration pres-
sure in guinea pigs suggest a 2.5-fold increase
within the first 2 postnatal months. This in con-
junction with maturational changes in permeabil-
ity of the filtration barrier and increase in available
surface area for filtration could account for a
20-fold increase in GFR [162]. GFR increases
during postnatal development and maturation in
various animal species [163, 164]. Barnett
et al. showed that GFR in preterm infants does
not increase postnatally as rapidly as in full-term
infants [165]. The overall developmental pattern
of postnatal GFR changes in the humans suggests
nonlinear increments in GFR that are much more
pronounced with postnatal age beyond 34 weeks
of gestation, coinciding with the time when
nephrogenesis is completed, as compared to
much slower GFR increases at lower gestational
15
questioned by recent estimates of GFR [167].
Overall, neonates constitute a high-risk group
requiring thoughtful fluid management and choice
2 Development of Glomerular Circulation and Function 49

Fig. 4 Creatinine CREATININE CLEARANCE

clearance measured within (ml/min/1.73 M2)
24–40 h of birth in 30-week
premature to 40-week r = 0.643
full-term infants p = < .001
(From Ref. [198]) 50

40

30

20

10

26 26 30 32 34 36 38 40
GESTATION (Weeks)

1.8
1− 30 days 31− 94 days
1.6 n = 52 n = 29
PLASMA CREATININE (mg/dl)

r = −0.575 r = 0.006
1.4 p < 0.001 PNS
1.2

1.0

0.8

0.6

0.4

0.2

10 20 30 40 50 60 70 80 90 100
AGE (Days)

Fig. 5 Plasma creatinine values during the first 3 months of life in low-birth-weight infants (less than 2,000 g). (From
Ref. [199])

and dosage of exogenous agents, aiming at
primum non nocere.
GFR: Basic Concepts
Our understanding of the GFR has followed a
distinct timeline with advances in evaluation of
renal hemodynamics and knowledge of glomeru-
lar physiology. The classical concept of glomeru-
lar filtration stems from Ludwig’s filtration theory
that states that water and solutes move across the
glomerular filtration barrier due to a hydrostatic
pressure difference that favors filtration [168].
50
creatinine clearance (ml / min)
5
28
Fig. 6 Increase in creatinine clearance with GA (gesta-
tional + postnatal ages). Infants studied at birth were
grouped for GA and are represented by mean values 
1 SD connected by the solid line (y = 0.170  4.95, r =
0.51, P <0.001). Infants studied during later extrauterine
This perhaps basic and widely accepted theory
was for some time refuted, and emphasis was
laid on tubular secretion as a primary mechanism
for excretion [169]. However, observations of the
direct passage of the dye indigo carmine from the
blood into the Bowman’s space [170] supported
the original filtration theory, also supported by
Starling [171].
The movement of water and solutes via ultra-
filtration and diffusion during glomerular filtra-
tion requires that the hydrostatic pressure within
the glomerular capillaries (PG) exceeds the sum of
the oncotic pressure (Po) plus the capsular pres-
sure (Pc), that is, the pressure existing within the
Bowman’s capsule and approximately equal to
interstitial pressure. Along the capillary, as hydro-
static pressure decreases and plasma oncotic pres-
sure increases, at some point, the net ultrafiltration
pressure dissipates and a filtration equilibrium is
achieved. This hydrostatic null point occurs in the
proximal portion of the efferent arteriole so as to
promote maximal GFR and maximal reabsorption
in the peritubular capillaries [149, 152]. Since the
mean glomerular pressure is ~ 50–60 % of the
mean systemic arterial pressure (~90 mmHg),
PG averages ~45 mmHg. At filtration equilib-
rium, this value should equal the oncotic pressure
A. Tufro and A. Gulati
30 32 34 36 38 40
gestational age (wks)
life are represented by a mass plot of data with GA, in
which studies from one infant at different ages are indi-
cated by the same symbol (y = 0.406  11.548, r = 0.68,
P <0.001) (Source: Ref. [166])
plus the capsular pressure. The equilibrium
oncotic pressure can be calculated as Po is the
initial oncotic pressure. FF averages ~20 % and
taking Po as 24 mmHg [172], at filtration equilib-
rium, the plasma proteins will have been concen-
trated 1.25 times, and Po raised to 30 mmHg. The
filtration equilibrium will in principle be
maintained so long as the glomerular circulation
is active and Bowman’s capsule contains free
fluid.
The Concept of Clearance
Renal clearance of a substance X is the volume of
plasma “cleared” of substance X by the kidneys in
a given time period (Cx; ml/min). The term “clear-
ance” entails a mathematical description of the
renal handling of various substances under phys-
iological and pathophysiological conditions and
thus is used to measure kidney functional proper-
ties such as GFR and the RBF [173, 174]. Clear-
ance is expressed as a rate (ml/min) and corrected
(normalized) by body weight, kidney weight, or
an estimate of surface area. These corrections are
indispensable in pediatrics, given the broad range
of normal patients size. Renal clearance of any
2 Development of Glomerular Circulation and Function
given substance results from glomerular filtration
and any combination of the following processes:
tubular secretion, tubular reabsorption, and
intrarenal metabolism.
Cx = (Ux * V)/Px; Ux and Px are the concen-
trations of X in urine and plasma (mg/ml), respec-
tively, and V (ml/min) is the urine flow. Thus, Cx
is expressed as ml/min.
Measurement of RBF
A plasma substance that is fully cleared on a
single passage through the kidneys can be used
as a marker of renal plasma flow (RPF). Para-
aminohippuric acid (PAH), an organic acid not
normally present in the body, is given intrave-
nously to achieve a low concentration (<12
mg/dl), and the clearance of PAH accurately esti-
mates RPF [175, 176]. Although conventionally a
constant infusion of PAH is infused and periodic
multiple plasma and urine samples are obtained, a
single injection technique using 131I-labeled PAH
is more practical. Indirect assessment of RBF by
Doppler ultrasound is more often used in clinical
practice. Similarly, Doppler RBF probes are
placed on the renal artery to assess pressure and
flow in experimental studies, in addition to the
measurement of intraglomerular pressure by
micropuncture of superficial glomeruli [149].
Measurement of GFR
The most frequent clinical use of measuring clear-
ance is in determining the GFR. While renal clear-
ance takes into account the cumulative
performance of all nephrons, techniques for mea-
surement of single-kidney and single-nephron
GFR have been employed in experimental set-
tings. In order for a substance to accurately mea-
sure GFR, it must meet a series of criteria, as
defined by Homer Smith. These include the fol-
lowing: (i) the substance in question is freely
filtered by the kidneys; (ii) it is neither secreted
nor reabsorbed by the tubules; (iii) it is physiolog-
ically inert and nontoxic; (iv) it is neither cleared
nor metabolized in extrarenal sites; (v) it is neither
synthesized, stored, nor metabolized within the
kidney; (vi) it is not bound to proteins and the
plasma level of the substance must remain reason-
ably constant; and (vii) it should be cleared at a
51
constant rate regardless of amount that is in solu-
tion within the plasma.
The all-time gold standard for GFR determina-
tion is the inulin clearance method [151]. Inulin is
a small, inert polysaccharide, is a fructose poly-
mer containing 32 fructose molecules, has a
molecular weight of 5.7 KDa, and fulfills all the
aforementioned criteria. Conventionally, GFR has
been measured following a constant intravenous
infusion of inulin with periodic blood and urine
sampling. This method is currently not used in
clinical practice but has value as a research tool
[175, 177]. Multiple variations of inulin clearance
have been developed. A bolus injection of inulin
and serial measurement of its plasma concentra-
tion enables accurate calculation of GFR and is
feasible [178, 179] but not frequently used. Inulin
availability for human use and a somewhat cum-
bersome assay are major limiting factors for its
clinical use. Inulin still remains as the gold stan-
dard validation tool against which all other
methods can be tested and compared.
Radioactive (3H)-labeled inulin is commonly
used experimentally in rodents and the only avail-
able method for direct measurement of single-
nephron GFR (SNGFR) in micropuncture studies.
A surrogate for radiolabeled inulin is 1251-
iothalamate, which can be used in humans
because of minimal radiation exposure, has been
shown to have comparable accuracy
[180–182]. However, logistics of radioactive iso-
topes used in medicine are making these methods
commonly out of reach. The methods based on
renal clearance of exogenous markers such as
51
Cr-EDTA, 99Tc-DTPA, 1251-iothalamate, and
iohexol are accurate but cumbersome with less
practical utility for clinical use. These methods
involve multiple periodic blood samples (4) as
the plasma clearance is calculated by the injected
total dose divided by the total area under the curve
(AUC) obtained by a number of blood samples
after injection of the tracer. The simplified version
using less or a single blood sample is less accurate
but more feasible. However, clinical imprecision
especially for low GFRs further limits its utility.
The tracer molecules are further inaccurate in
edema states when they get distributed into the
extracellular volume.
52
Fig. 7 Analysis of 100
log-transformed height/Scr 90
and iGFR, showing that 80
65.0 % of the variation in 70
log(iGFR) can be explained
60
by log(height/Scr).
Regression line and 50
nonparametric spline
iGFR
depicted by dashed curve 40
are superimposed (Source:
Fig. 1 of Ref. [184])
30
20
1/2
Creatinine as Endogenous Marker of GFR
Methods utilizing endogenous markers such as
serum creatinine are the most commonly used in
clinical practice. Creatinine is a small (113 Da)
molecule resulting from creatine and phosphocre-
atine metabolism, produced and excreted in the
urine at a steady rate (20  5 mg/kg/day) propor-
tional to muscle mass. Serum creatinine is not
bound to proteins and is freely filtered and also
secreted actively by the proximal tubule. Tubular
secretion of creatinine amounts to ~16 % in nor-
mal adults, but increases up to 92 % at low GFRs;
therefore the overestimation of GFR as assessed
by creatinine clearance is considerable in patients
with GFR <40 ml/min [183]. Another limitation
of creatinine as a marker of GFR is its dependence
on muscle mass, which renders it not meaningful
in patients with decreased muscle mass, such as
those with chronic malnutrition, malignancy,
liver, heart, or neurologic disease. This limitation
is also relevant to healthy children, as muscle
mass varies drastically with growth and develop-
ment, as well as gender postpuberty. The measure-
ment of creatinine clearance entails timed urine
collection, which is cumbersome and potentially
impossible in small children as outpatients. Serum
creatinine is useful for population studies and for
the longitudinal follow-up of kidney function in
individual patients deemed to have normal muscle
mass. Current serum creatinine assays are
A. Tufro and A. Gulati
R-square = 65.0%
iGFR= 41.2 [ht/Scr]0.775
2/3 1 3/2 2
height [meters] / Scr
enzymatic or by mass spectrometry, and standard-
ized values are approximately 25 % lower than
previously used colorimetric assays.
Pediatric GFR prediction models based on
serum creatinine and height are reasonably accu-
rate and widely used in clinical practice. The most
used and recommended is the Schwartz equation
for the calculation of estimated GFR, which nor-
malizes the calculated GFR to a standard adult
surface area and has been updated taking into
account the enzymatic creatinine assay [184]
Fig. 7.
Creatinine clearance (ml/min/1.73 m2) = k 
height (cm)/plasma creatinine (mg/dl); where k is
a constant = 0.413.
Cystatin C and Beta-Trace Protein
as Endogenous Markers of GFR
Plasma cystatin C, an endogenous small-
molecular-weight protein secreted by most tis-
sues, freely filtered, and almost completely
degraded in the tubules, has been used as a marker
of GFR and validated in large studies including
children and by meta-analysis [185, 186]. It is
generally considered as accurate as serum creati-
nine to estimate GFR and probably more accurate
when GFR is below 75 ml/min (when the percent-
age of tubular secretion of creatinine increases)
[167, 185–187]. The main current drawback for
cystatin C use is the lack of standardization of
2 Development of Glomerular Circulation and Function
reference values using two different assays avail-
able [167] and cost. Beta-trace protein is an
endogenous circulating low-molecular-weight
protein independent of size, gender, or muscle
mass and is considered a potential surrogate
GFR marker promising in pregnant women and
neonates [188].
Newer Methods for GFR Measurement
More recently, a fluorescence-based assay using
fluorescein isothiocyanate-inulin (FITC-inulin)
has been developed [189]. The rapid development
of fluorescent microscopy has led to some addi-
tional novel approaches to determining GFR
in vivo. Yu et al. used two-photon excitation
microscopy in rats with superficial glomeruli to
directly visualize GFR at near real-time speeds
[190]. The potential for such real-time GFR meth-
odology is provocative, as it is portable to the
bedside and does not depend on numerous
assumptions seen in some other methodologies.
The two-compartment system controls for itself
and does not require taking multiple blood sam-
ples. It is highly likely that the use of fluorescence
to accurately determine GFR in humans will ulti-
mately be accomplished noninvasively using sen-
sors that do not need to penetrate the skin or enter
the blood stream [191, 192].
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 Renal Tubular Development
3
Michel Baum

Contents Developmental Features of Proximal

Organization of the Nephron . . . . . . . . . . . . . . . . . . . . . . . 62
Principals of Membrane Transport . . . . . . . . . . . . . . . . 64
Maturation of Na+/K+-ATPase Along
the Nephron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Proximal Tubule Transport . . . . . . . . . . . . . . . . . . . . . . . . . 66
Developmental Glucose Transport . . . . . . . . . . . . . . . . . 67
Tubule Water Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Developmental Transport in the Thick
Ascending Limb . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Developmental Transport in the Distal
Convoluted Tubule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Development of Urinary Concentration
and Dilution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

Transport Amino Acid: Developmental Developmental Aspects of Distal Tubule

Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Acidification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

Development of Organic Acid Transport . . . . . . . . . . 70 Developmental Features of Cortical Collecting

Tubule Sodium Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Development of Phosphate Transport . . . . . . . . . . . . . . 70
Developmental Aspects of Tubular Potassium
Development of Proximal Tubular Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Acidification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
Development of Proximal Tubule NaCl
Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

M. Baum (*)
Departments of Pediatrics and Internal Medicine,
University of Texas Southwestern Medical Center at
Dallas, Dallas, TX, USA
e-mail: Michel.Baum@UTSouthwestern.edu

# Springer-Verlag Berlin Heidelberg 2016 61

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_76
62
Organization of the Nephron
The nephron is faced with the enormous task of
maintaining a constant composition and volume
of the extracellular fluid. The amount of electro-
lytes and water ingested and absorbed must be
eliminated, and the waste products from metabo-
lism must also be excreted. This challenge is all
the more complex as our dietary intake is quite
variable from day to day. Despite this variable
intake, there is virtually no change in the volume
or composition of the extracellular fluid volume
from day to day.
There are two possible ways that our kidney
could balance ingestion and excretion. The kidney
could be a secretory organ where all the excess
solutes and water ingested would be excreted by
tubular secretion. This would be very inefficient
and require an enormous amount of energy. In
addition, in times of a disturbance in the extracel-
lular fluid volume such as a high salt intake or
volume loss from diarrhea, the regulatory systems
necessary to maintain a constant extracellular
fluid volume and composition while excreting
waste products would be very complex. On the
other hand, the kidney could filter an enormous
quantity of extracellular fluid, which would be
very efficient in removing waste products, and
reclaim the desired salt, organic solutes, and
water. With volume depletion, a greater fraction
of salt and water could be reabsorbed, and when
one has a dietary indiscretion such as the salt
intake from a pepperoni pizza, there could be a
reduction in sodium reabsorption leading to an
increase in excretion to bring us back into balance.
The mammalian kidney actually uses both mech-
anisms to perform its job which is necessary for
our survival on land. The adult kidney filters
~150 l of isotonic fluid a day and reclaims most
of it, leaving the waste produces to be excreted in
about 1.5 l of urine. In addition, there are secretory
processes for solutes such as organic anions and
cations in the proximal tubule and secretory
mechanisms to excrete the excess acid generated
from metabolism in the distal nephron which aid
in maintaining homeostasis.
To accomplish the remarkable task of reclama-
tion of the necessary solutes and water to maintain
M. Baum
a constant composition of the extracellular fluid
volume, the mammalian kidney has evolved into a
highly specialized organ with thousands of units
called nephrons. Each human kidney has approx-
imately one million nephrons. Each nephron is a
tube consisting of epithelial cells and is divided
into 12 specialized segments as shown in Fig. 1.
The epithelial cells allow for the vectorial trans-
port of solutes. The proximal tubule is responsible
for the bulk reclamation of solutes and water and
for the secretion of organic cations and anions.
Approximately two-thirds of the glomerular fil-
trate is reabsorbed by the proximal tubule in an
isotonic fashion. Virtually all of the organic sol-
utes, as well as the majority of bicarbonate, phos-
phate, and chloride, are reabsorbed in this
segment. The proximal tubule is divided into S1,
S2, and S3 segments based on the rates of transport
of some solutes and morphological changes that
occur down the proximal tubule. The proximal
tubule transitions into the thin descending limb
which can be of variable length and then makes a
hairpin turn into the thin ascending limb which
aids in the generation of a concentrated urine. The
length of the thin ascending and descending limb
is variable among species with dessert rodents
having very long thin limbs as they need to con-
serve water and excrete very concentrated urine.
The length of the thin ascending and descending
limbs increase as one goes from the superficial
cortex down to the medulla. The thin descending
limb expresses aquaporin 1, a water channel, on
the apical and basolateral membranes making the
thin descending limb very permeable to water
[1]. This segment does not actively transport sol-
utes and has a very low solute permeability. This
results in a concentrated fluid in the medulla with
a very high sodium chloride content in the luminal
fluid providing a passive driving force for sodium
chloride diffusion in the thin ascending limb. The
thin ascending limb is impermeable to water but
has a high permeability to NaCl [2]. The chloride
channel (CLC-K1) in the thin ascending limb is
developmentally regulated [3]. There is no
expression in the fetus and in the first few days
of life until the end of the first week in rats. There
is a correlation between CLC-K1 and urinary
osmolality suggesting an important role of this
3 Renal Tubular Development
S1PCT
DCT
PT
60%
S2PCT
S3PCT
Thin DL
Thin AL
Fig. 1 This cartoon depicts the nephron with its 12
segments. Shown in the figure are the nephron segments.
S1, S2, and S3 PCT depict the three segments of the prox-
imal tubule. The loop of Henle consists of the thin
descending limb (thin DL) and thin ascending limb (thin
AL) and the medullary (MTAL) and cortical thick ascend-
ing limb (CTAL). The distal convoluted tubule is com-
prised of the (DCT), connecting tubule (CT) and the
channel in generating a hypertonic medulla
[3]. Diffusion of NaCl causes a high interstitial
osmolality. This loop structure along with the
thick ascending limb generates the countercurrent
multiplication system that results in a medullary
osmolality far greater than that of blood [4, 5]. The
importance of passive properties of the thin limbs
in this countercurrent system is exemplified in
mice which are deficient in aquaporin 1 [6]. The
urine osmolality of aquaporin 1-deficient mice is
greater than plasma but far less than control mice
that have normal aquaporin 1 expression [6]. In
addition, aquaporin 1 knockout mice cannot
increase their urine osmolality in response to
water deprivation unlike control mice.
63
DCT
7%
CT
TAL
30% CCT
CTAL
OMCD
CCD
1-3%
MTAL
IMCD
1%
initial cortical collecting tubule. The collecting duct is
made up of the cortical collecting tubule (CCT) and outer
medullary (OMCD) and inner medullary collecting duct
(IMCD). Shown in yellow is the percentage of sodium
reabsorbed by the proximal tubule (PT), thick ascending
limb (TAL), distal convoluted tubule (DCT), and cortical
collecting duct (CCD). One percent of the filtered sodium
is excreted
The thick ascending limb is the segment
responsible for ~30 % of sodium chloride trans-
port and has a vital role in generating a concen-
trated medulla. Apical sodium chloride absorption
is mediated by the bumetanide-sensitive
cotransporter. One of the unique features of this
segment is the fact that this segment is imperme-
able to water and thus the fluid leaving this seg-
ment is hypotonic to blood. In addition, this
segment has a very high paracellular permeability
to cations and is responsible for much of calcium
and magnesium reabsorption. The distal convo-
luted tubule is responsible for ~5–10 % of NaCl
transport. NaCl transport in this segment is medi-
ated by the thiazide-sensitive cotransporter.
64
Active transcellular calcium and magnesium
transport also occurs in this segment.
The rest of the distal tubule is separated into the
connecting tubule and the cortical, outer, and
inner medullary collecting tubule. These seg-
ments are responsible for sodium absorption,
potassium secretion, final urinary acidification,
and water absorption, the latter mediated by vaso-
pressin, which causes the insertion of aquaporin
2 water channels into the apical membrane. While
the fraction of salt transport and renal acidification
by the collecting tubule (or collecting duct) is a
small fraction of the capabilities of some of the
upstream nephron segments of that in other neph-
ron segments, the collecting tubule is responsible
for the final modulation of the tubular fluid. Thus,
the final composition of urine and significant reg-
ulation of transport occur in these last segments.
Principals of Membrane Transport
The cells along the nephron are quite different in
the various nephron segments, which will be
discussed in the subsequent sections. The cells in
each nephron segment are poised for vectorial
transport. The apical and basolateral membranes
are, by and large, a lipid bilayer which would be
impermeable to water and solutes if there were not
specific proteins to facilitate transport across the
apical and basolateral membranes. In addition,
many transporters are regulated to adjust their
rate of transport to meet the physiologic changes
in volume status or concentration of solutes in the
extracellular milieux.
The reabsorption of solutes along the nephron
is characterized by active and passive transport
processes. A typical cell is shown in Fig. 2. It
should be appreciated that if all active transport
was inhibited along the nephron, we would
excrete urine with the composition and volume
of the glomerular ultrafiltrate. Passive
transepithelial transport is, by and large, the result
of gradients generated by active transport. Most
active transport is the result of the basolateral
Na+/K+-ATPase. This transporter pumps three
sodiums out of the cell in exchange for two potas-
sium ions. The pump utilizes ATP and it is an
M. Baum
Na+
Na+
3HCO3−
H+
Na+
ATP
Glucose 3 Na+
OH−
2 K+
Cl− ADP+Pi
Cl−
Fig. 2 A proximal tubule renal cell which shows the Na+/
K+-ATPase on the basolateral membrane, an example of
primary active transport. Na+/K+-ATPase decreases the
intracellular sodium to about 10 mEq/l and increases the
intracellular potassium to approximately 140 mEq/l. The
pump is electrogenic with potential of about 60 mV when
one compares the cell to the extracellular fluid. The sodium
gradient provides the driving force for the apical Na+/H+
exchanger, and both the sodium gradient and the potential
difference provide the driving force for the apical sodium
glucose transporter. The secretion of protons via the Na+/
H+ exchanger results in the driving force for the Cl/OH
exchanger which is an example of tertiary active transport.
Chloride is shown transversing the paracellular pathway.
Bicarbonate is exiting the basolateral membrane via a
sodium bicarbonate cotransporter
example of primary active transport. This pump
is vital to the generation of the low intracellular
sodium and high intracellular potassium concen-
tration as well as the negative intracellular poten-
tial difference across the apical and basolateral
membranes. Both the low intracellular sodium
and this potential difference can provide a
driving force for secondary active transport. For
example, in Fig. 2, the reabsorption of glucose via
a sodium-dependent transporter utilizes both
the sodium gradient and the relative negative cell
potential to bring glucose in the cell. The Na+/H+
exchanger on this cell is electroneutral and
utilizes the sodium gradient to secrete protons
and reabsorb sodium. The sodium glucose and
the Na+/H+ exchanger are secondary active trans-
port processes dependent on the basolateral
3 Renal Tubular Development
Na+/K+-ATPase. The secretion of protons will
cause the luminal pH to drop providing a favor-
able driving force for the Cl/OH exchanger, an
example of tertiary active transport. Thus, in sec-
ondary and tertiary active transport, the trans-
porters do not directly utilize ATP directly;
however, inhibition of the ATP dependent
Na+/K+-ATPase would bring these transport pro-
cesses to a halt.
In addition to active transport, a substantive
amount of passive transport occurs between cells
across the tight junction. Active transport along
the nephron will generate ion and solute gradients
between the lumen and peritubular fluid.
Depending on the permeability properties of the
tight junction, passive absorption or secretion can
occur. In the cell depicted in Fig. 2, there is pas-
sive chloride transport across the paracellular
pathway. It has become apparent that the charac-
teristics of the tight junction vary along the neph-
ron. The tight junction creates the primary
permeability barrier to diffusion of solutes across
the paracellular pathway. Occludin and claudin
proteins are localized to junctional fibrils and are
transmembrane components of tight junctions
[7–9]. These tight junction fibrils or strands are a
major factor determining the permeability proper-
ties of the paracellular pathway [7, 10, 11]. The
claudin family of tight junction proteins now
numbers 24. Occludin has a ubiquitous distribu-
tion and is not responsible for the differential
permeability properties in the various nephron
segments. The claudin isoforms present at the
tight junction of various epithelia determine the
resistance and the permeability properties of the
epithelia [7, 10, 11]. The distribution of claudin
isoforms varies along the nephron and is respon-
sible for the unique permeability properties of
each nephron segment.
The final form of passive transport is called
solvent drag. Solvent drag has been postulated to
be responsible for a small fraction of transport in
the proximal tubule. The reabsorption of solutes
could result in water movement that could entrain
or carry solutes with it. For this to occur the solute
would have to have a low reflection coefficient or
high sieving coefficient (sieving coefficient =
1refection coefficient). In other words when a
65
solute is entrained in fluid and hits a membrane or
tight junction, it could pass through it and be
transported or bounce off and not be transported.
Direct measurements of solute drag in the proxi-
mal tubule of neonates and adults have shown that
it contributes a negligible fraction to transport
[12–15].
Maturation of Na+/K+-ATPase Along
the Nephron
The Na+/K+-ATPase is located on the basolateral
membrane of most tubules in the kidney [16]. It is
a heterodimer composed of an α- and β-subunit.
There are four different α-subunits and three beta
subunits on mammalian cells which have different
functional properties [17]. There is also evidence
for a small γ-subunit that is not required for
Na+/K+-ATPase activity [17, 18]. The γ-subunit
binds to the α-subunit, stabilizes the enzyme, and
plays a regulatory role in enzyme activity [17, 18].
The adult kidney expresses the α1 and β1 isoforms
of the Na+/K+-ATPase [19, 20]. The α-subunit is
the catalytic subunit and has the cation, ATP, and
ouabain binding sites [17]. The β-subunit is the
regulatory subunit and is essential for the function
of the enzyme [17, 21]. Several hormones that
regulate sodium transport along the nephron act,
at least in part, by regulating the Na+/K+-ATPase
activity [17, 22–34]. Dopamine and atrial
natriuretic factor inhibit, while alpha agonists
and angiotensin II stimulate, Na+/K+-ATPase
activity [16]. Some hormones such as dopamine
have a direct effect to regulate the Na+/K+-ATPase
and are independent of an effect on transporters on
the apical membrane [22].
The Na+/K+-ATPase is responsible for lower-
ing the intracellular sodium concentration and
establishing the negative cell potential difference.
The Na+-K+-ATPase provides the driving force
for sodium transport across the nephron. There is
a direct relationship between sodium transport
and Na+/K+-ATPase activity factored per millime-
ter of tubule along the nephron [35]. Indeed, an
increase in intracellular sodium concentration
increases Na+/K+-ATPase activity [36]. Neonates
have a lower renal Na+/K+-ATPase activity than
66 M. Baum

Fig. 3 Na+/K+-ATPase
activity is shown in nephron 25
segments of neonates and NEONATAL
adults. As is demonstrated,

Na-K-ATPase activity (mol Pi /kg dry weight/h)

there is a maturational MATURE
increase in Na+/K+-ATPase
20
activity in every nephron
segment [39]

15

10

10 8 7 7 9 10 1111 9 7 7 14
0
PCTSN PCTJM CTAL MTAL CCD MCD

do adults [25, 37–41]. As will be discussed, there

is a developmental increase in sodium transport in Proximal Tubule Transport
each nephron segment, which is paralleled by an
increase in Na+/K+-ATPase activity as shown in Proximal tubule transport is characterized by a
Fig. 3. phenomenon called threshold, which is depicted
The parallel maturational increase in sodium on Fig. 4. It is the threshold that keeps our serum
transport with Na+/K+-ATPase activity suggests bicarbonate at 25 mEq/l. If we were to ingest
that the maturational increase in apical sodium bicarbonate and try to raise our serum bicarbonate
transport may contribute to the postnatal increase level, we would have a bicarbonaturia and our
in Na+/K+-ATPase [35, 40]. In cell culture serum levels would return to 25 mEq/l as long as
studies an increase in intracellular sodium caused we were euvolemic. Our serum glucose is set by
a stimulation in Na+/K+-ATPase activity [42, 43] other factors well below the threshold level. As
as well as an increase in the α-subunit mRNA and shown in Fig. 4, if we increased the serum glucose
membrane pump density [44]. In addition to level, we would reabsorb more glucose until the
in vitro studies, there is evidence that a chronic load of glucose delivered to the proximal tubule
increase in Na+/H+ exchanger activity induced by exceeded its ability to reabsorb glucose and we
metabolic acidosis increased Na+/K+-ATPase would have glucosuria.
activity, an effect that was blocked by In the adult kidney there is a parallel change in
coadministration of the Na+/H+ exchange inhibi- proximal tubule transport with alterations in glo-
tor, amiloride [45]. Finally, there is a postnatal merular filtration rate. This phenomenon has been
increase in both serum thyroid hormone and designated glomerular tubular balance. If the rate
glucocorticoid levels with age [46–49]. of proximal tubular transport was fixed, an
Both glucocorticoids and thyroid hormone have increase in glomerular filtration rate would
been shown to increase Na+/K+-ATPase activity swamp the distal nephron with solutes and water,
[28, 38, 50–53]. and there would be a huge natriuresis and diuresis.
3 Renal Tubular Development
Transport Maximum
Fig. 4 This figure depicts the concept of renal threshold.
As the delivered load increases to the tubule either by an
increase in the serum concentration or an increase in glo-
merular filtration rate, the amount of solute absorbed
A similar phenomenon must occur during postna-
tal development where there is a developmental
increase in glomerular filtration rate. If there was
not a parallel increase in proximal tubule transport
with the maturational increase in glomerular
filtration rate, the neonate would die of dehydra-
tion when the glomerular filtration rate increased
after birth.
While during postnatal development there is a
concomitant increase in tubular transport to
accommodate or balance the increase in glomeru-
lar filtration rate [54–56], glomerular tubular bal-
ance is not present in the fetus [57]. Renal
development is characterized by centrifugal mat-
uration. The surface nephrons are relatively
immature compared to the juxtamedullary neph-
rons. These immature nephrons with short proxi-
mal tubules have glomerular tubular imbalance
[57]. This is clinically relevant as neonates born
before 34 weeks of gestation can have glucosuria
and very premature neonates can have significant
renal salt wasting [58].
The proximal tubule reabsorbs 60 % of the
glomerular filtrate in an isoosmotic fashion. Due
to the fact that the proximal tubule has a relatively
high permeability to many ions, even solutes
which are not actively transported by this segment
may get absorbed by diffusion across the
paracellular pathway. For example, since the
proximal tubule reabsorbs over half of the glo-
merular filtrate, the luminal concentration of a
solute such as magnesium, which is not actively
67
Reabsorbed Solute
Excreted Solute
increases. At some point, the renal tubular absorption
reaches a maximum, called the threshold for that solute,
and any further increase in the filtered load is excreted
transported by this segment, would increase over
twofold. However, this does not happen because
as the magnesium concentration rises above that
in the peritubular capillaries, magnesium is pas-
sively reabsorbed across the paracellular pathway
down the concentration gradient generated by the
reabsorption of other solutes.
Developmental Glucose Transport
Glucose is reabsorbed solely by the proximal
tubule. Physiologic studies have demonstrated
that the S1 proximal tubule reabsorbs glucose by
a high-capacity low-affinity transporter, while in
the late proximal tubule (S3), glucose transport is
via a low-capacity high-affinity transporter
[59]. Similar axial heterogeneity of glucose trans-
port kinetics was validated using cortical brush
border membrane vesicles to measure apical
membrane transport and outer medullary brush
border membrane vesicles which contain vesicles
from the S3 segment [60]. The high-capacity
low-affinity sodium-dependent glucose trans-
porter on the apical membrane designated is
SGLT-2 [61]. SGLT-2 removes the bulk of the
glucose from the glomerular ultrafiltrate. The
low-capacity high-affinity transporter is desig-
nated SGLT-1 [62, 63]. The glucose that is
transported by the tubule exits across the
basolateral membrane by facilitative diffusion.
As shown in Fig. 5, SGLT-2 transports one
68
Fig. 5 Diagram of glucose Early Proximal Tubule
transport in the proximal
tubule. The early proximal
tubule has SGLT-2 on the
apical membrane which is a
high-capacity, low-affinity SGLT-2
transporter, while in the late
proximal tubule, the
low-capacity high-affinity
transporter, SGLT1, is on
the apical membrane.
Glucose exits the cell across
the basolateral membrane
by passive diffusion
Late Proximal Tubule
SGLT-1
sodium with one glucose molecule while SGLT-1
transports two sodiums with each glucose mole-
cule. A genetic mutation in SGLT-1 resulting in
loss of function causes glucose-galactose malab-
sorption as this transporter is also present in the
intestine [64–67]. Some patients with familial
glucosuria, a benign condition, have a mutation
in SGLT-2 [68, 69]. This axial arrangement of
glucose transporters, where there is a high-
capacity, low-affinity transporter followed by a
low-capacity, high-affinity glucose transporter,
results in reabsorption of virtually all of the fil-
tered glucose.
Sodium-dependent glucose reabsorption results
in a positive charge entering the proximal tubule
cell. This charge results in a lumen-negative
transepithelial potential difference. This negative
potential provides a driving force for the absorption
of an anion or the back diffusion of a cation
(sodium) across the paracellular pathway. Thus
glucose transport can result in a net absorption of
sodium chloride with sodium moving into the cell
with glucose and chloride across the paracellular
M. Baum
ATP
3 Na+
Na+
2 K+
Glucose
ADP+Pi
Glucose
ATP
2Na + 3 Na+
Glucose 2 K+
ADP+Pi
Glucose
pathway. Whether sodium is recycled or chloride is
reabsorbed is dependent on the relative sodium/
chloride permeability of the paracellular pathway.
Numerous studies using various techniques
and animal species have shown that the fetus and
neonate transport glucose at a slower rate than the
adult [57, 70–73]. These studies are of clinical
relevance as premature neonates can have
glucosuria [58, 74, 75]. Despite the fact that the
glomerular filtration rate of a premature neonate is
about 1 % of that of the adult and thus the filtered
load delivered to the neonatal proximal tubule is
also about 1% of that of the adult, the filtered load
exceeds the reabsorptive capacity for glucose
transport in the premature neonate. Thus, there is
a time during development where glomerular
tubular balance is not present. Kinetic studies
demonstrate that the Km for the glucose trans-
porter is comparable between the fetus and adult;
however, the Vmax is much lower in the neonate
[76]. This indicates that the developmental
increase in transport is not due to a glucose trans-
porter isoform change but due to a maturational
3 Renal Tubular Development
increase in the number of glucose transporters on
the apical membrane.
Transport Amino Acid: Developmental
Features
All amino acid transport occurs in the proximal
tubule. Unlike most cells which have amino acid
transporters to provide substrates for protein syn-
thesis, the proximal tubule mediates the vectorial
transport of amino acids from the glomerular fil-
trate to the blood. The basic principal for amino
acid transport is similar to glucose. The uptake of
amino acids is sodium dependent and electrogenic
with the basolateral exit mediated by facilitated
passive diffusion. While there are 20 amino acids
that are utilized in the synthesis of proteins, there
are not 20 different amino acid transporters on the
apical and basolateral membranes. Since some
amino acids are similar in structure and/or charge,
there is promiscuity among the classes of trans-
porters. We will briefly discuss the three major
classes of amino acid transporters.
The neutral amino acids include leucine,
valine, isoleucine, methionine, phenylalanine,
tyrosine, cysteine, glutamine, alanine, glycine,
serine, histidine, tryptophan, and proline. Trans-
port of these amino acids is electrogenic with one
sodium ion being transported with the amino acid
across the apical membrane. B0AT1 (SLC6A19)
is expressed on the proximal tubule [77–79] and
transports all neutral amino acids, though there is
greater affinity for valine, leucine, isoleucine, and
methionine [80]. Mutation of SLC6A19 causes
Hartnup disease which is an autosomal recessive
disorder of variable severity characterized by a
pellagra-like rash, cerebellar ataxia, and psycho-
logical and neurological disturbances [77, 79,
81]. The symptoms of Hartnup disease are likely
the result of impaired intestinal and renal transport
of tryptophan [82]. There are other neutral amino
acid transporters but their transport properties
have been less well characterized [81, 83].
The acidic amino acids are aspartate and glu-
tamate. They are transported across the apical
membrane of the proximal tubule in an electro-
genic fashion where two sodium ions are
69
transported for each of these negatively charged
amino acids [84, 85]. Brush border membrane
vesicle studies have shown that there are at least
two apical transporters for glutamate: one with a
high substrate affinity and one with a low affinity
[86]. The high-affinity transporter has been cloned
and designated SLC1A1 (EAAC1) [87]. SLC1A1
is expressed on the apical membrane of the prox-
imal tubule [88]. Eaac-1 knockout mice have a
dicarboxylic aciduria proving the importance of
this transporter in acidic amino acid transport
[89]. Recently, patients with SLC1A1 mutations
have been found to have dicarboxylic aminoacid-
uria and mental retardation likely due to the
importance of SLC1A1 for glutamate transport
in the brain [90]. The basolateral transport of
glutamate is via a sodium-dependent
cotransporter indicating that the intracellular glu-
tamate levels must be very high in the proximal
tubule to provide an adequate driving force for
sodium exit across the basolateral membrane
[91]. In addition there is a sodium-independent
aspartate/glutamate transporter which is localized
to the basolateral membrane designated
AGT1 [92].
The lysine and arginine are basic amino acids
which utilize the same amino acid transporter as
cystine. There are a number of basic amino acid
transporters [93]. rBAT is a cystine/dibasic amino
acid transporter expressed along the proximal
tubule but predominantly in the S3 segment [94,
95]. rBAT protein is undetectable in the fetal
kidney and is expressed at very low levels even
after weaning [96]. Mutations in SLC3A1, the
gene encoding rBAT, result in type I cystinuria
[97–100]. B0+AT is also a cystine transporter/
dibasic amino acid transporter [101, 102]. Unlike
rBAT, B0+AT is predominantly expressed in the
early proximal convoluted tubule but it overlaps
with the expression of rBAT [95, 103]. While both
rBAT and B0+AT can function as cystine/dibasic
amino acid transporters, they likely function
in vivo as a heterodimer [104].
Neonates have a generalized aminoaciduria
which is more pronounced in premature neonates
[105–107]. While there have been numerous stud-
ies examining amino acid transport using a num-
ber of species, most have utilized kidney slices or
70
suspensions of renal tubules. These studies are
complicated by the fact that one is looking simul-
taneously at cellular uptake, often from a col-
lapsed tubule, metabolism of the amino acid, and
basolateral exit. Examination of glycine transport
in tubule suspensions and kidney slices of neo-
nates and adults have provided disparate results.
Glycine uptake has been shown to be the same in
neonates and adults in some studies [108, 109]
and lower in neonates than adults in others [110,
111]. The few studies looking at the brush border
membrane during development, a more direct way
of studying uptake transport across the apical
membrane, have shown lower rates of transport
in the neonate compared to the adult [112, 113].
Development of Organic Acid
Transport
In addition to filtration and reclamation, the kid-
ney has the ability to secrete some substances
through organic anion transport. There are five
organic anion transporters (OATS) on the
basolateral membrane of the proximal tubule
[114–118]. Different OATS have different sub-
strate specificities. Organic acids transported
by OATS include prostaglandins, uric acid,
nonsteroidal anti-inflammatory drugs, β-lactam
antibiotics, antiviral medications, para-
aminohippurate, probenecid, uric acid,
bumetanide, salicylates, methotrexate, and many
others [115–117]. Many of these substances are
protein bound in the blood which limits their
excretion by glomerular filtration. The basolateral
uptake of OATS is an example of tertiary active
transport [115, 116]. OATS take up organic anions
into the cell predominantly in exchange for
α-ketoglutarate. The α-ketoglutarate that is
exchanged for the organic acid enters the cell via
an α-ketoglutarate transporter. The energy for this
whole process is the basolateral Na+/K+-ATPase.
The organic acid transported inside the cell must
exit across the apical membrane to enter the pri-
mordial urine. The mechanism for this is less well
understood but includes members of the
multidrug resistance protein family [115–117],
M. Baum
which has been localized to the apical membrane
of the proximal tubule [119].
Para-aminohippurate is almost totally removed
from the blood with one pass through the kidney
and is used as a measure of renal blood flow.
Para-aminohippurate has been used to assess the
maturation of renal blood flow in humans and to
determine the maturational changes in organic
anion transport. Studies in humans have shown
that there is a maturational increase in para-
aminohippurate secretion with adult values being
attained at about 2 years of age [120, 121]. Para-
aminohippurate secretion is less in premature than
term neonates [122].
There are a number of factors that could con-
tribute to the maturational increase in organic
anion secretion. Since organic anion secretion
requires an organic anion transporter (OAT), a
sodium-dependent organic acid cotransporter,
and the Na+/K+-ATPase to mediate intracellular
transport of the organic acid and an apical secre-
tory mechanism to secrete the organic acid, a
paucity in any of these transporters in neonates
(compared to the adult) could be the rate-limiting
step. OAT1 and OAT2 have been shown to be
present in the late gestation fetus, and mRNA
and protein expression increases during postnatal
development [123, 124].
One of the unique features of induction of
organic anion transport is that it can be induced
prematurely during renal development by itself or
another organic anion [125–128]. This is not true
of adult animals where organic anions do not
cause a stimulation in transport [125]. In vitro
microperfusion studies have demonstrated that
there was an intrinsic increase in the rate of trans-
port with postnatal age in rabbits and that
pretreatment with penicillin increased the rate of
para-aminohippurate secretion in vitro [128]. The
mechanism of this induction of organic anion
transport by organic acids is unclear.
Development of Phosphate Transport
The adult ingests approximately 1–1.5 g of phos-
phorus a day and of that 80 % is absorbed. The
adult must be in neutral phosphorus balance so
3 Renal Tubular Development
that the amount of phosphorus absorbed daily
must equal the amount excreted. The phosphorus
in the human body is predominantly in the form of
phosphate. At a pH of 7.4, there is a 4:1 ratio of
HPO42/ H2PO41. The kidney maintains phos-
phate balance by the ability to regulate phosphate
transport which predominantly occurs in the prox-
imal tubule. The main factors that regulate renal
phosphate transport are dietary intake itself and a
number of hormones including parathyroid hor-
mone, FGF-23, and growth hormone. Phosphate
is essential for bone growth and 85 % of our
phosphate is in bones. In addition, phosphate is
involved in a myriad of enzymatic reactions and is
present in nucleotides, phospholipids, and pro-
teins. Unlike the adult, the neonate must be in
positive phosphate balance for growth. The
serum phosphate level is higher in the neonate
than the adult. This section will review phosphate
transport and then discuss developmental changes
which occur in transport and its regulation which
allows the neonate to be in positive phosphate
balance.
The transporters involved in the regulation of
phosphate transport are shown in Fig. 6. The first
phosphate transporter cloned, designated NaPi-1,
did not have the characteristics previously identi-
fied in physiologic studies, and its function is still
not clear [129, 130]. There are two sodium-
dependent phosphate transporters on the apical
membrane of the proximal tubule: one designated
NaPi-IIa [131] and the other NaPi-IIc [132]. NaPi-
IIa is an electrogenic transporter that transports
three sodium ions with one phosphate (HPO42),
while NaPi-IIc transports two sodium ions with
every phosphate and is electroneutral [132,
133]. NaPi-IIa is regulated by PTH and dietary
phosphate intake [134–137]. Phosphate exits the
proximal tubule by a transporter which has not yet
been identified and characterized. NaPi-IIb is the
phosphate transporter on the intestinal apical
membrane responsible for absorption of dietary
phosphate [138].
The serum phosphate levels are higher in neo-
nates than adults [139, 140]. Since the glomerular
filtration rate in the neonate is only a fraction that
of the adult, it is possible that this is the factor that
is responsible for the relative hyperphosphatemia
71
3Na+
IIa ATP
HPO42−
3 Na+
2 K+
ADP+Pi
2Na+
IIc HPO42−
HPO42−
Fig. 6 A proximal tubule cell reabsorbing phosphate is
shown. The top apical phosphate transporter NaPi-IIa is the
predominant phosphate transporter in adult rodents. NaPi-
IIc is the predominant phosphate transporter on the apical
membrane of neonatal rodents. NaPi-IIa is electrogenic
while NaPi-IIc is electroneutral. NaPi-IIc is the main phos-
phate transporter on the apical membrane of the proximal
tubule in humans
in neonates. However, early studies in human
neonates found that the fraction of phosphate
reabsorbed compared to the glomerular filtration
rate was higher in neonates and infants than that in
adults [139–142]. In the first 24 h of life, human
neonates reabsorb over 95 % of the filtered phos-
phate [140]. This level drops to 90 % later in the
first week of life [139, 140]. This fractional
reabsorption of phosphate meets or exceeds the
reabsorptive capacity of adults and older children
ingesting a normal phosphate diet [142].
Animal studies demonstrated that young rats
had a greater rate of phosphate reabsorption than
adults [143]. This was seen in rats that received
parathyroidectomy indicating that an altered
response to parathyroid hormone was not the fac-
tor that caused the disparity in renal phosphate
uptake [143]. Finally both young and adult rats
responded to a low-phosphate diet with an
increase in the fractional reabsorption of phos-
phate; the magnitude of phosphate absorption
was again higher in young animals than
adults [143].
72
REABSORPTION RATE OF Pi / KW (mg/min/g KW)
Newborn
60
Adult
50
40
30
20
10
0
10 20 30 40 50
FILTETED LOAD OF Pi (mg/min)
Fig. 7 The rate of phosphate absorption by isolated per-
fused kidney preparation. As the filtered load increases,
there was an increase in the reabsorptive rate in both the
neonate and the adult kidney. At all filtered loads the rate of
phosphate reabsorption per gram of kidney weight was
higher in the neonate (From Johnson and Spitzer [144],
with permission)
While the above studies are consistent with an
enhanced tubular reabsorptive capacity in neo-
nates, this could be due to a higher reabsorptive
capacity of the neonatal tubule, a diminished
response to a phosphaturic factor, or the result of
an increased response to a substance which
increases phosphate transport. To determine if
there was an inherent increase in tubular phos-
phate transport, Johnson and Spitzer examined
phosphate absorption in neonatal and adult kid-
neys perfused in vitro [144]. As shown in Fig. 7,
neonatal kidneys had a higher phosphate
reabsorptive rate at any filtered load of phosphate
which can only be due to an increased inherent
rate of phosphate transport. In addition, they
found that while addition of parathyroid hormone
to the perfusate caused a phosphaturia in adult
kidneys, there was no increase in phosphate
excretion in neonatal kidneys. These studies
directly demonstrate that neonates also have an
attenuated effect of the primary factor regulating
phosphate transport, parathyroid hormone. A
blunted effect of parathyroid hormone on
M. Baum
phosphate reabsorption has also been demon-
strated in young rats compared to adult rats
in vivo [145, 146].
Micropuncture studies of neonatal and adult
guinea pigs and rats have also demonstrated that
there is a higher intrinsic rate of phosphate trans-
port in young animals [147, 148]. Studies using
brush border membrane vesicles have demon-
strated that the maximal rate (Vmax) of phosphate
transport was severalfold higher in neonates than
adults, while there was no difference in the Km,
the phosphate concentration at half maximal
velocity [149]. A low-phosphate diet increased
the Vmax of the sodium phosphate transporter in
adult guinea pigs while a high-phosphate diet had
60 the opposite effect. There was no significant dif-
ference in Vmax in brush border membranes from
neonates gavaged with different phosphate-
containing diets [149]. As shown in Fig. 8, the
3-week-old rat has a maximal rate of phosphate
absorption that was higher than that of adult rats
[150]. At all ages there was an augmentation in the
maximal capacity of phosphate reabsorption with
a low-phosphate diet [150]. A greater effect of
phosphate deprivation on brush border membrane
vesicle phosphate uptake and NaP-IIa protein
abundance was demonstrated in 4-week-old rats
compared to older adult rats [148]. Finally, the
driving force for phosphate entry across the apical
membrane may be greater in neonates. The intra-
cellular phosphate concentration was almost 40 %
lower in kidneys measured using NMR [151].
Maturational studies examining the changes in
NaPi-IIa expression have revealed that NaPi-IIa
mRNA and protein are not detected in developing
nephrons until there is a distinct brush border
membrane [152]. NaPi-IIa protein abundance
was greater in brush border membranes from
13-day-old rats compared to 22-day-old rats
[152]. Others however have found that brush bor-
der membrane vesicle from suckling and adult rats
had a slower rate of phosphate uptake than wean-
ling rats (21 days old) [153]. There was no change
in NaPi-IIa mRNA abundance, but NaPi-IIa pro-
tein abundance from brush border membrane ves-
icles confirmed the transport findings that
21-day-old rats had the highest NaPi-IIa protein
expression [153]. Studies comparing 28-day-old
3 Renal Tubular Development
14
* Diet
10
Max RPl/GFR (umol/ml)
8 *
6
4 *
2
0
IMMATURE
Fig. 8 Age-dependent maximal phosphate reabsorption is
seen in 3–4-week-old rats (immature), 6–7-week-old rats
(young), and 12–13-week-old rats (adult). All rats were
parathyroidectomized. A low dietary phosphate intake
rats to adult rats have also demonstrated greater
brush border membrane NaPi-IIa protein abun-
dance in young rats than adults [148].
The high rates of phosphate transport in wean-
ling rodents suggested that there may be a
developmentally regulated transporter with
greater expression at that time during develop-
ment [154]. Indeed, NaPi-IIc is a brush border
phosphate transporter with its highest expression
at the time of weanling in the rat [132]. NaPi-IIc,
like NaPi-IIa, is regulated by dietary phosphate
uptake [132]. The relative importance of NaPi-IIa
and NaPi-IIc may be quite different in humans.
Patients with hereditary hypophosphatemic rick-
ets with hypercalciuria, a rare autosomal recessive
characterized by hypophosphatemia secondary to
renal phosphate wasting and high vitamin D
levels, have a mutation, in the gene encoding
NaPi-IIc. This suggests that NaPi-IIc may be the
predominant renal phosphate transporter in
humans and that NaPi-IIa cannot compensate for
the loss of NaPi-IIc [155].
Growth hormone increases phosphate trans-
port via stimulation of IGF-1 in the proximal
tubule [156]. Brush border membrane vesicle
phosphate transport is increased in dogs that
were administered growth hormone compared to
vehicle-treated controls [157]. While growth
73
Low Pi
Normal Pi
High Pi
*
* *
YOUNG ADULT
stimulated phosphate absorption in all age groups. The
maximal capacity for phosphate absorption was in the
immature groups which need phosphate for growth
(From Mulroney and Haramati [150], with permission)
hormone is not a significant regulator of phos-
phate transport in the adult, this may not be the
case in the growing animal. Administration of a
growth hormone-releasing factor antagonist,
which suppresses growth hormone secretion, has
no effect on phosphate transport in adult rats but
significantly reduces phosphate absorption in
young growing rats [158–160].
There are a number of hormones that have been
shown to regulate phosphate transport including
insulin [161], fibroblast growth factor-23
[162–164], frizzled-related protein 4 [165], and
Klotho [166, 167]. These hormones may have
differential effects on phosphate transport in the
neonate and adult, but this is yet to be determined.
Development of Proximal Tubular
Acidification
The proximal tubule reabsorbs 80 % of the filtered
bicarbonate. Luminal proton secretion is via the
Na+/H+ exchanger and the H+-ATPase. In the
adult, one-third of proton secretion is via the
luminal H+-ATPase, and two-thirds is mediated
by the luminal Na+/H+ exchanger that is desig-
nated NHE3 [168, 169]. The secreted proton
titrates the filtered HCO3 to generate H2CO3
74
a NHE8 Expression on BBMV
NHE8
β-actin
1D 7D 14D 26D AD
1.2
*+
1.0
*
NHE-8/β-actin
0.8
0.6
0.4
0.2
0.0
1D 7D 14D 26D AD
Fig. 9 Immunoblots of rat brush border membrane
vesicles depict the changes in NHE8 (A) and NHE3 (B)
protein abundance. As is seen there, there is higher expres-
sion of NHE8 in the neonate than the adult brush border
which is converted to CO2 and H2O by luminal
carbonic anhydrase (carbonic anhydrase IV). CO2
diffuses into the cell and combines with H2O
which is facilitated by intracellular carbonic
anhydrase (carbonic anhydrase II) to regenerate
H2CO3. H2CO3 dissociates into a proton that can
be secreted across the apical membrane and bicar-
bonate that exits the basolateral membrane via the
basolateral Na(HCO3)3 cotransporter. The sodium
that enters the cell via the Na+/H+ exchanger exits
the basolateral membrane by the Na+/K+-ATPase,
which provides the driving force for luminal pro-
ton secretion by the Na+/H+ exchanger. There are
maturational changes in most of these processes
that will be described below.
Neonates have a lower serum bicarbonate con-
centration than adults, which is the result of a
lower threshold for bicarbonate [170]. Premature
neonates can have physiologic bicarbonate con-
centrations as low as 15 mEq/l [171]. The lower
bicarbonate threshold is mediated in large part by
the lower rate of proximal tubule acidification.
The fine-tuning of renal acidification is mediated
M. Baum
b NHE3 Expression on BBMV
NHE3
β-actin
1D 7D 14D 26D AD
1.2
1.0
NHE-3/β-actin
0.8
#
0.6
0.4
^ ^
0.2 # #
0.0
1D 7D 14D 26D AD
membrane. The expression of NHE3 is highest in the adult.
The higher NHE3 protein abundance at 1 day of age is
likely the result of the surge of glucocorticoids at the time
of birth (From Becker et al. [176], with permission)
in the distal nephron which, by and large, is
responsible for the secretion of acid from metab-
olism and new bone formation. Studies have
shown that there is a maturational increase in
bicarbonate absorption during postnatal develop-
ment [73, 172], which accounts for the mutational
increase in the bicarbonate threshold.
The greatest developmental changes in proxi-
mal tubule acidification occur on the apical mem-
brane. There is a fourfold increase in Na+/H+
exchanger activity during postnatal maturation
and an even greater increase in apical H+-ATPase
activity [168, 173, 174]. Low levels of Na+/H+
exchanger activity have been measured in the
fetus as well [175]. Despite the fact that there
was Na+/H+ exchanger activity on the apical
membrane of the neonatal rat at 1–2 weeks of
age, as shown in Fig. 9, there was virtually no
NHE3 on the brush border membrane [174, 176],
the Na+/H+ exchanger on the apical membrane of
the adult proximal tubule [177, 178].
NHE3 knockout mice have been shown to
have substantive proximal tubule apical
3 Renal Tubular Development
membrane Na+/H+ exchanger activity [179]. The
apical Na+/H+ exchanger likely responsible for
this non-NHE3 Na+/H+ exchanger activity is
NHE8, a recently discovered NHE isoform
([180, 181]). NHE8 has sodium-dependent proton
extrusion capabilities, which made it a potential
candidate for the developmental isoform
[182]. As shown in Fig. 9, apical NHE8 was
predominantly present in neonatal proximal
tubules at a time when NHE3 was almost
undetectable [176]. Thus there is a developmental
Na+/H+ exchanger isoform.
The Na(HCO3)3 symporter mediates bicarbon-
ate exit in both the neonatal and adult proximal
tubule. While there is a maturational increase in
basolateral membrane Na(HCO3)3 symporter
activity, it is relatively small compared to the
magnitude of the change in Na+/H+ exchanger
activity on the apical membrane [183]. In addition
to bicarbonate exit, the basolateral membrane Na
(HCO3)3 symporter plays an important role in pH
regulation of the proximal tubule cell [183].
Carbonic anhydrase increases the rate of the
interconversion of CO2 and H2O to H2CO3.
Carbonic anhydrase II is located intracellularly
in proximal and distal tubule acidifying cells and
comprises ~95 % of total cellular carbonic
anhydrase activity. Carbonic anhydrase IV is on
the apical and basolateral membrane of renal acid-
ifying cells and comprises ~5 % of carbonic
anhydrase activity [184]. Both carbonic
anhydrase II and IV increase during maturation
of proximal and distal acidification, but neither is
likely a limiting factor causing the maturational
increase in renal acidification in the proximal or
distal tubule [185–187].
Since the developmental increase in renal acid-
ification is due primarily to apical proton secre-
tion, many studies have examined the cause for
the increase in Na+/H+ exchanger activity. There
is a substantive increase in both thyroid hormone
and glucocorticoids during postnatal develop-
ment, and both hormones increase in parallel
with the increase in proximal tubule Na+/H+
exchanger activity [46–49]. Administration of
either glucocorticoids or thyroid hormone prior
to the maturational increase in either hormone
results in a precocious increase in exchanger
75
Na+/H+ activity and NHE3 protein abundance
[46, 172, 188]. Both thyroid hormone and
glucocorticoids increase NHE3 activity by
increasing transcription [189, 190]. Glucocorti-
coids have also recently been shown to increase
the insertion of NHE3 into the apical membrane of
proximal tubular cells by a posttranscriptional
mechanism [191].
Interestingly, neither prevention of the matura-
tional increase in glucocorticoids or thyroid hor-
mone alone can totally prevent the postnatal
increase in Na+/H+ exchanger activity and NHE3
mRNA and protein abundance [46, 172, 192,
193]. Thus, there appears to be some redundancy
in the postnatal maturational triggers for NHE3.
As demonstrated in Fig. 10, prevention of the
maturational increase in both hormones results
in the prevention of the postnatal increase in
NHE3 mRNA, protein abundance, and Na+/H+
exchanger activity [194].
Development of Proximal Tubule NaCl
Transport
The glomerulus generates an ultrafiltrate of
plasma and delivers the fluid to the proximal
tubule. In the early proximal tubule, the
transcellular reabsorption of sodium with glucose
and amino acids results in a lumen-negative
potential difference. This lumen-negative poten-
tial provides a driving force for either chloride
absorption or sodium secretion across the
paracellular pathway. Whether chloride is
absorbed or sodium is secreted is dependent on
the relative permeabilities of chloride to sodium in
the proximal tubule. The relative chloride to
sodium permeability is higher in the adult than
the neonate, so passive chloride transport second-
ary to the lumen-negative potential difference in
the early proximal tubule does not occur to a
significant degree in the neonate [14, 195].
The reabsorption of bicarbonate in preference
to chloride by the early proximal tubule causes the
luminal fluid in the late proximal tubule to have a
higher chloride and lower bicarbonate concentra-
tion than that of the peritubular fluid [196,
197]. The axial changes in luminal fluid
76
1000
* greater than 9 day (unpaired T-test)
+ less than 9 day (unpaired T-test)
** greater than 9 day, Adx and Adx-HypoT
800
JH (pmol/mm.min)
600
400
200
0
9 Day
Fig. 10 Apical Na+/H+ exchanger activity in perfused
tubules in 9-day-old rats and adults that were adrenalecto-
mized as neonates, adults that were adrenalectomized and
hypothyroid since the neonatal period, and adults that were
adrenalectomized and hypothyroid (adx-HypoT) since the
neonatal period but given thyroid and glucocorticoid
replacement before study and sham controls. The
composition are depicted in Fig. 11. The chloride
permeability is greater than that for bicarbonate so
that the concentration gradient is a driving force
for passive chloride diffusion across the
paracellular pathway. The diffusion of chloride
across the paracellular pathway results in a
lumen-positive potential difference and a driving
force for the paracellular transport of sodium
resulting in net passive NaCl absorption.
In the adult proximal tubule, approximately
half of sodium chloride is active and transcellular
and half is passive and paracellular [193, 198,
199]. Active chloride transport is mediated by
parallel action of the Na+/H+ and Cl/base
exchangers on the apical membrane [193,
200–202]. It is still somewhat unclear what the
nature of the base is as there is evidence for
chloride exchange for hydroxyl, formate, and oxa-
late ions [193, 203–207]. The rate of active
transcellular chloride transport is lower in the
neonate than the adult. This is due to the lower
rate of the apical Cl/base exchanger [193, 208]
and the Na+/H+ exchanger [168, 173, 174]. The
M. Baum
**
**
*
+
Adx- Adx-HypoT
Adx Sham
HypoT +Replacement
9-day-old rats had a lower rate of Na+/H+ exchanger activ-
ity than the sham adult. Neonatal adrenalectomy did not
totally prevent the maturational increase in Na+/H+
exchanger activity, but the maturational increase in
Na+/H+ exchanger activity was abrogated in the adrenal-
ectomized hypothyroid group (From Gupta et al. [194],
with permission)
driving force for active transcellular NaCl trans-
port is the basolateral Na+/K+-ATPase that has
lower activity in the neonatal proximal tubule
[39, 40, 209]. There are a number of factors that
regulate proximal tubule NaCl transport including
renal nerves, dopamine, and angiotensin II which
are shown in Table 1. The serum levels of most
hormones that regulate sodium absorption are
equal or higher in the neonate than the adult, but
in general there is a blunted response to the action
of most regulatory hormones.
There are also changes in the properties of the
paracellular pathway during postnatal develop-
ment [14, 195, 210, 211]. Most importantly, the
permeability of the proximal tubule to chloride
ions is less in the neonate than the adult [14,
195, 211]. The low permeability to chloride ions
results in almost no passive paracellular chloride
transport in the neonate [14, 195]. As previously
noted, the permeability properties of an epithe-
lium are determined by the expression of a family
of proteins called claudins. The claudin proteins in
the tight junction change during the postnatal
3 Renal Tubular Development
2.0
1.8 Inulin
1.6
1.4 Cl−
1.2
Na+
TF
1.0
P Osm
0.8
0.6 HCO3−
0.4
Amino
acids
0.2
Glucose
0
0 25 50 75 100
% Proximal tubule length
Fig. 11 The axial changes in proximal tubular transport
are depicted on this figure. The early proximal tubule
preferentially reabsorbs glucose, amino acids, and bicar-
bonate leaving the luminal chloride solution higher than
the blood in the peritubular capillaries. The early proximal
tubule has a lumen-negative transepithelial potential dif-
ference due to sodium-dependent glucose and amino acid
reabsorption. The higher luminal chloride concentration in
the late proximal tubule provides a driving force for pas-
sive chloride absorption across the paracellular pathway
causing a lumen-positive potential difference (From Rector
[197], with permission)
development. Claudins 6, 9, and 13 are present in
the neonatal proximal tubule but not in the adult
[210]. The claudin isoform responsible for the low
paracellular chloride permeability in the neonate
and the factors that cause the claudin isoform
changes during development have yet to be
determined.
Of the potential factors that cause the maturational
changes in paracellular chloride transport, only thy-
roid hormone has been examined [212]. Administra-
tion of thyroid hormone prior to the normal
maturational increase results in an increase in chlo-
ride permeability. On the other hand, maintaining a
hypothyroid state into adulthood prevents the matu-
rational increase in chloride permeability. It is as yet
to be determined if thyroid hormone is the factor that
causes the proximal tubule claudin isoform changes
during postnatal development.
77
Developmental Features of Proximal
Tubule Water Transport
The proximal tubule reabsorbs most of the glo-
merular filtrate without a significant change in the
luminal osmolality. For this to occur, the proximal
tubule must be very permeable to water. Water
movement is predominantly through the cell and
not across the paracellular pathway [213,
214]. The constitutively water-permeable proxi-
mal tubule and thin descending limb have water
channels on the apical and basolateral membranes
[1]. The isoform of this water channel is aquaporin
1 [215–218]. Direct evidence of transcellular
water transport comes from aquaporin 1 knockout
mice which have a marked decrease in proximal
tubule sodium absorption [219]. There is a paucity
of water channels in the fetal kidney, and an
increase in expression of aquaporin 1 does not
occur until birth [220, 221].
Direct measurements of water permeability
have demonstrated that the neonatal rabbit proxi-
mal tubule has a higher water permeability than
that of the adult [222]. To determine the mecha-
nism for the higher water permeability in neonatal
rabbit tubules, studies were performed examining
the water permeability of apical and basolateral
membrane vesicles [223–225]. Despite the higher
transepithelial water permeability, the water per-
meability of the apical and basolateral membrane
was lower in the neonate than the adult and there
was less aquaporin 1 expression on both the apical
and basolateral membranes of the proximal tubule
of the neonate [224, 225]. The apparent paradox
between the higher water permeability in the neo-
natal proximal tubule and the lower water perme-
ability of the apical and basolateral membranes
was resolved with measurements of the contribu-
tion of the intracellular compartment to water
movement in the neonatal and adult proximal
tubule. The intracellular compartment was found
to cause a large resistance to water flow, and
neonatal proximal tubule intracellular compart-
ment was less of a constraint to transcellular
water movement than that of the adult [214].
The postnatal increase in glucocorticoids is a
likely factor in mediating the above noted matu-
rational changes in water permeability and
78 M. Baum

Table 1 Regulation of Sodium Transport

Effect on urinary Effect of hormone on sodium in
sodium excretion in Neonatal serum level compared neonatal sodium excretion
Hormone adult to adult compared to adult References
Renin Level responds appropriately in [327, 330,
neonate 342, 343]

Aldosterone Increases distal [325–331]

sodium absorption

Atrial Increases sodium Probably [344] [345–348]

natriuretic excretion
peptide

Dopamine Decreases proximal [25,

tubule sodium 349–351]
absorption

PGE-2 Causes natriuresis Blunted synthesis in the [353, 354]

and diuresis medullary and cortical
collecting tubule [352]

aquaporin 1 expression. Administration of gluco-
corticoids to neonatal rabbits resulted in an
increase in brush border membrane water perme-
ability and aquaporin 1 expression [226]. Of inter-
est, the developmental increase in thyroid
hormone was not shown to be a factor mediating
these postnatal maturational changes in water
transport [227].
Developmental Transport in the Thick
Ascending Limb
The thick ascending limb (TAL) is responsible for
the reabsorption of 30 % of filtered NaCl. The
thick ascending limb is impermeable to water.
The reabsorption of NaCl without water by the
cortical and medullary TAL along with the distal
convoluted tubule generates a luminal fluid with
an osmolality of 50 mOsm/kg water. In the
absence of ADH action on the collecting tubule,
this dilute urine will be excreted. The medullary
interstitial hypertonicity is largely generated by
active NaCl reabsorption without water transport
in that tubular segment.
The reabsorption of NaCl is due to secondary
active transport with the basolateral Na+/K+-
ATPase generating a low intracellular sodium to
provide a driving force for luminal sodium entry.
A cartoon of a thick ascending limb cell is
shown in Fig. 12. Sodium enters the cell via the
furosemide- or bumetanide-sensitive Na+/K+/
2Cl cotransporter [228, 229]. The Na+/K+/2Cl
cotransporter is electroneutral, yet there is a
lumen-positive transepithelial potential difference
in the thick ascending limb [230]. The positive
luminal potential is generated by apical potassium
recycling via the potassium channel ROMK1
[231, 232]. With recycling of potassium back
into the lumen, there is the net reabsorption of
1 sodium and 2 chloride ions which exit the cell
across the basolateral membrane. Sodium exits
the cell via the Na+/K+-ATPase. Chloride predom-
inantly exits the cell via a chloride channel desig-
nated ClC-Kb [233–235]. A subunit of this
chloride channel designated barttin is important
in the function of ClC-Kb [236]. The lumen-
positive potential difference, generated by potas-
sium secretion into the lumen, generates a driving
force for the passive reabsorption of cations
including Mg2+ and Ca2+. The thick ascending
limb has a very high permeability to magnesium
and calcium ions which results in a substantial
fraction of filtered calcium and magnesium being
reabsorbed passively across the paracellular path-
way in this segment [237–241]. The unique
3 Renal Tubular Development
Cl−
Na+
2Cl−
K+ ATP
3Na+
2K+
ADP+P
K+
K+
K+
Cl−
Fig. 12 The figure depicts a thick ascending limb cell. On
the apical membrane is the Na+/K+/2Cl cotransporter that
is sensitive to loop diuretics and the potassium channel
designated ROMK that is depicted as the green arrow.
The basolateral membrane has a Na+/K+-ATPase; a KCl
cotransporter; a chloride channel designated ClC-Kb, with
its accessory channel designated barttin in yellow; and a
potassium channel. A loss of function mutation in the
Na+/K+/2Cl cotransporter, ROMK, ClC-Kb, or barttin
results in Bartter’s syndrome
permeability properties of the thick ascending
limb are due to the expression of claudin
16 which is mutated in familial hypomagnesemia
with hypercalciuria and nephrocalcinosis where
there is renal magnesium and calcium
wasting [242].
Mutations of the Na+/K+/2Cl cotransporter
[243], ROMK [244, 245], ClC-Kb [246], and
barttin [236, 247] all cause Bartter’s syndrome
(see this text, ▶ Chap. 38, “Renal Tubular
Disorders of Electrolyte Regulation in Children”).
Barttin is also expressed in the ear where it is a
subunit of ClC-Ka and ClC-Kb. Mutations
in barttin cause sensory neural hearing loss
[236, 247].
Micropuncture studies studying fluid from the
early distal tubule showed that the osmolality of
the fluid was significantly lower in the adult than
the neonatal rat [248]. This is consistent with
lower rates of sodium transport in the thick
ascending limb, but this study did not examine
79
the latter parts of the diluting segment. Human
neonates are able to dilute their urine to the same
level as an adult (50 mOsm/kg water). This is vital
since neonates ingest a hypotonic fluid, mother’s
milk. Sodium transport in the thick ascending
limb has been directly examined in vitro and
shown to be fivefold lower in the neonate than in
the adult [249].
The Na+/K+/2Cl cotransporter can first be
detected in the mid-late gestation rat thick ascend-
ing limb and macula densa [250]. In the rat there is
a postnatal maturational increase in Na+/K+/2Cl
cotransporter, ROMK, Na+/K+-ATPase mRNA,
and protein abundance but no change in ClC-K
mRNA abundance [251]. Administration of dexa-
methasone before the normal maturational increase
at the time of weaning resulted in a premature
increase in urinary concentrating ability and
increase in Na+/K+/2Cl cotransporter, Na+/K+-
ATPase mRNA, and protein abundance but no
change in ROMK protein abundance [251]. There
is also a postnatal maturational increase in thick
ascending limb Na+/K+-ATPase activity [39, 252,
253]. The maturational increase in thick ascending
limb Na+/K+-ATPase activity could be accelerated
precociously by glucocorticoids and prevented by
neonatal adrenalectomy [252, 253].
Developmental Transport in the Distal
Convoluted Tubule
The distal convoluted tubule reabsorbs approxi-
mately 7 % of the filtered sodium. This segment is
water impermeable, and further reabsorption of
salt without water occurs in this segment resulting
in the nadir of luminal fluid osmolality. The trans-
porters responsible for NaCl transport in the distal
convoluted tubule are shown in Fig. 13. Sodium
entry is via the thiazide-sensitive cotransporter
[254–257]. Mutations in the thiazide-sensitive
cotransporter cause Gitelman’s syndrome
[258–260] (see this text, ▶ Chap. 38, “Renal
Tubular Disorders of Electrolyte Regulation in
Children”). There is also evidence for parallel
Na+/H+ and Cl/base exchange mediating sodium
entry in the rat [261]. The isoform of this Na+/H+
exchanger is NHE2 [262]. Chloride exits the cell
80
Cl−
K+
Na+
ATP
3 Na+
Cl−
2 K+
ADP+Pi
Cl−
K+
Fig. 13 Distal convoluted cell showing the transporters
involved in active sodium reabsorption. The apical NaCl
transporter is the thiazide-sensitive cotransporter.
Inactivating mutations in the thiazide-sensitive
cotransporter lead to Gitelman’s syndrome
via a basolateral chloride channel ClC-K [263]
and a KCl cotransporter KCNQ1 [264,
265]. There is also a basolateral potassium chan-
nel [266]. There is active transcellular calcium
and magnesium transport in the distal convoluted
tubule, but there is no information about the
developmental expression and regulation of
these transporters [267, 268].
The distal convoluted tubule is very difficult to
study in vitro and in vivo. No studies have been
performed to date examining the relative abun-
dance of the thiazide-sensitive cotransporter in
the neonate and adult. A study has been preformed
that suggests that there is increased sodium
absorption in 24- compared to 40-day-old rats
[269, 270]. Before detailing this study, it is impor-
tant to know that studies have shown that the
neonate is less able to excrete a salt load compared
to the adult [270–272]. For example, administra-
tion of isotonic saline equal to 10 % of the dogs
weight to an adult dog resulted in excretion of
50 % of the salt load over 8 h, but only 10 %
of the salt load was excreted in the 1–2-week-old
M. Baum
dog [270]. This limited ability to excrete a sodium
load was not due to the lower glomerular filtration
rate in the neonate [270]. Since sodium transport
is less in the proximal tubule, thick ascending
limb, and collecting tubule, the nephron segment
where there was avid neonatal sodium
reabsorption was unclear. It must be stated that it
was never considered that the volume of distribu-
tion of the saline infusion was greater in the neo-
nate than the adult to account for these findings. A
classical micropuncture study in developing rats
suggested that the sodium retention in the neonate
may be the result of avid sodium reabsorption in
the distal convoluted tubule [269].
Development of Urinary
Concentration and Dilution
The urine exiting the distal convoluted tubule and
entering the collecting duct has an osmolality of
~50 mOsm/kg water. Whether the urine will dilute
or is maximally concentrated depends on the pres-
ence or absence of vasopressin (ADH). Neonates
can dilute their urine to nearly the adult level of
50 mOsm/kg water [273–275]. Thus the neonate
that drinks mother’s milk can excrete free water
[274–276]. However, unlike the normal adult
where it is almost impossible to drink enough
water to cause hyponatremia (>30 l/day), neo-
nates have a rather limited ability to generate and
excrete free water. Therefore improperly mixed
and dilute formula or administration of excessive
amounts of dilute fluid via NG or G tube can result
in hyponatremia [274].
The maximum urine osmolality of the term
human neonate is ~400–600 mOsm/kg water
[277–279]. The adult urine osmolality of 1,200
mOsm/kg water is achieved at ~1.5–2 years of age
[279]. There are many potential developmental
factors that could limit the ability of the neonate
to generate maximally concentrated urine. The
factors involved in urine concentration include:
1. The ability to sense an increase in serum osmo-
lality or decrease in extracellular volume and
secrete vasopressin.
3 Renal Tubular Development
2. There must be functional vasopressin receptors
on the basolateral membrane of the
collecting duct.
3. The collecting duct must be able to
generate cAMP.
4. The loop of Henle must have generated a con-
centrated interstitium.
5. The architecture of the medulla must not limit
the concentrating ability.
6. The collecting tubule must have aquaporins on
the apical and basolateral membrane.
7. There must not be extracellular or intracellular
mechanisms upregulated in the neonate which
limit urinary concentrating abilities.
During prenatal and postnatal maturation,
there are anatomical changes that occur affecting
urinary concentrating ability. There is an increase
in the medullary capillary density, a decrease in
medullary interstitial connective tissue, and an
increased presence and length of the thin limbs,
and the tubules become more tightly packed with
cells in the loop decreasing in height with matu-
ration [280, 281]. The length of the papilla
increases linearly from day 10 to day 40 in the
rat [282]. All these developmental anatomical
changes are necessary for the countercurrent mul-
tiplication system to be maximally efficient.
Accompanied by these anatomical changes are
concomitant increases in the medullary sodium
and urea concentration [282, 283]. Administration
of a high-protein diet or urea to human neonates
results in an increased ability to concentrate urine
implying that the ability of urea to accumulate in
the medulla can be limited by dietary intake [284,
285]. Adrenalectomy in neonatal rats prevented
the maturational increase in urine osmolality
while administration of glucocorticoids prior to
the maturational increase in serum glucocorti-
coids caused a premature increase in urinary con-
centrating ability [282].
Principal cells in the collecting tubule and the
cells in the medullary collecting duct express
aquaporin 2 on the apical membrane and
aquaporins 3 and 4 on the basolateral membrane
[286–290]. Aquaporin 3 null mice have diabetes
insipidus whereas aquaporin 4 null mice only
have a small concentrating defect after water
81
deprivation indicating that basolateral water
movement is primarily via aquaporin 3 [291,
292]. Vasopressin causes the intracellular vesicles
containing aquaporin 2 to fuse with the apical
membrane resulting in the insertion of aquaporin
2 into the apical membrane [290]. There is a
developmental increase in aquaporin 2 expression
[286, 287, 293–295]. The maturational increase in
aquaporin 2 is accelerated by administration of
synthetic glucocorticoids prior to the normal post-
natal increase in plasma glucocorticoids
[295]. Neither aquaporin 3 nor aquaporin 4 are
factors impairing urinary concentration in the neo-
nate [286, 296].
The fetus and neonate respond to increases in
serum osmolality, stress, and hypovolemia with
appropriate increases in plasma vasopressin
levels. Fetal sheep have an increase in plasma
vasopressin and plasma osmolality with an infu-
sion of hypertonic saline and increase in plasma
osmolality [297–299]. Plasma vasopressin also
increases in fetal sheep in response to volume
depletion induced by hemorrhage or diuretics
[297, 300, 301]. Despite the fact that fetal sheep
can increase serum vasopressin levels, infusion of
vasopressin resulted in a blunted increase in urine
osmolality compared to adult sheep [302]. It
is difficult to directly relate these experimental
findings to humans. Comparison between a rela-
tively stressful vaginal delivery and a cesarean
section has consistently demonstrated higher
vasopressin levels in neonates born vaginally
[303–306]. However, there was no correlation
with vasopressin levels and the degree of perinatal
asphyxia in one study [305], while there was a
correlation in another [303]. The balance of data
suggests that the human fetus and neonate can
respond to appropriate stimuli with vasopressin
secretion.
There is a developmental increase in vasopres-
sin receptors in the kidney; however, vasopressin
receptor abundance does not appear to be a limit-
ing factor in urinary concentration in the neonate
[307, 308]. Exogenous administration of vaso-
pressin has been shown to increase the urine
osmolality in fetal sheep showing that vasopressin
action on the collecting tubule is a prenatal event
[302]. Vasopressin acts in the collecting tubule by
82
increasing cAMP. There is some discrepancy
about the extent of cAMP production during post-
natal maturation in comparison to adults. The
balance of data supports that conclusion; although
vasopressin stimulates cAMP production in the
neonate, the amount of cAMP generated is less
than that of the adult [307, 309–311].
The response of the collecting tubule to vaso-
pressin has been examined in vitro
[312–315]. Neonatal CT water permeability did
not increase in response to vasopressin to the same
extent as that seen in the adult [312–315]. Studies
have shown that the phosphodiesterase activity is
greater in the neonatal collecting tubule
[313]. The water permeability of the neonatal
collecting tubule was identical to the adult in the
presence of a phosphodiesterase inhibitor demon-
strating that the predominant factor limiting the
action of vasopressin in the collecting tubule was
the enhanced degradation of cAMP generated by
vasopressin in the neonatal collecting tubule
[313]. Vasopressin increases prostaglandin pro-
duction in the collecting tubule which attenuates
the vasopressin-mediated increase in cAMP pro-
duction [316, 317]. Vasopressin-mediated cAMP
production, while less in the neonatal collecting
tubule, increases to adult levels in the presence of
indomethacin, a prostaglandin synthesis inhibitor
[317]. Thus, prostaglandin production by the
collecting tubule may attenuate the effect of vaso-
pressin in the neonatal tubule to a greater extent
than the adult tubule [317].
Developmental Aspects of Distal
Tubule Acidification
The distal nephron makes the final adjustment in
renal acidification. The distal nephron, under nor-
mal circumstances, secretes the protons equal to
that generated from metabolism and in children
the protons liberated from the formation of bone.
The cortical collecting tubule has two types of
cells that are involved in renal acidification, as
shown in Fig. 14. The α-intercalated cell is
responsible for proton secretion. The
β-intercalated cell can secrete bicarbonate when
the animal is alkalotic or eats a diet with an alkali
M. Baum
content [318]. This is unusual for humans but not
for some mammals which ingest an alkali diet
[319]. Both α- and β-intercalated cells have dif-
ferences in the polarized localization of both the
Na+/K+-ATPase and the Cl/HCO3 as shown in
Fig. 14.
The neonatal cortical collecting tubule has
fewer and less differentiated intercalated cells
than that of the adult segment [320, 321]. The
rate of both bicarbonate secretion and luminal
acidification is less in the neonate than in the
adult [319, 321, 322]. The intercalated cells in
the inner medullary collecting duct were much
more differentiated in appearance and secreted
protons at a rate comparable to the adult [319,
322]. The α-intercalated cell of the CT, as noted
above, has a luminal H+-K+ ATPase which causes
proton secretion and potassium absorption from
the luminal fluid. It is activated under states of
metabolic acidosis and hypokalemia [323]. The
H+-K+ ATPase can function at comparable rates in
the neonatal cortical collecting tubule as that of
the adult [324].
Developmental Features of Cortical
Collecting Tubule Sodium Transport
While relatively little sodium is reabsorbed by the
cortical collecting tubule compared to the proxi-
mal nephron segments, it is the final nephron
segment responsible for regulating sodium
absorption and thus is vitally important for the
regulation of sodium homeostasis. Sodium
absorption occurs in the principal cell of the
collecting tubule which is shown in Fig. 14.
Sodium transport is through the epithelial sodium
channel designated ENaC which has three sub-
units. The driving force for sodium entry across
ENaC is the low intracellular sodium concentra-
tion and the potential difference across the luminal
membrane generated by the basolateral Na+/K+-
ATPase. The Na+/K+-ATPase undergoes a matu-
rational increase in this segment but is not the
limiting factor of the maturational increase in
sodium absorption [39]. The abundance of
ENaC on the apical membrane is by and large
determined by aldosterone in the adult, but
3 Renal Tubular Development 83

Fig. 14 Three cells of the

cortical collecting tubule
are demonstrated. The Principal Cell
principal cell is shown ATP
above with sodium entering
Na+
the cell down its 3 Na+
electrochemical gradient
via a channel designated
ENaC. This generates a ADP+Pi
lumen-negative K+
transepithelial potential K+
difference. Potassium is
secreted into the lumen
down its electrochemical Cl−
gradient via ROMK.
Chloride is moving through
the paracellular pathway
down the electrical gradient α-Intercalated Cell
generated by the lumen- ADP+Pi
negative potential
Cl−
difference. The cell below is
an alpha intercalated cell H+
which secretes protons into HCO3−
the cell lumen via a H+- ATP
ATPase. The bicarbonate Cl−
generated in this process is
secreted across the
basolateral membrane via a
Cl/HCO3 exchanger. The
third cell is a beta
intercalated cell which
secretes bicarbonate ions in
the face of metabolic β-Intercalated Cell Cl−
alkalosis. This cell is the
reverse of an alpha
ATP
intercalated cell, but the
HCO3−
Cl/HCO3 exchanger is a
H+
different isoform
Cl−
ADP+Pi

aldosterone has a blunted effect in the neonate
despite the fact that there are higher serum levels
in the neonate and ample aldosterone receptors
[325–331].
Sodium transport in the cortical collecting
tubule increases with postnatal development
[331, 332]. This increase in sodium transport is
paralleled by an increase in the apical expression
of ENaC, since ENaC expression is the limiting
factor in sodium absorption in this segment [333].
ENaC is composed of α-, β-, and γ-subunits, and
there is a developmental increase in mRNA and
protein abundance of each during postnatal matu-
ration of this segment [333–337].
Developmental Aspects of Tubular
Potassium Transport
Neonates have higher serum potassium levels
than adults. Potassium is the predominant intra-
cellular cation, and neonates must be in positive
84
potassium balance for growth unlike adults which
excrete the quantity of potassium absorbed from
their diet in their urine [323]. Adult animals are
more readily able to excrete an exogenous potas-
sium load than a neonate [338]. Approximately
half of the filtered potassium is reabsorbed in the
proximal tubule of the adult and neonate by pas-
sive diffusion across the paracellular pathway
[339]. The loop of Henle reabsorbs 80 % of the
delivered potassium in the adult and only 45 % in
the neonate [339]. Thus, the adult delivers approx-
imately 10 % of the filtered potassium to the distal
nephron while the neonate delivers 25 %. The
distal convoluted tubule, connecting tubule, and
cortical collecting duct are the sites of potassium
secretion and final modulation of urine potassium
excretion.
Potassium secretion in a principal cell is
depicted in Fig. 14. Sodium enters the principal
cell through the apical sodium channel down the
favorable electrochemical gradient generated by the
Na+/K+-ATPase on the basolateral membrane. This
results in a lumen-negative potential difference and
a favorable electrochemical gradient for potassium
secretion. While there is a maturational increase in
the sodium channel and the Na+/K+-ATPase, these
are not the limiting factors for potassium secretion.
There are two potassium channels in the collecting
tubule: a channel designated ROMK is on the apical
membrane of principal cells and a flow-dependent
channel that is activated by stretch designated maxi-
K channel [323]. There is a maturational increase in
potassium secretion in cortical collecting tubules
perfused in vitro [332]. The secretion of potassium
by principal cells is paralleled by the maturational
increase in potassium channels (ROMK) on the
apical membrane of the principal cell [337,
340]. There is also a developmental increase in the
maxi-K channel [341]. Potassium secretion is regu-
lated by aldosterone, and there is resistance to the
action of aldosterone despite higher serum levels in
the neonate [325–331].
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 Clinical Perinatal Urology
4
David A. Diamond and Richard S. Lee

Contents Diagnosis
Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Diagnostic Findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 In a large prospective Swedish study, the inci-
Rationale for Prenatal Intervention . . . . . . . . . . . . . . . 105
dence of prenatally detected renal anomalies was
0.28 % in which two-thirds (0.18 %) were
Clinical Experience with Intervention
hydronephrosis. A British study, in which 99 %
for Prenatal Hydronephrosis . . . . . . . . . . . . . . . . . . . . . . 106
of the pregnant population in Stoke-on-Trent were
Postnatal Management of Infants with Prenatally scanned at 28 weeks’ gestation, demonstrated
Diagnosed Urologic Renal Abnormality . . . . . . . . . . 108
Unilateral Hydronephrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 hydronephrosis prenatally in 1.40 % of cases,
Bilateral Hydronephrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 which was confirmed postnatally in 0.65 % [2].
Renal Agenesis, Renal Ectopia, and Unilateral Others have reported a 1–3% incidence of
Multicystic Dysplastic Kidney . . . . . . . . . . . . . . . . . . . . . . 109 ANH [1–5]. These authors defined prenatal
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 hydronephrosis as an anteroposterior (A-P) diam-
eter of the renal pelvis greater than 5 mm but noted
the lack of consensus on the definition of antenatal
hydronephrosis [6–8]. With the rapid improve-
ment of ultrasound technology, the incidence of
detection of renal anomalies may be changing.
In a more recent prospective cohort study
(1999–2003), a 0.76 % incidence of urinary tract
abnormalities was detected, which was increased
as compared to an earlier cohort from the same
institution (0.3 %, 1989–1993) [9]. Many varia-
tions in the definition and management of ANH
exist in the literature and clinical practice, includ-
ing method and frequency of in utero testing,
radiographic documentation, classification, or
postnatal management [10–16].
When an abnormality of the urinary tract is
D.A. Diamond • R.S. Lee (*) determined by maternal-fetal ultrasound, several
Department of Urology, Harvard Medical School, Boston
questions should be raised by the ultrasonogra-
Children’s Hospital, Boston, MA, USA
e-mail: david.diamond@childrens.harvard.edu; richard. pher and consulting urologist. Combinations of
lee@childrens.harvard.edu

# Springer-Verlag Berlin Heidelberg 2016 97

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_3
98 D.A. Diamond and R.S. Lee

Table 1 Major diagnostic findings in prenatal imaging

Finding Comment
Kidney Hydronephrosis Assess degree
Unilateral and May be different
bilateral degrees
Parenchymal Should be less than
echogenicity spleen or liver; if
increased suggests
congenital kidney
disease. If associated
with moderate to
severe renal
enlargements suggests
genetic polycystic
kidney disease (PKD)
Fig. 1 Ultrasound appearance of normal fetal kidney with
Duplication Often with dilation of echolucent medullary pyramids distinguishable from the
upper pole; may be more echogenic cortical parenchyma. The cortical paren-
lower pole dilation chyma should be of lower echogenicity than adjacent liver
Cysts Small cysts associated or spleen
with dysplasia.
Simple cyst of upper
pole suggests
duplication with Diagnostic Findings
ureterocele, or ectopic
ureter may suggest
genetic cystic disease Kidney
(PKD or phenocopy) There are critical elements to an antenatal US
Urinoma Perinephric or exam that may help identify urologic pathology.
subcapsular A constellation of aberrations may indicate
Ureter Dilation/ Obstruction or reflux
pathology, particularly when placed in context
tortuosity
Bladder Distended Variation with time
with other clinical findings. Specific details of
Wall thickness In relation to filling the US need to be reported to assist antenatal
status counseling, which include: number, location,
Intravesical Ureterocele size, duplication, renal parenchyma
cystic structure (echogenicity), pelvic dilation, calyceal dilation,
“Keyhole” Dilated posterior urothelial thickening, and cystic disease. Kidneys
pattern urethra; PUV
should be appropriate in size for gestational age
Not visible Exstrophy
and relatively symmetric [17]. Large differences
Amniotic Absence; Impaired urine output
fluid oligohydramnios in size may indicate contralateral compensatory
Polyhydramnios May be seen with growth. Absence of the kidney in the normal
mild-moderate location may represent ectopia or agenesis/dys-
hydronephrosis plasia. The normal kidney should be elliptical in
Gender Penis/scrotum/ Sex-associated shape and have distinctive internal echolucency,
testes conditions (e.g., PUV)
representative of normal medullary pyramids
Spine Meningocele Neural tube defect
(Fig. 1). The appearance of the medullary pyra-
PUV posterior urethral valves
mids should not be confused with renal calyceal
dilation. The echogenicity of the kidney should be
slightly less than the corresponding spleen or
specific findings direct the differential diagnosis liver. Abnormalities of echogenicity with or with-
and permit more accurate prognosis and tailoring out hydronephrosis may indicate congenital renal
of postnatal evaluation. The principal findings and disease (genetic, environmental, syndromic, or
their implications are listed in Table 1. due to intrauterine insult).
4 Clinical Perinatal Urology
Fig. 2 Unilateral hydroureteronephrosis at 35 weeks.
Note the dilated ureter (U ) and bladder (B). Postnatal
imaging confirmed this to be a ureterovesical junction
obstruction
Ureter, Bladder, and Urinoma
Findings of ureteral dilation, bladder filling and
emptying, bladder wall thickness, intravesical
cystic structures, dilation of the posterior urethra
(keyhole sign), urinoma, amount of amniotic
fluid, intra-abdominal/pelvic mass, and external
genitalia should be noted. Hydroureter is best
seen in cross section of a full bladder but is often
difficult to detect (Fig. 2). Duplication and ureteral
ectopia may also make this a difficult diagnosis.
Although the bladder is sometimes difficult to
image well, visualization of the bladder can be
very informative, as a full bladder implies renal
function. Inability to identify the bladder on repeat
studies should raise the question of bladder
exstrophy. Increased bladder wall thickness may
indicate outlet obstruction, and dilation of the
posterior urethra (keyhole sign) is strongly sug-
gestive of posterior urethral valves (PUV) (Fig. 6).
In duplex systems, ureteroceles may present as an
intravesical cystic structure and a dilated
upper pole.
Perirenal urinoma may indicate an obstructive
condition [18] (Figs. 3c and 4). Typically it will
have the appearance of an anechoic structure
around the kidney or be in a subcapsular location.
When identified, a urinoma or urinary ascites is
often associated with cases of severe bladder
obstruction, or PUV, in which the urinoma may
99
indicate a pop-off mechanism, which may protect
the kidney, particularly in cases of lower urinary
tract obstruction [19].
Amniotic Fluid
Critical to the evaluation of the urinary tract is the
assessment of the amniotic fluid (AF) level and
changes during pregnancy. After 16 weeks, AF
production shifts from placental transudate to fetal
urine, and by 20–22 weeks the vast majority of AF
is fetal urine [20]. Consequently, reduced AF or
oligohydramnios identified after 18–20 weeks
gestation may be a result of urinary tract obstruc-
tion or poor renal development [21].
External Genitalia
Proper identification of the external genitalia can
also be very valuable in cases of gender-specific
diagnosis, such as PUV. In cases of virilization
(e.g., congenital adrenal hyperplasia), a clitoris
may appear as a small phallus; and therefore the
presence of scrotal testes is critical for male gen-
der assignment [22–24]. Megalourethra, or a
dilated, elongated penile urethra, may be an iso-
lated anomaly or associated with prune belly syn-
drome [25] (Fig. 5). A female fetus with bilateral
renal obstruction and bladder outlet obstruction
would suggest a cloacal anomaly [26–28].
Hydronephrosis
Hydronephrosis, or dilation of the renal pelvis, is
the most common urologic abnormality found on
US. Numerous grading systems have been devel-
oped; however, there is no consensus on the best
and most consistent method of reporting ANH
(Fig. 6). Measurement of APD has been used
widely; however, there have been no formal stud-
ies to determine the inter- and intra-observer
reproducibility of ANH measurement, and APD
fails to describe pelvic configuration, calyceal
dilation, and the laterality of findings, which
should be included. APD can be affected by ges-
tational age, hydration status of the mother, blad-
der hypertonicity, and degree of bladder
distention. Since the dimensions of the renal pel-
vis may normally increase with gestational age,
most investigators have adjusted threshold APD
values for early and later gestational age.
100
Fig. 3 Images of a fetus with posterior urethral valves. (a)
Depicts bilateral echogenic kidneys with hydrouretero-
nephrosis (arrow). (b) Demonstrates the associated thick-
ened bladder wall (black arrow) and hydroureter (white
Fig. 4 Appearance of a fetal perinephric urinoma flatten-
ing the contour of the kidney
Unfortunately, a simple threshold APD value
which separates normal from abnormal does not
exist, as even severe cases of ANH have the
potential to resolve without incident, while mild
D.A. Diamond and R.S. Lee
arrow). (c) Further imaging revealed a perinephric
urinoma (white arrow) surrounding the hydronephrotic
kidney (black arrow)
degrees of ANH have the potential to
progress [29].
Varying the minimal APD threshold can sig-
nificantly alter the specificity and sensitivity of
APD as a measure of ANH and postnatal pathol-
ogy. To date there is no consensus on the optimal
APD threshold for determining the need for post-
natal follow-up. An APD cutoff of 15 mm for
determining obstruction yielded a postnatal sensi-
tivity of 73 % and specificity of 82 % [30]. A late
gestational age APD cutoff of 10 mm would
detect approximately 23 % of abnormal kidneys,
whereas a cutoff of 7 mm detected 68 % [31]. One
large systematic review estimated that only
11.9 % of total pathology presented with late
gestational age APD less than 9 mm while 39 %
of total pathology was noted at APD levels less
than 15 mm [16]. Nearly identical results have
been demonstrated by other investigators [32].
4 Clinical Perinatal Urology
Fig. 5 Fetal ultrasound appearance of a male with a
dilated and patulous urethra typical of a megalourethra.
This may be seen in prune belly syndrome as well as in
isolation. This child had marked vesicoureteral reflux and
bilateral hydroureteronephrosis
Fig. 6 Unilateral renal pelvis dilation with no ureteral
dilation in 37-week-old fetus
101
What appears certain is that lower cutoffs will be
more sensitive in detecting postnatal pathology
but will incur a higher false-positive rate.
Categorizing ANH by APD
There is near agreement that APD greater
than 15 mm represents severe or significant
hydronephrosis, and some would also agree that
a lower threshold of 4–5 mm is an appropriate
value for considering APD to be abnormal
[16, 32–36]. Taking the above-noted limitations
into consideration, we define ANH in the 2nd and
3rd trimester using APD thresholds for which
the best available evidence provides prognostic
information (Table 2).
Diagnostic Accuracy
Predicting accurate postnatal diagnosis and out-
come, regardless of the prenatal information, still
remains challenging. The importance of accurate
diagnosis is particularly critical in cases where
fetal intervention is considered, such as posterior
urethral valves (PUV). In other cases, the ability
to identify some degree of ANH is adequate to
permit a postnatal evaluation if deemed clinically
appropriate.
A recent systematic review of the ANH litera-
ture attempted to determine the risk of a patholog-
ical diagnosis for patients with varying severities
of ANH [16]. In this review of 1,308 patients with
any ANH and postnatal radiographic follow-up,
36 % had a postnatal pathological diagnosis. The
degree of ANH was defined by the anterior-
posterior diameter (APD) identified in a particular
trimester (Table 2) [16]. The overall risk for any
pathology increased with the degree of
hydronephrosis, except for vesicoureteral reflux
which remained consistent regardless of the
degree of ANH (Table 3) [16].
Although the risk of pathology with degrees
of ANH appears to be increased, accurately
determining the diagnosis remains difficult [16].
Earlier studies reported false-positive rates up to
22 %. The majority of false positives were
nonobstructive causes of hydronephrosis, such
as high-grade reflux, large, nonobstructed,
extrarenal pelves, or transient hydronephrosis
[6, 37]. Similarly, the diagnosis of vesicoureteral
102 D.A. Diamond and R.S. Lee

reflux is extremely challenging to make in utero, In another population-based series, the sensitivity
as evidenced by the fact that the risk for in detecting valves was as low as 23 % [6].
vesicoureteral reflux is the same regardless of the Increased renal echogenicity and decreased
degree of ANH [16]. amniotic fluid have been suggested to be indica-
As another example, the accurate diagnosis of tive of obstructive conditions [39]. Although
posterior urethral valves (PUV), in which inter- the hallmark signs of an in utero diagnosis of
vention might be considered, has proven difficult. posterior urethral valves have been described
In one series, the false-positive rate was as high as (oligohydramnios, dilated posterior urethra, thick-
58 %, but the criteria for diagnosing valves were ened bladder, and hydroureteronephrosis), there
quite liberal and perhaps inappropriate [38]. are very few studies that have prospectively
examined the clinical urologic implications of
Table 2 Classification of antenatal hydronephrosis these findings alone or in combination [16].
(ANH) by anterior-posterior diameter (APD)
APD Ureteropelvic Junction Obstruction
ANH 2nd trimester 3rd trimester The basic features of ureteropelvic junction
classification (mm) (mm) obstruction (UPJO) in the fetus include dilation
1. Mild 7 9 of the renal pelvis and collecting system with no
2. Mild/ <10 <15 evidence of ureteral dilation (Fig. 6). The thresh-
moderate old for recommending postnatal follow-up is
3. Moderate 7–10 9–15
largely arbitrary, and currently there are no long-
4. Moderate/ 7 9
term prospective studies to best determine the
severe
5. Severe 10 15
degree of postnatal evaluation. Increasing severity
of ANH increased the risk of identifying postnatal

Table 3 Risk of pathology by degree of antenatal hydronephrosis

Degree of antenatal hydronephrosis
Postnatal pathology Mild Mild-moderate Moderate Moderate-severe Severe Trend
[% (95 % CI)/a] (N = 587) (N = 213) (N = 235) (N = 179) (N = 94) P-valueb
Any pathology 11.9 (4.5, 39.0 (32.6, 45.1 (25.3, 72.1 (47.6, 88.0) 88.3 <0.001
28.0) 45.7) 66.6) (53.7,
98.0)
UPJ 4.9 (2.0, 13.6 (9.6, 18.9) 17.0 (7.6, 36.9 (17.9, 61.0) 54.3 <0.001
11.9) 33.9) (21.7,
83.6)
VUR 4.4 (1.5, 10.8 (7.3, 15.7) 14.0 (7.1, 12.3 (8.4, 17.7) 8.5 (4.7, 0.10
12.1) 25.9) 15.0)
PUV 0.2 (0.0, 0.9 (0.2, 3.7) 0.9 (0.2, 6.7 (2.5, 16.6) 5.3 (1.2, <0.001
1.4) 2.9) 21.0)
Ureteral obstruction 1.2 (0.2, 11.7 (8.1, 16.8) 9.8 (6.3, 10.6 (7.4, 15.0) 5.3 (1.4, 0.025
8.0) 14.9) 18.2)
Otherc 1.2 (0.3, 1.9 (0.7, 4.9) 3.4 (0.5, 5.6 (3.0, 10.2) 14.9 (3.6, 0.002
4.0) 19.4) 44.9)
a
Pointwise 95 % confidence intervals were estimated by logistic regression with robust standard errors based on
generalized estimating equations with a working independence correlation structure to adjust for clustering by study
for all degrees of antenatal hydronephrosis except mild to moderate. Because only one study had subjects with mild-
moderate antenatal hydronephrosis, the pointwise 95 % confidence intervals had to be estimated using logistic regression
with unadjusted standard errors
b
Testing for trend in risks with increasing degree of antenatal hydronephrosis using logistic regression with robust
standard errors based on generalized estimating equations with a working independence correlation structure
c
Includes prune belly syndrome, VATER syndrome, solitary kidney, renal mass, and unclassified
4 Clinical Perinatal Urology
Fig. 7 Two different fetal images (a) at 19 weeks and (b) at 26 weeks of a multicystic dysplastic kidney with multiple
noncommunicating cysts
UPJO [16]. In the case of significant unilateral
hydronephrosis, currently, there is little rationale
for in utero intervention. In a few cases with
massive dilation, therapeutic aspiration has been
recommended for dystocia. In the case of bilateral
UPJO, the efficacy of in utero intervention is
difficult to assess.
Attempts to correlate prenatal ultrasound
appearance with postnatal outcomes have been
complicated by the long-standing controversy
regarding postnatal evaluation and management
of UPJO. Grignon et al. developed a system of
grading hydronephrosis secondary to UPJO based
on the APD and degree of calyceal dilatation
[40, 41]. Mandell et al. attempted to correlate the
degree of APD relative to gestational age with
subsequent need for postnatal surgical interven-
tion [42]. They found the “at-risk” diameter to be
greater than or equal to 5 mm at 15–20 weeks’
gestation, greater than or equal to 8 mm at 20–30
weeks’ gestation, and greater than 1 cm at over
30 weeks’ gestation. An alternative system pro-
posed by Kleiner et al. defined hydronephrosis as
the ratio of APD to A-P diameter of the kidney as
being greater than 0.5 cm [43]; caliectasis was
later added as an additional indicator of significant
hydronephrosis. Others have suggested that
regardless of the degree of ANH, moderate or
severe postnatal hydronephrosis with evidence
of decreased renal function should be the
103
indications for surgical intervention [44]. Mild
degrees of renal pelvic dilatation may resolve in
utero. Mandell et al. noted this occurred in 23 % of
cases, with 66 % remaining stable and 9 % wors-
ening over the course of the pregnancy [42]. Sim-
ilarly, Lee et al. noted that 88.1 % of mild ANH
were found to have no postnatal pathology
[16]. Severe forms of UPJO may be associated
with urinary ascites or perinephric urinomas,
which may be a predictor of nonfunction of the
kidney [19, 45].
Cystic Kidneys
The distinction between severe unilateral
hydronephrosis and a multicystic dysplastic kid-
ney may occasionally be unclear. The findings of
multiple noncommunicating cysts, minimal or
absent renal parenchyma, and the absence of a
central large cyst are diagnostic of a multicystic
dysplastic kidney (MCDK) (Fig. 7). Real-time
examination of the kidneys to help determine the
communication of the calyces is essential. MCDK
may be present in any ectopic location but typi-
cally are in the normal position. In addition,
MCDK can be present in a duplex kidney, typi-
cally the upper pole. If detected early in the preg-
nancy, the MCDK will involute over a period of
time either prenatally or postnatally [45].
Bilaterally enlarged echogenic kidneys, partic-
ularly if associated with hepatobiliary dilatation or
104
Fig. 8 Bilateral markedly enlarged echogenic (“bright”) kidneys (a) with a small cystic lesion (b) in a fetus with
oligohydramnios, secondary to autosomal recessive polycystic kidney disease
oligohydramnios, suggests autosomal recessive
polycystic kidney disease (Fig. 8). Typically this
is identified prior to 20 weeks’ gestation, but later
development has been reported [42, 46, 47]. A
more challenging finding is normal-sized, dif-
fusely echogenic kidneys that are not associated
with other urologic lesions [48, 49]. Estroff
et al. described 19 cases (14 bilateral),
including 10 with normal function who survived
and 4 with autosomal recessive polycystic kid-
neys who died [50]. In a separate multicenter
retrospective study of 93 fetuses with
hyperechogenic kidneys and a later diagnosis of
nephropathy of varying etiology, only 1/3 of the
fetuses had renal cysts irrespective of their
diagnosis. Twenty-eight had ARPKD (only
3 with cysts) and 31 had ADPKD (9 with cysts).
Typically, those with ADPKD appeared to have
moderately enlarged hyperechogenic kidneys
with increased corticomedullary differentiation.
Additionally, as opposed to renal cyst character-
istics, associated malformations were the most
helpful clue to identify a diagnosis [51]. See also
▶ Chap. 36, “Childhood Polycystic Kidney
Disease.”
Ureterovesical Junction Obstruction
Less common than UPJO, ureterovesical obstruc-
tion (UVJ) is characterized by ureteral dilation
D.A. Diamond and R.S. Lee
along with varying degrees of renal pelvic and
calyceal dilation (Fig. 2). The best way to detect
ureteral dilation is at the level of the bladder,
preferably in the transverse view. It is not uncom-
mon for the distal ureter to be more dilated as
compared proximally. More extreme cases may
be confused with single system ectopic ureters,
particularly in males. In general, the differentia-
tion is made postnatally.
Duplication Anomalies
Duplication anomalies are often recognized on the
basis of upper pole hydroureteronephrosis, asso-
ciated with either an obstructing ureterocele
within the bladder or ectopic ureter inserting out-
side of the bladder [52] (Fig. 9). The upper pole
may appear as a cystic dysplastic unit without
hydronephrosis and a concomitant ureterocele.
This is often termed ureterocele disproportion
[53]. Occasionally, lower pole dilation is due to
ureteral obstruction by a very large ureterocele of
the upper pole ureter. In cases of a very large
ureterocele, the ureterocele may be mistaken for
the bladder. Of note, single system ureteroceles
are more likely to occur in boys and have variable
degrees of hydronephrosis of the entire kidney.
Lower pole hydronephrosis may also be present
as a result of vesicoureteral reflux or more rarely a
lower pole UPJO.
4 Clinical Perinatal Urology
Fig. 9 Fetal image of duplex kidney with marked upper pole hydronephrosis (arrow) in contrast to a normal lower pole
(a). Associated with this image is the finding of a ureterocele (arrow) within the bladder (b)
Vesicoureteral Reflux
A firm diagnosis of vesicoureteral reflux (VUR)
based on prenatal ultrasound is extremely difficult
to make, although intermittent hydronephrosis or
hydroureter is suggestive of VUR. Vesicoureteral
reflux may be present in as many as 38 % of
children with prenatal hydronephrosis [54]. Reflux
occurred in 42 % of children in whom postnatal
imaging revealed persistent upper tract abnormali-
ties and in 25 % of those with normal findings on
postnatal ultrasound but having a history of prena-
tal dilation. Tibballs and Debrun reported that in
patients with prenatal dilation and postnatally nor-
mal renal units by ultrasound, 25 % had grade III–V
reflux [55]. The incidence of high-grade reflux was
greater in males than in females as noted in previ-
ous studies. In two systematic reviews of the ANH
literature, a 10–15 % incidence of VUR was iden-
tified regardless of the degree of ANH [16, 56],
indicating that ANH is not indicative of VUR and
may not be the appropriate trigger for postnatal
evaluation. In a neonate with prenatally detected
hydronephrosis, the importance of diagnosing
vesicoureteral reflux remains controversial, as
VUR diagnosed on postnatal evaluation for ANH
is associated with earlier resolution of VUR [57].
Posterior Urethral Valves
Perhaps the most important diagnosis to be made
prenatally is that of posterior urethral valves
(PUV) in the male fetus. PUV, at the very least,
105
mandates prompt postnatal intervention, and in
some cases, prenatal intervention may be
warranted. Fetal sonographic findings of PUV
include bilateral hydroureteronephrosis, a thick-
walled bladder with a dilated posterior urethra,
and, in more severe cases, dysplastic renal paren-
chymal changes with perinephric urinomas
and urinary ascites (Fig. 3) [58]. When character-
istic sonographic findings are present, the differ-
ential diagnosis includes prune belly syndrome
(with or without urethral atresia), massive
vesicoureteral reflux, and certain cloacal anoma-
lies (in genetic females) [59–61]. Prenatal diag-
nostic accuracy for PUV is far from perfect but is
probably better than what was previously reported
(40 %) [38].
Rationale for Prenatal Intervention
The scientific rationale for prenatal treatment of
hydronephrosis is to maximize normal develop-
ment of renal and pulmonary function. These two
aspects of fetal development are closely linked
because urine comprises 90 % of amniotic fluid
volume, and oligohydramnios during the third
trimester has been causally related to pulmonary
hypoplasia. Before embarking on prenatal surgi-
cal intervention for obstructive uropathy, it is crit-
ical to assess the risk-benefit ratio. The time of
onset of oligohydramnios has been shown to be an
106 D.A. Diamond and R.S. Lee

Table 4 Prenatal assessment of renal functional prognosis

Good Poor
Amniotic fluid Normal to moderately decreased Moderate to severely decreased
Sonographic appearance of kidneys Normal to echogenic Echogenic to cystic
Fetal urine Glick Johnson Glick Johnson
et al. [64] et al. [65] et al. [64] et al. [65]
Sodium (mEq/L) <100 <100 >100 >100
Chloride (mEq/L) <90 – >90 –
Osmolarity (mOsm/L) <210 <200 >210 >200
Calcium (mg/dL) – <8 – >8
β2-Microglobulin (mg/L) – <4 – >4
Total protein (mg/dL) – <20 – >20
Output (mL/h) >2 – <2 –
Sequential improvement in urinary – X – –
values
X, only in this series was the criterion used

important determinant of outcome [62, 63]. In
fetuses in which adequate amniotic fluid was
documented at up to 30 weeks’ gestation in asso-
ciation with a urologic abnormality, pulmonary
outcomes were satisfactory, and postnatal clinical
problems were related to renal disease. It seems
inappropriate to recommend late urinary tract
decompression from a pulmonary or renal basis.
It is unclear whether early delivery, to permit earlier
postnatal urinary decompression, is beneficial.
The most widely accepted indicator of salvage-
able renal function is analysis of fetal urine. When
the urinary sodium is less than 100 mg/dL and
urine osmolarity less than 200 mOsm/dL, renal
function appears to be salvageable with in utero
intervention (Table 4) [64]. Serial aspirations of
fetal urine have been reported to yield more valu-
able results [65]. Guez et al. reported ten fetuses
who underwent multiple urine samplings and in
whom severe obstruction reduced sodium and
calcium reabsorption [66]. They concluded that
fetal urinary chemistries were reasonably predic-
tive of severe but not moderate postnatal renal
impairment. Other investigators have suggested
the use of fetal urinary β2-microglobulin as an
indicator of tubular damage. Using this parameter,
poor renal outcome has been predicted with a
specificity of 83 % and sensitivity of 80 % [67].
The accuracy of these predictors has been chal-
lenged. A 2007 systematic review of fetal urine as
a predictor of postnatal renal outcome demon-
strated that there was insufficient evidence to sup-
port its effectiveness to predict renal function
[68]. From 23 studies, they identified
572 women, from which the two most accurate
fetal urine tests were urinary calcium and sodium.
β2-microglobulin was found to be less
accurate [68].
Findings on antenatal ultrasound have also
been examined to predict long-term outcome
postnatal renal function. In a systematic review
of 13 articles encompassing 251 women,
oligohydramnios and the appearance of the renal
cortex (increased echogenicity or cystic changes)
at the diagnosis of LUTO were the best factors to
predict poor renal function (defined as Cr > 1.2
mg/dl) [69]. In this particular study, gestational
age at diagnosis (< 24 weeks) was not predictive
of renal function, which may be more of a reflec-
tion of the inherent variability of the available
literature.
Clinical Experience with Intervention
for Prenatal Hydronephrosis
The ability to diagnose severe prenatal
hydronephrosis and advances in fetal intervention
helped develop prenatal surgery for obstructive
uropathy. In 1982, Harrison et al. described the
4 Clinical Perinatal Urology 107

initial report of fetal surgery in a 21-week-old Table 5 Prenatal intervention for hydronephrosis
fetus with bilateral hydroureteronephrosis sec- Indications (prerequisites) Contraindications
ondary to PUV [70]. After the 1986 report of the Presumed obstructive Unilateral hydronephrosis
International Fetal Surgery Registry in which out- hydronephrosis, persistent with an adequately
comes did not seem to justify risk, a de facto or progressive, bilateral or functioning contralateral
in solitary unit kidney
moratorium on in utero urinary tract shunting
Otherwise healthy fetus Chromosomal
evolved [71]. abnormalities or presence
With improved technology and renewed inter- of associated severe
est in fetal shunting, most cases have been referred anomalies
to a small number of highly specialized centers Oligohydramnios Bilateral hydronephrosis
without oligohydramnios
actively engaged in prenatal surgery. The initial
No overt renal dysplasia Severely dysplastic
method of decompression with open surgery has kidneys
largely been replaced by in utero shunt placement. Adequate renal functional Evidence or urethral
Current practice uses the Rodeck shunt which lies potential based on urinary atresia
flat against the abdomen to minimize shunt dis- indices (see text)
lodgement. Complications included shunt dis- Informed consent Presence of a normal twin
lodgement and, in the case of the double-J shunt,
bowel herniation [72]. Amnioinfusion may be
needed to improve visualization for shunt place-
ment; however, this may lead to excessive fetal prenatal obstructive uropathy (Table 5) [79]. Serial
movement. Occasionally the fetus needs to be bladder sampling over 3 days has been used to
paralyzed for accurate placement. Very large blad- help determine if the fetus is a viable candidate.
ders may cause the shunt to be placed too high in The serial nature of the procedure allows one to
the abdomen resulting in dislodgement from the see the response of the fetal kidneys to bladder
bladder after it decompresses. decompression [65]. The principal reason for con-
Some investigators have explored the use of sidering vesicoamniotic shunting is to prevent
fetoscopic methods for direct intervention to pro- early neonatal pulmonary insufficiency and
vide prolonged bladder drainage, whereas others death. The risks that one accepts with intervention
have attempted direct endoscopic valve ablation include induction of premature labor, perforation
[73–77]. The proposed advantage of fetoscopic of fetal bowel and bladder, and fetal and/or mother
intervention over vesicoamniotic shunting is to hemorrhage and infection.
improve drainage and to restore normal cycling More recently, the ability to influence renal
of the bladder. There are no studies to determine if outcome in male patients with PUV but without
this method of decompression is adequate in the oligohydramnios has been suggested as a possible
face of significant prenatal bladder dysfunction. indication for in utero intervention. The principal
Furthermore, fetoscopic intervention also intro- goal of intervention is not to prevent pulmonary
duces the additional potential for iatrogenic injury hypoplasia and deaths but to prevent or delay
to the urethra, bladder neck, or external urethral end-stage renal failure. Although some reports
sphincter. In a systematic review of the literature, have shown promise in the ability to distinguish
four papers totaling 63 patients identified that fetal those fetuses with likely early renal failure from
cystoscopy as compared to vesicoamniotic shunts those with later-onset failure, the specificity and
had no significant improvement in perinatal sur- accuracy of methods using a combination of ultra-
vival [78]. Overall, experience with fetoscopic/ sound and urinary chemistries (sodium, β2-
endoscopic valve ablation is currently at the case microglobulin, and calcium) has not been well
report and experimental level, and long-term out- defined [80–82]. In summary, precise identifica-
come data is unknown. tion of those situations in which intervention may
Harrison et al. have clearly outlined the indi- benefit the fetus with obstructive uropathy
cations and contraindications of intervention for remains unclear.
108
To date, the reported long-term outcomes of
antenatal intervention for severe obstructive
uropathy (e.g., PUV, prune belly syndrome, ure-
thral atresia) are mixed [83–93]. Variability in
patient selection and assessment of outcome
within these studies has limited the ability to
determine if prenatal intervention has altered the
postnatal course. A large systematic review of the
prenatal intervention for obstructive uropathy
showed a statistically significant perinatal sur-
vival advantage with shunting [89]; however,
lack of randomization of patient selection in the
trails reviewed may have biased the results. Of the
studies that have reported long-term outcomes of
in utero vesicoamniotic shunting, many of the
children have renal insufficiency (57 %) and
growth impairment (86 %) [83, 85, 86]. Recently,
Baird et al. reported on long-term follow-up (5.8
years) of patients who have survived in utero
shunting [83]. They noted acceptable renal func-
tion in 44 %, 22 % with mild impairment, and
33 % with renal failure. Prune belly patients had
the best renal outcome (57 %), followed by PUV
(43 %) and then urethral atresia (25 %).
The European-based multicenter randomized
clinical trial (PLUTO Trial: Percutaneous
Shunting for Lower Urinary Tract Obstruction)
was recently conducted [94]. The initial goal
was to enroll 150 singleton pregnancies with
ultrasound evidence of LUTO to evaluate the
safety and effectiveness of vesicoamniotic shunt
(VAS) as compared to conservative management.
The study was stopped early because of poor
enrollment, as only 31 patients were enrolled
and randomized (16 VAS and 15 controls). In
the VAS group, there were 12 live births and
4 postnatal deaths at 28 days and and 12 live births
and 8 postnatal deaths in the control group. All
postnatal deaths were from pulmonary hypopla-
sia. Although underpowered, the study demon-
strated a nonsignificant increase in survival in
the VAS group. Consistent with the results of the
systematic review of existing prenatal interven-
tion data, there was minimal likelihood of surviv-
ing with long-term normal renal function.
Overall, it appears that in utero intervention for
the appropriate patient may reduce the risk of
neonatal mortality and may potentially improve
D.A. Diamond and R.S. Lee
renal function. To further improve outcomes,
more sensitive and specific markers to better iden-
tify which fetus will benefit from in utero shunting
need to be defined.
Postnatal Management of Infants
with Prenatally Diagnosed Urologic
Renal Abnormality
A child with a prenatal diagnosis of a urologic
renal abnormality such as ANH should be care-
fully evaluated and followed by a pediatri-
curologist from birth. The vast majority of these
children appear entirely healthy and, in the
absence of prenatal ultrasound findings, would
not have any indications for regular urologic
follow-up. Parental anxiety is common and
should be addressed directly with prenatal
counseling and education.
Unilateral Hydronephrosis
The presence of unilateral dilation of the kidney
warrants postnatal evaluation in a timely but
nonurgent fashion (3–8 weeks of life) with an
ultrasound [80]. The most common diagnoses
associated with this finding are UPJO, VUR, and
UVJO/megaureter. Early ultrasound is unlikely to
miss a significant abnormality. A normal postnatal
ultrasound indicates that obstructive uropathy is
not present; however, it does not clarify if the
child has VUR [55].
The decision to perform a voiding cystoure-
throgram (VCUG) or initiate prophylactic antibi-
otics in the newborn period is unclear. Although
some groups advocate postnatal VCUG in any
child with a history of antenatal hydronephrosis,
others have questioned the value of this approach
[95]. Although various guidelines and recommen-
dations have been proposed with regard to VCUG
usage, there are no definitive studies that have
rigorously studied the likelihood of VUR based
on consistent ultrasound findings or other clinical
characteristics. The current trend in management
of ANH minimizes the postnatal prophylactic
antibiotics and testing for VUR in cases of
4 Clinical Perinatal Urology
resolved ANH or mild to moderate cases of per-
sistent postnatal ANH [96].
In general, infants with severe ANH should be
placed on a prophylactic antibiotic (amoxicillin,
10–15 mg/kg/day) and undergo a VCUG. Severe
ANH may possibly indicate a higher grade of
VUR and may worsen the clinical presentation
of a child with a febrile urinary tract infection
[97, 98]. As for mild ANH, a recent prospective
study of 192 infants with ANH noted that the
majority of patients with mild ANH had no sig-
nificant events during infancy [34]. In another
study, female infants with a history of ANH and
postnatal uropathy had a higher risk of febrile
urinary tract infection [33]. Regardless, no appro-
priate prospective studies with coordinated and
comprehensive postnatal follow-up have exam-
ined this question in a rigorous fashion to provide
consensus guidelines [16, 56].
At our institution, children with moderate or
severe ANH are placed on prophylactic antibiotics
at birth. One possible exception is the circumcised
male. Children with moderate or severe ANH
undergo renal bladder ultrasound and VCUG post-
natally. Diuretic renography is reserved for those
with no VUR and persistent moderate or severe
postnatal hydronephrosis on serial US. Infants
with mild ANH and mild or no postnatal
hydronephrosis are observed and followed clini-
cally. Infants with a significant degree of antenatal
or postnatal ureteral dilation undergo ultrasound,
VCUG, and possibly diuretic renography (after
3 months) if clinically indicated.
Bilateral Hydronephrosis
Infants with bilateral hydroureteronephrosis may
have PUV, bilateral VUR, bilateral UPJO or
UVJO, or a combination of the above. For the
child with bilateral hydroureteronephrosis sug-
gestive of bladder outlet obstruction, an ultra-
sound and VCUG should be performed
promptly. In boys, PUV is the most important
diagnosis to be ruled out. In girls, an obstructing
ectopic ureterocele would be the most likely cause
for bladder outlet obstruction. In the event that an
obstructive lesion is discovered, it should be
109
corrected promptly. For children with suspected
lower urinary tract obstruction (e.g., PUV),
prompt bladder decompression and antibiotic pro-
phylaxis should be initiated prior to radiographic
intervention.
Renal Agenesis, Renal Ectopia,
and Unilateral Multicystic Dysplastic
Kidney
Infants born with solitary kidneys (renal agenesis),
renal ectopia, or unilateral multicystic dysplasia
should be evaluated postnatally by US and
VCUG. Functional studies such as a dimercapto-
succinic acid study (DMSA) are occasionally
needed to confirm the diagnosis. The need for
further screening is controversial. It has been
reported that of infants with a solitary kidney,
30 % have VUR, 11 % UPJO, and 7 % UVJO
[99, 100]. Similarly those with renal ectopia (sim-
ple or crossed fused ectopia) may also be at risk for
VUR in the ectopic or contralateral kidney (30 %)
[101–103]. However, there are others that report a
very low incidence of associated urologic anoma-
lies and do not recommend screening [104].
As for MCDK, these lesions are primarily uni-
lateral, isolated, and associated with a good prog-
nosis. If at birth the US findings are not conclusive
of MCDK, a DMSA study can be used to confirm
the diagnosis, as there should be no uptake. Patients
with MCDK are often thought to be similar to those
born with a solitary kidney. Additionally, patients
with MCDK have a reported increased frequency of
VUR and UPJO in the contralateral normal kidney
[105, 106], but utilization of routine screening by
VCUG remains controversial [107].
Acknowledgments The authors acknowledge the assis-
tance of Carol E. Barnewolt, M.D., for providing many of
the fetal ultrasound images.
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759–63.
 Renal Dysplasia/Hypoplasia
5
Paul Goodyer and Indra R. Gupta

Contents Overview
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Clinical Presentation of Renal Hypoplasia/ Congenital renal hypoplasia/dysplasia refers to
Dysplasia In Utero . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 the end result of a variety of pathogenic mecha-
Renal Agenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 nisms that disturb the primary induction of meta-
Unilateral Renal Agenesis (URA) . . . . . . . . . . . . . . . . . . . 118 nephric progenitor cells (renal agenesis),
Bilateral Renal Agenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 dysregulation of nephron number (disorders of
Renal Hypoplasia/Dysplasia . . . . . . . . . . . . . . . . . . . . . . . 120 progenitor pool size or ureteric bud branching),
Renal Hypoplasia and Defects in Ureteric Bud aberrant progenitor cell differentiation (dysplastic
Branching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
tissue elements), disturbance of the renal vascula-
Renal Hypoplasia and Dysregulation of the
Nephron Progenitor Pool . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 ture (causing tubular dysgenesis), organ structure
Renal Tubular Dysplasia with Disturbance of (multicystic/dysplastic kidney), and tissue migra-
Embryonic Kidney Perfusion . . . . . . . . . . . . . . . . . . . . . . . . 123 tion (horseshoe kidney, ectopic kidney). Many of
Renal Hypoplasia Associated with Lower Tract
these disturbances are linked to mutation of genes
Anomalies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
Environmental Causes of Renal Hypoplasia/ in molecular cascades involved in the develop-
Dysplasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 ment of other organs; thus, renal hypoplasia/dys-
Subtle Renal Hypoplasia . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 plasia can be seen with a variety of non-renal
malformations. It has been useful to assemble
Multicystic Dysplastic Kidney (MCDK) . . . . . . . . . . 126
large heterogeneous pediatric cohorts under the
Renal Ectopia and Fusion Anomalies . . . . . . . . . . . . . 129 umbrella term of congenital anomalies of the kid-
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 ney and urinary tract (CAKUT) as a way to study
classification and identification of underlying
genes without bias [1]. In this chapter we consider
forms of CAKUT associated with reduced func-
tional renal mass at birth.
P. Goodyer (*)
Division of Pediatric Nephrology, Montreal Children’s
Hospital, McGill University, Montreal, QC, Canada Clinical Presentation of Renal
e-mail: paul.goodyer@muhc.mcgill.ca
Hypoplasia/Dysplasia In Utero
I.R. Gupta
Department of Pediatrics and Department of Human
With the advent of routine antenatal ultrasono-
Genetics, Division of Pediatric Nephrology, Montreal
Children’s Hospital, McGill University, Montréal, QC, graphic screening, renal size and shape can be
Canada evaluated prior to birth (Fig. 1), and pediatric
# Springer-Verlag Berlin Heidelberg 2016 115
E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_4
116
Fig. 1 Magnetic resonance imaging showing normal fetal
kidney (arrow) and amniotic fluid (asterisk)
nephrologists are increasingly involved in the
management of pregnancies with renal abnormal-
ities. Optimal management is best accomplished
by a team that also includes the obstetrician, radi-
ologists, geneticists, and neonatologists. Pediatric
nephrologists should offer opinion regarding the
implications of antenatal renal hypoplasia/dyspla-
sia and meet with the family before birth to coun-
sel them about the rapid, often difficult, clinical
decisions that may unfold in the early postnatal
period.
Using transvaginal ultrasound, the urine is first
detected in the bladder by about 9 weeks of ges-
tation; renal size and amniotic fluid volume can be
assessed by transabdominal ultrasonography from
15 to 16 weeks of gestation onward. The diagno-
sis of antenatal renal hypoplasia (less than 5th
percentile) is usually made during the second
and third trimester using nomograms for renal
length and volume [2]; approximate mean and
5th percentile at selected gestational ages are sum-
marized in Table 1.
By 16 weeks, fetal skin has become
keratinized, and the amniotic fluid (about
P. Goodyer and I.R. Gupta
Table 1 Kidney size by gestational age. Sagittal kidney
length and renal volumes at various gestational ages; mean
(50th percentile) and lower limit of normal (5th percentile)
are shown. Adapted from Ref. [2]
Gestational Kidney Kidney
age (weeks) length (mm) volume (ml)
50th % 5th % 50th % 5th %
15 12.5 10 – –
20 20 16 2 1.5
25 26 23 4 2.5
30 33 28 7 4
35 38 34 12 7.5
40 44 38 17 11
175 ml) is generated primarily by fetal urine [3].
Thus, amniotic fluid volume may be reduced in
fetuses with reduced functional renal mass. How-
ever, regulation of amniotic fluid volume is com-
plex and difficult to estimate by ultrasonography.
By term, urine volume varies widely, from 400 to
1200 ml/day; about half is eliminated via the fetal
gastrointestinal tract while the remainder of the
(relatively hypotonic) fluid is transferred directly
to the placental compartment by diffusion and
bulk flow. Despite rapid renal growth in the third
trimester, amniotic fluid volume increases by only
35–70 ml/week during this period until the 38th
week of gestation and then declines by
125 ml/week to an average of 800 ml by
40 weeks [3]. Since accurate measures of amniotic
fluid volume cannot be made by ultrasonography,
oligohydramnios is often tracked by serial mea-
surements of amniotic fluid index (AFI). With the
transducer held in the sagittal plane and perpen-
dicular to the floor, the largest vertical diameter
(cm) in each of four quadrants is measured and
summed [4]. Nomograms for AFI in healthy preg-
nancies from 16 to 42 weeks were reported by
Moore et al. [5]; approximate values for mean and
2.5th percentile for AFI at various gestational ages
are summarized in Table 2. Although
oligohydramnios represents a clinically signifi-
cant consequence of bilateral renal hypoplasia, it
should be noted that it can also be seen with
premature rupture of membranes, fetal growth
restriction, and fetal hypoxia (thought to cause
fetal vasopressin release). Furthermore, a modest
5 Renal Dysplasia/Hypoplasia
Table 2 Amniotic fluid index at various gestational ages.
Amniotic fluid index (AFI) is calculated by antenatal ultra-
sonography as the sum of the largest vertical diameter
(cm) of amniotic fluid in each of the four quadrants in the
image. Mean (50th percentile) and lower limit (2.5th per-
centile) for various gestational ages are adapted from
Moore et al., Am J Ob Gyn (1990)
AFI(cm) 50th percentile 2.5th percentile
16 12.1 7.3
20 14.1 8.6
25 14.7 8.9
30 14.5 8.2
35 14.0 7.0
40 12.3 6.3
decline in AFI (from 14.7 to 12.3 cm) is normal
during the third trimester (Table 2).
When renal hypoplasia is extreme, inadequate
amniotic fluid production causes a characteristic
pattern of facial compression and pulmonary
hypoplasia (Potter syndrome) that may compro-
mise postnatal viability (Fig. 2) [6, 7]. By
compromising fetal respiratory movements that
optimize fetal lung development, severe
oligohydramnios may lead to pneumothorax in
the immediate postnatal period, and limited gas
exchange may require intubation and intensive
respiratory support. Antenatal ultrasonographic
assessment of lung volume is difficult and, when
suspected, is better examined by magnetic reso-
nance imaging. In fetuses with falling AFI, an
argument is often made for early induction of
labor once reasonable lung maturity can be
expected. On the other hand, if some amniotic
fluid is present in the third trimester and mechan-
ical compromise of lung development is less
extreme, it may be preferable to have the preg-
nancy go to term. This allows maximal somatic
and renal growth in the intrauterine environment,
making dialysis technically easier and reducing
the extrauterine growth needed to achieve trans-
plantable body size.
In babies who survive the immediate postnatal
respiratory crisis, the decision to embark on
chronic peritoneal dialysis is usually based on:
(a) whether the baby can achieve sufficient lung
recovery to be weaned off respiratory support;
(b) evidence of some functional renal parenchyma
117
Fig. 2 Magnetic resonance imaging showing case of Fra-
ser syndrome with the absence of the right kidney, hypo-
plastic left kidney (arrow), and anhydramnios (asterisk)
(identifiable by mercaptoacetyltriglycerine imag-
ing); (c) urine volume sufficient to permit minimal
long-term oral nutrition (100 kcal/kg/day); and
(d) family and institutional resources that can
sustain the dialytic therapy that successfully
achieves growth and development prior to renal
transplantation. Although the immediate compli-
cations of pulmonary hypoplasia dominate initial
care, some degree of pulmonary recovery can be
expected, and most babies who survive the first
week do not succumb to respiratory failure unless
oliguria leads to marked fluid overload. Judicious
fluid restriction and diuretic therapy are critical in
the first days of life to minimize edema that could
compromise gas exchange and make dialysis
catheter technically difficult to insert. Toward the
end of the first week, the baby’s urine volume
(on diuretics) should be estimated to inform fam-
ily and medical team whether survival on dialysis
is feasible.
When amniotic fluid is absent at an early stage,
families should be offered formal counseling
about the anticipated postnatal viability of the
infant and to prepare them for the rapid series of
decisions which may arise regarding the extent of
“heroic” interventions ranging from intubation, to
resuscitation, to dialysis. Because of the risk of
118
pneumothorax, fetuses with renal hypoplasia
and/or low AFI should be delivered in tertiary
care hospitals where neonatal intensive care is
available, ideally with prior awareness of the neo-
natology, nephrology, and genetics teams. When a
monogenic cause of renal hypoplasia is suspected,
families should be offered genetic counseling.
Renal Agenesis
Unlike lower species such as the fishes, human
nephron formation ends prior to birth, and babies
are born with a surfeit of nephrons (about
900,000/kidney) that are usually sufficient for
life. However, human nephron number varies
widely (>10-fold) among normal newborns
(210,000–2,700,000/kidney), and at the low end
of this spectrum are babies with clinically impor-
tant renal hypoplasia [8]. In the 1980s, Brenner’s
group postulated that suboptimal nephron endow-
ment induces compensatory glomerular
hyperfiltration, leading to slow but inexorable
glomerulosclerosis in postnatal life [9–11]; they
hypothesized that this increases the risk of essen-
tial hypertension and that reduced renal reserve
accelerates progressive renal insufficiency in
response to acquired renal injury later in life.
Although the mechanisms that set final neph-
ron are not well understood, it is clear that the
kidneys, adrenals, and gonads are derived from
progenitor cells in the posterior columns of inter-
mediate mesoderm which run along the
rostrocaudal axis of the embryo (see also
▶ Chap. 1, “Embryonic Development of the
Kidney” of this text). Key transcription factor
genes (e.g., WT1, CITED1) prime some of these
progenitors for nephrogenesis, while another sub-
set of cells express genes (e.g., GATA3 and
PAX2) transform them into nephric ducts on
each side of the embryo. At about 4–5 weeks of
gestation, ureteric buds emerge from the two cau-
dally migrating nephric ducts and grow outward
into the flanking column of intermediate meso-
derm. The left and right ureteric buds are induced
to arborize by glial-derived neurotrophic factor
(GDNF), and branching is further regulated by
other growth factors [12]. WNT signals from
P. Goodyer and I.R. Gupta
each ureteric bud branch tip induce adjacent neph-
ron progenitor cells to transform into the proximal
segments (glomerulus, proximal tubule, loop of
Henle, and distal convoluted tubule) of individual
nephrons. Each emerging nephron fuses to the
collecting duct formed by its parental ureteric
bud branch. By 36 weeks of gestation, the ureteric
bud has undergone 22–23 successive branching
events yielding 900,000 nephrons linked to a
common outflow tract in each kidney. Renal
hypoplasia/dysplasia arises through disturbance
of one or more of these embryonic processes.
Unilateral Renal Agenesis (URA)
URA occurs in about 1 in 2000 births [13]
(Fig. 3). It is estimated that about 5 % of URA
can be attributed to a unilateral multicystic/dys-
plastic kidneys which have involuted prior to
evaluation. The incidence of URA in males
(63 %) is slightly higher than in females, and the
left kidney is more often absent (63 %) than the
right [14]. Westland et al. reviewed 2684 URA
patients from 43 reports (pediatric and adult
cohorts) and found that about one third had vari-
ous urinary tract anomalies, including
vesicoureteral reflux (24 %), ureteropelvic junc-
tion obstruction (6 %), megaureter (7 %),
ureterocoele (1 %), and posterior urethral vales
(1 %) [13]. One third were associated with
Fig. 3 DMSA scan showing agenesis of the left kidney
5 Renal Dysplasia/Hypoplasia
syndromic extrarenal anomalies. From analysis of
an informative subset of the reports, it was esti-
mated that mean GFR was 100 ml/min/1.73 m2
and that the majority of patients with an isolated
solitary functioning kidney can be expected to
have stable renal function for many decades.
However, 21 % had microalbuminuria, 16 % had
hypertension, and 10 % had glomerular filtration
rate <60 ml/min/1.73 m2 [13]. It is unclear
whether this renal dysfunction is the consequence
of congenital nephron deficit or instead reflects
associated dysplasia in the remaining kidney. In a
pediatric cohort of URA/MCDK patients from the
Netherlands [15], hypertension was seen in 31 %,
and microalbuminuria was present in 23 %, indi-
cating that these issues are generally evident
before the age of 18 years.
URA obviously reduces total nephron mass
and usually induces compensatory renal hypertro-
phy in the remaining kidney during fetal life. In an
ultrasonographic study of a mixed URA/MCDK
cohort, length of the single functioning kidney
was estimated to be 23 % longer than normal
from the 20th week of gestation onward [2]. Cho
et al. proposed that the ratio of anterior-posterior
to transverse kidney dimensions discriminates
appropriately hypertrophied kidneys (1.0) from
the ratio in babies with two normal kidneys
(0.8) [16].
The majority of children with unilateral kidney
malformations are expected to retain stable renal
function for most of their adult lives. However,
not all babies with URA display appropriate com-
pensatory hypertrophy of the single remaining
kidney. In a study of postnatal renal hypertrophy
among 125 babies with various forms of solitary
functioning kidney, all but six had renal length
(as a function of body length) or renal volume
(as a function of body surface area) greater than
the 50th percentile for normal, and about one third
had kidney size greater than the 95th percentile for
normal [17]. Importantly, the babies with renal
size less than the 50th percentile developed renal
insufficiency (<60 ml/min/1.73 m2) [17]. Recent
observations by Sanna-Cherchi suggest that the
lifelong risk for URA may be underestimated
[18]. Of 71 patients with a solitary functioning
kidney followed for 30 years, 21 eventually
119
progressed to end-stage renal failure. The risk of
renal failure among URA babies was similar to
that in babies with bilateral hypoplasia/dysplasia.
This presumably reflects the associated lower tract
anomalies or contralateral renal dysplasia.
URA is frequently associated with ipsilateral
malformations of the reproductive tract. Among
girls, URA is seen in the Herlyn-Werner-
Wunderlich syndrome (HWWS), characterized
by bifid uterus (uterus didelphys), bifid vagina
(obstructed hemi-vagina in 60 %), and ipsilateral
URA [19, 20]. Women may be fertile after surgi-
cal repair of the uterus [21]. HWWS should be
suspected when antenatal ultrasonography reveals
URA in association with a fluid collection behind
the uterus [22]. URA is rarely associated with
Mayer-Rokitansky-Kuster-Hauser (MRKH) syn-
drome in which amenorrhea is linked to absence
of Mullerian ducts [23]. Among boys, URA can
be associated with cryptorchidism or ipsilateral
absence of the vas deferens [24]. However, most
males of unilateral absence of the vas deferens
(0.5–1.0 %) have normal kidneys. The presence
of URA does not exclude bilateral absence of the
vas deferens, and this should be ruled out, since it
is a cause of male infertility.
URA is also associated with a variety of other
malformation syndromes, particularly VACTERL
syndrome (1 per 10–40,000 births) involving a
variable combination of vertebral anomalies,
anal atresia, cardiac defects, tracheoesophageal
fistula with esophageal atresia, renal anomalies,
and limb defects [25]. About one quarter of
VACTERL patients have URA, while others
have vesicoureteral reflux (27 %) or a multicystic
dysplastic kidney (18 %) [26]. Other rare URA
associations include Barakat [27], Fraser [28],
Kabuki makeup [29], and Manitoba-oculo-
tricho-anal (MOTA) syndromes [30].
Recent genetic screens in large CAKUT kin-
dreds have yielded information regarding specific
genes associated with non-syndromic URA. In
these studies, URA has been associated with het-
erozygous (dominant) mutations of CDC5L [31]
and RET [31, 32] and with homozygous or com-
pound heterozygous (recessive) mutations of
FRAS1 and FREM2 [33]. Recessive mutations
of FRAS1 [33] and TRAP1 [26] genes were
120 P. Goodyer and I.R. Gupta

identified in several unrelated children with URA mutations of integrin alpha 8 (ITGA8) [38]. BRA
and VACTERL anomalies; recessive mutations of was noted in a family with recessive mutations of
GATA3 were reported in a child Barakat syn- the FGF20 gene [39] in a large kindred with
drome (deafness, seizures, and hypoparathyroid- X-linked Kallmann syndrome due to a KAL-1
ism) and URA [27]. The genetic basis for most gene mutations [40] and is seen occasionally
cases of isolated and syndromic URA is not yet with heterozygous RET mutations [41].
known.
Parents of a child with URA commonly ask
about whether contact sports should be avoided to
prevent loss of the functioning kidney. While the Renal Hypoplasia/Dysplasia
decision is ultimately an individual one, it should
be recognized that loss of a kidney due to trauma Renal hypoplasia/dysplasia (RHD) (Fig. 4) may
is extremely rare and is far less likely than the risk be unilateral or bilateral and may occur in associ-
of lethal car accident en route or serious brain ation with minor urinary tract malformations (e.g.,
injury incurred during the contact sport. vesicoureteral reflux) or in association with more
complex syndromes. In one large CAKUT series,
renal hypoplasia/dysplasia accounted for about
Bilateral Renal Agenesis one third of the cohort [18]. In a prospective
antennal ultrasound screen of 4000 healthy Japa-
Bilateral renal agenesis (BRA) is rare – about nese pregnancies, Hiraoko et al. noted renal hypo-
10 per 100,000 births in a report on congenital plasia in about 1:400 births [42].
anomalies among 601,545 births in the United Among 112 children with unilateral (URHD)
Kingdom [34]. BRA uniformly leads to or bilateral (BRHD) renal hypoplasia/dysplasia
anhydramnios and the Potter sequence in which analyzed by Sanna-Cherchi et al., 13 % had pro-
mechanical compression causes wide-set eyes, teinuria and 2 % had hypertension at the time of
flattened nose, receding chin, and large low-set diagnosis [18]. After 8–15 years of follow-up, 9 of
ears lacking cartilage [7]. Newborns also have
severe lung hypoplasia and clubbed feet and are
generally nonviable. However, Bienstock
et al. recently reported successful use of weekly
amnioinfusion from 24 weeks of gestation onward
to avert lethal lung hypoplasia. The affected child
required minimal postnatal respiratory support
and underwent successful peritoneal dialysis [35].
In most cases, the cause of BRA is not known.
However, reports of absent renal arteries and ure-
ters in the second trimester suggest arrest of kid-
ney development at an early stage. Roodhooft
et al. examined 40 siblings of 41 index cases
with BRA and found that 10 % had URA,
suggesting a heritable cause [36]. Bankier
et al. confirmed in an Australian cohort that
URA was found in sibs of babies with isolated
BRA but not in sibs of BRA associated with
multiple malformations [37]. Humbert
et al. recently reported a consanguineous Roma
Gypsy family in which 3 couples had multiple
pregnancies with BRA associated with recessive Fig. 4 Hypoplastic/dysplastic kidney
5 Renal Dysplasia/Hypoplasia
Fig. 5 Peritubular fibrous
cuff in an area of renal
dysplasia
Fig. 6 Ectopic cartilage in
an area of renal dysplasia
the children had progressed to end-stage renal
disease. In RHD, the presence of ureters, some
functional renal cortex, and basic reniform archi-
tecture reflects the fact that early formation of
ureteric buds and priming of the renal progenitor
population have occurred. However, functional
nephrons may be interspersed with patchy dys-
plastic tissue such as expanded stromal cells
around tubules (Fig. 5) or islands of cartilage
amid the functional nephrons (Fig. 6). Renal
hypoplasia necessarily means that nephron num-
ber is reduced, making it likely that there has been
dysregulation of the nephron progenitor pool, ure-
teric bud arborization, or both.
121
Renal Hypoplasia and Defects
in Ureteric Bud Branching
Hwang et al. screened 120 CAKUT patients with
renal hypoplasia/dysplasia and 90 with unilateral
renal agenesis [31]. Dysfunctional missense
mutations in eight genes were associated with
reduced renal mass (with or without anomalies
of the lower urinary tract). In the same cohort,
next-generation sequencing of 12 candidates
(drawn from mouse models of unilateral renal
agenesis) identified biallelic mild missense muta-
tions of 6 Fraser-syndrome-related genes [33]; a
renal hypoplasia phenotype was associated with
122
four of the six genes. Interestingly, many of the
mutant CAKUT genes linked to reduced renal
mass in these two studies are implicated in the
regulation of ureteric bud branching (e.g., RET,
HNF1B, BMP7, FRAS1, etc). The unique pheno-
type of ureteric bud tip cells is crucially dependent
on expression of RET (the GDNF receptor); the
transcription factor HNF-1B [43]; and the growth
factor, BMP7 [44]. Fraser syndrome genes encode
proteins belonging to a complex that is essential
for GDNF expression in the metanephric mesen-
chyme [33]. Other reports identify patients with
renal/hypodysplasia associated with mutations of
genes involved in fibroblast growth factor-
induced branching (e.g., FGF20 and
DSTYK) [26].
Autosomal dominant renal-coloboma syn-
drome (RCS) is a form of primary renal hypopla-
sia causing progressive renal insufficiency in
childhood associated with colobomas of the
optic nerve, reduced ureteric bud branching dur-
ing embryogenesis, and low nephron number at
birth [45]. RCS is caused by heterozygous null
mutations of PAX2, encoding a transcription fac-
tor expressed both in the ureteric bud and in the
metanephric mesenchyme [46]. Retrospectively,
children with “oligomeganephronia” who lacked
obvious ocular colobomas were found to have
PAX2 mutations, underscoring the wide variabil-
ity in extrarenal manifestations [47]. Typically,
children who inherit a heterozygous null PAX2
allele present with relatively small kidneys at
birth, proteinuria in the first months, and progres-
sive renal insufficiency during the first years of
life [48]. Renal biopsy shows that glomeruli are
greatly enlarged suggesting compensatory glo-
merular hyperfiltration. Most patients develop
end-stage renal disease by young adulthood.
Vesicoureteral reflux is present in about half of
RCS patients; mild high-frequency hearing loss is
less common [48].
The nephron deficit in mutant Pax2 mice
has been attributed to a primary defect in ureteric
bud branching during embryogenesis [49, 50].
Pax21Neu mice were found to have increased
programmed cell death of ureteric bud cells; loss
of Pax2 reduces expression of the antiapoptotic
gene, Naip, in ureteric bud cells [50, 51].
P. Goodyer and I.R. Gupta
A plausible explanation for the congenital neph-
ron deficit is that stochastic loss of cells might
slow the extension of ureteric bud branches
required before the next round of branching
events can occur.
Recently, a PAX2 missense mutation (p.
Gly189Arg) was identified in a family with dom-
inant focal segmental glomerulosclerosis (FSGS),
presenting in adulthood without any ocular phe-
notype [52]. Screening of a familial FSGS cohort
identified similar missense mutations in 4 % of
families [52]. Although the missense mutations
found in these patients are predicted to be patho-
genic, they presumably permit some residual
PAX2 function, accounting for late onset of renal
disease [53]. The finding in this family supports
Brenner’s hypothesis that individuals born with
suboptimal nephron number exhibit compensa-
tory hypertrophy and that glomerular
hyperfiltration early in life gradually leads to
focal glomerulosclerosis.
Renal Hypoplasia and Dysregulation
of the Nephron Progenitor Pool
In the subset of CAKUT patients with renal
hypoplasia, Hwang et al. identified heterozygous
(autosomal dominant) mutations of several genes
implicated in sustaining the nephron progenitor
cell pool [31]. These included dysfunctional
mutations of EYA1, SIX1 which cause the auto-
somal dominant branchio-oto-renal (BOR)
syndrome (OMIM 113650), pairing renal
hypoplasia with preauricular pits or branchial
fistulae, and variable degrees of deafness [54].
An expanded list of features includes a mixed
form of conductive/sensorineural hearing loss,
preauricular pits, a “cup-shaped” deformity of ear
pinnae, external auditory canal stenosis, branchial
fistulae, and renal anomalies including primary
renal hypoplasia, ureteropelvic junction obstruc-
tion, and vesicoureteral reflux. Renal hypoplasia
ranges from slightly reduced kidney size to bilat-
eral renal agenesis with perinatal death [55] and
may affect one kidney more than the other. Only
5–10 % of BOR patients develop end-stage renal
disease [56]. The prevalence of BOR syndrome is
5 Renal Dysplasia/Hypoplasia 123

about 1:40,000 and affects 2 % of children with Table 3 Syndromic renal hypoplasia/dysplasia genes.
profound deafness [56]. The cochlea is hypoplastic Syndromic renal hypoplasia/dysplasia associated with het-
erozygous mutations of various genes
and maybe simplified; dilatation of the vestibular
duct may be evident by MRI [57]. Gene Syndrome OMIM
About 40 % of BOR syndrome patients have PAX2 Renal-coloboma syndrome 120330
heterozygous inactivating mutations of the tran- EYA1 Branchio-oto-renal syndrome 113650
SIX1 Branchio-oto-renal syndrome 113650
scription factor EYA1 gene (8q13) [58]. EYA1 is
SIX5 Branchio-oto-renal syndrome 610896
expressed in progenitor cells of the embryonic
SALL1 Townes-Brocks syndrome 107480
kidney and first branchial arch that gives rise to
HNF1β Renal cortical cysts and diabetes 137920
the auricles, auditory canal, and ossicles of the
KAL1 Kallmann Syndrome 308700
ear; EYA1 is also expressed in hair cells of the FRAS1 Fraser syndrome 219000
cochlea [59]. Thus, EYA1 mutations appear to FREM2 Bifid nose, anorectal, renal 608980
cause renal hypoplasia, outer ear deformities, GATA3 Hypoparathyroidism with 146255
and conductive hearing loss by compromising deafness
progenitor cell fate in the embryonic kidney and GLI3 Pallister-Hall syndrome 146510
ear. Between 5 % and 10 % of BOR syndrome, LRP4 Cenani-Lenz syndrome 212780
patients have mutations of genes belonging to the RET Hirschsprung and CAKUT 142623
SIX transcription factor family. Several families BMP4 Isolated RHD –
bear mutations of SIX1 [60], and nearly 5 % have REN Renal tubular dysgenesis 179820
mutations of SIX5 [61]. Although loss of SIX2 in AGT Renal tubular dysgenesis 106150
mutant mice reduces nephron number, Hwang AGTR1 Renal tubular dysgenesis 106165
ACE Renal tubular dysgenesis 106180
et al. identified only one patient with a SIX2
mutation in their CAKUT cohort, and this did
not involve significant renal hypoplasia.
In 1972, Townes and Brocks described a patient Renal Tubular Dysplasia
with imperforate anus, sensorineural deafness, with Disturbance of Embryonic Kidney
minor anomalies of the external ear (preauricular Perfusion
tags and deformity of the superior helix), and mul-
tiple anomalies of the digits including missing Autosomal recessive renal tubular dysgenesis
bones and supernumerary thumbs [62]. Clinical (RTD) is a severe disorder in which development
features of the TBS syndrome are now recognized of renal tubules is compromised by hypoperfusion
to be more complex and include renal hypoplasia/ of the embryonic kidney [67]. Affected fetuses
dysplasia [63]. About half of TBS patients have have oligohydramnios with Potter sequence, peri-
heterozygous mutations of the transcription factor natal death due to pulmonary hypoplasia, and
gene, SALL1, which can sometimes lead to ESRD; refractory arterial hypotension and osseous
dominant negative SALL1 mutations produce a defects of the skull. Although the kidneys may
slightly more severe disease [64]. In developing be hyperechoic, renal size is usually normal. Glo-
mouse kidneys, SALL1 is expressed in the meta- meruli have normal structure and the surrounding
nephric mesenchyme surrounding branches of the tubules stain primarily for collecting duct
ureteric bud; Sall1 knockout mice lack outgrowth markers; proximal tubules are sparse and rudi-
of the ureteric bud and die perinatally of renal mentary, lacking a brush border and devoid of
agenesis [65]. Studies by Basta indicate that typical markers that are largely absent [68]. With
SALL1, like SIX genes, opposes differentiation saline infusion, intensive perinatal support and/or
of renal progenitor cells [66]. Thus, the mutant peritoneal dialysis, 10–15 % may survive the
genes above exemplify settings in which perinatal period. RTD is caused by mutations of
dysregulation of the embryonic renal progenitor genes encoding components of the renin-
cell pool compromises congenital nephron number angiotensin system (RAS): AGT
in humans (Table 3). (angiotensinogen), REN (renin), ACE
124
(angiotensin-converting enzyme), and AGTR1
(angiotensin II receptor type 1) [69].
Renal Hypoplasia Associated
with Lower Tract Anomalies
Mackie and Stephens examined autopsy specimens
from infants with duplex kidneys and noted an
association between a hypoplastic or dysplastic kid-
ney and the position of the ureteral orifice within the
bladder [70]. Ureters that inserted lateral to the
trigone or at the bladder neck were usually attached
to a malformed kidney. They then went on to pro-
pose the ureteric bud theory in which they postu-
lated that if the ureteric bud arose from a more
caudal location along the nephric duct, the ureter
would insert lateral to the trigone, while if it arose
from a more cranial location, it would insert at the
bladder neck. The ureteric bud is the primordial
tissue that gives rise to both the kidneys and the
ureters; therefore, if the ureteric bud emerges from
the wrong location from the nephric duct, it likely
does not receive or transmit the proper inductive
signals required for the development of these struc-
tures. From human studies, it is known that in
duplicated systems, the ureter that inserts lateral to
the trigone has a high likelihood of facilitating
vesicoureteric reflux. Similarly, the ureter that
inserts at the bladder neck is frequently associated
with urinary tract obstruction. While the initial
Mackie and Stephens studies were performed on
infants with duplex systems in which two ureteric
buds emerge, their theory likely applies to infants
with renal hypoplasia and single urinary tracts that
exhibit VUR or urinary tract obstruction.
While the ureteric bud theory is plausible, it is
difficult to prove. The best evidence that supports
the theory comes from the discovery of mouse
models with vesicoureteric reflux and renal
malformations [71, 72]. From these models, it
has been shown that the ureteric bud does emerge
from a more caudal location, and this results in a
delay in urinary tract development. The final
ureter has a short intravesical ureter and exhibits
vesicoureteric reflux. Using a mouse model in
which the receptor FGFR2 was deleted from the
P. Goodyer and I.R. Gupta
ureteric bud lineage, Hains et al. demonstrated
that a ureteric bud that emerges more cranially
results in ureteral insertion in the bladder neck
and these mice also exhibited VUR [73]. In sum-
mary, any child with evidence of renal hypopla-
sia warrants a thorough evaluation of the urinary
tract to rule out VUR and/or urinary tract
obstruction, and similarly, any child with evi-
dence of high-grade VUR and/or urinary tract
obstruction is likely to have a malformed kidney
on the same side as the urinary tract defect.
In Japanese perinatal screening studies, renal
hypoplasia (about 1:400 normal births) was
strongly correlated with vesicoureteral reflux [5].
Among neonates investigated for vesicoureteral
reflux, renal hypoplasia (reduced renal length by
ultrasonography and reduced DMSA uptake by
scintigraphy) was frequent, being symmetric in
about one third of cases or involving patchy
“scars” of the renal parenchyma in others [47,
48]. Among children evaluated for febrile urinary
tract infection in Texas, 15 % showed focal corti-
cal defects on DMSA scans; an additional 4 % had
evidence of reduced renal function without corti-
cal defects [74]. In Ireland, DMSA defects were
seen in 37 % of children with grade IV VUR and
67 % of those with grade V VUR [75]. Among
173 children with grade IV–V reflux (bilateral or
unilateral) vesicoureteral reflux in Taiwan, half
had developed CKD II or greater by about
140 months [76]. One third of children with bilat-
eral high-grade reflux developed hypertension
[76]. Controversy continues as to the best way to
distinguish congenital renal hypoplasia/dysplasia
from acquired defects attributable to infection, but
some clinicians have suggest a “top-down”
approach to assessing renal prognosis, while
focusing on VUR (“down-top” approach) to
guide the approach to recurrent infection.
Environmental Causes of Renal
Hypoplasia/Dysplasia
In the 1940s and 1950s, Wilson reported that
severe vitamin A deficiency caused renal agenesis
[77]. Observations in rodents indicate that
5 Renal Dysplasia/Hypoplasia 125

Table 4 Polymorphic gene variants associated with congenital renal hypoplasia. Single nucleotide polymorphisms
(SNPs) associated with reduced newborn kidney volume
Gene SNP MAF% SNP type Decrease in newborn kidney volume
PAX2 rs11599825(A) 18.5 % Noncoding 10 %
rs11190688(A)
rs11190702(A)
rs11190739(T) 6.5 % Noncoding 9 %
RET rs1800860(A) 25 % Exonic splice enhancer 10 %
OSR1 rs12329305(T) 6% Exonic splice enhancer 12 %
BMPR1A rs7922846(A) 31.6 % Noncoding 13 %
ALDH1A2 rs7169289(G) 22 % Noncoding +22 %

maternal vitamin A deficiency (50 % reduction in

circulating retinol levels) causes significant reduc-
tion (24 %) in postnatal kidney weight associated
with a reduction (20 %) in nephron number
[78]. Normally, the fetus acquires retinol from
the maternal circulation and converts it to the
active metabolite, all-trans-retinoic acid, in
kidney and other tissues [79]. In cultured fetal
rat kidneys, all-trans-retinoic acid
accelerates new nephron formation two- to three-
fold [80, 81]. Vitamin A deficiency is widespread
in developing countries; in North America and
Europe, vitamin A deficiency has been
documented in unsupplemented newborns born
prematurely before nephrogenesis is complete.
In a cohort of 46 pregnant Indian women,
Goodyer et al. noted that half had retinol levels
below the accepted limit of normal (0.9 umol/L)
and newborn kidney volume (adjusted for body
size) was only 62 % of adjusted newborn kidney
size in vitamin A-replete women from
Montreal [82].
In rats with streptozotocin-induced diabetes
mellitus, offspring have renal hypoplasia and
reduced nephron number [83]. In fetal mice
exposed, embryonic E14.5 kidneys had 45 %
fewer ureteric bud branches than controls to
maternal diabetes [84]. Among human offspring
of Type I diabetic mothers, Khalil et al. noted
reduced renal reserve in response to amino acid
infusion [85], and Cappuccini et al. found that
renal cortex volume was significantly reduced
compared to controls [86].
Subtle Renal Hypoplasia
Common completely dysfunctional alleles for
crucial developmental genes are rare, since they
produce malformations with a major disadvan-
tage. However, mild mutations of these genes
exert subtler effects on renal mesenchyme/ureteric
bud interactions and may be fairly common
among normal children (Table 4). If measured by
ultrasonography in the newborn period before
postnatal compensatory hypertrophy is complete,
kidney size was shown to correlate with nephron
number at autopsy [87]. Using this approach,
Quinlan et al. identified a signature (AAA) of
several tightly linked intronic PAX2 SNPs, pre-
sent in 18.5 % of normal Canadian newborns, that
was associated with a subtle (10 %) reduction in
newborn kidney size (adjusted for body surface
area). In a heterozygous renal cell carcinoma line,
the AAA allele was associated with a 50 % reduc-
tion in PAX2 transcript level [88]. No common
variants of the human GDNF gene affect kidney
size, but a polymorphism of the RET (GDNF
receptor) gene (RET1476 G/A, rs1800860) was
associated with a 10 % reduction in newborn
kidney volume (normalized for body surface
area) and 9 % increased serum cystatin C com-
pared to the wild-type (RET1476G) allele
[87]. Although fairly common in the healthy
European Caucasian population (MAF = 25 %),
the RET1476A allele alters a splicing enhancer site
in exon 7, permitting frequent transcription of a
126
truncated mRNA [87]. Interestingly, among the
15 % of infants who inherited both a RET1476A
and a PAX2AAA allele, newborn kidney size was
reduced by 25 %. Taken together, these observa-
tions suggest that hypomorphic RET and PAX2
gene variants act in combinatorial fashion to affect
ureteric bud branching and, thus, newborn kidney
size. In a separate cohort of Polish babies, new-
born kidney size was reduced by 13 % in infants
bearing a common variant of the BMPR1A gene
(encoding a bone morphogenetic protein receptor
on ureteric bud cells) [89]. Conversely, 22 % of
Canadian newborns inherit a variant of the
ALHD1A2 gene [rs7169289 (G)] associated
with increased production of all-trans-retinoic
acid metabolism in fetal tissues; this retinoid is
known to enhance RET expression in the ureteric
bud, and newborns with the G allele were shown
to have a 22 % increase in newborn kidney size
compared to the wild-type allele [90].
While human nephron number is affected by
genes regulating the extent of ureteric branching,
animal studies also indicate that the size of the
renal progenitor cell pool can also be rate-
limiting. One of the earliest transcription factors
marking the nephron progenitor cells of the inter-
mediate mesoderm is OSR1; Osr1 knockout mice
lack nephrogenic mesenchyme and are anephric at
birth [91]. Recently, a variant of the human OSR1
gene which interferes with an exon 2 splice
enhancer site was identified in about 6 % of nor-
mal Caucasians [92]. This OSR1rs12329305(T) vari-
ant was associated with a 12 % reduction in
newborn kidney size and 12 % increase in serum
cystatin C level [92]. Again, the effects of the
OSR1rs12329305(T) were additive with RET1476G
in infants who also inherited both alleles; kidney
volume/body surface area was reduced by 17 %
and serum cystatin C was increased by 22 %
[92]. Recently, Lozic et al. reported association
between OSR1rs12329305(T) and stillborn/neonatal
death among babies with congenital kidney
malformations [93]. Taken together, these obser-
vations suggest that human nephron endowment
may represent a complex polygenic trait deter-
mined by the additive effects of multiple genes
regulating either the renal progenitor cell pool or
the extent of ureteric branching during fetal life.
P. Goodyer and I.R. Gupta
Evidence is emerging that the common gene
variants associated with congenital renal hypopla-
sia predict accelerated renal deterioration in the
setting of acquired kidney disease later in life. In a
preliminary study, Cosgrove identified a common
(MAF = 6.5 %) noncoding PAX2 polymorphic
variant (rs11190739T) that was associated with
increased serum creatinine in a cohort of patients
with Type I diabetes for 10–30 years [94]. Esti-
mated GFR was 70.7 ml/min/1.73 m2 in diabetics
with the wild-type allele, but was 60.1 ml/min/
1.73 m2 (15 % decrease) in diabetics with one or
more rs11190739T alleles. These observations
support Brenner’s hypothesis that progressive
renal disease may be accelerated among individ-
uals with suboptimal nephron number and limited
renal reserve.
Multicystic Dysplastic Kidney (MCDK)
MCDK is a form of severe renal dysplasia in
which the kidney is occupied by tense
noncommunicating macrocyst; the largest cyst is
not usually located medially and, typically, there
is no functional parenchyma (Fig. 7). The renal
pelvis and calyces cannot usually be identified,
and the ureteropelvic junction and ureter are usu-
ally atretic; ipsilateral vesicoureteral reflux is rare.
The incidence of MCDK ranges from 1 per 2200
to 4300 births; it is slightly more common among
males (55 %) and slightly more common on the
right (53 %) than on the left [95–98]. Among
males, 13 % have ipsilateral or bilateral unde-
scended testes [97] (Fig. 8). With widely available
antenatal ultrasound screening, MCDK is now
increasingly diagnosed before birth (Fig. 9), but
follow-up postnatal ultrasonography is needed to
distinguish MCDK from high-grade urinary tract
obstruction.
Since MCDK is typically unilateral, the long-
term renal prognosis depends primarily on the
integrity of the contralateral kidney. With long-
term follow-up, 2/40 Italian and 2/34 Indian
MCDK patients were seen to develop end-stage
renal failure [18, 99]. In most cases, estimated
glomerular filtration rate is normal, ranging
from 86 to 122 ml/min/1.73 m2 in the pediatric
5 Renal Dysplasia/Hypoplasia 127

Fig. 7 Multicystic
dysplastic kidney. (a) Renal
ultrasound; (b) multicystic
kidney (left) and normal
kidney (right) en bloc; (c)
multicystic dysplastic
kidney in situ

Fig. 8 Antenatal magnetic resonance imaging showing the left multicystic dysplastic kidney

period [100]. About one quarter of the contralat-
eral kidneys have vesicoureteral reflux (most
often grade I–III). Although this is occasionally
associated with recurrent urinary tract infection,
the presence of mild-moderate vesicoureteral
reflux is of little clinical significance and does
not warrant routine cystourethrogram. Less
often, the contralateral kidney has ureteropelvic
junction obstruction (3–5 %) and ureterovesical
junction obstruction (2 %) smaller in number. In
several large series, there are a small number of
contralateral kidneys (2–5 %) which exhibit
severe reflux, remain small and hyperechoic or
fail to show normal compensatory hypertrophy,
and have highest risk of progressing to end-stage
renal failure [17]. Between 5 % and 30 % of cases
have low-grade proteinuria [95, 99]. Since the
MCDK is usually nonfunctional, this mild pro-
teinuria presumably arises from the contralateral
kidney. Hypertension occurs in a small fraction
128
Fig. 9 Ultrasound showing ipsilateral mid-canal unde-
scended testis (arrow)
(1–6 %) of cases [95, 101], usually associated
with minimal residual functional parenchyma
(noted by renal scan) in the MCDK. Importantly,
the hypertension is often reported to resolve when
the MCDK is resected [99].
The etiology of MCDK is unclear but presum-
ably involves embryonic disturbance of the mech-
anisms that generate functional nephrons linked to
the renal collecting system. Significantly
increased gene copy number variation is seen in
>10 % of MCDK patients [102]. In large screens
of CAKUT cohorts, MCDK has been associated
with dominant mutations of CHD1L, ROBO2,
and SALL1 genes [33]. Interestingly, the MCDK
phenotype has also been linked to heterozygous
null mutations of the TCF2 (HNF1β) gene. Hasui
reported a family in which one son with a TCF2
mutation presented with bilateral MCDK,
P. Goodyer and I.R. Gupta
whereas the other son had unilateral MCDK
[103]. In an antenatal cohort of 63 cases with
bilateral hyperechoic kidneys and normal amni-
otic fluid volume, 18 were found to have TCF2
mutations, most commonly a heterozygous TCF2
deletion [104]. However, the presentation is quite
variable and some affected kidneys develop wide-
spread cysts only in the postnatal period,
distinguishing this from typical MCDK. Further-
more, in this form of MCDK there may also be
pancreatic hypoplasia detectable by antenatal
ultrasound [105].
Whereas multicystic kidneys were commonly
resected in the past, most authors currently rec-
ommend a conservative approach. While it
remains important to distinguish MCDK from
cystic Wilms tumors, the risk of malignant trans-
formation in MCDK tissue may approximate the
risk in normal kidneys [100]. Early postnatal
ultrasound should be performed to exclude
obstruction, hyperechogenicity, or other overt
signs of dysplasia in the contralateral kidney.
Serial ultrasonography should document appro-
priate compensatory hypertrophy in the contralat-
eral kidney and involution of the MCKD. Spira
et al. showed that renal length of the
hypertrophied kidney should be greater than the
50th percentile for body length, and one third
excedes the 95th percentile [17]. In a separate
cohort of 323 MCDK cases, 10 % of antenatally
diagnosed MCDKs had involuted by birth, 35 %
by 2 years, 47 % by 5 years, and 62 % by 10 years
[97]. Blood pressure should be checked in the first
years of life; when hypertension is detected, renal
scanning is indicated since the presence of resid-
ual functional renal parenchyma may support a
role for nephrectomy. Serum creatinine and urine
protein excretion should also be monitored in the
first years of life to detect progressive renal dys-
function, particularly in those children with evi-
dence of overt dysplasia or suboptimal size of the
contralateral kidney. Hayes et al. have argued that
costly monitoring might be curtailed after the first
year in children who exhibit MCDK involution
and normal compensatory hypertrophy and have
no overt complications [97].
5 Renal Dysplasia/Hypoplasia
Fig. 10 Horseshoe
kidneys: (a) computerized
tomography scan showing
isthmus of horseshoe
kidney fused in midline,
(b) DMSA scan showing
pancake kidney fused in
midline
Renal Ectopia and Fusion Anomalies
During embryogenesis, the two kidneys ascend
from a lower pelvic position to their normal
upper intra-abdominal location. Disturbance of
renal ascent leads to renal ectopia in about 1:900
births and is usually (90 %) unilateral. The asso-
ciated ureter nearly always enters the bladder nor-
mally, but about half are associated with
hydronephrosis. This may be due to ureteropelvic
junction obstruction (16 %), ureterovesicular
junction obstruction (8 %), or vesicoureteral
reflux (25 %) [106]. In about 40 %, the ectopic
kidney is found in the pelvis. While most ectopic
kidneys are asymptomatic, lumbar and pelvic
129
kidneys are sometimes associated with poorly
localized pain. Rarely, ectopic kidneys have
been reported in the thorax and are identified as
an incidental nonfunctional chest mass [107].
When aberrant ascent brings them into contact,
the two kidneys may fuse. Most commonly, this
occurs in the midline and is referred to as a horse-
shoe kidney (Fig. 10). In 95 %, the horseshoe
kidney is low-lying (L3-L4) where further ascent
has been blocked by the inferior mesenteric artery.
The two moieties are fused via a parenchymal
isthmus bridging their lower poles and may
develop a variety of vascular arrangements, includ-
ing a single artery [108]. About half of horseshoe
kidneys exhibit vesicoureteral reflux and about one
130
third report recurrent urinary tract infection [109].
About 20 % of horseshoe kidneys develop stones at
some point in life [110]. There are occasionally
VACTERL (3 %) and reproductive tract (5 %)
anomalies [111]. Although there are case reports
of renal cell carcinoma arising within horseshoe
kidneys, there is no evidence that this occurs
more frequently than in the general population
[106]. On the other hand, patients have horseshoe
kidneys have a twofold increased risk of Wilms
tumor compared to normal [112]. Between 15 %
and 30 % of girls with Turner syndrome have
horseshoe kidneys [113, 114].
Crossed ectopia occurs when one kidney
crosses the midline in relationship to the side
where its ureter enters the bladder; in 90 % of
cases, the upper pole of the ectopic kidney is
fused to the lower pole of the orthotopic kidney
but sometimes occurs without fusion, and the two
kidneys may even exchange sides [106,
115]. Abnormalities of the reproductive tracts
are seen in about half of the cases; these include
cryptorchidism or absence of the vas deferens in
males and vaginal atresia or unilateral uterine
abnormalities in females [106].
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 Developmental Syndromes
and Malformations of the Urinary Tract 6
Chanin Limwongse

Contents Introduction
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Birth defects involving the kidney and urinary
Prevalence of Urinary Tract Anomalies . . . . . . . . . . 157
system are often encountered and frequently
Approach to the Child with a Urinary Tract occur in association with other structural abnor-
Anomaly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
malities. A congenital urinary tract anomaly may
Overview of Normal Embryogenesis of the provide the first clue to the recognition of
Urinary System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 multiorgan developmental abnormalities. Never-
Anomalies Involving the Urinary Tract . . . . . . . . . . 163 theless many renal anomalies remain asymptom-
Kidney Defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 atic and undiagnosed. Therefore it is critical, not
Ureter Defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Bladder Defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
only for pediatric nephrologists but also for pedi-
Urethral Defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 atricians in general, to be familiar with the com-
mon anomalies involving the kidney and urinary
Associations and Sequences Involving the
Urinary Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 system and the more complex disorders with
VATER Association . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 which they may be associated.
CHARGE Syndrome (CHARGE Association) . . . . . 168 The kidney is a pivotal organ in
MURCS Association . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 dysmorphology. Although the number of single
Oligohydramnios Sequence . . . . . . . . . . . . . . . . . . . . . . . . . 169
Urethral Obstruction Sequence . . . . . . . . . . . . . . . . . . . . . . 169 malformations involving the kidney is limited,
Sirenomelia Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 combinations of these malformations in conjunc-
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
tion with anomalies involving other organ sys-
tems are found in more than 500 syndromes. In
addition, many well-known sequences and asso-
ciations involve the kidney and urinary tract. This
chapter discusses common malformations,
sequences, and associations involving the kidney
and urinary tract and provides a summary of con-
ditions that have these anomalies as one of their
features. In addition, Tables 1, 2, and 3 summarize
more detailed information about a large number of
C. Limwongse (*) disorders, including their phenotypic features,
Department of Medicine, Division of Medical Genetics,
reported urinary tract anomalies, pattern of inher-
Faculty of Medicine, Siriraj Hospital, Mahidol University,
Bangkoknoi, Bangkok, Thailand itance, causative genes, and related references.
e-mail: chanin.lim@mahidol.ac.th; siclw@mahidol.ac.th These tables can be used both to provide readily
# Springer-Verlag Berlin Heidelberg 2016 135
E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_5
 136

Table 1 Syndromes and disorders that have urinary tract anomalies as a frequent feature
Urinary tract abnormalities

Other
associated

Renala genesis
Ectopia=horseshoe
Cystic=dysplasia
Duplication
Hypoplasia
Hydronephrosis=ureter
Diverticula
Atresia=stenosis
Reflux
Nephritis=sclerosis
Tumor=nephromegaly
Syndromes Other associated anomalies anomalies Gene(s) References
Abruzzo-Erickson H Coloboma, cleft palate, XR Unk [48]
hypospadias, deafness,
short stature
Acrocephalopolydactylous + + + Acrodactyly, hand AR Unk [49]
dysplasia (Elejalde hexadactyly, overgrowth,
syndrome) visceromegaly, globular
body, redundant neck skin
Acrorenal (Dieker) 1 E + + + Ua Ectrodactyly, oligodactyly, Sporadic Unk [50]
hypoplastic carpal/tarsal
bones
Acrorenal (Johnson- 1, U, Aphalangy, hemivertebrae, AR Unk [50]
Munson) 2 Ua genital/intestinal/anal
dysgenesis
Acrorenal (Siegler) E + U + Short stature, hypoplastic Uncertain Unk [50]
radii/ulnae/humeri,
oligodactyly
Acrorenal-mandibular 1 + Ectrodactyly, hypoplastic AR Unk [51]
mandible
Acrorenal-ocular 1 E + B Hypoplastic thumb, optic AD SALL4 [52]
coloboma, cleft lip/palate
Adrenoleukodystrophy, See Pseudo-Zellweger
neonatal
Aglossia-adactylia 1 Micrognathia, cranial nerve Sporadic Unk [53]
palsy
Agnathia- H + Arrhinencephaly, situs Sporadic Unk [54]
holoprosencephaly inversus, midline defects
C. Limwongse
 6

Alagille 1 + + + Cholestasis, peripheral AD JAG1 [55]

pulmonic stenosis,
characteristic face
Alport + Nephritis, proteinuria, AD, AR, XL COL4 [56]
deafness
Alsing + Nephritis, AR Unk [57]
nephronophthisis, optic
coloboma, hip dislocation
Alstrom + Diabetes mellitus, AR ALMS1 [58]
retinopathy, short stature,
deafness
Amelogenesis imperfecta + Enamel hypoplasia, AR Unk [59]
type IG nephrolithiasis, enuresis
Amyloidosis type 5 + Nephropathy, proteinuria, AD GELSOLIN [60]
cranial nerve palsy, cutis
laxa
Angiotensin-converting + IUGR, oligohydramnios, Sporadic none [61]
enzyme (ACE) inhibitor, patent ductus arteriosus,
maternal use limb anomalies, renal artery
stenosis, progressive renal
failure
Aniridia-Wilms tumor + Aniridia, ambiguous AD WT1 [62]
(WAGR) genitalia, hypospadia, short
Developmental Syndromes and Malformations of the Urinary Tract

stature
Axial mesodermal 1 Bladder exstrophy, Uncertain Unk [63]
dysplasia vertebral anomalies,
Goldenhar-like
Baldellou 1 Hypoparathyroidism, Uncertain Unk [64]
ocular coloboma, MR,
seizures
Baller-Gerold + E + + Craniosynostosis, radial AR RECQL4 [65]
aplasia, malformed ear, anal
atresia
Barakat + Mesangial sclerosis, MR, AD GATA3 [66]
optic atrophy, nystagmus
Bardet-Biedl + + + + + + Obesity, polysyndactyly, AR BBS1-12 [67, 68]
MR, retinopathy,
hypogonadism
137

(continued)
 138

Table 1 (continued)
Urinary tract abnormalities

Other
associated

Renala genesis
Ectopia=horseshoe
Cystic=dysplasia
Duplication
Hypoplasia
Hydronephrosis=ureter
Diverticula
Atresia=stenosis
Reflux
Nephritis=sclerosis
Tumor=nephromegaly
Syndromes Other associated anomalies anomalies Gene(s) References
Beckwith-Wiedemann E + + + + Overgrowth, macroglossia, Sporadic, AD CDKN1C, [69–72]
omphalocele, embryonal NSD1, H19
tumors
Berardinelli + + Insulin resistance, AR BSCL2 [73]
lipodystrophy, acanthosis
nigricans, MR
Brachymesomelia-renal + Micrognathia, corneal Uncertain Unk [74]
opacity, craniofacial
dysmorphism
Branchio-oculo-facial 1 Philtrum hypertrophy, cleft AD TFAP2A [75]
lip/palate, branchial
remnant
Branchio-oto-renal 1, E + + + + + Branchial remnant, AD EYA1, SIX5 [76]
2 preauricular pit/tag,
microtia, deafness
Braun-Bayer + U + Deafness, bifid uvula, Uncertain Unk [77]
digital anomalies
Cat eye See Table 3 Chromosomal
Caudal duplication 1, E + + + Duplication of colon, Sporadic AXIN1 [78]
2 sacrum, genitalia, vertebral
defects
Caudal regression 1, E, + + Ua + Atresia of colon, anus, AD, AR VANGL1 [79, 80]
2 H genitalia, vertebral defects,
TE fistula
Cerebro-hepato-renal + See Zellweger syndrome AR PEX [81]
(Passarge)
C. Limwongse
 6

Cerebro-oculo-hepato- + Cerebellar hypoplasia, AR Unk [82]

renal hepatic fibrosis, Leber
amaurosis
Cerebro-osteo-nephro + + Rhizomelic limb AR Unk [83]
dysplasia shortening, cerebral
atrophy, MR, seizures
CHARGE + E + + + + U + See text for details Sporadic, AD CHD7 [33–37]
Chondroectodermal 1 + + + Acromelic dwarfism, AR EVC, EVC2 [84, 85]
dysplasia (Ellis van polydactyly, nail dystrophy,
Creveld) tooth hypoplasia narrow
thorax, CHD
Cocaine, maternal use 1, + + Ua + Vascular disruption Sporadic none [86]
2 anomalies affecting
multiple organs
Cornelia de Lange 1 + + + + SS, microcephaly, limb Sporadic NIPBL, [87]
defect, hirsutism, synophrys SMC1L1
Crossed renal ectopia- E + U Clubbing of fingers, Uncertain Unk [88]
pelvic lipomatosis gynecomastia
Czeizel + U Ectrodactyly, spina bifida, AD Unk [89]
megacystis
Diabetic mother, infant of 1, E, + + + + Ua + Neural tube defect, cardiac/ Sporadic none [90, 91]
2 H limb anomalies, sacral
agenesis
Developmental Syndromes and Malformations of the Urinary Tract

DiGeorge/velocardiofacial 1, E + + + + + See Table 3 Chromosomal

3
Denys-Drash E + + + Pseudohermaphroditism, Sporadic WT1 [92]
Wilms tumor, proteinuria
Down See Table 3 Chromosomal
Ectrodactyly-ectodermal 1 + + + + Ectrodactyly, hypohidrosis, AD Unk [93]
dysplasia-clefting (EEC) sparse hair, cleft lip/palate
Elejalde See
acrocephalopolydactylous
dysplasia
(continued)
139
 140

Table 1 (continued)
Urinary tract abnormalities

Other
associated

Renala genesis
Ectopia=horseshoe
Cystic=dysplasia
Duplication
Hypoplasia
Hydronephrosis=ureter
Diverticula
Atresia=stenosis
Reflux
Nephritis=sclerosis
Tumor=nephromegaly
Syndromes Other associated anomalies anomalies Gene(s) References
Epstein + Thrombocytopenia, nerve AD MYH9 [94]
deafness, cataract
Facio-cardio-renal H + U Cardiomyopathy, AR Unk [95]
conduction defect, MR,
typical face
Fanconi anemia 1 E, + + + + Pancytopenia, limb defects, AR FANCA-N [96, 97]
H leukemia, lymphoma
Fetal alcohol 1 E, + + + + IUGR, DD, microcephaly, Sporadic none [98]
H short palpebral fissure
Fibromatosis, infantile + + Multiple myofibromatosis, AR Unk [99]
myositis ossificans
Fraser cryptophthalmos 1, + U Fused eyelids, ear/genital AR FRAS1 [100]
2 anomalies, syndactyly
Frasier + + Male pseudohermaphrodite, AD WT1 [101]
amenorrhea, ovarian cysts
Goeminne + + Congenital torticollis, XR Unk [102]
keloids, cryptorchidism
Goldenhar (oculo-auriculo- 1 E + + + + + Hemifacial microsomia, ear Sporadic, AD Unk [103]
vertebral) anomalies, vertebral defects
Goldston + Dandy-Walker Uncertain NPHP3 [104]
malformation, cerebellar
malformation
Graham + + Cystic hamartoma of lung Sporadic Unk [105]
and kidney
C. Limwongse
 6

Hemifacial microsomia See Goldenhar syndrome

(oculo-auriculo-vertebral)
Hemihyperplasia + + Asymmetry, vascular Sporadic LIT1, H19 [106]
malformation, embryonal
tumors
Hepatic fibrosis + Congenital hepatic fibrosis Sporadic PKHD1 [107]
Holzgreve 2 Potter sequence, cardiac Uncertain Unk [108]
defect, polydactyly, cleft
palate
Hypertelorism-microtia- E + Microcephaly, cleft AR Unk [109]
clefting lip/palate, MR
Ivemark 1 + + + Polysplenia/asplenia, Sporadic, AR Unk [110]
complex CHD, laterality
defects
Jeune + Narrow chest, short limbs, AR EVC, EVC2 [84, 85]
polydactyly,
glomerulosclerosis
Joubert + Vermis aplasia, apnea, jerky AR JBTS1–7 [111]
eyes, retinopathy, ataxia
Juberg-Hayward H + Microcephaly, cleft AR Unk [112]
lip/palate, abnormal
thumbs/toes
Kabuki H + + U + MR, characteristic Kabuki- Uncertain Unk [113]
Developmental Syndromes and Malformations of the Urinary Tract

like face, large ears, cleft

palate
Kallmann 1 + Anosmia, cleft lip/palate, AD, AR, XR KAL1–4 [114]
hypogonadotropic
hypogonadism
Kaufman-McKusick E + + + Ua Hydrometrocolpos, AR GLI3 [115]
polydactyly, anal/urogenital
sinus defect
Kivlin E + Short stature, Peters’ AR B3GALTL [116]
anomaly, MR, genital/
cardiac defects
(continued)
141
Table 1 (continued)
142

Urinary tract abnormalities

Other
associated

Renala genesis
Ectopia=horseshoe
Cystic=dysplasia
Duplication
Hypoplasia
Hydronephrosis=ureter
Diverticula
Atresia=stenosis
Reflux
Nephritis=sclerosis
Tumor=nephromegaly
Syndromes Other associated anomalies anomalies Gene(s) References
Klippel-Feil 1 E + Short neck, cervical Sporadic, AD Unk [117]
vertebral fusion, low
posterior hairline
Kousseff 1 Sacral meningocele, AR Unk [118]
hydrocephalus, cardiac
defects
Leprechaunism (Donohue) + + Insulin resistance, AR INSR [119]
lipodystrophy, hirsutism
Limb-body wall complex 1 E + U, Lateral body wall defect, Sporadic Unk [120]
Ua limb reduction, CHD
Mammo-renal + Ipsilateral supernumerary Sporadic Unk [121]
breasts/nipples
Marden-Walker + + Microcephaly, AR Unk [122]
blepharophimosis,
micrognathia, contractures
Meckel-Gruber + + U, Encephalocele, cardiac AR MKS1–4 [123]
Ua defects, cleft lip/palate,
polydactyly
Megacystis-microcolon + U Large bladder, intestinal AR Unk [124]
hypoperistalsis,
oligohydramnios
Melnick-Needles + U Bowing long bones, short XR FLNA [125]
osteodysplasty upper limbs, micrognathia
Mendenhall + Insulin resistance, AR INSR [126]
acanthosis nigricans
Microgastria-upper limb 1 E + Hypoplastic spleen, limb Uncertain Unk [127]
anomaly reduction defects
C. Limwongse
 6

Miranda + + Brain malformation, liver Uncertain Unk [128]

dysplasia
Moerman 1 + + + + Short limbs, brain Uncertain Unk [129]
malformation, cleft palate
MURCS association 1 E + U See text for details Sporadic Unk
Nager acrofacial dysostosis + + + + Facial bone hypoplasia, AD Unk [130]
cleft eyelid, radial ray defect
Nail-patella + Absent/hypoplastic nails AD LMX1B [131]
and patellae
Neu-Laxova 1 IUGR, lissencephaly, CHD, AR,sporadic Unk [132]
pterygia, ichthyosis
Neurofaciodigitorenal 1 Prominent forehead with Uncertain Unk [133]
vertical groove, MR, ear
anomaly
Neurofibromatosis type I 1 Ua + renal artery stenosis, AD NeUnkro [134]
hypernephroma, cafe-au- fibromin
lait spots
Nezelof + + Arthrogryposis, hepatic AR Unk [135]
impairment, hypotonia,
club feet
Noonan + + + Webbed neck, short stature, AD NS1 [136]
MR, pulmonic stenosis
Developmental Syndromes and Malformations of the Urinary Tract

Occipital horn + B Ua Cranial exostosis, XR cUnkATPase [137]

hyperextensibility, cutis
laxa
Ochoa + + B U, Ua + Facial grimacing with Uncertain Unk [138]
lateral displacement of
mouth
Oculo-auriculo-vertebral See Goldenhar syndrome
Oculo-hepato-encephalo- + + Encephalocele, hepatic AR Unk [82]
renal fibrosis, coloboma
Oculorenal-cerebellar + MR, spastic diplegia, AR Unk [139]
choreoathetosis,
retinopathy
(continued)
143
 144

Table 1 (continued)
Urinary tract abnormalities

Other
associated

Renala genesis
Ectopia=horseshoe
Cystic=dysplasia
Duplication
Hypoplasia
Hydronephrosis=ureter
Diverticula
Atresia=stenosis
Reflux
Nephritis=sclerosis
Tumor=nephromegaly
Syndromes Other associated anomalies anomalies Gene(s) References
Oculorenal See Pierson syndrome
Oculorenal (Karcher) 1 + + Optic nerve coloboma AD Unk [140]
OEIS (omphalocele- + + Ua + Malrotation of colon, sacral Sporadic Unk
exstrophy of bladder- defect, tethered spinal cord,
imperforate anus-spinal pelvic bone abnormalities
dysraphism) complex
Otorenal + + + U Renal pelvis diverticula, AD Unk [141]
nerve deafness
Pallister-Hall 1, E, + + Hypothalamic AD GLI3 [142, 143]
2 H hamartoblastoma,
polydactyly
Pierson + Hypoplastic retina, cataract, AR LAMB2 [144]
anterior chamber anomalies
Penoscrotal transposition E + + + B U Abnormal placement of Sporadic Unk [145]
external genitalia
Perlman + + + + + Early overgrowth, typical AR Unk [146]
face, nephroblastomatosis
Polydactyly-obstructive + Ua + Post-axial polydactyly of Uncertain Unk [147]
uropathy hands and feet
Potter (oligohydramnios) 1, + + Ua See text for details Sporadic Unk
2
Prune belly + + + Ua + See text for details Sporadic Unk
Pseudo-Zellweger + + Hypotonia, seizures, MR, AR PTS1 [148–150]
typical face, FTT,
hepatomegaly
Pyloric stenosis H + + + + Cystic kidney Sporadic Unk [151]
C. Limwongse
 6

Raas-Rothschild + + Klippel-Feil anomaly, Uncertain Unk [152]

sacral agenesis,
cryptorchidism
RAPADILINO + Radial/patella hypoplasia, AR RECQL4 [153]
diarrhea, short stature, long
nose
Renal/Mullerian H + + Absent uterus, broad AR Unk [154]
hypoplasia forehead, DD, large
fontanel
Renal dysplasia or 1 E + + + Ua + Abnormal uterus in some AD RET, [155]
adysplasia patients UnkPK3A
Renal-hepatic-pancreatic + Pancreatic cysts, AR EVC, EVC2 [84, 85]
dysplasia extrahepatic biliary atresia,
Caroli disease
Retinoic acid, maternal use + + U Ear anomalies, CHD, cleft Sporadic none [156, 157]
palate, neural tube defect
Roberts 1 H + + Limb reduction, oligo/ AR ESCO2 [158]
syndactyly, CHD,
dysmorphic face
Robson + MR, macrocephaly, XR COL4A [159]
deafness, proteinuria,
Alport-like
Rokitansky-Mayer-Kuster- 1 E + U + Absence of vagina, uterine Sporadic Unk [160]
Developmental Syndromes and Malformations of the Urinary Tract

Hauser anomalies, amenorrhea

Rubella, congenital 1 + + CHD, MR, deafness, Sporadic none [161]
cataract, growth retardation
Rubinstein-Taybi 1 E + + + Ua SS, MR, broad thumbs and Sporadic CREBBP [162, 163]
great toes, typical face
Russell-Silver + Ua SS, triangular face, Sporadic,AD Unk [164]
asymmetry, clinodactyly,
hypoglycemia
Santos 1 Hirschsprung disease, AR Unk [165]
hearing loss, postaxial
polydactyly
(continued)
145
 146

Table 1 (continued)
Urinary tract abnormalities

Other
associated

Renala genesis
Ectopia=horseshoe
Cystic=dysplasia
Duplication
Hypoplasia
Hydronephrosis=ureter
Diverticula
Atresia=stenosis
Reflux
Nephritis=sclerosis
Tumor=nephromegaly
Syndromes Other associated anomalies anomalies Gene(s) References
Say + SS, microcephaly, AD Unk [166]
micrognathia, large ear,
cleft palate
Schimke + SS, spondyloepiphyseal AR SMARCAL1 [167]
dysplasia,
immunodeficiency
Schinzel-Giedion E U + U CHD, distinctive face, AR Unk [168]
figure 8 head shape, eyelid
groove
Senior-Loken + + Nephronophthisis, AR NPHP1,4,5 [169]
tapetoretinal degeneration
Setleis + U Cutis aplasia with temporal AD,AR Unk [170]
scarring, abnormal
eyelashes
Short rib, Beemer Langer + + + U Hydrops, cleft lip, bowed AR Unk [171]
long bones, atretic ear canal
Short rib-polydactyly, type 1 + Ua Urethral fistula, CHD, AR Unk [171, 172]
1–3 cloacal/urogenital sinus
anomalies
Silverman (dyssegmental + U SS, flat face, cleft palate, Uncertain HSPG2 [173]
dwarfism) generalized skeletal
dysplasia
Simopoulos + Hydrocephalus, AR Unk [174]
polydactyly
Simpson-Golabi-Behmel + + + Overgrowth, polydactyly, XR GPC3 [175]
typical face, arrhythmia
C. Limwongse
 6

Sirenomelia sequence 1, E + + + + + + + See text for details Sporadic Unk

2
Smith-Lemli-Opitz 1 + + SS, ambiguous genitalia, AR DHCR7 [176, 177]
2–3 toe syndactyly, brain
anomalies
Sommer 1 Iris aplasia, corneal opacity, AD Unk [178]
glaucoma, prominent
forehead
Sorsby (coloboma- 1 Ocular coloboma, AD Unk [179]
brachydactyly) brachydactyly type B, bifid
thumbs
Sotos Ua + Overgrowth, MR, Sporadic NSD1 [180]
embryonal tumors,
advanced bone age
Supernumerary nipples- E + + + + Familial polythelia AD Unk [181]
renal anomalies
Thalidomide, maternal use 1, E, + + + + + Limb reduction, Sporadic none [182, 183]
2 H phocomelia, neural tube
defect
Thymic-renal-anal-lung + + U SS, absent thymus, AR Unk [184]
parathyroid agenesis,
urethral fistula
Tolmie + Lethal multiple pterygia, XR CHRN [185]
Developmental Syndromes and Malformations of the Urinary Tract

long bone abnormalities

Townes-Brock 1 + + Ua + Triphalangeal thumb, AD SALL1 [186]
imperforate anus, skin tag,
deafness
Trimethadione, maternal 1 U SS, CHD, omphalocele, Sporadic none [187]
use distinctive face
Tuberous sclerosis + + MR, seizures, cortical tuber, AD TS1, TS2 [188]
facial angiofibroma
Turner See Table 3 Chromosomal
Ulnar-mammary 1 Oligodactyly, ulnar ray AD TBX3 [189]
defect, nipple aplasia,
genital defects
(continued)
147
 148

Table 1 (continued)
Urinary tract abnormalities

Other
associated

Renala genesis
Ectopia=horseshoe
Cystic=dysplasia
Duplication
Hypoplasia
Hydronephrosis=ureter
Diverticula
Atresia=stenosis
Reflux
Nephritis=sclerosis
Tumor=nephromegaly
Syndromes Other associated anomalies anomalies Gene(s) References
Urogenital adysplasia 1, + Absent uterus, vaginal AD RET, [190]
2 atresia, hydrometrocolpos UnkPK3A
VATER (VACTERL) 1 E, + + + + + + + See text for details
association H
Velocardiofacial 1, E, + + + + + See Table 3
2 H
von Hippel-Lindau + + Cerebello-retinal AD VHL [191, 192]
angiomatosis,
pheochromocytoma
Weinberg-Zumwalt + + Multiple lung cysts, ascites, Uncertain Unk [193]
accessory spleen
Wenstrup 1 + + + U Female Uncertain Unk [194]
pseudohermaphrodite,
imperforate anus
Weyers H + + + U Oligodactyly, pterygia, AR EVC [195]
sternal defect, cleft palate
Williams E + + B, U See Table 3 Chromosomal
U
Wilms tumor-horseshoe H + Possible association Sporadic WT1 [196]
kidney
C. Limwongse
 6

Wilms tumor- + Ipsilateral vascular Sporadic WT1 [106]

hemihypertrophy malformation, cafe-au-lait
spots
Wilms tumor-radial aplasia + Hypoplastic fibula/tibia, Sporadic Unk [197]
abnormal thumbs
Winter (oto-renal-genital) 1, + Middle ear anomalies, AR Unk [198]
2 deafness, vaginal atresia
Wolfram (DIDMOAD) + + Diabetes mellitus/insipidus, AR, WFS1–2 [199]
optic atrophy, nerve mitochondrial
deafness
Zellweger 1 + + Hypotonia, seizures, AR PEX [148–150]
hepatosplenomegaly,
growth delay
U ureter, B bladder, Ua urethra, 1 unilateral renal agenesis, 2 bilateral renal agenesis, E ectopia, H horseshoe, ACC agenesis of corpus callosum, SS short stature, MR mental
retardation, DD developmental delay, CHD congenital heart disease, FTT failure to thrive, IUGR intrauterine growth retardation, Unk gene unknown
Developmental Syndromes and Malformations of the Urinary Tract
149
Table 2 Well-known syndromes associated with occasional urinary tract anomalies
150

Inheritance

Renal agenesis
Ectopia=horseshoe
Cystic=dysplasia
Duplication
Hypoplasia
Hydronephrosis=ureter
Diverticula
Atresia=stenosis
Reflux
Nephritis=sclerosis
Tumor=nephromegaly
Syndromes Other associated anomalies pattern Gene(s) References
Aase Ua Triphalangeal thumb, AD,AR RPS19, 24 [200]
hypoplastic anemia
Achondrogenesis + + Micromelic dwarfism, short AR COL2A1 [201]
trunk, fetal hydrops
Acrocallosal 1 + ACC, macrocephaly, AR GLI3 [202]
polymicrogyria, polydactyly,
CHD
Acro-facial dysostosis H + Abnormal thumb/toe, facial AR Unk [203]
bone defect, ear anomalies
Acromelic frontonasal E + Polydactyly, ACC, Sporadic Unk [204]
dysplasia encephalocele, Dandy-
Walker anomaly
Adrenogenital 1 + + Ambiguous genitalia, AR CYP11,21 [205]
vomiting, salt losing, UPJ
obstruction
Adrenal hypoplasia-MR + U + Aminoaciduria, MR, X-linked NROB1 [206]
muscular dystrophy, visual
abnormality
Antley-Bixler H + Craniosynostosis, radio- AR FGFR2 [207]
humeral synostosis, cardiac
defects
Apert (acrocephalo + + Acrocephaly, AD FGFR2 [208]
syndactyly) craniosynostosis, syndactyly
Bloom + Short stature, telangiectasias, AR BLM [209]
leukemia, lymphoma
Bowen-Conradi H + Micrognathia, AR Unk [210]
arthrogryposis, cloudy
cornea, brain anomaly
C. Limwongse
 6

Brachydactyly type E 1 + Vertebral anomalies, narrow Uncertain HOXD13 [211]

auditory canal
3C (Ritscher-Schinzel) + SS, Dandy-Walker anomaly, AR Unk [212]
typical face, CHD
C-trigonocephaly 1 Polysyndactyly, abnormal AR CD96 [213]
ear, hypospadias, dislocated
joints
Campomelic dysplasia 1 + + Tibial bowing, pretibial AD SOX9 [214]
dimples, ambiguous
genitalia
Carbohydrate deficient + FTT, abnormal fat pad, AR PMM2, [215]
glycoprotein hepatosplenomegaly, MPI
neurodegeneration
Carpenter + Aminoaciduria, AD RAB23 [216]
polysyndactyly,
craniosynostosis
CHILD 1,2 + Unilateral erythroderma, X-linked NSDHL [217]
ipsilateral limb defect
Chondrodysplasia + Flat face, microcephaly, AR Unk [218]
punctata, non-rhizomelic cataract, short femora/
humeri, stippled epiphyses
Coffin-Siris 1 + MR, sparse scalp hair, AR Unk [219]
hirsutism, coarse face, thick
Developmental Syndromes and Malformations of the Urinary Tract

lips
Cutis la + a type I B GI tract diverticula, AR FBLN5 [220]
emphysema, diaphragmatic
defect
Disorganization-like + Polydactyly, duplication of Sporadic Unk [221]
lower limbs, skin
appendages
Duane anomaly-radial 1 Limited ocular abduction, AD SALL4 [222]
defects radial defects,
blepharophimosis
Ehlers-Danlos + Joint hypermobility, skin AD, AR, XR COL5A, [223]
hyperextensibility, easy COL1A1
bruising
(continued)
151
 152

Table 2 (continued)

Inheritance

Renal agenesis
Ectopia=horseshoe
Cystic=dysplasia
Duplication
Hypoplasia
Hydronephrosis=ureter
Diverticula
Atresia=stenosis
Reflux
Nephritis=sclerosis
Tumor=nephromegaly
Syndromes Other associated anomalies pattern Gene(s) References
Epidermolysis bullosa + U Skin blistering, pyloric AR LAMA3, [224–245]
stenosis, dystrophic nails, LAMB3
sparse hair
Femoral hypoplasia- 1 Various leg deformities, Sporadic, AD Unk [226]
unusual facies abnormal genitalia, typical
face
Floating-Harbor E SS, typical face, DD, Uncertain Unk [227]
delayed bone age
Focal dermal hypoplasia H Atrophy/linear skin X-linked PORCN [228]
pigmentation, hand/vertebral
anomalies
Freeman-Sheldon + + Whistling face, ulnar AD TNNT3, [229]
deviation of hands, talipes TNNI2
equinovarus
Frontometaphyseal + + Prominent supraorbital X-linked FLNA [230]
dysplasia ridges, contractures,
deafness
Frontonasal dysplasia + E Hypertelorism, broad nasal Sporadic Unk [231]
tip, median cleft nose
Fryns + Digital hypoplasia, AR Unk [232]
diaphragmatic defect, cleft
palate
G (Opitz/BBB) + + + See Opitz (G/BBB)
syndrome
Glutaric aciduria, type II + Cerebral anomalies, AR ETFA, B, [233]
pancreatic dysplasia, biliary DH
dysgenesis
C. Limwongse
 6

Grieg cephalopoly + Macrocephaly, polydactyly, AD GLI3 [234]

syndactyly hypertelorism
Hajdu-Cheney + SS, Wormian bones, acro- AD Unk [235]
osteolysis, osteoporosis
Hydrolethalus + Ua Hydrocephalus, polydactyly, AR HYLS1 [236]
polyhydramnios, cleft lip
Jarcho-Levin + Spondylothoracic dysplasia, AR DLL3, [237]
fused ribs, hemivertebrae MESP2
Johanson-Blizzard + Pancreatic insufficiency, AR UnkBR1 [238]
spiky hair, small alae nasi
Killian-Pallister See Table 3 Chromosomal
Lacrimo-auriculo-dento- 1 Nasolacrimal duct stenosis, AD FGFR2, 3 [239]
digital (LADD) malformed ears/enamel/
digits
Larsen 1 + Multiple joint dislocations, AD, AR FLNB [240, 241]
flat face
Lenz microphthalmia 1 + + Ocular coloboma, ear/facial X-linked Unk [242]
anomalies, syndactyly/
camptodactyly
LEOPARD (multiple 1 Hypertelorism, deafness, AD PTPN11, [243]
lentigines) abnormal ECG, genital RAF1
anomalies
Developmental Syndromes and Malformations of the Urinary Tract

Marfan + + Tall thin habitus, aortic root AD FBN1 [244, 245]

dilatation, lens
sublu + ation,
arachnodactyly
Marshall-Smith + MR, FTT, accelerated bone Sporadic Unk [246]
maturation, broad phalanges
Miller-Dieker 1 + See Table 3 Microdeletion LIS1
(lissencephaly)
Moebius-peripheral + Peripheral neuropathy, Sporadic Unk [247]
neuropathy anosmia, hypogonadism
Mohr-Majewski See oro-facio-digital
syndrome type IV
Multiple pterygium + Multiple soft tissue AR CHRNG [248]
contractures, camptodactyly
(continued)
153
 154

Table 2 (continued)

Inheritance

Renal agenesis
Ectopia=horseshoe
Cystic=dysplasia
Duplication
Hypoplasia
Hydronephrosis=ureter
Diverticula
Atresia=stenosis
Reflux
Nephritis=sclerosis
Tumor=nephromegaly
Syndromes Other associated anomalies pattern Gene(s) References
Myotonic dystrophy + Myotonia, muscle weakness, AD DMPK [249, 250]
cataract, arrhythmia,
diabetes
Nijmegen breakage + MR, SS, microcephaly, AR NBS1 [251]
immunodeficiency
Opitz (G/BBB) Hypertelorism, hypospadias, AD, X-linked MID1 [252, 253]
cleft lip/palate, dysphagia
Oro-facio-digital, type I + + Midline cleft lip, multiple X-linked CXORF5 [254]
frenula, polydactyly, tongue
nodules
Oro-facio-digital, type IV 1 + Cleft palate, multiple AR Unk [254, 255]
frenula, polysyndactyly,
lobed tongue
Oro-facio-digital, type VI 1 + Midline cleft lip, multiple AR Unk [254]
frenula, polydactyly, tongue
nodules
Pallister-Killian See Table 3 Chromosomal
Peutz-Jeghers + Hamartomatous intestinal AD STK11 [256, 257]
polyposis, lip
hyperpigmentation
Poland anomaly 1 + + Hypoplastic pectoralis, Sporadic Unk [258, 259]
ipsilateral upper limb
reduction
Restrictive dermopathy U Aplasia cutis, rigid skin, AR LMNA, [260]
contractures, typical face ZMPSTE24
Robinow + + Mesomelic dwarfism, typical AD, AR ROR2 [261]
face, abnormal genitalia
C. Limwongse
 6

Rothmund-Thomson + Poikiloderma, alopecia, AR RECQL4 [262]

dysplastic nails,
photosensitivity
Serpentine fibula + Elongated curved fibulae, Uncertain Unk [263]
hirsutism, hypertelorism
Spondylocostal 1 + Sacral agenesis, anal atresia, Uncertain Unk [264]
dysostosis bifid thumb, skin tags
Spondyloepimetaphyseal + U Joint laxity, kyphoscoliosis, AR COL2A1 [265]
dysplasia talipes equinovarus, CHD
Spondylometaphyseal + SS, platyspondyly, co + a AD Unk [266]
dysplasia vara, vertebral/long bone
anomalies
Syndactyly, type V E + Bladder exstrophy, fusion of AD HOXD13 [267]
4th and 5th metacarpal bones
U ureter, B bladder, Ua urethra, 1 unilateral renal agenesis, 2 bilateral renal agenesis, E ectopia, H horseshoe, ACC agenesis of corpus callosum, SS short stature, MR mental
retardation, DD developmental delay, CHD congenital heart disease, FTT failure to thrive, IUGR intrauterine growth retardation, Unk gene unknown
Developmental Syndromes and Malformations of the Urinary Tract
155
Table 3 Chromosomal disorders and their consistent associated urinary tract anomalies
156

Reported

Renal agenesis
Ectopia=horseshoe
Cystic=dysplasia
Duplication
Hypoplasia
Hydronephrosis=ureter
Diverticula
Atresia=stenosis
Reflux
Nephritis=sclerosis
Tumor=nephromegaly
Chromosomal disorders Other associated anomalies familial cases References
3p deletion E, + MR, growth delay, ptosis, postaxial polydactyly, No [270]
H micrognathia
3q duplication H + + + MR, SS, seizures, hirsutism, typical face, cardiac No [271]
defects
Williams syndrome (7q deletion) E + + B, U SS, typical face, supravalvar aortic stenosis, Yes [268]
U hypercalcemia
Trisomy 9 mosaicism + + B MR, joint contractures, cardiac defects, brain No [269]
anomalies
10q duplication H + MR, ptosis, short palpebral fissures, Yes [270]
camptodactyly
Aniridia-Wilms tumor (WAGR) + Ambiguous genitalia, hypospadias, short stature AD [271]
(11p13 deletion)
Pallister-Killian syndrome + SS, MR, hypogonadism, seizures, diaphragmatic No [271]
(tetrasomy 12p) defect
Patau syndrome (trisomy 13) 1,2 H + + + Holoprosencephaly, midline anomalies, cleft No [269]
lip/palate
Miller-Dieker syndrome (17p13 1 + MR, lissencephaly, microgyria, agyria, typical No [272]
deletion) face, seizures
Edward syndrome (trisomy 18) + E, + + + IUGR, CHD, clenched hands, rocker bottom feet Yes [269]
H
18q deletion H SS, MR, microcephaly, narrow external ear Yes [273]
canals, long hands
Down syndrome (trisomy 21) 1 H + + MR, hypotonia, CHD, typical face, clinodactyly Yes [274]
Cat eye syndrome (tetrasomy 22p) 1 H + + U MR, CHD, colobomas, anal/digital anomalies Yes [275]
Velocardiofacial syndrome (22q11 1,2 E, + + + + + + Conotruncal CHD, thymic aplasia, typical face, Yes [276, 277]
deletion) H cleft palate
Turner syndrome (45,+ or 46,+,i 1 E, + + + + + SS, amenorrhea, webbed neck, cubitus valgus, No [278]
(+q)) H hypogonadism
Triploidy H + + Large molar placenta, IUGR, syndactyly of 3rd No [271]
and 4th digit, others
U ureter, B bladder, Ua urethra, 1 unilateral renal agenesis, 2 bilateral renal agenesis, E ectopia, H horseshoe, ACC agenesis of corpus callosum, SS short stature, MR mental retardation, DD developmental
delay, CHD congenital heart disease, FTT failure to thrive, IUGR intrauterine growth retardation
C. Limwongse
6 Developmental Syndromes and Malformations of the Urinary Tract
available information about potential urinary tract
anomalies for patients with a diagnosed genetic
syndrome and to suggest a differential diagnosis
when anomalies are identified. Readers interested
in additional details about a specific syndrome are
referred to standard reference textbooks and data-
bases about syndromes and malformations for
further reading (e.g., [5–7]). Online Mendelian
Inheritance in Man (OMIM; https://www.omim.
org) is currently considered the most updated
online information for cataloging genetic syn-
dromes and can lead readers to many informative
sites through its various links.
To understand the pathophysiologic basis of
structural abnormalities, it is important to be
familiar with the meaning of certain terms as
they are used in describing malformations and
syndromes:
Malformation refers to a single structural anomaly
that arises from an error in organogenesis.
Such an error may be due to the failure of
cells or tissues to form, to die (programmed
cell death), or to induce others. Examples
include renal agenesis, horseshoe kidney, and
bladder exstrophy.
Deformation refers to a single structural anomaly
that arises from mechanical forces, such as
intrauterine constraint. Examples include
many cases of metatarsus adductus, torticollis,
and congenital scoliosis. The underlying tissue
may be normal or abnormal, and sometimes a
malformation (e.g., renal agenesis) can predis-
pose patients to a deformation (e.g., Potter’s
sequence from oligohydramnios).
Disruption refers to a single structural anomaly
that results from a destructive event after nor-
mal morphogenesis. Such events can be caused
by lack of vascular supply, an infectious pro-
cess, or mechanical factors. Examples include
limb amputation from amniotic bands and
abdominal wall defect from vascular insuffi-
ciency related to maternal cocaine use.
Dysplasia refers to a single structural anomaly
occurring in one or more organs or tissues
that result from abnormal organization of
structural components. The term “dysplasia”
frequently implies a process that is dynamic
157
and phenotypically evolves over time, thus
making this designation distinct from
“malformation.”
Sequence refers to a cascade of abnormalities that
result from a single initiating anomaly.
Sequences can be malformational, deforma-
tional, or disruptive, and they sometimes rep-
resent more than one of these categories.
Obstruction of urine flow at the level of the
ureter during early gestation, for example, can
cause malformation of the kidneys, intestines,
and abdominal wall – a malformation
sequence. At the same time, decreased urine
flow will produce oligohydramnios, fetal com-
pression, and multiple deformities of the face,
limbs, and chest wall – a deformation
sequence.
Syndrome refers to a consistently observed pattern
of anomalies found in an individual, whether
malformation, deformation, or disruption.
Anomalies comprising a syndrome are thought
to have a single cause, although in many cases,
their causes are still unknown. Examples
include Turner syndrome and fetal alcohol
syndrome.
Association refers to a constellation of anomalies
that occur together more often than expected
by chance alone but cannot be explained by a
single cause or sequence of events, and so do
not represent a syndrome or sequence. VATER
association, which is discussed later in this
chapter, is a common example.
Prevalence of Urinary Tract Anomalies
The true incidence of urinary tract anomalies is
difficult to ascertain because many anomalies are
asymptomatic and therefore undetected. Many
reported statistics have apparent bias of ascertain-
ment because they are derived from symptomatic
individuals. Furthermore, inconsistent terminol-
ogy and clustering of data have decreased the
power of much of the epidemiologic data. Long-
term analysis of data collected through major
national birth defect registries showed increasing
prevalence of reported statistics for many congen-
ital birth defects, not only from an actual
158
increment but also from increased use of prenatal
screening and increased tendency to report several
isolated and associated anomalies [1–4]. For this
reason, the practical use of the derived prevalence
seems not to be meaningful. However, there cur-
rently are quite a number of reliable estimates for
prevalence of specific isolated anomalies and of
those associated with a specific syndrome. A large
number of European birth cohorts during
1996–1998 (EUROSCAN) was prenatally studied
and recently reported [4]. Table 4 shows a com-
parison of prevalence figures from various
studies.
Approach to the Child with a Urinary
Tract Anomaly
The approach to the child with a urinary tract
anomaly is similar to that for other birth defects.
The initial step is to make a specific diagnosis if
possible, based on a complete history, physical
examination, and laboratory investigation. A thor-
ough family history for both urinary tract anoma-
lies and for any other type of congenital or
developmental anomalies that may have occurred
in the family should be obtained for at least two
generations, if possible. A careful environmental
exposure history should be part of this history.
Many genetic disorders have variable expression
even within the same family. A careful physical
examination looking specifically for major and
minor anomalies should be performed. Some-
times, a pattern of multiple anomalies can be
recognized immediately as a well-described syn-
drome. Patterns of anomalies that cannot be rec-
ognized may require a literature or database
search or referral to an expert in syndrome recog-
nition, such as a clinical geneticist. The search for
a specific diagnosis is optimally accomplished by
identifying the least common and most distinctive
anomalies, for which the list of differential diag-
noses is limited. Many excellent textbooks,
atlases, and databases are available [5–7]. To aid
in this effort, refer to Tables 1, 2, and 3 in addition
to the tables listing the differential diagnosis that
accompanies the description of each of the major
urinary tract anomalies as noted (Tables 5, 6, 7, 8,
C. Limwongse
9, 10, 11, 12, 13, 14, and 15). For example, it is
preferable to search for syndromes with urethral
agenesis (22 syndromes) rather than renal dyspla-
sia (more than 80 syndromes) when the two
anomalies coexist. A search based on the more
common anomalies can be performed if the first
search does not reveal a match. Even after careful
evaluation, a substantial number of children with
multiple congenital anomalies remain
undiagnosed.
When a suspected syndrome is known to be
caused by a gene mutation, confirmatory molecu-
lar genetic testing can be performed. DNA-based
testing is currently available on either a clinical
service or research basis. Knowledge regarding a
pathogenic mutation specific for each proband
may potentially be useful for genetic counseling
and future reproductive options in order to avoid
intrafamilial recurrence. Tables 1 and 2 list cur-
rently known causative genetic mutations for a
number of disorders.
A chromosome analysis is indicated in any
child who has at least two major congenital anom-
alies or one isolated anomaly that is a pertinent
feature of a well-characterized chromosomal
abnormality, such as aniridia (microdeletion
11p). Growth or developmental delay with dys-
morphic features or lack of familial resemblance
should also prompt a chromosome analysis. Chro-
mosome abnormalities are found in approxi-
mately 10–12 % of all renal anomalies [3, 8].
Table 3 lists common, distinct chromosomal
disorders with their associated urinary tract
anomalies.
If a cluster of anomalies is present in a patient
but a definitive diagnosis cannot be given, that
patient is designated an unknown syndrome and
should be followed periodically. At each follow-
up visit, a further literature review may reveal
similar cases that will eventually lead to delinea-
tion of a new syndrome. Presently, state-of-the-art
sequencing technology such as whole exome,
whole genome, or next-generation DNA sequenc-
ing (NGS) can be attempted to delineate patho-
genic culprit mutation in a previously
undiscovered causative gene (see this text,
▶ Chap. 17, “Genomic Methods in the Diagnosis
and Treatment of Pediatric Kidney Disease”).
6 Developmental Syndromes and Malformations of the Urinary Tract 159

Table 4 Prevalence of urinary tract anomalies detected by various surveys

Rates per 1,000 births
ICHBDS EUROSCAN CBDMP WSBDR MACDP
Anomalies 2011a 1996–1998b 1983–1994c 1987–1989d 1983–1988e
Total renal malformation ~1.6 ~2.31 ~2.33 ~1.5
Unilateral renal agenesis 0.08
Bilateral renal agenesis/ 0.13
dysplasia
Unilateral multicystic 0.14
dysplasia
All renal agenesis /dysplasia 0.2–0.6 0.36 0.48 0.58 0.47
Horseshoe/ectopic kidney 0.04 0.04 0.16 No data
Cystic kidney 0.2–0.5 0.04 0.03 0.05 No data
Obstruction of kidney/ureter No data 0.43 1.27 1.27 0.8
Double ureter No data No data 0.004 0.05 No data
Exstrophy of bladder 0.02 0.03 0.03 0.02 0.03
Cloacal exstrophy 0.005 No data No data No data No data
Obstruction of bladder/urethra No data 0.04 0.16 0.2 0.2
VATER, CHARGE, and No data No data 0.21 No data No data
MURCS associations
Sirenomelia 0.0098 No data 0.09 No data No data
a
International Clearinghouse for Birth Defects Surveillance and Research (total 26,355,094 births) [2]
b
European Renal Anomaly Detection Program (total 709,030 births) [4]
c
California Birth Defect Monitoring Program [1]
d
Washington State Birth Defect Registry
e
Metropolitan Atlanta Congenital Defects Program

For a child with no known urinary tract anom-
aly, findings that should prompt an evaluation of
the urinary tract are oligohydramnios, undefined
abdominal mass, abnormal genitalia, aniridia,
hypertension, preauricular pits or tags, branchial
cleft cyst or sinus, imperforate anus, or symptoms
indicative of renal dysfunction, urinary tract infec-
tion, or obstructive uropathy [3]. For patients with
known syndromes, the associated urinary tract
anomalies are listed in Tables 1 and 2.
The best initial evaluation to screen for urinary
tract anomalies is arenal and bladder ultrasound
examination. This study is noninvasive and gives
good anatomic information about the urinary
tract. It is also the only method routinely used
for the prenatal diagnosis of urinary tract anoma-
lies. Specific imaging studies such as an intrave-
nous pyelogram, voiding cystourethrogram,
radionuclide renal and urinary system scan, and
specialized genetic testing may then be
performed, depending on the working diagnosis
(see this text, ▶ Chap. 17, “Genomic Methods in
the Diagnosis and Treatment of Pediatric Kidney
Disease”). The type of anomaly generally guides
treatment. Corrective or reparative treatments are
available for many anomalies (stenosis or atresia,
bladder exstrophy, duplication, diverticula, and
tumors). Symptomatic treatment for complica-
tions is often necessary.
Families who have a child with a urinary tract
anomaly should be informed of the diagnosis. A
search for a related anomaly in first-degree rela-
tives is indicated only when the proband has renal
agenesis by ultrasound examination [9]. Other-
wise, the decision to investigate family members
should be based on a thorough family history
and/or physical examination and whether the
child’s disorder is a well-described, inherited syn-
drome. Genetic counseling should be provided to
the family and should include a discussion of the
manifestations of the disorder, the natural history,
complications, available treatments, cause, and
160 C. Limwongse

Table 5 Syndromes associated with unilateral renal Table 6 Syndromes associated with unilateral or bilateral
agenesis renal agenesis
Acrocallosal syndrome Acrorenal, Johnson-Munson type
Acrorenal syndrome, Dieker type Alkylating agent, maternal use
Acrorenal-mandibular syndrome Caudal duplication syndrome
Acrorenal-ocular syndrome Caudal regression syndrome
Adrenogenital syndrome Cocaine, maternal use
Aglossia-adactylia syndrome CHARGE syndrome
Alagille syndrome (arteriohepatic dysplasia) Diabetic mother, infant of
Branchio-oto-renal syndrome DiGeorge syndrome
C-trigonocephaly syndrome
Fraser (cryptophthalmos) syndrome
Campomelic dysplasia
Holzgreve syndrome
Cat eye syndrome
Pallister-Hall syndrome
Chondroectodermal dysplasia
Potter (oligohydramnios) sequence
Coffin-Siris syndrome
Sirenomelia sequence
Cornelia de Lange syndrome
Thalidomide embryopathy
Ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome
Femoral hypoplasia-unusual facies syndrome Urogenital adysplasia
Fetal alcohol syndrome Velocardiofacial syndrome
Goldenhar syndrome Winter syndrome
Ivemark syndrome
Kallmann syndrome
Klippel-Feil anomaly
Lacrimo-auriculo-dento-digital syndrome recurrence risk when these are known. Reproduc-
Larsen syndrome tive options should be discussed in a nondirective
Lenz microphthalmia syndrome fashion. For an isolated anomaly without a family
LEOPARD syndrome (multiple lentigines)
history of similar or related anomalies, an empiric
Limb-body wall complex
risk can be provided. Accurate risk figures can be
Miller-Dieker syndrome
MURCS association
determined for Mendelian disorders, and esti-
Neu-Laxova syndrome mated risks are available for associations.
Oro-facio-digital syndrome, types IV and VI All children with congenital anomalies need
Pfeiffer syndrome long-term, periodic follow-up to detect new
Poland anomaly abnormalities or complications of their birth
Renal dysplasia defects. This is especially the case for children
Roberts syndrome with undiagnosed multiple congenital anomalies,
Rokitansky-Mayer-Kuster-Hauser syndrome for whom follow-up examination may lead to a
Rubella syndrome, congenital
specific diagnosis. Additional relevant family
Rubinstein-Taybi syndrome
information should be specifically sought for any
Russell-Silver syndrome
Short rib-polydactyly syndrome, types 1–3
newly affected member. Finally, for patients with
Smith-Lemli-Opitz syndrome a urinary tract anomaly who reach reproductive
Sorsby coloboma-brachydactyly syndrome age, the recurrence risk for their offspring and
Spondylocostal dysostosis reproductive options should be discussed.
Townes-Brocks syndrome The remainder of this chapter contains descrip-
Trisomy 22 tions of the major types of urinary tract anomalies,
Turner syndrome including the etiology, pathogenesis, and associ-
Ulnar-mammary syndrome ated disorders. First, a review of the embryology
VATER (VACTERL) association
of the normal urinary tract will be useful in under-
Zellweger syndrome
standing structural urinary tract abnormalities.
6 Developmental Syndromes and Malformations of the Urinary Tract 161

Table 7 Syndromes associated with ectopic kidney Table 8 Syndromes associated with horseshoe kidney
Acromelic frontonasal dysplasia Acro-facial dysostosis syndrome
Acrorenal syndrome, Dieker type Agnathia-holoprosencephaly syndrome
Acrorenal syndrome, Siegler type Antley-Bixler syndrome
Acrorenal-ocular syndrome Bowen-Conradi syndrome
Baller-Gerold syndrome Caudal regression syndrome
Beckwith-Wiedemann syndrome Diabetic mother, infant of
Branchio-oto-renal syndrome Fanconi anemia syndrome
Caudal regression syndrome Fetal alcohol syndrome
CHARGE syndrome Focal dermal hypoplasia
Crossed ectopia-pelvic lipomatosis syndrome Juberg-Hayward syndrome
DiGeorge syndrome Kabuki syndrome
Drash (Denys-Drash) syndrome Pallister-Hall syndrome
Fanconi anemia syndrome Pyloric stenosis
Fetal alcohol syndrome Roberts syndrome
Floating-Harbor syndrome Thalidomide embryopathy
Frontonasal dysplasia Trisomy 13, 18, 21, and 22
Goldenhar syndrome Turner syndrome
Kaufman-McKusick syndrome VATER (VACTERL) association
Klippel-Feil anomaly Weyers syndrome
Limb-body wall complex Wilms tumor
MURCS association
Pallister-Hall syndrome
Penoscrotal transposition mesodermal ridge, the urorectal septum that
Renal adysplasia divides the cloaca into the anterior portion, the
Rokitansky-Mayer-Kuster-Hauser syndrome primitive urogenital sinus, and the posterior por-
Rubinstein-Taybi syndrome tion, the cloacal sinus or anorectal canal. The
Schinzel-Giedion syndrome mesonephric ducts open into the urogenital sinus
Sirenomelia sequence and later become the ureters. The urorectal septum
Turner syndrome develops caudally and fuses with the cloacal
VATER (VACTERL) association
membrane, dividing it into the urogenital mem-
Velocardiofacial syndrome
brane (anterior) and the anorectal membrane (pos-
Williams syndrome
terior) by the end of the seventh week. The
primitive perineal body forms at the site of fusion.
The primitive urogenital sinus develops primar-
Overview of Normal Embryogenesis ily into the urinary bladder. The superior portion,
of the Urinary System originally continuous with the allantois, later
becomes a solid fibrous cord, the urachus, or median
Renal organogenesis is reviewed in ▶ Chap. 1, umbilical ligament, which connects the bladder to
“Embryonic Development of the Kidney”. the umbilicus. The inferior portion of the urogenital
Embryogenesis of the lower urinary tract includes sinus in the male divides into a pelvic portion,
development of the mesonephric duct and urogen- containing the prostatic and membranous urethra,
ital sinus. The mesonephric duct from which the and the long phallic portion, containing the penile
ureteric bud arose inserts into the lower allantois, urethra. The inferior portion in the female forms a
just above the terminal part of the hindgut, the small portion of the urethra and the vestibule. At the
cloaca. During the fourth to seventh weeks, meso- same time, the distal portion of the mesonephric
derm proliferates and forms the transverse ducts is incorporated into the endodermal
162 C. Limwongse

Table 9 Syndromes associated with renal dysplasia/cystic Table 9 (continued)

kidney Thalidomide embryopathy
Acrorenal-mandibular syndrome Trisomy 8, 9, 13, 18, 21, and 22
Alagille syndrome (arteriohepatic dysplasia) Tuberous sclerosis
Baller-Gerold syndrome VATER (VACTERL) association
Bardet-Biedl syndrome Von Hippel-Lindau disease
Beckwith-Wiedemann syndrome Zellweger and pseudo-Zellweger syndromes
Branchio-oto-renal syndrome
Campomelic dysplasia
Carbohydrate deficient glycoprotein syndrome Table 10 Syndromes associated with hydronephrosis or
CHARGE syndrome hydroureter
Chondrodysplasia punctata, non-rhizomelic Acrocephalo-polysyndactylous dysplasia
Cloacal exstrophy Acrorenal syndrome, Dieker and Johnson-Munson types
Cornelia de Lange syndrome Bardet-Biedl syndrome
Diabetic mother, infant of Branchio-oto-renal syndrome
Ectrodactyly-ectodermal dysplasia-clefting (EEC) Campomelic dysplasia
syndrome
Caudal duplication and regression syndromes
Fanconi anemia syndrome
CHARGE syndrome
Fetal alcohol syndrome
Cloacal exstrophy
Fraser (cryptophthalmos) syndrome
Coffin-Siris syndrome
Fryns syndrome
Crossed ectopia-pelvic lipomatosis syndrome
Glutaric aciduria, type II
Cornelia de Lange syndrome
Goldenhar syndrome
Diabetic mother, infant of
Hajdu-Cheney syndrome
Ectrodactyly-ectodermal dysplasia-clefting (EEC)
Ivemark syndrome syndrome
Jeune syndrome Fanconi anemia syndrome
Joubert syndrome Fetal alcohol syndrome
Kaufman-McKusick syndrome Goldenhar syndrome
Lenz microphthalmia syndrome Hydrolethalus syndrome
Leprechaunism (Donohue) syndrome Kabuki syndrome
Limb-body wall complex Kaufman-McKusick syndrome
Marden-Walker syndrome Megacystis-microcolon syndrome
Marfan syndrome Noonan syndrome
Marshall-Smith syndrome Ochoa syndrome
Meckel-Gruber syndrome Omphalocele-Sxstrophy of bladder-Imperforate anus-
MURCS association Spinal dysraphism (OEIS) complex
Noonan syndrome Pallister-Hall syndrome
Omphalocele-Exstrophy of bladder-Imperforate anus- Polydactyly-obstructive uropathy syndrome
Spinal dysraphism (OEIS) complex Pyloric stenosis
Oral-facial-digital syndrome, types I and VI Roberts syndrome
Pallister-Hall syndrome Sirenomelia sequence
Pallister-Killian syndrome Schinzel-Giedion syndrome
Potter (oligohydramnios) sequence VATER (VACTERL) association
Prune belly syndrome
Renal adysplasia
Roberts syndrome
vesicoureteral primordium, forming the trigone of
Rokitansky-Mayer-Kuster-Hauser syndrome
Rubella syndrome, congenital
the bladder. A part of the distal end of both meso-
Short rib-polydactyly syndrome nephric ducts just proximal to the trigone develops
Smith-Lemli-Opitz syndrome into the seminal vesicles and ductus deferens in the
(continued) male. Finally, at the end of the 12th week, the
6 Developmental Syndromes and Malformations of the Urinary Tract 163

Table 11 Syndromes associated with duplication of ure- Table 13 Syndromes associated with urethral agenesis
ters or collecting systems
Aase syndrome
Achondrogenesis Acrorenal syndrome, Dieker and Johnson-Munson types
Acromelic frontonasal dysplasia Adrenogenital syndrome
Adrenogenital syndrome Caudal regression syndrome
Antley-Bixler syndrome Cocaine, maternal use
Bardet-Biedl syndrome Diabetic mother, infant of
Bowen-Conradi syndrome Hydrolethalus syndrome
Branchio-oto-ureteral syndrome Kaufman-McKusick syndrome
Braun-Bayer syndrome Limb-body wall complex
Caudal duplication syndrome Meckel-Gruber syndrome
Diabetic mother, infant of Occipital horn syndrome
Drash (Denys-Drash) syndrome Ochoa syndrome
Ectrodactyly-ectodermal dysplasia-clefting (EEC) Omphalocele-Exstrophy of bladder-Imperforate anus-
syndrome Spinal dysraphism (OEIS) complex
Fanconi anemia syndrome Potter (oligohydramnios) Sequence
Fetal alcohol syndrome Prune belly syndrome
Frontometaphyseal dysplasia Renal adysplasia
G (Opitz-Frias) syndrome Russell-Silver syndrome
Goldenhar syndrome Short rib-polydactyly syndrome, types 1–3
Kabuki syndrome Sirenomelia sequence
Kaufman-McKusick syndrome Sotos syndrome
Mammo-renal syndrome Townes-Brocks syndrome
Noonan syndrome Trisomy 21
Ochoa syndrome
Perlman syndrome
Poland anomaly Table 14 Syndromes associated with urethral
Prune belly syndrome duplication
Robinow syndrome Amniotic band disruption sequence
Rubinstein-Taybi syndrome Limb-body wall complex
Trisomy 8, 9, 13, 18, and 21 Omphalocele-Exstrophy of bladder-Imperforate anus-
Turner syndrome Spinal dysraphism (OEIS) complex
Weyers syndrome Prune belly syndrome

epithelium of the superior portion of the prostatic

urethra proliferates to form buds that penetrate the
surrounding mesenchyme. In the male, these buds
form the prostate gland; in the female, they form the
Table 12 Syndromes associated with bladder exstrophy urethral and paraurethral glands.
Axial mesodermal dysplasia
Caudal duplication syndrome
Caudal regression syndrome Anomalies Involving the Urinary Tract
Cloacal exstrophy
Frontonasal dysplasia Kidney Defects
Omphalocele-Exstrophy of bladder-Imperforate anus-
Spinal dysraphism (OEIS) complex
Renal Agenesis
Trisomy 18
By definition, renal agenesis refers to complete
Sirenomelia sequence
Syndactyly, type V
absence of one of both kidneys without identifi-
able rudimentary tissue. Renal agenesis is usually
164
Table 15 Syndromes associated with posterior urethral
valves
Acrorenal syndrome, Johnson-Munson type
Caudal regression syndrome
Diabetic mother, infant of
Kaufman-McKusick syndrome
Limb-body wall complex
Neurofibromatosis, type I
Ochoa syndrome
Omphalocele-Exstrophy of bladder-Imperforate anus-
Spinal dysraphism (OEIS) complex
Polydactyly-obstructive uropathy syndrome
Potter (oligohydramnios) sequence
Prune belly syndrome
Renal adysplasia
Rubinstein-Taybi syndrome
Sirenomelia sequence
Townes-Brocks syndrome
VATER (VACTERL) association
associated with agenesis of the ipsilateral ureter.
The pathogenesis of renal agenesis is failure of
formation of the metanephros. Causal heterogene-
ity has been shown, by both animal studies and
human observations [10–12], including failure of
ureteric bud formation, failure of the bud to reach
the metanephric blastema, or failure of the bud
and the metanephric blastema to induce interac-
tion. In addition, interruption in vascular supply
and regression of a multicystic kidney can lead to
renal agenesis in the fetal period [11].
Unilateral renal agenesis is usually asymptom-
atic and found incidentally, whereas bilateral renal
agenesis results in severe oligohydramnios and
fetal or perinatal loss. Renal agenesis can occur
on either side equally. Birth prevalence in the
United States for renal agenesis/hypoplasia ranges
between 0.30 and 9.61 per 10,000 live births
[3]. Several studies have demonstrated that unilat-
eral renal agenesis is associated with an increased
frequency of anomalies in the remaining kidney
[9, 13]. Moreover, renal agenesis is often detected
in conjunction with anomalies of other organ sys-
tems. These anomalies can occur both in contigu-
ous structures (e.g., vertebrae, genital organs,
intestines, and anus) and also in noncontiguous
structures (e.g., limbs, heart, trachea, ear, and
central nervous system). The diagnosis of renal
C. Limwongse
agenesis is made by abdominal ultrasound. Care
must be taken to exclude the possibility of ectopic
kidney. Intravenous pyelography, computerized
tomography, and radionuclide studies can be help-
ful in equivocal cases.
The recurrence risk for renal agenesis can be
provided if the pattern of inheritance is known or
if the proband has a recognizable syndrome. For
nonsyndromic renal agenesis, an empiric risk of
3 % can be used for families in which renal anom-
alies in first-degree relatives (siblings, parents)
have been excluded [3]. First-degree relatives of
patients with nonsyndromic renal agenesis have
an increased prevalence of related urogenital
anomalies. It has been reported that 9 % of first-
degree relatives of infants with agenesis or dys-
genesis of both kidneys had a related urogenital
anomaly, and 4.4 % had an asymptomatic renal
malformation [13]. In a retrospective review,
empiric risks were 7 % in offspring, 2.5 % in
siblings, and 4.5 % in parents [14]. Moreover,
offspring of an individual with unilateral agenesis
appears to be at an increased risk for bilateral renal
agenesis. Therefore renal ultrasound is
recommended for the first-degree relatives of the
proband unless renal agenesis in the proband is
clearly sporadic or a specific cause without an
increased recurrence risk is identified.
Tables 5 and 6 list the syndromes commonly
associated with unilateral and bilateral renal agen-
esis, respectively. See also Tables 1, 2, and 3 for
more information about these disorders and other
less known conditions with renal agenesis.
Ectopic Kidney
The ectopic kidney derives from an abnormality
of normal ascent. Most are pelvic kidneys that fail
to ascend out of the pelvic cavity. Rare case
reports of thoracic kidney exist [15]. Ectopic kid-
neys can be unilateral or bilateral. In bilateral
pelvic kidneys, the kidneys often fuse into a mid-
line mass of renal tissue, with two pelves and a
variable number of ureters, which is referred to as
a pancake or discoid kidney. Fused pelvic kidney
may in fact be due to fusion of ureteric buds or
metanephric blastema. Crossed renal ectopia
refers to an ectopic kidney whose ureter crosses
the midline. It often fuses with the normal kidney.
6 Developmental Syndromes and Malformations of the Urinary Tract
The embryogenesis of crossed renal ectopia is not
well understood, but presumably involves abnor-
mal migration of the ectopic kidney to the contra-
lateral side. An ectopic kidney is usually
hypoplastic and rotated and has numerous anom-
alous small blood vessels and associated ureteric
anomalies. Ectopic kidneys may be asymptomatic
and incidentally found, but complications from
ureteral obstruction, infection, and calculi can
occur. Ectopic kidney without hypoplasia or
hydronephrosis does not appear to be associated
with an increased frequency of associated anom-
alies. In such cases, further urologic investigation
is not indicated [16]. Table 7 provides a list of
syndromes that include ectopic kidney. These are
described in Tables 1, 2, and 3.
Horseshoe Kidney
Horseshoe kidney refers to a condition in which
both kidneys are fused at the lower poles with a
renal parenchymal or, less commonly, fibrous
isthmus. The embryogenesis of horseshoe kidney
with parenchymal isthmus is thought to be migra-
tion of nephrogenic cells across the primitive
streak before the fifth gestational week. Horse-
shoe kidney with fibrous isthmus is believed to
originate from mechanical fusion of the two
developing kidneys at or after the fifth week
before renal ascent [17]. The concept of a narrow
vascular fork leading to approximation and fusion
of the two kidneys is no longer considered valid.
Most horseshoe kidneys are located in the pelvis
or at the lower lumbar vertebral level because
ascent is further prevented when the fused kidney
reaches the junction of the aorta and inferior mes-
enteric artery.
Complications of horseshoe kidneys include
obstructive uropathy related to ureteropelvic junc-
tion obstruction, calculi, and urinary tract infec-
tion. Similar to other urinary tract anomalies,
horseshoe kidneys are often associated with
other genitourinary anomalies. In addition, there
is an increased risk of renal tumors developing in
horseshoe kidneys compared with normal kidneys
[18]. Renal cell carcinoma is the most common,
but Wilms tumor, adenocarcinoma, transitional
cell carcinoma, malignant teratoma, oncocytoma,
angiomyolipoma, and carcinoid have all been
165
reported. Horseshoe kidneys also carry an
increased risk for renal pelvis carcinoma and
renal squamous cell carcinoma [19].
Table 8 lists syndromes associated with horse-
shoe kidney. See Tables 1 and 2 for more details of
these disorders.
Dysplasia and Polycystic Kidney
Renal dysplasia is the most common congenital
urinary tract anomaly and the most common cause
of an abdominal mass in children [3]. (see also in
this text, ▶ Chap. 5, “Renal Dysplasia/Hypopla-
sia”). Unilateral dysplasia is reported to occur in
1:1,000, whereas the prevalence of bilateral dis-
ease is estimated to be 1:5,000 [20]. It may be
unilateral or bilateral and diffuse, segmental, or
focal. Symptoms are variable and range from a
symptomatic unilateral or focal dysplasia to the
progressive renal dysfunction of diffuse or bilat-
eral dysplasia. Dysplasia refers to abnormal dif-
ferentiation or organization of cells in the tissue.
Renal dysplasia is characterized histologically by
the presence of primitive ducts and nests of meta-
plastic cartilage [20, 21]. Although cysts are not
always present in a dysplastic kidney, the dysplas-
tic process often results in the formation of cysts
that are variable in size and number. Several
hypotheses have been proposed for the embryo-
genesis of the dysplastic kidney. The most likely
pathogenesis is an error of the inductive interac-
tion between the ureteric bud and the metanephric
blastema (see ▶ Chap. 1, “Embryonic Develop-
ment of the Kidney”, in this text). The molecular
pathogenesis of cystic kidney, especially polycys-
tic kidney, has been one of the most extensively
studied aspects of nephrology, and recent studies
have discovered distinct genes and pathways crit-
ical for renal cyst formation as reviewed in
▶ Chap. 36, “Childhood Polycystic Kidney Dis-
ease”. Dysplastic kidneys are usually identified
as enlarged echogenic kidneys on prenatal ultra-
sonography. If there is associated functional renal
impairment, alteration in amniotic fluid volume
may be detected and signifies a poor prognosis
[22]. Unilateral dysplasia carries an overall better
postnatal prognosis than that of bilateral disease.
However, up to 30–50 % of those with unilateral
dysplasia have associated contralateral urinary
166
anomalies [22]. Multicystic renal dysplasia is the
most common cause of renal dysplasia and is
usually unilateral. Polycystic kidney diseases,
both autosomal dominant (ADPKD) and autoso-
mal recessive (ARPKD) forms, are in general far
more common than other syndromic causes of
renal dysplasia. See this text, ▶ Chap. 36, “Child-
hood Polycystic Kidney Disease”. Table 9 sum-
marizes well-known syndromes with renal
dysplasia/cystic kidney. See also Tables 1, 2,
and 3.
Obstruction and Hydronephrosis
Urinary obstruction is a complication of a pri-
mary anomaly, which can be stenosis or atresia of
the ureteropelvic junction, ureter, or urethra; a
poorly functional bladder causing reflux; a mal-
formed dilated ureteral end (ureterocele); or
extrinsic compression by other structures, such
as anomalous blood vessels or tumors.
Hydronephrosis and pyelectasis (dilated renal
pelvis) are the most common urinary tract abnor-
malities detected by prenatal ultrasound exami-
nation. Early diagnosis of collecting system
dilatation can be achieved by ultrasound exami-
nation in the second trimester [23], although
spontaneous remission by birth is not uncom-
mon. Persistent dilatation generally indicates an
underlying anomaly. Isolated obstructive
uropathies diagnosed prenatally may not require
antenatal or immediate postnatal surgical inter-
vention. Postnatally diagnosed obstructive
uropathies are almost always symptomatic and
require thorough investigation to delineate the
anatomy of the urinary tract and to exclude asso-
ciated anomalies.
Table 10 provides a list of syndromes com-
monly associated with obstruction and
hydronephrosis. See also Tables 1, 2, and 3.
Ureter Defects
Duplication
Double ureters or collecting systems are caused
by duplication of the ureteric bud. Early duplica-
tion results in duplicated kidney, which is usually
smaller and fused with the ipsilateral kidney and
C. Limwongse
has ureters that enter into the bladder separately.
Duplication that occurs later results in double
ureters that may have separate openings into the
bladder or may join each other before the opening.
On rare occasion, one of the ureters may have an
ectopic opening into the vagina, vestibule, or ure-
thra. In most double ureters, the two ureters cross
each other, and that from the higher pelvis enters
the bladder more caudally. Duplication anomalies
are common but usually asymptomatic; therefore
they often remain undetected. One autopsy study
reported the prevalence of duplication anomalies
to be as high as 1 in 25, with females about four
times more likely to be affected than males
[24]. Unilateral duplication is 5–6 times more
common than bilateral duplication [3]. Double
ureters are commonly associated with
vesicoureteral reflux due to their ectopic opening
into the urinary bladder and/or the ureterocele
[23]. In addition, ureteric obstruction can occur
at the level of vesicoureteric junction or that of
ureteropelvic junction.
Table 11 summarizes syndromes associated
with duplication, and Tables 1, 2, and 3 provide
clinical information about these disorders.
Hydroureter
Hydroureter, or megaloureter, is caused by distal
obstruction and is usually found with
hydronephrosis, except in ureteropelvic junction
obstruction. Hydroureter has the same etiology as
hydronephrosis (see section “Obstruction and
Hydronephrosis”).
Bladder Defects
Anomalies of the bladder are rare. These include
agenesis, hypoplasia, diverticula, and dilatation
or megacystis with or without distal obstruction.
Agenesis of the bladder is usually associated with
severe developmental anomalies of the urinary
tract, such as those seen in sirenomelia and cau-
dal regression syndrome. Hypoplastic bladder
can be found in conditions associated with bilat-
eral renal agenesis because no urine is produced.
Bladder diverticula have heterogeneous causes.
They result from an intrinsic defect in the bladder
6 Developmental Syndromes and Malformations of the Urinary Tract
wall, as seen in cutis laxa (or Ehlers-Danlos),
Ochoa, occipital horn, and Williams syndromes.
They can also be caused by increased
intravesicular pressure from distal obstruction
or by a persistent urachus. See Tables 1, 2, and 3
for information about specific syndromes associ-
ated with bladder diverticula.
Bladder Exstrophy
Bladder exstrophy refers to a urinary bladder that
is open anteriorly because of the lack of an
abdominal wall closure. It is usually associated
with anomalies of the contiguous structures
including epispadias and separation of the pubic
rami. This anomaly is thought to result from an
overdeveloped cloacal membrane that interferes
with inferolateral abdominal mesenchymal clo-
sure. Therefore, when the cloacal membrane rup-
tures, the inferior abdominal wall has not
completely closed and the bladder cavity is
exposed. It has been suggested that bladder
exstrophy belongs to the spectrum of
omphalocele-cloacal exstrophy-imperforate
anus-spinal dysraphism (OEIS) complex
[25–27]. The extent of anomalies is determined
by the timing of the cloacal membrane rupture.
Rupture that occurs after the separation of cloaca
by the urorectal septum results in bladder
exstrophy, whereas one that occurs before the
separation results in the more severe cloacal
exstrophy and OEIS complex. Bladder exstrophy
is six times more common in males than females.
Table 12 lists syndromes associated with blad-
der exstrophy, and Tables 1, 2, and 3 provide
information about these disorders.
Urethral Defects
Agenesis and Atresia
Urethral agenesis is rare, and its predominant
occurrence in males probably reflects the greater
complexity of embryogenesis of the male urethra.
Urethral agenesis is often associated with bladder
obstruction sequence. Table 13 lists syndromes
associated with urethral agenesis, and clinical
information about these disorders is summarized
in Tables 1, 2, and 3.
167
Duplication
Duplication refers to complete or partial duplica-
tion of the urethra, which is a rare anomaly found
only in a few syndromes. Those syndromes asso-
ciated with urethral duplication are listed in
Table 14, and their findings are provided in
Tables 1, 2, and 3.
Posterior Urethral Valves
Posterior urethral valves refer to abnormal
mucosal folds that function as a valve to obstruct
urine flow. This is the most common childhood
cause of obstructive uropathy leading to renal
failure. Posterior urethral valves can be
suspected prenatally when a dilated bladder is
seen in association with obstructive uropathy.
A “keyhole” sign has been demonstrated in pre-
natal ultrasound of fetuses with subsequently
confirmed posterior urethral valves [28].
A voiding cystourethrogram or endoscopy is
usually required for a definitive diagnosis. The
embryogenesis of posterior urethral valves is
unknown. Proposed hypotheses include an
overdeveloped posterior urethral fold, a remnant
of the mesonephric duct, and an anomalous
opening of the ejaculatory duct. Table 15 lists
syndromes in which posterior urethral valves can
be seen, and the other findings in these disorders
are provided in Tables 1, 2, and 3.
Associations and Sequences Involving
the Urinary Tract
A number of associations and sequences involve
anomalies of the urinary tract that may be impor-
tant to both diagnosis and management. For this
reason, such conditions are described in more
detail in this section, in addition to the information
presented in Tables 1 and 2.
VATER Association
VATER association is an acronym used to desig-
nate a nonrandom occurrence of vertebral defects,
imperforate anus, tracheoesophageal fistula, and
radial and renal anomalies [29, 30]. An acronym
168
VACTERL has been proposed to broaden the
spectrum of VATER to include cardiac defects
and limb anomalies. The term VATER is not a
diagnosis per se, but the designation provides
clues for potentially associated anomalies and
for recurrence risk counseling when no specific
syndromic diagnosis can be made. Patients with
VATER association need a careful evaluation for
multiorgan anomalies. A specific diagnosis
should be sought. Causes of VATER association
include: chromosomal disorders, such as trisomy
18; genetic syndromes, such as Goldenhar and
Holt-Oram syndromes; and teratogenic expo-
sures, such as infants of diabetic mothers and
fetal alcohol syndrome. A family with a mito-
chondrial DNA mutation was identified in which
the daughter was born with VACTERL associa-
tion, and her mother and sister had classic mito-
chondrial cytopathy [31]. Thus all patients
suspected to have VATER association should
have undergone a chromosomal analysis, a care-
ful family and prenatal exposure history, and a
thorough examination for dysmorphic features.
The spectrum of anomalies seen in VATER is
broad. Associated renal anomalies usually
include agenesis, ectopy, or obstruction [29, 30].
Because there is causal heterogeneity for
VATER association, the inheritance pattern and
recurrence risk vary with the cause. VATER asso-
ciation is usually sporadic with an empirical recur-
rence risk of 1–3 % when a specific cause cannot
be identified [32]. Autosomal recessive and
X-linked inheritance have been reported for sub-
sets of patients, such as for VATER with hydro-
cephalus, and recurrence risk in these families can
be as high as 25 % [32].
CHARGE Syndrome (CHARGE
Association)
CHARGE syndrome – previously designated as an
association but now recognized to have a major
causative gene – is an acronym used to designate
an association of coloboma of iris, choroid or retina,
heart defects, atresia choanae, retarded growth and
development, genital anomalies or hypogonadism,
and ear anomalies or deafness [33–36]. In addition,
C. Limwongse
unilateral facial palsy is a common finding. Renal
anomalies found in CHARGE syndrome may
include ectopy, dysplasia, renal agenesis, and/or
ureteric anomalies. The presence of two or more
anomalies associated with CHARGE syndrome
should prompt a search for the others. To prevent
overuse of the term, it has been suggested that at
least three anomalies should be required for the term
CHARGE to be applied and one of the anomalies
should be either coloboma or choanal atresia
[34]. To date, consistent features in CHARGE syn-
drome have been ocular coloboma, choanal atresia,
and semicircular canal hypoplasia [37]. Conditions
with anomalies in the spectrum of CHARGE
include trisomy 13, trisomy 18, and Wolf-
Hirschhorn (deletion 4p), cat-eye, Treacher-Collins,
velocardiofacial, Apert, Crouzon, and Saethre-
Chotzen syndromes. Therefore, a careful physical
examination for malformations and dysmorphic
features should be conducted. Recently, CHD7
mutations have been identified to be the cause of
this syndrome in about 60 % of typical patients, thus
making a molecular confirmation possible. In those
without a CHD7 mutation, chromosome analysis
including specific fluorescence in situ hybridization
(FISH) probes for velocardiofacial syndrome (dele-
tion 22q) and 4p deletion should be performed.
Because most cases of CHARGE association are
sporadic, the empirical recurrence risk in siblings is
low [33, 36].
MURCS Association
MURCS association refers to a rare occurrence of
Mullerian duct aplasia, renal aplasia, and
cervicothoracic somite dysplasia [38]. Anomalies
include absence of the proximal two-thirds of the
vagina, uterine hypoplasia or aplasia, unilateral
renal agenesis, ectopic kidney, renal dysplasia,
C5-T1 vertebral anomalies (hypoplasia of verte-
brae, fusion, hemivertebrae, and butterfly verte-
brae), and short stature. Additional anomalies are
common, including rib defects, facial asymmetry,
limb anomalies, hearing loss, and brain anomalies,
such as encephalocele and cerebellar cyst [39].
The pathogenesis of MURCS association is
unknown, but is thought to be related to defects in
6 Developmental Syndromes and Malformations of the Urinary Tract
the paraxial mesoderm, which gives rise to the
cervicothoracic somites and the adjoining interme-
diate mesoderm. Most patients are diagnosed
because of primary amenorrhea or infertility asso-
ciated with normal secondary sexual characteris-
tics, followed by recognition of reproductive organ
atresia. MURCS association is usually sporadic. A
report of vertebral and renal anomalies associated
with azoospermia has been proposed to represent a
male version of MURCS association [40].
Oligohydramnios Sequence
Oligohydramnios of any cause leads to a recurrent
pattern of abnormalities known as the
oligohydramnios sequence [3, 6].
Oligohydramnios may be caused by decreased pro-
duction of fatal urine from bilateral renal agenesis
or dysplasia or by urinary obstruction, or it can
result from amniotic fluid leakage. When the
oligohydramnios is prolonged and severe, the con-
dition is lethal because of pulmonary hypoplasia.
Moderate oligohydramnios from amniotic fluid
leakage may result in a live-born child with multi-
ple congenital anomalies. These anomalies are both
malformations and deformations. Intrauterine con-
straint leads to mechanical compression that leads
to the characteristic flat facial profile (Potter’s
facies), limb deformities (e.g., talipes equinovarus),
and intrauterine growth retardation (IUGR).
Decreased fetal movement as a result of intrauterine
constraint causes multiple joint contractures
(arthrogryposis). Breech presentation is common.
Pulmonary hypoplasia can be the consequence of
compression of the chest cavity coupled with
decreased inspiration of amniotic fluid. Live-
borns have respiratory distress caused by pulmo-
nary hypoplasia, and the lungs may have insuffi-
cient volume to support life.
Because the initial defect has many causes,
recurrence risk is based on the underlying defect.
When oligohydramnios is due to nonsyndromic
bilateral renal agenesis or dysgenesis, related
renal malformations occur at an increased fre-
quency in first-degree relatives [13], and recur-
rence risk can be as high as 4–9 %. The
recurrence risk can be as high as 25 % for an
169
autosomal recessive disorder causing bilateral
renal agenesis or dysplasia.
Urethral Obstruction Sequence
The initial defect in this sequence is obstruction of
the urethra leading to dilation of the proximal
urinary tract, bladder distension, and hydroureter
[3, 6, 41]. Obstruction of urine flow interferes with
normal nephrogenesis, resulting in renal dyspla-
sia. Other potential anomalies related to bladder
distension include cryptorchidism, malrotation of
the colon, persistent urachus, and limb deficiency
caused by iliac vessel compression. In addition,
oligohydramnios results from lack of urine and
leads to the oligohydramnios sequence.
Prune belly syndrome [41, 42] is a rare entity
referring to a constellation of anomalies that
includes megacystis, abdominal wall muscle defi-
ciency, hydroureter, renal dysplasia, and charac-
teristic wrinkled abdominal skin. This condition,
previously thought to be a form of urethral
obstruction sequence, is in fact a non-obstructive
cause of bladder distension that results from a
malformation, thus now being properly desig-
nated a syndrome [28].
The most common cause of urethral obstruction
is posterior urethral valves, but urethral agenesis/
atresia or bladder neck obstruction can also be the
cause. This anomaly occurs mostly in males, with a
male to female ratio of 20:1. Survival is rare in
fetuses with complete obstruction, and severe uri-
nary tract dysfunction is always present in those
that are live-born. Prenatal diagnosis by ultrasound
examination can detect the abnormally dilated
bladder at the beginning of the second trimester
[43], and intrauterine urinary decompression pro-
cedures, such as vesicoamniotic shunts, are options
for treatment in order to decrease the occurrence of
pulmonary hypoplasia, although their benefits have
not been unequivocal [44, 45].
Sirenomelia Sequence
Sirenomelia is a malformation characterized by
the presence of a single lower extremity with
170
posterior alignment of the knees and feet, sacral
agenesis and other lower vertebral defects, imper-
forate anus and rectal agenesis, and absence of
external and internal genitalia [46]. The current
view of the embryogenesis is that sirenomelia
results from a vascular steal phenomenon
[47]. This is supported by the presence of abnor-
mal vasculature in the caudal part of affected
embryos. A single large vessel originating from
the aorta, a derivative of the vitelline artery com-
plex, connects the iliac arteries to the placenta
rather than the two normal umbilical arteries.
The area caudal to the origin of this vessel has
minimal blood supply because of the lack of aortic
branches. Therefore, a “vascular steal phenome-
non” is generated, leading to a vascular disruption
sequence. Alternatively, since sirenomelia shares
a number of anomalies with caudal regression
syndrome, it is thought to potentially be causally
similar and represent different patterns in the same
spectrum.
Sirenomelia is a rare condition and has a broad
spectrum of anomalies. Virtually any urinary tract
anomaly can occur in sirenomelia sequence.
Renal agenesis occurs in two-thirds of cases, and
a variable degree of renal dysplasia is present in
one-third of cases [3]. Absence of the ureter and
bladder are common. All cases of sirenomelia are
sporadic and almost uniformly fatal because of
pulmonary hypoplasia. Sirenomelia has been
noted with an increased frequency among mono-
zygotic twins in which only one of the twins in
usually affected.
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 Part II
Homeostasis
 Physiology of the Developing Kidney:
Sodium and Water Homeostasis and Its 7
Disorders

Nigel Madden and Howard Trachtman

Contents Sodium Balance Disturbances: Deficit and

Excess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 Sodium Deficit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Body Fluid Compartments and Their Sodium Excess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 Water Balance Disturbances: Deficit and
Total Body Water and Its Compartments . . . . . . . . . . . . 182 Excess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Composition of Body Water Compartments . . . . . . . . 183 Hyponatremia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Serum Osmolality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184 Hypernatremia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
Maintenance Sodium and Water References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
Sodium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Distinct Roles of Sodium and Water
in Body Fluid Homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . 187
Sensor Mechanisms: Sodium and Water . . . . . . . . . 189
Sodium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
Efferent Mechanisms: Sodium and Water . . . . . . . 191
Sodium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
Effector Mechanisms: Sodium and Water . . . . . . . . 197
Sodium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Laboratory Assessment of Sodium and Water
Balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Sodium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
Overview of the Evaluation of Fluid and Water
Abnormalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202

N. Madden (*) • H. Trachtman (*)

NYU Langone Medical Center and NYU School of
Medicine, New York, NY, USA
e-mail: howard.trachtman@nyumc.org

# Springer-Verlag Berlin Heidelberg 2016 181

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_6
182
Introduction
Let there be work, bread, water, and salt for all.
Nelson Mandela
Salt and water are the stuff of life [1, 2]. The
ancient and the modern voices have been
invoked in the past to demonstrate that human
beings intuitively appreciate the critical role
played by sodium and water balance for the
integrity of the plasma compartment and for
continued existence on land. The third time
around, I turn to a towering contemporary figure
of our age as inspiration for this chapter. The
material will review the physiological mecha-
nisms involved in the control of sodium and
water homeostasis. This knowledge will provide
a basis for the analysis of the diseases that arise
when these systems malfunction and a guide to
the optimal therapy of conditions associated
with excessive or deficient total body sodium
and water.
Body Fluid Compartments and Their
Composition
Total Body Water and Its
Compartments
Water is vital for the maintenance of life. It pro-
vides an aqueous environment for cytosolic chem-
ical reactions; a solvent for elimination of waste
products; a medium for transport of nutrients, key
molecules, and gases; and thermoregulation via
sweat production [1]. On average, water com-
prises 60 % of the total body weight in adults.
This proportion is higher in infants and even
greater in babies born prematurely and very low-
birth-weight neonates. It declines during early
infancy and reaches the adult value by the end of
the first year [3]. The percentage of body weight
that is water is lower in postpubertal girls because
they have a higher percentage of body mass that is
fat. It is altered in disease states that are associated
with altered salt handling such as cystic fibrosis
and endocrinopathies.
N. Madden and H. Trachtman
Total Body Water
1:2
ECW ICW
Sodium sequestering, water
independent portion of the interstitial
compartment
IVS INT
1:3
Fig. 1 Graphic illustration of the total body water com-
partments and the relative size of the intracellular water
(ICW) to the extracellular water (ECW) spaces and intra-
vascular (IVS) to interstitial water space
Total body water is divided into two principle
components – the intracellular (ICW) and the
extracellular water (ECW) spaces [4]. These
spaces are apportioned in a 2:1 ratio. When there
is an increase in the total body water, this is
clinically manifested by an increase in the ECW
space because the ICW compartment is not acces-
sible to direct assessment. The ECW compartment
is further divided into the interstitial and intravas-
cular spaces, which are separated in a 3:1 ratio.
Thus, the intravascular space constitutes 1/12 of
the total body water, i.e., 1/3 X 1/4 (Fig. 1). A
component of the ECW, namely, the interstitial
fluid in the skin and connective, may serve as a
reservoir that can mobilize water into the plasma
volume to sustain circulation during conditions of
hypovolemia (see below [5]). Finally, there are
separate transcellular water compartments, such
as the gastrointestinal lumen or cerebrospinal
fluid. They are not in direct contact with the
rest of the fluid spaces and are separated by an
epithelial membrane. Water and electrolytes enter
these spaces via tightly regulated active transport
processes.
7 Physiology of the Developing Kidney: Sodium and Water Homeostasis and Its Disorders
Composition of Body Water
Compartments
All of the major fluid compartments in the body
are separated by semipermeable membranes
[4, 5]. This type of barrier permits free passage
of the aqueous solvent but limits movement of
selective solutes across the membrane. Water
moves down its concentration gradient to ensure
that the osmolality of the solution is the same on
both sides of the membrane. Water channels
called aquaporins are selectively expressed in spe-
cific cell membranes such as the erythrocytes and
distinct nephron segments and facilitate water
movement in response to an osmotic gradient
[6]. Upregulation of aquaporin-1 channels can
occur in disease states such as peritoneal dialysis,
resulting in increased water permeability [7].
Because of the presence of active transporters
and selective channels for various solutes within
the cell membrane, there is an uneven distribution
of solutes in the ICW and ECW compartments.
The presence of the Na-K ATPase pump in the
basolateral membrane domain ensures that potas-
sium and sodium are the principle cations in the
ICW and ECW spaces, respectively [8]. The sec-
ondary movement of Na+ and K+ through other
pathways such as the apical amiloride-sensitive
epithelial sodium channel (ENaC) or ROMK
channel is driven by the primary operation of the
Na-K-ATPase [9]. Limitations in membrane per-
meability to chloride and bicarbonate confine
these anions almost exclusively to the ECW
space, while proteins and phosphate comprise
the major intracellular anions. The differences in
cell permeability and binding characteristics of
specific solutes are reflected in the coefficients
that are used to determine their volume of distri-
bution. For example, because of the permeability
of cell membranes to water, the volume of distri-
bution for sodium is equal to the entire body water
compartment even though sodium is confined to
the ECW space. In contrast, the volume of distri-
bution of bicarbonate is 0.3  total body water.
These considerations are important in formulating
therapeutic regimens to treat specific disorders of
sodium and water homeostasis [10].
183
Besides distinctive membrane permeability
characteristics for specific solutes, the unequal
distribution of ions across the membrane is in
part due to the Gibbs-Donnan effect, which arises
because of the presence of impermeant, nega-
tively charged proteins, primarily albumin, in the
intravascular space [3].
Recent findings underscore the importance of
nonvascular body compartments in the regulation
of total body sodium content and balance, inde-
pendent of water, specifically the interstitial space
in the skin and muscle. The skin interstitium has
been identified as a storage site for sodium using
animal models and sodium-MRI measurements of
sodium content in humans [11]. This storage
occurs without compensatory water retention by
the kidneys. This implies that there is a need for
alternate mechanisms of electrolyte regulation in
the skin (Fig. 1).
Sodium storage in the skin is directly related to
the production of negatively charged, sulfated gly-
cosaminoglycans (GAGs). These negatively
charged molecules trap cations without water, pro-
ducing a hypertonic environment in the skin
interstitium relative to blood plasma. Macrophages
in the blood and tissue contain a tonicity response
element and sense this hypertonicity. They migrate
to the skin interstitium where they release vascular
endothelial growth factor-C (VEGF-C). VEGF-C
induces the hyperplasia of the cutaneous lymph
capillary network and thus enhances the local clear-
ance of skin electrolytes [12].
Disturbances in this extrarenal mechanism for
sodium storage can lead to salt-sensitive hyper-
tension, emphasizing the role of this regulatory
system in local hemostatic control of electrolyte
composition. The importance of interstitial
sodium content to body homeostasis is likely to
have expanding clinical relevance as methods are
developed to assay sodium in this compartment.
Sodium-MRI studies have demonstrated that
hemodialysis can remove tissue sodium from
this compartment in the skin and muscle. Patient
age and levels of circulating VEGF-C determined
the extent of removal during a standard hemodi-
alysis session. Older individuals had more sodium
storage in the skin and muscle and lower levels of
184
VEGF-C. Additionally, patients with lower levels
of VEGF-C showed higher skin sodium storage
after dialysis than those with higher VEGF-C
levels [13].
Serum Osmolality
Despite the significant differences in the compo-
sition of the various body water compartments,
under equilibrium conditions, there is
electroneutrality and tonicity or osmolality, i.e.,
the sum of all osmotically active particles, is equal
in all body water compartments. Under normal
conditions, the serum osmolality is 286 
4 mosm/kg water. Because sodium is the major
cation in the ECW, osmolality can be closely
estimated by the formula:
Serum osmolality  2  ½serum sodium concentration
(1)
A reflection coefficient of 1.0 indicates a totally
non-permeant solute, while freely permeable mol-
ecules have a reflection coefficient of zero. The
reflection coefficient for urea is approximately
0.4. Similarly, in the absence of insulin, the reflec-
tion coefficient for glucose is 0.5. When there is a
pathological elevation in the serum urea nitrogen,
e.g., acute kidney injury (AKI), or glucose con-
centration, e.g., diabetic ketoacidosis, these sol-
utes will contribute to osmolality, albeit less
effectively than sodium. Therefore, the following
formula should be used to more accurately calcu-
late serum osmolality:
Serum osmolality ¼
2  ½serum sodium concentration
(2)
þ ½serum urea nitrogen=2:8
þ ½serum glucose concentration=18
This formula is based on the molecular weights of
urea nitrogen (28 Da) and glucose (180 Da) and
the standard practice of reporting the serum con-
centrations as mg/100 ml. The calculated serum
osmolality is normally within 1–2 % of the value
obtained by direct osmometry in clinical
N. Madden and H. Trachtman
chemistry laboratories. If the calculated serum
osmolality is significantly lower than the value
obtained by measurement with an osmometer,
there is an “osmolal gap” and reflects the accumu-
lation of unmeasured osmoles. Clinically relevant
examples are the organic solutes that are produced
after an ingestion of ethanol or ethylene glycol
(antifreeze) [14, 15].
Maintenance Sodium and Water
Requirements
Sodium
The total body sodium content in adults is approx-
imately 80 mmol/kg of fat-free body weight. This
proportion is higher in newborns, infants, and
young children. Sodium is an essential dietary
component in newborns that is required for nor-
mal growth, especially in very low-birth-weight
infants. In adults, 30–50 % of sodium is contained
in the skeleton. Because this proportion is smaller
in infants, a positive sodium balance is required to
build the skeleton and promote adequate growth
[16]. Sodium stimulates cell growth and prolifer-
ation, possibly through the Na+ -H+ antiporter-
mediated alkalinization of the cell interior. It
causes protein synthesis by promoting nitrogen
retention, thereby increasing muscle mass, and is
necessary for proper neural development
[17]. The growth-permissive role of sodium has
been studied in animal models, which demon-
strate that deprivation of NaCl results in a reduc-
tion in weight, height, muscle mass, and brain
protein, RNA, and lipid content [18]. This effect
is independent of the protein or calorie content of
the diet. Interestingly, in contrast to chronic potas-
sium deficiency induced by diuretics,
compromised sodium intake does not cause struc-
tural damage to the kidney [19]. Although sodium
supplementation restored the rate of growth to
normal and reversed the brain abnormalities, it
was not possible for these animals to catch up to
the healthy controls [20]. The impact of sodium
depletion has also been studied in children with
salt-wasting renal diseases and in premature
infants, both of which are particularly vulnerable
7 Physiology of the Developing Kidney: Sodium and Water Homeostasis and Its Disorders
Table 1 Mean daily sodium intake and recommended
amounts
Recommended
Age group Actual sodium sodium intake
(years) intake (g/day) (g/day)
1–5 4 2
6–10 6 4
11–20 8 5
to hyponatremia. In these cases, salt supplemen-
tation is necessary to promote normal growth and
cognitive development [21].
Balance studies indicate that the daily sodium
requirement is 2–3 mmol/kg body weight. This
quantity is nearly two to threefold higher in term
and very low-birth-weight premature infants
[22]. This reflects the immaturity in renal tubular
function coupled with the increased need for
sodium to achieve the high rate of growth during
early life. It will be exaggerated by intrinsic (diar-
rhea, increased losses via chronic peritoneal dial-
ysis, genetic defect in tubular sodium transport) or
exogenous (administration of diuretics) factors
that promote sodium loss. In most developed
countries, the daily sodium intake in childhood
is in excess of the amount needed to promote
growth or maintain body function. Table 1 sum-
marizes the difference between actual sodium
intake and current recommendations in young
children and adolescents.
There has been extensive discussion about the
daily sodium intake that yields optimal health in
adults. An emerging consensus is that a daily salt
intake in the range of 3–6 g is desirable to prevent
hypertension and minimize cardiovascular mor-
bidity and mortality [23–25]. A J curve, with
extremely low or high sodium intake leading to
worsening cardiovascular outcomes, appears to
define the relationship in adults. This recommen-
dation is based on large international cohort stud-
ies with extended follow-up, accurate assessment
of sodium intake, and detailed characterization of
the clinical outcomes. It is worth noting that even
in adults with nondiabetic chronic kidney disease
(CKD), reducing daily salt intake to below the
3–6 g range is not associated with improved
renal or cardiovascular outcomes [26]. There are
no comparable studies in pediatric patients who
185
do or do not have CKD. Therefore, it is not pos-
sible to define a daily sodium intake during child-
hood that will prevent cardiovascular and renal
disease in adulthood.
Under normal circumstances, the principal
anion that accompanies sodium is chloride. The
identity of the anion that accompanies sodium and
a variety of other dietary constituents impacts on
the adverse consequences of excessive sodium
intake such as hypertension [27]. In certain dis-
ease states such as renal tubular acidosis, meta-
bolic acidosis associated with CKD, or
urolithiasis, it may be advisable to provide a por-
tion of the daily sodium requirement as the bicar-
bonate or the citrate salt.
Water
The daily requirement for water is traditionally
expressed as ml per metabolic kg [28]. However,
in clinical practice, this is a very cumbersome and
impractical method and all calculations are based
on body weight or body surface area (BSA). There
are three methods that are currently utilized to
estimate the daily fluid requirement. The first is a
direct extension of the use of metabolic kilogram
and utilizes the following formula:
1. Daily water requirement = 100 ml/kg for a
child weighing less than 10 kg + 50 ml/kg for
each additional kg up to 20 kg + 20 ml/kg for
each kg in excess of 20 kg
The second method is based on BSA and
utilizes the following formula:
2. Daily water requirement = 1500 ml/m2 BSA
The last method is a refinement of the sec-
ond and utilizes the following formula:
3. Daily water requirement = Urine output +
insensible water losses
Based on clinical experience, under normal
circumstances, urine output is approximately
1,000 ml/m2 per day and insensible losses amount
to 500 ml/m2 per day.
Example: For a child weighing 30 kg and
123 cm in height with a BSA of 1.0 m2, according
to the first method, the daily water requirement is
186
1,700 ml, while the second method yields
1,500 ml per day. The first method is easier to
apply, but it tends to overestimate the water
requirement as body weight increases. The third
method is the most precise and should be applied
in more complicated circumstances such as the
patient in the intensive care unit with oliguria
secondary to AKI or the child with increased
insensible losses, e.g., diarrhea, increased ambient
temperature, tachypnea, burns, or cystic fibrosis
[29]. In addition to the daily energy requirement
and insensible losses that are represented in the
formulas, the amount of water excreted by
the kidney on a daily basis is dependent upon
the solute load. Because urine has a minimum
osmolality, approximately 50 mosm/kg H2O,
even in the absence of arginine vasopressin
(AVP), increased dietary intake of solute will
result in a larger obligatory urine volume to
accommodate the larger solute load [30, 31].
Provision of adequate water intake is espe-
cially important in select medical conditions
such as urolithiasis and CKD where higher water
intake can prevent disease recurrence or progres-
sion, respectively [32].
The daily sodium and water requirement are
generally provided enterally. Intravenous admin-
istration of fluids and electrolytes should be
resorted to only under clinical circumstances that
interfere with normal feeding such a persistent
vomiting, gastrointestinal tract surgery, or states
of altered consciousness.
The choice of parenteral fluid for maintenance
therapy in children has long been debated in the
literature and by clinicians. Holliday and Segar
developed early guidelines based on a linkage
between metabolic needs and fluid requirements
in healthy children. They suggested that a hypo-
tonic maintenance solution containing 0.2 %
saline in 5 % dextrose water should be adminis-
tered to sick children [33]. This guideline has been
repeated in several recent reports [34–36]. Since
then, many nephrologists have criticized this rec-
ommendation claiming that the Holliday and
Segar guidelines were developed based on the
study of healthy children. They assert that it does
not apply to children with acute illness who often
have reduced energy expenditure and lower levels
N. Madden and H. Trachtman
of sensible and insensible water loss. Addition-
ally, acutely ill children often present with
overproduction of AVP, which causes free water
retention and renders the patient vulnerable to
hyponatremia [37]. Adding hypotonic solution in
the setting of inappropriate AVP secretion may
place the child at even greater risk of serious
complications of low serum sodium including
headache, nausea, vomiting, muscle cramps,
depressed reflexes, disorientation, seizures, respi-
ratory arrest, and cerebral edema, which can lead
to permanent brain damage and death [38]. Chil-
dren are at even greater risk of developing neuro-
logical symptoms from hyponatremia because the
size of their brain relative to their skull is greater
than adults. Opponents of the use of hypotonic
maintenance fluids have documented the occur-
rence of hyponatremia and neurological compli-
cations in hospitalized children who receive these
fluids parenterally [37, 39]. To prevent cerebral
edema and neurological consequences of
hyponatremia, they advocate routine administra-
tion of isotonic saline (0.9 % NaCl), with or with-
out dextrose, to all pediatric patients who require
intravenous maintenance fluids [40]. Moritz and
Ayus report more than 50 cases of neurologic
damage and death from 1993 to 2003 as a result
of iatrogenic hyponatremia due to administration
of hypotonic maintenance fluid [37]. In large ran-
domized control trials of critically ill and postop-
erative pediatric patients, Choong et al. [41, 42]
support this view by demonstrating that the use of
hypotonic parenteral maintenance solution results
in significantly increased risk of hyponatremia as
compared to isotonic fluids.
In a meta-analysis of randomized control trials,
hypotonic maintenance fluids were associated
with an increased risk of hyponatremia in ICU
and postoperative patients [43]. Opponents to iso-
tonic saline point to the risk of hypernatremia with
isotonic solutions, yet results from Choong
et al. suggest that isotonic maintenance fluids do
not result in increased risk for hypernatremia
[41, 42]. Several groups in the UK, Canada, and
the USA, including the Institute for Safe Medica-
tion Practices, have warned against the use of
hypotonic saline in the pediatric population. This
issue will require well-designed interventional
7 Physiology of the Developing Kidney: Sodium and Water Homeostasis and Its Disorders 187

Fig. 2 This scheme Clinical Disorders of

illustrates the importance of Sodium/Water Homeostasis
independent assessment of
sodium and water handling
in managing patients with Sodium Disturbance Water Disturbance
clinical disorders of
sodium-water homeostasis
Altered Sodium Balance Altered Water Distrubution

Abnormal ECF Volume Abnormal ICW/ECW Ratio

Clinical Assesment Laboratory Testing

Integrate Evaluation

studies to supplement practice guidelines [44].
Finally, it is worth noting that in a prospective,
open-label pilot study in 760 adults, implementa-
tion of a strategy to restrict chloride content of
intravenous maintenance fluids resulted in a
reduced incidence of AKI and the need for renal
replacement therapy [45]. The impact of chloride-
containing solutions on kidney function in pedi-
atric patients has not been studied. In the interim,
it is important to emphasize that in the face of
clinically significant acute ECW contraction,
there is universal agreement that isotonic fluids
are necessary to effectively replete the intravascu-
lar compartment. Careful clinical and laboratory
monitoring is key to ensuring good outcomes in
all children who are given maintenance fluids and
electrolytes parenterally.
Distinct Roles of Sodium and Water
in Body Fluid Homeostasis
Sodium and water are inextricably linked in the
determination of the serum sodium concentration.
However, it is critical to recognize that sodium
and water serve two distinct functions within the
body. Sodium is instrumental in the maintenance
of the size of the ECF space and the vascular
perfusion compartment, while water is critical to
the maintenance of the size of individual cells.
The regulation of sodium and water homeostasis
represents two distinct processes with discrete
sensing and effector mechanisms. Although
these systems overlap, from a physiological and
clinical perspective, a complete understanding of
body fluids and electrolytes mandates separate
evaluation of sodium and water (Fig. 2).
Because sodium is the principal cation in the
ECW compartment, disturbances in total body
sodium content are reflected by expansion or con-
traction of this space. Adequacy of the ECW
compartment is essential to maintain the intravas-
cular space and sustain perfusion of vital organs.
In terrestrial mammals living in an environment
where ECW volume depletion is a constant threat,
the kidney is designed for maximal sodium
reabsorption as the default mode unless physio-
logical signals instruct it to respond otherwise.
The primary step in the pathogenesis of distur-
bances in ECW compartment size is a perturba-
tion in sodium balance. When total body water
and sodium content are within the normal range,
the net sodium balance is zero and the daily intake
of sodium is matched by losses in the urine, stool,
and insensible losses. Provided kidney function is
normal, the daily dietary sodium intake can be as
low as 0.1 mmol/kg or in excess of 10 mmol/kg
without any derangement in ECF compartment
size. If the alterations in diet are not abrupt, then
sodium balance is maintained, even when kidney
188 N. Madden and H. Trachtman

Table 2 Clinical diseases of sodium and water homeosta- deuterium dilution [48]. Fluid overload often indi-
sis: relationship between ECW size and tonicity cates worsening disease severity in many clinical
Tonicity conditions including CKD, heart failure, and liver
ECW volume Low Normal High cirrhosis. As such, a reliable and efficient tool for
Low Addison’s Isotonic Hypertonic measuring volume status is important in the man-
disease diarrheal diarrheal
agement of these diseases [47]. Measurement of
Salmonella Dehydration Dehydration
diarrhea ECW fluid content is particularly important in
Mannitol Diabetes patients undergoing dialysis. Blood pressure reg-
infusion insipidus ulation in these individuals has proven difficult
Normal SIADH No disease Acute and abnormalities, including both hypotension
sodium and hypertension, are common. Bioimpedance
bicarbonate
infusion
has been established as a useful tool in monitoring
High Acute renal Nephrotic Salt
the body fluid content of patients on dialysis and
failure syndrome intoxication yields important information about their volume
Nephrotic Salt-water status, which can be used to reduce the cardiovas-
syndrome drowning cular risks associated with chronic renal replace-
Cirrhosis ment therapy [49].
Congestive Water homeostasis is a prerequisite for the nor-
heart
mal distribution of fluid between the ICW and
Failure
ECW compartments. Cell function is dependent
on stabilization of cell volume in order to keep the
cytosolic concentration of enzymes, cofactors,
function is markedly impaired [46]. In contrast, if and ions at the appropriate level and to prevent
the daily input of sodium exceeds losses, there is molecular crowding. Perturbations in water bal-
expansion of the ECF space with edema, while if ance result in fluctuations in serum osmolality.
the input does not match the daily losses, there Because cell membranes permit free movement
will be symptoms and signs related to ECW space of water down its osmolal gradient, this causes
contraction. These disturbances are not associated obligatory shifts in water between the cell and
with any obligatory parallel changes in the serum the ECW space. Any disorder that alters the 2:1
sodium concentration (Table 2). ratio of water volume in the ICW/ECW spaces
The determination of ECW compartment size will be reflected by changes in cell size and sub-
and adequacy has generally been based on a clin- sequent cellular dysfunction. Under hypoosmolal
ical assessment (see below). Recent advances conditions, water will move from the intravascular
have highlighted new technological methods to compartment into the cell, causing relative or
measure this space that may facilitate this evalua- absolute cell volume expansion. Conversely, if
tion and provide greater accuracy and validity. the serum osmolality is elevated, water will exit
Bioelectrical impedance is an emerging technique from the cell to the ECW space resulting in abso-
for the measurement of body fluid content. It is an lute or relative cellular contraction [50].
easy, affordable, and noninvasive method and Disturbances in cell function related to abnor-
potentially has great utility in many clinical malities in cell size are most prominent in cerebral
areas. This tool applies an electrical current of cells for two reasons. First, the blood-brain barrier
various frequencies to measure the reactance and limits the movement of solute between the ICWand
resistance of the space between electrodes placed ECW compartments while permitting unrestricted
on the skin. The reactance value is proportional to flow of water down an osmolal gradient [51]. Sec-
body mass and the resistance is inversely related ond, the brain is contained within the skull, which is
to total body water (TBW) [47]. The correlation a closed, noncompliant space, and is tethered to the
between bioimpedance analysis and TBW is cranial vault by bridging blood vessels, which
strong as assessed by measurements made by limits its tolerance of cell swelling or contraction.
7 Physiology of the Developing Kidney: Sodium and Water Homeostasis and Its Disorders
Thus, alterations in water balance and serum osmo-
lality are dominated by clinical findings of central
nervous system dysfunction, including lethargy,
seizures, and coma [50].
In the same way that disturbances in sodium
balance do not necessarily predict specific abnor-
malities in serum sodium concentration, the pres-
ence of a disturbance in water balance and serum
osmolality is not linked to a specific abnormality in
the ECW compartment size. The independent
nature of disturbances in sodium and water balance
is illustrated in Table 2. Alterations in ECW size
can occur in patients with hypotonicity, isotonicity,
or hypertonicity. Similarly, each alteration in serum
osmolality can develop in patients with contraction
or expansion of the ECW compartment.
The presence of disturbances in sodium and
water homeostasis must be addressed separately
in the clinical evaluation of patients with derange-
ments in ECF volume or tonicity. This assessment
must be integrated to obtain a comprehensive
view of what is abnormal and determine how to
effectively restore sodium and water homeostasis
with minimal side effects (Fig. 2).
Sensor Mechanisms: Sodium
and Water
For both sodium and water homeostasis, the sen-
sor mechanisms that maintain the equilibrium
state are primarily designed to be responsive to
the consequences of abnormalities in sodium or
water balance, i.e., changes in ECW and cell size,
respectively, rather than measuring the primary
variable. In this regard, they differ from the acid-
base sensor that is directly responsive to changes
in pH [52]. They operate using negative feedback
loops in which deviations from normal are
detected, counter-regulatory mechanisms are acti-
vated that antagonize the initiating event, and the
system is restored to the normal state.
Sodium
The net sodium deficit is detected as a decrease in
ECW space size, while the net sodium excess is
189
perceived as an obligatory expansion of the ECW
space. These receptors in the venous (low-
pressure) and arterial (high-pressure) circulation,
which are influenced by the filling pressure within
the circulation, are called baroreceptors or mech-
anoreceptors. These signals are supplemented in
certain instances by chemoreceptors that respond
directly to changes in the serum sodium concen-
tration and trigger adaptive modifications in renal
sodium handling. These receptors may effect
change by altering nervous system activity or by
activating upstream promoter elements and stim-
ulating the expression of relevant genes [53].
Atrial receptors: Within the right atrium, sen-
sors possess the distensibility and compliance
needed to detect alterations in intrathoracic
blood volume provoked by increasing negative
intrathoracic pressure or head-out water immer-
sion. Both of these maneuvers, which increase the
central blood volume and raise central venous and
right atrial pressures, are followed by a brisk
natriuresis and diuresis [54]. These relative
changes are triggered even in the absence of a
concomitant change in the total ECW space size.
Neural receptors that respond to mechanical
stretch or changes in right or left atrial pressure
convey the signal via the vagus nerve [54, 55].
Hepatic receptors: The enhanced renal sodium
excretion triggered by saline infusions directly into
the hepatic vein versus the systemic circulation
suggests that there are low-pressure sensors within
the portal vein or hepatic vasculature. The hepatic
responses to changes in sodium balance have been
divided into two categories [56]. The “hepatorenal
reflex” involves direct activation of sodium chemo-
receptors and mechanoreceptors in the hepatoportal
region, via the hepatic nerve, and causes a reflex
decrease in renal nerve activity. The “hepatoin-
testinal reflex” utilizes chemoreceptors to respond
to changes in sodium concentration and modulate
intestinal absorption of sodium via signals con-
veyed along the vagus nerve. Activation of these
hepatic volume sensors may contribute to the
sodium retention and edema states that develop
secondary to chronic liver disease and cirrhosis
with the associated intrahepatic hypertension.
Pulmonary receptors: There may also be pres-
sure sensors within the pulmonary circulation that
190
are activated by changes in pulmonary perfusion
or mean airway pressure [57]. The receptors in the
lung may be located in the interstitial spaces and
influence the physical forces that modulate
paracellular absorption of sodium and water.
They resemble receptors in the renal interstitium
that also influence paracellular absorption of fluid
and solutes along the nephron, especially in the
proximal tubule segment [58].
Carotid arch receptors: There are also volume-
dependent sensors on the high-pressure arterial
side of the circulation including the carotid arch,
the brain, and the renal circulation. Thus, occlu-
sion of the carotid leads to increased sympathetic
nervous system activity and alterations in renal
sodium handling [55]. The responsiveness of the
carotid arch receptors may be modulated by
chronic changes in ECF volume. For example, a
head-down bed position and a high salt diet blunt
carotid baroreceptor activity [59].
Cerebral receptors: Increases in the sodium
concentration of the CSF or brain arterial plasma
promote renal sodium excretion [60]. Lesions in
discrete anatomic areas of the brain such as the
anteroventral third ventricle alter renal sodium
reabsorption, confirming that there are central
mechanisms of sensing changes in sodium bal-
ance and ECF volume. Derangements in the sens-
ing system within the brain in patients with long-
standing central nervous system diseases may
contribute to the cerebral salt-wasting syndrome.
Intracerebral expression of angiotensin-
converting enzyme isoforms contributes to
peripheral sodium and water handling [61].
If arterial sensors perceive underfilling of the
vascular space, this activates counter-regulatory
mechanisms to restore the ECW compartment size
even if the receptors in the venous system detect
adequate or even overfilling of the venous tree.
This implies that despite normal or even excess
total body sodium and net positive sodium bal-
ance, there are conditions in which the body per-
ceives an inadequate circulating plasma volume.
This has given rise to the notion of the “effective”
intravascular volume, a concept that is applicable
in the edema states such as congestive heart fail-
ure, cirrhosis, and nephrotic syndrome [62]. For
example, in patients with cardiac pump failure,
N. Madden and H. Trachtman
perceived underfilling of the arterial tree may
occur despite significant venous distention [40].
Similarly, women who develop edema during
pregnancy may have primary peripheral vasodila-
tation and excess total body sodium [62, 63].
Water
The receptors that are responsible for regulating
water homeostasis are primarily osmoreceptors
and are sensitive to alterations in cell size
[63, 64]. These osmoresponsive cells are located
in the circumventricular organs and anterolateral
regions of the hypothalamus, adjacent to but dis-
tinct from the supraoptic nuclei. They shrink or
swell in response to increases or decreases in
plasma tonicity and this change in cell size trig-
gers the release of AVP and/or the sensation of
thirst. The anatomic configuration of these cells
enables them to be exposed to circulating peptides
that are involved in water homeostasis.
AVP: AVP is a peptide containing nine amino
acids and has a molecular weight of 1,099 Da. It is
synthesized by magnocellular neurons in the
hypothalamus, transported down the axon, and
stored in the posterior pituitary in conjunction
with larger proteins, called neurophysins
[65]. The gene for AVP is located on chromosome
20 and has a cAMP response element in the pro-
moter region. Prolonged stimulation of AVP
release leads to upregulation of the AVP gene;
however, the synthesis does not keep up with the
need for the peptide because pituitary levels of
AVP are usually depleted in states such as chronic
salt loading and hypernatremia [66].
The principle solute that provokes the release
of AVP is sodium. Infusion of sodium chloride to
increase plasma osmolality results in increased
secretion of AVP in the absence of parallel
changes in ECW volume. This underscores the
primary role of plasma osmolality per se in stim-
ulating AVP release [64]. Mannitol, an exogenous
solute that is used in clinical practice to treat
increased intracranial pressure, is nearly as effec-
tive as sodium in stimulating AVP release. Urea
and glucose are less than 50 % as effective as
sodium in provoking AVP secretion because
7 Physiology of the Developing Kidney: Sodium and Water Homeostasis and Its Disorders
they are more permeable than sodium and cause
less pronounced changes in osmoreceptor cell
volume. However, in disease states such as AKI
or diabetic ketoacidosis, in which urea or glucose,
respectively, acts as osmotically active molecules
or following the exogenous administration of
mannitol, these solutes also stimulate increases
in AVP release. There is coupling between
mechanical changes in membrane structure and
hormone release. However, the exact mechanism
and the neurotransmitters that mediate the actions
of the osmoreceptors on the cells of the posterior
pituitary have not been identified.
A variety of non-osmotic stimuli to AVP
release may contribute to water handling in vari-
ous disease states [64, 65]. Vomiting and acute
hypoglycemia promote AVP release by neural-
hormonal pathways that are not well defined.
Stress associated with pain or emotional anxiety,
physical exertion, high body temperature, acute
hypoxia, and acute hypercapnia are other condi-
tions that lead to increased secretion of AVP in the
absence of a primary disturbance in water balance.
Numerous drugs directly influence the hypotha-
lamic release of AVP including carbamazepine,
cyclophosphamide, and vincristine. Finally,
hemodynamic changes arising from primary alter-
ations in sodium balance and the ECW space can
trigger AVP release. If the ECW volume distur-
bance is mild, then the stimulation of AVP release
is modest. However, in the face of severe ECW
volume contraction, there is marked secretion of
AVP. Under these circumstances, the imperative
to protect the effective circulating blood volume
takes precedence over the need to maintain
plasma osmolality and ECW volume is restored
at the expense of hypoosmolality. This clinical
observation indicates the intellectual attempts to
separate sodium and water homeostatic mecha-
nisms; these two factors are closely linked
in vivo and there can be significant overlap in
sensor and effector mechanisms in the regulation
of ECW and ICW compartment size. Table 3 sum-
marizes the factors that modulate AVP release.
In addition to AVP release, the osmoreceptor
cells also respond to the changes in serum osmo-
lality in an independent manner to stimulate thirst
and increase drinking [67]. The stimuli for thirst
191
Table 3 Factors that increase AVP release
"Plasma osmolality
Hemodynamic
#Blood volume
#Blood pressure
Emesis
Hypoglycemia
Stress
Elevated body temperature
Angiotensin II
Hypoxia
Hypercapnia
Drugs
are generally the same as those for AVP release,
with hypernatremia being the most potent trigger.
The osmotic threshold for thirst in humans
appears to be higher than for AVP secretion,
namely, 295 mosm/kg. The sensing mechanism
that leads to this increase in water intake is even
more obscure than that for AVP release. It is likely
that changes in ECW volume are also involved in
this process because angiotensin II, which rises in
states of ECW volume contraction, is a potent
dipsogen [68]. Recent studies suggest that the
day-to-day regulation of thirst by osmoreceptors
is under the control of dopamine-mu opioid neu-
rotransmitters in the brain, while angiotensin II
may be activated under more stressful conditions.
Efferent Mechanisms: Sodium
and Water
The efferent mechanisms involved in maintaining
sodium and water balance include the neural and
endocrine-humoral systems. There often is an
overlap in the action of these effectors, with an
individual effector having distinctive effects on
both sodium and water balance.
Sodium
Renin-angiotensin-aldosterone axis: The major
components of this system – renin,
angiotensinogen, and angiotensin-converting
enzyme (ACE) – are found within the kidney
192
and the vasculature of most organs. These ele-
ments are linked in a large feedback loop involv-
ing the liver, kidney, and lung as well as smaller
loops within individual organs. This accounts for
the often disparate data about plasma renin activ-
ity (PRA) and the expression of individual com-
ponents within the kidney during disturbances in
ECW compartment size.
Angiotensin II is the major signal generated by
this axis [69]. There are two distinct forms of ACE
and the ACE2 isoform may metabolize angioten-
sin II to non-pressor breakdown products that
react with specific receptors and that are less likely
to promote the development of hypertension
[70]. This introduces another layer of complexity
in the regulation of sodium balance by the renin-
angiotensin axis. Angiotensin II interacts with two
different receptors, and most of its biological
activity is mediated by the angiotensin type
1 (AT1) receptor. The AT2 receptor is more prom-
inently expressed in the fetal kidney; however,
interaction of angiotensin II with the AT2 receptor
postnatally stimulates the release of molecules
such as nitric oxide that counteract the primary
action of the peptide [71]. In addition, angiotensin
I can be processed to the heptapeptide angiotensin
1-7, which interacts with a separate mas receptor
and modulates the biological effects of angioten-
sin II [70]. More research is needed to elucidate
the role of alternate forms of angiotensin such as
angiotensin 1-7 on sodium and water balance in
children.
The best-known effects of angiotensin II
include peripheral vasoconstriction to preserve
organ perfusion and stimulation of adrenal syn-
thesis of aldosterone to enhance renal sodium
reabsorption. Angiotensin II also has direct
actions on tubular function and stimulates both
proximal and distal sodium reabsorption. The
proximal tubule cells contain all of the elements
needed to synthesize angiotensin II locally and the
peptide increases the activity of the sodium-
hydrogen exchanger [72]. In the distal tubule,
angiotensin II modulates this exchanger as well
as the amiloride-sensitive sodium channel [69]. It
is worth noting that in the context of glomerular
disease and podocyte injury, the major source of
increased renal angiotensin II is derived from
N. Madden and H. Trachtman
increased filtration of liver-derived
angiotensinogen rather than conversion of
angiotensinogen produced within the kidney [73].
Aldosterone: The effects of aldosterone on the
renal tubule include an immediate effect to
increase apical membrane permeability to sodium
and more extended effects that involve enhanced
gene transcription and de novo synthesis of Na-K-
ATPase. Aldosterone stimulates the synthesis of
other enzymes involved in renal cell bioenergetics
such as citrate synthase that are needed to sustain
maximal tubular sodium transport [74]. Aldoste-
rone induces a state of glucose-6-phosphate dehy-
drogenase deficiency in endothelial cells which
may contribute to oxidant stress and altered reac-
tivity of blood vessels in response to disturbances
in sodium balance [75]. It is important to note that
increased salt intake can activate the mineralocor-
ticoid receptor via a pathway that is independent
of aldosterone. In salt-sensitive animal strains,
high dietary salt intake activates Rac1, a member
of the Rho-guanine triphosphate hydroxylase
family, which stimulates activity of the mineralo-
corticoid receptor. This may represent a novel
pathway by which high salt intake promotes
hypertension, vascular injury, and cardiovascular
disease [76].
Endothelin: This vasoactive molecule is part of
a family of three peptides of which endothelin-1
(ET-1) is the most important in humans [77]. It is
converted in two steps from an inactive precursor
to a biologically active 21-amino acid peptide.
Endothelins react with two receptors, ETA and
ETB, and cause vasoconstriction, resulting in a
decrease in renal blood flow and GFR. With
regard to sodium balance, the primary effect of
endothelin is sodium retention mediated by the
reduction in GFR. This suggests that endothelin
acts in concert with angiotensin II to protect ECW
compartment size under conditions of sodium
deficit. However, the situation may be more com-
plicated because direct exposure of proximal
tubule and medullary collecting duct cells to
endothelin in vitro inhibits sodium absorption.
Renal nerves: There is abundant sympathetic
nervous innervation of the renal vasculature and
all tubular segments of the nephron [78]. The
efferent autonomic fibers are postganglionic and
7 Physiology of the Developing Kidney: Sodium and Water Homeostasis and Its Disorders
originate in splanchnic nerves. The renal innerva-
tion is primarily adrenergic and involves α1
adrenoreceptors on blood vessels and both α1
and α2 receptors along the basolateral membrane
of the proximal tubule. Renal sympathetic ner-
vous activity is inversely proportional to dietary
salt intake [78]. Renal sympathetic nervous sys-
tem activity contributes to preservation of ECF
volume by [1] promoting renal vasoconstriction
and lowering GFR and [2] increasing sodium
reabsorption. Among the catecholamines
involved in adrenergic transmission, norepineph-
rine exerts an antinatriuretic effect. Dopamine,
another sympathetic nervous system neurotrans-
mitter, promotes a natriuresis, suggesting that
there is internal regulation of the effect of nerve
activation on renal sodium handling [79]. Some of
the genes involved in dopamine metabolism
including dopamine receptors and their regula-
tors, G-protein-coupled receptor kinase 4 in the
proximal tubule cell, may contribute to salt sensi-
tivity in patients with hypertension and the sus-
ceptibility to CKD [80].
The sympathetic nervous system also influ-
ences sodium handling in the distal nephron. In
animal models of salt-sensitive hypertension, acti-
vation of the sympathetic nervous system leads to
reduced expression of WNK4 (see below). Alter-
ation in this regulatory protein results in enhanced
activity of the sodium-chloride co-transporter
in the distal convoluted tubule leading to
hypertension [76].
Drug-induced sodium retention and volume-
dependent hypertension, e.g., with the use of
cyclosporine, is mediated in part by activation of
the sympathetic nervous system [81]. Increased
adrenergic nervous signaling within the kidney is
instrumental in the initiation of hypertension in
experimental animals by causing a right shift
in the pressure-natriuresis curve [79]. Alterations
in WNK4 activity may be operative in calcineurin
inhibitor-induced hypertension [82]. However,
sodium balance is normal and ECF volume is
maintained in the denervated transplanted kidney,
implying that the role of the sympathetic nervous
system in maintaining sodium homeostasis is
redundant and can be taken over by other regula-
tory mechanisms [78].
193
Atrial natriuretic peptide (ANP): ANP is a
28-amino acid peptide that is a member of a
group of proteins that includes C-type natriuretic
peptide [83]. It is synthesized as a prohormone
that is stored in granules in the cardiac atria. There
are other molecular isoforms of the hormone
including brain natriuretic peptide (BNP) whose
circulating levels are altered and which can be
monitored at diagnosis and in response to treat-
ment in conditions such as congestive heart
failure [84].
Increases in right atrial pressure provoke cleav-
age and release of the mature peptide. For each
1 mmHg rise in central venous pressure, there is a
corresponding 10–15 pmol/L increase in circulat-
ing ANP levels. Conversely, declines in atrial
pressure secondary to sodium depletion or hem-
orrhage inhibit ANP release. There are two recep-
tors for ANP and both are coupled to guanylate
cyclase. The activation of this enzyme results in
cytosolic accumulation of cGMP, which in turn
diminishes agonist-stimulated increases in intra-
cellular calcium concentration. The principle
effects of ANP are to promote an increase in
GFR, diuresis, and, most importantly, natriuresis.
The augmented renal sodium excretion is, in part,
mediated by an increased filtered load secondary
to the rise in GFR. However, ANP also exerts
direct actions on renal tubular cells to diminish
sodium reabsorption including inhibition the Na-
K-Cl co-transporter in the loop of Henle and the
amiloride-sensitive ENaC in the medullary
collecting duct. Finally, ANP antagonizes the
action of several antinatriuretic effectors, includ-
ing sympathetic nervous system activity, angio-
tensin II, and endothelin. The overall effects of
ANP to counteract increases in ECW compart-
ment have been demonstrated by short-term stud-
ies in which acute infusions of ANP improved
cardiac status in patients with congestive heart
failure and promoted a diuresis in patients with
acute renal failure [85].
Prostaglandins: The kidney contains the
enzymes required for constitutive (COX-1) and
inducible (COX-2) cyclooxygenase activity that
convert arachidonic acid to prostaglandins
[86]. The major products of these pathways are
PGE2, PGF2α, PGD2, prostacyclin (PGI2), and
194
thromboxane (TXA2). In the cortical regions,
PGE2 and PGI2 predominate, while PGE2 is the
major prostaglandin metabolite in the medulla.
These two compounds increase GFR and promote
increased urinary sodium excretion. In addition,
they antagonize the action of AVP. These actions
may mediate the adverse effects of hypercalcemia
and hypokalemia on renal tubular function
[86]. The natriuretic effects of prostaglandins, in
response to normal alterations in dietary sodium
intake, are unclear. The role of prostaglandins as
efferent signals is more apparent in conditions
associated with increased vasoconstrictor tone
such as congestive heart failure or reduced renal
perfusion, where prostaglandins counteract the
vasoconstrictor and sodium-retaining effects of
high circulating levels of angiotensin II and nor-
epinephrine. Inhibition of prostaglandins with
cyclooxygenase inhibitors is associated with dra-
matic declines in GFR and profound sodium
retention and edema [87].
Prostaglandins also modulate sodium handing
in the collecting tubule. Inactivation of the B1
proton pump in β-intercalated cells results in
increased urinary losses of NaCl, potassium, and
water leading to hypovolemia, hypokalemia, and
polyuria. Inhibition of PGE2 synthesis restores
activity of the ENaC and reverses the electrolyte
abnormalities [88]. These novel findings under-
score the importance of prostaglandins in the reg-
ulation of sodium homeostasis. Moreover, they
indicate that in the collecting duct, both principal
and intercalated cells are instrumental in
maintaining sodium balance.
Nitric oxide (NO): The kidney contains all
three isoforms of nitric oxide synthase (NOS) –
neuronal NOS in the macula densa, inducible
NOS in renal tubules and mesangial cells, and
endothelial NOS in the renal vasculature –
involved in NO synthesis. The neuronal and endo-
thelial isoforms are calcium-dependent enzymes
and produce small, transient increases in NO syn-
thesis. The inducible isoform is upregulated by
various cytokines and inflammatory mediators,
resulting in large sustained elevations in NO
release.
Activation of eNOS within the kidney
increases the activity of soluble guanylate cyclase
N. Madden and H. Trachtman
and causes vasodilatation and an increase in GFR.
In addition to its effect on renal blood flow and
GFR, NO directly inhibits Na-K-ATPase in cul-
tured proximal tubule and collecting duct cells
[89, 90]. The specific isoform of NOS that is
responsible for modulating urinary sodium excre-
tion is not as well defined. Studies with inducible
NOS, neuronal NOS, and endothelial NOS
knockout mice suggest that only the first two
isoforms are involved in the regulation of sodium
and water reabsorption in the proximal tubule
[64]. A role of NO in maintaining sodium balance
under normal conditions is suggested by the
observation that alterations in dietary salt intake
are associated with parallel changes in urinary
excretion of nitrite, the metabolic byproduct of
NO [91]. In normotensive Wistar-Kyoto rats and
spontaneously hypertensive rats, increased die-
tary sodium intake is associated with a modest
increase in urinary nitrite excretion [92]. This
effect is not well documented in pediatric patients.
Along with ANP and bradykinin, NO is part of the
defense system against sodium excess and expan-
sion of the ECW compartment. Derangements in
renal NO synthesis and responsiveness to cGMP
may be instrumental in the pathogenesis of salt-
dependent hypertension in experimental animals
[92]. There are no drugs in current use that mod-
ulate sodium handling based on alteration of
intrarenal NO production.
Kinins: Kinins are produced within the kidney
and act via B1 and B2 receptors. Because the half-
life of kinins in the plasma is very short, in the
range of 20–40 s, it is likely that their actions in
the kidney are regulated locally through produc-
tion and proteolytic processing in the tissue
[93]. Their principal action is to promote renal
vasodilatation and natriuresis. The kinins act pri-
marily in the distal tubule to reduce sodium
reabsorption [93, 94].
Insulin: Insulin promotes sodium reabsorption
in the proximal and distal segments of the neph-
ron. It has direct effects on the Na-H antiporter in
the proximal convoluted tubule [95] and on the
NaCl co-transporter in the distal tubule [96]. Part
of the effect of insulin may be mediated by mod-
ulation of the sympathetic nervous system and
WNK kinase activity [97]. The antinatriuretic
7 Physiology of the Developing Kidney: Sodium and Water Homeostasis and Its Disorders
Fig. 3 Graphic illustration
summarizing sensor and
effector mechanisms
involved in the regulation of
sodium balance and ECF
volume. ANP atrial
natriuretic peptide, NO
nitric oxide, PG
prostaglandins
Sympathetic
nervous system
effect of insulin contributes to the hypertension
observed in children with hyperinsulinemia and
the metabolic syndrome. In the context of insulin
and sodium, it is worth noting that a new class of
drugs that have been introduced for the treatment
of diabetes, namely, sodium-glucose
co-transporter inhibitors, is associated with
increased natriuresis and a modest reduction in
blood pressure [98].
FGF23: Recent data suggest that FGF23, an
important bone-derived hormone involved in
phosphate homeostasis, may contribute to sodium
balance [99]. It appears to act in the distal tubule
to promote sodium reabsorption by the Na-Cl
co-transporter via a pathway that involves the
FGF23 receptor/Klotho complex, extracellular
signal-regulated kinase 1/2, serum
glucocorticoid-regulated kinase 1, and WNK4.
This observation may account, in part, for the
association of FGF23 and cardiovascular disease
in children with CKD.
Adrenomedullin: Adrenomedullin is a
52-amino acid peptide that was isolated from
human pheochromocytoma cells [100]. It reacts
with a G-protein cell receptor and causes vasodi-
latation, an effect that may be mediated by
195
Disturbance in sodium
balance
Clinical assessment
Volume sensing
mechanisms
Arterial and venous
EFFECTOR SYSTEMS
Renin- Circulating mediators
Angiotensin- Endothelin, insulin,
Aldosterone Axis ANP, NO, PG, kinins
Restoration of sodium balance
Resolution of hypovolemia or
edema
increased synthesis of NO. The resultant natriure-
sis secondary to the increase in GFR is accompa-
nied by direct inhibition of tubular sodium
reabsorption. Its role in sodium balance is under
investigation.
The WNK, with no lysine protein kinase fam-
ily, are serine threonine kinases. They are respon-
sible for the regulation of several membrane
proteins in the kidney that mediate Na+ handling
in the distal tubule. This family senses intracellu-
lar and extracellular ion concentrations and
accordingly alters the activity of channels and
transport proteins in the nephron via SPS1-related
proline/alanine-rich kinase (SPAK) or oxidative
stress-responsive kinase 1 (OSR1) [101]. Of par-
ticular importance are the cation-chloride
co-transporters (CCCs) found throughout the
tubule. The WNK family serves to help maintain
appropriate blood pressure by acting on these
co-transporters. WNK inhibition acts to lower
blood pressure by decreasing NaCl reabsorption
in the distal collecting tubule and thick ascending
limb while maintaining serum K+ levels by reduc-
ing K+ secretion [101]. Figure 3 summarizes the
sensor and effector mechanisms that are involved
in the regulation of sodium homeostasis.
196
Water
AVP: The primary efferent mechanism in the
maintenance of water homeostasis is AVP. This
peptide fosters water retention by the kidney and
stimulates thirst. The plasma AVP concentration
is approximately 1–2 pg/ml under basal condi-
tions [64, 65]. It is unknown whether there is
tonic release of AVP or whether there is pulsatile
secretion in response to minute fluctuations in
plasma osmolality. The set point, or osmotic
threshold for AVP release, ranges from 275 to
290 mosm/kg H2O. The circulating hormone con-
centration rises approximately 1 pg/mL for each
1 % increase in plasma osmolality. The sensitivity
of the osmoreceptors in promoting AVP release
varies from person to person with some individ-
uals capable of responding to as small as a 0.5
mosm/kg H2O increase in osmolality and others
requiring greater than a 5 mosm/kg H2O incre-
ment to stimulate AVP release. Patients with
essential hypernatremia possess osmoreceptors
that have normal sensitivity but the osmotic
threshold for AVP release is shifted to the right.
Because the relative distribution of water between
the ECW and ICW compartments is undisturbed,
these patients are unaffected by their abnormally
high serum sodium concentration. Although there
may be sex-related differences in AVP secretion in
response to abnormal water homeostasis with
increased sensitivity in women, this is not a rele-
vant clinical concern in prepubertal children.
Reliable measurement of AVP is hindered by
several factors including high level (>90 %) of
binding to platelets. Thus, plasma AVP measure-
ments are influenced by the number of platelets,
incomplete removal of platelets, or pre-analytical
processing steps. Copeptin (CTproAVP), a
39-amino acid glycopeptide, is a C-terminal part
of the precursor pre-provasopressin (pre-
proAVP). Activation of the AVP system stimu-
lates copeptin secretion into the circulation from
the posterior pituitary gland in equimolar amounts
with AVP. Therefore, CTproAVP directly reflects
AVP concentration and it has been used clinically
as a surrogate biomarker of AVP secretion [102].
Angiotensin: Angiotensin II serves as an effer-
ent system in water homeostasis primarily by
N. Madden and H. Trachtman
acting as a potent dipsogen and stimulating drink-
ing [68]. Its role in water handling within the
kidney is minor and may be related to modulation
of the renal response to AVP. However, it is a
potent dipsogen and stimulates thirst and drinking
behavior.
Thirst: Thirst, or the consciously perceived
desire to drink, is a major efferent system in
water homeostasis [67]. It is estimated that for
each 1 pg/mL increase in the circulating plasma
AVP level, there is parallel rise of 100 mosm/kg
H2O in urinary concentration. If the basal plasma
osmolality and AVP concentration are approxi-
mately 280 mosm/kg H2O and 2 pg/mL, respec-
tively, and the steady state urine osmolality is
200 mosm/kg H2O, then as soon as the plasma
osmolality and AVP concentration reach
290 mosm/kg H2O and 12 pg/mL, respectively,
the urine is maximally concentrated. Beyond this
point, the only operational defense against a fur-
ther rise in plasma osmolality is increased free
water intake, underscoring the essential role of
thirst as an efferent mechanism in water homeo-
stasis. It highlights the increased risk of
hyperosmolality in patients who do not have free
access to water such as infants, the physically or
mentally incapacitated, or the elderly [69].
Thirst is a biological function that is difficult to
quantitate because it is an expression of a drive
rather than an actual behavior. At present, visual
analog scales using colors or faces are the most
useful tools for quantitating thirst under controlled
condition. There can be dissociation between
water intake and the sensation of thirst as in
patients with psychogenic polydipsia (e.g.,
schizophrenia, neurosis). It is not known whether
specific drugs directly stimulate the dipsogenic
response. The role of diet, e.g., high salt intake,
in the regulation of thirst is also unknown. The
osmotic control of thirst may be suboptimal in
newborn infants and in the elderly [103].
Thirst and drinking behavior are stimulated by
significant contraction of the ECF space or by
hypotension. In addition, thirst and drinking
behavior are modulated by signals that originate
in the oropharynx and upper gastrointestinal tract.
Animals with hypernatremia who are given access
to water as the sole means of correcting the
7 Physiology of the Developing Kidney: Sodium and Water Homeostasis and Its Disorders
Fig. 4 Graphic illustration
summarizing sensor and
effector mechanisms
involved in the regulation of
water homeostasis,
osmolality, and cell volume.
AVP arginine vasopressin
Urinary concentrating
mechanism
hyperosmolal state stop drinking sooner than
animals corrected in part with supplemental intra-
venous fluid. This is most likely due to oropha-
ryngeal stimuli that curtail drinking prior to
complete normalization of plasma osmolality
[104]. Figure 4 summarizes the sensor and effec-
tor mechanisms that are involved in the regulation
of sodium homeostasis.
Effector Mechanisms: Sodium
and Water
The kidney is the principal organ that acts in
response to sensory input, delivered via neural or
humoral signals, to restore ECW volume size to
normal following the full range of clinical prob-
lems. Absorption of sodium across the gastroin-
testinal tract, modulated by chemoreceptors in the
hepatic vasculature, plays a minor in body sodium
homeostasis.
Sodium
GFR: In children with normal kidney function,
changes in GFR are associated with parallel
197
Disturbance in serum osmolality
Laboratory determination
Hypothalamic sensors
Peripheral sodium sensors
EFFECTOR MECHANISMS
Thirst
AVP
Normalization of serum osmolality and cell
volume
Resolution of cell shrinkage or expansion
alterations in sodium balance. This is accom-
plished by glomerular-tubular balance in which
load-dependent proximal tubule sodium absorp-
tion and delivery of filtrate to the distal tubule is
modulated in response to GFR [105]. Changes in
GFR also lead to changes in the oncotic pressure
in the peritubular capillaries that influence sodium
reabsorption [106]. Thus, an increased GFR is
associated with higher hydrostatic pressures in
the peritubular capillary network that retard fluid
and solute reabsorption in the proximal tubule.
Finally, tubuloglomerular feedback is activated
by alterations in solute delivery to the distal neph-
ron to bring GFR in line with alterations in tubular
function. Many of the efferent signals including
renin, angiotensin, NO, adenosine, and prosta-
glandins participate in this particular pathway.
The release of these effector molecules is acti-
vated via myogenic stretch receptors and chemo-
receptors located in the macula densa region of the
distal nephron. Even in children with
compromised renal function (GFR <30 ml/min/
1.73 m2), glomerulotubular balance is maintained
in the face of gradual changes in GFR. However,
they are unable to respond to abrupt changes in
sodium balance and ECF volume changes as rap-
idly as healthy children and are susceptible to
198
volume contraction or hypervolemia if sodium
intake is substantially reduced or increased over
a short period of time [46].
Neural and humoral factors that lower GFR act
predominantly on the vascular tone of the afferent
arteriole and reduce renal blood flow and the
filtration fraction. Agents in this category include
adrenergic nerve stimulation and endothelin. In
contrast, angiotensin II acts primarily on efferent
arteriolar tone. This tends to preserve GFR more
than renal blood flow and the filtration fraction
(RBF/GFR) is increased. This pattern is most
evident in states of compromised effective perfu-
sion such as congestive heart failure, cirrhosis,
and nephrotic syndrome [62, 63, 107]. The critical
role of angiotensin II in maintaining GFR and
sodium excretion in these conditions is manifest
during the acute reversible functional decline in
GFR that occurs after the administration of ACE
inhibitors [108]. This phenomenon also explains
the reduction in kidney function and sodium
retention that are observed in patients with a crit-
ical renal artery stenosis in a kidney transplant
following initiation of ACE inhibitor
therapy [108].
Proximal tubule: Nearly 60–70 % of the fil-
tered sodium and water load are reabsorbed in the
proximal tubule. Sodium and fluid reabsorption
are isosmotic in this nephron segment. These pro-
cesses are driven by Na-K-ATPase activity along
the basolateral membrane surface with secondary
active transport of solute across the apical mem-
brane. The bulk of sodium reabsorption is driven
by the sodium-hydrogen exchanger, with a lesser
contribution by other co-transport systems for
glucose, phosphate, organic anions, and amino
acids. The linkage between disturbances in ECF
volume and sodium reabsorption in the proximal
tubule is created, in part, by changes in the phys-
ical forces that govern fluid and solute movement.
These include changes in peritubular capillary
hydrostatic pressure, peritubular capillary protein
concentration, and oncotic pressure and changes
in renal interstitial pressure that modulate water
and solute movement across cells (transcellular)
and along the paracellular pathway.
Sympathetic nervous stimulation, norepineph-
rine release, and both filtered and locally
N. Madden and H. Trachtman
synthesized angiotensin II stimulate the activity
of the sodium-hydrogen antiporter and promote
sodium reabsorption in conditions associated with
decreased ECF volume. Conversely, ANP and the
kinins act on proximal tubular cells to inhibit
sodium reabsorption and limit expansion of the
ECW space.
Distal nephron including collecting duct: This
portion of the nephron is responsible for the
reabsorption of approximately 10–25 % of the fil-
tered sodium and water load. Under most circum-
stances, it adapts to changes in delivery arising
from alterations in proximal tubule function. This
segment of the nephron is responsive to virtually all
of the humoral efferent signals and accomplishes
the final renal homeostatic response to fluctuations
in sodium balance. Sodium reabsorption in the
distal tubule and connecting segment is responsive
to circulating levels of aldosterone [74]. In the
collecting tubule, mineralocorticoid-responsive
sodium reabsorptive pathways in the principal cell
achieve the final modulation of sodium excretion in
response to alterations in sodium intake. Aldoste-
rone enhances sodium reabsorption by inducing a
number of transport proteins whose synthesis is
triggered by activation of SGK1, serum, and
glucocorticoid-inducible kinase [109]. The most
prominent of these is the epithelial sodium channel
(ENaC). This transepithelial protein is composed of
three distinct chains – α, β, and γ – each of which is
encoded by a separate gene [110]. The complete
protein has two membrane-spanning domains with
an amino and carboxyl terminus within the cell.
The α-chain appears to constitute the actual
sodium-conducting pathway, while the β- and
γ-chains represent regulatory components that con-
trol the open/closed status of the channel. Genetic
defects in each individual component have been
described and linked to human disease. Thus,
pseudohypoaldosteronism has been mapped to
mutations in the α, β, and γ chains, and Liddle’s
syndrome has been attributed to truncation in the
β-chain with increased ubiquitinylation and
proteasomal degradation of the abnormal protein
[109–111]. As noted above, recent evidence indi-
cates that β-intercalated cells in the collecting duct
also modulate sodium, potassium, and water
excretion [88].
7 Physiology of the Developing Kidney: Sodium and Water Homeostasis and Its Disorders
Water
AVP: AVP acts along several segments of the
nephron. However, its primary site of action for
maintenance of water homeostasis is the
collecting tubule [112]. In that segment of the
nephron, AVP reacts with the V2 receptor, a
371-amino acid protein that is coupled to a
heterotrimeric G protein, along the basolateral
membrane of the distal tubule and collecting
duct cells. The V2 receptor gene has been local-
ized to region 28 of the X chromosome. This
epithelial cell receptor is distinct from the V1
receptor in the vasculature that is linked to Ca
activation of the inositol triphosphate cascade
and which mediates vasoconstrictor response to
the hormone [112].
The binding of AVP to the V2 receptor acti-
vates basolateral adenylate cyclase and stimulates
the formation of cAMP within the cytosol. This
intracellular second messenger then interacts with
the cytoskeleton, specifically microtubules and
actin filaments, and promotes fusion of
intramembrane particles that contain preformed
water channels with the apical membrane of prin-
cipal cells in the collecting duct. The
AVP-induced entry of preformed water channels
involves clathrin-coated pits. The withdrawal of
AVP leads to endocytosis of the membrane seg-
ment containing the water channels into vesicles
that are localized to the submembrane domain of
the cell. This terminates the hormone signal.
Recycling of water channels from vesicles to the
apical membrane and then back into vesicles has
been demonstrated in freeze-fracture studies of
cells exposed to AVP [112]. The importance of
the V2 receptor in water homeostasis is confirmed
by genetic mutations and corresponding abnor-
malities in protein structure in children with
X-linked congenital nephrogenic diabetes
insipidus [113]. Conversely, activating mutations
in the V2 receptor lead to increased water absorp-
tion and hyponatremia in the absence of AVP,
resulting in a condition called nephrogenic syn-
drome of inappropriate antidiuresis [114, 115].
The water channels that mediate transmembrane
movement of water across the collecting tubule in
response to AVP are called aquaporins [116]. There
199
are nine known members of this group of proteins,
all of which contain six membrane-spanning
domains. The first member to be identified was
aquaporin-1 (originally called channel-forming
integral membrane protein of 28 kD or CHIP-28),
which mediates water movement across the eryth-
rocyte membrane and along the proximal tubule.
Mice that do not express AQP-1 have a normal
phenotype and concentrate their urine normally.
Aquaporin-2 (AQP-2) is the major AVP-sensitive
water channel in the collecting tubule
[117]. Immunogold electron microscopy studies
have confirmed that AQP-2 represents the water
channel in the cytosolic vesicles which fuse with
the apical membrane following exposure of princi-
pal cells to AVP. The contribution of AQP-3 and
AQP-4 to the normal urinary concentrating mech-
anism has been confirmed in mice that have been
genetically manipulated and which do not express
these two proteins [118].
The importance of AQP-2 in mediating the
normal response to AVP has been verified by the
discovery of mutations in the AQP-2 gene in
children with non-X-linked, autosomal-recessive
forms of nephrogenic diabetes insipidus [119].
Moreover, alterations in AQP-2 protein expres-
sion have been documented in other states associ-
ated with a urinary concentrating defect such as
lithium exposure, urinary tract obstruction, hypo-
kalemia, and hypercalcemia [116]. There are per-
sistent challenges in the development of better
modulators of the aquaporins including
druggability of the target and the suitability of
the assay methods used to identify the
modulators [120].
Water reabsorption in the collecting duct is not
completely dependent upon the presence of AVP.
In animals that are genetically deficient in AVP
(Brattleboro rats) or in patients with central dia-
betes insipidus, urinary osmolality increases
slightly above basal levels in the face of severe
ECF volume contraction. This may be the conse-
quence of reduction in urinary flow rate along the
collecting duct that enables some passive equili-
bration between the luminal fluid and the hyper-
tonic medullary interstitium.
Although the collecting duct is the primary site
of regulation of net water reabsorption, the
200
proximal tubule contributes to water balance
under circumstances of decreased ECW compart-
ment size. Whereas the proximal tubule normally
reabsorbs approximately 60 % of the filtered
water load, this proportion may exceed 70 %
when the ECF volume is diminished. Further-
more, by decreasing fluid delivery to the distal
nephron, this enhances the AVP-independent
reabsorption of water along the collecting tubule.
These effects may explain the clinical benefit
achieved by the administration of thiazide
diuretics to patients with nephrogenic diabetes
insipidus [121]. The utility of hydrochlorothiazide
in the management of nephrogenic diabetes
insipidus also reflects increased synthesis and
expression of aquaporins and sodium transporters
such as ENaC and sodium-chloride
co-transporter [122].
Countercurrent Mechanism
The primary locus of the urinary concentrating
mechanism is the medulla and involves the thin
descending limb of Henle, medullary thick
ascending limb of Henle, cortical thick ascending
limb of Henle, and collecting duct [123]. Sodium
and water reabsorption are isosmotic in all seg-
ments of the nephron proximal to the loop of
Henle. In order to concentrate or dilute the urine,
water and solute must be separated to enable
excretion of free water or urine that is
hyperosmolal relative to plasma. This process
begins in the medullary and cortical thick ascend-
ing limb of Henle where NaCl is reabsorbed inde-
pendently of water, generating a hypotonic
luminal fluid. This action is linked in series to
the low water permeability of the distal tubule
and the connecting segment, which together with
continued sodium reabsorption, enhances the
hypotonicity of the urine in this segment. In a
secondary step, the permeability to water along
this segment of the nephron is much lower than in
the descending limb of the loop of Henle. This
enables water to move down its osmolal gradient
from the tubule lumen into the interstitium as it
enters the medulla in the descending limb. Finally,
the third critical component of the countercurrent
mechanism is the presence of vasa recta, which
perfuse the inner medulla via vascular bundles
N. Madden and H. Trachtman
Table 4 Factors that contribute to the countercurrent
mechanism
Na-Cl-K-mediated solute absorption in the medullary
thick ascending limb of Henle
Low water permeability of the distal tubule and
connecting segment
High water permeability in the descending limb of Henle
Vasa recta and elimination of interstitial water volume
AVP responsiveness of the collecting tubule
that contain hairpin loop-shaped blood vessels.
This facilitates efficient removal of water that
exits the descending limb of Henle from the med-
ullary interstitium without washing out the solute
gradient that passively drives water reabsorption
in the collecting tubule. The final effector mecha-
nism is the alteration in the water permeability of
the collecting tubule in response to AVP and gen-
eration of a concentrated urine or the excretion of
solute-free water in the absence of AVP (Table 4).
Conditions that disturb the normal architectural
relationship between the vasculature and the
tubules in the medulla such as chronic allograft
nephropathy, acute interstitial nephritis, and
obstructive uropathy disrupt the countercurrent
mechanism and lead to a urinary concentrating
defect.
Osmoprotective molecule (compatible
osmolytes): Besides the presence of effector
mechanisms to maintain water balance, cells pos-
sess a wide range of adaptive mechanisms to
counteract the undesirable movement of water
between the cell and the ECW during hypotonic
and hypertonic states and to prevent neurological
dysfunction. These include early response genes
that mediate the prompt accumulation of chaper-
one molecules to counteract the adverse effects of
altered cell size on protein function [50]. This is
followed temporally by the uptake or extrusion of
electrolytes as an acute response to altered size
cell. Because there are inherent limits on the abil-
ity to regulate cell volume exclusively with inor-
ganic electrolytes, the more extended response
involves membrane transport and/or synthesis/
degradation of a variety of compatible solutes,
called osmolytes, whose cytosolic concentration
can be safely altered without perturbing enzy-
matic activity and cell function. These
7 Physiology of the Developing Kidney: Sodium and Water Homeostasis and Its Disorders 201

osmoprotective molecules include carbohydrates demyelinating syndrome following rapid reversal

(sorbitol, myoinositol), amino acids (taurine, glu-
tamate), and methylamines (betaine, glyceropho-
sphorylcholine) [50]. They accumulate in the
cytosol to preserve cell function during chronic
osmolal disturbances. The cell volume regulatory
response can be activated by electrolytes such as
sodium or neutral molecules, e.g., urea and glu-
cose [50]. The adequacy of the cell volume regu-
latory response and the accumulation of
osmoprotective molecules in cerebral and renal
cells depend on the rate of rise in osmolality as
well as the magnitude of the absolute
change [124].
Experimental data in animals and clinical
experience in premenopausal women suggest
that estrogens may impair the cell volume regula-
tory response to disturbances in plasma osmolal-
ity. This increases the risks associated with both
the untreated abnormalities and therapy
[125]. The cell volume regulatory adaptation is
fully operational during maturation. The accumu-
lation of osmoprotective molecules in the face of
chronic hypernatremia is normal in pre-weanling
rats, albeit with a higher set point to preserve the
increased brain cell water content [126].
The failure to adequately account for the cell
volume regulatory response to osmolal disorders
contributes to some of the adverse effects associ-
ated with inappropriately rapid correction of
abnormalities in plasma osmolality. Under these
circumstances, there is disruption in the blood-
brain barrier and activation of glial cells in the
central nervous system. These lead to neurologi-
cal dysfunction, specifically seizures, during
the treatment of hypernatremia, osmotic
of hyponatremia, dialysis disequilibrium syn-
drome after the initiation of dialysis in patients
with AKI or CKD, and cerebral edema and brain
herniation in patients with a first episode of acute
diabetic ketoacidosis [127, 128].
Laboratory Assessment of Sodium
and Water Balance
There are no normal values for sodium and water
intake or excretion, a reflection of the wide range
of normal daily dietary intake for both sodium and
water. Healthy individuals are in balance and the
excretion of sodium and water matches the daily
intake. Therefore, laboratory assessment of
sodium and water homeostasis is confined to dis-
ease states in which the clinician must determine
whether renal sodium and water handling are
appropriate for the clinical circumstances.
Sodium
The urine sodium concentration is not a valid
index of sodium balance because the value may
vary depending upon the volume and concentra-
tion of the sample. Therefore, renal handling of
sodium is best evaluated using the fractional
excretion of sodium (FENa). After obtaining a
random urine sample and a simultaneous blood
sample and measuring the sodium and creatinine
concentrations in both specimens, the FENa is
calculated using the following formula:

FENa ¼ excreted sodium=filtered sodium

¼ urinary sodium concentration  urine flow rate=plasma sodium concentration  GFR
urine sodium concentration=plasma sodium concentration (3)
¼
urine creatinine concentration=plasma creatinine concentration

This formula is based on the insertion of the denominator. The determination of the FENa is
creatinine clearance as a measurement of GFR in useful in clinical practice because it can be done
the second equation and the cancelation of the using spot samples and does not require timed
urine flow rate term in the numerator and urine collections. In healthy individuals, the
202
FENa varies depending upon the daily sodium
intake. However, in patients with ECF volume
contraction who are responding appropriately,
the FENa is less than 1 % (<3 % in neonates).
Conversely, in patients with ECW compartment
expansion, the FENa will exceed 3 % unless there
is concomitant renal disease.
Water
The determination of the urine specific gravity or
osmolality in a random sample varies depending
Free water clearance ¼ urine volume  osmolal clearance
¼ urine volume  ½urine osmolality  urine flow rate=plasma osmolality
If the free water clearance is a positive number,
then the urine/plasma osmolality ratio is less than
1, the urine is dilute, and the kidney is in a diuretic
mode. When a water diuresis is maximal, the free
water clearance measures the capacity of the kid-
ney to excrete free water. Under these circum-
stances, urine volume is directly related to the
dietary solute intake [31]. In contrast, patients
who are in an antidiuretic mode have a urine/
plasma osmolality ratio greater than 1 and a neg-
ative free water clearance. As the solute excretion
rate increases, both the maximum values for free
water clearance and free water reabsorption
increase. At any given solute excretion rate, the
free water clearance greatly exceeds the free water
reabsorption. This indicates that the renal water
homeostatic mechanisms designed to protect
against overhydration and dilution of the ECW
are more robust than those used to defend against
water deficit and dehydration.
Overview of the Evaluation of Fluid
and Water Abnormalities
In practice, the clinical information and laboratory
data used to evaluate patients overlap with one
another. However, in view of the different physi-
ological roles of sodium and water balance in
N. Madden and H. Trachtman
upon the water intake in the past 2–4 h. Therefore,
the assessment of water handling is best judged by
determining these values under more controlled
conditions such as in the water-deprived state or
following administration of a water load (10–20
ml/kg body weight) to evaluate the urinary con-
centrating or diluting capacity, respectively.
The functional aspects of renal water handling
are best assessed by determining the free water
clearance. This represents the amount of solute-
free water excreted by the kidney. It is calculated
using the following formula:
(4)
body fluid homeostasis, the distinct regulatory
mechanisms activated to control these factors,
and the varied therapeutic strategies that must be
employed to restore sodium and water balance in
disease states, it is essential that disturbances in
sodium-waterand water balance be evaluated sep-
arately. These dual assessments are done in paral-
lel, a reflection of the body’s own method of
operation.
When confronted with a child with a sodium
and water disturbance, the first question is
whether the size of the ECF volume is altered –
is it decreased or expanded? – to the extent that
perfusion of vital organs or pulmonary gas
exchange is jeopardized. Depending on the sever-
ity of the problem, such patients have a life-
threatening condition and may require emergency
therapy such as volume resuscitation or acute
dialysis.
After making this acute determination and
instituting appropriate emergency therapy, it is
important to grade the magnitude of the distur-
bance in ECF volume. There is no single or com-
bination of laboratory tests that substitute for
clinical judgment. Because acute changes in
body weight always reflect alterations in sodium
balance and ECW compartment size, serial mea-
surements in body weight are the most reliable
indicator of the presence and severity of
7 Physiology of the Developing Kidney: Sodium and Water Homeostasis and Its Disorders
disturbances in ECF volume. However, these
measurements are often unavailable or will have
been done in different locations using different
equipment. Therefore, the evaluation of sodium
balance is based upon a wide range of clinical
findings including changes in mental status, level
of alertness, irritability, presence of thirst, pulse
rate, blood pressure, orthostatic changes, fullness
of the anterior fontanel in infants, presence of
tears, dryness of the mucus membranes, skin
color, elasticity of the skin or tenting, capillary
refill or turgor, peripheral edema, shortness of
breath, and presence of rales on auscultation of
the chest. Capillary refill may be the most useful
test to rapidly and accurate assess ECF volume
and the response to treatment [129]. Other find-
ings on clinical examination include urinary spe-
cific gravity and central venous pressure.
Laboratory investigations include BUN, serum
creatinine, and bicarbonate concentrations.
It is important to emphasize that assessment of
ECF volume is a clinical determination. There is
no single or combination of laboratory tests that is
a valid surrogate marker. Moreover, despite the
frequency of clinical disturbances in sodium bal-
ance, especially ECF volume contraction, no suit-
able scoring has been devised to accurately and
reliably distinguish different degrees of ECF vol-
ume contraction or expansion. This contrasts to
the Glasgow coma score or the PRISM score that
have been successfully applied to the initial
assessment of patients with acute neurological or
multisystem organ failure.
The third step in the evaluation of a patient
with a sodium and water disturbance is to measure
the plasma osmolality, which indicates an abnor-
mal distribution of water between the ECW and
ICW compartments. The most likely symptoms
include confusion, irritability, lethargy,
obtundation, and seizures. These manifestations
overlap significantly in patients with
hyperosmolality or hypoosmolality. In contrast
to disorders of ECF volume, disturbances in
water balance and distribution require a labora-
tory determination for confirmation and grading.
The steps involved in the initial evaluation of a
child with a disturbance in sodium and water
balance are summarized in Table 5. The following
203
Table 5 Steps in the initial evaluation and treatment of a
child with a disturbance in sodium and/or water balance
Step 1: determine if there is a life-threatening alteration in
ECF volume
Volume resuscitation if there is ECF volume
contraction
Consider dialysis if there is ECF volume expansion
Step 2: grade severity of defect in sodium balance
Clinical determination of ECF volume
Step 3: determine if there is a defect in water balance
Laboratory measurement of plasma osmolality
sections describe the individual clinical entities
responsible for derangements in sodium and
water balance and outline the general treatment
of these conditions.
Sodium Balance Disturbances: Deficit
and Excess
Sodium Deficit
Diagnosis and evaluation: Sodium deficits and
ECF volume contraction are dangerous because
decreased size of the intravascular space leads to
reduced perfusion and ischemia of vital organs,
namely, the brain, heart, and kidneys. In children,
the absence of concomitant atherosclerosis dis-
ease or endothelial dysfunction secondary to
essential hypertension, smoking, hyperlipidemia,
and diabetes decreases this risk. However, there
are pediatric patients who are more susceptible to
the adverse consequences of hypovolemia includ-
ing newborn babies in whom high circulating
levels of vasoconstrictor hormones and impaired
autoregulation render the glomerular microcircu-
lation sensitive to reduced perfusion [130]. In
addition, underlying diseases or medications
may hinder the counter-regulatory responses to
ECF volume contraction and heighten the risks
of hypovolemia.
Diseases that cause sodium deficiency can
originate outside the kidney or within the kidney.
It is unfortunate that the word dehydration is rou-
tinely used to describe these states because it
suggests that water deficit is the major pathophys-
iological problem. It deflects attention from the
204 N. Madden and H. Trachtman

primary defect, namely, a net negative sodium bal- Table 6 Causes of net sodium deficit
ance [131, 132]. The critical role of the ECF space Renal causes
and sodium balance in the pathogenesis of clinical Compromised GFR
states of volume contraction is highlighted by com- Acute decrease in sodium intake or increased losses
paring the situation that occurs in patients with Tubular disorders
diabetes insipidus. When sodium balance is Osmotic diuresis
perturbed, >30 % of the fluid loss is derived from Diabetic ketoacidosis
the ECW compartment provoking the rapid onset Renal tubular acidosis
of symptoms. In contrast, only 1/12 or 8 % of the Renal Fanconi syndrome
pure water loss that occurs in diabetes insipidus is Pseudohypoaldosteronism
derived from the ECF (1/3 X 1/4), accounting for Obstructive uropathy
Bartter’s syndrome
the rare evidence of ECF volume contraction in
Renal dysplasia/hypoplasia
children with central or nephrogenic diabetes
Central nervous system
insipidus (Fig. 1). The term “denatration” may
Cerebral salt wasting
provide a more accurate depiction of what is occur-
CSF drainage procedures
ring in patients with primary deficits in sodium Hepatobiliary system
balance and contraction of the ECF volume [131]. Biliary tract drainage
Extrarenal causes can occur from losses of Gastrointestinal tract
sodium in any body fluid or across any epithelial Infectious diarrhea
surface including the CSF, pleural fluid, biliary Chloride diarrhea
tree, gastrointestinal losses, or skin. They can be Laxative abuse
the result of medical treatment. CKD can cause Malignancy (carcinoid, tumor related)
sodium deficit because the lower GFR compro- Adrenal diseases
mises the homeostatic capacity of the renal Salt-losing congenital adrenal hyperplasia
tubules. Alternatively, there may be primary Addison’s disease
renal sodium loss that is not the consequence of Skin losses
a decrease in kidney function. Finally, renal Cystic fibrosis
sodium reabsorption may be diminished because Neuroectodermal diseases
of reduced circulating levels or unresponsiveness Burns
to aldosterone. The major causes of sodium defi-
ciency are summarized in Table 6.
Identifying the cause of a disturbance in If the losses are extrarenal, then renal sodium
sodium balance is accomplished based on a thor- reabsorptive mechanisms will be activated and the
ough history and physical examination. Previ- urinary specific gravity will be >1.015 and the
ously, the degree of ECF volume contraction FENa will be low, generally <1 % except in
was categorized as mild, moderate, or severe if infants. The failure to increase the urine concen-
the changes in body weight were estimated to be tration and lower FENa in the face of clinical signs
<5 %, 5–10 %, or >10 %, respectively. Life- of ECF volume contraction points toward a renal
threatening ECF volume contraction was thought or adrenal cause for the disorder. A renal ultra-
to represent >15 % decrease in weight. Recent sound documenting the presence of small,
data, based upon systematic body weights at the misshaped kidneys or hydronephrosis may be
time of hospitalization and immediately after cor- indicative of congenital abnormalities of the kid-
rection of the sodium deficit, suggest that these ney such as dysplasia or obstructive uropathy.
numbers overestimate the degree of sodium defi- Adrenal insufficiency is suggested by concomi-
cit and that the ECF volume contraction is better tant hyponatremia and hyperkalemia.
estimated to be <3 %, 3–6 %, and >6 % with Treatment: If the ECW compartment size is so
>9 % change in body weight representing an severely contracted that vital organ perfusion is
emergency [133]. compromised, based upon an altered mental
7 Physiology of the Developing Kidney: Sodium and Water Homeostasis and Its Disorders
status, orthostatic changes, and azotemia, then
fluid resuscitation must be initiated on an emer-
gent basis. This is necessary to prevent the devel-
opment of AKI, which may occur if there is
sustained hypotension and renal ischemia second-
ary to ECF volume contraction. The risk of AKI is
higher in children with preexisting renal disease,
who are receiving nephrotoxic medications or
who have hemoglobinuria or myoglobinuria, e.
g., crush injury or compartment syndrome. If
there is no evidence of cardiac or pulmonary dis-
ease, then the optimal therapy under these condi-
tions is infusion of isotonic crystalloid (0.9 %
NaCl, Ringer’s lactate), 20 ml/kg body weight.
This fluid is appropriate regardless of the initial
serum sodium or osmolality. Concerns about
infusing Ringer’s lactate are misplaced in view
of the low potassium concentration (4 mmol/L)
in this solution. Transfusion of whole blood is
optimal for treatment of hemorrhagic shock.
However, in all other circumstances, infusion of
crystalloid solutions avoids difficulties caused by
extravasation of the colloid into the interstitial
compartment. A systematic review of the litera-
ture does not support the use of colloid solutions
for volume replacement in critically ill patients
[134]. Thus, in general, there is no proven benefit
of providing colloid versus crystalloid solutions
for the initial treatment of shock and organ
hypoperfusion [135]. In addition, a meta-analysis
has demonstrated that the use of synthetic colloids
such as hydroxyethyl starch is associated with a
higher risk of AKI and the need for implementa-
tion of renal replacement therapy compared to any
other alternative fluid [136]. The infusion should
be as rapid as possible and the fluid dose should be
repeated as often as necessary to achieve some
evidence of clinical improvement such as
improved mental status, decrease in pulse, rise in
blood pressure, or improved capillary refill. A
catheter should be placed in the bladder to facili-
tate monitoring of urine output, especially in
patients with preexisting renal or cardiopulmo-
nary disease who may be more sensitive to sudden
changes in ECF volume.
After correcting life-threatening
hypoperfusion, a fluid repletion plan should be
initiated as soon as possible. Preference should
205
be given to correcting sodium and electrolyte
deficits with oral rehydration solutions (ORS).
In general, patients can be repleted with a rapid
(1–2 h) intravenous infusion to restore ECF
volume followed immediately by initiation of
ORS [137]. The only conditions that represent
contraindications to the use of ORS are impaired
neurological status, persistent vomiting, or dis-
eases associated with mucosal damage in the gas-
trointestinal lumen.
ORS fluids introduced by the World Health
Organization contain sodium 90, potassium
20, bicarbonate 30, chloride, and glucose
111 mmmol/L (20 g/L). The sodium and glucose
are present in a molar ratio that maximizes the
secondary active uptake of these solutes via the
sodium-glucose co-transporter across the gastro-
intestinal epithelium. These transporters retain
activity in the context of secretory diarrhea and
only become impaired when there is extensive
mucosal damage. Water is absorbed passively
down its osmolal gradient. The presence of other
solutes such as potassium and bicarbonate are not
critical to the successful utilization of ORS. These
fluids have been utilized for over 30 years. They
can be administered ad libitum in response to the
child’s own thirst and they are effective and safe
with minimal occurrence of hypernatremia or
hyperkalemia. Various alternatives to glucose
such as rice-syrup or amylase-resistant starch
may facilitate sodium and water reabsorption
from ORS, decrease fecal fluid loss, and shorten
recovery time after an episode of cholera [138,
139]. Clinical studies are needed to confirm the
utility of these additives because they may
increase the cost and decrease the shelf life of
ORS. These are important considerations in
developing countries where there is a high inci-
dence of infectious diarrhea in infants and
children.
When parenteral therapy is required to correct
sodium and water deficits, the following guide-
lines can be applied when devising a therapeutic
plan. First, in the absence of reliable data regard-
ing the acute weight loss, it is easiest to calculate
maintenance and deficit therapy based on the cur-
rent weight and the clinical estimate of the per-
centage decrease in body weight. Second, it is
206
advisable to discount any emergency fluid ther-
apy, such as bolus infusions of isotonic saline, in
computing the total corrective fluid prescription.
Third, if the clinical problem has developed in less
than 48 h, then it should be considered an acute
process and the sodium and fluid losses are
derived from the ICW and ECW in the ratio of
80–20 %. If the patient has been ill for more than
48 h and the process is chronic, then the sodium
and fluid losses are derived from the ICW and
ECW in the ratio of 60–40 %. Under most condi-
tions in which the sodium and water losses are
isotonic, the ECW portion of the loss, in liters, can
be multiplied by 140 mmol/L to determine the
absolute sodium loss. Similarly, the ICW portion
of the loss, in liters, can be multiplied by
140 mmol/L to determine the absolute potassium
deficit. If the ECW and ICW fluid losses together
with the respective sodium and potassium deficits
are added to the maintenance requirements, then a
total fluid volume can be determined. After
selecting a fluid that most closely approximates
the total sodium and water losses, the total fluid
volume is divided by the time frame of the cor-
rection to determine the intravenous infusion rate.
It is important to monitor the child and replace
ongoing losses to insure that there is full resolu-
tion of the underlying clinical problem.
Example: If a 10 kg child is judged to have a
5 % decrease in body weight over 36 h, then the
total fluid deficit of 500 ml can be divided into two
components – 400 ml ICW with 56 mmol potas-
sium and 100 ml ECW containing 14 mmol
sodium. The daily maintenance water and sodium
requirements are 1,000 ml and 30 mmol sodium.
Adding these two quantities together results in a
fluid that closely resembles 0.25 % saline
(37 mmol NaCl/L) containing 30 mmol KCl/L
and the infusion rate is approximately 60 ml/h if
the correction is designed to occur over 24 h.
Sodium Excess
Diagnosis and evaluation: These conditions,
which generally are less common than sodium
deficit, are evidenced by clinical signs of ECF
volume expansion. They can arise secondary to
N. Madden and H. Trachtman
exogenous addition of sodium or abnormal reten-
tion of endogenous sodium. Because the normal
kidney has the adaptive capacity to rapidly excrete
a sodium load, in order for the excess sodium to
cause clinical symptoms and signs, there must be
a concomitant factor that limits natriuresis.
Common causes of excess exogenous sodium
are salt-water drowning, ingestion of a diet abnor-
mally high in sodium, or therapeutic infusion of
sodium-containing intravenous solutions, such as
sodium bicarbonate during the resuscitation after
a cardiopulmonary arrest. Examples of dietary
excess of sodium include errors in the preparation
of infant formulas. The widespread use of
premixed baby formulas may decrease the inci-
dence of these accidents.
Conditions associated with excessive endoge-
nous sodium retention include AKI and the edema
states, namely, nephrotic syndrome, cirrhosis,
congestive heart failure, and gastrointestinal dis-
ease (malabsorption or protein-losing enteropa-
thy). In the first condition, which may occur due
to glomerulonephritis or acute tubular necrosis,
the sodium retention is directly related to the
decrease in GFR and diminished filtration of
sodium. In the later four states, renal sodium and
water reabsorption are activated by a combination
of mechanisms that are termed, underfill and over-
fill. A critical review of the evidence in patients
with nephrotic syndrome suggests that those dis-
eases that are associated with an inflammatory
infiltrate in the kidney develop primary sodium
retention and overfilling of the ECF volume. This
histopathological feature is absent in minimal
change nephrotic syndrome and the underfill
mechanism predominates [140]. Thus, in
nephrotic syndrome, total body sodium excess
may be coupled with a diminished, normal, or
expanded effective ECF volume. This explains
the normal distribution of PRA values and wide
range of measured plasma volume in these
patients. The causes of net total body sodium
excess are summarized in Table 7.
The major clinical problems that accompany
these conditions are related to pulmonary venous
congestion, impaired gas exchange, and dyspnea.
In idiopathic nephrotic syndrome, the intra-
alveolar pressure is often sufficient to prevent
7 Physiology of the Developing Kidney: Sodium and Water Homeostasis and Its Disorders
Table 7 Causes of net sodium excess
Exogenous sodium
Salt-water drowning
Errors in formula preparation
Infusion of hypertonic sodium solutions
For example, after cardiopulmonary arrest
Endogenous sodium
Acute renal failure (glomerulonephritis, ATN)
Nephrotic syndrome
Cirrhosis
Congestive heart failure
frank pulmonary edema. However, in other cir-
cumstances, intrinsic capillary leak together with
lowered plasma oncotic pressure promotes the
development of pulmonary interstitial fluid.
Peripheral edema may be associated with skin
infection, peritonitis, or thromboembolic events.
Conditions associated with sodium excess
should be evident on physical examination. The
FENa will be high if there is sodium loading and
renal function is normal. In contrast, in the states
characterized by retention of endogenous sodium,
the FENa will be very low. The FENa is more
useful than the urinary specific gravity because
the later is likely to be elevated in all cases of
sodium excess.
Therapy: The optimal therapy is targeted at
correcting the underlying disease. This is most
important in patients with cirrhosis, congestive
heart failure, or gastrointestinal disease. Ancillary
therapies include administration of diuretics to
facilitate urinary sodium excretion. Although thi-
azide diuretics may be adequate, more potent loop
diuretic may be required if the GFR is diminished.
Potassium-sparing diuretic such as amiloride or
spironolactone may be added to attenuate
diuretic-induced hypokalemia. Patients with
nephrotic syndrome may require combinations
of diuretic agents that act in the proximal (e.g.,
metolazone) and distal (e.g., furosemide) seg-
ments of the nephron to promote an adequate
natriuresis and diuresis. Infusions of albumin
(1 g/kg body weight) can augment the
medication-induced diuresis, especially in those
with severe ECF volume contraction, reduced
GFR, and azotemia [141]. In the most severe
207
cases, acute dialysis using peritoneal or hemodi-
alysis techniques may be necessary to foster
rapid clearance of excess sodium. Finally, contin-
uous hemofiltration may be a safe and effective
means to rapidly remove sodium and water in
severely ECF-overloaded children with nephrotic
syndrome [142].
Water Balance Disturbances: Deficit
and Excess
Hyponatremia
Diagnosis and evaluation: Patients with
hyponatremia have relative or absolute expansion
of the ICW compartment. If renal function is
normal, the excess free water is rapidly eliminated
within 2–4 h. Therefore, for hyponatremia to
occur, there must be continued AVP release that
is inappropriate to the serum sodium concentra-
tion and the patient must have continued access to
free water. The symptoms and signs of
hyponatremia, which generally involve central
nervous system dysfunction, are vague and
nonspecific. Laboratory confirmation is required
to diagnose this abnormality. The presence of
hyponatremia is not informative about the status
of the ECF volume, and this later issue must be
assessed clinically to determine whether the child
has hypovolemic hypotonicity, isovolemic hypo-
tonicity, or hypervolemic hypotonicity.
Hyponatremia is not uncommon in newborns,
especially in preterm very low-birth-weight
infants. Most often, hyponatremia is a result of
impaired water excretion caused by excessive
arginine vasopressin (AVP). It can also be caused
by the immaturity of the renal tubular sodium
reabsorption mechanisms, which result in exces-
sive urinary sodium loss [143]. Hyponatremia is
dangerous in newborns because sodium is a per-
missive growth factor and chronic deficiency is
associated with impaired muscle, skeletal, and
neural tissue growth and development [144].
Hyponatremia has also been linked to cerebral
palsy, sensorineural hearing impairment, and
intracranial hemorrhages. When the hyponatremia
208
is severe, less than 120 mEq/L, there is a risk of
hyponatremic encephalopathy and possibly death
[143, 145]. As such, it is important to recognize
hyponatremia in infants early and institute the
appropriate treatment to ensure proper growth
and neurocognitive development.
In older children, hyponatremia represents one
of the common electrolyte disorders in hospital-
ized patients. The incidence ranges from 12 % in
patients with intracranial tumors who undergo
neurosurgical procedures [146] to 38 % in patients
with cirrhosis and refractory ascites [147]. In all
clinical circumstances hyponatremia is an indica-
tor of a heightened risk for adverse neurological
outcomes and mortality.
“Physiological” sodium concentration ¼ measured serum sodium concentration þ 1:6 
½each 100 mg=dl increment in serum glucose above 100 mg=dl
The most common clinical causes of
hyponatremia are classified by the concomitant
ECF volume status in Table 8. In some diseases,
such as congestive heart failure, the degree of
hyponatremia reflects circulating AVP levels and
sympathetic nervous system activation and pro-
vides a marker of disease severity. A relationship
between the degree of hyponatremia and the clin-
ical disturbance has not been demonstrated in
patients with nephrotic syndrome or cirrhosis.
The syndrome of inappropriate AVP or ADH
(SIADH) release causes hyponatremia with mild
to modest ECF volume expansion. It can occur as
a consequence of central neurological lesions,
pulmonary disease, or tumors. In addition, drugs
can result in abnormal secretion or action of AVP
and lead to chronic hyponatremia. A list of these
agents is provided in Table 9. The diagnosis of
SIADH requires confirmation that the urine is
excessively concentrated relative to the plasma
osmolality without evidence of ECF volume con-
traction or adrenal or thyroid insufficiency. These
two hormones are required to maintain the low
water permeability of the collecting duct in the
absence of AVP. Deficiencies of either hormone
impair free water clearance leading to euvolemic
N. Madden and H. Trachtman
All biochemical analyzers currently in use at
large medical centers assay sodium concentration
in the aqueous phase of serum and yield an accu-
rate determination of the sodium level. Therefore,
the entity called pseudohyponatremia is no longer
clinically relevant. In contrast, the reduced serum
sodium concentration noted in patients with
increased circulating levels of an impermeant sol-
ute such as mannitol, urea, contrast media, or
glucose in patients with diabetic ketoacidosis is
genuine and reflects osmotic redistribution of
water from the ICW to the ECW space. The phe-
nomenon is reflected in the following formula
which enables adjustment of the serum sodium
in patients with severe hyperglycemia:
(5)
hyponatremia. In practice, diagnosing SIADH
requires comparison of the urine specific
gravity or osmolality with the concurrent serum
osmolality. The urine should normally be
maximally dilute if the serum sodium concentra-
tion is <130 mmol/L or the plasma osmolality
<270 mosm/kg H2O. A urinary sodium concen-
tration >40 mmol/L is an adequate evidence
against ECF volume contraction.
Treatment: In all cases, the first line of therapy
should be directed at the underlying cause of the
low serum sodium concentration. However,
hyponatremia often warrants specific corrective
treatment. Much ink has been spilled in detailing
the appropriate therapy of this electrolyte abnor-
mality. At times, this issue has been quite conten-
tious and the world has been divided into two
supposedly distinct camps – those who advocate
“rapid” versus “slow” correction of
hyponatremia. The former group asserts that
hyponatremia has direct adverse effects on central
nervous system function including impaired oxy-
genation that can lead to seizures or cardiopulmo-
nary collapse prior to initiation of therapy
[148]. Such a sequence of events has been
documented in experimental animals with acute
7 Physiology of the Developing Kidney: Sodium and Water Homeostasis and Its Disorders 209

Table 8 Causes of hyponatremia Table 9 Drugs that cause water retention and the syn-
drome of inappropriate AVP release according to mode of
Hypovolemic: ECF volume contraction
action
Renal
Mineralocorticoid deficiency Increasing water permeability of the nephron
Mineralocorticoid resistance AVP (arginine or lysine vasopressin)
Diuretics Vasopressin analogs, e.g., 1-deamino, 8-D-arginine
vasopressin (DDAVP)
Polyuric acute renal failure
Oxytocin
Salt-wasting renal disease
Promoting AVP release
Renal Fanconi syndrome
Barbiturates
Renal tubular acidosis
Carbamazepine
Metabolic alkalosis
Clofibrate
Bartter’s syndrome/Gitelman’s syndrome
Colchicine
Gastrointestinal
Isoproterenol
Diarrheal dehydration
Nicotine
Gastrointestinal suction
Vincristine
Intestinal fistula
Inhibition of prostaglandin synthesis
Laxative abuse
Salicylates
Transcutaneous
Indomethacin
Cystic fibrosis
Acetaminophen (paracetamol)
Heat exhaustion
Other nonsteroidal anti-inflammatory drugs
“Third-space” loss with inadequate fluid replacement
Potentiation of the action of AVP
Burns
Chlorpropamide
Major surgery, trauma
Cyclophosphamide
Septic shock
Euvolemic: normal ECF volume
Glucocorticoid deficiency
Hypothyroidism hyponatremia is more closely related to the acuity
Mild hypervolemia: ECF volume expansion of the change rather than the absolute size of
Reduced renal water excretion the drop in serum sodium concentration [151].
Antidiuretic drugs Thus, therapy should be guided by the time
Inappropriate secretion of ADH
frame in which hyponatremia has developed. If
Hypervolemic: ECF volume expansion
the hyponatremia is acute, i.e., <12 h in duration,
Acute renal failure (glomerulonephritis, ATN)
then the brain will behave as a perfect osmometer
Chronic renal failure
leading to potentially life-threatening cerebral cell
Nephrotic syndrome
Cirrhosis
swelling. Under these circumstances, there is an
Congestive heart failure urgent need to rapidly reverse the hyponatremia to
Psychogenic polydipsia/compulsive drinking counteract cell swelling. Clinical experience indi-
cates that infusion of a 3 % NaCl solution
(513 mmol/L) in a volume designed to raise the
hyponatremia [149]. This risk may be especially sodium concentration by 3–5 mmol/L is sufficient
prominent in premenopausal women. In contrast, to halt central nervous system dysfunction
there are others who emphasize the cerebral cell [152]. The benefits and lack of adverse effects of
volume regulatory response to hyponatremia and acute correction have been confirmed in a series of
highlight the risk of brain cell dehydration and 34 infants and children with acute water intoxica-
osmotic demyelinating syndrome in patients who tion caused by the ingestion of dilute infant
are corrected too quickly [150]. formula [153]. After this is achieved, the
Taking into account the entire literature on the hyponatremia can be corrected more slowly.
subject, current evidence suggests that the risk of There is preclinical evidence that administration
210
of urea but not steroids can attenuate the adverse
neurological consequences of rapid correction of
hyponatremia with hypertonic saline or a vaso-
pressin antagonist [154]. This requires confirma-
tion in children with hyponatremia.
Example: If a 6-year-old child weighing 20 kg
develops a seizure after a tonsillectomy and is
noted to have a serum sodium concentration of
115 mmol/L, then 36–60 mmol of sodium are
needed to raise the sodium concentration by 3–5
mmol/L. This is accomplished by infusing
72–120 ml of the hypertonic saline infusion.
If hyponatremia has developed over more than
12 h or if the duration of the problem is unclear,
especially in the absence of signs of neurological
dysfunction, then slow correction is the prudent
course of action [150, 151]. The current definition
of slow correction includes two features: (1) the
rate of rise in serum sodium concentration should
be <0.6 mmol/L throughout the correction phase
and (2) the total increment and/or the final serum
sodium concentration after 48 h of treatment
should not exceed 25 mmol/L or 130 mmol/L,
respectively. The more cautious criterion should
be applied depending on the initial serum sodium
level. If a child develops acute changes in mental
status or new neurological findings, during or
shortly after the fluid treatment, then a serum
sodium concentration should be checked. Imag-
ing studies, specifically an MRI of the brain, may
reveal the changes of osmotic demyelinating
syndrome.
There are several therapeutic options for
patients with SIADH whose underlying cause
cannot be corrected. Restriction of free water
intake to match insensible losses and urine output
may be adequate to stabilize the serum sodium
concentration. If this is not well tolerated, then the
administration of furosemide 1–2 mg/kg per day
to promote a hypotonic diuresis together with oral
administration of NaCl 1–2 g per day may correct
the hyponatremia. If these measures fail, consid-
eration can be given to treatment with lithium or
demeclocycline, two drugs that interfere with
AVP action in the collecting tubule to foster excre-
tion of free water and raise the serum sodium
concentration [155].
N. Madden and H. Trachtman
Finally, non-peptide vasopressin receptor
antagonists, the vaptans that bind to the V2 recep-
tor in the collecting duct and block AVP action,
have been developed to treat chronic
hyponatremia [156, 157]. This results in diuresis
without electrolyte loss, termed aquaresis, reduc-
ing total body water and raising the serum
concentration of sodium, thus correcting
hyponatremia.
Vaptans have been approved for use in the
treatment of hypervolemic or euvolemic
hyponatremia in adults. They are not indicated in
patients with hypovolemic hyponatremia. Vaptans
have proven useful in the treatment of congestive
heart failure and chronic liver failure, both of
which are frequently associated with
hypervolemic hyponatremia, and SIADH when
associated with euvolemic hyponatremia.
Conivaptan was the first FDA-approved agent in
this class and is useful for short-term intravenous
use. Tolvaptan is an oral agent that has been
demonstrated to safely correct hyponatremia in
several dose-response studies involving adults
with euvolemic hyponatremia or hypervolemic
hyponatremia [158]. Safety and efficacy of these
drugs have not been assessed in pediatric patients.
Randomized control trials of the use of vaptans in
children with euvolemic hyponatremia are under-
way. The results may demonstrate that vaptans
have an important role in treating newborns and
children with hyponatremia due to excessive AVP.
Hypernatremia
Diagnosis and evaluation: Hypernatremia may
arise due to excessive intake of sodium and ECF
volume expansion. However, excessive water loss
relative to the sodium deficit with hypovolemia
is far more common. In a recent survey of
hypernatremia in hospitalized children, the vast
majority had significant underlying medical prob-
lems and 76 % of the cases were secondary to
inadequate water intake [159]. The prevalence of
this electrolyte abnormality is much lower than
hyponatremia. One of the common causes is diar-
rheal illness in infants; however, the reduction in
7 Physiology of the Developing Kidney: Sodium and Water Homeostasis and Its Disorders
the sodium concentration of most baby formulas
to match the level in human breast milk has
resulted in a dramatic decrease in the incidence
of hypernatremic dehydration. Nonetheless,
changes in medical practice with early discharge
of newborn infants after delivery have resulted in
the steady occurrence of hypernatremic dehydra-
tion in breastfed babies [160]. Patients with
hypernatremia have relative or absolute contrac-
tion of the ICW compartment. Similar to the situ-
ation with hyponatremia, the clinical clues to the
presence of hypernatremia are nonspecific and
laboratory confirmation is mandatory to diagnose
this abnormality. Moreover, like hyponatremia,
hypernatremia must be evaluated in light of the
clinically determined ECF volume status.
Patients with hypernatremia and ECF volume
expansion are easy to diagnose. The children who
represent a serious problem are those with
hypovolemia. They may have some distinct fea-
tures including a marked irritability, a high-
Water deficitðin mlÞ ¼ 0:6  body weight  ½actual serum sodium concentration=140  1
This formula may overestimate the water deficit
and it has been recommended that the following
alternative equation be used to estimate the
Change in sodium concentration ¼ ½infusate Naþ  serum Naþ =total body water þ 1
Finally, with regard to the rate of correction, the
standard practice is to correct hypovolemic
hypernatremia gradually over at least 48 h. After
the groundbreaking work of Finberg et al. [162],
the risk of cerebral edema following rapid correc-
tion of chronic hypernatremia is now attributed to
the inability of brain cells to extrude the
osmoprotective solutes that accumulate during
sustained hyperosmolal conditions in parallel
with the decline in plasma osmolality during
fluid therapy [163, 164]. Therefore, the osmolal
gradient will be reversed with plasma osmolality
lower than cerebral cell osmolality leading to ICW
211
pitched cry, and a doughy skin texture. Because
the hyperosmolality of the ECW compartment
provokes movement of water from the ICW
down its osmolal gradient, these patients preserve
ECF volume until late in the disease course. Their
illness is usually chronic and there is a greater
contribution of the ICW to the water and electro-
lyte deficits. Assessment of the FENa is useful in
assessing the ECF volume in these patients. The
causes of hypernatremia are listed in Table 10.
Treatment: Because children with
hypovolemic hypernatremia are usually very ill,
they often require bolus infusions of isotonic
saline to restore organ perfusion. Once this is
accomplished, then the fluid regimen should
include the maintenance fluids, the estimated def-
icit with the assumption that 60 % is derived from
the ECW and 40 % from the ICW. In addition,
there is a free water deficit. This can be calculated
from the following formula:
(6)
increase in serum sodium concentration that will
be achieved following the infusion of 1 l of a
given solution [161]:
(7)
expansion and clinical signs of cerebral edema,
findings that have been confirmed in NMR spec-
troscopy studies of the brain [164]. The experi-
mental observations were confirmed in a
randomized trial, which demonstrated that the
safest and most effective fluid therapy for
hypovolemic hypernatremia is 0.18 % NaCl
given slowly over 48 h compared to 0.45 % saline
given slowly or rapidly over the same time period
[165]. Because hyperosmolality impairs insulin
and PTH release, patients should be monitored
for hyperglycemia and hypocalcemia during the
correction period.
212 N. Madden and H. Trachtman

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 Physiology of the Developing Kidney:
Potassium Homeostasis and Its Disorder 8
Lisa M. Satlin and Detlef Bockenhauer

Contents Potassium is the most abundant intracellular

Potassium Homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 cation. Approximately 98 % of the total body
potassium content is located within cells, primar-
Regulation of Internal Potassium Balance . . . . . . . 220
Plasma Potassium Concentration . . . . . . . . . . . . . . . . . . . . 222
ily muscle, where its concentration ranges from
Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 100 to 150 mEq/l; the remaining 2 % resides in the
Acid–Base Balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 extracellular fluid, where the potassium concen-
Other Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 tration is tightly regulated within a narrow
Regulation of External Potassium Balance . . . . . . . 224 range (3.5–5.0 mEq/l in the adult). The ratio of
Renal Contribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 the intra- to extracellular potassium concentration
Sites of Potassium Transport Along the Nephron . . . 225
determines, in large part, the resting membrane
Luminal and Peritubular Factors Affecting potential and is thus critical for normal function of
Potassium Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 electrically excitable cells, including nerve and
Sodium Delivery and Absorption . . . . . . . . . . . . . . . . . . . 231
Tubular Flow Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232 muscle. Maintenance of a high intracellular
Potassium Intake and Cellular Potassium potassium concentration is essential for many
Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232 cellular processes, including DNA and protein
Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 synthesis, cell growth and apoptosis, mitochon-
Acid–Base Balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
Circadian Control of Renal Potassium Excretion . . . 235 drial enzyme function, and conservation of cell
volume and pH [1–7]. Because of the many vital
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
processes dependent on potassium homeostasis,
multiple complex and efficient mechanisms have
developed to regulate the internal distribution of
potassium between the intra- and extracellular
compartments and the external balance between
intake and excretion by the kidney and GI tract.
L.M. Satlin (*)
Department of Pediatrics, Division of Pediatric
Nephrology, Icahn School of Medicine at Mount Sinai, Potassium Homeostasis
New York, NY, USA
e-mail: lisa.satlin@mssm.edu
Total body potassium content reflects the balance
D. Bockenhauer between intake and output, the latter regulated
Great Ormond Street Hospital, Institute of Child Health,
primarily by renal and, to a lesser extent, fecal
University College London, London, UK
e-mail: d.bockenhauer@ucl.ac.uk; excretion; the amount of potassium lost through
detlef.bockenhauer@nhs.net sweat is normally negligible. The homeostatic
# Springer-Verlag Berlin Heidelberg 2016 219
E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_7
220
goal of the healthy adult is to remain in zero
potassium balance. Thus, ~90 % of the daily
potassium intake, which typically averages
1 mEq/kg body weight, is eliminated by the kid-
neys and the residual ~10 % is lost through the
stool [8]. Approximately 85 % of ingested potas-
sium is reabsorbed by the gut in the adult [9].
Total body potassium content increases from
approximately 8 mEq/cm body height at birth to
>14 mEq/cm body height by 18 years of age [10,
11] (Fig. 1), with the rate of accumulation of body
potassium per kilogram body weight in the infant
exceeding that in the older child and adolescent.
The increase in total body potassium content cor-
relates with an increase in cell number and potas-
sium concentration (at least in skeletal muscle)
with advancing age [11–13]. The robust somatic
growth early in life requires the maintenance of a
state of positive potassium balance [14, 15], as has
been demonstrated in growing infants greater than
approximately 30 weeks of gestational age
(GA) [16, 17]. The tendency to retain potassium
early in postnatal life is reflected, in part, in the
higher plasma potassium values in infants and
particularly in preterm neonates [17, 18].
Studies in rats suggest that the accumulation of
potassium in the growing fetus is facilitated by the
active transport of potassium across the placenta
Fig. 1 Relationship
between total body
potassium (gms) and height
(cm) for infants and
children. The rate of
accretion of body potassium
in the neonate is faster than
Total Body K (gms)
in later childhood, likely
reflecting both an increase
in cell number and
potassium concentration, at
least in skeletal muscle,
with advancing age (From
Flynn et al. [11])
L.M. Satlin and D. Bockenhauer
from mother to fetus [19]. This notion is further
supported by studies in dog [20] and rat [21] that
show that fetal plasma potassium concentrations
are maintained during maternal hypokalemia, an
adaptation proposed to be due to directed potas-
sium transport towards the fetus.
Regulation of Internal Potassium
Balance
The daily dietary intake of potassium in the adult
(~50–100 mEq) approaches or exceeds the total
potassium normally present within the extracellular
fluid space (~70 mEq in 17 L of extracellular
fluid with a potassium concentration averaging ~4
mEq/l). This potassium load must ultimately be
excreted. However, potassium excretion by the
kidney is sluggish. Only 50 % of an oral or intra-
venous load of potassium is excreted during the
first 4–6 h after it is administered [22, 23]. Life-
threatening hyperkalemia is not generally observed
during this period because of the rapid (within
minutes) hormonally mediated translocation of
~80 % of the retained potassium load from the
extracellular space into cells [24]. The buffering
capacity of the combined cellular storage
reservoirs, which includes muscle, bone, liver, and
MALES
100
FEMALES
10
1
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190
Height (cm)
8 Physiology of the Developing Kidney: Potassium Homeostasis and Its Disorder
red blood cells (RBCs), is capable of sequestering
up to approximately 3,500 mEq of potassium and is
vast compared with the extracellular pool [8].
Cells must expend a significant amount of
energy to maintain the steep potassium (and
sodium) concentration gradients across their cell
membranes. This is accomplished by the ubiqui-
tous Na-K-ATPase which transports three sodium
ions out of and two potassium ions into the cell at
the expense of the hydrolysis of adenosine
triphosphate (ATP). The unequal cation exchange
ratio produces a charge imbalance across the cell
membrane, and thus the Na-K-ATPase is defined
as an electrogenic pump. Positively charged
potassium ions, present in high concentration
within the cell, passively leak out of cells down
a concentration gradient through ubiquitously
expressed potassium-selective channels. A steady
state is reached at which the outward movement of
positively charged potassium is opposed by the
negative cell potential (generally, 30 to 70
mV, cell interior negative, in mammalian cells).
At this cell equilibrium potential, the net trans-
membrane flux of potassium is zero.
The basic functional unit of the Na-K-ATPase
is comprised of a catalytic α- and β-subunits; the
β-subunit acts as a molecular chaperone that
directs the correct membrane insertion of the
α-subunit [25]. Na-K-ATPase is regulated by
changes in its intrinsic activity, subcellular distri-
bution, and cellular abundance. Long-term stimu-
lation of pump activity is generally mediated by
changes in gene and protein expression, whereas
short-term regulation typically results from
changes in the intracellular sodium concentration,
alterations in the phosphorylation status of the
pump and/or interaction with regulatory
proteins, or changes in membrane trafficking of
preexisting pumps [26–28]. The α/β-heterodimer
can complex with the γ-subunits phospholemman
in heart and skeletal muscle [26] or FXYD2 in
the kidney [29], which modulate pump activity.
The cardiac glycoside digoxin binds to the
catalytic α-subunit of the enzyme, inhibiting its
activity. Thus, digoxin overdose can be associated
with hyperkalemia, especially in the presence of a
concomitant perturbation of potassium
homeostasis.
221
Regulation of internal potassium balance in the
neonate is influenced by developmental stage-
specific expression of potassium transporters,
including the Na-K-ATPase, as well as channels,
receptors, and signal transduction pathways [30,
31]. Thirty to fifty percent of very low-birth
weight and premature infants <28 weeks GA
exhibit nonoliguric hyperkalemia during the first
72 h of life, defined as a serum potassium concen-
tration of >7.0 mEq/l, in the presence of urinary
output of >1 ml/kg/h, despite the intake of negli-
gible amounts of potassium [32–37]. This phe-
nomenon, not observed in mature infants or
VLBW infants after 72 h, has been proposed to
reflect failure of the Na-K pump as well as a
limited secretory capacity of the kidney for potas-
sium [35, 36, 38]. Prenatal steroid treatment may
prevent this nonoliguric hyperkalemia via induc-
tion of Na-K-ATPase activity (see below) in the
fetus [39, 40].
The chemical, physical, and hormonal factors
that acutely influence the internal balance of
potassium are listed in Table 1 and are discussed
below. Potassium uptake into cells is acutely stim-
ulated by insulin, β2-adrenergic agonists, and
alkalosis and is impaired by α-adrenergic ago-
nists, acidosis, and hyperosmolality. Generally,
deviations in extracellular potassium concentra-
tion arising from fluctuations in internal
Table 1 Factors that regulate internal potassium balance
Effect on cell uptake of
potassium
Physiologic factors
Plasma K
concentration
Increase Increase
Decrease Decrease
Insulin Increase
Catecholamines
α-Agonists Decrease
β-Agonists Increase
Pathologic factors
Acid–base balance
Acidosis Decrease
Alkalosis Increase
Hyperosmolality Enhances cell efflux
Cell breakdown Enhances cell efflux
222
distribution are self-limited as long as the endo-
crine regulation of internal balance and mecha-
nisms responsible for regulation of external
balance are intact.
Plasma Potassium Concentration
Active cellular potassium uptake by the ubiqui-
tous Na-K-ATPase in large part determines the
intracellular pool of potassium. An increase in
potassium input into the extracellular fluid space,
which may arise from exogenous and endogenous
sources or result from a chronic progressive loss
of functional renal mass, decreases the concentra-
tion gradient against which the Na-K-ATPase
must function and thus favors an increase in cel-
lular potassium uptake. Sources of exogenous
potassium input may not be readily apparent and
include not only diet but also potassium-
containing drugs (potassium penicillin G), other
parenteral potassium supplements (including total
parenteral nutrition), salt substitutes, herbal med-
ications, and packed RBCs [41]. The extracellular
fluid potassium concentration can also increase in
the absence of change in total body potassium
content in response to endogenous release of
potassium during tissue breakdown (intravascular
hemolysis, rhabdomyolysis, tumor lysis syn-
drome) and severe exercise, the latter mediated
by ATP depletion and opening of
ATP-dependent potassium channels. In epithelial
cells of the kidney and colon specifically respon-
sible for potassium secretion, an increase in intra-
cellular potassium maximizes the concentration
gradient between cell and tubular lumen, thereby
promoting potassium diffusion into the urinary
space and thus potassium excretion.
Hormones
Insulin, the most important hormonal regulator of
internal potassium balance, stimulates Na-K-
ATPase-mediated cellular potassium uptake and
thus the rapid transfer of potassium from the
extracellular to the intracellular fluid space of
insulin-responsive cells in liver, skeletal muscle,
L.M. Satlin and D. Bockenhauer
adipocytes, and brain, a response that is
independent of the hormonal effects on glucose
metabolism [42]. The mechanism of insulin
action in these tissues differs, in part, because of
differences in the isoform composition of the
catalytic α-subunit of the pump. Insulin stimulates
Na-K-ATPase activity by promoting the
translocation of preformed pumps from intracel-
lular stores to the cell surface [43–47] and/or
increasing cytoplasmic sodium content [48–50]
or the apparent affinity of the enzyme for
sodium [51].
Basal insulin secretion is necessary to maintain
fasting plasma potassium concentration within the
normal range [22]. An increase in plasma potas-
sium in excess of 1.0 mEq/l in the adult induces a
significant increase in peripheral insulin levels to
aid in the rapid disposal of the potassium load, yet
a more modest elevation of approximately 0.5
mEq/l is without effect [22, 52, 53]. In the setting
of insulin deficiency (diabetes), there is a reduc-
tion in uptake of potassium by muscle and
liver [24].
Catecholamines enhance the cell uptake of
potassium via stimulation of Na-K-ATPase activ-
ity in skeletal muscle and hepatocytes through β2-
adrenergic receptors [54, 56]. The effect of epi-
nephrine on potassium balance in the adult is
biphasic and is characterized by an initial
increase, followed by a prolonged fall in plasma
potassium concentration to a final value below
baseline. The initial transient rise in plasma potas-
sium results from α-adrenergic receptor stimula-
tion which causes release of potassium from
hepatocytes and impairs cell uptake of potassium
[54, 57–59]. β2-Receptor stimulation, via stimu-
lation of adenylate cyclase leading to generation
of the second messenger cyclic adenosine
monophosphate (cAMP) and activation of down-
stream protein kinases, stimulates the sodium
pump and thus promotes enhanced uptake of
potassium by skeletal and cardiac muscle, effects
that are inhibited by nonselective β-blockers
including propranolol and labetalol [54,
59–63]. The observation that the potassium-
lowering effects of insulin and epinephrine are
additive suggests that their responses are mediated
by different signaling pathways.
8 Physiology of the Developing Kidney: Potassium Homeostasis and Its Disorder
Plasma Potassium,
mmol/L
Bicarbonate
0 Aldosterone is best known for its effect on
Epinephrine
−0.5
Insulin/glucose
−1.0
Dialysis
−1.5 p < 0.01
0 10 20 30 40 50 60
Fig. 2 Changes in plasma potassium concentration
(mmol/L) during intravenous infusion of bicarbonate
(8.4 %), epinephrine, or insulin in glucose and during
hemodialysis in adult patients on maintenance hemodialysis
(From Blumberg et al. [83])
The effects of these hormones on the distribu-
tion of potassium between the intracellular and
extracellular compartments have been exploited
to effectively treat disorders of homeostasis
(Fig. 2). Administration of β2-adrenoreceptor ago-
nists (albuterol or salbutamol via nebulizer),
which promote potassium uptake by cells, has
been used to treat acute hyperkalemia in neonates,
children, and adults [64–66]. A single dose of
nebulized albuterol can lower serum potassium
by as much as 0.5 mEq/l [67, 68]. Transient side
effects associated with this class of drugs include
mild tachycardia, tremor, and mild vasomotor
flushing [69]. Administration of glucose alone
(to induce endogenous insulin release) or glucose
plus insulin is efficacious, although patients must
be monitored for complications of hyperglycemia
(without insulin) or hypoglycemia (with insulin),
especially in neonates [70–72]. It should be kept
in mind that the treatment options discussed thus
far are only temporizing. To remove potassium
from the body, potassium excretion must be
enhanced, either by the kidney by stimulating
223
potassium secretion in the distal nephron (see
below) or, in the presence of renal insufficiency,
by the gut (using cation exchange resins) or by
dialysis.
transporting tissue, increasing potassium secre-
tion in distal segments of the nephron and colon
(see below). Triiodothyronine (T3) also promotes
Na-K-ATPase-mediated potassium cellular
uptake in skeletal muscle [27]. Whereas T3 had
been thought to act as a direct transcriptional
activator of target genes, nongenomic effects are
also important, including the stimulation of trans-
location of Na-K-ATPase to the plasma mem-
brane by a pathway that requires activation of
MAPK and phosphatidylinositol 3-kinase (PI3K)
[73, 74]. The postnatal increases in Na-K-ATPase
expression in kidney, brain, and lung depend on
normal thyroid hormone status [75].
Acid–Base Balance
It is well known that the transcellular distribution
of potassium and acid–base balance are interre-
lated [76–79]. Whereas acidemia (increase in
extracellular hydrogen ion concentration) is asso-
ciated with a variable increase in plasma potas-
sium secondary to potassium release from the
intracellular compartment, alkalemia (decrease in
extracellular hydrogen ion concentration) results
in a shift of potassium into cells and a consequent
decrease in plasma potassium. However, the
reciprocal changes in plasma potassium that
accompany acute changes in blood pH differ
widely among the four major acid–base disorders;
metabolic disorders cause greater disturbances in
plasma potassium than do those of respiratory
origin, and acute changes in pH result in larger
changes in plasma potassium than do chronic
conditions [76].
Acute metabolic acidosis after administration
of a mineral acid that includes an anion that does
not readily penetrate the cell membrane, such as
the chloride of hydrochloric acid or ammonium
chloride, consistently results in an increase in
plasma potassium. As excess extracellular pro-
tons, unaccompanied by their impermeant anions,
224
enter the cell where neutralization by intracellular
buffers occurs, potassium is displaced from the
cells, thus maintaining electroneutrality and lead-
ing to hyperkalemia. However, comparable
acidemia induced by acute organic anion acidosis
(lactic acid in lactic acidosis, acetoacetic and
β-hydroxybutyric acids in uncontrolled diabetes
mellitus) may not elicit a detectable change in
plasma potassium [76, 80, 81]. In organic
acidemia, the associated anion diffuses more
freely into the cell and thus does not require a
shift of potassium from the intracellular to the
extracellular fluid.
In respiratory acid–base disturbances, in which
carbon dioxide and carbonic acid readily perme-
ate cell membranes, little transcellular shift of
potassium occurs because protons are not
transported in or out in association with potassium
moving in the opposite direction [76].
Changes in plasma bicarbonate concentration,
independent of the effect on extracellular pH, can
reciprocally affect plasma potassium concentra-
tion [82]. Movement of bicarbonate (out of the
cell at a low extracellular bicarbonate concentra-
tion and inward at a high extracellular bicarbonate
concentration) between the intracellular and
extracellular compartments may be causally
related to a concomitant transfer of potassium.
This relationship may account for the less marked
increase in plasma potassium observed during
acute respiratory acidosis, a condition
characterized by an acid plasma pH with an ele-
vated serum bicarbonate (hence inward net bicar-
bonate and potassium movement), as compared
with acute metabolic acidosis with a low serum
bicarbonate concentration (hence outward net
bicarbonate and potassium movement). Though
recommended as a mainstay of therapy, alkalini-
zation of the extracellular fluid with sodium bicar-
bonate to promote the rapid cellular uptake of
potassium may not be useful in patients on main-
tenance hemodialysis for end-stage renal disease
(Fig. 2) [83]. However, this maneuver remains
valuable if metabolic acidosis is at all responsible
for the hyperkalemia. The major toxicities of
bicarbonate therapy include sodium overload
and precipitation of tetany in the face of
preexisting hypocalcemia.
L.M. Satlin and D. Bockenhauer
Other Factors
A number of other pathologic perturbations alter
the internal potassium balance. An increase in
plasma osmolality secondary to severe dehydra-
tion or administration of osmotically active agents
causes water to shift out of cells. The consequent
increase in intracellular potassium concentration
exaggerates the transcellular concentration gradi-
ent and favors movement of this cation out of
cells. The effect of hyperosmolality on potassium
balance becomes especially problematic in dia-
betic patients with hyperglycemia, in whom the
absence of insulin exacerbates the hyperkalemia.
Succinylcholine, a depolarizing paralytic agent
and an agonist of nicotinic acetylcholine recep-
tors, which are found predominantly in skeletal
myocyte membranes, may lead to efflux of potas-
sium from myocytes into the extracellular fluid
under certain pathologic states associated with
upregulation and redistribution of the receptors,
including states characterized by physical or
chemical upper or lower motor neuron denerva-
tion, immobilization, infection, muscle trauma or
inflammation, and burn injury [84, 85].
Finally, parenteral administration of cationic
amino acids such as lysine, arginine, or epsilon-
aminocaproic acid (used to improve hemostasis in
patients undergoing cardiac surgery) may lead to
electroneutral exchange of cell potassium for the
cationic amino acid in skeletal myocytes and thus
hyperkalemia [86–88].
Regulation of External Potassium
Balance
Renal Contribution
The kidney is the major excretory organ for potas-
sium. In the adult, urinary potassium excretion
generally parallels dietary intake. However, as
stated earlier, the renal adaptation to variations in
dietary intake is rather sluggish. Extreme adjust-
ments in the rate of renal potassium conservation
cannot be achieved as rapidly as for sodium, nor
are the adjustments as complete; whereas urinary
sodium can be virtually eliminated within
8 Physiology of the Developing Kidney: Potassium Homeostasis and Its Disorder
3–4 days of sodium restriction, there is a minimum
urinary potassium loss of about 10 mEq/day in the
adult, even after several weeks of severe potassium
restriction [89]. An increase in dietary potassium
intake is matched by a parallel increase in renal
potassium excretion within hours, yet maximal
rates of potassium excretion are not attained for
several days after increasing potassium intake.
Children and adults ingesting an average
American diet that contains sodium in excess of
potassium excrete urine with a sodium-to-potas-
sium ratio greater than one [17, 90]. Although
breast milk and commercially available infant for-
mulas generally provide a sodium-to-potassium
ratio of approximately 0.5–0.6, the urinary
sodium-to-potassium ratio in the newborn up to
4 months of age generally exceeds 1. This high
ratio may reflect the greater requirement of potas-
sium over sodium for growth. In fact, some pre-
mature (<34 weeks GA) newborns may excrete
urine with a sodium-to-potassium ratio greater
than 2, a finding suggesting significant salt
wasting and a relative hyporesponsiveness of the
neonatal kidney to mineralocorticoid activity.
This is consistent with the physiological postnatal
reduction in relative size of the extracellular fluid
compartment from approximately 60 % of body
weight at 23 weeks of gestation to 40 % at term
[17, 91, 92].
Net urinary potassium excretion in the fully
differentiated kidney reflects the sum of three
processes: glomerular filtration, reabsorption
along the proximal tubule and the loop of Henle,
and bidirectional transport (secretion and
reabsorption) in the distal nephron [8, 93]. Most
of the factors known to modulate potassium
excretion do so by altering the rate of potassium
secretion.
Renal potassium clearance is low in newborns,
even when it is corrected for their low glomerular
filtration rate [16–18]. Infants, like adults, can
excrete potassium at a rate that exceeds its glo-
merular filtration, reflecting the capacity for net
tubular secretion. However, they are unable to
excrete an exogenously administered potassium
load as efficiently as the adult [94]; specifically,
the rate of potassium excretion normalized to
body weight or kidney weight is less in newborn
225
than older animals [95, 96]. Comparison of the
fractional delivery of potassium to the early distal
tubule with that present in the final urine reveals
that the distal nephron of the young (2-week-old)
rat secretes approximately fivefold less potassium
than the older (5-week-old) rat [97]. Similarly,
clearance studies in saline-expanded dogs also
provide indirect evidence of a diminished secre-
tory and enhanced reabsorptive capacity of the
distal nephron to potassium early in life [98]. Fur-
thermore, premature neonates studied weekly
after birth exhibited a 50 % reduction in the frac-
tional excretion of potassium between 26 and
30 week GA, in the absence of significant change
in absolute urinary potassium excretion [16]. To
the extent that the filtered load of potassium
increases almost threefold during this same time
interval, the constancy of renal potassium excre-
tion could be best explained by a developmental
increase in the capacity of the kidney for potas-
sium reabsorption [16]. It is important to note that,
in general, the limited potassium secretory capac-
ity of the immature kidney becomes clinically
relevant only under conditions of potassium
excess.
Sites of Potassium Transport Along
the Nephron
Proximal Tubule
Potassium is freely filtered at the glomerulus.
Thus, the concentration of potassium entering
the proximal convoluted tubule (PCT) is similar
to that of plasma. Approximately 65 % of the
filtered load of potassium is reabsorbed along the
initial two-thirds of the proximal tubule in the
adult, a fractional rate of reabsorption similar to
that of sodium and water (Fig. 3) [100–102].
A similar fraction (50–60 %) of the filtered load
of potassium is reabsorbed along the proximal
tubules of suckling (13–15-day-old) rats
[97, 103].
Reabsorption of potassium along the early
proximal tubule is passive, closely following that
of sodium and water [104], and has been proposed
to occur by solvent drag via the paracellular path-
way and diffusion [105–107]. Solvent drag
226
Fig. 3 Potassium transport along the nephron. (a) The
percentages of filtered potassium reabsorbed along the
proximal tubule and thick ascending limb of the loop of
Henle (TALH) are indicated for the adult (A). Arrows show
direction of net potassium transport (reabsorption or secre-
tion). GFR glomerular filtration rate. (b) Simplified cell
depends on active sodium reabsorption, which
generates local hypertonicity in the paracellular
compartment, providing an osmotic force driving
water reabsorption that entrains potassium in the
reabsorbate. Potassium diffusion is driven by the
lumen-positive transepithelial voltage in the sec-
ond half of the proximal tubule and the slightly
elevated concentrations of potassium in the
lumen [108].
In the proximal tubule, as in all other nephron
segments discussed below, transepithelial sodium
reabsorption requires the coordinated function of
apical sodium transport proteins and the
basolateral Na-K-ATPase which actively extrudes
intracellular sodium into the interstitium and
thereby maintains the low intracellular sodium
concentration and steep sodium concentration
gradient critical to the driving force for apical
sodium entry (Fig. 3). There is no evidence for
specific regulation of potassium reabsorption
along the proximal tubule, and most observed
L.M. Satlin and D. Bockenhauer
models of potassium transport along the nephron, showing
apical transporters unique to discrete nephron segments
and cells therein and basolateral transporters which are
similar in all nephron segments (Adapted from Giebisch
[99], Fig. 3. Publisher: Nature Publishing Group Date: Nov
1, 2002)
modulation of proximal reabsorption of this cat-
ion can be accounted for by alterations in sodium
transport.
Electrogenic sodium-coupled entry of sub-
strates such as amino acids and glucose across
the luminal cell membrane of the proximal tubule
as well as bicarbonate exit across the basolateral
cell membrane is driven by the potential differ-
ences across the respective cell membranes,
which are maintained by potassium flux through
potassium channels [109]. Electrophysiological
studies in isolated perfused proximal tubules sug-
gest that potassium movement from the cell to
lumen maintains the electrical driving force for
sodium-coupled cotransport in the proximal
tubule. Immunohistochemical studies reveal that
KCNE1, a modulatory β-subunit, and the pore-
forming α-subunit KCNQ1, which together con-
stitute the slowly activated component of the
delayed rectifying potassium current in the heart,
colocalize in the luminal membrane of the
8 Physiology of the Developing Kidney: Potassium Homeostasis and Its Disorder
proximal tubule in mouse kidney, as does the
cyclic nucleotide-gated, voltage-activated potas-
sium channel KCNA10 [110]. The observation
that KCNE1 knockout mice exhibit an increased
fractional excretion of fluid (with accompanying
volume depletion), sodium, chloride, and glucose
compared to their wild-type littermates supports a
role for KCNE1 in repolarizing the membrane
potential in proximal tubule in response to
sodium-coupled transport [111]. However,
although mutations in KCNQ1 [112] and
KCNE1 [113, 114] give rise to the long QT syn-
drome, affected patients have not been described
to have proximal tubular transport defects,
suggesting that the role of these channels in
human kidney is not clinically significant.
Thick Ascending Limb of the Loop
of Henle (TALH)
Approximately 10 % of the filtered load of potas-
sium reaches the early distal tubule of the adult rat
[100], an observation that implies that significant
reabsorption of this cation occurs beyond the ini-
tial two-thirds of the proximal tubules that are
accessible to micropuncture. Sites responsible
for this additional avid potassium reabsorption
are the later, deeper part of the proximal tubule,
which is inaccessible to micropuncture analysis,
and the TALH where potassium reabsorption is
mediated, at least in part, by the apical Na-K-2Cl
cotransporter (NKCC2) that translocates a single
potassium ion into the cell accompanied by one
sodium and two chloride ions (Fig. 3). This sec-
ondary active transport is ultimately driven by the
low intracellular sodium concentration,
established by the basolateral Na-K-ATPase,
which drives sodium entry from the lumen into
the cell. The diuretics furosemide and bumetanide
specifically inhibit NKCC2 and thus block
sodium, potassium, and chloride reabsorption at
this site.
Critical to the function of NKCC2 is the pres-
ence of a secretory potassium channel in the uri-
nary membrane which provides a pathway for
potassium, taken up into the cell via the
cotransporter, to recycle back into the lumen.
This “recycling” of potassium counteracts net
potassium reabsorption through NKCC2 but
227
ensures that a continuous supply of substrate is
available for the apical cotransporter. Moreover,
potassium secretion into the urinary space creates
a lumen-positive transepithelial potential differ-
ence, which in turn provides a favorable electrical
driving force that facilitates paracellular
reabsorption of sodium, potassium, calcium, and
magnesium.
The luminal potassium secretory channel in the
TALH has been identified as ROMK, a channel
originally cloned from the TALH in the rat outer
medulla [115–117]; this low-conductance
ATP-sensitive potassium channel is encoded by
the KCNJ1 gene. Loss-of-function mutations in
ROMK lead to antenatal Bartter syndrome
(type 2), also known as the hyperprostaglandin E
syndrome, which is characterized by severe renal
salt and fluid wasting, electrolyte abnormalities
(hypokalemia, hypomagnesemia, and
hypercalciuria), and elevated renin and aldoste-
rone levels [118]. The clinical picture observed
is similar to that of chronic administration of loop
diuretics. The typical presentation of antenatal
Bartter syndrome includes polyhydramnios,
premature delivery, life-threatening episodes of
dehydration during the first week of life, and
profound growth failure [119]. It should be
noted that mutations in either NKCC2
(SLC12A1), or the basolateral chloride channel
CLC-Kb (CLCNKB), or its associated subunit
Barttin (BSND) can also give rise to Bartter syn-
dromes types 1, 3, and 4, respectively [119–121].
In contrast to the situation in the adult, up to
35 % of the filtered load of potassium reaches the
early distal tubule of the young (2-week-old) rat
[97], suggesting that the TALH undergoes a sig-
nificant postnatal increase in its capacity for
reabsorption. Consistent with this premise are
the observations from studies in rats of significant
increases in the (a) fractional reabsorption of
potassium along the TALH, expressed as a per-
centage of delivered load, between the second and
sixth weeks of postnatal life [97], and
(b) osmolarity of early distal fluid between the
second and fourth weeks of life [122]. While
these findings provide compelling evidence for a
developmental maturation of potassium absorp-
tive pathways and diluting capacity of the
228
TALH, respectively, direct functional analysis of
the transport capacity of this segment in the devel-
oping nephron has not been performed.
Molecular studies in rat kidney indicate that
mRNA encoding NKCC2, absent at birth, is first
expressed on postnatal day 8 in rat, coincident
with completion of nephrogenesis [31], and
increases, at least in medulla, between postnatal
days 10 and 40 [123]. Na-K-ATPase activity in the
TALH of the neonatal rabbit is only 20 % of that
measured in the adult when expressed per unit of
dry weight [124]. The postnatal increase in pump
activity is associated with a parallel increase in
expression of medullary Na-K-ATPase mRNA
[123]. Although the balance of the studies sum-
marized above identifies a functional immaturity
of the TALH early in life and would thus predict
limited effects of inhibitors of NKCC2 on
transepithelial transport, administration of furose-
mide (2 mg/kg) to newborn lambs leads to a
natriuretic response equivalent to that observed
in adult animals [125]. Similarly, furosemide is
one of the most commonly prescribed medica-
tions in premature neonates with a demonstrated
natriuretic response [126].
Distal Nephron
Within the aldosterone-sensitive distal nephron
(ASDN) of the fully differentiated kidney, the
late distal convoluted tubule (DCT), connecting
tubule (CNT), and cortical collecting duct (CCD)
are considered to be the primary sites of potassium
secretion, and thus urinary potassium excretion,
which can approach 20 % of the filtered load [100,
102, 127, 128] (Fig. 3). The DCT secretes a con-
stant small amount of potassium into the urinary
fluid [129]. An elevation in plasma potassium
concentration inhibits the thiazide-sensitive
NaCl cotransporter NCC expressed at the apical
membrane of the DCT [130, 131], thereby
increasing the delivery of fluid and sodium into
the distal nephron. This observation may account
for the ability of high potassium diets to lower
blood pressure, in a fashion similar to that of
thiazide administration [132].
Regulated bidirectional potassium transport
occurs in the CNT and CCD, comprised of two
major populations of cells, each with distinct
L.M. Satlin and D. Bockenhauer
functional and morphologic characteristics. In
general, CNT/principal cells reabsorb sodium
and secrete potassium, whereas intercalated cells
regulate acid–base balance but can reabsorb
potassium in response to dietary potassium
restriction or metabolic acidosis (Fig. 3) [133,
134]. Thus, the direction and magnitude of net
potassium transport in these segments depend on
the metabolic needs of the organism and reflect
the balance of potassium secretion and absorption.
Potassium Secretion
Potassium secretion in the CNT and CCD is crit-
ically dependent on the reabsorption of filtered
sodium delivered to these segments. Sodium pas-
sively diffuses into the CNT/principal cell from
the urinary fluid down a favorable concentration
gradient through the luminal amiloride-sensitive
epithelial Na channel (ENaC) and is then
transported out of the cell at the basolateral mem-
brane in exchange for the uptake of potassium via
the basolateral Na-K-ATPase (Fig. 3). The accu-
mulation of potassium within the cell and the
lumen-negative voltage, created by movement of
sodium from the tubular lumen into the cell and its
electrogenic extrusion, creates a favorable electro-
chemical gradient for intracellular potassium to
diffuse into the urinary space through apical
potassium-selective channels. The magnitude of
potassium secretion is determined by its electro-
chemical gradient and the apical permeability to
this cation. Basolateral potassium channels in
these same cells provide a route for intracellular
potassium ions to recycle back into the
interstitium, thereby maintaining the efficiency
of the Na-K pump. Any factor that increases the
electrochemical gradient across the apical mem-
brane or increases the apical potassium permeabil-
ity will promote potassium secretion. An apical
electroneutral potassium chloride cotransporter
has also been functionally identified in the CCD
[135, 136].
Two apical potassium-selective channels have
been functionally identified in the distal nephron:
the small-conductance secretory potassium
(SK) channel and the high-conductance BK chan-
nel. The density of these channels appears to be
greater in the CNT than in the CCD [137].
8 Physiology of the Developing Kidney: Potassium Homeostasis and Its Disorder
The SK channel, restricted to the
CNT/principal cell, mediates baseline constitutive
potassium secretion [138, 139]. ROMK, origi-
nally cloned from the TALH (described in the
section above on the TALH), is considered to
constitute the SK channel. Indeed, there is
absence of SK channel activity in apical mem-
branes from both TALH and collecting duct in
ROMK null mice [140]. This is also consistent
with the clinical observation of initial
hyperkalemia in neonates with Bartter syndrome
type 2 due to loss-of-function of ROMK [141]. A
complex interplay of hormones, second messen-
gers, and kinases/phosphatases regulates the
SK/ROMK channel in the distal nephron, thereby
allowing the kidney to respond appropriately to
the metabolic needs of the organism [142]. Protein
kinase A (PKA)-induced phosphorylation of the
channel is essential for its activity [143, 144] and
may account for the well-documented stimulatory
effect of vasopressin on renal potassium secretion
[145]. Protein tyrosine kinase (PTK) mediates the
endocytosis of ROMK channels in the rat CCD in
the face of dietary potassium restriction [146,
147]. Tyrosine phosphorylation of ROMK
enhances channel internalization and thus the
removal of channels from the plasma membrane
[148], leading to a reduction in surface expression
of channels and net potassium secretion. Tyrosine
phosphorylation of ROMK channels decreases in
response to dietary potassium loading [149].
The “with-no-lysine” kinases, or WNK
kinases, comprise a recently discovered family
of serine/threonine kinases that act as molecular
switches that direct differential effects on down-
stream ion channels, transporters, and the
paracellular pathway to allow either maximal
sodium chloride reabsorption or maximal potas-
sium secretion in response to hypovolemia or
hyperkalemia, respectively [150]. WNK4 inhibits
sodium and chloride absorption in the DCT by
reducing the surface expression of the apical
thiazide-sensitive NaCl cotransporter NCC
[151], an effect that would be expected to increase
sodium delivery to and reabsorption by the CCD,
in turn augmenting the driving force for potassium
secretion. However, WNK4 decreases surface
expression of ROMK by enhancing endocytosis
229
of this channel [152], thereby negating the effect
of the enhanced electrochemical gradient on stim-
ulation of net potassium secretion. Mutations in
the genes encoding WNK1 or WNK4 lead to
pseudohypoaldosteronism type II (PHA II; famil-
ial hyperkalemic hypertension, or FHHt), an auto-
somal dominant disorder characterized by
hypertension sensitive to thiazide diuretics,
hyperkalemia, and metabolic acidosis
[153]. WNK4 harboring PHAII-type mutations
lead to increased apical expression of NCC and
stimulation of sodium absorption in the DCT
[151, 154]. The consequent reduction in sodium
delivery to the CNT and CCD would be expected
to reduce potassium secretion. However, the same
mutations in WNK4 that relieve the inhibition of
NCC further decrease surface expression of
ROMK and thus reduce potassium secretion in
the CCD. Together, these effects explain the car-
dinal symptoms of PHAII (hypertension and
hyperkalemia). WNK1 suppresses the activity of
WNK4. Therefore, a gain-in-function mutation in
WNK1 will also produce the clinical signs and
symptoms of PHA II [153]. Finally, a kidney-
specific short form of WNK1, KS-WNK1,
inhibits WNK1 and can thereby abrogate
WNK-1-mediated inhibition of WNK4
[155]. More recently, mutations in ubiquitin-
ligase elements Kelch-like 3 and Cullin 3 have
been identified as further genetic causes of PHAII
[156, 157], and these proteins are thought to be
involved in ubiquitination and degradation of
WNK4 [158].
The BK channel, present in CNT/principal and
intercalated cells, is considered to mediate flow-
stimulated potassium secretion [159, 160]. In the
CNT and CCD, the density of conducting BK
channels is greater in intercalated than in
CNT/principal cells [161, 162]. The BK channel
is comprised of two subunits: a channel pore-
forming α-subunit and a regulatory β-subunit.
This channel is rarely open at the physiologic
resting membrane potential but can be activated
by cell depolarization, membrane stretch, and
increases in intracellular Ca2+ concentration
elicited by increases in urinary flow rate
[162–165]. The role of the BK channel in flow-
stimulated urinary potassium secretion has been
230
confirmed in mouse models with targeted deletion
of the BKα pore-forming subunit or accessory β1
subunit; the fractional excretion of potassium in
knockout mice subjected to high distal urinary
flow rates is significantly lower than that observed
in wild-type mice [166, 167].
The BK channel assumes great importance in
regulating potassium homeostasis under condi-
tions where SK/ROMK channel-mediated potas-
sium secretion is limited. Thus, adult animals with
genetic ablation of ROMK (i.e., Bartter pheno-
type) are not hyperkalemic, as would be expected
in the absence of a primary potassium secretory
channel, but instead lose urinary potassium
[168]. The sensitivity of distal potassium secre-
tion in this rodent model of Bartter syndrome to
iberiotoxin, a specific inhibitor of BK channels,
presumably reflects recruitment of the latter chan-
nels to secrete potassium in response to high distal
flow rates consequent to loss-of-function of the
TALH NKCC2 cotransporter activity [168]. Simi-
larly, although neonates with antenatal Bartter
syndrome due to loss-of-function mutations in
ROMK (Bartter type 2) may exhibit severe
hyperkalemia during the first few days of life
[169], the hyperkalemia is not sustained. In fact,
these patients typically exhibit modest hypokale-
mia beyond the neonatal period [170, 171]. Of
note is that increases in renal tubular flow rate in
the CCD stimulate PGE2 release and inhibitors of
PGE2 production (indomethacin) inhibit flow-
induced potassium secretion in the CCD
[172]. Indomethacin has long been used as a ther-
apeutic option in polyuric loop disorders, such as
Bartter Syndrome, to mitigate the salt and volume
losses [280].
Emerging evidence now suggests that WNK
kinases regulate apical BK channel expression in
the ASDN. Specifically, WNK4 reduces expres-
sion of the α-subunit of the BK channel, targeting
the protein for degradation through an ubiquitin-
dependent pathway [173].
Potassium Absorption
In response to dietary potassium restriction or
metabolic acidosis, the distal nephron may reverse
the direction of net potassium transport from
secretion to absorption. Potassium reabsorption
L.M. Satlin and D. Bockenhauer
is mediated by an H-K-ATPase, localized to the
apical membrane of acid–base transporting inter-
calated cells, that couples potassium reabsorption
to proton secretion [133, 174–176]. Two isoforms
of the H-K-ATPase are found in the kidney: the
gastric isoform, HKAg, is normally found in gas-
tric parietal cells and is responsible for acid secre-
tion into the lumen whereas the colonic HKAc
isoform is a structurally related H-K-ATPase
found in distal colon that mediates active potas-
sium reabsorption [175]. Expression of the apical
gastric-like H-K-ATPase in the rat and rabbit
intercalated cell is increased in response to dietary
potassium restriction and metabolic acidosis [133,
174, 177, 178].
A reduction in potassium intake leads to a fall
in potassium secretion by the distal nephron
within 5–7 days in rat [179]. This adaptation is
associated with a decrease in the number of apical
SK/ROMK [180] and BK [181] channels and
stimulation of H-K-ATPase-mediated potassium
reabsorption in intercalated cells in the distal
nephron [182]. The reduction in number of
SK/ROMK channels in potassium-restricted ani-
mals is mediated by the effect of dietary potassium
on circulating levels of aldosterone and other
effectors, such as PTK, as described above. Stim-
ulation of luminal H-K-ATPase activity in inter-
calated cells results not only in potassium
retention but also in urinary acidification and met-
abolic alkalosis.
Developmental Regulation of Distal
Potassium Transport
Potassium secretion in the distal nephron, and spe-
cifically in the cortical collecting duct (CCD) stud-
ied in vitro, is low early in life and cannot be
stimulated by high urinary flow rates [128]. Indeed,
basal potassium secretion cannot be detected in the
rabbit CCD until after the third week of postnatal
life, just about the time of weaning, with potassium
secretory rates increasing thereafter to reach adult
levels by 6 weeks of age [128]. Consistent with the
relatively undifferentiated state of the newborn
CCD are the ultrastructural and morphometric find-
ings in neonatal principal cells of few organelles,
mitochondria, and basolateral infoldings, the site of
Na-K-ATPase [183, 184].
8 Physiology of the Developing Kidney: Potassium Homeostasis and Its Disorder
The limited capacity of the CCD for potassium
secretion early in life could be explained by either
an unfavorable electrochemical gradient across
the apical membrane or a limited apical perme-
ability to this ion. Cumulative evidence suggests
that the electrochemical gradient is not limiting
for potassium secretion in the neonate. Activity of
the Na-K-ATPase, present along the basolateral
membrane of corticomedullary collecting ducts in
the neonatal rabbit [185], is 50 % of that measured
in the mature nephron; the observation that the
intracellular potassium concentration in this seg-
ment is similar in the neonate and adult presum-
ably reflects a relative paucity of membrane
potassium channels in the distal nephron early in
life [124, 184, 186]. Concordant with the mea-
surements of sodium pump activity, the rate of
sodium absorption in the CCD at 2 weeks of age
is approximately 60 % of that measured in the
adult [128]. However, electrophysiologic analysis
has confirmed the absence of functional
SK/ROMK channels in the luminal membrane of
the neonatal rabbit CCD with the number of open
channels per patch increasing progressively after
the second week of life [187]. Thus, the postnatal
increase in the basal potassium secretory capacity
of the distal nephron appears to be due primarily
to a developmental increase in number of
SK/ROMK channels, reflecting an increase in
transcription and translation of functional channel
proteins [187–189].
The appearance of flow-stimulated net potas-
sium secretion is a relatively late developmental
event. Flow-stimulated potassium secretion can-
not be elicited in rabbit CCDs until the fifth week
of postnatal life, which is approximately 2 weeks
after basal net potassium secretion is first detected
[128, 190]. The absence of flow-stimulated potas-
sium secretion early in life is not due to a limited
flow-induced rise in net sodium absorption and/or
intracellular calcium concentration, each of which
is equivalent to that detected in the adult by the
second week of postnatal life [190]. The observa-
tion that mRNA encoding the BK channel
α-subunit and immunodetectable channel protein
cannot be demonstrated until the fourth and fifth
weeks of postnatal life, respectively [190], sug-
gests that flow-dependent potassium secretion is
231
determined by the transcriptional/translational
regulation of expression of BK channels.
While the neonatal distal nephron is limited in
its capacity for potassium secretion, indirect evi-
dences suggest that this nephron segment absorbs
potassium. As indicated above, saline-expanded
newborn dogs absorb 25 % more of the distal
potassium load than do their adult counterparts
[95]. Functional analysis of the rabbit collecting
duct has shown that the activity of apical H-K-
ATPase in neonatal intercalated cells is equivalent
to that in mature cells [133]. The latter data alone
do not predict transepithelial potassium absorp-
tion under physiologic conditions, as the balance
of transport will depend on the presence and
activity of apical and basolateral potassium con-
ductances and the potassium concentration of the
tubular fluid delivered to this site. The high distal
tubular fluid potassium concentrations, as mea-
sured in vivo in the young rat, may facilitate
lumen-to-cell potassium absorption mediated by
the H-K-ATPase [97].
Luminal and Peritubular Factors
Affecting Potassium Transport
The major factors that influence the external bal-
ance of potassium are listed in Table 2 and are
discussed in the following sections.
Sodium Delivery and Absorption
The magnitude of sodium reabsorption in the dis-
tal nephron determines the electrochemical
Table 2 Factors that regulate external potassium balance
Renal factors
Potassium intake/plasma potassium concentration
Tubular (urinary) flow rate
Distal sodium delivery and transepithelial voltage
Hormones (mineralocorticoids, vasopressin)
Acid–base balance
Gastrointestinal tract factors
Stool volume
Hormones (mineralocorticoids)
232
driving force favoring potassium secretion into
the luminal fluid, as described above. Processes
that enhance distal sodium delivery and increase
tubular fluid flow rate, such as extracellular vol-
ume expansion or administration of a variety of
diuretics (osmotic diuretics, carbonic anhydrase
inhibitors, loop and thiazide diuretics), lead to an
increase in urinary excretion of both sodium and
potassium. The kaliuresis is due not only to the
increased delivery of sodium to the distal nephron
but also to the increase in tubular fluid flow rate,
which maximizes the chemical driving forces, as
described below, favoring potassium secretion.
Processes that decrease sodium delivery to less
than 30 mM in the distal tubular fluid [191, 192]
and/or sodium reabsorption sharply reduce potas-
sium secretion in the CCD and can lead to
hyperkalemia. In vivo measurements of the
sodium concentration in distal tubular fluid gen-
erally exceed 35 mEq/l both in healthy adult and
suckling rats and thus should not restrict distal
sodium secretion [97, 122, 191, 193]. However,
in edema-forming states, including congestive
heart failure, cirrhosis, and nephrotic syndrome,
the urinary sodium concentration typically falls to
less than 10 mEq/l; a reduction in potassium
excretion in these patients may be ascribed to the
low rates of distal sodium delivery as well as
urinary flow. The potassium-sparing diuretics,
amiloride and triamterene, inhibit ENaC and
thus block sodium absorption, thereby
diminishing the electrochemical gradient favoring
potassium secretion [194, 195]. Trimethoprim and
pentamidine can also limit urinary potassium
secretion via the same mechanism [196, 197].
Sodium delivered to the distal nephron is gen-
erally accompanied by chloride. Chloride
reabsorption occurs in large part via the
paracellular pathway. The movement of the nega-
tively charged chloride out of the lumen dissipates
the lumen-negative potential, creating a less
favorable driving force for luminal potassium
secretion [198]. When sodium delivered to the
distal nephron is accompanied by an anion less
reabsorbable than chloride, such as bicarbonate
(in proximal renal tubular acidosis),
β-hydroxybutyrate (in diabetic ketoacidosis), or
carbenicillin (during antibiotic therapy), luminal
L.M. Satlin and D. Bockenhauer
electronegativity is maintained, thereby eliciting
more potassium secretion than what occurs with a
comparable sodium load delivered with
chloride [199].
Tubular Flow Rate
High rates of urinary flow in the mature, but not
the neonatal or weanling, distal nephron, as
elicited by extracellular fluid volume expansion
or administration of diuretics or osmotic agents,
stimulate potassium secretion [128]. There are a
number of factors responsible for the flow stimu-
lation of potassium secretion. First, increases in
tubular fluid flow rate in the distal nephron
enhance sodium reabsorption due to an increase
in the open probability of ENaC (time the channel
spends in the open state), which augments the
electrochemical gradient favoring potassium
secretion [195, 200]. Second, the higher the uri-
nary flow rate in the distal nephron, the slower the
rate of rise of tubular fluid potassium concentra-
tion because secreted potassium is rapidly diluted
in urine of low potassium concentration
[201]. Maintenance of a low tubular fluid potas-
sium concentration maximizes the potassium
concentration gradient (and thus the chemical
driving force) favoring net potassium secretion.
Finally, increases in luminal flow rate transduce
mechanical signals (circumferential stretch,
shear stress, hydrodynamic bending moments on
the cilium decorating the apical surface of
virtually all renal epithelial cells) into increases
in intracellular calcium concentration, which in
turn activate apical BK channels to secrete
potassium, thereby enhancing urinary potassium
excretion [160, 190].
Potassium Intake and Cellular
Potassium Content
The kidney adjusts potassium excretion to match
input, in large part by regulating the magnitude of
potassium secretion and reabsorption in the distal
nephron. Thus, for example, an increase in dietary
potassium intake stimulates whereas a decrease in
8 Physiology of the Developing Kidney: Potassium Homeostasis and Its Disorder
intake reduces net potassium secretion [102]. An
increase in potassium concentration in the extra-
cellular fluid space increases potassium entry into
principal cells via the basolateral Na-K-ATPase,
which in turn maximizes the concentration gradi-
ent favoring apical potassium secretion into the
urinary fluid. Simultaneously, the increase in
circulating levels of plasma aldosterone that
accompanies potassium loading enhances the
electrochemical driving force favoring potassium
secretion in the distal nephron by stimulation of
ENaC-mediated sodium absorption and its elec-
trogenic absorption via the Na-K-ATPase. Within
6 h of an increase in dietary potassium intake, the
density of apical ROMK channels increases in
principal cells in rats due to activation of a previ-
ously “silent” pool of channels or closely associ-
ated proteins [202]. Chronic potassium loading
also increases BK channel message, apical pro-
tein, and function in the distal nephron
[181]. Finally, the inhibition of proximal tubule
and loop of Henle salt and water reabsorption in
response to a potassium load [203, 204] increases
tubule fluid flow rate which in turn stimulates
potassium secretion via activation of BK channels
and increased distal delivery of sodium to the
distal nephron.
The trigger for the renal adaptation to dietary
potassium loading remains uncertain. It is now
believed that after a potassium-rich meal, a reflex
increase in potassium excretion is initiated by
sensors in the splanchnic bed (gut, portal circula-
tion, and/or liver) that respond to local increases in
potassium concentration that occur in the absence
of changes in plasma potassium concentration or
before changes in plasma potassium concentra-
tion are detected [205, 206]. In support of the
notion of potassium sensing, intraportal delivery
of potassium chloride to rats leads to an increase
in hepatic afferent nerve activity and urinary
potassium excretion, responses that are unaccom-
panied by increases in plasma potassium concen-
tration [207]. Increases in the intraportal
concentration of glucagon, following ingestion
of a protein- and potassium-rich meal, also
increase renal excretion of potassium [208, 209],
a response proposed to be due to the release of
cAMP, the second messenger of glucagon in the
233
liver, into the circulation and its uptake by
kidney [206].
Chronic potassium loading leads to potassium
adaptation, an acquired tolerance to an otherwise
lethal acute potassium load [8]. Potassium adap-
tation, which begins after a single potassium-rich
meal, includes increases in the rate of skeletal
muscle uptake of potassium from the extracellular
fluid [56] due to stimulation of Na-K-ATPase
activity [210] and secretion of potassium by the
distal nephron and colon. The process is facili-
tated by the increase in circulating levels of aldo-
sterone elicited by the increase in serum
potassium concentration [211]. A similar adaptive
response is seen in renal insufficiency such that
potassium balance is maintained during the course
of many forms of progressive renal disease, as
long as potassium intake is not excessive
[212]. The molecular mechanisms underlying
this adaptation in the distal nephron (and colon)
include not only an increase in the density of
apical membrane potassium channels but also an
increase in the number of conducting ENaC chan-
nels and activity of the basolateral Na-K pump.
The latter two processes result in increases in
transepithelial voltage and the intracellular potas-
sium concentration, events that enhance the driv-
ing force favoring potassium diffusion from the
cell into the urinary fluid.
Hormones
Mineralocorticoids are key regulators of renal
sodium absorption and potassium excretion and
thus of circulating volume, blood pressure, and
sodium and potassium homeostasis. There are two
major physiologic stimuli for aldosterone release
from the zona glomerulosa in the adrenal gland
[213]: angiotensin II (AII) and elevation in serum
potassium concentration [214]. AII, generated in
response to intravascular volume depletion by
activation of the renin-angiotensin system, binds
to the type 1 AII receptor (AT1) to increase aldo-
sterone production. ACE inhibitors, by reducing
the conversion of angiotensin I to angiotensin II
and thus aldosterone secretion by the adrenal
gland, may lead to hyperkalemia as the ability of
234
the distal nephron to secrete potassium is
impaired. AII receptor blockers (ARBs), by com-
petitively binding to AT1 receptors and thus
antagonizing the action of AII on aldosterone
release, may have similar effects.
Aldosterone stimulates sodium reabsorption
and potassium secretion in principal cells of the
fully differentiated ASDN [215, 216]. Circulating
mineralocorticoids bind to their cytosolic recep-
tors in the ASDN; the aldosterone antagonists
spironolactone and eplerenone competitively
inhibit this binding. The hormone–receptor com-
plex translocates to the nucleus where it promotes
the transcription of aldosterone-induced physio-
logically active proteins (e.g., Na-K-ATPase).
Among the cellular and molecular effects of an
increase in circulating levels of aldosterone are
increases in density of ENaC channels, achieved
by the recruitment to and retention of intracellular
channels at the apical membrane, de novo synthe-
sis of new ENaC subunits, and activation of
preexistent channels, as well as stimulation
of Na-K-ATPase activity by translocation of
preformed transporters to the membrane and
translation of new sodium pump subunits
[217–222]. The sum effect of the stimulation of
apical sodium entry and Na-pump-mediated
reabsorption is an increase in lumen-negative
transepithelial voltage and thus electrochemical
driving force favoring potassium exit across the
apical membrane [217, 220, 223].
The effects of aldosterone on ENaC and, to
some extent, the Na-K pump appear to be indirect,
mediated by aldosterone-induced proteins,
including serum and glucocorticoid-inducible
kinase (sgk). Aldosterone rapidly induces Sgk1
in the distal nephron [224]. Phosphorylated Sgk
stimulates sodium reabsorption, in large part by
inhibiting ubiquitin-ligase Nedd4-2-mediated
endocytic retrieval of ENaCs from the luminal
membrane [225]. Renal water and electrolyte
excretion is indistinguishable in sgk1-knockout
(sgk1/) and wild-type (sgk1+/+) mice fed a nor-
mal diet [226], indicating that the kinase is not
necessary for basal sodium absorption. However,
dietary sodium restriction reveals an impaired
ability of sgk1/ mice to reduce sodium excre-
tion despite increases in plasma aldosterone levels
L.M. Satlin and D. Bockenhauer
and proximal tubular sodium and fluid
reabsorption [227]. Sgk1/ mice exhibit an
impaired upregulation of renal potassium excre-
tion in response to potassium loading, presumably
due to the impact of the loss of SGK1 function on
ENaC and/or Na-K-ATPase activity and thus the
electrochemical gradient favoring potassium
secretion [228]. Corticosteroid hormone-induced
factor (CHIF) is another aldosterone-induced pro-
tein that is expressed in the basolateral membrane
of the collecting duct where it increases the affin-
ity of Na-K-ATPase for sodium [229–232].
Plasma aldosterone concentrations in prema-
ture infants and newborns are higher than those
measured in the adult [17, 233]. Yet, clearance
studies in fetal and newborn animals demonstrate
a relative insensitivity of the immature kidney to
the hormone [17, 234–236]. The density of aldo-
sterone binding sites, receptor affinity, and degree
of nuclear binding of hormone receptor are
believed to be similar in mature and immature
rats [236].
The transtubular potassium gradient (TTKG)
provides an indirect, semiquantitative measure of
the renal response to mineralocorticoid activity in
the aldosterone-sensitive cortical distal nephron
and is calculated using the equation: TTKG =
U[K]/P[K] divided by Uosmolality/Posmolality, where
[K] equals the potassium concentration in mEq/l
[237–239]. Measurements of TTKG have been
reported to be lower in 27- than 30-week GA
preterm infants followed over the first 5 weeks
of postnatal life [240]. The low TTKG has been
attributed to a state of relative hypoaldosteronism
[240] but may also reflect the absence of potas-
sium secretory transport pathways (i.e., channel
proteins).
Acid–Base Balance
Disorders of acid–base homeostasis induce
changes in tubular potassium secretion in the dis-
tal nephron [198]. In general, acute metabolic
acidosis causes the urine pH and potassium excre-
tion to decrease, whereas both acute respiratory
alkalosis and metabolic alkalosis increase urine
pH and potassium excretion [198, 241]. Chronic
8 Physiology of the Developing Kidney: Potassium Homeostasis and Its Disorder
metabolic acidosis has variable effects on urinary
potassium excretion.
Acute metabolic acidosis reduces cell potas-
sium concentration and leads to a reduction in
urine pH, which in turns inhibits activity of the
SK/ROMK channel and thereby limits potassium
secretion in the distal nephron [139,
241–243]. This makes physiological sense, as
the electrical balance for sodium reabsorption by
either potassium or proton secretion is thus
maintained by increased proton secretion to
restore acid–base homeostasis.
The effect of chronic metabolic acidosis on
potassium secretion is more complex and may be
influenced by modifications of the glomerular fil-
trate (e.g., chloride and bicarbonate concentra-
tions), tubular fluid flow rate, and circulating
aldosterone levels [8, 198]. The latter two factors
may lead to an increase rather than a decrease in
potassium secretion and excretion.
The alkalosis-induced stimulation of potas-
sium secretion reflects two direct effects on prin-
cipal cells: (1) an increase in net sodium
absorption [244], which enhances the electro-
chemical gradient for net potassium secretion,
and (2) an increase in the permeability of the
apical membrane to potassium resulting from an
increase in duration of time the potassium-
selective channels remain open [8]. Alkalosis
also decreases acid secretion in intercalated cells,
thereby reducing H-K-ATPase-mediated
countertransport of potassium.
Potassium deficiency stimulates proton secre-
tion in the distal nephron, increases the production
of the urinary buffer ammonia [245], and may
stimulate bicarbonate generation by increasing
expression of H-K-ATPase in the distal
nephron [246].
Circadian Control of Renal Potassium
Excretion
Renal potassium excretion varies during the 24-h
day in the adult [247, 248]. Although some of this
variation can be attributed to timing of meals, a
pattern of low rates of potassium excretion at
night and in the morning and high rates in the
235
afternoon emerges, even when potassium intake
ingestion is spread evenly throughout the day and
night [247, 248]. As with other circadian rhythms,
evidence suggests that the periodicity depends on
both central and peripheral pacemakers. Notably,
the DCT and CCD express several regulatory
“clock” genes, indicating operation of a local
intrarenal pacemaker. ENaC, aquaporin 2, and
vasopressin receptors have marked circadian
rhythms, which could influence indirectly the
excretion of potassium [249, 250]. Moreover,
recent data from space flight simulation suggest
that there is also an infradian rhythmicity to aldo-
sterone secretion [251]. It is unknown whether a
circadian cycle of urinary potassium excretion
prevails in infancy.
Contribution of the Gastrointestinal
Tract
Under normal conditions in the adult, 5–10 % of
daily potassium intake is excreted in the stool. The
colon is considered to be the main target for reg-
ulation of intestinal potassium excretion
[252]. Potassium transport in the colon represents
the balance of secretion and absorption
[253]. Under baseline conditions, net potassium
secretion predominates over absorption in the
adult, whereas the neonatal gut is poised for net
potassium absorption [252].
Potassium secretion requires potassium uptake
by the Na-K-ATPase and furosemide-sensitive
Na-K-2Cl cotransporter located in the basolateral
membrane of colonocytes; potassium is then
secreted across the apical membrane through
potassium channels, including a calcium-
activated BK channel similar to that found in the
distal nephron [254–258]. Potassium absorption
is mediated by two H-K-ATPases localized to the
apical membrane of the distal colon [259].
Stool potassium content can be enhanced by
any factor that increases colonic secretion, includ-
ing aldosterone, epinephrine, and prostaglandins
[24, 260, 261]. Indomethacin and dietary potas-
sium restriction reduce potassium secretion by
inhibiting the basolateral transporters and apical
potassium channels. Diarrheal illnesses are typi-
cally associated with hypokalemia, presumably
due to the presence of nonreabsorbed anions
236
(which obligate potassium), an enhanced electro-
chemical gradient established by active chloride
secretion, and secondary hyperaldosteronism due
to volume contraction [262].
Potassium adaptation in the colon is demon-
strated by increased fecal potassium secretion
after potassium loading and in the face of renal
insufficiency. Fecal potassium excretion may
increase substantially to account for 30–50 % of
potassium excretion in patients with severe
chronic renal insufficiency [24, 263–265]. The
enhanced colonic potassium secretion character-
istic of renal insufficiency requires induction
and/or activation of apical BK channels in surface
colonic epithelial cells [266].
Net colonic potassium absorption is signifi-
cantly higher in young compared to adult
rats [252]. The higher rate of potassium absorp-
tion during infancy is due to robust activity of
apical K-ATPases and limited activity of the
basolateral transporters that mediate secretion
[252, 256, 267].
Mendelian Disorders Associated
with Altered Plasma Potassium Levels
There are several inherited disorders, which can
affect plasma potassium levels either by altering
internal potassium balance between the intra- and
extracellular fluid compartments or renal potas-
sium handling. These disorders are summarized in
Table 3. Most of these disorders are discussed in
detail in their respective chapters.
Clinical Approach to the Patient
with Hyperkalemia
Cardiac arrhythmias are feared consequences of
severe hyperkalemia and constitute a medical
emergency. Immediate treatment usually focuses
on enhancing potassium uptake by cells. This can
be accomplished by administration of insulin/glu-
cose or β-sympathomimetics, such as albuterol. In
asymptomatic patients, more time is available to
consider potential causes, allowing for specific
treatment.
Pseudohyperkalemia
Pseudohyperkalemia refers to elevated in vitro
potassium concentrations that do not reflect the
L.M. Satlin and D. Bockenhauer
true plasma level but occur during or after blood
drawing. This is a common cause of
hyperkalemia, especially in small children,
where obtaining a free-flowing blood specimen
is difficult. Small needle size, squeezing of the
extremity, and use of a tourniquet can all contrib-
ute to cell lysis with subsequent release of potas-
sium [268–270]. In addition, release of potassium
after phlebotomy due to delayed processing of the
specimen, the presence of an increased cellular
component of blood, or altered potassium trans-
port activity of the cell membrane can lead to
pseudohyperkalemia [271, 272]. Most laborato-
ries will alert a healthcare provider as to the pres-
ence of hemolysis. A repeat sample with prompt
processing will establish a normal plasma level in
most cases. In the interim, an ECG may help
assess the degree of hyperkalemia, as judged by
the height of the T-waves, although the sensitivity
of this tool is reportedly poor [273].
Altered Internal Balance
Hyperkalemia due to an altered internal balance
should be suspected if there are suggestive factors,
such as metabolic acidosis and/or poorly con-
trolled diabetes mellitus (see also Table 1). Treat-
ment in these cases should focus on correcting the
underlying abnormality.
Altered External Balance
Hyperkalemia due to an altered external balance
may be due to excessive potassium intake,
impaired renal potassium secretion, or a combina-
tion of both. With normal kidney function, even a
very high potassium intake should not lead to
hyperkalemia, due to the rapid hormonally medi-
ated translocation of potassium into cells, unless
administered as an acute bolus [274]. Commonly
used tools to assess renal potassium secretion
include the fractional excretion of potassium
(FEK), the ratio of urinary potassium to sodium,
and the TTKG, described earlier in this chapter.
The TTKG is considered to be specific for the
assessment of distal mineralocorticoid-sensitive
potassium secretion independent of the glomeru-
lar filtration rate [237, 238, 275]. In one study in
children, a TTKG value of 4.1 corresponded to the
3rd percentile for TTKG results and 13.1 to the
8 Physiology of the Developing Kidney: Potassium Homeostasis and Its Disorder 237

Table 3 Genetic disorders associated with altered plasma potassium levels

Disease OMIM# Gene(s) Mode Mechanism
Hyperkalaemic disorders
Hyperkalaemic periodic paralysis 170500 SCN4A AD Acute release of potassium from muscle
cells
Pseudohypoaldosteronism type 1 264350 SCNN1A AR Impaired renal potassium secretion
SCNN1B AR
SCNN1G AR
600983 NR3C2 AD
Pseudohypoaldosteronism type 614492 WNK1 AD Impaired renal potassium secretion
2 (Fhht; Gordon syndrome) 614491 WNK4 AD
614495 KLH3 AD
614496 CUL3 AD
or
AR
Congenital adrenal hyperplasia 201910 CYP21A2 AR Impaired renal potassium secretion due to
(salt wasting forms) deficiency in mineralocorticoids
Hypokalaemic disorders associated with low or normal blood pressure
Hypokalaemic periodic paralysis 170400 CACNA1S AD Acute potassium uptake by muscle cells
613345 SCN4A AD
Renal Fanconi syndromes 134600 ? AD Increased renal potassium secretion
613388 SLC34A1 AR
615605 EHHADH AD
Bartter syndrome 601678 SLC12A1 AR Increased renal potassium secretion
241200 KCNJ1 AR
607364 CLCKNB AR
602522 BSND AR
Gitelman syndrome 263800 SLC12A3 AR Increased renal potassium secretion
EAST (SeSAME) syndrome 612780 KCNJ10 AR Increased renal potassium secretion
Hypokalaemic disorders associated with elevated blood pressure
Congenital adrenal hyperplasia 202010 CYP11B1 AR Increased renal potassium secretion due to
(salt retaining forms) excess mineralocorticoids
Liddle syndrome 177200 SCNN1G AD Increased renal potassium secretion
Apparent mineralocorticoid excess 218030 HSD11B2 AR Increased renal potassium secretion
Familial hyperaldosteronism 605635 ? AD Increased renal potassium secretion
613677 KCNJ5 AD
Glucocorticoid-remediable 103900 CYP11B1 AD Increased renal potassium secretion
hyperaldosteronism
Distal renal tubular acidosis 267300 ATP6V1B1 AR Increased renal potassium secretion
ATP6V0A4 AR
179800 SLC4A1 AD
Listed are Mendelian disorders associated with altered plasma potassium levels, their respective OMIM numbers, mode of
inheritance and the key mechanism for dyskalemia. The disorders listed in the upper panel of the table are associated with
hyperkalemia, whereas those in the lower panels with hypokalemia, the latter grouped according to expected blood
pressure
Not listed are disorders leading to end-stage kidney disease and associated hyperkalemia, nor metabolic disorders with
secondary tubular dysfunction (e.g., cystinosis, mitochondrial cytopathies), nor intestinal salt wasting disorders with
secondary hyperaldosteronism (e.g., congenital chloride diarrhea)
AR autosomal recessive, AD autosomal dominant, ? gene unidentified yet
238
97th percentile [240]. Accordingly, in
hyperkalemia, a TTKG value below 4.1 would
be inappropriate and indicate low mineralocorti-
coid activity, suggesting impaired renal potassium
secretion. Conversely, a TTKG value above 13.1
is consistent with high mineralocorticoid activity
and would thus suggest excessive potassium
intake as the underlying etiology of the
hyperkalemia.
Treatment varies depending on the etiology of
the hyperkalemia. Intake of potassium should be
reduced if indicated. In those with impaired potas-
sium secretion, the cause should be identified. If
clinical evidence of volume depletion is present,
hyperkalemia may simply reflect inadequate distal
sodium delivery and treatment should be aimed at
volume expansion. This approach would also be
appropriate for the rare patients with type I
pseudohypoaldosteronism (PHA), who may
require supplementation with sodium chloride or
sodium bicarbonate and, possibly, treatment with
potassium-binding resins [276]. In patients who
present with hyperkalemia and hypertension and
low TTKG, PHAII (FHHt, see above) should be
suspected. Administration of calcineurin inhibi-
tors can result in a clinical picture similar to
PHAII and, like PHAII, responds to thiazide
diuretics [277]. Type III PHA is typically transient
and secondary to other pathologies such as
obstructive uropathy or urinary tract
infection [278].
Clinical Approach to the Patient
with Hypokalemia
Cardiac arrhythmias, muscle weakness, and even
paralysis are major morbidities associated with
severe hypokalemia, and emergency treatment usu-
ally consists of potassium administration. In asymp-
tomatic patients, more time is available to consider
potential causes, allowing for specific treatment.
Altered Internal Balance
Hypokalemia due to an altered internal balance
should be suspected if there are suggestive factors,
such as metabolic alkalosis or evidence of
increased insulin activity (see also Table 1). Treat-
ment in these cases should focus on correcting the
underlying abnormality.
L.M. Satlin and D. Bockenhauer
Altered External Balance
Hypokalemia due to an altered external balance
can be either from excessive potassium loss from
the intestine or kidney, insufficient intake, or a
combination of these factors. As with
hyperkalemia, assessment of the urinary potas-
sium excretion can clarify the role of the kidney
in the etiology. A low TTKG suggests renal potas-
sium conservation and thus points towards intes-
tinal losses and/or insufficient intake. Again,
treatment should focus on the underlying abnor-
mality. Conversely, a high TTKG points to renal
potassium wasting. In the context of volume
depletion, this finding would likely reflect
aldosterone-mediated attempts to maintain extra-
cellular volume at the expense of potassium
homoeostasis and volume repletion is the appro-
priate treatment. Hypokalemia with high TTKG in
the presence of hypervolemia and hypertension
suggests primary hyperaldosteronism or the rare
forms of primary increased distal sodium
reabsorption/potassium secretion (“pseudohyper-
aldosteronism”) as seen in some inherited disor-
ders, such as Liddle syndrome or apparent
mineralocorticoid excess (see Table 3). This
pseudohyperaldosteronism can also be acquired,
e.g., with licorice abuse [279]. Treatment should
focus on the underlying defect. In the short term,
or if the underlying problem cannot be targeted,
distal potassium secretion can be reduced either
by administration of inhibitors of ENaC
(amiloride) and/or mineralocorticoid receptor
(spironolactone or eplerenone).
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 Physiology of the Developing Kidney:
Acid-Base Homeostasis and Its Disorders 9
Peter D. Yorgin, Elizabeth G. Ingulli, and Robert H. Mak

Contents Interpretation of Acid–Base Status

from Blood Gas Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248 General Approach to Evaluating
General Acid–Base Concepts . . . . . . . . . . . . . . . . . . . . . . 248 Acid–Base Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248 pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
The Henderson–Hasselbalch Equation . . . . . . . . . . . . . . 248 Bicarbonate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
Buffering Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 Base Excess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

Proximal Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 Metabolic and Respiratory Derangements . . . . . . . 260

Metabolic Acidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
NHE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 Respiratory Acidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
Discriminating Between Acute and Chronic
V-ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Respiratory Acidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
Na+/K+-ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Metabolic Alkalosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
Carbonic Anhydrase (CA) . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
Respiratory Alkalosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Na+/HCO 3 Cotransporter (NBC) . . . . . . . . . . . . . . . . . . . . 252
Simple Acid–Base Disorders . . . . . . . . . . . . . . . . . . . . . . . . 265
Non-proximal Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . 252 Mixed Acid–Base Disorders . . . . . . . . . . . . . . . . . . . . . . . . . 265
Loop of Henle and Thick Ascending Loop . . . . . . . 252 Nutrition and Acid–Base Balance . . . . . . . . . . . . . . . . . 267
Distal Convoluted Tubule . . . . . . . . . . . . . . . . . . . . . . . . . . 253 Acid–Base Balance in Neonates, Infants,
and Children . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
Ammonia Production and Transport . . . . . . . . . . . . . 254
Mechanisms of Growth Retardation in
Chronic Acidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
Effect of Acidosis on Bone and Calcium
Homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
P.D. Yorgin • R.H. Mak (*)
Acid–Base Balance in CKD . . . . . . . . . . . . . . . . . . . . . . . . 272
Pediatric Nephrology, University of California, San Diego,
CA, USA References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
Pediatric Nephrology Division, Rady Children’s Hospital,
San Diego, CA, USA
e-mail: romak@ucsd.edu
E.G. Ingulli
Department of Pediatrics, University of California,
San Diego, CA, USA
Division of Nephrology, Rady Children’s Hospital
San Diego, San Diego, CA, USA
Kidney Transplant Program, Rady Children’s Hospital,
San Diego, CA, USA

# Springer-Verlag Berlin Heidelberg 2016 247

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_8
248
Introduction
The body is highly dependent on acid–base control
by the kidneys, lungs, and buffer systems to
provide a cellular environment suitable for normal
health, growth, and development. The acid and
alkali loads from ingesting food and fluid must be
managed so that the extracellular hydrogen
ion (H+) concentration is maintained within a very
narrow range. There are serious consequences
from acid–base perturbations. Patients with severe
acidemia, high blood levels of H+, may have
problems with hyperkalemia, increased susceptibil-
ity to cardiac dysrhythmias, osteopenia, recurrent
nephrolithiasis, skeletal muscle atrophy, and
growth retardation in children. Conversely, patients
with severe alkalemia, low blood H+ concentration,
may experience arteriolar constriction, refractory
dysrhythmias, hypoventilation, hypokalemia,
decreased ionized calcium, paresthesias, and even
coma. The following information will serve as
practical assistance to physicians who manage
acid–base problems in children.
General Acid–Base Concepts
An acid is a compound, which donates a H+, often
referred to as a proton, to a base, which is a proton
acceptor. Protons react easily and rapidly with
other molecules in serum and particularly with
water (H2O). There are strong and weak acids.
Strong acids, like hydrogen chloride and sulfuric
acid, are good at donating hydrogen ions, and they
ionize fully when dissolved in water. Weak acids,
like carbonic acid, do not ionize fully when
dissolved in water. Solutions of weak acids and
salts of their conjugate bases form buffer solu-
tions. An acid is considered to be dissociated,
after the H+ has been donated. An alkali is a base
that is soluble in water and donates hydroxide ions
(OH).
pH
pH provides a convenient means of expressing the
concentration of H+ in water. pH utilizes a
P.D. Yorgin et al.
logarithmic scale to express H+ molality in a solu-
tion. The equation for pH is:
pH ¼  log ½Hþ 
A solution with a pH of 6 has 10 times the amount
of H+ than a solution with a pH of 7. In water, the
pH of an acidic solution is less than 7 (H+ concen-
tration = 107 mmol/L), and a pH greater than
7 is alkaline. In a healthy individual, the extracel-
lular fluid pH is maintained within a narrow range,
pH 7.38–7.42 (H+ concentration = 107.38–7.42 M).
Serious health consequences are associated with
abnormal blood pH values.
The Henderson--Hasselbalch Equation
The pH of blood can be directly measured, using a
pH probe (and the Nernst equation), by photo-
chemical sensors (optodes) [1] and semiconductor
sensor [2] methods, or can be calculated using a
modified version of the Henderson–Hasselbalch
equation [3] which describes the relationship
between carbonic acid and bicarbonate:
H2 CO3 $ Hþ þ HCO3 
The equation can be modified so that the con-
centration of H+ can be determined by knowing
both the HCO3 and H2CO3 concentrations.
log½Hþ  ¼ pH ¼ pK þ log ð½HCO3  =½H2 CO3 Þ
The pK, in this equation, represents the pH
value at which HCO3 and H2CO3 are found in
equal concentrations and represents the pH at
which there is the greatest amount of buffering
capacity. The pK of the CO2 – HCO3 buffering
system is 6.1. At a pH of 7.40, carbonic acid
concentrations are very low and cannot be mea-
sured easily. In most equations, the carbonic acid
value is replaced by a solubility coefficient multi-
plied by the partial pressure of carbon dioxide
(PaCO2), which can be easily measured. pH can
then be defined:
pH ¼ pKa þ logð½HCO3  =½ð0:0308ÞðCO2 ÞÞ
9 Physiology of the Developing Kidney: Acid-Base Homeostasis and Its Disorders
Buffering Systems
CO2/Bicarbonate Buffering System
Buffers, found within cells and in blood, quickly
limit the change in extracellular H+ concentrations
by combining with an acid or a base. An
acid–base buffer is usually composed of two or
more chemical compounds.
The carbonic acid + bicarbonate extracellular
buffering system has two buffering molecules:
carbonic acid (H2CO3) and bicarbonate (HCO3).
H+ and bicarbonate can combine to form carbonic
acid. Carbonic acid, in a reversible process, disso-
ciates to yield water and carbon dioxide:
Hþ þ HCO3  $ H2 CO3 $ H2 O þ CO2
If an acid is added to the carbonic acid + bicar-
bonate buffering system, more carbonic acid is
generated as shown in the following reaction:
Hþ þ HCO3  $ H2 CO3
If a base is added to the carbonic acid + bicarbon-
ate buffering system, more bicarbonate is gener-
ated as shown in the following reaction:
OH þ H2 CO3 $ H2 O þ HCO3 
Phosphate and Protein Buffering
Systems
The phosphate buffering system functions like the
carbonic acid + bicarbonate buffering system.
Two compounds, dihydrogen phosphate
(H2PO4) and hydrogen phosphate (HPO4), act
as buffers. When a strong acid is added, the fol-
lowing reaction occurs:
HCl þ Na2 HPO4 ! NaH2 PO4 þ NaCl
In this reaction, a strong acid, hydrochloric acid, is
converted into a weak acid, NaH2PO4, thereby
only causing a minor change in the pH. If a strong
base is added to the buffer system, the following
reaction occurs:
NaOH þ NaH2 PO4 ! Na2 HPO4 þ H2 0
249
With this reaction, a strong base is exchanged for a
weak base, causing only a minor shift toward an
alkaline pH.
The phosphate buffering system has a pKa of
6.8, which means that there are similar amounts of
H2PO4 and HPO4 at the pH of 7.40. Therefore
the phosphate buffering system has its best buff-
ering capacity in the normal blood pH range. Yet,
the concentration of H2PO4 and HPO4 is much
less than that of bicarbonate system and therefore
contributes less buffering capacity.
Intracellular Buffering
Cells and calcium salts in the bone contribute to
the buffering of extracellular fluid. Intracellular
proteins including hemoglobin, imidazole, and
histidine act as potent buffers and perhaps account
for as much as three-quarters of all chemical buff-
ering in the body [4]. Lysosomes and mitochon-
dria may also play a role in intracellular buffering
of H+. Intracellular H+ concentrations are primar-
ily affected by carbon dioxide concentrations
which rapidly diffuse through the cell membrane
to affect intracellular pH [5]. Bicarbonate, which
can move into the cell via an electrogenic
Na–HCO3 cotransport mechanism, also influ-
ences intracellular pH [6].
Pulmonary Regulation
The pulmonary compensatory response to acute
metabolic acidosis or alkalosis is swift. If a strong
acid is added to the blood, the pH begins to drop as
bicarbonate is consumed and PaCO2 begins to
rise. Blood pH cannot directly influence the cen-
tral respiratory center due to the blood–brain bar-
rier. Instead, as PaCO2 increases, CO2 carbonic
acid and H+ levels increase in the cerebral spinal
fluid. Hydrogen ions directly stimulate the central
respiratory center causing ventilation to increase.
Additional ventilatory stimuli are supplied by
peripheral chemoreceptors which respond to the
rising H+ concentrations. Because the lungs rep-
resent an open system by which CO2 can be
rapidly disposed, the blood pH can be rapidly
corrected to 7.40 by decreasing the PaCO2 value
to less than 40 mmHg.
When a strong base is added to the blood, the
pH rises rapidly and carbonic acid and PaCO2
250
levels decrease. Ventilatory drive decreases in
response to decreased serum and cerebrospinal
fluid H+ levels, causing an increase in PaCO2.
However, the effectiveness of hypoventilation as
a compensatory response is limited, as patients
will not hypoventilate to the point where they
become hypoxic.
Renal Regulation
The kidneys play a major role in acid–base
homeostasis through reabsorption of filtered
bicarbonate, secretion of hydrogen ions, and
ammonia (NH3) excretion. The proximal tubule
reabsorbs 80 % of the filtered bicarbonate, the
thick ascending limb of the nephron reabsorbs
15 %, and the distal nephron reabsorbs the
remaining 5 %. Very little of the filtered HCO3
is excreted in the urine.
The second function of the kidney is to secrete
hydrogen ions, in order to cope with dietary or
endogenous metabolic acids. These acids come
from dietary carbohydrate and fat metabolism,
which yields large quantities of CO2 and carbonic
acid. Dietary and endogenous acids amount to
about 1 mEq/kg body weight per day in adults
ingesting a typical Western diet. Oxidation of
dibasic and sulfur-containing amino acids gener-
ates small quantities of hydrogen ions. Hydrogen
ions secreted by the distal tubule react with
(1) dihydrogen phosphate (H2PO4) and hydro-
gen phosphate (HPO4) to form phosphoric acid
and (2) NH3 to form ammonium NH4+. The term
titratable acid (TA) refers to acid excreted that has
been bound to urinary buffers. It equals the
amount of acid (H+) that is added to the tubular
fluid by the nephron and is a function of both urine
pH and buffering capacity. Titratable acids
(TA) found in the urine include phosphoric and
sulfuric acid, but not ammonium.
The production of new bicarbonate, which is
not filtered, by the kidney is quantitated by urinary
net acid excretion (NAE), calculated as:
 þ  its final secretion in the distal tubule/collecting
NAE ¼ NH4 þ TA  HCO3 
Urinary NH3 excretion produces new bicarbonate
in the distal tubule cell by the metabolism of
P.D. Yorgin et al.
glutamine to bicarbonate and NH3. The bicarbon-
ate moves out the distal tubule cell into the extra-
cellular fluid by a chloride–bicarbonate
exchanger.
Proximal Mechanisms
Even though the proximal tubule of the nephron is
responsible for reabsorbing approximately 80 %
of the filtered bicarbonate, there is no evidence of
direct bicarbonate reabsorption [7]. Bicarbonate
reabsorption in the proximal tubule involves the
integrated function of multiple exchanger pro-
teins. The process of bicarbonate reabsorption
begins with H+ secretion by both the Na+/H+
exchangers (NHE) (SLC9A3) and H+-ATPase
(V-ATPase a2 isoform). Most of the H+ secreted
by the proximal tubule is used to reclaim bicar-
bonate. The remaining secreted H+ will be com-
bined with phosphate as titratable acid (Fig. 1) [4].
NHE
Sodium–hydrogen ion exchangers (NHE) are
members of a large monovalent cation–proton
antiporter family. NHE isoforms 2, 3, and 8 are
expressed along the apical membrane and extrude
hydrogen ions utilizing a high inward sodium
gradient [8]. NHE2 (SLC9A2), which is
expressed from the proximal tubule to the distal
nephron, is thought to have a minor role in urinary
acidification. NHE3 (SLC9A3) is expressed in the
proximal tubule and thick ascending limb (TAL).
NHE3 generates most of the urinary H+ needed
for the reabsorption of bicarbonate in the proximal
tubule and TAL. NHE8 (SLC9A8) has been iden-
tified in the proximal tubule of younger animals,
but its role during early development is not
known. NHE1 (SLC9A1) and NHE4 (SLC9A4),
which localize to the basolateral membrane, may
mediate NH4+ reabsorption in the TAL, prior to
duct. Murine studies have suggested that
two-thirds of the bicarbonate reabsorption in the
proximal tubule is achieved through the NHE
[9–12].
9 Physiology of the Developing Kidney: Acid-Base Homeostasis and Its Disorders
Fig. 1 Bicarbonate reabsorption in the proximal tubule.
Proximal tubule HCO3 reabsorption involves the inte-
grated function of multiple proteins. H+ ions are secreted
by both the Na+/H+ exchanger NHE3 and H+-ATPase and
titrate luminal HCO3 to H2CO3. Luminal H2CO3 dehy-
dration to H2O and CO2 is accelerated by luminal carbonic
anhydrase (CA) activity mediated by CA IV. CO2 enters
the cell via aquaporin-1 (AQP1) and, most likely, via
diffusive movement, where its hydration to H2CO3 is
V-ATPase
The remaining one-third of bicarbonate
reabsorption is achieved through V-ATPase a2 iso-
form which is similar to V-ATPase B1 isoform, the
distal nephron [13]. The H+-ATPase [9–12, 14] is
an apically located, multi-subunit vacuolar type
[15–17]. There is a maturational increase in both
NHE activity [18–22] and basolateral Na+/K+-
ATPase activity [23, 24] resulting in an increase in
proximal tubule acidification during development.
251
accelerated by cytoplasmic CA II. H2CO3 rapidly dissoci-
ates to H+ and HCO3, thereby replenishing the secreted
cytosolic H+. Cytosolic HCO3 exits across the basolateral
plasma membrane primarily by the Na+ (HCO3)
cotransporter NBCe1. Basolateral CA II and CA IV facil-
itate HCO3 transport (Weiner DI, Verlander JW. Renal
acidification mechanisms. Brenner & Rector’s The Kidney,
9th Edition, 2012, Chapter 9, pp 293–325. Reproduced
with permission from Ref. [4])
Na+/K+-ATPase
Sodium can move passively into the proximal
tubular cell facilitated by a concentration gradient
generated within the cell. The sodium concentra-
tion gradient in the tubular cell is generated by the
basolateral Na+/K+-ATPase (ATP1A1). This is an
active process, which extrudes three sodium
ions in exchange for two potassium ions.
The sodium concentration inside the proximal
tubule is usually low, around 19 mM [25].
252
The Na+/K+-ATPase pump indirectly provides the
energy that allows most of the transport proteins to
translocate filtered solutes in the proximal tubule.
Carbonic Anhydrase (CA)
H+ in the urinary lumen reacts with HCO3 to
form carbonic acid (H2CO3). The rate of luminal
dehydration of H2CO3 to CO2 and H2O is dramat-
ically increased by the luminal carbonic
anhydrase isoenzyme type IV (CA IV), a member
of zinc metalloenzyme family. This zinc molecule
enhances H+ transfer to and from the active site by
weakening the hydrogen–oxygen bond. Luminal
H2O and CO2 move across the apical plasma
membrane via aquaporin (AQP)-1. Cytosolic
CO2 is then hydrated in the presence of carbonic
anhydrase (CA) II, forming carbonic acid, which
then rapidly dissociates to H+ and HCO3,
thereby replenishing the secreted cytosolic H+26.
Currently, there are 15 known CA isoenzymes
which enhance H+/HCO3 transport [26]. CA II
accounts for 95 % of the CA activity in the kidney
and resides primarily within the cytosol. CA IV
and CA XII account for the remaining 5 % and are
membrane associated. CA II interacts with a num-
ber of transporters including AE1, AE2, and AE3
and proximal tubule NBCe1, which facilitate
movement of HCO3 [27]. Basolateral CA II
and CA IV also facilitate HCO3 transport.
Na+/HCO3 Cotransporter (NBC)
In general, there are two main groups of
bicarbonate transporters: chloride–bicarbonate
exchangers (anion exchange transporter) and
sodium–bicarbonate cotransporter (NBC). Cyto-
solic HCO3 exits across the basolateral plasma
membrane primarily by the Na cotransporter
NBCe1 (gene: SLC4A4) in a 3 HCO3 to 1 Na+
ratio. The chloride–bicarbonate exchangers,
SLC26A6 and SLC26A7, transport oxalate, sul-
fate, chloride, and bicarbonate out of tubule cells,
across the basal lamina. SLC26A6 is distributed
widely in the proximal tubule, TAL, macula densa,
and distal convoluted tubules. SLC26A7 localizes
P.D. Yorgin et al.
to the basolateral membrane of α-intercalated cells
in the collecting duct. mRNA levels of SLC26A7
increase in response to water deprivation.
The sodium–bicarbonate cotransporter 1,
NBC1 (SLC4A7), is an electrogenic sodium–
bicarbonate transporter located on the basolateral
membrane of the proximal tubule [28]. NBC1 is
responsible for transporting three bicarbonate
molecules and one sodium molecule into the
blood [29]. NBC1 appears to be the dominant
pathway for HCO3 reabsorption in the S1 and
S2 segments of the proximal tubule, but a
basolateral Cl-HCO3 exchanger plays a role in
the S3 segment [30–32].
Non-proximal Mechanisms
The distal nephron is responsible for net acid
excretion. As in the proximal tubule, processes
are at work to reabsorb the remaining filtered
bicarbonate. In addition, protons are actively
transported to the tubular lumen where they com-
bine with NH3 and HPO42 to form H2PO4 and
NH4+ resulting in urinary acidification.
Loop of Henle and Thick
Ascending Loop
The TAL of the loop of Henle contributes to
acid–base homeostasis through its roles in
reabsorbing approximately 15–20 % of the fil-
tered bicarbonate load and in contributing to the
development of the medullary interstitial ammo-
nia gradient that is critical for collecting
duct ammonia secretion. H+ is secreted by
apical Na+/H+ exchange activity and vacuolar
H+-ATPase. Quantitatively, apical Na+/H+
exchange activity is the predominant apical H+
mechanism; vascular H+-ATPase is also present.
Two Na+/H+ exchanger isoforms are present in the
TAL, NHE2 (SLC9A2), and NHE3 (SLC9A3).
Cytoplasmic CA II catalyzes CO2 hydration to
form H2CO3, which dissociates to H+ and
HCO3, thereby recycling the H+ secreted across
the apical plasma membrane. Cytosolic HCO3
exists via a basolateral Cl/ HCO3 exchanger
9 Physiology of the Developing Kidney: Acid-Base Homeostasis and Its Disorders
Fig. 2 Intercalated cell subtypes in the distal nephron. The
distal nephron – that is, the connecting segment, initial
collecting tubule, cortical collecting duct (CCD), outer
medullary collecting duct (OMCD), and inner medullary
collecting duct (IMCD) – has multiple distinct cell types.
Three intercalated cell types can be distinguished based on
differential expression in different plasma membrane
domains of several proteins involved in renal acid–base
(AE1) and possibly AE2, via an electroneutral
sodium–bicarbonate cotransporter (NBCn1,
SLC4A7), and possibly via basolateral Cl chan-
nels. An apical K+-dependent HCO3 transporter
mediates coupled K+ and HCO3 transport and
functions in parallel with apical Na+/H+ exchange
and, because of a cytoplasmic to luminal K+ gra-
dient, may secrete HCO3. NBCn1 is expressed
in the basolateral membrane in the TAL and in the
apical membranes of the outer (OMCD) and inner
(IMCD) medullary collecting duct intercalated
cells. Both metabolic acidosis and hypokalemia
increase TAL NBCn1 expression.
Distal Convoluted Tubule
Urine acidification occurs mainly in the collecting
duct [33]. Two major cell types are found inter-
spersed in the collecting duct: the principal cell
and the intercalated cell. The principal cell is
primarily involved with Na+ reabsorption and K+
secretion and plays a little role in acid–base bal-
ance. The intercalated cell is primarily involved in
acid–base transport and regulation. There are two
types of intercalated cells that display different
functions. The α-intercalated cells secrete pro-
tons, while the β-intercalated cells secrete bicar-
bonate. The cortical collecting duct (CCD)
253
transport, including H+–adenosine triphosphatase
(ATPase), anion exchanger 1 (AE1), pendrin, Rh B glyco-
protein (Rhbg), and Rh C glycoprotein (Rhcg) (Weiner DI,
Verlander JW. Renal acidification mechanisms. Brenner &
Rector’s The Kidney, 9th Edition, 2012, Chapter 9, pp
293–325. Modified from an original drawing by of
Ki-Hwan Han, M.D., Ph.D., Ewha Womans University,
Seoul, Korea. Reproduced with permission from Ref. [4])
contains both α- and β-intercalated cells, while
the OMCD contains mostly α-intercalated cells
(Figs. 2 and 3) [4].
The α-intercalated cell actively transports H+
into the urine via a vacuolar H+-ATPase and to a
lesser extent a H+/K+-ATPase on the luminal sur-
face [34]. Intracellular CA II is required to gener-
ate intracellular H+ for active transport. An apical
Na+/H+ exchanger (NHE2) also secretes H+ into
the tubule [35, 36]. Proton secretion in the
collecting duct serves to reabsorb the remaining
HCO3– that has evaded earlier transport
mechanisms of the nephron. In addition, secreted
H+ combines with HPO42 to form H2PO4 and
NH3 to trap NH4+. HCO3 is transported from
the cell to the interstitium via a basolateral
Cl/HCO3 anion exchanger (AE1). Other
exchangers such as AE2, AE4, and SLC26A7
(pendrin) have been identified on the basolateral
membrane of selective cells within the IMCD or
OMCD cells contributing to HCO3 exchange
[37–41].
The β-intercalated cell is opposite in polarity to
the α-intercalated cell. The function is to actively
secrete HCO3 into the urine coupled to Cl
reabsorption but independent of Na+. HCO3 is
moved from the lumen into the cell through
pendrin (SLC26A4), a Cl/HCO3 exchanger
on the apical membrane [42].
254
Fig. 3 Bicarbonate reabsorption by the type A interca-
lated cell. The type A intercalated cell is present from the
connecting segment through the initial inner medullary
collecting duct (IMCD). Two families of H+ transporters,
H+–adenosine triphosphatase (ATPase), and H+-K+-
ATPase are present in the apical plasma membrane.
Secreted H+ ions titrate luminal HCO3 – to form H2CO3 –
which dehydrates to H2O and CO2. Luminal carbonic
anhydrase activity, most likely mediated by carbonic
anhydrase IV (CA IV), is variably present in the collecting
duct. Cytosolic H+ and HCO3 are formed from CA
II-accelerated hydration of CO2 and rapid dissociation of
Ammonia Production and Transport
NH3 is produced by almost all renal epithelial
cells. The proximal tubule is the primary site
for physiologically relevant ammoniagenesis
(Fig. 4) [4]. The glomeruli proximal tubule seg-
ments, descending thin limb of the loop of Henle,
medullary and cortical TAL of the loop of Henle,
distal convoluted tubule, CCD, OMCD, and
P.D. Yorgin et al.
H2CO3. Cytosolic HCO3 exits across the basolateral
plasma membrane via the kidney isoform of anion
exchanger 1, kAE1. Cl that enters via kAE1 recycles via
a basolateral Cl channel. K+ that enters via apical H+-K+-
ATPase can either recycle via an apical Ba+-sensitive K+
channel or be reabsorbed via a basolateral Ba+-sensitive K+
channel. A basolateral Na+/H+ exchanger is present but
does not contribute to bicarbonate reabsorption and is not
shown (Weiner DI, Verlander JW. Renal acidification
mechanisms. Brenner & Rector’s The Kidney, 9th Edition,
2012, Chapter 9, pp 293–325. Reproduced with permission
from Ref. [4])
IMCD, all have the capability to synthesize
NH3, with glutamine being the primary meta-
bolic substrate [43]. Phosphate-dependent gluta-
minase (PDG) is involved in this process in each
of these segments [44]. The proximal tubule
accounts for 60–70 % of the total renal NH3
production under basal conditions and at least
70–80 % of production in response to metabolic
acidosis [43].
9 Physiology of the Developing Kidney: Acid-Base Homeostasis and Its Disorders
Fig. 4 Summary of renal ammonia metabolism. The prox-
imal tubule produces ammonia, as NH4+, from glutamine.
NH4+ is then secreted preferentially into the luminal fluid,
primarily by Na+/H+ exchanger isoform 3 (NHE3). It is
then reabsorbed by the thick ascending limb of the loop of
Henle (TAL), primarily by Na+/K+-2Cl cotransporter
type 2 (NKCC2). This results in the delivery of ammonia
to the distal nephron, which accounts for approximately
Multiple conditions simulate renal ammonia-
genesis. Both acute and chronic metabolic acidoses
stimulate ammoniagenesis [45] and appear to do so
by stimulating the PDG pathway. NH3 produced in
the proximal tubule is secreted preferentially into
the tubule lumen, although there is some transport
across the basolateral membrane. The proximal
tubule also can reabsorb luminal NH3; this appears
to occur primarily in the late proximal tubule [46].
The apical Na+/K+-2Cl cotransporter NKCC2
mediates the majority of NH3 reabsorption.
Some of the NH3 absorbed by the medullary
TAL of the loop of Henle undergoes recycling into
255
20 % of final urinary ammonia; the remaining approxi-
mately 80 % is secreted in the collecting duct through
parallel NH3 and H+ transport. Numbers in red indicate
the proportion of total urinary ammonia present at the
indicated sites under baseline conditions (Weiner DI,
Verlander JW. Renal acidification mechanisms. Brenner
& Rector’s The Kidney, 9th Edition, 2012, Chapter 9, pp
293–325. Reproduced with permission from Ref. [4])
the thin descending limb of the loop of Henle,
which results in countercurrent amplification of
medullary interstitial ammonia concentration.
NH3 recycling predominantly involves NH3 trans-
port, with a smaller component of NH3
transport [47].
NH3 movement across collecting duct cell api-
cal and basolateral membrane appears to involve
both diffusive and transporter-mediated NH3
transport. The Rh glycoprotein Rhcg plays a crit-
ical role in renal NH3 excretion. Rhcg is an NH3-
specific transporter with no identifiable affinity for
solutes other than NH3.
256
Interpretation of Acid–Base Status
from Blood Gas Results
General Approach to Evaluating
Acid–Base Status
To effectively assess acid–base status, the clini-
cian is greatly assisted by reviewing simultaneous
blood chemistry and blood gas results. The fol-
lowing steps are most helpful in yielding a
diagnosis:
1. Assess the serum pH as it generally indicates
whether a patient is acidemic (pH < 7.38) or
alkalemic (pH > 7.42).
2. Assess the PaCO2 and serum total CO2/HCO3
values relative to the pH to determine the pri-
mary cause of the pH disturbance.
3. Calculate the expected respiratory or renal
compensation. If memorization of the compen-
sation rules seems overwhelming, the modified
version of the Henderson–Hasselbalch equa-
tion can be used to determine the appropriate
respiratory or renal response when one
assumes a normal pH.
4. Calculate the anion gap, delta anion gap, and
delta ratio to distinguish the cause for meta-
bolic acidosis and to determine if there is an
additional acid–base disorder.
pH
The first step in analyzing a blood gas is to deter-
mine whether the patient is acidemic or alklemic.
The normal pH of extracellular fluids is
7.38–7.42. Acidemia is present whenever the
blood pH is lower than 7.38 and will be attributed
to an increase in PaCO2 or decrease in HCO3.
Any uncompensated decrease in PaCO2 or
increase in HCO3 such that the blood pH is
higher than 7.42 is characterized as an alkalemia.
When there is a perturbation in the blood pH, the
etiology is either respiratory or metabolic.
Any acute change in the H+ concentration in
the blood causes a defense of the pH by compen-
satory mechanisms. For example, if there is a
perturbation in PaCO2, then the serum
P.D. Yorgin et al.
bicarbonate will increase or decrease to compen-
sate, thereby normalizing the pH. When a primary
respiratory or metabolic process is so severe that it
overwhelms the compensatory systems or the
compensatory response is inadequate to the per-
turbation, the blood pH will not be normal.
Example
A neonate with respiratory distress has the follow-
ing blood gas: pH: 7.16, PaCO2: 70 mmHg,
HCO3: 26 mmol/L. What is the primary
disturbance?
Answer In this particular case, the patient is
acidemic. The PaCO2 is very high due to a pri-
mary respiratory acidosis. Note that the HCO3 is
slightly above normal so it is not the cause of the
acidemia.
PaCO2
The partial pressure of carbon dioxide (PaCO2) in
the blood (normal range 35–35 mmHg) has pro-
found effects on blood pH. The arterial PaCO2
will vary inversely with alveolar ventilation in a
linear manner:
pa CO2  K  V’CO2 =V’A
where V’CO2 is the volume of carbon dioxide
produced by the body’s metabolism per minute
(l/min) and V’A is the alveolar ventilation (l/min).
Thus, assuming a constant metabolic produc-
tion of CO2, a reduction in alveolar ventilation by
half will lead to a doubling of PaCO2 from
40 mmHg to 80 mmHg causing a respiratory
acidosis. In the context of a low blood pH, high
PaCO2 values typically point to a primary respi-
ratory acidosis.
Bicarbonate
Serum bicarbonate (HCO3) or total CO2 values
are routinely measured using analytical clinical
laboratory or blood gas equipment. The actual
bicarbonate, standard bicarbonate, and base
excess are then computed from the pH and
9 Physiology of the Developing Kidney: Acid-Base Homeostasis and Its Disorders 257

Fig. 5 Scales for pH, Total-CO2 mMol/l plasma.

PaCO2, base excess of 60
HCO3− mEq/l plasma.
whole blood of different 60
hemoglobin concentrations, Base Excess
plasma bicarbonate, plasma 50 mEq/l blood or plasma.
CO2 by Siggaard-Andersen 50 +30
(Strnad Z. [Numerical
calculation of basic
+25 pH
indicators of blood acid- 40
40 8.0
base balance using an
equilibration method]. Vet +20
35 35 7.9
Med 1986;31:557–64.
Reproduced with
permission from Ref. [48]) 30 +15 7.8
30

+10 7.7
25 25
0 +5 7.6

5 7.5
20
20 −5
7.4

7.3
−10
15
7.2
15
7.1
−15
7.0

10 6.9
25
9 20
−20 6.8
10
8 15
6.7
9 10
7
00 ml.

6.6
8
lobin g/1

6 −25 5

7
Hemog

6
4
0
−30

PaCO2 using the Siggaard-Andersen nomogram acid (H2CO3), dissolved CO2, carbonate, and
(Fig. 5) [48]. Some laboratories will report serum CO2 bound to amino acids. Total CO2 can be
total CO2 values instead of HCO3. The total CO2 assayed by adding a strong acid to the blood.
value includes bicarbonate (HCO3), carbonic Because 95 % of the total CO2 value is HCO3,
258
the total CO2 value is usually 2 mmol/L higher
than the calculated HCO3 value. Serum HCO3
levels can be estimated from total CO2 values.
Serum bicarbonate and total CO2 can be deter-
mined from known pH and PaCO2 values using
the Henderson–Hasselbalch equation.
Serum total CO2 and HCO3 values change in
response to blood CO2 levels due, in part, to the H+
accepting capacity of hemoglobin and other pro-
teins. When CO2 is bubbled through a bicarbonate
solution without hemoglobin, the bicarbonate con-
centration does not change. Therefore, hemoglobin
plays a role in mediating bicarbonate response to
PaCO2. If sodium bicarbonate is infused into the
blood, PaCO2 values increase due to the carbon
dioxide + water $ carbonic acid $ hydrogen +
bicarbonate buffer system.
Example
A 17-year-old girl with renal tubular acidosis has
a serum bicarbonate of 6 mmol/L, a pH of 7.10,
and a PaCO2 of 17.5 mmHg. After intravenous
fluids and 100 mEq of sodium bicarbonate were
infused, a repeat blood gas shows a bicarbonate
value of 11 mmol/L, PaCO2 of 29, and a pH of
7.17. Why is the PaCO2 rising?
Answer As sodium bicarbonate is infused, the
PaCO2 rises because of a decrease in respiratory
drive from the rising pH and due to the conversion
of H+ and HCO3 to H2O and CO2.
In children and adults, the normal serum
bicarbonate value is 24 mmol/L (range 22–29
mmol/L). In newborns the values are significantly
lower due to an expanded extracellular volume, a
decreased renal tubule ammonia production,
a lower bicarbonate reabsorption threshold, and
a limited ability to excrete hydrogen ions
[49]. Mean serum bicarbonate values are lower
in preterm infants (18–20 mmol/L) than those
born at term (20–22 mmol/L).
Base Excess
Base excess or the base deficit is characterized by
the amount of base that is required to normalize
P.D. Yorgin et al.
the pH of the blood. Normal values range from 2
to +2 mEq/L. Base excess can be determined by
plotting the values on the Siggaard-Andersen
nomogram (Fig. 5) or by calculating the formula
where the base excess is based on PaCO2 and pH
or PaCO2 and HCO3:

Base excess ¼ 0:9287 ðHCO3  24:4Þ
þ ð14:83 ðpH  7:4ÞÞ
Positive base excess numbers are seen when
there is a metabolic alkalosis, whereas negative
numbers indicate the presence of a metabolic aci-
dosis. The base deficit can be used to calculate the
amount of bicarbonate (or other base) needed to
normalize the pH. Since the volume of distribu-
tion of bicarbonate is 0.4–0.5 [50, 51], the formula
calculating the amount of base that is required to
normalize the pH of the blood is as follows:
Base required ¼ ðBase deficitÞ  ðWeight in kgÞ
 0:4  0:5
Example
A 12-year-old male with chronic renal failure
(estimated GFR = 25.3 mL/min/1.73 m [2]) aspi-
rates after having a hypertension-induced seizure
and is now intubated and on a ventilator. His
blood gas shows a pH of 7.04, PaCO2 of
39 mmHg, and a HCO3 of 12 mmol/L. His
hemoglobin is 10.1 g/dL. The base excess is
16.5 mEq/L. How much sodium bicarbonate
per kilogram would be needed to normalize the
HCO3?
Answer The amount of bicarbonate per kilogram
needed to correct the metabolic acidosis is deter-
mined by multiplying the base deficit by the bicar-
bonate volume of distribution (0.4–0.5). The
patient would need 6.6–8.3 mEq per kilogram of
sodium bicarbonate to achieve a serum total CO2
of 24 mmol/L.
As with the serum anion gap, serum albumin
concentrations impact base excess calculations
[52]. If the serum albumin is low, the base excess
9 Physiology of the Developing Kidney: Acid-Base Homeostasis and Its Disorders
Cations
Sodium
ANION GAP
Potassium
Calcium
Magnesium
Fig. 6 Serum anion gap. Due to the demands of electro-
chemical neutrality, the concentration of positive and neg-
ative ions in serum must be equal. The serum sodium value
is greater than that of chloride and bicarbonate combined.
The normal ion gap, 12  4 mmol/L, is composed of
albumin, phosphates, and amino acids. If the anion gap is
elevated, bicarbonate has been replaced by other anions,
will be more positive than the reported value, as
demonstrated in this equation:
Baseexcesscorrection ¼0:25
 ð4:2  serumalbuming=dLÞ
Serum Anion Gap
The serum anion gap is useful in determining the
source of a metabolic acidosis. Electrochemical
neutrality (e.g., the sum of all the cations equals
the sum of the anions) insures that there are equal
concentrations of positive and negative ions in
body fluids (Fig. 6) [53]. The equation for the
serum anion gap is:
þ
Anion gap ¼ Unmeasured cations þ Na
¼ Unmeasured anions þ HCO3 and Cl
Rearranged equation looks like this:
Anion gap ¼ Naþ  ðHCO3 and Cl Þ
¼ Unmeasured anions  Unmeasured cations
The normal anion gap is 12  4 mmol/L. Patients
who have high anion gaps may have increased
unmeasured anions or decreased unmeasured
259
Anions
Chloride
Bicarbonate
Albumin
Other Proteins
Amino acids
making the anion gap greater than normal (Andersen
OS. Blood acid-base alignment nomogram. Scales for
pH, pCO2 base excess of whole blood of different hemo-
globin concentrations, plasma bicarbonate, and plasma
total-CO2. Scand J Clin Lab Invest 1963;15:211–7.
Reproduced with permission from Ref. [53])
cations. Under normal conditions, the anion gap
is predominantly constituted of negative charges
on serum proteins including albumin [54, 55]. The
anion gap falls by 2.5 mmol/L for every 1 g/L
reduction in serum albumin [56]. A low anion gap
can be due to hypoalbuminemia, hypercalcemia,
hyperkalemia, hypermagnesemia, or lithium
ingestion.
Some patients have more than one metabolic
derangement affecting the serum HCO3 value.
The delta anion gap assesses the change in the
anion gap relative to a normal anion gap of
12 mmol/L. The delta anion gap is calculated
using the following equation:
Delta anion gap ¼ ðcurrent anion gap  12Þ
After solving for the delta anion gap, proceed to
solve the delta gap which is of use to determine if
the anion gap is sufficient to explain the entire
metabolic acidosis:
Delta Gap ¼ delta anion gap
 delta HCO3 ð24 mmol  current HCO3 Þ
A significantly positive delta gap (more than
6 mmol/L positive) is associated with a concurrent
metabolic alkalosis. Conversely, if significantly
260
negative delta gap (more than 6 mmol/L nega-
tive), then a hyperchloremic metabolic acidosis
is also present.
Some clinicians have found that a delta anion
gap ratio calculation is useful.
Delta anion gap ratio: delta anion gap/delta
HCO3 (24 mmol/L – current HCO3 mmol/L).
Ratios less than 0.4 are seen with hyperchloremic
normal anion gap acidosis; 0.4–1 are common
with high anion gap and normal anion gap acido-
sis; 1–2 are seen with a high anion gap acidosis,
and >2 are found when there is a high anion gap
acidosis and a metabolic alkalosis or a preexisting
compensated respiratory acidosis.
Example
A 3-year-old male with cerebral palsy who had an
exacerbation of his respiratory disease was
brought to the emergency department. His blood
gas showed a pH of 7.14, PaCO2 of 50 mmHg,
and a HCO3 of 18 mmol/L. His serum sodium
was 144 mEq/L, chloride 102 mEq/L, and serum
total CO2 16 mmol/L. Calculate the delta
anion gap.
Solution The anion gap is 26 mmol/L and the
delta anion gap is 14 mmol/L. The delta gap
is + 6, consistent with a concurrent metabolic
alkalosis. The delta anion gap is 2.33, indicating
the patient has at least two factors influencing
his metabolic acidosis – either a metabolic alka-
losis or a previously compensated respiratory aci-
dosis. The respiratory compensation is not
appropriate given the severity of metabolic
acidosis.
Metabolic and Respiratory
Derangements
Metabolic Acidosis
There are three mechanisms that can cause serum
bicarbonate levels to fall, resulting in metabolic
acidosis: (1) acid is added to or increased in body
fluids, (2) bicarbonate is lost (through the gastro-
intestinal tract [57] or by the kidneys), or
P.D. Yorgin et al.
(3) serum bicarbonate levels are decreased by
dilution with a non-bicarbonate-containing solu-
tion [58]. The compensatory response to both
acute and chronic metabolic acidoses is respira-
tory alkalosis.
Any evaluation of any metabolic acidosis
should include a determination of the serum
anion gap because it can be used to determine
the cause of the metabolic acidosis [59].
Most patients with a metabolic acidosis either
have an increased or normal anion gap. If, for
example, hydrochloric acid is infused into the
blood, then there is a milliequivalent per
milliequivalent replacement of bicarbonate for
chloride yielding a normal anion gap acidosis.
The same is true for intestinal or kidney losses of
HCO3. However, if HCO3 is replaced by an
unmeasured anion, like lactic acid, the anion gap
will increase. A list of conditions which cause an
increased anion gap is shown in Table 1. Increases
in the anion gap induce a respiratory compensa-
tion; for every 1 mmol/L rise in serum anion gap,
the PaCO2 should decrease by 1 mmHg.
Most of the acute change in serum bicarbonate
is repaired by intracellular buffering processes
[60]. Hemoglobin, phosphorous, protein, and the
bone [61, 62] all contribute to buffering H+.
Serum chloride levels rise in response to the
decrease in HCO3 levels.
Intravenous sodium bicarbonate infusions might
seem to be appropriate therapy for all forms of
metabolic acidosis. Sodium bicarbonate treatment
is appropriately used in the treatment of a
hyperchloremic metabolic acidosis caused by con-
ditions including renal tubular acidosis, diarrhea,
ureterosigmoidostomy, and amino acid or chole-
styramine infusions, but not in the treatment of
critically ill patients with a high anion gap metabolic
acidosis [4, 64, 65]. In addition to sodium bicarbon-
ate, sodium acetate (in intravenous solutions), oral
sodium citrate, and potassium citrate may be used to
correct a persistent hyperchloremic acidosis.
Since the injectable solution of sodium bicar-
bonate has a sodium content of 1,000 mEq/L,
large doses can cause significant hypernatremia
[64]. Intravenous use of diluted sodium bicarbon-
ate solution in concentrations of 140 mEq/L
in patients with normal kidney excretory
9 Physiology of the Developing Kidney: Acid-Base Homeostasis and Its Disorders 261

Table 1 Causes of an anion gap acidosis THAM ðmL of 0:3 mol=L solutionÞ
Toxins and drugs Unmeasured anion ¼ lean body weight ðkgÞ
Ethanol Lactic acid  base deficit ðmmol=LÞ:
Ethylene glycol Oxalic acid
Isoniazid toxicity Lactic acid Serum potassium values inversely correlate with
Methanol Formic acid serum bicarbonate values. Serum potassium con-
Paraldehyde Acetic acid centrations fall in response to the infusion of
Salicylates Lactic acid bicarbonate due to the movement of potassium
Isopropyl alcohol Oxalic acid into cells in exchange for hydrogen ions. A pH
Lactic acidosis Lactic acid increase of 0.1 leads to a decrease in serum potas-
Uremia Uric, oxalic, succinic, sium of 0.2–0.4 mEq/L. Conversely, for every
pimelic, adipic acid
decrease in blood pH of 0.1, the serum potassium
Amino acidopathies
rises by 0.6 mEq/L.
Maple syrup urine α-Ketoisocaproic,
disease α-keto-β-methylvaleric,
α-ketoisovaleric,
indoleacetic, acetoacetic, Respiratory Acidosis
β-hydroxybutyric acid
Isovaleric acidemia Isovaleric acid
Any process that interferes with ventilation can
Glutaric acidemia Glutaric, lactic, isobutyric,
isovaleric, α-methylbutyric cause respiratory acidosis. The causes of ventila-
acid tory failure include chronic obstructive pulmo-
Propionyl coenzyme Propionic, methylcitric nary disease, drugs, an extreme ventilation
A carboxylase deficiency propionylglycine, perfusion mismatch, an extensive infiltrative pro-
acetoacetic,
cess, exhaustion, neuromuscular disorders, or
β-hydroxypropionate,
β-hydroxybutyric acid excessive CO2 production.
Methylmalonic Methylmalonic acid The compensatory response to respiratory aci-
aciduria dosis is metabolic alkalosis. Hypercapnia stimu-
Defects in lates the secretion of hydrogen ions by the distal
carbohydrate tubule (Fig. 5) which lowers urine pH [66–68].
metabolism
Serum bicarbonate values also increase because
Diabetic ketoacidosis Acetoacetic,
β-hydroxybutyric acid bicarbonate secretion in the distal tubule is
Fructose-1,6- Lactic, pyruvic acid inhibited [69]. The higher bicarbonate concentra-
diphosphatase deficiency tion is maintained by enhanced kidney bicarbonate
Glucose-6- Lactic acid reabsorption in both proximal and distal tubules.
phosphatase deficiency As serum bicarbonate levels increase, the serum
Pyruvate carboxylase Lactic, pyruvic acid chloride levels must decrease. Hypercapnia leads
deficiency
to a decrease in proximal sodium chloride
Succinyl-CoA- Acetoacetic,
transferase deficiency β-hydroxybutyric acid reabsorption and induces chloruresis. The compen-
satory response of metabolic alkalosis, to chronic
hypercarbia, usually takes 1–2 days.
function rarely causes hypernatremia. In situa-
tions where a patient is acidemic but is Example
significantly hypernatremic, tris-hydroxymethyl A 16-year-old male with a pulmonary hemorrhage
aminomethane (THAM) can be used [63]. due to Wegener granulomatosis has a pH of
THAM has been safely used in patients with 7.32, a PaCO2 of 52 mmHg, and a HCO3 of
renal acidosis, salicylate or barbiturate intoxica- 26 mmol/L.
tion, and increased intracranial pressure associ-
ated with cerebral trauma [65]. THAM dosing is Diagnosis The elevated bicarbonate could not
based on the following formula: have caused the acidemia, so the principal cause
262 P.D. Yorgin et al.

of the acidemia is a respiratory acidosis. Yet, the
renal compensatory process is either incomplete
or has not had time to correct the pH to normal.
If there were no compensatory process the
recognition of the inciting problem would be
quite easy. Formulas have been derived that
describe the relationship between carbon dioxide
and bicarbonate in acute and chronic acid–base
disorders (Tables 2 and 3). These formulas can
assist with the determination of the primary event
and the subsequent compensatory process. In
most cases, the direction of the pH deviation sug-
gests the primary process.
Discriminating Between Acute
and Chronic Respiratory Acidosis

Acute respiratory acidosis is typically caused by

Table 2 Rules of acute respiratory compensation an abrupt decline in ventilation, which causes
blood PaCO2 to rise and pH to fall. A child with
Change Rule Example
acute respiratory acidosis frequently is hypoxic
"PaCO2 For every 1 mmHg PaCO2
increase in PaCO2, 40 ! 60 and presents with tachypnea, dyspnea and
the pH will decrease pH hyperpnea.
by 0.008 pH unit 7.40 ! 7.24
Compensation The HCO3 will HCO3 Example
to "PaCO2 increase by 0.1 24 ! 26 An arterial blood gas obtained from a child with
mmol/L for every
1 mmHg increase in known severe asthma, now presenting to the
PaCO2 emergency room, with status asthmaticus reveals
#PaCO2 For every 1 mmHg PaCO2 a low pH (7.16) with a high PaCO2 (74 mmHg)
decrease in PaCO2, 40 ! 20 and a slightly elevated serum bicarbonate
the pH will increase pH (27 mmol/L).
by 0.007 pH unit 7.40 ! 7.54
Compensation The HCO3 will HCO3
to #PaCO2 decrease by 0.25 24 ! 19
Diagnosis These results are consistent with a
mmol/L for every child who has impending respiratory
1 mmHg decrease failure. Most asthmatics have a mild respiratory
in PaCO2 alkalosis at the time of presentation. The elevated

Table 3 Rules of chronic acid–base compensation

Chronic change Rule Example
"PaCO2 For every 1 mmHg increase in PaCO2, the pH decreases by 0.0025 PaCO2 40 ! 60
pH unit pH 7.40 ! 7.35
Compensation for The HCO3 will increase by 0.4 mmol/L for every 1 mmHg increase HCO3 24 ! 28
"PaCO2 in PaCO2
#PaCO2 For every 1 mmHg decrease in PaCO2, the pH increases by 0.003 pH PaCO2 40 ! 20
unit pH 7.40 ! 7.46
Compensation for The HCO3 will decrease by 0.5 mmol/L for every 1 mmHg HCO3 24 ! 14
#PaCO2 decrease in PaCO2
"HCO3 For every 1 mmHg increase in HCO3, the pH increases by HCO3 24 ! 34
0.003–0.008 pH unit pH7.40 ! 7.43–48
Compensation for The PaCO2 will increase by 0.2–0.9 mmHg for every 1 mmHg PaCO2 40 ! 48
"HCO3 increase in HCO3
#HCO3 For every 1 mmHg decrease in HCO3, the pH decreases by 0.012 HCO3 24 ! 14
pH unit pH 7.40 ! 7.28
Compensation for The PaCO2 will decrease by 1.25 mmHg for every 1 mmHg PaCO2 40 ! 28
#HCO3 decrease in HCO3
9 Physiology of the Developing Kidney: Acid-Base Homeostasis and Its Disorders
bicarbonate is attributable to buffering by
intracellular buffering mechanisms. The increase
in serum bicarbonate by 1 mmol/L for
every 10 mmHg increase in PaCO2 [70] is
immediate.
Renal buffering does not noticeably impact the
pH until 12–24 h after the respiratory acidosis
begins [71]. Treatment of the patient with acute
respiratory acidosis involves rapid recognition
and correction of the inciting cause coupled with
oxygen administration. Sodium bicarbonate,
which can cause a rise in PaCO2, should be used
only to preclude the serious cardiovascular effects
of acidosis.
The basis for chronic respiratory acidosis is
typically a decrease in alveolar ventilation.
Plasma CO2 values are elevated; however, unlike
in the patient with acute respiratory acidosis,
effective renal compensation has occurred. There-
fore, serum pH values are only slightly below
normal. For each increase in the PaCO2 of
1 mmHg, the bicarbonate value increases by
0.41 mmol/L [72].
Example
A 3-month-old infant with bronchopulmonary
dysplasia on diuretic therapy has the following
venous blood gas and laboratory results: pH,
7.38; PaCO2, 68 mmHg; PO2, 53 mmHg; HCO3,
38 mmol/L; serum sodium, 136 mEq/L; serum
potassium, 2.9 mEq/L; serum total CO2,
39 mmol/L; and serum chloride, 86 mEq/L.
Solution This blood gas obtained from a child
with stable bronchopulmonary dysplasia is
remarkable for the normal to slightly low pH, a
markedly elevated PaCO2, and a hypochloremic
metabolic alkalosis. This gas shows an effective
renal compensation for the chronic respiratory
acidosis.
Metabolic Alkalosis
Metabolic alkalosis, characterized by an increased
concentration of serum bicarbonate, can be caused
by three major mechanisms: (1) intravascular
263
volume contraction where chloride is lost dispro-
portionately to bicarbonate, (2) loss of hydrogen
ion, or (3) net addition of HCO3 to the extracel-
lular space.
Chloride loss, without concurrent H+ loss, most
commonly occurs with gastrointestinal disease,
chemical diuretic use [73], cystic fibrosis [74, 75],
Bartter syndrome [76], or Gitelman syndrome
[77, 78]. With intractable chloride depletion, the
kidney increases the reabsorption of bicarbonate by
increasing distal tubule H+ secretion. A metabolic
alkalosis can develop when chloride is lost in
excess of sodium from the gastrointestinal tract.
Patients with acute diarrhea may have chloride
losses that range from 10 to 110 mEq per liter of
stool [79]. Chemical diuretics, including loop
(furosemide, bumetanide, ethacrynic acid) and thi-
azide (including chlorothiazide, hydrochlorothia-
zide, and metolazone) diuretics, can cause a
profound loss of chloride by the kidney.
Bicarbonate values rise acutely with the use of
loop and thiazide diuretics due to at least four or
five mechanisms. First, contraction of the intra-
vascular volume, without net loss of bicarbonate,
causes an increase in serum bicarbonate levels.
Second, increased salt delivery to the distal
tubule stimulates potassium and hydrogen ion secre-
tion independent of an aldosterone effect [80]; thus
contributing to higher serum bicarbonate values.
Third, extravascular volume contraction causes
the GFR to decrease. As a result, more sodium and
water are reabsorbed in the proximal tubule.
Because sodium delivery is decreased to the
collecting duct, the juxtaglomerular apparatus
secretes renin into the afferent arteriole. A renin-
mediated increase of angiotensin II leads to an
increase in aldosterone levels. Aldosterone
decreases the H+ concentration in serum by increas-
ing distal tubule H+ secretion via the H+/K+-ATPase
luminal pump. As plasma potassium levels begin to
decrease due to diuretic mediated potassium deple-
tion, aldosterone levels decrease. This represents an
important feedback mechanism to prevent severe
hypokalemia and metabolic alkalosis.
Fourth, profound hypokalemia due to potas-
sium depletion causes K+ to egress from somatic
cells. With significant potassium depletion, potas-
sium moves out of the cell in order to maintain the
264
ratio of intracellular and extracellular potassium,
and hydrogen ions move into the cell maintain
electrochemical neutrality. Since all cells have a
K+–Na+ pump that keeps intracellular levels of
sodium low, sodium cannot move into the cell in
exchange for the potassium leaving the cell. The
loss of H+ from the extracellular space into intra-
cellular space causes the rise in serum HCO3.
Finally, thiazide diuretic-induced inhibition of
NDBCE in the intercalated cells may also block
the loss of HCO3 in the urine, contributing to the
metabolic alkalosis.
Treatment of the hypokalemic, hypochloremic
metabolic alkalosis caused by diuretics typically
consists of potassium chloride or potassium-
sparing diuretics. Potassium chloride therapy is
advantageous in that both potassium depletion
and chloride deficiency are corrected. Sodium
chloride therapy can be used to correct the chlo-
ride depletion, but its use is controversial.
Example
A 4-month-old female with bronchopulmonary
dysplasia has chronic CO2 retention and is receiv-
ing furosemide and metolazone therapy. Arterial
blood gas results show a pH of 7.43, PaCO2 of
58 mmHg, PO2 of 68 mmHg, and HCO3 of
36 mmol/L. Her serum sodium is 137 mEq/L,
serum potassium is 2.4 mEq/L, serum chloride is
86 mEq/L, and serum total CO2 is 35 mmol/L.
How would one describe this acid–base problem?
Diagnosis In this particular case, the child is
slightly alkalemic due to an excessive compensa-
tory metabolic alkalosis in the context of chronic
respiratory acidosis. The metabolic alkalosis is
excessive because of the excessive diuretic-
induced urinary chloride loss.
Hydrogen ion can be lost through gastrointes-
tinal or renal mechanisms. The secretion of
hydrochloric acid in the stomach leaves behind a
cation and bicarbonate in the serum. If the H+ ion
is lost from the body (e.g., vomiting), there will be
a net increase in serum bicarbonate.
In the administration of bicarbonate or citrate,
acetate or lactate will cause a rise in serum HCO3
levels. Because the kidney has the capacity to
P.D. Yorgin et al.
excrete bicarbonate and decrease urinary acidifi-
cation in response to an alkali load [81], the alka-
losis that develops in response to exogenous alkali
is typically mild.
There are three mechanisms that act to return the
pH toward 7.40 when a metabolic alkalosis
develops. There are intracellular buffering mecha-
nisms by which H+, derived from intracellular pro-
teins and phosphate, is released into the
extracellular space. Lactic acid levels also increase
and provide additional H+ for buffering [50]. The
kidney has the capacity to excrete bicarbonate via
β-intercalated cells in the distal tubule [82]. Com-
pensatory respiratory acidosis is the third process by
which the pH is returned toward normal. Decreased
ventilatory drive, limited by hypoxia, increases
PaCO2 values to approximately 55 mmHg. With
respiratory compensation, the PaCO2 should
increase by 0.25–1 times the bicarbonate change.
There are factors including volume depletion
and aldosterone and potassium depletion that
interfere with renal bicarbonate wasting, thus
maintaining the alkalosis [83]. Volume depletion
decreases the amount of glomerular filtrate deliv-
ered to the proximal tubule; therefore, sodium and
bicarbonate reabsorptions are increased. Aldoste-
rone increases distal tubule H+ secretion via the
H+/K+-ATPase luminal pump [84, 85]. Potassium
depletion increases the rate of bicarbonate
reabsorption [86], thus helping to sustain meta-
bolic alkalosis.
Occasionally, metabolic alkalosis can occur in
patients who experience a rapid resolution of their
chronic hypercapnia but cannot excrete bicarbon-
ate rapidly enough. Conversion of large amounts
of lactic acid due to shock can also cause a meta-
bolic acidosis [87].
The effective treatment of a metabolic alkalosis
depends upon the recognition of chloride-
responsive and chloride-resistant processes.
Sodium or potassium chloride replacement will
lead to rapid resolution of the chloride depletion-
induced metabolic alkalosis [88]. Blocking the
reabsorption of bicarbonate in the urine by inhi-
bition of carbonic anhydrase can be very effective.
The use of even a single dose of acetazolamide
can lead to a rapid improvement of a metabolic
alkalosis [89, 90].
9 Physiology of the Developing Kidney: Acid-Base Homeostasis and Its Disorders 265

Individuals who have sodium chloride- Table 4 Simple acid–base disorders

resistant metabolic alkalosis may have Type of disorder pH PaCO2 HCO3
hyperaldosteronism, pseudohyperaldosteronism Metabolic acidosis # # #
(Liddle’s syndrome, type 1 pseudohyperaldos- Metabolic alkalosis " " "
teronism), aldosterone receptor gain of function Acute respiratory acidosis # " "
mutation (type 2 pseudohyperaldosteronism), Chronic respiratory acidosis # " "
apparent mineralocorticoid excess (AME) or per- Acute respiratory alkalosis " # #
haps potassium depletion [91]. Patients with Chronic respiratory " # #
alkalosis
AME experience growth failure, hypertension,
and a chronic hypokalemic metabolic alkalosis From Schrier RW. Renal and Electrolyte Disorders,
ed. 3, Boston, Little Brown and Company, 1986, p. 146;
[92]. Excessive aldosterone activity can be with permission
blocked with spironolactone [78, 93, 94],
triamterene [95], or amiloride [94].
Although not commonly used, the intravenous
infusion of hydrochloric acid has also been used Simple Acid–Base Disorders
to correct acute and severe metabolic alkalosis
[96–98]. Acid–base problems fall into two broad catego-
ries: those with a single primary disorder coupled
with a compensatory response, a simple acid–base
Respiratory Alkalosis disorder, and those in which two or more primary
disorders occur together, a mixed acid–base dis-
Respiratory alkalosis is caused by a process in order. The type of acid–base disorder can be deter-
which the pH rises in response to a decreasing mined by evaluating the pH, PaCO2, and the
PaCO2. The PaCO2 falls when ventilatory losses HCO3. In a simple acid–base problem, the
of carbon dioxide exceed CO2 production. Venti- PaCO2 and the HCO3 always move in the same
lation can be increased due to central or peripheral direction (Table 4). Movement of the PaCO2 and
neural stimulation, mechanical ventilation, or vol- the HCO3 in opposite directions indicates a
untary effort. mixed acid–base disorder. Assessment of the
The compensatory mechanism is a metabolic appropriateness of the compensation using
acidosis. Buffering by H+ release from intracellu- Tables 2 and 3 and Fig. 7 can also be helpful in
lar sources constitutes the first defense against uncovering a mixed acid–base problem.
respiratory alkalosis [99]. Buffering is complete
in minutes and persists for at least 2 h [100]. Example
In response to acute respiratory alkalosis, the A 2-year-old male with hypochloremic metabolic
HCO3 decreases by 1–3 mmol/L for every alkalosis is diagnosed as having Bartter syn-
10 mmHg decrease in PaCO2. drome. His initial blood gas result showed a pH
The kidney compensates in response to respi- of 7.41, PaCO2 54 mmHg and a HCO3 of
ratory alkalosis by reducing the amount of new 33 mmol/L.
HCO3 generated and by excreting HCO3
[101]. The process of renal compensation occurs Diagnosis This is an example of simple
within 24–48 h [102]. The stimulus for the renal acid–base disorder with a primary metabolic alka-
compensatory mechanism is not pH but PaCO2 losis and a compensatory respiratory acidosis.
[103, 104]. In chronic respiratory alkalosis, the
plasma HCO3 is decreased by 2–5 mmol/L for
every 10 mmHg decrease in PaCO2. The only Mixed Acid–Base Disorders
means to treat a respiratory alkalosis is to correct
the underlying disorder responsible for causing A mixed acid–base disorder is a combination of
the disorder. two primary disorders [55]. Recognition of a
266 P.D. Yorgin et al.

Fig. 7 Acid–base Arterial blood [H+] (nmol/L)

nomogram (or map).
Shaded areas represent the 100 90 80 70 60 50 40 30 20
95 % confidence limits of 60
120 110 100 90 80 70 60 50 40
normal respiratory and 56
metabolic compensations
for primary acid–base 52
disturbances. Data falling 35
48
outside the shaded areas METABOLIC
denote a mixed disorder if a 44
Arterial plasma [HCO3−] (mmol/L)
ALKALOSIS
CHRONIC 30
laboratory error is not RESPIRATORY
present (Dubose 40 ACIDOSIS
TD. Disorders of acid–base 25
36
balance. In The Kidney 8th
ed, Brenner B (ed.), 32
Philadelphia, Elsevier ACUTE 20
Saunders 2007;505–546. 28 RESPIRATORY
ACIDOSIS
Reproduced with 24 NORMAL
permission from Ref. [105]) ACUTE
15
20 RESPIRATORY
ALKALOSIS
16 CHRONIC
10
RESPIRATORY
12 ALKALOSIS
METABOLIC
8 ACIDOSIS
Pco2(mm Hg)
4

0
7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8
Arterial blood pH

mixed acid–base disorder is dependent on deter- respiratory acidosis. Note that the PaCO2 and
mining whether the compensatory process was HCO3 do not move in the same direction. With
adequate and appropriate. In mixed acid–base the high PaCO2, an increase in the total CO2
disorders, the blood gas result will fall outside of would be the normal compensatory response.
the predictive bands found in an acid–base nomo-
gram (Fig. 7). When there is a significant devia- The anion gap and delta anion gap can also
tion of the pH, one of the two primary disorders point to a mixed acid–base disorder.
blocks the compensation for the other
(Fig. 7) [105]. Example 2
A 13-month-old female presents with a 2-day
Example 1 history of non-bilious vomiting and diarrhea.
A 9-month-old male with renal tubular acidosis The child has vomited 10 times over the last
is unable to take his medication due to the respi- 36–48 h. Initial laboratory tests show a Na of
ratory distress associated with respiratory syncy- 132 mmol/L, chloride of 87 mmol/L, and total
tial virus. His admission arterial blood gas results CO2 of 18 mmol/L.
show a pH of 7.03, PaCO2 of 52 mmHg, PO2 of
67 mmHg, and HCO3 of 16 mmol/L. Diagnosis The anion gap is 27 mmol/L with a
delta gap of +11 mmol/L. This is an example of a
Solution This is an example of a mixed acid–base mixed acid–base disorder because the patient has
disorder with a metabolic acidosis and acute a metabolic acidosis but also has metabolic
9 Physiology of the Developing Kidney: Acid-Base Homeostasis and Its Disorders
alkalosis due to vomiting with loss of HCl.
The metabolic alkalosis is not obvious due to the
severity of the metabolic acidosis. Without the
vomiting, the metabolic acidosis would have
been much worse.
Occasionally the amount of compensation is
excessive given the clinical situation. For exam-
ple, children presenting with aspirin intoxication
will typically have PaCO2 values lower than
expected based upon the acidosis caused by the
aspirin alone [106]. The excessive respiratory
alkalosis is attributable to the stimulatory effect
aspirin has upon ventilation.
Patients with chronic lung disease typically
have high PaCO2 values and an appropriate
compensatory metabolic alkalosis. The addition
of diuretics, which are frequently used to
decrease alveolar interstitial edema, causes
serum HCO3 values to increase to levels greater
than expected on the basis of the respiratory
acidosis alone. The blood gas results are fre-
quently consistent with a simple metabolic alka-
losis. A patient’s history may be the only means
by which the mixed acid–base disorder may be
suspected.
Example
A 15-month-old female with bronchopulmonary
dysplasia receives furosemide at a dose of
1 mg/kg/day. Her typical blood gases on 1/2 l of
nasal O2 are as follows: pH, 7.49; PaCO2,
50 mmHg; PaO2, 87 mmHg; and HCO3,
40 mmol/L.
Diagnosis Mixed acid–base disorder, chronic
respiratory acidosis, and compensatory metabolic
alkalosis + metabolic alkalosis due to a severe
contraction alkalosis. The child develops a viral
pneumonia. The arterial blood gas is as follows:
pH, 7.35; PaCO2, 80 mmHg; PaO2, 62 mmHg;
and HCO3, 41 mmol/L. If one were not familiar
with the case, this would appear to be an acute
respiratory acidosis with an incomplete compen-
satory metabolic alkalosis; however, with an
increase of the PaCO2 value by 40 mmHg
(above the normal 40 mmHg), one would antici-
pate an acute HCO3 compensation of 4 mmol/L
267
(1 mmol/L per 10 mmHg rise in PaCO2). More-
over, the blood gas value falls into the chronic
respiratory acidosis prediction band.
Nutrition and Acid–Base Balance
Dietary intake with daily generation of organic
acids (lactic acid, pyruvic acid, and acetic acid)
and excretion of bicarbonate in the stool result in
net daily acid production of approximately 1 mEq
hydrogen ions per kilogram body weight in adults
[107]. Dietary intake contributes to the majority of
this daily acid production, with the latter two
processes making a minimal contribution. With
the development of agriculture and animal hus-
bandry, our diets changed from net base or alkali
producing to net acid producing. This occurred as
net acid-producing animal foods and cereal grains
replaced alkali-rich fruits and vegetables.
The typical modern Western diet produces a net
acid load or hydrogen ion in an adult of approxi-
mately 50–100 mEq per day [107, 108]. Vegetarian
diets that consist of vegetables, fruits, and nuts gen-
erate net base, whereas nonvegetarian diets includ-
ing meat, seafood, and dairy products generate net
acid [109]. This load is markedly increased in
infants who are solely fed with cow milk-based
infant formula, which produces a net acid load of
approximately 2 mEq/kg/day compared to about 0.8
mEq/kg/day when fed with human milk [110]. The
dietary net acid load in children in the West is
roughly 15–80 mEq/day or roughly 1–3 mEq/kg/
day, data obtained from population-based studies,
being higher in adolescents than younger children
and in males compared to females [111–113].
Metabolism of sulfur-containing amino acids
cysteine and methionine, cationic amino acids
arginine and lysine, and phosphorus produces
acid. These substances are found in high concen-
trations in animal proteins, cereals, nuts, and dairy
products. Conversely, metabolism of anionic
amino acids, aspartic acid, and glutamic acid,
found in wheat, soy protein, cow’s milk, vegeta-
bles, and potassium- and magnesium-containing
foods, like fruits, vegetables, nuts, and dairy prod-
ucts, consumes hydrogen ion [113].
268
Physiologically, two types of acid are impor-
tant: carbonic acid, which is a volatile acid and
produced by the metabolism of dietary carbohy-
drates and fats, and non-carbonic acids, produced
from the metabolism of proteins. Metabolism
of dietary carbohydrates and fats results in gener-
ation of carbon dioxide that can be excreted by
alveolar ventilation. Approximately 15,000 mmol
of volatile acid is produced per day [112]. On the
other hand, renal urinary excretion eliminates
excess non-carbonic or nonvolatile acids, mainly
sulfuric acid (H2SO4) that is generated endoge-
nously or from dietary intake of sulfur-containing
amino acids.
The nonvolatile acid produced from metabo-
lism and dietary intake is balanced with renal net
acid excretion, thus maintaining acid–base equi-
librium. The normal healthy kidney easily handles
dietary net acid, but in the setting of reduced renal
function, excess dietary load provides a challenge
for excretion. In fact, there is evidence that even
with normal aging, gradual reduction in glomeru-
lar filtration rate (GFR) results in metabolic aci-
dosis on a normal diet [114].
Goodman, Lemann, and Lennon demonstrated
that despite minor daily fluctuations, the cumula-
tive acid balance in normal individuals is approx-
imately zero [115]. Typical acid production in
adults, estimated from extrapolation of urinary
sulfate and organic anion, is equal to urinary
acid output. With chronic renal disease (CKD),
serum CO2 content reaches the acidotic level over
a period of 6 days after withdrawal of bicarbonate
therapy. Net acid production exceeds
compromised or reduced net acid excretion,
resulting in a positive balance of 21–30 mEq of
acid per day [115]. Withdrawing the alkaline ther-
apy of a patient with CKD may lead to metabolic
acidosis resulting primarily from hydrogen ion
retention.
Acid–Base Balance in Neonates,
Infants, and Children
Newborns and infants have a lower serum bicar-
bonate of about 20 mEq/L compared to older
children and adults, whose serum bicarbonate is
P.D. Yorgin et al.
24 mEq/L. The imbalance between increased acid
load and decreased ability for net acid excretion
results in increased susceptibility of neonates and
infants to the development of metabolic acidosis.
The acid load from diet and metabolism in infants
is approximately one hundred percent higher than
that of adults, adjusted for body weight. The
increased acid load is exaggerated in infants fed
with cow’s milk formula. In small preterm babies,
renal acid excretion capacity is low compared to
term newborns. In both preterm and term infants,
maximum renal net acid excretion improves rap-
idly during the first few weeks of life (Fig. 8)
[110]. Insipient late metabolic acidosis is common
in premature infants in the first year of life, and its
pathophysiology is multifactorial (Fig. 9) [110].
The growing bones of neonates and children
results in production of additional acid as a result
of hydroxyapatite production for bone minerali-
zation. Approximately 0.92 mmol of protons is
released into the extracellular circulation for every
1 mmol of calcium incorporated into the skeleton
[116]. Broadly speaking, renal regulation of
hydrogen ion balance involves (a) glomerular fil-
tration; (b) reclamation of filtered bicarbonate,
thus repleting body stores; and (c) excretion of
hydrogen ion as titratable acid or ammonium,
which excretes the nonvolatile acid from metabo-
lism, dietary intake, and, in children, bone growth.
Net urinary acid excretion consists of the sum of
titratable acid (hydrogen ion buffered by phos-
phate and sulfate) plus ammonium.
Since the beginning of the twentieth century, it
was observed that urine pH is higher in preterm
and term neonates and gradually decreases after
several weeks. When preterm and term infants fed
human milk or formula were challenged with
acute or chronic acid loading using ammonium
chloride or calcium chloride, to determine maxi-
mal net renal acid excretion, the results revealed
lower net acid excretion in preterm compared to
term infants. Furthermore, there was an increase
in maximum net renal acid excretion during the
first few weeks in term and preterm infants
(Fig. 8).
Animal studies point to decreased activity and
immaturity of almost every transporter involved
in bicarbonate reabsorption and acid secretion as
9 Physiology of the Developing Kidney: Acid-Base Homeostasis and Its Disorders 269

Fig. 8 Maximum renal Maximum renal net

excretion of net acid and acid excretion
ammonium in preterm and 9.0
term infants on nutrition
with human milk or (mmol/kg/d) Cow’s milk / Formula NAE
formulas (Kalhoff H, Manz 7.5 NH4
F. Nutrition, acid-base
Human milk NAE
status and growth in early
NH4
childhood. Eur J Nutr 6.0
2001;40:221–30.
Reproduced with
permission from Ref. [110]) 4.5

3.0

1.5

0
30 35 40 45 50 65 70
Mean developmental age (weeks)

Nutrition

“Inner milieu”
of the body

Ventilation

Growth
Incipient late Weight gain
metabolic
acidosis (ILMA)
Na-retention
Aldosterone Ca,P-retention
Renal acid excretion N-assimilation
capacity Arg. -vasopressin

Genetic
features

Urinary excretion

Fig. 9 Pathophysiological mechanisms in premature in early childhood. Eur J Nutr 2001;40:221–30.

infants with incipient late metabolic acidosis (ILMA) Reproduced with permission from Ref. [110])
(Kalhoff H, Manz F. Nutrition, acid-base status and growth

causes for the reduced ability of the neonatal potential mechanisms. Less bicarbonate reclama-
kidney to excrete acid. While it may not be possi- tion occurs in the proximal convoluted tubule of
ble to study these mechanisms in humans, animal infants compared to adults. Whereas in adults,
studies have provided substantial insights into approximately 85 % of filtered bicarbonate is
270
Fig. 10 The apical protein expression of the Na+/H+
exchanger during postnatal development. As shown,
NHE8 is highly expressed in the neonate, while NHE3 is
the predominant Na+/H+ exchanger in the adult proximal
tubule. The isoform change occurs at the time of weaning
and is likely mediated by the increase in thyroid hormone
reabsorbed proximally, only 65 % of the bicar-
bonate is reabsorbed proximally in infants [110].
The Na+/H+ exchanger (NHE) is directly or
indirectly responsible for most sodium bicarbon-
ate and NaCl reabsorption by the adult proximal
tubule [117]. There are 10 NHE isoforms, but
NHE3 is the predominant isoform on the apical
membrane of the adult proximal tubule [12]. The
abundance of NHE3 is far less on the brush border
of the neonate compared to the adult proximal
tubule [118]. However, unlike the adult proximal
tubule where all of NHE activity could be attrib-
uted to NHE3, there is a discordance between
NHE activity and NHE3 mRNA and protein
abundance in the neonate. There is far greater
NHE activity than could be explained by NHE3
alone in the neonatal proximal tubule [22]. NHE8
was found to be this developmental NHE, which
is highly expressed in neonatal proximal tubules
P.D. Yorgin et al.
and glucocorticoids that occur during that time
(Twombley K, Gattineni J, Bobulescu IA, Dw
arakanath V, Baum M. Effect of metabolic acidosis on
neonatal proximal tubule acidification. Am J Physiol
Regul Integr Comp Physiol 2010;299:R1360-R68.
Reproduced with permission from Ref. [118])
but barely detectable in adults, as shown in Fig. 10
[118]. NHE8 was found to have significantly dif-
ferent properties than NHE3 in terms of its regu-
lation and its sensitivity to amiloride analogues,
which inhibit NHEs [119]. Metabolic acidosis in
neonates in vivo and in cells expressing NHE8
in vitro increase NHE8 activity consistent with
this isoform playing an important role in neonatal
proximal tubule acidification.
Animal studies show that the lower neonatal
bicarbonate transport is due to lower activities of
all the transporters involved in proximal bicarbon-
ate reabsorption, that is, the apical Na+/H+
antiporter, apical H+-ATPase, the basolateral
Na+/3HCO3 transporter, and the basolateral
Na+/K+-ATPase [19, 20, 120, 121]. The activity
of all these transporters reached adult levels by the
age of 6–7 weeks in these studies. One of the
major hormones that stimulate the development
9 Physiology of the Developing Kidney: Acid-Base Homeostasis and Its Disorders
of bicarbonate transport is glucocorticoid. Neo-
nates are relatively glucocorticoid deficient during
the first weeks of life. There is a developmental
increase in glucocorticoids which precedes the
increase in bicarbonate transport. Indeed, if glu-
cocorticoids are administered to pregnant animals
prior to giving birth, the neonates have proximal
tubular bicarbonate transport rates which are com-
parable to those in adults [20]. Thyroid hormone
also affects the development of bicarbonate trans-
port but plays a much less important role.
The limited ability for urinary acidification
may in part be due to carbonic anhydrase II defi-
ciency in immature renal tubules. This isoform of
carbonic anhydrase is found in the cytoplasm of
proximal and distal tubular cells. Karashima
et al. found that carbonic anhydrase II levels
increased from 14 % of adult levels in 1-week-
old rats to 40 % at age 3 weeks and 97 % at age
7 weeks [122]. Carbonic anhydrase IV is the iso-
form that is membrane bound on the luminal
surface of tubular cells. There is decreased activity
of carbonic anhydrase IV in neonatal rabbit kid-
neys compared with adults [123].
However, human studies provided contradic-
tory data. Studies of kidney tissue from human
embryos showed positive staining for carbonic
anhydrase in proximal and distal tubules from
12 to 15 weeks gestation. The catalytic activity
and the amount of carbonic anhydrase increased
as gestational age increased from 19 to 26 weeks,
being comparable to those of adult renal cortex at
26 weeks [124]. Thus, in humans, carbonic
anhydrase may not play a major role in the matu-
rational changes associated with renal acid–base
homeostasis.
α-Intercalated and β-intercalated cells in the
collecting duct are responsible for distal urinary
acidification. There are fewer intercalated cells in
the rabbit neonatal kidney and the activity of the
apical H+-ATPase is reduced [125]. Additionally,
the capacity for ammonium excretion is reduced
in newborn animals [126, 127]. Thus, the ability
for distal urinary acidification by excreting hydro-
gen ions is limited in newborns.
Therefore, maturational deficiencies in almost
every transporter involved in acid–base homeo-
stasis throughout the renal tubule put the neonate
271
at higher risk for developing metabolic acidosis.
This limited capacity for acid excretion, in con-
junction with increased acid load from the diet and
bone formation in growing children, is an impor-
tant factor in acid–base homeostasis.
Mechanisms of Growth Retardation in
Chronic Acidosis
Poor growth occurs in many chronic disorders
such as chronic kidney disease (CKD), cystic
fibrosis, rheumatologic conditions, and inflamma-
tory bowel disease. Growth retardation is a fre-
quent, long-recognized complication of chronic
metabolic acidosis [128].
Studies in acidotic children with classic renal
tubular acidosis have shown a blunted release of
growth hormone in response to provocative
stimuli. The growth retardation associated with
acidosis can be reversed by systemic bicarbonate
therapy [129]. The release of growth hormone
under acidotic conditions has been studied [130].
Exploration in rats with normal anion gap acidosis
from ingestion of ammonium chloride demon-
strated aberrant growth hormone (GH) secretion.
Significant inhibition of pulsatile GH secretion
was present in the acidotic rats such that both the
amplitude of the GH secretory pulse and the area
under the curve were significantly smaller than in
controls. These reductions in pulse amplitude and
area were correlated to decreased growth (weight)
in the acidotic rats [130]. Evaluation of tibial
epiphyseal growth plate insulin growth factor-I
(IGF-I) gene expression in acidotic and control
rats revealed that IGF-I messenger RNA abun-
dance was lower in the acidotic growth plates.
IGF-I peptide was predominantly localized to the
hypertrophic zone of chondrocytes and was
weakly detectable in the proliferative zone in
both the acidotic and control rats’ growth plates.
Anthropomorphic measurements demonstrated
that acidotic rats grew less than did control rats
in both length and weight, and these physical
measurements were reflected in the size of the
tibial epiphyseal growth plates being significantly
smaller in the acidotic rats compared with the
control group [131]. Taken together, these
272
observations suggest that metabolic acidosis
reduces IGF-I message abundance and induces
resistance to IGF-I peptide action within the tibial
epiphyseal growth plate. The use of GH to stimu-
late growth in normal anion gap acidosis has been
ineffective experimentally, despite enhancement
of IGF-I- and IGF-binding protein immunoreac-
tivity within the stem cell chondrocyte zone of the
tibial epiphyseal growth plate [131].
Effect of Acidosis on Bone and Calcium
Homeostasis
The renal response to acidosis, which consists of
H+ excretion, is relatively slow and takes days.
The initial response to acidosis is buffering by
extracellular bicarbonate and by cellular and
bone buffers. This role of the skeleton as buffer
occurs at the expense of bone mineral content
resulting in loss of calcium and phosphate
[132]. Bone sodium and potassium are lost in
exchange for hydrogen ion, and carbonate con-
sumed as a buffer. Positive acid and negative
calcium balance (due to hypercalciuria) occurs in
healthy individuals made chronically acidotic by
ammonium chloride loading [133, 134]. A similar
profile is found in patients with renal tubular aci-
dosis. Correction of the metabolic acidosis with
bicarbonate therapy results in return of acid bal-
ance to zero and less negative calcium balance
[135]. Furthermore, bicarbonate administration
corrects bone mineral loss in patients with acido-
sis [136, 137].
Acutely, bone resorption is primarily due to
physicochemical mineral dissolution, while cell-
mediated mechanisms predominate after 24 h
[132]. Chronic metabolic acidosis stimulates cal-
cium efflux from the bone due to increased osteo-
clastic bone resorption and decreased osteoblastic
activity. The negative calcium balance observed in
acidotic patients is due to calcium mobilization
from the bone, resultant hypercalciuria [138,
139], and lack of concomitant increase in intestinal
calcium absorption [136, 140]. There is net loss of
body calcium. The hypercalciuria predisposes to
osteoporosis, nephrocalcinosis and nephrolithiasis.
The latter renal effects result in renal impairment as
P.D. Yorgin et al.
evidenced in the study by Goodman et al., in which
few adult patients with RTA had normal
GFR [115].
Patients with CKD with concomitant meta-
bolic acidosis and hyperparathyroidism are at an
increased risk for skeletal demineralization.
Krieger et al. studied the effect of acidosis with
and without parathyroid hormone on calvariae
[133]. They found that acidosis and parathyroid
hormone (PTH) independently stimulated cal-
cium efflux from the bone, inhibited osteoblastic
collagen synthesis, and stimulated osteoclastic
beta-glucuronidase secretion. Furthermore, the
effects of acidosis and PTH on bone resorption
were additive.
Acid–Base Balance in CKD
CKD is characterized by a progressive deteriora-
tion of kidney function eventually leading to
end-stage renal disease necessitating either dialy-
sis or renal transplantation. As renal function dete-
riorates due to a progressive loss in nephron mass,
the ability of the kidney to excrete the daily acid
load produced as either hydrogen ions or ammo-
nium deteriorates. Endogenous net acid produc-
tion in infants and children is variable and has
been estimated to be between 1 and –3 mEq/kg/
day [141]. In adolescents, endogenous net acid
production is similar to that of adults, 1 mEq/kg/
day [142].
Metabolic acidosis is a common complication
of CKD. In the initial stages of CKD, the inability
to excrete hydrogen ions is compensated by an
increase in ammonium excretion, and balance is
maintained [143]. However, as nephrons are lost
and GFR falls, ammonium excretion cannot keep
up and acidosis ensues [143–149]. Metabolic aci-
dosis usually appears when GFR falls below
25 mL/min/1.73 m2 [144, 146, 150]. However, it
can appear earlier in the course of CKD, particu-
larly if additional defects in tubular acid secretion
are present or damage to the collecting duct is
present [149, 151].
Even in the late stages of CKD, the severity of
the acidosis is usually mild to moderate (serum
bicarbonate 12–23 mEq/L) [144, 152, 153].
9 Physiology of the Developing Kidney: Acid-Base Homeostasis and Its Disorders
The reasons for the variability in the onset and
severity of metabolic acidosis are not well under-
stood but may reflect differences in tubular func-
tion. An additional contributing factor may be
differences in dietary animal protein intake, a
major contributor to the endogenous net acid pro-
duction. Controlled studies have suggested that the
loss of the buffering ability of the bone may be an
important factor leading to acidosis [115, 140,
154]. Uribarri et al [155] have looked at adults on
dialysis and not found that to be the case. Thus the
role of the bone in playing a major role in acidosis
in ESRD patients remains in dispute.
The anion gap in CKD patients with the meta-
bolic acidosis can be elevated or normal
(normochloremic versus hyperchloremic)
[143]. It had been suggested that a normal anion
gap pattern characterizes early CKD, but as GFR
declines anion gap increases [149]. However, sev-
eral studies have shown that a normal anion gap
pattern can occur at any stage of CKD [149,
153]. Patients with significant tubular damage
and hyporeninemic hypoaldosteronism are more
likely to have normal anion gap levels [156]. The
accumulation of anions such as phosphate, sul-
fate, urate, and hippurate is usually exacerbated
by a diet high in animal proteins and reduced in
diets rich in fruit (high in citrate) [157, 158].
Despite its lack of severity, persistent acidosis
can have an adverse impact on cellular function
and contribute to increased morbidity and mortal-
ity in this patient population [143, 159, 160].
Symptoms resulting from metabolic acidosis are
more prominent when GFR drops below 25 % of
normal [161]. These patients are in negative nitro-
gen balance and experience (a) increased catabo-
lism of muscle protein resulting in muscle
wasting; (b) metabolic bone disease with
increased bone resorption, osteopenia, and wors-
ening hyperparathyroidism; (c) depletion of the
body’s buffering systems; (d) impaired myocar-
dial function; (e) endocrine abnormalities such as
resistance to growth hormone and impaired glu-
coneogenesis; (f) hypertriglyceridemia; and
(g) systemic inflammation [162]. Metabolic aci-
dosis is postulated to activate the complement
system and the renin–angiotensin system and
increase production of endothelin-1, all of which
273
result in chronic inflammation in the kidney and
progressive renal fibrosis [163].
Treatment of children with acidosis and CKD is
primarily with bicarbonate therapy. KDIGO guide-
lines recommend alkali therapy to be used to main-
tain serum bicarbonate levels within the normal
range [164]. Sodium bicarbonate or sodium citrate
is typically used at doses of 0.5–1 mEq/kg/day.
Citrate is rapidly metabolized to bicarbonate in
the liver and thus should be avoided in patients
with liver failure. In patients on maintenance dial-
ysis, the option to increase the bicarbonate concen-
tration in the dialysate is an option. Correction of
the acidosis in children with CKD can often result
in improved appetite and growth.
Successful renal transplantation has resulted in
reduced mortality and improvement in quality of
life for patients with ESRD on dialysis. Although
renal transplantation may restore GFR, patients
may demonstrate overt or subclinical metabolic
acidosis. Several mechanisms may account for
metabolic acidosis after kidney transplantation
[165]. Reduced nephron mass being the most obvi-
ous. Patients have low serum bicarbonate levels
and impaired renal acid excretion. In a study of
over 800 adults posttransplant, approximately
60 % of patients had low serum bicarbonate levels
[166]. Serum bicarbonate levels strongly correlated
with GFR. However, low bicarbonate levels were
also observed in patients where GFR was near
normal. The authors found an association between
serum bicarbonate levels with higher serum phos-
phate and PTH concentrations and lower serum
calcium levels. They postulate that the low bicar-
bonate levels induce calcium excretion, reduce
vitamin D synthesis, and stimulation of PTH
release. The relationship between posttransplant
acidosis and disturbances in mineral metabolism
needs further investigation. Other known causes of
metabolic acidosis posttransplant include the
development of renal tubular acidosis most often
caused by calcineurin inhibitor use (cyclosporine
and tacrolimus). It is postulated that calcineurin
inhibitors impair regulation of tubular transport
proteins involved in renal acid handling on a
molecular level. It is also speculated that a defect
in proximal tubule ammonia synthesis due to insu-
lin resistance is at play.
274
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 Bone Developmental Physiology
10
MH Lafage-Proust

Contents Introduction
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
Ossification is a key stage of bone development
Embryonic Bone Development . . . . . . . . . . . . . . . . . . . . 279
and growth. This complex process combines
Ossification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280 cell–cell contacts, cell proliferation and apoptosis,
Intramembranous Ossification . . . . . . . . . . . . . . . . . . . . . . . 282
cell migration and differentiation, vasculogenesis,
Endochondral Ossification . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
Periosteum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 matrix synthesis, and mineralization. The com-
plete ossification of the skeleton is a very long
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286
process that begins as early as the first weeks of
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 fetal development and ends at the end of growth,
some 20 years later. The local molecular control of
bone growth and development is summarized.
Hormonal regulation and the effects of genetic
mutations are beyond the scope of this review.

Embryonic Bone Development

Bones derive from the mesoderm embryonic

layer. The axial skeleton is formed from the
sclerotome compartment of the somites and the
limbs originate from the lateral plate mesoderm.
The skull arises from neural crest-derived mesen-
chymal cells. The site and the shape of each bone
are determined early during embryonic develop-
ment patterning, which is under tight genetic con-
trol. Briefly, the spine patterning follows the
anterior–posterior axis and results from the for-
mation of segmented mesodermal symmetric
structures, the somites, which develop succes-
M. Lafage-Proust (*)
sively, according sequential bursts of gene expres-
INSERM U 1059, Université de Lyon, Saint-Etienne,
France sion. These waves, which are rhythmically
e-mail: lafagemh@univ-st-etienne.fr activated, act as a molecular oscillator called the
# Springer-Verlag Berlin Heidelberg 2016 279
E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_9
280 M. Lafage-Proust

Fig. 1 Control of limb bud

development: (a) Limb
patterning and growth along
the anterior/posterior and
proximal/distal axes and
illustration of feed-back
loops between FGFS and
Shh signaling pathways.
AER apical ectodermal
ridge, ZPA zone of
polarizing activity, BMP
bone morphogenetic
protein, FGF fibroblast
growth factor, Shh sonic
hedgehog, Grem1 gremlin
1. See text for explanations.
(b) Limb patterning and
growth along the ventral/
dorsal axis, illustration of the
Wnt7a/BMP signaling loop

“segmentation clock” ([1]). They involve the
Notch, Wnt/β-catenin and fibroblast growth factor
(FGF) signaling pathways, which exert specific
perimeters of influence. Vertebrae and ribs will
develop from the sclerotome, at the ventral part
of the somites.
Limb patterning follows an even more com-
plex scheme according to three axes: the limb is
elongated along a proximal–distal axis (PD: from
the shoulder to the last phalanx of the digits), it
flattens along a dorsal–ventral axis (DV: from the
back of the hand to the palm), and it becomes
asymmetric along an anterior–posterior axis (AP:
from the thumb to the little finger; Fig. 1). After
mesenchymal cell condensation at the presump-
tive site of the limb, a bud outgrowth takes place,
whose tip consists of the apical ectodermal ridge
(AER). The AER is a key region that controls the
limb PD elongation via the balance of two major
signaling pathways: Wnt/beta cateninsignaling is
a positive regulator, while BMP signaling is an
inhibitor of limb elongation. In addition, many
members of the FGF family interact at this level,
especially via an FGF10–FGF8 feedback loop.
The zone of polarizing activity (ZPA), which is
located at the posterior part of the limb bud, close
to the AER, controls the AP hand patterning. The
early events are mediated via the combination of
transcription factors, namely Alx 4, Gli 3, Twist
1, and dHAND, followed by the action of sonic
hedgehog (Shh) [2]. Finally, the remaining
non-AER ectoderm region of the limb bud con-
trols limb development along the DV axis mainly
via Wnt and BMP signaling control. Briefly, BMP
activates the expression of En1, an homeotic gene,
in the ventral ectoderm [3], which leads to the
suppression of Wnt7a expression in this region.
Thus, expression of Wnt7a is restricted to the
dorsal ectoderm, where it induces, in the underly-
ing mesoderm, expression of Lmx1b, another
homeobox transcription factor that establishes
the dorsal identity (Fig. 1) [4]. Conversely,
BMPs may act directly in an En-1-independent
manner via inhibition of Lmx1b expression. The
coordination of the three controlling zones is
mediated by inter-connected feed-back loops,
which lead to harmonious limb development.
FGFs expressed in the AER, control Shh expres-
sion in the ZPA, which in turn, regulates FGFs.
Shh is also able to antagonize the inhibitory action
of BMP on AER FGF expression, via Gremlin
1, and in turn, high FGF levels in AER inhibit
Gremlin1 [5].
Ossification
Bone can develop from two different mecha-
nisms: endochondral ossification or
intramembranous ossification. The term
10 Bone Developmental Physiology 281

Fig. 2 Fetal human woven bone. (a) Goldner’s trichrome observed under polarized light, same section as in (a),
stained section. Magnification 100, green mineralized illustrating the woven aspect of primary bone. (c) Adult
bone, pink osteoid tissue. (b) Bone histological sections bone with lamellar collagen
282
Fig. 3 Schematic illustration of endochondral bone ossification
ossification is used because it results from the
transformation of a pre-existing mesenchymal
template, which is cartilage in the case of endo-
chondral ossification and a condensation of
non-cartilaginous mesenchymal cells in the case
of membranous ossification. The skull, the jaw,
and a large part of the scapula undergo membra-
nous ossification. Skeletal ossification results
from the successive appearance of ossification
centers, which take place sequentially in each
bone-to-be, according to a pattern that is tightly
regulated in time and space.
Intramembranous Ossification
The mesenchymal cells proliferate and condense
into nodules. Some cells differentiate into osteo-
blast precursors and then into osteoblasts. Others
become endothelial precursor cells and then endo-
thelial cells, which migrate and form capillaries.
The matrix synthesized by osteoblasts,
called”osteoid”, which surrounds the cells,
M. Lafage-Proust
mineralize progressively. Some osteoblasts get bur-
ied in the matrix and differentiate into osteocytes.
The ossification progresses in a centrifugal way, the
most proliferating and immature cells being at the
periphery. The matrix deposited at this early step is
not well organized. When observed under polar-
ized light, the collagen fibers appear woven
(Fig. 2). Later, it is resorbed by osteoclasts and
lamellar collagen is synthesized by a second set
of osteoblasts. At the skull, the specialized struc-
tures called “sutures,” which connect the various
cranial bones, ossify more progressively during
growth. Morphogenesis and phenotypic preserva-
tion of the sutures are regulated by complex tissue
interactions, especially with the underlying dura
mater, thus allowing the harmonious development
of both the skull and the brain.
Endochondral Ossification
The ossification process summarized here takes a
long bone as an example (Fig. 3).
10 Bone Developmental Physiology
The Cartilage Template
The first step consists of the differentiation of
condensed mesenchymal cells into chondrocytes
and the formation of a cartilage anlage surrounded
by perichondrium [6]. Chondrocytes proliferate
and synthesize a matrix mainly comprising type
II collagen. Then, the deeper chondrocytes
located at the middle of the future diaphysis stop
proliferating and become pre-hypertrophic. These
cells then undergo hypertrophy, increasing their
volume and secreting extracellular matrix
containing type X collagen, which will eventually
mineralize. Meanwhile, the periosteal cells, which
surround the zone of hypertrophic chondrocytes,
differentiate into osteoblasts, leading to the for-
mation of a hollow bony cylinder called the “peri-
osteal collar.” Then, the hypertrophic
chondrocytes facing the bone collar undergo apo-
ptosis, which leads to the collapse of the cartilage
matrix surrounding them, leaving a cavity with
few intact septa.
Formation of the Primary Ossification
Center
Sprouting blood vessels preceded by osteoclasts
(multinucleated bone-resorbing cells) penetrate
the periosteal collar and invade the underlying
mineralized cartilage, allowing the colonization
of the cavity by osteoblast precursors [7], which
differentiate into osteoblasts and rapidly deposit
woven type I collagen matrix, constituting the
primary ossification center (Fig. 4a) [8]. Ossifica-
tion extends from the center of the diaphysis
toward the epiphyses as vascularization develops
and the mineralized cartilage matrix is progres-
sively replaced by bone. According to a similar
mechanism, secondary centers of ossification are
formed at each epiphysis. Vascular invasion is
stimulated by VEGF expression by hypertrophic
chondrocytes owing to activation of the Hif-1
alpha signaling pathway [9].
The Growth Plate Allows Bone
Elongation
The growth plates are located between the diaph-
ysis and the epiphyses of long bones. The growth
plate allows bone elongation until the end of
growth. It comprises several zones (Fig. 4):
283
• The resting zone is adjacent to the trabeculae of
the epiphysis, and contains a thin layer of
small, non-oriented, reserve chondrocytes.
• The proliferation zone consists of stacks of flat
oval cycling chondrocytes organized in longi-
tudinal parallel rows.
• The zone of hypertrophy comprises round and
large chondrocytes within individual lacunae.
This step is preceded by a phase where
pre-hypertrophic chondrocytes exit the cell
cycle. In the lowest layer, the walls of the
lacunae are mineralized and some
chondrocytes exhibit signs of apoptosis
• The zone of ossification contains osteoclasts
that resorb the mineralized cartilage matrix.
Their precursors are recruited at this specific
site via trafficking through the walls of the
numerous arch-shaped vessels that develop at
contact with the hypertrophic zone. Then, a
first set of osteoblasts differentiate and deposit
woven bone on the top of the remnants of
calcified cartilage matrix, using them as scaf-
folds. These trabeculae directly “hanging”
below the hypertrophic zone form “the primary
spongiosa.” They undergo remodeling [10,
11], including a first step of resorption by oste-
oclasts, which remove the woven bone
followed by a second set of osteoblasts that
replace it with lamellar bone, leading to the
formation of the “secondary spongiosa.”
Regulation of Cell Proliferation
and Differentiation
During bone elongation each zone “feeds” the
adjacent one according to a complex regulation
that allows harmonious bone growth [12]. This
progression is tightly regulated by a number of
interacting signaling pathways that balance chon-
drocyte proliferation and hypertrophy [13, 14].
Briefly, Indian hedgehog (Ihh) is synthesized
by the prehypertrophic chondrocytes. It diffuses
toward the proliferating chondrocytes and main-
tains them in a proliferating state by stimulating
PTHrP expression [15]. PTHrP promotes chon-
drocyte cycling and inhibits hypertrophy
[16]. This stimulus stops when chondrocytes
progress and leave the area of PTHrP
diffusion. Ihh also modulates hypertrophy in a
284 M. Lafage-Proust

Fig. 4 (continued)
10 Bone Developmental Physiology
Fig. 4 Growth plate organization. (a) Schematic illustra-
tion of the growth plate. (b, c) Growth plate section in a
growing rat. (b) Magnification 100, Goldner’s trichrome-
stained histological sections. Note the green (i.e., mineral-
ized) walls of the lacunae of hypertrophic chondrocytes. (c)
PTHrP-independent manner [17]. The Wnt
canonical and noncanonical signaling pathways
interact with the Ihh/PTHrP-negative feedback
and contribute to the regulation of the pace
of chondrocyte proliferation, survival, and hyper-
trophy [18]. The spatial organization of the pro-
liferation zone into columns is dependent on
the noncanonical frizzled signaling pathway
[19]. BMP signaling modulates Ihh expression,
chondrocyte proliferation and maturation. It has
a dual action on hypertrophic chondrocytes,
whereby it slows down hypertrophy and promotes
terminal maturation [20]. The major role of FGFs
in bone growth was revealed 20 years ago when
Shiang et al. [21] reported that mutations in the
FGFR3 transmembrane domain were responsible
for achondroplasia, one of the most common
forms of dwarfism caused by genetic mutations.
FGFR3 signaling inhibits chondrocyte prolifera-
tion and hypertrophy. FGFR1 and FGFR2 are also
involved in the regulation of bone growth. FGF18
is the major ligand in a number of cell types,
including hypertrophic chondrocytes and
perichondrial cells. It promotes both proliferation
and modulates initiation of chondrocyte hypertro-
phy at early stages of chondrogenesis, mediates
285
Magnification 250, toluidine blue staining, cartilage is
stained dark purple. (d) Tartrate-resistant acid phosphatase
(TRAP) histo-enzymological staining of osteoclasts at the
junction between the hypertrophic zone and the primary
spongiosa, aniline blue counter-staining, 400
bone vascularization as well as osteoclast and
osteoblast recruitment to the growth plate
[22]. FGF effects may also be down-regulated
by local specific inhibitors such as Sef, which is
expressed in the periosteum and at the
osteochondral junction in the growth plate of neo-
natal mice [23]. Basically, BMP and FGF signal-
ing pathways exert opposite effects in the growth
plate. For instance, it was recently shown that the
deletion of BMP type I receptor a (BMPR1a) in
FGFR3 knock-out mice rescued the bone over-
growth phenotype by reducing chondrocyte dif-
ferentiation, because FGFR3 activation induces
the proteasome degradation of Bmpr1a [24].
The master genes for chondrocyte and osteoblast
differentiation are Sox9 [25] and Runx2 [26, 27]
respectively. These transcription factors are essen-
tial molecular switches for the commitment of mes-
enchymal cells toward the cartilage and bone-
forming cell lineages. They also play a major role
in the differentiation and the maintenance of their
phenotype. Therefore, they are necessary targets of
the above-mentioned signaling pathways. Basi-
cally, activation of Wnt signaling leads to enhanced
ossification and suppressed chondrocyte formation,
whereas genetic inactivation of beta-catenin, the
286
main factor involved in intracellular canonical Wnt
signal transduction, causes ectopic formation of
chondrocytes at the expense of osteoblast differen-
tiation during both intramembranous and endo-
chondral ossification [28]. Sox9 is phosphorylated
and activated by PTHrP and it antagonizes the Wnt
canonical signaling pathway by promoting beta-
catenin proteasome degradation [29]. Runx2 exerts
complex effects according to the cells where it is
activated, namely chondrocytes or perichondrial
cells. Runx2 deficiency in mice decreases chondro-
cyte Ihh expression and retards chondrocyte hyper-
trophy [30]. In contrast, in perichondrial cells,
Runx2 activation (under the inhibitory control of
Twist-1) leads to FGF18 release and inhibition of
chondrocyte maturation [31]. At the level of the
primary and secondary spongiosae, the mecha-
nisms of osteoblast differentiation are not
completely understood. During long bone develop-
ment, all the populations of osteoblasts within peri-
osteum and osteoblasts at the endosteal and
trabecular bone surfaces within the marrow are
derived from the embryonic perichondrium. Differ-
entiation of osteoblast precursors, chondrocyte apo-
ptosis, and the development of vessels at contact
with the growth plate are functionally [32] and
spatially coupled. Skeletal stem cells borne by the
vessel walls may differentiate into osteoblasts.
Recently, Kusumbe et al. [33] described two sub-
types of bone vessels according to their high (H) or
low (L) level of CD31 and endomucin expression.
In young animals, H vessels contain more prolifer-
ating endothelial cells. They are abundant in the
primary spongiosa and run along the cortical end-
osteum. H vessel walls bear more osteoprogenitors
and express higher levels of HIF-1α. Their number
decreases with age. Inhibition or activation of the
HIF-1α signaling pathway in endothelial cells only,
modulates the number of perivascular osteopro-
genitors. In addition, Zhou et al. used mice in
which activation of gene promoters specific to
hypertrophic or nonhypertrophic chondrocytes
(i.e., type X collagen and aggrecan respectively)
could be observed and allowed cell lineage track-
ing. They showed that both chondrocytes before
initial ossification and growth plate chondrocytes
before or after birth could transdifferentiate into
osteoblasts [34].
M. Lafage-Proust
Periosteum
The periosteum is composed of two distinct
layers (Fig. 5): an outer fibrous layer and an
inner proliferative cambium layer in contact
with the underlying cortical bone. The outer
fibrous layer consists of fibroblasts and thick
collagen fibers. The inner layer is highly
vascularized and contains mesenchymal stem
cells that can differentiate into osteoblasts and
chondrocytes. During growth, periosteal bone
apposition increases the outer perimeter of the
bone, while resorption occurs at the endocortical
surface, leading to harmonious radial expansion
of both the bone cortex and the marrow cavity
[35]. However, progressive cortex thickening
occurs, due to an excess of outer formation over
inner resorption, and is associated with bone
tissue displacement away from the neutral axis
of the shaft, thus optimizing bone mechanical
resistance to load [36]. Periosteal apposition is
continuous in the growing skeleton, characteris-
tic of bone modeling [37]. Studies in various
mouse strains showed that cortical bone geome-
try (size and shape), which is partly determined
by periosteum activity, as seen above, is geneti-
cally controlled by different, although
overlapping, groups of polymorphic loci [38]. It
is highly dependent on mechanical load. The
biology of the periosteum has been less fre-
quently studied than that of longitudinal growth.
Recently, it was shown that periosteal cells
express periostin, an extracellular matrix protein,
also expressed by cortical osteocytes. This
expression is stimulated by load and PTH
[39]. BMP2 expression in osteoblasts controls
periosteal vascularization [40].
Conclusion
Our knowledge of bone development and growth
biology is not complete. Indeed, the physiology
of endochondral ossification has been exten-
sively studied over the past 20 years at the molec-
ular level, which has brought major insights into
the pathophysiology of a number of skeletal
10 Bone Developmental Physiology
Fig. 5 Periosteum. (a) Schematic illustration of the periosteum during growth. (b, c) Goldner’s trichrome-stained bone
histological sections of periosteum in growing humans, green mineralized bone, pink osteoid tissue. (b) 100, (c) 250
diseases that affect the pediatric population.
Furthermore, this information has been some-
how “recycled” in the research on osteoarthritis
(OA), which is a major public health concern,
since some of the events are recapitulated during
OA progression at the osteochondral junction. In
contrast, the role of the periosteum and the pre-
cise molecular mechanisms that control its func-
tions remain to be elucidated. Similarly, it is only
recently that major breakthroughs have been
reported in bone vessel heterogeneity and its
specific roles.
287
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 Physiology of the Developing Kidney:
Disorders and Therapy of Calcium 11
and Phosphorous Homeostasis

€ppner
Amita Sharma, Rajesh V. Thakker, and Harald Ju

Contents Jansen’s Metaphyseal Chondrodysplasia (JMC) . . . . 298

Williams Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 Infantile Hypercalcemia: CYP24A1 Deficiency . . . . 300
Regulators of Calcium and Phosphate Hypocalcemia and Hyperphosphatemia Due
Homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 to Reduced Parathyroid Hormone Activity . . . . . . 300
Parathyroid Hormone (PTH) and PTH-Related Hypoparathyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
Peptide (PTHrP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 Abnormalities at the Calcium-Sensing Receptor
Vitamin D and Its Metabolites . . . . . . . . . . . . . . . . . . . . . . . 293 (CaSR) and the Downstream Signaling
Fibroblast Growth Factor 23 (FGF23) and Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
Other Proteins with Phosphaturic Properties . . . . . . . . 294 Pseudohypoparathyroidism (PHP) . . . . . . . . . . . . . . . . . . 308
Hypercalcemia and Hypophosphatemia Due Blomstrand’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310
to Increased Parathyroid Hormone Secretion . . . 295 Hyperphosphatemic Disorders with Reduced
Parathyroid Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 Secretion of Biologically Active FGF23 . . . . . . . . . . . 311
Hyperparathyroidism in Chronic Kidney Hyperphosphatemic Familial Tumoral
Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296 Calcinosis (HFTC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
Non-syndromic Isolated Hyperparathyroidism . . . . . . 296 Hyperostosis with Hyperphosphatemia (HHS) . . . . . . 312
Hypercalcemic Disorders Without Elevated Hypophosphatemic Disorders . . . . . . . . . . . . . . . . . . . . . 312
Parathyroid Hormone Secretion . . . . . . . . . . . . . . . . . . 296 Hypophosphatemic Disorders with Increased
Disorders of the Calcium-Sensing Receptor (CaSR) FGF23 Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
and the Downstream Signaling Molecules . . . . . . . . . . 296 Hypophosphatemic Disorders with Normal or
Suppressed FGF23 Activity . . . . . . . . . . . . . . . . . . . . . . . . . 315
Other Hypophosphatemic Disorders . . . . . . . . . . . . . . . . 317
A. Sharma (*) Acute and Chronic Treatment of Calcium
Department of Pediatrics, Pediatric Nephrology Unit, and Phosphorus Disorders . . . . . . . . . . . . . . . . . . . . . . . . . 317
Massachusetts General Hospital and Harvard Medical Hypercalcemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
School, Boston, MA, USA Hypocalcemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
e-mail: asharma5@mgh.harvard.edu Hyperphosphatemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
R.V. Thakker Hypophosphatemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320
Radcliffe Department of Medicine, Academic Endocrine Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
Unit, University of Oxford, OCDEM (Oxford Centre for
Diabetes, Endocrinology and Metabolism), The Churchill References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
Hospital Headington, Oxford, UK
e-mail: rajesh.thakker@ndm.ox.ac.uk
H. J€uppner
Departments of Medicine and Pediatrics, Endocrine Unit
and Pediatric Nephrology Unit, Massachusetts General
Hospital and Harvard Medical School, Boston, MA, USA
e-mail: jueppner@helix.mgh.harvard.edu

# Springer-Verlag Berlin Heidelberg 2016 291

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_10
292
Introduction
The regulation of calcium and phosphate homeo-
stasis involves several different hormones that
act on the kidney, intestine, and bone. The most
important calcium-regulating peptide hormone is
parathyroid hormone (PTH). Its production and
secretion by the parathyroid glands increases
in response to a decrease in the extracellular
calcium concentration. PTH increases (1) the
1α-hydroxylase activity in the proximal renal
tubules to promote production of the biologically
active 1,25-dihydroxyvitamin D (1,25(OH)2D)
from its precursor 25-hydroxyvitamin D, thereby
enhancing intestinal absorption of calcium (and
phosphorus); (2) it stimulates bone resorption,
thus releasing calcium and (phosphorus); (3) it
enhances calcium reabsorption in the distal renal
tubules; and (4) it promotes urinary phosphate
excretion. These phosphaturic actions of PTH
occur within minutes by reducing the expression
of two sodium-dependent phosphate
cotransporters, NPT2a and NPT2c, in the proxi-
mal convoluted tubules. The long-term regula-
tion of phosphate homeostasis involves
fibroblast growth factor 23 (FGF23), a more
recently discovered hormone made by osteocytes
and probably osteoblasts. Like PTH, FGF23
reduces the expression of NPT2a and NPT2c,
but the time courses for the effects of both hor-
mones are very different [1–3]. Furthermore, in
contrast to the stimulatory actions of PTH on
1α-hydroxylase, FGF23 reduces the expression
of this enzyme in the proximal renal tubules,
and it enhances 24-hydroxylase, leading to two
different mechanisms to a reduction in serum
1, 25(OH)2D levels [4–8].
This chapter will review the physiological and
biochemical mechanisms underlying calcium and
phosphate homeostasis and the genetic basis of
rare inherited disorders that provided important
novel insights into the regulation of these
minerals [9].
A. Sharma et al.
Regulators of Calcium and Phosphate
Homeostasis
Parathyroid Hormone (PTH) and
PTH-Related Peptide (PTHrP)
The mature secreted form of PTH peptide com-
prises 84 amino acids, which is derived from a
longer prepropeptide (for review, see Ref. [10]).
PTH gene transcription (as well as PTH peptide
secretion) is regulated primarily by the extracel-
lular concentration of calcium and through a vita-
min D response element upstream of the
transcription start site [11, 12]. Phosphate affects
also parathyroid gland activity, independent of
concomitant changes in ambient calcium concen-
tration, parathyroid gland activity; however, these
effects take several hours and may result from
secondary changes in hormone biosynthesis
rather than secretion [13–15].
PTH secretion by the parathyroid glands is
regulated through the calcium-sensing receptor
(CaSR), which recognizes remarkably small per-
turbations in calcium and responds quickly by
altering PTH secretion. This G protein-coupled
receptor is stimulated by extracellular calcium
and requires coupling to two closely related G
proteins, namely, Gαq and Gα11, to activate the
Ca+2/IP3/PKC signaling pathway. Besides being
expressed in the chief cells of the parathyroid
glands, the CaSR is expressed in the kidneys and
in several other tissues, albeit at lower abundance,
which are not directly involved in the regulation
of calcium homeostasis. In addition to regulating
PTH secretion, the CaSR plays an important role
in regulating aquaporin trafficking in the
kidneys [16].
The PTH-related peptide (PTHrP also known
as PTHrH, PTH-related hormone), which was
first isolated from tumors that cause the humoral
hypercalcemia of malignancy, shares significant
amino acid sequence homology with PTH
[17–19]. PTH and PTHrP, although distinct
11 Physiology of the Developing Kidney: Disorders and Therapy of Calcium and Phosphorous. . .
Multiple
tissues
PTHrP PTH
Ca++
Cartilage
Breast
Bone
Teeth Kidney
Pancreas
Skin Regulation of
other tissues ? calcium and phosphate
Fig. 1 Parathyroid hormone (PTH) mediates its endocrine
actions through the PTH/PTHrP receptor expressed in the
kidney and the bone to regulate mineral ion homeostasis
and bone metabolism. In numerous other tissues, this G
protein-coupled receptor mediates the paracrine actions of
PTHrP; particularly important is its role in the growth plate
products of different genes, exhibit considerable
functional and structural similarities, including
equivalent positions of the boundaries between
some of the coding exons and of the adjacent
introns; both may have evolved from a shared
ancestral gene [10, 20]. PTHrP is a larger, more
complex protein than PTH that functions as an
autocrine/paracrine rather than an endocrine fac-
tor with a little influence on calcium homeostasis
except when secreted in large concentrations
[21]. Its most prominent functions involve the
regulation of chondrocyte proliferation and dif-
ferentiation, and consequently bone elongation
and growth [22]. It also affects mammary gland
development and function [23, 24].
PTH and PTHrP both mediate their actions
through a common G protein-coupled receptor,
the PTH/PTHrP receptor [25, 26] (Fig. 1). In the
kidney and bone, it mediates the endocrine actions
of PTH. However, the most abundant expression
of the PTH/PTHrP receptor occurs in
chondrocytes of the metaphyseal growth plate
where it mediates the autocrine/paracrine actions
of PTHrP, i.e., it delays the hypertrophic
293
differentiation of growth plate chondrocytes
[22, 27]. A closely related receptor, the PTH2
receptor, shares more than 50 % homology with
the PTH/PTHrP receptor [28]. Its primary ligand,
TIP39 (tuberoinfundibular peptide of 39 residues),
is involved in nociception and reproduction
[29–31].
Vitamin D and Its Metabolites
Most of the vitamin D (D2 and D3) in healthy
individuals is derived from the precursor
7-dehydrocholestrol through exposure to ultravi-
olet light. It is made in the skin and is stored in
muscle or fat [32, 33]. Presumably through the
enzyme CYP2R1 [34, 35], the prohormone
undergoes hydroxylation in the liver to form the
25-hydroxyvitamin D metabolite (25(OH)D). In
the proximal convoluted tubular cells of the kid-
ney, 25(OH)D is further hydroxylated by the
enzyme 25-hydroxyvitamin D-1α-hydroxylase
(1α-hydroxylase; CYP27B) to yield the biologi-
cally active metabolite 1,25 dihydroxyvitamin D
(1,25(OH)2D) [32, 33]. The expression of
CYP27B is regulated by extracellular concentra-
tions of ionized calcium, inorganic phosphate,
PTH, and FGF23 [36]. 1,25(OH)2D binds in tar-
get organs (e.g., the intestine, bones, kidneys,
and parathyroids) to the intracellular vitamin D
receptor (VDR) [37] and thereby activates the
transcription of genes in the bone, kidney, and
enterocytes that help increase gut absorption of
calcium, reduce urinary calcium losses, and
increase bone resorption, thereby ensuring ade-
quate extracellular concentration of calcium and
phosphate [33, 37]. Homozygous or compound
heterozygous mutations in CYP27B, CYP2R1,
or VDR lead to hypocalcemia, which increases
PTH secretion, thus enhancing urinary phosphate
excretion, which leads to the development of rick-
ets/osteomalacia [32–35].
294
1 25
N-term.
C-term.
Fig. 2 The primary structure of FGF23. The C-terminal
and the N-terminal portions of FGF23 are connected by an
“RXXR” motif, which is the recognition site for a furin-
Fibroblast Growth Factor 23 (FGF23)
and Other Proteins with Phosphaturic
Properties
Fibroblast growth factor 23 (FGF23), which is
probably the most important phosphate-regulating
hormone, belongs to a large family of structurally
related proteins. FGF23 was discovered through
two independent approaches, namely, positional
cloning to define the molecular cause of autoso-
mal dominant hypophosphatemic rickets (ADHR)
[38] and sequence analysis of cDNAs derived
from rare mesenchymal tumors that cause tumor-
induced osteomalacia (TIO) [6, 39]. The FGF23
gene consists of 3 exons that encode a 251-amino
acid precursor protein comprising a hydrophobic
leader sequence (residues 1–24), thus allowing its
secretion into the blood circulation (Fig. 2). The
Golgi casein kinase Fam20C phosphorylates
Ser180 of FGF23, which reduces O-glycosylation
of Thr178 by the polypeptide N-acetylgalactosami-
nyltransferase 3 (GALNT3), thus making the hor-
mone prone to proteolytic cleavage between
Arg179 and Ser180 by the subtilisin-like proprotein
convertase SPC2 [40, 41]. Only full-length
FGF23 has phosphaturic activity [7], yet
C-terminal fragments appear to have some biolog-
ical activity since they can antagonize the actions
of the intact hormone [42] (Fig. 2).
FGF23 is most closely related to fibroblast
growth factors 21 and 19, but shows limited homol-
ogy also with other fibroblast growth factors [38,
43]. FGF23 mRNA could not be detected by North-
ern blot analysis in normal tissues, but it has been
A. Sharma et al.
~15 kDa ~8 kDa
176-9 251
FAM20c-dependent
phosphorylation
RXTR… SAEDD..
Cleavage by Furin-
like enzyme
like protease. Mutations at this site make FGF23 resistant
to the cleavage by a furin-like protease, thereby stabilizing
the intact bioactive FGF23 molecule
identified by reverse transcriptase (RT)-PCR in the
heart, liver, thymus, small intestine, and brain [38,
43]. The most abundant expression of FGF23
occurs in the bone cells, particularly in osteocytes,
which represent the largest population of bone cells
[44–47]. Its regulation remains incompletely under-
stood, but mice that are null for DMP1, ENPP1, or
Fam20C show an increased expression of FGF23,
suggesting that each of these proteins are negative
regulators of FGF23 expression and/or activity
[48–50]. Consistent with these findings, humans
with inactivating mutations in any of these three
genes show increased urinary phosphate excretion
leading to hypophosphatemia [48, 51–57].
In the presence of αKlotho, a protein associ-
ated with longevity [58, 59], FGF23 binds with
high affinity to FGFR1 [59, 60] (Fig. 3). Consis-
tent with a role of Klotho in the regulation of
phosphate homeostasis, mice that are “null” for
Klotho develop severe hyperphosphatemia due to
diminished urinary phosphate excretion. These
animals furthermore show elevated 1, 25(OH)2D
levels, i.e., findings that are similar to those
observed in the FGF23-null mice [4, 45,
46]. Klotho-null mice have dramatically elevated
FGF23 levels [60, 61].
FGF23-null mice (FGF23/) develop
hyperphosphatemia and elevated serum 1,25
(OH)2D concentration, and they die prematurely,
secondary to renal failure because of glomerular
capillary calcifications [45, 62, 63]. FGF23/
animals furthermore show reduced bone turnover,
an unexpected increase in osteoid, and diminished
osteoblast and osteoclast number and activity.
11 Physiology of the Developing Kidney: Disorders and Therapy of Calcium and Phosphorous. . .
sFRP4
FGF23 FGF7
Klotho
Other R?
FGFR1c (co-receptor)
FGFR1c
Inhibition
??
of 1 α-
hydroxylase
NaPi-IIa NaPi-IIc
Fig. 3 Effect of FGF23 and PTH on the proximal tubular
cells. (a) FGF23 binds to the FGFR1c and Klotho complex
and inhibits 1α-hydroxylase. At the same time, this cascade
inhibits the apical expression of NaPi-IIa and NaPi-IIc. (b)
PTH binds to the PTHR(GPCR) and inhibits the
Animals overexpressing FGF23 transgenically
show increased unmineralized osteoid because
of increased urinary phosphate excretion leading
to hypophosphatemia as well as significant wid-
ening of growth plates leading to deformities of
weight-bearing bones [45, 64].
In addition to FGF23, other cDNAs were over-
represented in libraries derived from tumors that
cause oncogenic osteomalacia [6, 65, 66]. These
included DMP1, MEPE, sFRP4, and FGF-7 and
were implicated in phosphate handling in vivo
and/or in vitro, suggesting that “phosphatonins”
other than FGF23 may be involved in the renal
regulation of phosphate homeostasis [66–68].
Their role needs to be elucidated more as the abla-
tion of sFRP4 in mice at least does not affect
phosphate homeostasis [69].
Hypercalcemia and
Hypophosphatemia Due to Increased
Parathyroid Hormone Secretion
Hypercalcemia can be observed in several differ-
ent nonfamilial (sporadic) or familial disorders.
Besides physical examination and a careful
review of the family history, the evaluation of
295
PTH
Second messengers
Stimulation of
1α-hydroxylase
NaPi-IIa NaPi-IIc
expression of NaPi-IIa and NaPi-IIc, whereas it simulates
1α-hydroxylase activity. Thus, FGF23 only acts as stimu-
late phosphaturia, and PTH acts both P and Ca homeostasis
simultaneously
several additional laboratory parameters is
required for diagnostic work-up (Fig. 4).
Parathyroid Tumors
Increased parathyroid gland activity can be seen in
patients with multiple gland hyperplasia, adenomas,
and, rarely, parathyroid carcinoma. Two principal
defects can lead to the development of parathyroid
tumors: (1) a heterozygous mutation that enhances
the activity of a gene (gain-of-function mutation),
which is therefore referred to as a proto-oncogene,
or (2) homozygous loss-of-function mutation in a
tumor-suppressor gene, which is therefore referred
to as a recessive oncogene. Parathyroid tumors usu-
ally occur as an isolated and sporadic
endocrinopathy or as part of inherited tumor syn-
dromes [70] such as the multiple endocrine neopla-
sias (MEN) or hereditary hyperparathyroidism with
jaw tumors [71, 72]. Sporadic parathyroid tumors
can be caused by single somatic mutations that lead
to the activation or overexpression of proto-
oncogenes, such as PRAD1 ( parathyroid adenoma
1) or RET (mutated in MEN2), or by mutations in
tumor-suppressor genes – predicted to be located on
several different chromosomes, for example, chro-
mosome 1p (RIZ1) and 11q3 (MEN1) – that allow
296
Hypercalcemia
Serum PTH↑
sP↓, uCa↑, ↓TRP sP↑, ↑TRP
Primary hyperparathyroidism CKD?
Sporadic
Familial
Adenoma
Carcinoma Multiple endocrine neoplasia Type I
Multiple endocrine neoplasia Type II
Hereditary hyperparathyroidism with jaw tumors
Neonatal severe hyperparathyroidism ( NSHPT)
Fig. 4 Flow diagram for the work-up of patients with hypercalcemia
for the clonal expansion of a single parathyroid cell
and its progeny (see Ref. [73] for a detailed review
of this topic in adults). In children, parathyroid
tumors are rare, with adenomas being the most
common abnormality [74].
Hyperparathyroidism in Chronic
Kidney Disease
Chronic overstimulation of the parathyroid glands
frequently occurs in patients with chronic kidney
disease (CKD), which may lead to the development
of hyperplasia transitioning to adenoma (tertiary
hyperparathyroidism) due to the clonal expansion
of one or several parathyroid cells [75], through a
process possibly involving the reduced expression
of different cyclin-dependent kinase inhibitors [76]
and an abnormal WNT β-catenin signaling path-
way [77]. Increased levels of biologically active
circulating FGF23 [78–81] and decreased levels
of 1,25(OH)2D [5, 82–84] contribute to the devel-
opment of hyperparathyroidism.
Non-syndromic Isolated
Hyperparathyroidism
PTH structural changes as the cause of hyperpara-
thyroidism appear to be rare. One patient, who
A. Sharma et al.
Serum PTH nl-↓
sP↓-nl, uCa↑ sP↓-nl, uCa↓
Williams Syndrome Familial Benign Hypercalcemia (FBH)
McCune-Albright Syndrome FBH1: loss-of-function mutation in CaSR
Jansen’s disease: PTH/PTHrP FBH2: gain-of-function mutation in Gα11
receptor activation FBH3: activating mutation in AP2σ2
(these three are associated with Autoimmune Hypocalciuric Hypercalcemia (AHH)
developmental abnormalities)
Infantile hypercalcemia
presented with hypercalcemia and
hypophosphatemia, yet undetectable levels of
PTH in the circulation, was reported. A single
parathyroid adenoma was identified that secreted
a mutant PTH molecule, which was truncated
after amino acid residue 52, thus explaining why
different two-site immunometric PTH assays had
been unable to detect elevated circulating levels of
this hormone [85]. After surgical removal of the
adenoma, clinical symptoms and biochemical
abnormalities resolved.
Hypercalcemic Disorders Without
Elevated Parathyroid Hormone
Secretion
Disorders of the Calcium-Sensing
Receptor (CaSR) and the Downstream
Signaling Molecules
CaSR is a G protein-coupled receptor (GPCR)
located on chromosome 3q21.1 [86] that couples
through Gαq and Gα11 to the Ca2+/IP3/PKC sig-
naling pathway. Three hypocalciuric hypercalce-
mic disorders are caused by abnormalities in
calcium-sensing receptor (CaSR) function:
(1) familial benign hypercalcemia (FBH), also
referred to as familial hypocalciuric hypercalce-
mia (FHH), (2) neonatal severe
11 Physiology of the Developing Kidney: Disorders and Therapy of Calcium and Phosphorous. . .
hyperparathyroidism (NSHPT), and (3) autoim-
mune hypocalciuric hypercalcemia (AHH) caused
by acquired autoantibodies against the CaSR.
Familial Hypocalciuric Hypercalcemia
(FHH): FHH1 (CaSR), FHH2 (Ga11),
FHH3 (AP2s1)
FHH is a genetically heterogeneous disorder
which may be inherited as an autosomal dominant
trait, although patients often have no family his-
tory as they may have developed a de novo muta-
tion. Affected patients are usually asymptomatic
or have nonspecific symptoms such as fatigue,
weakness, painful joints, and headache, and the
diagnosis is often only suspected after routine
biochemical screening shows elevated serum cal-
cium levels.
Three variants of FHH have been defined at the
molecular level; these are (1) loss-of-function
mutations in CaSR (FHH1) [87–92]; (2) loss-of-
function in Gα11, the signaling protein at the
CaSR (FHH2) [93, 94]; and (3) loss-of-function
mutations in AP2σ1 (adapter protein-2 sigma
subunit), a central component of clathrin-coated
vesicles (FHH 3) [95]. FHH2 and FHH3 families
were initially linked genetically to chromosome
19p (FHH19p) and 19q13 [96] (FHHOK), respec-
tively [97, 98]. Heterozygous loss-of-function
CaSR mutations were found in approximately
two-thirds of the affected members of investigated
FHH kindreds (FHH1) [87, 92, 99, 100]. CaSR
mutations cluster around low-affinity calcium-
binding sites (that are similar to calsequestrin)
containing aspartate- and glutamate-rich regions
(codons 39–300) within the extracellular domain
of the receptor [101, 102] and show gene-dosage
effect [87–92, 102]; for details of CaSR muta-
tions, visit http://www.casrdb.mcgill.ca. Another
heterozygous mutation in exon 4 of CaSR gene
(F180C, TTC > TGC) leads to FHH phenotype
only in vitamin D deficiency state. Vitamin D
deficiency probably impaired CaSR expression,
thus leading to increased PTH secretion; consis-
tent with this conclusion, vitamin D supplemen-
tation resulted in the normalization of PTH levels
[103]. Individuals in FHH families carrying the
CaSR mutation L137P have shown increased sus-
ceptibility for chronic pancreatitis, when
297
compounded with a mutation (N34S) in the pan-
creatic secretory trypsin inhibitor gene
(SPINK1) [104].
Neonatal Severe Hyperparathyroidism
(NSHPT)
Patients with severe neonatal hyperparathyroid-
ism usually have homozygous or compound het-
erozygous CaSR mutations that are both
inactivating [87, 88, 90, 105–107]. Infants with
NSHPT usually exhibit severe bone disease
driven by markedly increased PTH, leading to
significant mortality, if left untreated [108,
109]. Some patients with sporadic neonatal hyper-
parathyroidism have been reported to carry de
novo heterozygous CaSR mutations [89], thereby
suggesting the involvement of factors other than
the dosage of the mutant gene or dominant-
negative actions on the wild-type CaSR [105]. A
novel heterozygous de novo mutation (R551K)
was shown recently to cause NSHPT, which grad-
ually reverted to asymptomatic FBH without the
need for surgical intervention [110].
Autoimmune Hypocalciuric
Hypercalcemia (AHH)
AHH is an acquired disorder with circulating anti-
bodies directed against the extracellular domain of
the CaSR. Some of these antibodies stimulate the
release of PTH when tested with dispersed human
parathyroid cells in vitro, probably by reducing the
activation of the CaSR by extracellular calcium
[111]. AHH diagnosis should be considered for
patients who have hyperparathyroidism in combi-
nation with other autoimmune disorders [111, 112]
and lack mutations associated with FHH. AHH was
initially described in four individuals from two
unrelated kindreds. Among them, three patients
had antithyroid antibodies, and one had celiac
sprue with antigliadin and anti-endomyseal anti-
bodies [111]. Another patient described had an
IgG4 blocking autoantibody against CaSR.
Regression of biochemical abnormalities and auto-
immune dysregulation followed glucocorticoid
treatment [112]. An autoantibody isolated from a
patient with AHH caused distinct conformational
changes in CaSR favoring coupling to Gα11 and
uncoupling from Gi. These allosteric changes
298
Fig. 5 Patient with a severe form of Jansen’s metaphyseal
chondrodysplasia. (From [114], with permission)
occurred at a site in close proximity to the binding
site for calcimimetics [113].
Jansen’s Metaphyseal
Chondrodysplasia (JMC)
JMC is a rare autosomal dominant disease that is
characterized by short-limbed dwarfism caused by
an abnormal regulation of chondrocyte prolifera-
tion and differentiation in the metaphyseal growth
plate [114] (Figs. 5 and 6) that is usually associated
with severe hypercalcemia and hypophosphatemia,
despite normal or undetectable serum levels of
PTH or PTHrP [115]. JMC is caused by heterozy-
gous mutations in the PTH/PTHrP receptor that
lead to constitutive PTH- and PTHrP-independent
receptor activation [116–118]. Since the
PTH/PTHrP receptor is most abundantly expressed
in the kidney, bone, and growth plates, these find-
ings provided a likely explanation for the abnor-
malities observed in mineral homeostasis and for
the associated defects in chondrocyte growth and
differentiation. Several different heterozygous
mutations in the PTH/PTHrP receptor have been
identified in the severe form of JMC; these involve
A. Sharma et al.
codon 223 (His ! Arg), codon 410 (Thr ! Pro
or Arg), and codon 458 (Ile ! Arg or Lys). The
expression of these mutant receptors in suitable
cells resulted in constitutive agonist-independent
accumulation of cAMP, while the basal accumula-
tion of inositol phosphates was not measurably
increased. The H223R mutation is the most fre-
quent cause of JMC, while all other mutations were
found in only single patients [116–122]. Trans-
genic mice, in which the expression of a
PTH/PTHrP receptor carrying the H223R mutation
was targeted to the growth plate by the rat α1
(II) collagen promoter, showed a significant delay
in chondrocyte differentiation, supporting the con-
clusion that the defect in endochondral bone for-
mation in JMC patients is caused by the
constitutively active mutant receptor [123]. The
slowed differentiation of growth plate
chondrocytes was associated with an upregulation
of cyclin- and E2F-dependent gene expression,
indicating that the PTH/PTHrP receptor controls
the timing of cell cycle exit and the onset of differ-
entiation of chondrocytes [124]. The Thr410Arg
mutation in PTH/PTHrP receptor leads to less pro-
nounced skeletal and laboratory abnormalities, i.e.,
only mild skeletal dysplasia and relatively normal
stature and high-normal plasma calcium concentra-
tion associated with normal or suppressed serum
PTH levels and with hypercalciuria leading to
nephrolithiasis in two individuals [125]. In com-
parison to PTH/PTHrP receptors with the
Thr410Pro mutation, the Thr410Arg mutation
showed less pronounced agonist-independent
cAMP accumulation in vitro [117, 126].
Williams Syndrome
Williams syndrome (WS) is a hemizygous contin-
uous deletion syndrome caused by the deletion of
25–30 genes on chromosome 7 q11.23. WS is
characterized by vascular stenosis, hypertension,
elfin-like facies, developmental delay, and typical
behavioral features. Endocrine abnormalities
including hypercalcemia, diabetes mellitus, and
subclinical hypothyroidism might be present.
About 20 % of affected children may present
with congenital anomalies of the kidney and
11 Physiology of the Developing Kidney: Disorders and Therapy of Calcium and Phosphorous. . . 299

Fig. 6 Radio- and photographs of patients with a relatively mild form of Jansen’s disease (From [125] with permission)

urinary tract, hypercalciuria, or dysfunctional
voiding [127].
Of the deleted genes, elastin is the most impor-
tant. The ablation of elastin gene in mice results in
vascular abnormalities similar to those observed
in WS patients [128]. Homozygous deletion at the
elastin locus was found in over 90 % of patients
with the classical Williams phenotype [129], and a
series of 235 WS patients revealed submicro-
scopic deletions of the elastin gene in 96 % of
the investigated individuals [130], thus making it
an important diagnostic tool. Other deleted pro-
teins, like LIM-kinase [131], the cytoplasmic
linker protein-115 (CYLN2), and the transcription
factors GTF2I and GTF2IRD1 [132], may con-
tribute to some of the distinct neurological and
cognitive deficits. In contrast, NCF1 deletions are
protective against hypertension [133]. In addition,
genes flanking aneuploid genes, some of which
are located several megabases away from the
deletion, may contribute to the observed pheno-
typic variation [134, 135].
300 A. Sharma et al.

The etiology of hypercalcemia is less clear.
Although it is clinically not severe in most cases,
some affected individuals require therapeutic
interventions with bisphosphonates [136]. The
calcitonin receptor gene, located on chromosome
7q21, is not included in the deletions identified in
WS [137]. Increased calcium retention, sensitiv-
ity, or abnormal synthesis or degradation of 1,25
(OH)2 vitamin D has been postulated to be causal
[138, 139]. Claudin 3 and 4 though deleted were
not found to be causative for hypercalcemia
[140]. The transient receptor potential cation
channel 3 (TRPC3) overexpressed in the intes-
tines and kidneys of a WS patient has been
shown to increase calcium absorption in the gut
and reabsorption in the kidneys [141].
Infantile Hypercalcemia: CYP24A1
Deficiency
Initially referred to as idiopathic infantile hyper-
calcemia (IIH), this disorder was shown to be
caused by homozygous or compound heterozy-
gous mutations in the gene encoding the
24-hydroxylase (CYP24A1), the enzyme that
metabolizes and thus inactivates the biologically
active 1,25(OH)2D [142–146]. Affected infants
develop often severe hypercalcemia and
hypercalciuria leading to nephrocalcinosis and
failure to thrive. Besides elevated plasma and
urinary calcium levels, 1,25(OH)2D levels are
increased, which was shown in one child to dra-
matically enhance intestinal calcium absorption
[143], thereby suppressing circulating PTH con-
centrations through two mechanisms, i.e., ele-
vated plasma calcium and 1,25(OH)2 vitamin D
concentration.
Hypocalcemia and
Hyperphosphatemia Due to Reduced
Parathyroid Hormone Activity
As for hypercalcemic disorders, patients who
present with hypocalcemia require careful
clinical evaluation, as well as the assessment of
laboratory parameters in serum and urine, and a
detailed review of the family history [147]
(Fig. 7).
Hypoparathyroidism
Hypoparathyroidism comprises a heterogeneous
group of disorders presenting with hypocalcemia

Hypocalcemia

Serum PTH nl-↓ (sP↑; uCa nl-↑) Serum PTH ↑

Hypoparathyroidism
sP↓; uCa nl-↓ sP↑; uCa↓

Isolated Forms Complex syndromic forms

autosomal dominant DiGeorge syndrome ( TBX1) Vitamin D deficiency Pseudohypoparathyroidism (PHP)
autosomal recessive HDR (GATA3) (25 (OH ) ↓;1,25(OH) NL-↑)
X-linked Kenny-Caffey/Sanjad-Sakati syndrome ( TBCE; FAM111A)
Autoimmune polyglandular syndrome
VDDR type I
CaSR abnormalities
Genetic mutations in: (25 (OH ) NL;1,25(OH) ↓) Without AHO With AHO
autosomal dominant hypocalcemic hypercalciuria ( ADHH)
PTH PHP type Ib PHP type 1a
Bartter syndrome Type V
GCMB autoimmune acquired hypoparathyroidism (AH) VDDR type II PHP type 1c
PHP type II
GNA11 Mitochondrial disorders associated with hypoparathyroidism (25 (OH ) NL;1,25(OH) ↑)
Rare associations
congenital lymphoedema
Barakat syndrome

Fig. 7 Flow diagram for the work-up of patients with hypocalcemia

11 Physiology of the Developing Kidney: Disorders and Therapy of Calcium and Phosphorous. . .
of varying severity, inappropriately normal or
elevated urinary calcium excretion, and
hyperphosphatemia, yet normal or diminished
PTH levels. Hypoparathyroidism may be
nonfamilial (sporadic) or familial, and it may
occur as an isolated defect or as a component of
disorders with additional manifestations, such as
pluriglandular autoimmune disorder or various
developmental abnormalities, e.g., DiGeorge syn-
drome (DGS). Familial isolated hypoparathyroid-
ism can present anytime from early infancy well
into adulthood.
The main treatment options available for
patients with acute or chronic hypoparathyroid-
ism are calcium salts, vitamin D or vitamin D
analogues, and drugs that increase renal tubular
reabsorption of calcium (i.e., thiazides). The para-
thyroid hormone-dependent renal production of
1,25-dihydroxyvitamin D is deficient in all
hypoparathyroid states. Consequently, therapy
with a vitamin D analogue is used to improve
serum calcium levels. However, because of the
absence of functional PTH and the lack of
PTH-dependent calcium reabsorption in the distal
renal tubules, patients affected by hypoparathy-
roidism frequently develop hypercalciuria and
nephrolithiasis, and chronic kidney disease, even
if attempts are made to maintain plasma calcium
within the lower end of the normal range
[148]. Considerable efforts were therefore made
to treat patients with recombinant PTH(1-34) or
PTH(1-84) [149–155]. Compared with twice-
daily delivery, pump delivery of PTH(1-34)
appears to provide the most promising modality
for improving calcium homeostasis and bone
turnover in children with severe congenital hypo-
parathyroidism [156, 157].
Parathyroid Hormone (PTH) Gene
Abnormalities
Preproparathyroid hormone (preproPTH), the
PTH precursor, contains a 25-residue amino-ter-
minal signal sequence followed by a 6-residue
pro-specific peptide and the mature hormone.
The hydrophobic core of the human preproPTH
signal peptide is composed of 12 contiguous
uncharged amino acids (residues 5 to 16 of
the signal peptide).
301
Several different PTH gene mutations were
identified in patients with autosomal dominant or
autosomal recessive isolated hypoparathyroidism.
A single-base substitution (T ! C) resulted in the
substitution of arginine (CGT) at position 18 in
the signal peptide for cysteine (TGT), thus
disrupting the hydrophobic core of the signal
sequence, which is required by the secreted pro-
teins for efficient translocation across the endo-
plasmic reticulum [158]. Another single-point
mutation changes cysteine to arginine at the
8 position of the signal peptide, which causes
interference with the normal targeting and
processing of secretory proteins, including the
normal PTH precursor, suggesting that the mutant
gene product exerts a dominant-negative effect
in vitro by trapping the hormone intracellularly,
predominantly in endoplasmic reticulum
(ER) [159], thereby causing stress-induced cell
death [160]. In another family, a single-base sub-
stitution (T ! C) involving codon 23 of exon
2 was detected, which resulted in the substitution
of proline (CCG) for the normal serine (TCG) in
the signal peptide [161]. This mutation at the 3
position of the preproPTH protein cleavage site
most likely disrupts cleavage of the mutant pre-
cursor molecule and prevents the efficient forma-
tion and secretion of PTH [161]. The affected
individuals of one other kindred with autosomal
recessive isolated hypoparathyroidism showed a
single-base transition (G ! C) at position 1 of
intron 2 of the gene encoding PTH. This mutation
resulted in the deletion of exon 2, which encodes
the initiation codon and the signal peptide,
thereby causing parathyroid hormone deficiency
[162]. A nonsense mutation involving codon
23 (Ser23Stop) was recently reported in a girl
with autosomal recessive isolated hypoparathy-
roidism [163], and a heterozygous T to C point
mutation was identified that eliminates the initia-
tor methionine, thereby deleting the first six amino
acids of the signal peptide [164].
GCM2 Abnormalities
Glial cells missing 2 (GCM2), the mammalian
homolog of the Drosophila Gcm2 gene, encodes
a 506-amino acid parathyroid-specific transcrip-
tion factor, which contributes to the regulation of
302
PTH gene expression [165]. Mice that are homo-
zygous for the deletion of GCM2 (the murine
homolog) lack parathyroid glands, and these ani-
mals develop hypocalcemia and hyperpho-
sphatemia without a compensatory increase in
PTHrP or 1,25(OH)2 vitamin D levels [166].
GCM2-null mice revealed a small cluster of
PTH-expressing cells under the thymic capsule;
however, the amount of PTH was insufficient to
prevent hypocalcemia [167].
The gene encoding GCM2 is located on chromo-
some 6p23–24, and isolated hypoparathyroidism
can be caused by inactivating mutations on both
parental alleles or by a dominant-negative heterozy-
gous mutation. The first GCM2 mutation, a large
homozygous intragenic deletion, was identified in
the proband of an extended kindred with an autoso-
mal recessive form of isolated hypoparathyroidism
[168]. Subsequently, homozygous mutations were
identified as additional causes of hypoparathyroid-
ism; these affect amino acid residues 63 (G63S)
[169] or 47 (R47L) [170], as well as several other
residues [171–174]. Two closely related heterozy-
gous GCM2 mutations were furthermore identified
in two families in which hypoparathyroidism fol-
lows an autosomal dominant mode of inheritance
[175, 176]. Both mutations lead to a shift in the open
reading frame and the replacement of the putative
transactivation domain within the carboxyl-terminal
region by unrelated amino acid sequence, and both
mutant proteins have a dominant-negative effect
on the wild-type GCM2 protein. A similar mecha-
nism may apply to the Asn502His mutation, which
localizes to the nucleus and retains the ability to
bind to the GCM-consensus DNA recognition
motif but shows impaired gene transactivation and
a dominant-negative effect on the wild-type
protein [173]. For a large number of patients with
isolated hypoparathyroidism, however, no disease-
causing mutation has yet been identified [171],
making it likely that mutations in additional
as-of-yet unidentified genes can also cause
hypoparathyroidism.
X-Linked Recessive Hypoparathyroidism
X-linked recessive hypoparathyroidism has been
reported in two related multigenerational kindreds
[177]. The relatedness of these two kindreds was
A. Sharma et al.
established by demonstrating an identical mito-
chondrial DNA sequence, inherited via the mater-
nal lineage, in affected males from the two
families [178]. Affected males suffered from
infantile onset of epilepsy and hypocalcemia
[179]. Studies utilizing X-linked polymorphic
markers in these families localized the mutant
gene to chromosome Xq26-q27 [180].
Characterization of 906 kb region on Xq27 by
combined analysis of single nucleotide polymor-
phisms and sequence-tagged site identified a
23–25 kb deletion, which did not contain genes.
However, DNA fiber-FISH and pulsed-field gel
electrophoresis revealed an approximately 340 kb
insertion that replaced the deleted fragment and a
molecular deletional-insertion that involved chro-
mosome 2p25 and Xq27 [181]. This complex
deletion-insertion {del(X) (q27.1) inv ins (X;2)
(q27.1; p25.3)} is located 67 kb downstream of
the SOX3 gene. The developing parathyroid tis-
sue of mouse embryos has strong SOX expres-
sion. Thus, this mutation may have a position
effect on SOX3 expression. These findings
added SOX3 to the growing list of transcription
factors, including GCM2, GATA3, TBX1,
HOXA3, PAX1, and PAX9, that operate in para-
thyroid development [181].
Autoimmune Pluriglandular
Hypoparathyroidism
Autoimmune pluriglandular syndromes associ-
ated with hypoparathyroidism can be subdivided
into three subtypes: (1) APS1, (2) APS3 (which is
associated with thyroid autoimmunity), and
(3) APS4 (which is associated with other autoim-
mune disorders).
APS1 or autoimmune polyendocrinopathy-
candidiasis-ectodermal dystrophy (APECED) is
an autosomal recessive disease with a high inci-
dence in isolated subpopulations in Central and
Eastern Finland [182], which usually becomes
manifest during childhood. Establishing the diag-
nosis requires the presence of at least two of three
major components: hypoparathyroidism, candidi-
asis, and adrenal insufficiency; however, hypo-
parathyroidism may be the only manifestation of
the syndrome. APS1 is caused by mutations
within the autoimmune regulator (AIRE) gene
11 Physiology of the Developing Kidney: Disorders and Therapy of Calcium and Phosphorous. . .
located on chromosome 21 (21q22). As part of
this syndrome, autoantibodies against NACHT
leucine-rich repeat protein 5 (NALP5Abs) can
be detected. Non-APS forms of hypoparathyroid-
ism show an association with class I human leu-
kocyte antigen (HLA) allele A*2601 and with
class II HLA alleles DRB1*01 and
DRB1*09 [183].
Linkage studies of Finnish families mapped
the APECED gene to chromosome 21q22.3
[184]. Further, positional cloning approaches led
to the isolation AIRE (autoimmune regulator),
which encodes a 545-amino acid protein. Specific
domains of AIRE protein indicate that it is
involved in transcriptional processes, including
(i) the amino-terminal HSR domain, (ii) nuclear
localization signal (NLS), (iii) a SAND domain,
(iv) two plant homeodomain (PHD) type zinc
fingers, and (v) four LXXLL motifs [185]. To
date, more than 50 different APS1-causing muta-
tions have been established in affected patients.
Though mutations are distributed throughout the
coding region of the gene, most mutations are
located in the amino-terminal HSR domain. The
R257X mutation in SAND domain is the most
prevalent mutation among Finnish patients,
accounting for 83 % of the disease alleles [185,
186]. This particular mutation is also frequently
found in Central and Eastern European and North-
ern Italian populations, indicating an early intro-
duction of the mutation into Caucasian
populations [186, 187]. Characteristic mutations
have been reported also for various other ethnic-
ities [188–192]. Recently, a novel mechanism of
AIRE dysregulation has been described in which
miR-220b inhibits AIRE gene translation through
the 3’UTR [193].
AIRE has been shown to regulate the elimina-
tion of organ-specific T cells in the thymus and
thus failure to delete forbidden T cells [194] and to
disrupt of the nuclear organization [195], thereby
resulting in failure to establish immunologic tol-
erance. A unique G228W variant in the SAND
domain appears to inhibit wild-type AIRE from
reaching the sites of active transcription in med-
ullary thymic epithelial cells. This resulted in the
failure to delete T cells reactive against antigens
specific for the thymus, leading to autoimmunity.
303
Thus, the AIRE-mediated dominant-negative
effect may cause autoimmune predisposition to
phenotypes distinct from APS [196]. In addition,
there is a downstream abnormality as activated
CD4+ T cells of APECED demonstrate a selective
IL-22 defect [197].
APS1 patients with hypoparathyroidism can
have antibodies directed against the CaSR,
which leads to a decrease in PTH secretion and
thus hypocalcemia [198]. The NACHT leucine-
rich repeat protein 5 (NALP5), which is predom-
inantly expressed in the cytoplasm of parathyroid
chief cells, is another antigen, autoantibodies
against which can be responsible for some forms
of hypoparathyroidism. NALP5-specific autoan-
tibodies were detected in almost half of the
patients with APS1 and hypoparathyroidism, but
were absent in all APS1 patients without hypo-
parathyroidism [199]. However, the pathological
significance of these antibodies has been
questioned [200]. Instead of seeking AIRE muta-
tions, which is a cumbersome process,
IgG-neutralizing autoantibodies against type I
interferon can be used as a diagnostic criterion
[201] as high titers of these antibodies were
found in 100 % of Finnish and Norwegian patients
with two prevalent AIRE truncations [202]. Fur-
thermore, rising titers of anti-cytokine antibodies
may precede clinical presentation [203].
DiGeorge Syndrome
DiGeorge syndrome (DGS) is associated with the
absence or hypoplasia of the thymus and the para-
thyroid glands, cardiovascular anomalies, and cra-
niofacial dysmorphism [204]. Most DGS cases
that result from the deletion of the 250–3,000 kb
critical region that contains approximately
30 genes [205, 206] are designated as DGS type
1 (DGS1) [207]. DGS2 is associated with the
deletion locus on chromosome 10p [208]. Approx-
imately 17 % of patients with the phenotypic
features of DGS have no detectable genomic
deletion [209].
Hypocalcemia, considered one of the cardinal
features of DGS, is invariably due to hypopara-
thyroidism as originally described by DiGeorge in
1965 and documented by aplasia or hypoplasia of
parathyroid glands at surgery or autopsy [210].
304
Hypocalcemia was noticed in 203/340 subjects
from a large European cohort; the majority of
affected individuals were found to have transient
hypocalcemia [211]. Similar transient hypocalce-
mia occurs at birth due to the abrupt cessation of
active maternal calcium supply in combination
with birth stress. Increased demands on parathy-
roid reserve resolve as stress dissipates. Symp-
tomatic or latent hypocalcemia may also be
precipitated by the stress of surgery, pregnancy,
or puberty [212]. Point prevalence of hypocalce-
mia outside the neonatal period was 30 % in
27 subjects of all ages [213]. EDTA infusion can
be used to unmask latent hypocalcemia [214].
Treatment is aimed at keeping serum calcium
in low-normal range since the absence of PTH
leads to high or inappropriately normal urinary
excretion of calcium, which increases the risk of
renal calcification and nephrolithiasis.
The deleted region in DGS1 contains several
genes [207, 215–217]. The ablation of several of
these genes causes developmental abnormalities
of the pharyngeal arches in transgenic mice
[218–220]. Tbx1-ablated transgenic mice [220]
and humans with point mutations of TBX1 have
the DGS1 phenotype [221]; mutations in this gene
are therefore likely to cause DGS1 [222]. TBX1 is
a DNA-binding transcriptional factor of the T-Box
family with an important role in organogenesis
and pattern formation. Tbx1-null mutant mice
(Tbx1/) had all the developmental anomalies
of DGS1, while haploinsufficiency (Tbx1+/) was
associated with a milder phenotype. The spectrum
of DGS1 malformations is thus elicited in a dose-
dependent manner [223], suggesting that the Tbx1
dose can be modulated by other modifying genes,
thus causing phenotypic variability [224–227].
Hypoparathyroidism, Deafness,
and Renal Anomalies (HDR) Syndrome
The combined occurrence of hypoparathyroidism,
deafness, and renal dysplasia (HDR) as an auto-
somal dominant trait was reported in one family in
1992 [228]. Patients had asymptomatic hypocal-
cemia with undetectable or inappropriately nor-
mal serum concentrations of PTH and normal
brisk increases in plasma cAMP in response to
the infusion of PTH. The patients also had
A. Sharma et al.
bilateral, symmetrical, nonprogressive sensori-
neural deafness involving all frequencies. The
renal abnormalities consisted mainly of bilateral
cysts that compressed the glomeruli and tubules,
leading to renal impairment in some patients.
Cytogenetic abnormalities were not detected and
abnormalities of the PTH gene were excluded.
Deletion mapping studies in two unrelated
HDR patients showed cytogenic abnormalities
involving chromosome 10p14-10pter [229].
These two patients suffered from hypoparathy-
roidism, deafness, and growth and mental retarda-
tion; one patient also had a solitary dysplastic
kidney with vesicoureteric reflux and a uterus
bicornuis unicollis, and the other HDR patient
also had cartilaginous exostoses and was shown
to have a complex reciprocal insertional translo-
cation of chromosomes 10p and 8q. Neither of
these patients had immunodeficiency or heart
defects, which are key features of DGS2 (see
above), and further studies defined two
nonoverlapping regions; thus, the DGS2 region
was located on 10p13-14 and HDR on 10p14-
10pter. It is important to note that HDR patients
with GATA3 haploinsufficiency do not have
immune deficiency. Similarly, the facial
dysmorphism, growth, and developmental delay,
commonly seen in patients with larger 10p dele-
tions, were absent in the HDR patients carrying
GATA3 mutations, further indicating that these
features were likely due to other genes on 10p
[230]. Deletion mapping studies in two other
HDR patients further defined a critical 200 kb
region that contained GATA3 [230], which
belongs to a family of zinc finger transcription
factors that bind to the consensus DNA sequence
and are involved in vertebrae embryonic develop-
ment. DNA sequence analysis in other HDR
patients identified mutations that resulted in a
haploinsufficiency and loss of GATA3 function
[230–232]. GATA3 has 2 zinc fingers. The
C-terminal finger (ZnF2) binds DNA, while the
N-terminal finger (ZnF1) stabilizes this DNA
binding and interacts with other zinc finger pro-
teins, such as the Friends of GATA (FOG)
[233]. HDR-associated mutations involving
GATA3 ZnF2 or the adjacent basic amino acids
were found to result in a loss of DNA binding,
11 Physiology of the Developing Kidney: Disorders and Therapy of Calcium and Phosphorous. . .
while those involving ZnF1 either lead to a loss of
interaction with FOG2 ZnFs or altered
DNA-binding affinity [232]. These findings are
consistent with the proposed 3-dimensional
model of GATA3 ZnF1, which has separate
DNA and protein binding surfaces [232, 234].
Electrophoretic mobility shift assays (EMSAs)
revealed three classes of GATA3 mutations:
those that lead to a loss of DNA binding (over
90 % of all mutations), those that lead to a loss of
the carboxyl-terminal zinc finger and result in
reduced DNA-binding affinity, and those (e.g.,
Leu348Arg) that do not alter DNA binding or
the affinity but likely induce conformational
change in GATA3 [235]. Furthermore, a hetero-
zygous mutation, 432insG, has been described,
which introduces a premature stop codon at exon
4 (K302X), resulting in a loss of both zinc finger
domains of the GATA3 protein [236]. No muta-
tions were identified in patients with isolated
hypoparathyroidism, indicating that GATA3
abnormalities are more likely to result in two or
more of the phenotypic features of the HDR syn-
drome and not in isolated disease affecting only
the regulation of calcium homeostasis [235]. The
co-occurrence of the 22q11 deletion and the HDR
due to a de novo deletion in chromosome 10p14
has been described in a Japanese boy with severe
phenotype, including progressive renal failure and
severe intellectual disabilities [237].
The HDR phenotype is consistent with the
expression pattern of GATA3 during human and
mouse embryogenesis in the developing kidney,
otic vesicle, and parathyroids. Homozygous abla-
tion of GATA3 in mice leads to CNS defects, but
heterozygous GATA3-null animals were initially
reported to have no abnormalities [238]. However,
further studies have revealed that these heterozy-
gous mice have a progressive hearing loss associ-
ated with continuous morphological degeneration
of the cochlea [239, 240] and that they also have
hypoparathyroidism [241].
Mitochondrial Disorders Associated
with Hypoparathyroidism
Hypoparathyroidism has been reported to occur in
three disorders associated with mitochondrial dys-
function:(1) Kearns-Sayre syndrome (KSS),
305
(2) MELAS syndrome, and (3) mitochondrial
trifunctional protein deficiency syndrome
(MTPDS). Both the KSS and MELAS syndromes
have been reported to occur with insulin-
dependent diabetes mellitus and hypoparathyroid-
ism [242, 243]. A point mutation in the mitochon-
drial gene tRNA leucine (UUR) has been reported
in one patient with the MELAS syndrome who
also suffered from hypoparathyroidism and dia-
betes mellitus [101]. Large deletions, consisting
of 6,741 and 6,903 base pairs and involving
>38 % of the mitochondrial genome, have been
reported in other patients who suffered from
hypoparathyroidism and sensorineural deafness
[244]. Rearrangements and duplication of mito-
chondrial DNA have also been reported in KSS.
MTPDS is a disorder of fatty acid oxidation that is
associated with peripheral neuropathy, pigmen-
tary retinopathy, and acute fatty liver degeneration
in pregnant women who carry an affected fetus.
Hypoparathyroidism has been observed with
trifunctional protein deficiency [245, 246]. The
role of these mitochondrial mutations in the etiol-
ogy of hypoparathyroidism remains to be further
elucidated.
Kenny-Caffey and Sanjad-Sakati
Syndrome
Hypoparathyroidism has been reported to occur in
over 50 % of patients with the Kenny-Caffey syn-
drome (KCS), which is associated with short stat-
ure, osteosclerosis, and cortical thickening of the
long bones, delayed closure of the anterior fonta-
nel, basal ganglia calcification, nanophthalmos,
dental anomalies, and hyperopia [247]. Parathy-
roid tissue could not be found in a detailed
postmortem examination of one patient [248],
suggesting that hypoparathyroidism may be due
to an embryological defect of parathyroid devel-
opment. KCS, namely, KCS type 1 (KCS1), was
convincingly demonstrated in 16 affected children
in 6 unrelated sibships, born to healthy consan-
guineous parents of Bedouin ancestry [249].
In eight consanguineous Kuwaiti kindreds, link-
age to a locus in the 1q42–q43 region was found
for the autosomal recessive form of Kenny-Caffey
syndrome [250]. This disease has been
observed almost exclusively in the Middle East.
306
The only available data about its prevalence is
from Saudi Arabia, where its frequency is esti-
mated between 1:40,000 and 1:100,000 live births
[247]. An analysis of DNA from 14 children with
severe short stature of unknown etiology by whole
exome sequencing identified one subject with
Kenny-Caffey syndrome, highlighting the fact
that rare syndromic causes of short stature may
be under-recognized and underdiagnosed [251].
KCS1 is caused by mutations of the tubulin-
specific chaperone (TBCE), whereas patients
with KCS2 were recently shown to carry de novo
mutations in FAM111A (family with sequence
similarity 111A) [252]. There is close genotype-
phenotype correlation.
Five individuals with KCS2 and five with more
severe osteocraniostenosis (OCS) were found to
have heterozygous mutations in FAM111A. This
protein comprises 611 amino acids with homol-
ogy to trypsin-like peptidases. Molecular model-
ing revealed that these mutations are close to the
outer surface rather than on the active site, thus
raising the possibility that decreased binding of
FAM111A by its inactivating partner results in
increased and/or deregulated FAM111A activity,
a gain-of-function effect observed even in a het-
erozygous state [253]. FAM111A functions as a
host range restriction factor which plausibly plays
a role in embryonic morphogenesis and appears to
be crucial for parathyroid gland formation and
function [252]. Mother-to-daughter transmission
of KCS2 was shown to be associated with a recur-
rent dominant FAM111A mutation, namely,
pArg569His [254].
In the Sanjad-Sakati syndrome, hypoparathy-
roidism is associated with severe prenatal and
postnatal growth failure and dysmorphic features,
and this has been reported in 12 patients from
Saudi Arabia [255]. The presenting complaint in
all patients was hypocalcemic tetany or general-
ized convulsions, usually detected in the first few
days or weeks of life. Consanguinity was noted in
the families of 11 of the 12 patients, the majority
of which originated from the Western province of
Saudi Arabia. This syndrome, which is inherited
as an autosomal recessive disorder, has also been
identified in families of Bedouin origin, and
homozygosity and linkage disequilibrium studies
A. Sharma et al.
located this gene to chromosome 1q42–q43 [256].
Sanjad-Sakati syndrome resembles the autosomal
recessive form of KCS with similar manifesta-
tions but lacking osteosclerosis. Eight Sanjad-
Sakati families from Saudi Arabia were
genotyped with polymorphic short tandem repeat
markers from the SSS/KCS critical region. A
maximum multipoint LOD score of 14.32 was
obtained at marker D1S2649, confirming linkage
of Sanjad-Sakati syndrome to the same region as
autosomal recessive Kenny-Caffey syndrome.
Haplotype analysis refined the critical region to
2.6 cM and identified a rare haplotype present in
all the Sanjad-Sakati syndrome disease alleles,
indicative of a common founder. In addition to
the assignment of the Sanjad-Sakati syndrome in
Saudi families and of the Kenny-Caffey syndrome
in Kuwaiti families to overlapping genetic inter-
vals, comparison of the haplotypes unexpectedly
demonstrated that the diseases shared an identical
haplotype. This finding, combined with the clini-
cal similarity between the two syndromes, sug-
gests that the two conditions are not only allelic
but are also caused by the same ancestral mutation
[257]. Molecular genetic investigations led to the
conclusion that mutations of the tubulin-specific
chaperone (TBCE) are associated with both syn-
dromes [258]. TBCE encodes one of several chap-
erone proteins required for the proper folding of
α-tubulin subunits and the formation of α-β tubu-
lin heterodimers. In addition, deletion and trunca-
tion mutations in the gene encoding a tubulin-
specific chaperone cofactor E (TBCE) have been
shown to cause the hypoparathyroidism, mental
retardation, and facial dysmorphism (HRD) syn-
drome which is associated with extreme growth
failure. However, cryptic translational initiation at
each of three out-of-frame AUG codons upstream
of the genetic lesion can rescue a mutant HRD
allele by producing a functional TBCE protein
[259]. The defect in the tubulin folding and
assembly pathway also has grave consequences
on growth and PMN functions [260, 261].
Additional Familial Syndromes
Single familial syndromes in which hypoparathy-
roidism is a component have been reported. The
inheritance of the disorder in some instances has
11 Physiology of the Developing Kidney: Disorders and Therapy of Calcium and Phosphorous. . .
been established, and molecular genetic analysis
of the PTH gene has revealed no abnormalities.
Thus, an association of hypoparathyroidism, renal
insufficiency, and developmental delay has been
reported in one Asian family in whom autosomal
recessive inheritance of the disorder was
established [262]. An analysis of the PTH gene
in this family revealed no abnormalities [262].
The occurrence of hypoparathyroidism, nerve
deafness, and a steroid-resistant nephrosis leading
to renal failure, which has been referred to as
the Barakat syndrome [263], has been reported
in four brothers from one family, and an associa-
tion of hypoparathyroidism with congenital
lymphoedema, nephropathy, mitral valve pro-
lapse, and brachytelephalangy has been observed
in two brothers from another family [264]. Molec-
ular genetic studies have not been reported from
these two families.
Abnormalities at the Calcium-Sensing
Receptor (CaSR) and the Downstream
Signaling Molecules
Abnormalities at the CaSR are associated with
several different hypocalcemic disorders. These
include (1) autosomal dominant forms of hypo-
calcemic hypercalciuria (ADHH), (2) Bartter syn-
drome type V (i.e., ADHH with a Bartter-like
syndrome), and (3) autoimmune hypoparathy-
roidism (AH).
Autosomal Dominant Hypocalcemic
Hypercalciuria (ADHH) Due to CaSR
and Ga11 Mutations
ADHH, although rare, in index cases may com-
prise a sizeable fraction of cases of idiopathic
hypoparathyroidism, perhaps representing as
many as one-third of such cases [265]. CaSR
mutations that result in a loss-of-function are asso-
ciated with familial benign (hypocalciuric) hyper-
calcemia [87–92, 102]. It was therefore postulated
that gain-of-function mutations in CaSR lead to
hypocalcemia with hypercalciuria, and the inves-
tigation of kindreds with autosomal dominant
forms of hypocalcemia has indeed identified
such CaSR mutations [102, 266–270]. Soon
307
after the cloning of the CaSR, investigators
showed linkage of ADHH to a locus on chromo-
some 3q13 [267], i.e., the same locus as for the
gene encoding the CaSR. Shortly afterward, a
heterozygous missense mutation, Q127A, was
identified as the cause of ADHH in an unrelated
family [266]. The majority (>80 %) of CaSR
mutations that result in a functional gain are
located within the extracellular domain [102,
266–270], which is different from the findings in
other disorders that are the result of activating
mutations in G protein-coupled receptors. A sec-
ond hotspot for mutations is transmembrane
domains 6 and 7 and the intervening third extra-
cellular loop, a site which maintains the CaSR in
an inactive conformation and also is critical for the
binding and/or action of allosteric modulators
including calcimimetics of the phenylalkylamine
class to which Cinacalcet belongs [271]. In addi-
tion, two deletion mutations have been described.
Most ADHH patients are heterozygous for the
activating mutation. In one family, a homozygous
mutation was described, but it was not associated
with a more severe phenotype [272], and although
there is a spectrum of phenotypic severity for a
given genotype, the symptoms present in affected
members of the same family tend to be similar.
Thus, one mutated allele may be enough to shift
the set point to left, and the presence of second
mutated allele makes no change in the phenotype
perhaps due to the “dominant-positive” effect of
the mutant receptor.
In addition to heterozygous gain-of-function
mutations in the CaSR (ADHH1), ADHH cases
were recently identified that are associated with
heterozygous mutations in GNA11 (ADHH2), the
gene encoding Gα11 [93, 273]. Gα11 is one of
two heterotrimeric G proteins that couple the
CaSR to the Ca2+/IP3/PKC signaling pathway in
parathyroid cells [274]. Functional studies of the
Gα11 mutation R60L revealed a significantly
decreased EC50 and MAP kinase activity. Com-
pared with subjects with CaSR mutations, patients
with GNA11 mutations lacked significant
hypercalciuria and had normal serum magnesium
levels [274]. FHH3 is caused by loss-of-function
AP2σ1 mutations [95]; however, 19 patients
(including 6 familial cases) with ADHH, who
308
did not have CaSR or GNA11 mutations, were
found not to have AP2σ1 mutations [275], indi-
cating that gain-of-function AP2σ1 mutations are
not a cause of ADHH.
Bartter Syndrome Type V
Bartter syndrome is a heterozygous group of auto-
somal recessive disorders of electrolyte homeosta-
sis characterized by hypokalemic alkalosis, renal
salt wasting that may lead to hypotension, hyper-
reninemic hyperaldosteronism, increased urinary
prostaglandin excretion, and hypercalciuria with
nephrocalcinosis [276, 277]. Mutations of several
ion transporters and channels have been associated
with Bartter syndrome, and five types are now
recognized [277]. The CaSR-related cases of
Bartter’s syndrome identified to date have been
inherited in an autosomal dominant manner, unlike
other subtypes that are inherited as autosomal
recessive traits.
Bartter syndrome type V is due to activating
mutations of the CaSR. Activating mutations of
the CaSR gene in three patients involving L125P,
C131W, and A843E, which inhibited the activity
of the ROMK channel, provided the missing link
that explains why some activating mutations of
CaSR can cause the Bartter syndrome phenotype
[278, 279]. Another recent mutation in monozy-
gotic twins involving the K29E in the ECD of the
CaSR described mild hypokalemia, minimal aldo-
sterone and renin production, and absent alkalosis
but notable hypocalcemia [280]. The K29E muta-
tion is an activating mutation of the CaSR [281]
and buttresses previous observations that the phe-
notype of Bartter syndrome is variable and not
directly related to the in vitro potency of the
known genetic changes associated with this syn-
drome [282]. CaSR mutations causing type V
Bartter syndrome have been shown to increase
ER to cytosol calcium gradient, thus explaining
the higher sensitivity of CaSR gain-of-function
variants to external calcium [283].
Autoimmune Acquired
Hypoparathyroidism (AH)
Twenty percent of patients, who had acquired
hypoparathyroidism (AH) in association with
autoimmune hypothyroidism, were found to
A. Sharma et al.
have autoantibodies to the extracellular domain
of the CaSR [111, 112, 284]. The CaSR autoanti-
bodies are not persistent [284]. The majority of the
patients who had CaSR autoantibodies were
females, a finding that is similar to that found in
other autoantibody-mediated diseases. Indeed a
few AH patients have also had features of auto-
immune polyglandular syndrome type 1 (APS1).
These findings establish that the CaSR is an
autoantigen in AH [111, 284]. CaSR autoanti-
bodies bind to the CaSR ECD and have, in some
cases, been shown to enhance receptor function,
thereby reducing PTH secretion. Thus, hypopara-
thyroidism associated with activating CaSR auto-
antibodies results from activated parathyroid
CaSR function and not from immune-mediated
destruction of the parathyroid glands and is of
varying severity [284].
Pseudohypoparathyroidism (PHP)
The term pseudohypoparathyroidism (PHP) was
first introduced to describe patients with hypocal-
cemia and hyperphosphatemia due to PTH resis-
tance rather than PTH deficiency [285]. Affected
individuals show partial or complete resistance to
biologically active exogenous PTH as demon-
strated by a lack of increase in urinary cyclic
AMP and urinary phosphate excretion; this con-
dition is now referred to as PHP type I
[286–288]. If associated with other endocrine
deficiencies and characteristic physical stigmata,
now collectively termed Albright’s hereditary
osteodystrophy (AHO), the condition is referred
to as PHP type Ia. This latter syndrome is caused
by heterozygous inactivating mutations within
exons 1–13 of GNAS located on chromosome
20q13.3, which encode the stimulatory G protein
(Gsα) (for review, see [288, 289]). These muta-
tions were shown to lead to an approximately
50 % reduction in Gsα activity/protein in readily
accessible tissues, like erythrocytes and fibro-
blasts, and explain, at least partially, the resistance
toward PTH and other hormones that mediate
their actions through G protein-coupled receptors
[286–288]. A similar reduction in Gsα activity/
protein is also found in patients with
11 Physiology of the Developing Kidney: Disorders and Therapy of Calcium and Phosphorous. . .
pseudopseudohypoparathyroidism (PPHP), who
often show the same physical appearance as indi-
viduals with PHP-Ia, but lack endocrine abnor-
malities [286, 290–295]. However, contrary to
previous reports, recent studies have shown that
not all AHO features are observed in PPHP
patients. For example, obesity, which was thought
to be a hallmark of PHP-Ia and PPHP, was not
evident in a large number of PPHP patients [296].
Patients affected by PHP-Ia or PPHP are typi-
cally found within the same kindred, but not
within the same sibship, and hormonal resistance
is parentally imprinted, i.e., PHP-Ia occurs only if
the genetic defect is located on the maternal allele,
while PPHP occurs only if the defect is located on
the paternal allele [297, 298]. Observations con-
sistent with these findings in humans were made
in mice that are heterozygous for the ablation of
exons 1 or 2 of the Gnas locus [299–301]. Animals
that inherited the mutant allele from a female
showed much reduced Gsα protein in the renal
cortex and decreased plasma calcium concentra-
tion due to resistance toward PTH. In contrast,
offspring that had obtained the mutant allele
from a male showed no evidence for abnormali-
ties in the regulation of mineral ion homeostasis.
These findings in mice with ablation of Gnas exon
1 or 2 and in humans with inactivating mutations
in GNAS exons 1–13 indicate that certain tissues,
such as the proximal renal tubules, express Gsα
predominantly from the maternal allele; expres-
sion from the paternal allele is silenced through
some unknown mechanisms. These data provided
a reasonable explanation for the finding that het-
erozygous GNAS mutations result in a dominant
phenotype with regard to the mineral ion abnor-
malities, if inherited through females.
Progressive osseous heteroplasia (POH) is
caused also by heterozygous inactivating muta-
tions in the GNAS exons encoding Gsα
[302–305]. Interestingly, POH became apparent
predominantly when a Gsα mutation was inherited
from a male. Furthermore, the majority of muta-
tions occurred in GNAS exons 2–13, thus impli-
cating XLαs rather than Gsα in the pathogenesis of
POH. Recent evidence, however, indicated that
the abnormal bone formation process followed a
dermomyotomal distribution pattern, suggesting
309
that a somatic mutation is required in addition to
heterozygosity at the GNAS locus [306].
Mutations in the GNAS gene encoding Gsα
have not been detected in patients with PHP type
Ib (PHP-Ib), a disorder in which affected individ-
uals show PTH-resistant hypocalcemia and
hyperphosphatemia, but usually lack develop-
mental defects. Initially it was therefore thought
that this variant of PHP is caused by PTH/PTHrP
receptor mutations; however, mutations in its
gene and mRNA could not be identified
[307–310]. Furthermore, it was shown that there
is an increased incidence of TSH resistance in
PHP-Ib patients [261, 287, 311, 312] and that
some individuals affected by this disease variant
show some shortening of the fourth metacarpals,
suggesting some overlap between the develop-
mental features of PHP-Ia and PHP-Ib [313, 314].
A genome-wide search to identify the location
of the “PHP-Ib gene” mapped the PHP-Ib locus to
chromosome 20q13.3, which contains the GNAS
locus [315], and it was furthermore shown that the
genetic defect is parentally imprinted, i.e., it is
inherited in the same mode as the PTH-resistant
hypocalcemia in kindreds with PHP-Ia and/or
PPHP [297, 298]. Patients affected by PHP-Ib
furthermore show a loss of methylation on the
maternal allele, which is usually restricted to
GNAS exon A/B [316, 317]. In most families
with the autosomal dominant form of PHP-Ib
with parental imprinting (AD-PHP-Ib), affected
individuals and healthy carriers were shown to
carry a 3 kb deletion located with the syntaxin
16 (STX16) gene about 220 kb upstream of exon
A/B [318–321] (Fig. 8). Other AD-PHP-Ib kin-
dreds, in which the affected members show a loss
of A/B methylation alone, revealed either a 4.4 kb
deletion within STX16 overlapping with the 3 kb
deletion by 1,286 bp [322] or a 24.6 kb deletion
comprising most STX16 exons [323]. Methylation
changes at GNAS exon A/B alone were further-
more observed in the affected members of a fam-
ily with a 18.9 kb deletion comprising exon
NESP55 and a large part of AS intron 4 [324]. In
affected individuals with either mutation, the dele-
tion is always found on the maternal allele, while
it occurs on the paternal allele in unaffected
healthy carriers.
310 A. Sharma et al.

P11.23

P11.21

q11.21
q11.22
q11.23

q13.12

q13.13

q13.31
q13.32
q13.33
P12.3

P12.2

P12.1

q13.2
P13

q12
4.2–kb
18.9–kb
24.6–kb
4.0/4.7–kb
Gsα exons
4.4–kb
3.0
3.0–kb

1 1a 23 456 7 8 5 NESP554 3 21 XL A/B 1 2 3 N1 4 5 6 7 13

STX16 P + – – –
Control
M – + + +

3.0/4.4/24.6/18.9–kb del.
P + – – –
M – + + –
P + – – –
4.0/4.2/4.7–kb del.
M +/– – – –

Fig. 8 Genomic structure of PHP-Ib locus to chromosome mechanisms related to the defect of GNAS are described
20q13.3. This locus contains GNAS, encoding in the text
the stimulatory G protein (Gsα). The pathophysiological

The affected members of two families with
broader methylation changes within the GNAS
locus were shown to carry two distinct deletions
on the maternal allele that remove GNAS exon
NESP55 and the antisense exons 3 and 4 [325],
a third deletion removes only antisense exons
3 and 4 [326]. Although indistinguishable broad
methylation changes were observed in most
patients with sporadic PHP-Ib, no deletions or
point mutations have yet been identified in these
individuals [327]. In fact, only patients with pater-
nal uniparental isodisomy involving either the
long arm of chromosome 20 (patUPD20q) or the
entire chromosome (patUPD20) have provided a
molecular explanation for this variant of PHP-Ib
[328]. Taken together, these findings suggest that
several different deletions upstream or within the
GNAS locus or patUPD20q lead to indistinguish-
able clinical and laboratory findings. However, it
remains uncertain how the deletion affecting
STX16 results in a loss of exon A/B methylation
alone, while the deletion of GNAS exon NESP
results in a broader loss of methylation. Further-
more, it remains uncertain how the different
deletions affect signaling through the PTH/PTHrP
receptor in the proximal renal tubules, but not in
most other tissues. Mice lacking the murine
homolog of exon A/B were recently shown to
have biallelic and thus increased Gsα transcription
[329] (Fig. 8). Loss of exon A/B methylation on
the maternal allele allowing active transcription
from the promoters of both parental alleles and
this transcript therefore seems to have a prominent
role in suppressing Gsα expression.
Blomstrand’s Disease
Blomstrand’s chondrodysplasia is an autosomal
recessive human disorder characterized by early
lethality, dramatically advanced bone maturation,
and accelerated chondrocyte differentiation [330].
Affected infants are typically born to consanguin-
eous healthy parents (only in one instance did
unrelated healthy parents have two affected off-
spring) [331–335] and show pronounced
hyperdensity of the entire skeleton (Fig. 9) [336]
and markedly advanced ossification, and
11 Physiology of the Developing Kidney: Disorders and Therapy of Calcium and Phosphorous. . .
Fig. 9 Radiological findings in a patient with
Blomstrand’s disease. (From [333], with permission from
Loshkajian with permission). Note the markedly advanced
ossification of all skeletal elements, and extremely short
limbs, despite comparatively normal size and shape of
hands and feet. In addition, notice that the clavicles are
relatively long and abnormally shaped
particularly the long bones are extremely short and
poorly modeled. PTH/PTHrP receptor mutations
that impair its functional properties were identified
as the most likely cause of Blomstrand’s disease.
One of these defects is caused by a nucleotide
exchange in exon M5 of the maternal PTH/PTHrP
receptor allele, which introduces a novel splice
acceptor site and thus leads to the synthesis of a
receptor mutant that does not mediate, despite
seemingly normal cell surface expression, the
actions of PTH or PTHrP; the patient’s paternal
PTH/PTHrP receptor allele is, for yet unknown
reasons, only poorly expressed [336]. In a second
patient with Blomstrand’s disease, the product of a
consanguineous marriage, a nucleotide exchange
was identified that changes proline at position
132 to leucine [337, 338]. The resulting
PTH/PTHrP receptor mutant despite reasonable
cell surface expression had severely impaired bind-
ing to radiolabeled PTH and PTHrP analogues,
resulting in greatly reduced agonist-stimulated
cAMP accumulation and no measurable inositol
phosphate response. Additional loss-of-function
mutations of the PTH/PTHrP receptor have
recently been identified in three unrelated patients
311
with Blomstrand’s disease. Two of these frameshift
mutations secondary to a homozygous single
nucleotide deletion in exon EL2 [339] and 27 bp
insertion between exon M4 and EL2 led to a trun-
cated receptor protein [340]. The other defect, a
nonsense mutation at residue 104, also resulted in
the same receptor protein defect [341]. Affected
infants show abnormalities in other organs, includ-
ing secondary hyperplasia of the parathyroid
glands (presumably due to hypocalcemia), besides
the described skeletal defects. In addition, analysis
of fetuses with Blomstrand’s disease revealed
abnormal breast development and tooth impaction,
highlighting the involvement of the PTH/PTHrP
receptor in the normal development of breast and
tooth [342].
Hyperphosphatemic Disorders
with Reduced Secretion of Biologically
Active FGF23
Hyperphosphatemic Familial Tumoral
Calcinosis (HFTC)
HFTC needs to be differentiated from sporadic
and secondary TC. It is a rare autosomal recessive
disorder [343], which can be severe, sometimes
fatal, and is typically characterized by hyperpho-
sphatemia and often massive calcium deposits in
skin and subcutaneous tissues; in some patients,
however, only few minor abnormalities are noted
[344]. The biochemical hallmark of tumoral cal-
cinosis is hyperphosphatemia caused by loss-of-
function mutations in genes relevant to the pro-
duction of an intact bioactive form of FGF23, i.e.,
mutations in (1) FGF23 [40], (2) GALNT3 [345],
or resistance to the end-organ effects of FGF23
bioactivity (αKlotho) [346]. Homozygous or
compound heterozygous mutations in GALNT3,
which encodes a glycosyltransferase responsible
for initiating mucin-type O-glycosylation, appear
to occur more frequently. Furthermore, the disor-
der can be caused by loss-of-function mutations in
FGF23. Mutations in either gene lead to indistin-
guishable changes in FGF23, namely, low serum
intact FGF23, but significantly elevated
C-terminal FGF23 concentrations [347, 348].
312
In contrast, HFTC caused by mutations in αKL is
characterized by profound increases of both forms
of FGF23.
Hyperostosis with Hyperphosphatemia
(HHS)
HHS is an autosomal recessive disorder, which
shares several clinical and metabolic features
with HFTC. The combination of hyperostosis
with hyperphosphatemia was first described in
1970 [349, 350]. Most cases appear to be spo-
radic, but consanguineous parents were described
for some patients, implying that the disease can be
recessive. All affected individuals in two
unrelated Arab-Israeli HHS families were found
to harbor a previously described splice site muta-
tion in GALNT3, indicating that HFTC and HHS
are allelic variants [351]. Four novel GALNT3
gene mutations were found in additional unrelated
subjects with considerable phenotypic overlap,
again underscoring the fact that these two disor-
ders are variants of the same disease [352]. The
molecular reasons for the enhanced skeletal
involvement in HHS are currently unknown, but
could potentially be due to modifier gene
interactions.
Hypophosphatemic Disorders
The different forms of hypophosphatemia repre-
sent the most common cause of hereditary rickets,
which can be divided into two main groups
according to the predominant metabolic abnor-
mality [353, 354]. In the first group,
hypophosphatemia is the result of a renal tubular
defect, which may consist of either a single (iso-
lated) defect in renal phosphate handling, as it
occurs in the X-linked hypophosphatemia
(XLH), or of multiple tubular defects as encoun-
tered in Fanconi syndrome. In the second group,
vitamin D metabolism is abnormal, either because
of a defect in the 1α-hydroxylase enzyme or
because of defects in the 1,25-dihydroxyvitamin
D3 receptor (VDR) leading to end-organ resis-
tance. The application of molecular genetics
A. Sharma et al.
approaches has helped to elucidate some of the
mechanisms underlying these disorders of hered-
itary hypophosphatemic rickets (Table 1).
Hypophosphatemic Disorders
with Increased FGF23 Activity
Autosomal Dominant
Hypophosphatemic Rickets (ADHR)
Autosomal dominant hypophosphatemic rickets
is characterized by FGF23 excess due to “activat-
ing” mutations in the FGF23 gene. Genetic link-
age studies mapped the ADHR locus to
chromosome 12p13.3 [355] and defined a
1.5 Mb critical region that contained 12 genes.
Positional cloning in affected members of four
unrelated families identified three different mis-
sense mutations involving a new member of the
fibroblast growth factor (FGF) family [38].
The C-terminal and the N-terminal portions of
FGF23 are connected by an “RXXR” motif,
which is the recognition site for a furin-like pro-
tease. At this site, FGF23 can be cleaved and thus
inactivated. Missense mutations that replace the
arginine (R) residue at either position 176 or
179 with glutamine (Q) or tryptophan
(W) disrupt the cleavage site, thus making
FGF23 resistant to enzymatic cleavage (Fig. 2),
thereby stabilizing the intact bioactive FGF23
molecule [6, 39, 48, 356, 357]. Iron deficiency
stimulates FGF23 expression and leads mice
engineered to carry one of the ADHR mutations
to hypophosphatemia [358], and similar observa-
tions were made in human with ADHR [359].
Oncogenic Osteomalacia (OOM)
OOM (also referred to as tumor-induced osteoma-
lacia (TIO)) is a rare disorder characterized by
hypophosphatemia, phosphaturia, low circulating
levels of 1,25-dihydroxyvitamin D, and osteoma-
lacia that develops in previously unaffected indi-
viduals [360]. Similarities between OOM,
ADHR, and XLH suggested that they may
involve the same phosphate-regulating pathway,
and it is important to note that OOM tumors do
express PHEX [361, 362], which is mutated in
XLH (see below). The possibility that FGF23,
 11

Table 1 Biochemical findings in several inherited hypo- and hyperphosphatemic disorders and underlying genetic defects
TRP or Serum
FGF-23 TmP/GFR 1,25(OH)2D PTH calcium Urinary calcium Mutant gene
Hypophosphatemic disorders
FGF23 dependent
XLH Increased/ Low Low/ Normal/ Normal Normal PHEX
inappropriately inappropriately increased
normal normal
ADHR Increased/ Low Low/ Normal Normal Normal FGF23
inappropriately inappropriately
normal normal
ARHP Increased/ Low Low/ Normal Normal Normal DMP1, ENPP1, FAM20C
inappropriately inappropriately
normal normal
Hypophosphatemic disorders
FGF23 independent
HHRH Low/normal Low High Low Normal/ High NPT2c
increased
Renal Fanconi syndrome Low/normal Low High Low Normal/ High NPT2a
increased
Hyperphosphatemic disorders
Tumoral calcinosis Intact: low High Normal-high Low Normal/ Increased FGF23 or GALNT3
C-terminal: very increased (glycosyltransferase)
high
Extremely high High Normal-high Elevated Normal/ Increased Klotho
(intact and increased
C-terminal)
Isolated hypoparathyroidism Normal-increased Elevated Low-normal Low- Low Increased or Calcium-sensing receptor, PTH,
normal inappropriately GCM2, GNA11, and unknown genetic
Physiology of the Developing Kidney: Disorders and Therapy of Calcium and Phosphorous. . .

normal defects
Pseudohypoparathyroidism Normal-increased Elevated Low-normal High Low Low PHP-Ia: GNAS exons encoding Gsα
type Ia (PHP-Ia) or Ib PHP-Ib: microdeletions within or
(PHP-Ib) upstream of GNAS
313
314
which is mutated in ADHR, may also be
expressed in OOM tumors and that FGF23 may
be a secreted protein was therefore explored [39,
65, 356, 363]. Indeed, OOM tumors were found
by Northern blot analysis to contain high levels of
FGF23 mRNA and protein. Consistent with this
finding, FGF23 plasma concentrations can be
increased considerably in OOM patients, until
successful tumor removal [356, 357, 364, 365].
Tumors responsible for oncogenic osteomala-
cia produce two molecular forms of FGF23 (~32
and ~12 kDa), and both variants were also
observed when assessing conditioned medium
from different cell lines expressing full-length
wild-type FGF23 [39]. When conditioned
medium from cells expressing R176Q FGF23 or
R179Q FGF23 was investigated by Western blot
analysis, only the larger protein band was
observed [7, 8, 366]. These findings showed that
the ADHR mutations impair FGF23 degradation,
thus enhancing and/or prolonging its biological
activity. In addition to FGF23, these tumors have
also been shown to express other factors, includ-
ing DMP1, sFRP4, and MEPE [367].
X-Linked Hypophosphatemia (XLH)
Like ADHR, patients with XLH present with
hypophosphatemia, hyperphosphaturia, low cir-
culating 1,25-dihydroxyvitamin D levels, and
osteomalacia. XLH is caused by inactivating
mutations in PHEX, a gene located on Xp22.1
[368, 369], which shows significant amino acid
sequence homology to the M13 family of zinc
metallopeptidases. In the past, substrates for
PHEX considered were FGF23, matrix extracel-
lular phosphoglycoprotein (MEPE) [68, 370], and
secreted frizzled-related protein 4 (sFRP4) [65,
67]. Among these proteins, FGF23 levels were
found to be elevated in about two-thirds of XLH
patients [78, 261, 356] and more than tenfold in
Hyp mice, the murine homolog of XLH [63, 371].
Although PHEX-dependent cleavage of FGF23
was observed in one study in vitro [372], these
findings were not confirmed by others [40]. How-
ever, genetic ablation of FGF23 in male Hyp
mice, i.e., animals that are null for FGF23 and
PHEX, led to blood phosphate levels that are
indistinguishable from those in mice lacking
A. Sharma et al.
FGF23 alone [45, 46], indicating that FGF23
resides genetically downstream of PHEX. Fur-
thermore, recent studies have shown that Hyp
mice normalize their plasma phosphate concen-
tration and heal their rachitic changes, when
injected with inactivating antibodies to FGF23,
indicating that FGF23 is indeed the phosphaturic
principle in XLH [373]. XLH patients exhibited a
similar response to a single injection of anti-
FGF23 antibodies [374].
Autosomal Recessive
Hypophosphatemia (ARHP)
There are different forms of autosomal recessive
hypophosphatemia (ARHP) characterized by renal
phosphate wasting. ARHP can be caused by muta-
tions in DMP1 (Dentin matrix protein 1), which
belongs to the family of integrin-binding ligand
N-linked glycoproteins (SIBLING proteins) that
are secreted phosphoproteins involved in bone
mineralization [375]. DMP1 mutations lead to
increased FGF23 expression and thus increased
urinary phosphate excretion and defective osteo-
cyte maturation [376, 377]. ARHP was first
reported in consanguineous kindreds, suggesting
an autosomal recessive form of hypophosphatemia
[378–380]. Patients affected by ARHP have
FGF23 levels that are either elevated or inappro-
priately normal for the level of serum phosphorous
[48, 51]. Of the several different DMP1 mutations
identified thus far, one mutation alters the transla-
tion initiation codon (M1V), two mutations are
located in different intron-exon boundaries, and
three are frameshift mutations within exon 6. All
mutations appear to be inactivating, suggesting that
the loss of DMP1 causes increased FGF23 produc-
tion leading to hypophosphatemia. Accordingly,
Dmp1-null mice show severe defects in dentine,
bone, and cartilage, as well as hypophosphatemia
and osteomalacia [381, 382]. Furthermore, FGF23
levels in osteocytes and in serum are drastically
elevated in these animals [48]. Given the
established importance of DMP1 in osteoblast
function, loss of DMP1 actions in osteoblasts and
extracellular matrix may also contribute to the phe-
notype of patients with ARHR. Consistent with
this hypothesis, a high-calcium/high-phosphate
diet which is capable to rescue osteomalacia in
11 Physiology of the Developing Kidney: Disorders and Therapy of Calcium and Phosphorous. . .
VDR-null mice does not seem to prevent bone and
dentine mineralization defect in Dmp1-null mice
[383]. Other forms of ARHP are caused by muta-
tion in the gene encoding ENPP1 gene
(ectonucleotide pyrophosphatase/phosphodiester-
ase), which catalyzes phosphoester cleavage of
adenosine triphosphate, thereby generating pyro-
phosphate, a mineralization inhibitor. All forms of
hypophosphatemia are often associated with ossi-
fication of the posterior spinal ligaments, and
ENPP1 mutations can also be associated with cal-
cifications of the vascular endothelium, as
observed in GACI (generalized arterial calcifica-
tion of infancy) [51, 54, 384].
Hypophosphatemic Disorders
with Normal or Suppressed FGF23
Activity
Nephrolithiasis and Osteoporosis
Associated with Hypophosphatemia
Two different heterozygous mutations (A48P and
V147M) in NPT2a, the gene encoding a sodium-
dependent phosphate transporter, have been
reported in patients with urolithiasis or osteoporo-
sis and persistent idiopathic hypophosphatemia
due to decreased tubular phosphate reabsorption
[385]. When expressed in Xenopus laevis oocytes,
the mutant NPT2a showed impaired function and,
when co-injected, dominant-negative properties.
However, these in vitro findings were not con-
firmed in another study using oocytes and OK
cells, raising the concern that the identified
NPT2a mutation alone cannot explain the findings
in the described patients [386]. On the other hand,
additional heterozygous NPT2a variations
(in-frame deletion or missense change) have
recently been identified upon analyzing a large
cohort of hypercalciuric stone-forming kindreds;
however, these genetic variations do not seem to
cause functional abnormalities [387]. Homozygous
in-frame duplication of 21 bp in SLC34A1
resulting in complete loss of function of the mutant
NPT2a, one of two sodium-dependent phosphate
cotransporters expressed in the proximal renal
tubules, was identified in two siblings from a con-
sanguineous family with autosomal recessive
315
Fanconi syndrome and hypophosphatemic
rickets [388].
These findings are similar to those observed
in mice with homozygous ablation of Npt2a
(Npt2a/) [389]. Due to hypophosphatemia,
Npt2a/ mice show an appropriate elevation in
the serum levels of 1,25-dihydroxyvitamin D, lead-
ing to hypercalcemia, hypercalciuria and decreased
serum parathyroid hormone levels, and increased
serum alkaline phosphatase activity.
Hereditary Hypophosphatemic Rickets
with Hypercalciuria (HHRH)
Homozygous or compound heterozygous mutations
in the sodium phosphate cotransporter NPT2c
(SLC34A3) are the cause of HHRH, an autosomal
recessive disorder affecting renal tubular phosphate
reabsorption, leading to hypophosphatemia,
increased 1,25(OH)2 vitamin D levels, and
hypercalciuria, which lead in a considerable number
of patients to nephrolithiasis [390–393]; even indi-
viduals with heterozygous NPT2c mutations can
show laboratory abnormalities and nephrolithiasis.
Long-term phosphate supplementation as the sole
therapy leads, with the exception of persistently
decreased TmP/GFR, to reversal of the clinical and
biochemical abnormalities [390]. Treatment with
calcitriol is contraindicated since it can further
increase urinary calcium excretion.
Vitamin D-Dependent Rickets (VDDR)
Patients with VDDR type I (pseudo-vitamin
D-resistant rickets) show clinical and laboratory
findings that are similar to patients with vitamin
D-deficient rickets but do not respond to treatment
with 25(OH) vitamin D; instead treatment with
1,25(OH)2 vitamin D is required [394]. Clarifica-
tion of the abnormal vitamin D metabolism [395]
led to the recognition of two different forms of
rickets: VDDR type I (defect in the 1-
α-hydroxylation) and vitamin D-dependent rick-
ets type II (VDDR type II) (end-organ resistance
to 1,25(OH)2D).
1-Alpha Hydroxylase Deficiency
(VDDR Type I)
Patients affected by VDDR type I (autosomal
recessive) show almost all the clinical and
316
biochemical features of vitamin D-deficient rick-
ets except that vitamin D intake is usually ade-
quate. The permanent teeth show marked enamel
hypoplasia unlike in XLH [396]. The serum con-
centration of 1,25(OH)2 vitamin D is low in
untreated patients [397] and in vitamin
D-sufficient patients with normal or elevated
serum 25(OH)D3 levels, which provided the first
hint that the renal 1α-hydroxylase is deficient
[398–400]; molecular genetic studies later con-
firmed this conclusion. Indeed, genetic linkage
studies in affected French-Canadian families
mapped VDDR type I to a region on chromosome
12q13.3 [401], which is the location of the gene
encoding the 25(OH)D 1α-hydroxylase
(CYP27B). DNA sequence analysis of patients
affected by VDDR type 1 identified more than
20 different mutations of CYP27B [402–406] in
26 kindreds. All patients with VDDR type 1 were
found to carry homozygous or compound hetero-
zygous mutations, while the obligate carriers were
heterozygous for the mutant allele. Mutations that
confer partial enzyme activity in vitro were found
in the two patents with mild laboratory abnormal-
ities, suggesting that such mutations contribute to
the phenotypic variation observed in patients with
1α-hydroxylase deficiency [407].
End-Organ Resistance (VDR Mutations,
VDDR Type II)
Vitamin D-dependent rickets type II is an autoso-
mal recessive disorder caused by end-organ resis-
tance to 1,25(OH)2 vitamin D [408–410].
Alopecia, ectodermal defects like oligodontia,
and epidermal cysts might be present. The disease
varies in its clinical and biochemical manifesta-
tions, which suggest heterogeneity in the under-
lying molecular defects [410]. Most of the patients
have early-onset rickets, but the first reported
patient was a 22-year-old woman who had skele-
tal pain for 7 years [408], and another patient
presented at the age of 50 years following
5 years of symptoms [411]. Alopecia in some
cases is associated with poor therapeutic response
to the 1α-(OH) vitamin D [412, 413].
1,25(OH)2 vitamin D actions are mediated by
an intracellular receptor that binds DNA and con-
centrates the hormone in the nucleus [414],
A. Sharma et al.
analogous to the classical steroid hormones
[415]. The interactions between 1,25(OH)2 vita-
min D and its intracellular receptor have been
studied using cultured skin fibroblasts from con-
trol subjects and VDDR type II patients [416,
417]. Several defects were identified, including
absent receptors, a decreased number of receptors
with normal affinity, a normal receptor-hormone
binding but a subsequent failure to translocate the
hormone to the nucleus, and a post-receptor
defect, in which normal receptors are present,
but there is a deficiency in the induction of the
25-OHD-24 hydroxylase enzyme in response to
1,25(OH)2 vitamin D. Thus, the heterogeneity
suggested from clinical observations in VDDR
type II patients could be demonstrated at the cel-
lular level with various combinations of defective
receptor-hormone binding and expression. Vita-
min D receptor (VDR) is an intracellular protein,
which has a molecular weight of 60,000 Daltons.
The binding site for 1,25(OH)2 vitamin D resides
in the C-terminal part of the protein, while the
N-terminal part of the molecule possesses the
DNA-binding domain [418]. This hormone-
receptor complex binds to a DNA region, which
is located upstream of the promoter of genes
encoding calcium-binding proteins and other
proteins.
The availability of cDNAs encoding the avian
and human VDR [419] helped to clarify the
molecular basis of VDDR type II [420]. Nucleo-
tide sequence analysis of genomic DNA revealed
that the human VDR gene consists of nine exons;
exons 2 and 3 encode the DNA-binding domain,
while exons 7, 8, and 9 encode the vitamin
D-binding domain. The gene is located on chro-
mosome 12q12–q14 in man [401], i.e., in a region
that comprises the gene encoding the 1-
α-hydroxylase. Mutational analysis of the VDR
gene in VDDR type II patients demonstrated the
presence of nonsense and missense mutations
affecting different parts of the receptor. The
expression of these mutations in COS-1 monkey
kidney cells demonstrated that these mutations
result in a reduction or a loss of VDR function
similar to the heterogeneous effects observed in
cultured fibroblasts from VDDR type II patients.
Furthermore, null mutant, i.e., “knockout” mice
11 Physiology of the Developing Kidney: Disorders and Therapy of Calcium and Phosphorous. . .
for VDR produced by targeted gene disruption
[421, 422], was found to have features consistent
with those observed in patients with VDDR type
II. The severity of resistance to 1,25(OH)2 vitamin
D is variable, and some patients have improved
following therapy with very large doses of vita-
min D [423] or 1,25(OH)2 vitamin D
[424–426]. However, currently most patients are
treated with long-term nocturnal intravenous cal-
cium infusions followed by oral calcium supple-
mentation [427, 428]; cinacalcet to reduce PTH
secretion has been tried with some success [429].
Other Hypophosphatemic Disorders
There are several other genetic disorders associated
with hypophosphatemia and often with other
defects in proximal tubular function. These include
Dent’s disease, an X-linked recessive disorder,
which is caused by mutations in CLCN5 encoding
the chloride/proton antiporter, chloride channel
CLC-5 [430, 431], and Lowe syndrome (oculocer-
ebrorenal syndrome) [432], another X-linked reces-
sive disorder that is caused by mutations in OCRL1
[433, 434]. Other rare diseases that can be associ-
ated with severe hypophosphatemia are the
Fanconi-Bickel syndrome, which is caused by
homozygous or compound heterozygous mutations
in GLUT2 [435, 436], a glucose transporter local-
ized on the basolateral membrane of the proximal
renal tubules. The hallmarks of this disorder are
severe failure to thrive, hepatomegaly, and proximal
renal tubular dysfunction with massive glycosuria,
fasting hypoglycemia, and postprandial hypergly-
cemia, in addition to variable phosphaturia, acido-
sis, and aminoaciduria. Other hypophosphatemic
disorders include osteoglophonic dysplasia (OGD)
[437], an autosomal dominant disorder, which was
shown to be caused by different heterozygous mis-
sense mutations in the FGFR1 [438, 439], and
linear nevus sebaceous syndrome (LNSS), also
known as epidermal nevus syndrome (ENS) or
Schimmelpenning-Feuerstein-Mims syndrome, in
which elevated FGF23 was observed [440,
441]. Somatic activating mutations of HRAS or
NRAS (mitogen-activated protein kinase) associ-
ated with giant epidermal or melanocytic nevi
317
cause increased endogenous FGF23 from bone,
thus causing hypophosphatemia and osteomalacia
[442]. Furthermore, fibrous dysplasia can be asso-
ciated with increased urinary phosphate excretion.
This disorder is caused by heterozygous activating,
post-zygotic mutations in exon 8 of GNAS, the gene
encoding the alpha-subunit of the stimulatory G
protein (Gsα) [443, 444]. These mutations lead in
the dysplastic regions to a cAMP-dependent
increase in FGF23 production by osteoblasts/oste-
ocytes and fibrous cells [44, 445].
Acute and Chronic Treatment
of Calcium and Phosphorus Disorders
Goals of acute and chronic therapy are dependent
upon the specific clinical setting in which the dis-
turbance occurs, and management decisions usu-
ally require individual attention to the underlying
pathophysiology of each patient’s underlying dis-
order. Broad principles for correcting calcium and
phosphorus abnormalities are discussed.
Hypercalcemia
Acute Intervention
Hypercalcemia is the biochemical hallmark of
increased PTH or 1,25(OH)2 vitamin D levels,
less frequently in patients with fat necrosis or malig-
nancies. When severe it can be life-threatening and
requires urgent medical intervention. If cardiac and
renal function is normal, increasing sodium clear-
ance, maximizing glomerular filtration rate, and
correcting dehydration increase calcium excretion.
Other available pharmacological options are calci-
tonin, bisphosphonates, and cinacalcet. Calcitonin
causes rapid reduction in serum calcium level by
inhibiting bone resorption and enhancing urinary
calcium excretion, but its use is limited by the
requirement for frequent dosing and tachyphylaxis.
Bisphosphonates inhibit osteoclast activity. Typi-
cally, a clinical response is seen within 1–4 days,
with nadir in calcium observed by 4–7 days. Cau-
tion is required, because acute renal failure and
osteonecrosis of the jaw can be encountered as
rare side effects. Cinacalcet is an allosteric activator
318
of CaSR (calcimimetic) that increases the receptor’s
affinity for calcium, leading to a reduction in PTH
synthesis and an increase in renal calcium excretion
because of reduced distal tubular calcium
reabsorption. Peritoneal or hemodialysis with low
calcium concentration in the dialysis fluid might be
required in severe cases. Corticosteroids to reduce
intestinal calcium absorption are not always
effective.
Chronic Intervention
Chronic treatment is guided by the underlying dis-
order. Primary hyperparathyroidism (PHPT), e.g.,
parathyroid tumors, NSPHT, and MEN, respond
well to surgical removal of the parathyroid glands.
No effective medical therapy for the long-term
management of PHPT is currently approved for
children. In some NSHPT patients, pamidronate
[446] has been used to manage life-threatening
hypercalcemia and skeletal demineralization prior
to parathyroidectomy. Intraoperative PTH mea-
surements have been shown to be effective for
monitoring whether parathyroidectomy is com-
plete [447]. Cinacalcet has been used as well,
but with equivocal results, since CaSR mutations
can disrupt interaction with this drug. Heterozy-
gous loss-of-function mutations of the CaSR
(FHH) usually cause mild-to-moderate hypercalce-
mia that is not driven by excess PTH. FHH is a
benign disorder characteristically associated with
hypocalciuria, which unlike PHPT requires no
treatment except reassurance. Parathyroidectomy
is not indicated.
Hypercalcemia driven by increased intestinal
calcium absorption, e.g., Williams syndrome,
usually responds to a low-calcium diet with elim-
ination of vitamin D. Low-calcium milk (Locasol)
and protection from the sunlight can be useful
[148]. Corticosteroids may be a useful adjunct to
lower plasma calcium levels by decreasing
absorption from the gut. Steroids can be usually
discontinued after a few days when dietary
changes have shown to be effective. Treatment
of infantile hypercalcemia consists of calcium-
and vitamin D-restricted diets, steroids, and
bisphosphonates, if necessary. Cellulose phos-
phate has also been used to decrease calcium
absorption.
A. Sharma et al.
Hypercalcemia associated with AHH may
improve following glucocorticoid administration
in some, but not all, patients [118].
Hypocalcemia
A decrease in ionized calcium is frequently a
manifestation of defective vitamin D homeostasis
or inadequate PTH secretion or action. The
PTH-dependent renal production of 1,25-
dihydroxyvitamin D is deficient in all
hypoparathyroid states.
Acute Intervention
Symptomatic hypocalcemia or severe asymptom-
atic hypocalcemia is a medical emergency and
often requires intravenous 10 % calcium gluco-
nate or calcium chloride infusion. Concomitant
hypomagnesemia and hyperkalemia, if present,
should be corrected. The transition from acute
management of hypocalcemia to chronic mainte-
nance is highly dependent on the individual clin-
ical situation. It is often possible to begin oral
supplementation within a few days of intravenous
therapy with a few days of overlap. Some children
with abnormal parathyroid development, e.g.,
DGS, maintain normal calcium when healthy,
but have decreased parathyroid reserve and there-
fore develop symptomatic hypocalcemia when
stressed, for example, due to surgery or infections,
or when normal intestinal calcium intake is
reduced because of diarrhea or vomiting.
Chronic Intervention
The main treatment options available for patients
with acute or chronic hypoparathyroidism are cal-
cium salts, vitamin D or vitamin D analogues, and
drugs that increase renal tubular reabsorption of
calcium (i.e., thiazides). The selection of the dose
and type of the vitamin D analogue varies with the
underlying condition and the response to therapy.
Calcitriol is the preferred metabolite as it is consis-
tently effective at nontoxic doses and has a short
half-life, thus allowing rapid correction should
hypercalcemia develop. Monitoring of therapy is
essential to avoid hypercalcemia, hypercalciuria,
and nephrocalcinosis. Furthermore, recombinant
11 Physiology of the Developing Kidney: Disorders and Therapy of Calcium and Phosphorous. . .
PTH has been used in some cases of severe hypo-
parathyroidism. Compared with twice-daily deliv-
ery, pump delivery of PTH(1-34) appears to provide
the most promising modality for improving calcium
homeostasis and bone turnover in these children
[154]. The absence of functional PTH and the
resulting lack of PTH-dependent calcium
reabsorption in the distal renal tubules predispose
to hypercalciuria, nephrolithiasis, and chronic kid-
ney disease. Maintaining serum calcium at the lower
end of the normal range avoids clinical symptoms
and minimizes the risk of hypercalciuria. This is
exactly important, and if the hypocalcemia is not
symptomatic, maintenance of lower serum Ca level
is recommended. Hypercalciuria, if it occurs, may
respond to oral hydrochlorothiazide. Monitoring for
these complications at regular intervals is the key to
avoid complications.
Hypocalcemia due to vitamin D deficiency is
treated with cholecalciferol or related analogues.
It is important to ensure an adequate calcium
supply with the vitamin D replacement, as rapid
mineralization of the skeleton may follow initial
treatment, potentially causing a precipitous drop
in serum calcium (hungry bone syndrome). Treat-
ment of VDDR I (1α-hydroxylase deficiency)
requires lifelong replacement with calcitriol or
alphacalcidiol, thus bypassing the 1-
α-hydroxylation step. Some patients with VDDR
II (end-organ resistance to 1,25(OH2) vitamin D)
may respond to very high doses of calcitriol or
alphacalcidiol, but most pediatric patients require
regular intravenous calcium infusions or high
doses of oral calcium.
Hypocalcemia due to calcium-sensing receptor
(CaSR) mutations is due to increased CaSR sen-
sitivity (ADHH). These patients therefore have
serum PTH levels that are in the low-normal
range, which is different from patients with hypo-
parathyroidism, who often have undetectable
serum PTH levels or patients with pseudohypo-
parathyroidism, who typically have major PTH
elevations. Treatment with active metabolites of
vitamin D to correct the hypocalcemia can result
in marked hypercalciuria, nephrocalcinosis,
nephrolithiasis, and renal impairment, which
may be only partially reversible after cessation
of the vitamin D treatment.
319
Bartter syndrome is characterized by increased
prostaglandin E2 production, which stimulates the
1α-hydroxylase activity, thereby increasing 1,25
(OH)2D levels. Indomethacin (or other COX2
inhibitors) decreases prostaglandin levels, which
furthermore restricts intestinal calcium and vita-
min D absorption from the diet, thus helping to
correct hypercalciuria and nephrocalcinosis.
However, indomethacin itself possesses serious
nephrotoxicity; appropriate dose in each patient
should be considered.
Long-term therapy of hypocalcemia due to
pseudohypoparathyroidism (PHP) is similar to
other forms of hypoparathyroidism, namely, oral
calcium and calcitriol. However, PHP patients are
at little risk of developing increased urinary cal-
cium excretion, unless medications are not suffi-
ciently reduced after the skeleton is fully
mineralized after pubertal development. Treat-
ment with calcitriol can thus be usually more
aggressive than that of other forms of hypocalce-
mia, and normocalcemia and normal or close-to-
normal PTH should be achieved. Patients with
PHP-Ia (and PHP-Ib) should furthermore be rou-
tinely screened and eventually treated for any
associated endocrinopathy, in particular, hypothy-
roidism. In addition, careful physical examination
and, when necessary, specific psychological
investigations should be performed annually to
detect and follow up the presence/evolution of
specific AHO features (heterotopic ossifications,
cognitive delay, brachydactyly). Increasing evi-
dence suggests that children should be screened
with appropriate provocative tests for GH defi-
ciency [448]; note that PHP-Ia patients often
show “normal” growth rates early in life, but
fuse their growth plates prematurely, making it
necessarily to start treatment with rhGH as soon
as possible. Finally, there are no specific treat-
ments for the various manifestations of AHO.
Hyperphosphatemia
Acute Intervention
Acute hyperphosphatemia usually does not cause
symptoms unless reciprocal reduction in serum
calcium leads to hypocalcemia. In the setting of
320
normal kidney function, hyperphosphatemia is
usually self-limited because of the capacity of
kidney to excrete the phosphorus load. In the
setting of markedly compromised renal function,
dialysis can be required, in addition to oral phos-
phate binders.
Chronic Intervention
Chronic hyperphosphatemia management is
dependent on the underlying disorder. Acetazol-
amide has been used to treat hyperphosphatemia
and tumor-like extraosseous calcifications due to
inactivating mutations in FGF23 [449] or
GALNT3 in patients with FTC. This treatment
induces mild metabolic acidosis, thus improving
calcium-phosphate complex solubility. In severe
cases, regular hemodialysis has been prescribed
with reasonable success.
Secondary hyperparathyroidism seen in
chronic kidney disease is driven by hyperpho-
sphatemia, reduced 1,25(OH)2D production and
thus hypocalcemia, and possibly elevated FGF23
levels. The cornerstone of management is dietary
phosphate restriction, use of phosphate binders,
and dialysis to aid with phosphate removal.
Cinacalcet has been shown in EVOLVE trial to
decrease bone turnover and tissue fibrosis
[450]. Parathyroidectomy is often reserved for
ESRD patients, who have profound PTH eleva-
tions and thus increased bone resorption.
Hypophosphatemia
Acute Intervention
Acute severe hypophosphatemia particularly
presenting as hemolysis, rhabdomyolysis, flaccid
paralysis, or cardiac dysfunction is a medical
emergency that should be corrected promptly
with intravenous sodium- or potassium-
phosphate, as appropriate. Cumulative phosphate
deficit cannot be accurately predicted from serum
levels. Once the levels are above 1.5 mg/dL, tran-
sition to oral replacement should be attempted.
Chronic Intervention
The cornerstone of management is oral phospho-
rus replacement, which needs to be administered
A. Sharma et al.
in 4–5 daily doses; high doses of phosphate are
associated with diarrhea. Other adjunctive therapy
is guided by the underlying disorder. Furthermore,
anti-FGF23 antibodies may become available for
FGF23-dependent hypophosphatemic disorders.
In patients with oncogenic osteomalacia
(OOM), complete resection of the FGF23-
secreting tumor leads to rapid correction of the
biochemical abnormalities. Somatostatin and
cinacalcet has been tried with some success if
tumor resection is incomplete. Anti-FGF23 anti-
bodies are likely to ameliorate potentially harmful
effects of long-standing phosphorus and vitamin
D therapy before the tumor is found and resected.
The primary goal for the treatment or X-linked
hypophosphatemia (XLH) and other FGF23-
dependent forms of hypophosphatemia is to cor-
rect or minimize rachitic changes and aim for an
acceptable height velocity; in addition, meticu-
lous dental care is advised because of the high
incidence of dental abscesses. Serum alkaline
phosphatase is the most useful laboratory bio-
marker to gauge the success of therapy, in addition
to radiographs. The current therapy consists of the
oral administration of phosphate and calcitriol.
Cinacalcet and calcitonin have been tried with
some success as adjunctive therapies. Anti-
FGF23 antibodies were recently shown to normal-
ize blood phosphate levels in adult XLH patients
[374]. Treatment with recombinant human growth
hormone (rhGH) is controversial given the lack of
adequately controlled trials. Lower limb correc-
tive osteotomies or minimally invasive
hemiepiphysiodesis are needed for severe bony
deformities in older children. For all patients
with inherited forms of hypophosphatemia, iron
stores should be replenished, if deficient.
Hereditary hypophosphatemic rickets with
hypercalciuria (HHRH) is a hypophosphatemic
disorder characterized by normal or suppressed
FGF23 activity and thus elevated 1,25(OH)2 vita-
min D levels. This disorder is therefore managed
differently from the hypophosphatemia such as
XLH, ARHP, or ADHR. Oral phosphate supple-
ments are effective. Calcitriol is contraindicated,
as 1,25(OH)2 vitamin D levels are elevated.
Fanconi-Bickel syndrome is a disorder of gen-
eralized proximal tubular dysfunction that is
11 Physiology of the Developing Kidney: Disorders and Therapy of Calcium and Phosphorous. . .
caused by GLUT2 mutations. Treatment aims at
correcting acidosis, treating the hypophosphatemic
rickets, and using fructose-based diets to circum-
vent hypoglycemia. Long-term prognosis is
unknown, but there is a risk of atherosclerotic
vascular disease, repeated fractures from general-
ized osteopenia, and markedly short stature.
Concluding Remarks
Remarkable advances have been made in identify-
ing key proteins that are involved, either directly or
indirectly, in the regulation of calcium and phos-
phate homeostasis, the hormones that are involved
in these mechanisms, and receptors that mediate
these hormonal actions in the different target tissues.
Although most of these conditions are rare, their
molecular definition resulted in the identification of
several proteins that are involved in the normal
regulation of mineral ion homeostasis and/or bone
development. These advances have added to
targeted therapy for some of these disorders.
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 Nutrition Management in Childhood
Kidney Disease: An Integrative and 12
Lifecourse Approach

Lauren Graf, Kimberly Reidy, and Frederick J. Kaskel

Contents Common Phosphate Additives Used By

the Food Industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342 Hypoallergenic Diet as a Treatment for Idiopathic
A Life Course Approach to the Renal Diet: Nephrotic Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
Integration with Developmental Physiology . . . . . . 342 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
A Life Course Approach to the Renal Diet: References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
Integration with Etiology of Renal Disease . . . . . . . 347
A Life Course Approach to the Renal Diet:
Integration with Prevention of Cardiovascular
Disease and Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . 348
Effects of Diet on Inflammation and Heart Disease
and Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
The Anti-Inflammatory Diet for Kidney Disease:
A Paradigm Shift . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350
Fiber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
Phosphorus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
Dietary Guidelines for CKD and ESRD . . . . . . . . . . 354
Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
Phosphate Preservatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355

L. Graf (*)
Children’s Hospital at Montefiore, Albert Einstein College
of Medicine, Bronx, NY, USA
e-mail: Lgraf@montefiore.org
K. Reidy • F.J. Kaskel
Division of Pediatric Nephrology, Children’s Hospital at
Montefiore, Albert Einstein College of Medicine, Bronx,
NY, USA
e-mail: Kreidy@montefiore.org;
Frederick.kaskel@einstein.yu.edu

# Springer-Verlag Berlin Heidelberg (outside the USA) 2016 341

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_11
342
Introduction
Infants, children, and adolescents with chronic kid-
ney disease (CKD), end-stage renal disease
(ESRD), and kidney disorders such as nephrotic
syndrome face unique nutritional challenges. The
goals of this chapter are to outline the nutritional
problems associated with various forms of kidney
disease and describe interventions to correct them.
The approach to nutritional management varies
greatly based on the age of the child and type of
kidney disorder. The primary goals of nutrition in
infants and young children with CKD are to pro-
mote adequate weight gain, linear growth, and
hydration as well as maintain electrolyte homeo-
stasis. For older children and teenagers with CKD
and ESRD, the traditional nutritional goals have
focused on the management of potassium and
phosphorus, fluid balance, and growth. However,
as our understanding of nutrition evolved, it is now
recognized that diet (quantity and quality of food)
has far-reaching effects on overall health. Nutrition
is extremely important in modulating inflamma-
tion, immune function, and the gut microbiome,
all of which play a major role in preventing chronic
diseases including cardiovascular disease (CVD)
and cancer. A large body of research over the past
decade has led to a paradigm shift in nutrition and
kidney disease. The recommendations for protein
intake in the ESRD population as well as the most
effective way to manage dietary phosphorus have
changed dramatically. In addition, in some cases of
idiopathic nephrotic syndrome, diet therapy has
been effective as a treatment of the disease rather
than simply managing symptoms. The following
sections will identify nutritional problems in vari-
ous kidney disorders and describe the diet therapy
to best help manage them.
A Life Course Approach to the Renal
Diet: Integration with Developmental
Physiology
The approach to nutrition in chronic kidney dis-
ease is modified by developmental changes in
physiology. First, the nutritional needs of a grow-
ing infant or child are distinct from that of an
L. Graf et al.
adult. A healthy child doubles their height in the
first 4 years of life and requires significant net
calcium and phosphorus absorption for bone
accretion. Early childhood is a period of peak
growth velocity, with growth of 7–11 cm/year,
and over 50 % of adult height is attained in the
first 4 years of life. Thus, optimal nutritional sup-
port for infants and young children with CKD will
have far-reaching consequences for ultimate adult
height.
There are several unique challenges to opti-
mize nutrition in infants with CKD. First, the
major source of calories and nutrients in the first
year of life is liquid. In addition, caloric intake is
the major determinant of growth in the first year of
life and outweighs the effect of growth hormone
[1]. Thus, it is critical to optimize the caloric
intake of both infants and toddlers with CKD
[2, 3]. This often requires supplementation with
either oral feeds or gastrostomy tube feeding (see
Table 1 for nutritional formulas and supplements
frequently used in kidney disease). Estimates of
caloric needs can be based on estimated energy
requirements for age (see Table 2) [4]. These
should be used as a guide and increased if inade-
quate weight gain is achieved. These guidelines
are also intended for chronic care, and different
guidelines may apply to children in the intensive
care unit, for example.
Maintenance of euvolemia and serum potas-
sium is also particularly an issue for anuric
infants. Breast milk is the preferred source of
nutrition for infants with or without chronic kid-
ney disease. Especially for infants with
hyperkalemia, breast milk potassium content is
typically lower than commercially available for-
mulas. Similac PM 60/40 ® can be used for infants
who require formulas. In severe cases, potassium
content can be lowered further in formula or
breast milk by mixing sodium polystyrene with
formula, allowing it to settle for 30–45 min, and
decanting the supernatant (leaving the sediment
behind) [5, 6]. Potassium is exchanged for
sodium, so monitoring for hypernatremia is advis-
able. It is preferable to avoid direct administration
of sodium polystyrene to infants as it has been
associated with necrotizing enterocolitis. To limit
fluid overload, anuric infants can be given higher
 12

Table 1 Nutrition Formulas for Children with Renal Disease

Age range of Carbohydrates (g/L Na/K Ca/P mOsm/kg
use Manufacturer kcal/ml Protein (g/L) sources Fat sources sources) (mEq/L) (mg/L) water
Infant–toddler Similac Ross 0.67 16 (whey, caseinate) 38 g/L (soy, coconut) 69 (lactose) 7/15 380/ 250
PM products 190
60/40
Infant–toddler Amin- R&D 2 19 (free amino acids) 46 g/L (partially 365 (maltodextrin, <15/ – 700
Aid Laboratories hydrogenated soybean sucrose) <15
oil)
Infant–toddler MCT oil Multiple 7.7 – 14 g/15 mL –
Toddler–adult Suplena Ross product 1.8 44 (caseinates) 95 g/L (high oleic 199 (maltodextrin, 33/28 1,060/ 600
safflower and soy oil) sucrose, cornstarch) 700
Toddler–adult Renalcal Nestle 2 34 (essential L amino 82 g/L (MCT oil, 290 (maltodextrin, – – 600
Nutrition acids, select nonessential canola oil, corn oil, soy modified cornstarch)
amino acids, whey lecithin)
protein concentrate)
Toddler–adult Nepro Ross 1.8 81 (caseinates) 96 g/L (high oleic 167 (corn syrup, 46/27 1,060/ 585
with products safflower oil and soy sucrose, FOS) 700
Carb oil)
Steady
Nutrition Management in Childhood Kidney Disease: An Integrative and Lifecourse Approach
343
344 L. Graf et al.

Table 2 Equations to Estimate Energy Requirements for Children at Healthy Weights

Age Estimate energy requirement (EER) (kcal/day) = total energy expenditure + energy deposition
0–3 months EER = (89  weight (kg) – 50) + 175
4–6 months EER = (89  weight (kg) – 50) + 56
7–12 months EER = (89  weight (kg) – 50) + 22
13–35 months EER = (89  weight (kg) – 50) + 20
3–8 years Boys EER = 88.5 – 61.9  age (y) + PA  (26.7  weight (kg) + 903  height (m)) + 20
Girls EER = 125.3 – 30.8  age (y) + PA  (10  weight (kg) + 934  height (m)) + 20
9–18 years Boys EER = 88.5 – 61.9  age (y) + PA  (26.7  weight (kg) + 903  height (m)) + 25
Girls EER = 135.3 – 30.8  age (y) + PA  (10  weight (kg) + 934  height (m)) + 25
Physical Activity Coefficients for Determination of Energy Requirements in Children Ages 3–18 years (adapted from
KDOQI guidelines [4])
Gender Sedentary Low active Active Very active
Typical ADL + 30–60 min of ADL + ADL + 60 min of daily moderate activity
activities of daily moderate activity 60 min + an additional 60 min of vigorous activity
daily living (e.g., walking 5–7 km/h) daily or 120 min of moderate activity
(ADL) only moderate
activity
Boys 1.0 1.13 1.26 1.42
Girls 1.0 1.16 1.31 1.56
PA = physical activity

caloric feeding (up to 30 kcal/oz) by either pre-
paring more concentrated formula or adding either
PM 60/40 ® or medium chain triacyglyceol (MCT)
oil to breast milk.
Nutritional management of CKD must also
account for the changes in normal physiology of
mineral homeostasis, as it differs for infants, chil-
dren, and adults. A prime example of this is the
changes in calcium and phosphorus homeostasis.
The developmental regulation of calcium and
phosphorus during infancy and childhood reflects
the need for net calcium and phosphorus
reabsorption, for bone growth, and for minerali-
zation (Figs. 1 and 2). Before birth, the placenta
actively transports up to 300 mg/day of calcium to
the developing fetus, mostly during the third tri-
mester [7]. This is dependent on parathyroid
hormone-related protein (PTHrp), likely secreted
by the placenta [8]. Thus, calcium and phosphorus
requirements in premature infants born before this
critical window for bone growth and mineraliza-
tion may be higher than in full-term newborns [9].
After birth, the intestine becomes the major ave-
nue for calcium via passive absorption [7]. Although
calcium is best absorbed from breast milk [10],
infants have greater net absorption of calcium
from infant formula due to the much higher calcium
content in infant formulas (42–50 mg/100 mL
vs. 21–26 mg/100 mL in breast milk) [11]. There
is a transient drop in serum calcium after birth that
leads to increased parathyroid hormone secretion
(up to adult levels within 48 h), which in turn
stimulates synthesis of calcitriol (1,25-OH vitamin
D) in the kidney [7]. By later infancy, intestinal
calcium absorption becomes dependent on
calcitriol. On average, 30–60 % of ingested calcium
is absorbed, but up to 90 % may be absorbed in
preterm infants [11]. The recommended daily intake
of calcium varies with age (Table 3) (http://ods.od.
nih.gov/factsheets/Calcium-HealthProfessional/).
In children, 30–350 mg of calcium/day is
deposited in growing bones [7, 12]. In compari-
son, the average adult ingests 1,000 mg of calcium
per day. Of this, 350 mg is absorbed, but 800 mg
gets excreted in the stool (650 not absorbed and
150 lost through gut cell sloughing and bile secre-
tion), and 200 mg is excreted in the urine,
maintaining a net zero balance for homeostasis.
In the adult, 98% of the 7,500 mg filtered through
the kidney is reabsorbed. In the neonate calcium
excretion in the urine is higher, especially in the
premature infant [13, 14]. Despite this, there is net
12 Nutrition Management in Childhood Kidney Disease: An Integrative and Lifecourse Approach
Fig. 1 Developmental changes in calcium handling: a
positive calcium balance is required for bone growth in
infants and children, whereas adults maintain a net zero
calcium balance to maintain homeostasis. Increased intes-
tinal absorption during infancy and childhood contributes
calcium accretion throughout childhood. Figure 1
illustrates the distinct age-related normal physio-
logic changes in calcium absorption, renal excre-
tion, serum calcium concentration, and bone
deposition.
Similar to calcium, phosphorus balance is reg-
ulated by intestinal absorption, distribution of
phosphorus between the extracellular and bone/
intracellular storage pool, and renal phosphate
excretion (Fig. 2) [11]. In addition to being
required for bone mineralization, phosphorus is
required for basic cellular function [15]. Inside the
cell, phosphorus is predominantly in the form of
phosphate anion. Phosphates are the most abun-
dant intracellular anion, a major component of
345
to the positive calcium balance. Premature infants and
neonates have increased urinary calcium excretion, which
decreases with age. Serum calcium levels may be higher in
childhood than in adults
nucleic acids and lipid membranes, and the pri-
mary source of cellular energy, ATP. In addition,
phosphorylation (addition of phosphates to pro-
teins) is key for the regulation of multiple cell
signaling pathways. Phosphorus excretion in the
urine serves as a buffer and contributes to
acid–base homeostasis.
The average adult has an intake of 1,000–1,600
mg/day of phosphorus (mean 20 mg/kg/day), and
80 % of this is absorbed in the intestine
[11]. There is some secretion of phosphorus into
the gut as a component of bile (3 mg/kg/day), so
net phosphorus absorption is approximately
13 mg/kg/day. In the adult, approximately
3 mg/kg/day will enter the bone for bone
346 L. Graf et al.

Fig. 2 Developmental changes in phosphorus han-
dling: the majority of phosphorus is intracellular (as a
component of nucleic acids, ATP, or phospholipids) or
stored in the bone, and serum inorganic phosphorus repre-
sents <0.1 % of whole body phosphorus. Increased
absorption and decreased renal excretion contribute to the
higher serum phosphorus levels and positive phosphorus
balance in the infant. In the adult, renal excretion of
ingested phosphorus results in a net zero phosphorus bal-
ance and maintains homeostasis

Table 3 Recommended intake of calcium (Adapted from tubule. Fibroblast growth factor 23 (FGF-23)
http://ods.od.nih.gov/factsheets/Calcium-HealthProfessional/) and parathyroid hormone regulate phosphorus
Age Recommended daily intake (mg/day) excretion in the adult. In the steady state, the
0–6 months 200 adult maintains net zero phosphorus balance and
6–12 months 260 all the ingested phosphorus is excreted in the
1–3 years 700 urine.
4–8 years 1,000 In the infant, a net positive phosphorus balance
8–18 years 1,300 is required for bone accretion and growth. Thus,
Adult 1,000–1,200 normal serum phosphate levels vary with age,
with the highest levels in infants (up to 8.5
mg/dL) which decrease with age (4.5 mg/dL) in
adults. Human milk has a concentration of 11–17
remodeling. Phosphorus is fully filtered by the mg/dL phosphorus, while standard cow milk for-
glomerulus and the tubular fluid concentration is mulas contain 28–39 mg/dL of phosphorus
the same as the adult. In the adult, 85 % of [11]. As with calcium, the recommended guide-
filtered phosphorus is reabsorbed in the proximal lines for phosphorus intake vary by age (Table 4)
12 Nutrition Management in Childhood Kidney Disease: An Integrative and Lifecourse Approach
Table 4 Recommended minimum phosphorus intake
(Adapted from http://www.nal.usda.gov/fnic/DRI/DRI_
Tables/recommended_intakes_individuals.pdf)
Recommended minimum intake
Age (mg/day)
0–6 months 100
6–12 275
months
1–3 years 380
4–8 years 405
9–18 years 1,055
Adult 580
(http://www.nal.usda.gov/fnic/DRI/DRI_Tables/
recommended_intakes_individuals.pdf). How-
ever, the average diet likely greatly exceeds this
recommended minimum intake [15].
Up to 90 % of ingested phosphorus is absorbed in
the newborn via passive and active transport in the
intestine (predominantly jejunum) via paracellular
HPO4 transport and NaPi-IIb sodium-dependent
phosphate transporters, respectively [11]. The new-
born kidney tubules are relatively insensitive to
parathyroid hormone, leading to decreased phos-
phorus excretion and higher serum phosphate levels
[16]. Newborn kidneys also express a NaPi trans-
porter isoform that also favors phosphorus
reabsorption [16]. The role of FGF-23 in neonatal
phosphate excretion is still under investigation [17].
The developmental differences in calcium/
phosphorus homeostasis have significant implica-
tions for management of the CKD-induced meta-
bolic bone disease in childhood. First, the
recommended target goals for calcium and phos-
phorus are age based (Table 5). Impaired phos-
phorus excretion is an issue for the infant with
CKD. Breast milk remains the optimal nutrition,
again, due its lower content of phosphorus. Renal
formulas (Table 1) also have lower phosphorus
content than typical infant formulas (albeit still
higher than breast milk). Calcium carbonate
remains a commonly used phosphate binder in
infants. Calcium carbonate can be mixed with
daytime and nighttime feeds. However, in infants
with hypercalcemia, the newly available
sevelamer (Renvela ®) powder can be used as an
alternative [18]. Renvela ® is first reconstituted in
water and then mixed with the overnight feeds.
347
Table 5 Target Serum Calcium and Phosphorus Levels
By Age
Calcium Phosphorus
Age (mg/dL) (mg/dL)
0–5 months 8.7–11.3 5.2–8.4
6–12 8.7–11.0 5.0–7.8
months
1–5 years 9.4–10.8 4.5–6.5
6–12 years 9.4–10.3 3.6–5.8
13–20 years 8.8–10.2 2.3–4.5
A Life Course Approach to the Renal
Diet: Integration with Etiology of Renal
Disease
The approach to nutrition is also modified by the
etiology of chronic kidney disease. Congenital
abnormalities of the kidney and urinary tract
(CAKUT) are the most common cause of chronic
and end-stage kidney disease in childhood. Dys-
plasia and obstructive uropathy often result in
polyuria, with concomitant salt and water
wasting. Thus, infants and children with CKD
secondary to CAKUT require unique approach
to their nutritional management. Severely
polyuric infants have daily fluid requirements
that are 1.5–2 times that of a healthy infant.
Thus, for polyuric infants, it may be advisable
not to concentrate formulas to provide addi-
tional free water. When this is insufficient, water
boluses can be given either orally or via a
gastrostomy tube.
Sodium accretion is required for normal
growth. Sodium supplementation in infants with
salt wasting forms of CKD improves linear
growth [19]. Sodium may be administered as
sodium bicarbonate (in patients with concomitant
acidosis) or with commercially available sodium
chloride solutions. Typically, sodium chloride
solutions need to be given via G-tube as they
may not be tolerated orally. Close monitoring of
serum sodium is advisable. Other, rarer renal dis-
eases, such as nephropathic cystinosis with
Fanconi syndrome, often require electrolyte sup-
plementation, including phosphorus, orally or via
G-tube to support growth [20, 21].
348
A Life Course Approach to the Renal
Diet: Integration with Prevention
of Cardiovascular
Disease and Inflammation
Cardiovascular disease (CVD) is the leading
cause of death in both children and adults with
chronic and end-stage kidney disease (ESRD).
Good nutrition plays a major role in preventing
and reversing CVD in ESRD. Children with CKD
are at very high risk of developing premature
CVD during young adulthood. Adults with a his-
tory of childhood-onset ESRD have significantly
shorter life expectancies than their peers. Dialysis
patients live 40–50 years less, and transplant
patients live 20–25 years less than age-matched
peers in the general population. In addition, young
adults (25–34 years old) on maintenance dialysis
die at rates 100 times higher of cardiovascular
causes than age- and race-matched peers in the
general population [22]. Renal transplantation
significantly reduces cardiovascular mortality
[23]. However, even in these patients, the mortal-
ity rates are still 10 times higher than in the gen-
eral pediatric population secondary to CVD
factors.
The causes of early onset CVD in the pediatric
kidney disease population are multifactorial. The
prevalence of both traditional and uremia-related
cardiovascular risk factors is extremely high in
children with advanced kidney disease [24]. Tra-
ditional risk factors for heart disease include
hypertension, diabetes, dyslipidemia, obesity,
and insulin resistance. The Chronic Kidney Dis-
ease in Children (CKiD) study, an observational
cohort study of children aged 1–16 years with
stages 2–4 CKD, provides valuable insight into
the cardiac risk factors present in this population
[25]. Hypertension was present in 54 % of sub-
jects at enrollment. Even with antihypertensive
medications, 48 % of participants had poorly con-
trolled blood pressure. Dyslipidemia was present
in 45 % of children in the CKiD study and 15 %
were obese [26]. Although less common, meta-
bolic disturbances are also found in children with
CKD. In the CKiD cohort, 19 % of subjects were
insulin resistant. These risk factors tend to worsen
as CKD progresses.
L. Graf et al.
Traditional cardiac risk factors are compounded
by those secondary to uremia. The largest contrib-
utors to vascular changes in children with ESRD
are disturbances in mineral metabolism (high phos-
phorus, calcium–phosphorus product, and PTH)
[24]. Soft tissue calcification and coronary artery
calcification (CAC) are evident in pediatric patients
with advanced kidney disease. One study of young
adults with childhood-onset CKD found coronary
artery calcifications present in 92 % of patients
[27]. Markers of inflammation associated with
heart disease such as elevated C-reactive protein
(CRP) and homocysteine are often elevated in
ESRD and remained high even after renal trans-
plantation. Given the multitude of risk factors in
children with kidney disease, the high prevalence
of early onset cardiovascular disease in this popu-
lation is not surprising. An epidemiological report
found that young adults on dialysis (ages 25–34)
have the same cardiovascular mortality risk as an
average 80–85-year-old in the general
population [28].
Over the past 30 years, advances in medicine
have improved outcomes for children with CKD
and ESRD, with increasing numbers surviving
into adulthood. Patients with CKD no longer die
of kidney disease but of heart disease [29]. How-
ever, little progress has been made in reducing risk
factors for heart disease such as coronary artery
calcifications. This may be largely due to the fact
that little attention has been given to diet as a
therapy.
A large body of evidence suggests that many
causes of heart and blood vessel disease in chil-
dren can be greatly ameliorated with aggressive
nutritional intervention. Mineral metabolism and
fluid overload are directly related to nutrition. It is
increasingly recognized that nutrition also plays
a major role in improving inflammation, hyper-
tension, and metabolic acidosis in kidney disease
[30, 31].
The goal in pediatric nephrology is to improve
both quantity and quality of life. Pediatric
nephrology has had great success increasing the
survival rates of young children with kidney dis-
ease. A new goal now is to help these patients
transition into adolescence and young adulthood
with much lower cardiovascular risk factors,
12 Nutrition Management in Childhood Kidney Disease: An Integrative and Lifecourse Approach
which would place them on a path toward a
healthier and more productive life. A greater
focus on nutrition may be the most effective,
risk-free strategy to help prevent early onset
CVD in children with advanced kidney disease.
This requires careful attention to the overall qual-
ity of food and diet patterns rather than simply
focusing on individual nutrients.
Effects of Diet on Inflammation
and Heart Disease and Immunity
The standard American diet (characterized by a
high intake of refined carbohydrates, simple sugars,
red meat, and processed food and low intake of
fiber, fruits, and vegetables) is a major cause of
CVD, inflammation, hypertension, dyslipidemia,
and type 2 diabetes [32]. It is also well established
that a high-fiber diet based on unprocessed, plant-
based foods that is low in simple sugar drastically
reduces risk of CVD and in many cases is an
effective way to reverse it [33]. High fiber intake
is associated with lower levels of inflammation and
reduced mortality in the general population. Recent
studies suggest that a high-fiber diet may be even
more effective in reducing inflammation in CKD
than in the general population. One large cohort
study analyzed data from the National Health and
Nutrition Examination Survey (NHANES III) data-
base to examine the association between dietary
fiber intake and elevated CRP levels in CKD and
non-CKD populations [34]. Higher intake of die-
tary fiber was associated with lower CRP levels in
both the CKD and non-CKD groups, but the effect
was more dramatic in those with kidney disease.
For every 10-g increase in daily dietary fiber, the
odds of elevated CRP were decreased to 11 % in the
non-CKD group and 38 % in the CKD group. In
subjects without kidney disease, dietary total fiber
intake was not significantly associated with
reduced mortality. However, in the CKD group,
dietary fiber was inversely associated with mortal-
ity. Therefore, a high dietary fiber intake is associ-
ated with reduced inflammation and mortality
in CKD.
Diet has an enormous influence on the gut
microbiome. The human gut is home to more
349
than 100 trillion microbial cells, which play a
significant role in immunity and inflammation.
Both diet and diseases including CKD and
ESRD directly impact the quantity and type of
microbes present in the gut [35]. This in turn
impacts inflammation and CVD risk. The compo-
sition of bacteria in the gut can be manipulated by
changes in diet.
A diet high in whole, plant-based foods (fiber)
has been shown to favor the growth of beneficial
bacteria [30, 36]. High fiber intake results in short
chain fatty acid production by microbes in the gut,
which decrease the intestinal pH. This suppresses
the growth of harmful bacteria such as E. coli and
other members of Enterobacteriaceae family.
Studies have found that European children have
depleted levels of the beneficial bacteria
Bacteroidetes and higher levels of Enterobac-
teriaceae compared with rural African children,
which the authors believe is due to the lower
fiber intake among Europeans [37]. Analysis of
the fecal microbiota of hemodialysis patients
revealed that the total number of enterobacteria
was approximately 100 times higher in subjects
with ESRD compared to healthy controls
[38]. Hemodialysis patients also had lower num-
bers of bifidobacteria and higher Clostridium
perfringens than controls. The authors speculate
that the dysbiosis may be due to multiple factors
including uremia, frequent antibiotic use, as well
as decreased intake of fiber.
The production of short chain fatty acids from
fiber has many benefits that are protective to the
heart and vascular system [30]. They inhibit cho-
lesterol production by the liver and increase the
production of cholesterol-binding bile acids,
which aids in the elimination of lipids. Short
chain fatty acids also improve insulin sensitivity
and help quell uremic toxins.
On the other hand, high protein intake pro-
motes the growth of harmful bacteria that induce
inflammation. In the general population, a high
protein intake increases the activity of bacterial
enzymes that produce toxic metabolites that trig-
ger inflammation [30, 36]. The harmful effects of
proteolytic fermentation appear to be exaggerated
in CKD and ESRD. The fermentation of protein
by bacteria in the large intestine results in indoles
350
and phenols including p-cresyl sulfate and
indoxyl sulfate. These are gut-derived uremic
toxics that are absorbed into the blood and nor-
mally cleared by the kidney. However, they are
resistant to clearance by dialysis. Elevated levels
of p-cresyl sulfate and indoxyl sulfate are found in
CKD and ESRD and appear to accelerate the
progression of kidney disease. Elevated levels of
indoxyl sulfate are associated with vascular stiff-
ness, aortic calcification, and increased cardiovas-
cular mortality in patients with kidney disease
[39–41]. Evidence suggests that diet may play a
significant role in controlling the level of these
uremic toxins in the blood even as CKD pro-
gresses. Serum concentrations of indoxyl sulfate
and p-cresyl vary in subjects with similar esti-
mated glomerular filtration rates (GFR), which is
due in part to differences in fiber intake. Dietary
fiber suppresses colonic production of p-cresyl
and indoxyl sulfate, whereas protein exacerbates
it [30]. Therefore, maximizing intake of fiber from
whole foods as well as ensuring adequate but not
excessive protein intake will help keep inflamma-
tory uremic toxins at bay.
High-fiber, plant-based diets are also more
effective in keeping serum phosphorus within
the target range compared with animal protein-
based diet. One crossover trial compared the
effects of a vegetarian diet versus a meat-based
diet on serum phosphorus levels [42]. Although
both diets had equivalent amounts of phosphorus,
they found significantly lower serum phosphorus
in the vegetarian group. This is due to decreased
absorption from plant sources of phosphorus.
Plant foods naturally contain phytate, the storage
form of phosphorus, which is not digestible to
humans. Therefore, much of the phosphorus
from plant foods passes through undigested
[43–45]. It is important to take into account the
source (plants, meat, and processed food) rather
than simply the amount of phosphorus. The veg-
etarian group also had lower FGF-23 levels,
suggesting decreased inflammation.
A recent observational study of 3,972 adults
with CKD found that a diet high in processed
meat, fried food, and sugary beverages was asso-
ciated with higher mortality [46]. A plant-based
diet high in fruits and vegetables as well as fish
L. Graf et al.
was associated with a lower mortality risk over
time. One of the highlights of this study is that it
focused on diet patterns rather than individual
nutrients. The authors also drew attention to the
need for food-based guidelines rather than micro-
nutrient guidelines. The current guidelines focus
on micronutrients and macronutrients (sodium,
potassium, phosphorus, protein) with little focus
on actual food. There is a trend in nutritional
research to focus more on dietary patterns rather
than individual nutrients of supplements. This
comes with the recognition that many studies
looking at nutrients in isolation yield misleading
results because the larger picture is often not taken
into account. Nutrients are not eaten in isolation;
rather they are consumed as part of a whole food
with a vast array of nutrients and antioxidants that
act synergistically. We suggest that food-based
guidelines would make it easier for patients to
visualize and understand, which may improve
adherence.
The Anti-Inflammatory Diet for Kidney
Disease: A Paradigm Shift
Over the past decade, advances have been made in
our understanding of how food and nutrition
impact the health of patients with kidney disease.
The foundation of an anti-inflammatory diet for
kidney disease is plant-based foods
(low-potassium fruits, vegetables, unrefined
whole grains, nuts, and seeds) combined with
smaller amounts of unprocessed lean protein
such as turkey, chicken, or fish. The diet is also
low in processed food, dairy, and refined carbo-
hydrates such as sugar-sweetened beverages,
white-bread, sugary cereals, and deli meat. Avoid-
ance of processed foods containing phosphate
additives should be particularly emphasized.
Traditionally, the renal nutritional community
has taken a reductionist approach – evaluating
food simply based on the number of calories,
milligrams of sodium, or potassium and phospho-
rus content. The conventional nutritional goals in
children with kidney disease have been to pro-
mote adequate weight gain and linear growth,
manage serum phosphorus and potassium levels,
12 Nutrition Management in Childhood Kidney Disease: An Integrative and Lifecourse Approach
and maintain fluid balance. However, little
emphasis was placed on the healthfulness of the
diet as a whole or how nutrition influences risk of
developing cardiovascular disease. Moreover,
some traditional nutritional advice given to
patients, once thought to improve outcomes,
such as an emphasis on animal protein, may actu-
ally be detrimental. In addition, there are a number
of foods that may appear appropriate for a renal
diet because they are low in potassium and phos-
phorus such as a lemon–lime soda and other
sugar-sweetened beverages. However, these prod-
ucts are high in sugar and fructose, which may
lead to inflammation and dyslipidemia and
worsen hypertension [47–52]. As our understand-
ing of nutrition has evolved, two additional goals
have emerged: to reduce inflammation in the body
and reduce heart disease risk. These new goals
focus on prevention and should be included in
nutritional counseling for patients with kidney
disease.
Fiber
Fiber is a type of carbohydrate indigestible to
humans and is found exclusively in plants. Fiber
intake is a marker of the amount of plant foods in
the diet. There are two main types of fiber: soluble
and insoluble. Soluble fiber dissolves in water and
slows the movement of food through the gastro-
intestinal tracts. Insoluble fiber does not dissolve
in water and provide bulking which can help
prevent constipation. Both soluble and insoluble
fibers can be fermented in the large intestine into
active by-products such as prebiotics. Prebiotics
serve as food or fuel for the probiotics. Therefore,
adequate consumption of fiber is imperative to
maintain sufficient amounts of beneficial bacteria
in the gut. Despite the health benefits of dietary
fiber, more than 90 % of adults and children in the
general population fail to meet the minimum
recommended requirements [34]. The average
intake is 17 g per day, which is well below the
recommended intake of 25–30 g daily. In people
with advanced CKD, average dietary fiber intake
is even lower at 15 g per day [34]. This is not
surprising given that patients with CKD and
351
ESRD are often instructed to limit or avoid
many plant foods due to a high potassium or
phosphorus content. However, there are many
plant foods that do not contain an excessive
amount of potassium or phosphorus. Furthermore,
much of the phosphorus in whole plant foods is
poorly absorbed and can be tolerated in people
with advanced CKD without raising serum phos-
phorus. The importance of fiber is not usually
discussed in traditional diet counseling for renal
patients. In fact many of the standard nutritional
recommendations given to patients with advanced
kidney disease lead to much lower fiber intakes. In
cases where obtaining adequate fiber through food
alone is difficult, fiber supplements such as
Benefiber ® or Unifiber ® may be considered.
However, they should not serve as a replacement
for healthy, wholesome food. Nutritional educa-
tion for children and adolescents should empha-
size healthy, fiber-rich foods that can be
incorporated into a renal diet.
Phosphorus
It is well documented that limiting dietary phos-
phorus is crucial for patients with advanced kid-
ney disease. Phosphorus restriction helps prevent
elevations in serum phosphorus, which lead to
hyperparathyroidism, renal osteodystrophy, heart
and blood vessel calcifications, and increased
mortality. Maintaining serum phosphorus in the
target range is one of the most difficult goals to
achieve for patients with advanced kidney dis-
ease. Phosphorus often remains difficult to control
even with patients who are adherent with phos-
phate binders. There is increasing evidence that
excessive phosphorus intake is harmful even to
the general population without kidney disease.
Recent research suggests that excess dietary phos-
phorus is an arterial toxin [53]. One double-blind
crossover study found that high phosphorus
intake impairs flow-mediated dilation and causes
arteries to stiffen within 2 h of ingestion [54]. A
post hoc analysis report of 4,127 subjects found
that serum phosphorus levels at the high end of the
normal range (>3.5 mg/dL) are associated with
increased risk of heart failure and myocardial
352
infarction [55]. A recent report using the
NHANES III data of over 9,000 adults (ages
20–80 years) found that high phosphorus intake
is associated with increased mortality [56].
Phosphorus is present in food in both organic
and inorganic forms. Organic phosphorus is natu-
rally present in both plant and animal foods and is
bound to protein and other molecules naturally
found in protein-rich foods. Most animal protein
including meat, poultry, fish, and dairy are high in
phosphorus. High-protein plant foods such as
nuts, seeds, legumes, some whole grains, as well
as cacao and chocolate are also high in phospho-
rus. Organic phosphorus is broken down in the gut
and then absorbed into circulation as inorganic
phosphorus. The gastrointestinal tract absorbs
about 40–60 % of the phosphorus naturally pre-
sent in animal foods. Plant-based foods have an
even lower absorption rate of only about 20–50 %
[43]. Phosphorus from plant foods is mostly in the
storage form of phytic acid or phytate. Humans do
not contain the enzyme phytase, which is required
to degrade phytate. Therefore, the bioavailability
of phosphorus from most plant foods is low.
Within the plant kingdom, there are several factors
that alter phosphorus bioavailability. Yeast con-
tains phytase, which breaks down some of the
phytate in whole grains making the phosphorus
in leavened breads more readily absorbed than
flatbread, cereal, or other non-yeast-containing
grains [45]. The process of soaking and sprouting
grains and nuts removes a significant portion of
phytic acid. Sprouting a grain or nut involves
soaking it in water for a period of hours.
This moist environment begins the germination
process and activates enzymes, including
phytase, which is naturally present in some
grains. Wheat and rye are particularly high in
phytase. Thus, the soaking and sprouting process
renders the phosphorus more bioavailable.
Sprouted grain breads and nuts are increasingly
appearing on grocery store shelves and are being
marketed as health foods due to the higher nutri-
ent bioavailability. While these foods have health
benefits for the general population, those with
CKD and ESRD should avoid soaked and
sprouted grains and nuts due to the more efficient
phosphorus absorption.
L. Graf et al.
Food sources of inorganic phosphorus are
mainly additives in processed food. Phosphate
additives are found in a wide variety of products
including processed meats, cheeses, frozen foods,
soups, yogurts, cereals, nondairy milk, and sports
drinks. In contrast to organic phosphorus, the
absorption rate of phosphorus from food additives
is very high at 80–100 % [43, 45]. Phosphate
additives in processed food are the largest obsta-
cles to achieving adequate serum phosphorus con-
trol in patients with advanced kidney disease.
Phosphates are added to processed food to extend
shelf life as well as improve flavor and texture.
Over the past two decades, the amount of phos-
phate preservatives in a variety of food products
has risen dramatically. In the early 1990s, phos-
phate additives contributed about 500 mg per day
to the standard Western diet. Today, phosphate
additives alone contribute as much as 1,000 mg
per day to a typical Western diet [45, 57, 58]. In
the United States, 11 different phosphate salts are
allowed to be injected into meat and poultry to
improve tenderness and flavor and extend shelf
life [59]. This can increase phosphorus levels in
the meat by as much as 70 %. This practice is
currently banned in Europe.
Avoiding phosphate additives is challenging
given that they are ubiquitous in processed and
packaged food. Effectively limiting phosphate
additives is doable for most patients but requires
detailed nutritional education by a dietitian with
instructions to carefully read ingredient lists.
Given the wide variability in phosphorus absorp-
tion from different foods, understanding the
source of phosphorus is equally as important as
the amount present in a given food. Food com-
panies are not required by law to report the quan-
tity of phosphorus on the nutritional facts section
of the label. However, they are mandated to
report the presence of phosphate additives in the
ingredient list. All packaged and processed foods
contain complete ingredient lists in descending
order or predominance. Given that phosphate
additives are the most readily absorbed form of
phosphorus, paying careful attention to the often
overlooked ingredient list is one of the most
important tools to teach patients. Encouraging
patients to minimize processed food and
12 Nutrition Management in Childhood Kidney Disease: An Integrative and Lifecourse Approach
make fresh food and low-potassium produce
dietary staples will result in higher intake of
fiber, phytochemicals, and antioxidants. Limit-
ing processed food will not only restrict the
most efficiently absorbed form of phosphorus
but will simultaneously minimize other harmful
ingredients often found in packaged food such as
sugar, refined flour, and vegetable oil. Decreas-
ing processed food will have a myriad of health
benefits such as decreasing inflammation,
improving lipid profile and blood pressure, as
well as improved serum phosphorus control.
Exposure to a healthy, anti-inflammatory diet
during childhood will set the foundation for
healthy nutrition and lifestyle habits as patients
grow, receive kidney transplants, and transition
into adulthood.
Protein
Adequate protein intake is important to make
enzymes and hormones, build and repair tissue,
and support linear growth. Advanced CKD is
often accompanied by a decrease in both protein
and overall calorie intake, particularly in young
children and infants. Regular nutritional evalua-
tions that include diet recalls as well as measure-
ments of weight, linear growth, and growth
velocity are important determinants of nutritional
status.
Correcting protein deficiency can usually be
achieved easily through nutritional counseling
and in some cases through supplementation. Ade-
quate but not excessive protein intake appears to
be the most beneficial for patients with CKD.
There does not appear to be any benefit from
restricting protein lower than the dietary reference
intake (DRI) for age in terms of slowing the pro-
gression of kidney disease [4]. However, protein
intake significantly beyond recommended levels
increases nitrogenous waste products and serum
phosphorus and can have detrimental effects on
the bone [4, 60]. This is important to note because
it is easy to exceed protein needs especially when
supplements or tube feedings are used as a pri-
mary source of nutrition. The most recent pediat-
ric KDOQI nutritional guidelines from 2008
353
reduced the recommended protein intake for all
ages and stages of CKD based on the most current
literature [4]. Protein recommendations had pre-
viously been much higher for dialysis patients
than age-matched healthy children. This was
based on the knowledge that small amounts of
amino acids are lost through dialysis membranes
as well as the belief that dialysis induces catabo-
lism. While both peritoneal and hemodialysis ses-
sions result in some protein loss, the amount is
relatively small and can be easily regained
through diet or supplementation. Studies of chil-
dren on peritoneal dialysis have found that protein
losses decrease with age. Average protein losses
for infants on peritoneal dialysis are 0.28 g/kg per
day [61]. During adolescence the average protein
losses decrease to less than 0.1 g/kg/day [61]. Pro-
tein losses in hemodialysis have not been studied
extensively in children. Research in adults has
found that an average of 1–3 g of protein is lost
per hemodialysis session [62–65]. Children
would be expected to have slightly higher dialytic
protein losses than adults. The updated KDOQI
guidelines provide formulas for estimating protein
requirements for children based on the stage of
CKD, the type of renal replacement therapy, and
age. The formula takes into account the small
amount of protein lost through different dialysis
modalities.
The belief that dialysis induces catabolism
and that higher protein intake is required to
improve weight gain and linear growth is not
supported by the literature. Several clinical trials
have studied the effects of selectively increasing
protein intake by using amino acid-based dialysis
solutions rather than glucose in children on
peritoneal dialysis. Protein intake beyond
recommended levels did not improve nutritional
status or linear growth but did result in accumu-
lation of nitrogenous waste [66–70]. Too high
protein intakes appear to be harmful to patients
with advanced CKD in several ways. Excess
protein has been found to worsen metabolic aci-
dosis, increases bone loss, and is associated with
hyperphosphatemia. A DEXA study of 20 chil-
dren on peritoneal dialysis with a mean protein
intake that was 144 % of the recommended die-
tary allowance (RDA) was inversely correlated
354 L. Graf et al.

Table 6 Protein recommendations for children with CKD stages 3–5 (Adapted from KDOQI Guidelines)
DRI Recommendations for Recommendations for CKD Recommended Recommended
(g/kg/ CKD stage 3 (g/kg/day) stages 4–5 (g/kg/day) for HD (g/kg/ for PD (g/kg/
Age day) 100–140 % DRI (100–120 % DRI) day) day)
0–6 1.5 1.5–2.1 1.5–1.8 1.6 1.8
months
7–12 1.2 1.2–1.7 1.2–1.5 1.3 1.5
months
1–3 1.05 1.05–1.5 1.05–1.2 1.15 1.3
years
4–13 0.95 0.95–1.35 0.95–1.15 1.05 1.1
years
14–18 0.85 0.85–1.2 0.85–1.05 0.95 1.0
years

with bone mineral density, lean body mass, and
serum bicarbonate level [60]. Studies in adults
have found that diets rich in vegetables and fruit
lower acid levels and reduce kidney injury
(slowing the progression of disease) and help
improve blood pressure without inducing
hyperkalemia in patients with advanced kidney
disease [31, 71, 72] (Table 6).
calories is as important as the quantity. The goal
is to promote as many whole, plant-based foods
as possible and limit excess animal protein,
refined sugar, processed food, and chemical
additives.
The Following Low-Potassium Vegetables
and Fruit Can Be Consumed Regularly
on a Renal Diet

Dietary Guidelines for CKD and ESRD Low-Potassium Vegetables

Asparagus, broccoli, cabbage, carrots, cau-
liflower, celery, cucumbers, green beans, green
peas, eggplant, kale, lettuce, mushrooms,
onions, parsley, peppers, turnips, and water
chestnuts
Low-Potassium Fruit
Apple, applesauce, blackberries, blue-
berries, cherries, cranberries, lemons, limes,
grapes, peaches, pears, pineapple, plum, and
watermelon
Herbs and Spices: Enhance the Flavor of Food
and Are Low in Potassium and High in Anti-
oxidants and Phytochemicals
Allspice, basil, bay leaf, cardamom, chili
powder, cilantro, cinnamon, chives, coriander,
cumin, curry, garlic, ginger, fennel, horserad-
ish, mint, nutmeg, oregano, paprika, parsley,
pepper, peppermint, rosemary, saffron, sage,
thyme, turmeric, vanilla, and wasabi
As a child transitions to solid food, adequate
Healthiest Fats and Oils
calorie intake remains important. However, it is All oils are calorically dense and have
important to recognize that the quality of those 120 cal per tablespoon. Oils have a very low
12 Nutrition Management in Childhood Kidney Disease: An Integrative and Lifecourse Approach
mineral content so they are a good way to boost
calories without adding potassium or phos-
phorus. Healthy oils include olive oil, coconut
oil, and flaxseed oil. Although coconut oil is
high in saturated fat, research suggests it has a
very different effect on the body compared
with saturated fat from animal products [73].
Coconut oil is high in lauric acid, which is a
medium-chain triglyceride (MCT). MCTs are
easily digestible, have a favorable impact on
blood cholesterol, and contain antimicrobial
properties. Coconut oil also contains a variety
of protective phytochemicals including
phenolic acid [73]. It is advisable to limit or
avoid adding vegetable oils such as soybean
oil, corn oil, and sunflower oil as they are
high in omega-6 fats, which are inflammatory
when consumed in excess [74]. Trans fat (par-
tially hydrogenated oils) found in some mar-
garines and shortening should be avoided
because they are linked to inflammation
and CVD.
Guidelines
1. Avoid sugar-sweetened beverages including
natural fruit juice, fruit drinks, iced tea, and
soda regardless of the amount of potassium or
phosphorus. The high sugar content can
increase blood pressure and promote inflam-
mation in the body. Instead, drink water or
water with lemon, lime, or mint slices. Try
seltzer or natural herbal tea. Do not introduce
sugary beverages to young children.
2. Aim to eat low-potassium vegetables and fruit
with every meal. Expose young children to a
variety of vegetables and fruit regularly.
Repeated exposure helps develop and train
the palate.
3. Choose unrefined whole grains such as brown
rice, barley, buckwheat, oats, quinoa, and
100 % whole grain breads. Avoid sprouted
grains as the phosphorus is more readily
absorbed.
4. Be cautious with boxed cereal as many are not
whole grain and contain significant amounts
of added sugar and trans fat as well as
355
phosphates and other preservatives. Read the
ingredient list carefully. To add flavor to a
plain whole grain cereal, add cinnamon,
fruit, a small amount of maple syrup or
honey, chopped nuts, or seeds.
5. Avoid red and processed deli meat or limit to
special occasions. Limit animal protein to two
servings per day. Choose fresh meat such as
turkey, chicken, beef, fish, or eggs. If con-
suming meat, choose grass fed, organic
when possible.
6. Beans and legumes can be consumed once a
day. Serving size should be limited to about
½ cup due to the high potassium content
of most beans. Chickpeas and black beans
contain the lowest amount of potassium.
Avoid adzuki beans as they are very high in
potassium.
7. Avoid or limit cow’s milk and cheese to one
serving daily. Try a plant-based milk such as
almond milk or rice milk. Choose a brand that
does not contain phosphate additives. Hemp
milk and soy milk should be limited as they
are high in phosphorus.
8. Carefully read ingredient lists on all packaged
food including nondairy milk, meat, cereal,
bread, crackers, etc. Avoid products with
phosphate additives, partially hydrogenated
oils (trans fat), and high-fructose corn syrup.
9. Choose real food snacks when possible such
as fruit, nuts or nut butter, granola bar, vege-
tables, or whole grain cracker with hummus.
10. To add additional calories, try mixing extra
olive oil into pasta, rice, or other whole grain
or vegetable dishes. Coconut oil is a good
addition to smoothies or breakfasts such as
oats or cream of wheat cereal. Whole food
sources of fat, calories, and protein such as
peanut butter or almond butter can also be a
good way to boost calorie intake, but they do
come with some extra potassium and phos-
phorus as well.
Phosphate Preservatives
Phosphate additives sneak into food products and
are listed on the ingredient list in many forms. The
356 L. Graf et al.

examples below are not a complete list of all the

phosphate additives but give an idea of how to
identify them on a package. Food companies are
currently not required to report the quantity of
phosphorus present in a food on the nutritional
facts panel. However, they are required to report
any phosphate additives in the product, and this
will appear in the ingredient list. Given that phos-
phate preservatives are the most readily absorbed
form of phosphorus, it is important for patients to
be taught to read the ingredient list (Fig. 3,
Table 7).

Common Phosphate Additives Used By

the Food Industry

Phosphoric acid
Pyrophosphates
Polyphosphates
Hexametaphosphate
Dicalcium phosphate
Disodium phosphate
Sodium
Polyphosphate
Phosphate

Hypoallergenic Diet as a Treatment

for Idiopathic Nephrotic Syndrome

Over the past three decades, several clinical stud-

ies and case reports have documented the success-
ful treatment of nephrotic syndrome with a
hypoallergenic diet. Cow’s milk in particular and
more recently gluten have been implicated in
some cases of idiopathic nephrotic syndrome.
However, the use of elimination diets in the man- Fig. 3 Sample food label: low-sodium turkey breast
agement of nephrotic syndrome has received little from Deli
attention from clinicians [75].
To date, there have been five published reports
that document the successful treatment of children ages 10–13 years old with frequently
nephrotic syndrome with a hypoallergenic diet relapsing, steroid-responsive nephrotic syndrome.
[76–80]. The first study to investigate the link During the study period, prednisone was
between nephrotic syndrome and cow’s milk sen- discontinued and the subjects were placed on a
sitivity was conducted by Sandberg et al. in 1977 liquid elemental diet. Remission occurred quickly
[76]. Sensitivity to cow’s milk was studied in six within 10 days in three of the six patients. Protein
12 Nutrition Management in Childhood Kidney Disease: An Integrative and Lifecourse Approach
Table 7 Variation in phosphorus absorption from food
and preservatives
Gastrointestinal
Source of phosphorus absorption %
Animal protein (organic) 40–60
Cheese, milk, yogurt, beef, turkey,
chicken, fish, egg
Vegetarian protein 10–30
Bread, nuts, beans, vegetables,
chocolate
Food additives and 80–100
preservatives (inorganic)
Cola, packaged food, processed
meat
excretion decreased to less than 500 mg/24 h.
Significant proteinuria and edema returned when
cow’s milk was reintroduced. Four of the six
patients were able to maintain remission on a
milk protein-free diet, while the other two subjects
experienced relapses. When 20 mg of prednisone
was added and the diet was restricted further to
exclude grains, relapses were controlled. This
suggests a possible sensitivity to wheat or gluten
in these two subjects.
In 1989, Laurent et al. investigated the connec-
tion between idiopathic nephrotic syndrome and a
wide range of food allergies in 26 patients ages
7–72 years old [77]. The foods investigated were
cow’s milk, egg, chicken, beef, pork, and gluten.
Subjects were given skin testing with various food
allergens. Patients were instructed to follow spe-
cific diet restrictions based on these results. Six of
the 26 patients successfully responded to diet
treatment with resolution of nephrotic syndrome.
Two patients were successfully treated with the
elimination of gluten and one patient with the
exclusion of diary. Three patients required the
elimination of multiple foods to achieve
remission.
In the early 1990s, Sieniawska et al. evaluated
the effects of a dairy-free diet in 17 patients ages
1–15 years with steroid-resistant nephrotic syn-
drome [78]. Six of the 17 subjects responded to
the milk protein-free diet with remission of
nephrotic syndrome. In all six subjects, protein-
uria resolved quickly – within 3–8 days of starting
357
the diet. Full remission was achieved within
2–3 weeks. More recently, Rasoulpour described
four patients with steroid-dependent nephrotic
syndrome ages 4–10 years who responded to a
milk protein-free diet [80].
The resolution of nephrotic syndrome in
response to diet therapy supports the concept
that food sensitivities may trigger the disease in
some cases. Despite the promising results of these
studies, very little attention has been given to diet
in the treatment of nephrotic syndrome over the
past 20 years. There has been a renewed interest
within the nephrology community about the asso-
ciation between food allergy and sensitivity, par-
ticularly milk protein and gluten with the
development of nephrotic syndrome [75]. This
coincides with the large increase in awareness
and prevalence of food allergies. The prevalence
of food allegories has increased 18 % from
1997 to 2007 (http://www.cdc.gov/nchs/data/
databriefs/db10.pdf). In addition, children with
food allergies are two to four times more likely
to have other immune-mediated related health
problems such as asthma and eczema. Children
now require more time to outgrow the allergies
[81, 82]. There is increased attention from the
research community in nonceliac gluten sensitiv-
ity (NCGS), and the effects of NCGS on the
immune system are still elucidated [83]. The
effect of diet on the immune system is complex
and may be mediated in part by the amount and
type of bacteria in the gut. The influence of diet on
the gut microbiome may lead to the development
of allergies or immune system dysfunction.
More studies are needed to address the poten-
tial of using hypoallergenic diets as treatment for
idiopathic nephrotic syndrome. Clinical trials are
currently underway investigating the effects of
gluten on nephrotic syndrome. While the
published literature at this time is limited, diet
therapy is a treatment that confers no risk to the
patient and may decrease or eliminate their expo-
sure to potentially harmful and toxic medications.
Therefore, nutritional intervention with a hypoal-
lergenic is a therapy that should be offered to
patients with steroid-resistant and frequently
358 L. Graf et al.

Table 8 Diet modification for nephrotic syndrome

Age Responsible foods (n = patients Length of time between initiating
Studies range with sensitivity in various studies) Diet therapy diet therapy and remission of NS
Sandberg >3 Cow’s milk (10) Specific Remission occurred within 3 days
et al. [76] years avoidance to 1 month
of age
Laurent Possible gluten/wheat (4)
et al. [77]
Rasoulpour Egg, chicken, and cow’s milk (1)
et al. [80] Gluten, pork, and cow’s milk (1)
Beef, pork, and egg (1)
Sieniawska <3 Cow’s milk (7) Cow’s milk Remission occurred within 3 days
et al. [78] years protein-free to 1 month of starting the diet
Salazar de of age diet
Sousa
et al. [79]

relapsing forms of idiopathic nephrotic syndrome
(Table 8).
Conclusion
This chapter has reviewed the most recent
advances in nutrition and CKD over the past two
decades. There is an emphasis on maintaining
appropriate growth and electrolyte homeostasis
during infancy and childhood, as well as
preventing early onset cardiovascular disease.
The goal of the chapter is to expand the awareness
of the importance of nutrition in improving CKD
outcomes throughout the life course journey. The
topics covered in the chapter are in accordance
with current NIH research goals [84]. When pos-
sible, the focus should be not only on slowing the
progression of CKD but also on regression of
disease to the greatest extent possible.
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 Physiology of the Developing Kidney:
Fluid and Electrolyte Homeostasis and 13
Therapy of Basic Disorders (Na/H2O/K/
Acid Base)

Isa F. Ashoor and Michael J. G. Somers

Contents Oral Rehydration Schemes . . . . . . . . . . . . . . . . . . . . . . . . . . 381

Use and Acceptance of Oral Rehydration
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
Total Body Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 Choice and Volume of Parenteral Fluid . . . . . . . . . . . . . 383
Extracellular and Intracellular Fluid Colloid Solutions and Volume Resuscitation . . . . . . . . 384
Compartments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 Rapid Rehydration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
Effective Circulating Volume . . . . . . . . . . . . . . . . . . . . . . . . 365 Symptomatic Hyponatremia . . . . . . . . . . . . . . . . . . . . . . . . . 386
Asymptomatic Hyponatremia . . . . . . . . . . . . . . . . . . . . . . . 387
Water and Electrolyte Requirements . . . . . . . . . . . . . 366 Severe Hypernatremia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
Maintenance Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
Serum Osmolality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368 Fluid and Electrolyte Therapy with Renal
Water Homeostasis in Acutely Ill Children Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
or During the Perioperative Period . . . . . . . . . . . . . . . . . . 368 Impact of Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
Derangements in Urinary Concentrating Ability Fluid and Electrolyte Therapy in the Pediatric
Affecting Water Homeostasis . . . . . . . . . . . . . . . . . . . . . . . 370 Intensive Care Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
Nephrogenic Diabetes Insipidus . . . . . . . . . . . . . . . . . . . . . 370 Abnormalities in Serum Sodium Complicated
Special Considerations in Infants and Children by Kidney Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391
with Chronic Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . 371 Potassium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394
Maintenance Electrolyte Therapy . . . . . . . . . . . . . . . . . . . 371
Hyperkalemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394
Sodium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372 Causes of Hyperkalemia in Children . . . . . . . . . . . . . . . . 394
Alterations in Water Balance, Serum Sodium, Evaluation of Hyperkalemia in Children . . . . . . . . . . . . 398
and Cell Volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372 Treatment of Hyperkalemia in Children . . . . . . . . . . . . . 399
Hyponatremia: Initial Approach . . . . . . . . . . . . . . . . . . . . . 373
Hypernatremia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374 Hypokalemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
Causes of Hypokalemia in Children . . . . . . . . . . . . . . . . . 401
Fluid Replacement Therapy . . . . . . . . . . . . . . . . . . . . . . . 376 Diagnostic Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405
Assessment of Volume Depletion . . . . . . . . . . . . . . . . . . . 376 Treatment of Hypokalemia in Children . . . . . . . . . . . . . 407
Oral Rehydration with Fluids Other Than ORS . . . . . 380
Oral Rehydration and Serum Sodium
Abnormalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381

I.F. Ashoor
Division of Nephrology, Children’s Hospital, New
Orleans, LA, USA
e-mail: iashoor@chnola.org
M.J.G. Somers (*)
Division of Nephrology, Boston Children’s Hospital,
Harvard Medical School, Boston, MA, USA
e-mail: michael.somers@childrens.harvard.edu

# Springer-Verlag Berlin Heidelberg 2016 361

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_12
362 I.F. Ashoor and M.J.G. Somers

Acid–Base Homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407

Introduction
Perturbations in Acid–Base Balance . . . . . . . . . . . . . . . . 408
Simple Acid–Base Disorders . . . . . . . . . . . . . . . . . . . . . . . . 408 The spectrum of clinical conditions requiring the
Respiratory Acid–Base Disorders . . . . . . . . . . . . . . . . . . . 409 prescription of fluid and electrolyte therapy by
Metabolic Acidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
pediatric clinicians is vast, ranging from
Respiratory Compensation for Metabolic
Acidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410 rehydrating otherwise healthy children with
Serum Anion Gap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410 acute gastrointestinal illness to correcting life-
Urine Anion Gap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411 threatening abnormalities in children with com-
Urine Osmolal Gap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
plex chronic disease. Recognition of each child’s
Metabolic Alkalosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
Mixed Acid–Base Disorders . . . . . . . . . . . . . . . . . . . . . . . . . 412 individual clinical situation, notably any changes
The Δ Anion Gap/Δ HCO3 Ratio . . . . . . . . . . . . . . . . . . . 413 in normal physiology and homeostatic mecha-
Clinical Application and Approach . . . . . . . . . . . . . . . . . . 413 nisms that accompany the acute or chronic illness,
General Considerations for Therapy of
and each situation’s ultimate goal with respect to
Acid–Base Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414
volume resuscitation or electrolyte correction is
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415 crucial for the provision of the correct combina-
tion of fluid and electrolytes in the proper amount
of time.
Historically, an understanding of the morbidity
and mortality that accompanies significant pertur-
bations in fluid and electrolyte balance dates to
clinical observations made in epidemics of chol-
era and other diarrheal illness in the eighteenth
century [1]. The recognition that moribund
patients could be saved with provision of salt
solutions spurred interest in defining the fluid
and electrolyte needs in healthy individuals and
in developing clinical parameters for fluid and
electrolyte therapy. Over time, this work helped
to define the threshold for the minimum daily
provision of fluid and electrolytes – so-called
maintenance requirements – as well as a threshold
of maximal tolerance.
In the early twentieth century, clinicians began
to try to restore circulation in children with vol-
ume compromise by intraperitoneal injection of
saline or intravenous infusion of isotonic solu-
tions [2, 3]. Fluid spaces were defined in terms
of intracellular and extracellular compartments,
and the kidney’s role in the regulation of overall
body volume and specific solute gradients
between these fluid spaces became better appreci-
ated [4, 5].
By the middle of the twentieth century, simple
equations to link average daily metabolic rate to
daily fluid requirements were devised, and the
practice of calculating daily fluid and electrolyte
needs for an ill child based on consideration of
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
both “maintenance” needs and past and current
losses or “deficits” was taught as the best
approach to minimize complications and improve
outcomes [6–8]. Despite caveats by the founders
of this approach to individualize therapy and
be cognizant of clinical situations where they
may not pertain, emphasis on these empiric
equations led to formulaic hydration protocols
that became problematic in the setting of reduced
urine output or non-osmotically stimulated ADH
release.
Reassessment of this approach in the late
twentieth and early twenty-first centuries has lead
to more widespread understanding that such
elaborate maneuvers are often unnecessary and
that there needs to be ongoing focus on the
child’s response to initial fluid and electrolyte ther-
apy with consideration for adjustments to optimize
outcome [9]. More widespread recognition that oral
rehydration solutions are simple, safe, and effica-
cious alternatives for most children has also
impacted this tradition grounded on arbitrarily
calculated intravenous fluid volumes [10].
In more complex disorders of fluid and
electrolyte pathophysiology, such as seen in
critically ill children with sepsis, burns, trauma,
or postoperatively or in children and adolescents
with conditions such as chronic pulmonary
disease or complex congenital cardiac disease,
an understanding of the distribution of body
fluids, usual fluid and electrolyte requirements,
and the effect of disturbances in normal homeo-
static balance remains vital to the correct prescrip-
tion of therapy and the proper assessment of
response. To this extent, there continues to be a
place for an understanding of these precise calcu-
lations and the ability to apply them when appro-
priate [11]. Similarly, such an understanding
becomes paramount when approaching the care
of a child with renal disease in whom normal
homeostatic mechanisms may not be able to
come into play and with whom a standard
approach to fluid and electrolyte therapy may not
be appropriate. In these circumstances, the clini-
cian needs to combine a systematic approach with
attention to individual clinical circumstance,
thereby reducing the chances that any intervention
363
exacerbates preexisting alterations in body fluid
volume, composition, or distribution or creates a
new problem.
The sections that follow provide information
on fluid and electrolyte homeostasis in
children, most notably the regulation of water,
sodium, potassium, and acid–base balance.
There is additional focus on therapeutic
approaches to correct basic imbalances as well
as discussions on how chronic perturbations
in their homeostasis may affect usual approaches
to therapy.
Total Body Water
Extracellular and Intracellular Fluid
Compartments
Water makes up a large component of body
weight, the exact proportion varying in a child
by age, body size, and body composition.
In early gestation, up to 90 % of the developing
fetus may be water. By the early third trimester,
total body water (TBW) of the developing fetus
approximates 80 % of body weight in kilograms.
After birth, this percentage falls further, from
70–75 % in term infants to 65–70 % in toddlers
and young children and, eventually, to 60 % in
older children and adolescents. Body habitus also
affects body water composition, with lean chil-
dren having higher TBW than obese and more
muscular children and adolescents manifesting
higher TBW than their less well-conditioned
peers. Although 60 % is the usual benchmark for
estimating TBW in older children and adults,
there may be the need to adjust the estimated
percentage based on specific individual somatic
characteristics [12].
Body water is distributed across intracellular
and extracellular compartments (Fig. 1). The
intracellular compartment consists of all the
water within the cells of the entire body, compris-
ing about two-thirds of TBW or 40 % of body
weight. The extracellular compartment is smaller,
comprising one-third of TBW or 20 % of body
weight. The extracellular compartment consists of
364
Fig. 1 Total body water:
fluid compartments and
solute composition
Intracellular
Fluid
the interstitial fluid that bathes all cells as well as
the intravascular plasma water. The increased
TBW as a proportion of body weight that is seen
in young children is the result of a relatively
increased surface area as compared to body
weight, resulting in an overall increased extracel-
lular compartment [13].
The cell membrane separates the intracellular
and extracellular compartments. Input or output
from the body proceeds via some interface with
the extracellular compartment, so there needs to
be net transport of fluid or solute across the cell
membrane for changes in the composition of the
intracellular compartment. For instance, intrave-
nous electrolyte solutions are infused into the
extracellular intravascular space, and the subse-
quent delivery of the fluid and electrolyte in the
solution into the intracellular compartment
depends on a host of factors that influence trans-
port across the cell membrane.
Most cell membranes are readily permeable
to water, and the distribution of water
between the intracellular and extracellular spaces
comes to reflect osmotic forces. Each body
space contains a solute that is primarily seques-
tered within that compartment and that maintains
its osmotic gradient [14, 15]. For instance,
sodium–potassium adenosine triphosphatase
activity (sodium–potassium pump) found in
all human cell membranes leads to an increased
intracellular concentration of potassium and
I.F. Ashoor and M.J.G. Somers
Plasma
Water
Extracellular
Fluid
Interstitial
Fluid
an increased sodium concentration in the
interstitium. Thus, potassium serves as the effec-
tive osmol intracellularly and sodium intersti-
tially. Similarly, plasma proteins such as albumin
exert osmotic force to maintain water intravascu-
larly. Hydrostatic perfusion pressure counterbal-
ances this osmotic force by pushing water across
the capillary from the lumen to the interstitium.
Changes in the distribution of the effective
osmoles or in hydrostatic perfusion can result in
redistribution of water between intracellular and
extracellular spaces.
The equilibrium between the intracellular and
extracellular spaces is dynamic. Diffusional
gradients, osmotic forces, and cellular transporter
activities all combine to establish and maintain
differences in body compartment composition.
Because the intracellular space cannot be directly
accessed, its composition can be altered only
by affecting the extracellular space and its subse-
quent communication with the cell. Any intake by
ingestion or infusion requires a new equilibrium
to be established as solute and fluid comes to be
exchanged from the extracellular space into
the cells. The final equilibrium is a result of all
of these complex biochemical, electrical, and
physical interactions.
Communication between the cellular spaces can
be bidirectional so there can also be exchange from
the intracellular space to the extracellular space,
allowing for transfer or release of cell metabolites.
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
Since the extracellular space can communicate with
the external milieu, output from the extracellular
space to the external milieu results in effective
excretion from the body. Because no direct com-
munication exists between the intracellular space
and the external milieu, any output from the cells
themselves is mediated via the cell’s direct ability to
interface with the extracellular compartment by
way of interstitial fluid or plasma water.
Impairment in the patient’s normal fluid and
electrolyte homeostatic mechanisms will strikingly
impact the patient’s TBW and its extracellular and
intracellular constituents. The development of
hypertension in chronic kidney disease exemplifies
this disruption of normal balance. As GFR falls, the
kidney’s ability to excrete free water (CH2O) also
declines, generally in the setting of a decreasing
number of functioning nephrons with concomitant
impairment of overall tubular solute excretion, most
notably salt. Superimposed on this dysregulation of
salt and water balance may be other clinical factors
such as decreased effective arterial volume from
circulatory failure, leading to further renal salt and
water retention. This salt and water overload leads
to chronic TBW expansion and mediates systemic
hypertension with expansion of the extracellular
volume compartment. Appropriate therapy would
include sodium and fluid restriction and use of
diuretics to reduce the total body burden of salt
and water and to restore the total body water to a
more physiologic state. Failure to appreciate the
preexisting expansion of the total body water
because of chronic salt and water overload could
prove deleterious if management did not include
some measure to address this perturbation. This
example also highlights that fluid and electrolyte
therapy in children may involve the removal of
solute and water, in contrast to the more common
clinical scenario that focuses on correction of elec-
trolyte and volume deficits.
Effective Circulating Volume
Effective circulating volume is a more abstract
concept than the division of body water into
intracellular and extracellular fluid compartments.
As the vascular volume circulates, oxygen and
365
nutrients get delivered to the intracellular space
and cellular metabolites get cleared from the
intracellular space. Effective circulating volume
refers to that portion of the extracellular vascular
space that actually perfuses the tissues and accom-
plishes such an exchange.
The body attempts to maintain effective perfu-
sion through the intravascular space since any
compromise proves deleterious to cell homeosta-
sis. Feedback mechanisms include baroreceptors
that respond to the stretch of specialized areas of
the carotid arteries and the atrium. Hypoperfusion
decreases stimulation of these stretch receptors,
triggering the secretion of vasopressin to increase
distal nephron water reabsorption with subsequent
expansion of the vascular volume. Similarly, in
response to glomerular hypoperfusion, there is not
only decreased afferent arteriolar stretch but also
decreased glomerular filtration and presentation
of filtered sodium to the macula densa. These
stimuli lead to the secretion of renin from the
juxtaglomerular cells of the afferent arteriole.
Renin release initiates a cascade ultimately increas-
ing aldosterone-mediated sodium and water
reabsorption from the kidney as well as increasing
angiotensin-mediated vasoconstriction and sodium
and water uptake.
Effective circulating volume is impacted by
multiple factors, not the least of which include
the size of the vascular space and the influence
of various regulatory hormones. As a component
of the extracellular body water, the size of the
vascular space often parallels the size of the extra-
cellular space. The size of the vascular space and
the adequacy of the effective circulating volume
do not, however, always vary coordinately. The
extracellular space may be replete or expanded
and the actual effective circulating volume
decreased. For instance, children with significant
liver disease are often edematous, due to sodium
retention and expansion of the interstitial compo-
nent of the extracellular space. The intravascular
component of their extracellular space may also
be expanded as a result of factors resulting in avid
salt and water reabsorption by the kidney. But,
because of portal hypertension, splanchnic vessel
congestion, and multiple arteriovenous spider
angiomas that are seen with this condition, much
366
of the expanded intravascular volume is ineffec-
tive – it does not serve to perfuse the tissues and
accomplish effective cellular exchange. Thus,
these children act as if they are volume depleted:
they avidly reabsorb any filtered sodium and
excrete small volumes of urine, and they
vigorously continue to expand their already
overexpanded extracellular space by reabsorbing
even more salt and water in response to the effects
of renin and ADH. Similarly, this paradoxical
state of sodium avidity and ADH-mediated water
reabsorption characterizes children with nephrotic
syndrome or with cardiac failure despite their
preexisting expansion of the extracellular space.
In prescribing fluid and electrolyte therapy, the
clinician needs to assess the patient’s current
extracellular volume status along with the effec-
tive circulating volume and then reconcile these
with potential causes of volume loss. At all times,
the first maneuver should be to maintain an effec-
tive circulating volume and, to accomplish this
properly, therapeutic decisions should be based
on the patient’s unique clinical circumstances at
that point in time. Consequently, effective man-
agement may require rather disparate therapeutic
interventions. For instance, expansion of the
extracellular volume with vigorous rehydration
may be called for in a child with poor perfusion
secondary to dehydration from diarrhea, whereas
another child with equally poor perfusion due to
cardiodynamic compromise may be intravascu-
larly replete and require the initiation of pressor
therapy, and yet another child with edema from
relapsed nephrotic syndrome may actually
require fluid and salt restriction. These examples
underscore that loss of effective circulating
volume generally arises as a result of one or
more broad perturbations in the extracellular
fluid compartment that impacts effective
perfusion (Table 1). In hospitalized children
where there may be both aberrant disease-related
physiology as well as iatrogenic derangements
of regulatory response, the causes of effective
volume perturbations may be even more complex.
The clinical signs and symptoms of effective
circulating volume loss may be subtle. There may
even be preservation of effective circulating vol-
ume in the face of an overall depleted extracellular
I.F. Ashoor and M.J.G. Somers
Table 1 Alterations in effective circulating volume
Cause Mechanism
Contracted Water or sodium chloride
extracellular fluid deficit
space
Massive Loss of vascular tone
vasodilatation sustaining perfusion pressure
Loss of intravascular Osmotic fluid losses into the
osmotic pressure interstitium
Overfill of the Hydrostatic fluid losses into
intravascular space the interstitium
Hemorrhage Direct loss of blood and
plasma water
fluid compartment. In such a circumstance, failure
to initiate appropriate fluid and electrolyte therapy
may result in eventual compromise of the effec-
tive circulating volume.
Important initial clinical signs to assess in any
patient being evaluated for fluid therapy include
pulse rate and capillary refill. Tachycardia and
sluggish refill generally precede more obvious
signs of ineffective circulation such as hypoten-
sion and oliguria. Clinical symptoms may also be
nonspecific and include fatigue and lethargy that
are often attributed to an underlying illness rather
than volume depletion. Proper restoration of
effective circulating volume or extracellular fluid
compartment depletion requires both an under-
standing of baseline fluid and electrolyte needs
as well as consideration of extenuating clinical
circumstances unique to the patient.
Water and Electrolyte Requirements
Maintenance Therapy
Maintenance therapy denotes the amount of water
and electrolytes required to replace usual daily
losses and to maintain an overall balance of
water and electrolytes with no net gain or loss;
as noted below, “maintenance” fluid therapy
should not be conflated with simple estimation
formulas taught in medical school and residency.
Maintenance needs are a function of homeostatic
and environmental factors and vary from day to
day and from child to child. In the average child
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . . 367

with adequate access to water and food, these Table 2 Relationship of body weight to metabolic and
maintenance needs are generally readily met maintenance fluid needs
[6, 16]. In the ill or hospitalized child who requires Maintenance
therapeutic intervention and in whom there may Body weight Metabolic needsa fluid needsb
be ongoing aberrant physiology, these needs must (kg) (kcal/day) ml/day ml/h
be considered more carefully. 5 500 500 20
10 1,000 1,000 40
Maintenance fluid and electrolyte requirements
15 1,250 1,250 50
are typically calculated based on weight or surface
20 1,500 1,500 60
area, but individual clinical circumstance must be
30 1,700 1,700 70
considered when making such calculations
40 1,900 1,900 80
[17]. For instance, the 20-kg child who is well 50 2,100 2,100 90
will require a far different “maintenance” quantity 60 2,300 2,300 95
of fluid and electrolytes than the 20 kg child who is 70 2,500 2,500 105
tachypneic and febrile or the 20 kg child who is As described in Holliday and Segar [6]
anuric and on a ventilator in the pediatric intensive a
Based on 100 kcal/kg for the first 10 kg of body weight +
care unit. Careful assessment of the patient’s vol- 50 cal/kg for the next 10 kg of body weight + 20 cal/kg
ume status and attention to the balance of overall for the next 50 kg of body weight
b
Based on need of 1 ml of water to metabolize 1 kcal of
daily input and output will prove more useful at energy
arriving at a correct estimate of daily fluid and
electrolyte needs than using mathematical equa-
tions without clinical correlation. Despite these
caveats, it is widespread clinical practice to make
Table 3 Factors affecting insensible water losses
certain empirical assumptions regarding daily
needs for water and the major electrolytes. Decreased
Increased Losses % change losses % change
Historically, daily maintenance water needs
Prematurity 100–300 Enclosed 25–50
were first derived based on measurements in hos- incubator
pitalized patients suggesting that water needs Radiant warmer 50–100 Humidified 15–30
could be linked to energy expenditure (Table 2) air
[6, 16]: for the first 10-kg of body weight, 100 ml Phototherapy 25–50 Sedation 5–25
of water per kg is provided daily; for the next Hyperventilation 20–30 Decreased 5–25
10 kg of body weight, 50 ml of water per kg is activity
provided daily; and for every kg of body weight in Increased 5–25 Hypothermia 5–15
activity
excess of 20 kg, 20 ml of water per kg is provided
Hyperthermia 12 %/ C
daily. In addition, in the process of oxidation of
carbohydrate and fat, approximately 15 ml of
water is generated for every 100 kcal of energy Clinical factors can have a striking impact on
produced. This water of oxidation contributes sig- insensible water losses (Table 3). Fever increases
nificantly to overall water balance. insensible losses by >10 % for each degree Cel-
Maintenance water losses occur from urine sius. Premature infants with relatively increased
output and from insensible sources that are almost surface areas for size can have insensible losses
exclusively evaporative and respiratory losses. In two- to threefold higher than baseline, especially
the child with average metabolic demands, for if they are on open warmers or under photother-
every 100 kcal of energy expended, 100 ml of apy. On the other hand, children on ventilators
water must be ingested. Of this 100 ml of water, providing humidified oxygen may have half the
40 ml is lost insensibly and 75 ml is lost as urine insensible losses of a non-ventilated child.
output. Since oxidative metabolism generates Similarly, urinary water output can vary tre-
15 ml of water in the course of producing the mendously. A child with a renal concentrating
100 kcal of energy, net water balance is zero. defect or ADH unresponsiveness may have
368
obligate urinary water losses of several liters per
day, whereas an oligoanuric child will have no
appreciable urinary water losses regardless of
input. In any child with normal renal function,
even in the setting of maximal ADH stimulation,
there is a minimal volume of urinary water neces-
sary to excrete the osmotic load arising from diet
solute and basal metabolism. As a result, even the
child concentrating urine to 1,200–1,400 mOsm/L
will lose nearly 25 ml of urinary water per
100 kcal of energy expended [6].
Recently, there has been recognition that the
relationships between energy expenditure and
water requirements demonstrated by stable hospi-
talized children at bed rest do not apply to anesthe-
tized or critically ill children [18]. For instance, in
infants and children studied during general anes-
thesia, energy expenditure was half that of awake
inactive children. On the other hand, water needs
for cell metabolism were increased over baseline by
about 60%, leaving the overall relationship
between water needs and caloric expenditure sim-
ilar in both situations. In critically ill children,
however, the overall relationship may be affected
and volumes may need to be reduced by 40–50% to
prevent positive water balance [19].
Serum Osmolality
Serum osmolality is determined by concentrations
of sodium, glucose, and urea nitrogen in the serum
and maintained by water homeostasis [20]. Osmo-
lality is estimated by the equation: (2  serum
Na) + (serum glucose/18) + (BUN/2.8), where
the serum sodium is measured in mEq/L and the
glucose and BUN in mg/dl. In the majority of
children with no functional renal impairment and
normal glucose metabolism, the contributions of
BUN and glucose to the effective osmolality are
negligible, and the serum osmolality can be
estimated by doubling the serum sodium concen-
tration [21, 22]. Most children have a serum
osmolality between 270 and 290 mOsm/kg,
corresponding to normal serum sodium values
between 135 and 145 mEq/L.
Chemoreceptors in the hypothalamus constantly
monitor serum osmolality, responding to even
I.F. Ashoor and M.J.G. Somers
small variations outside of the normal range by
adjusting posterior pituitary ADH release. With
hypovolemia, changes in osmolality augment
ADH release further. ADH effect on water perme-
ability of the collecting tubule is a principal influ-
ence on the regulation of water balance.
Sustained alterations in water intake or excre-
tion result in hypo- or hyperosmolality developing
as the usual ratio of extracellular solute to water is
perturbed. Since sodium is the largest component
of extracellular osmolality, its concentration can
be influenced profoundly by changes in water
metabolism. An understanding of this link
between water regulation and serum sodium
values is crucial when prescribing fluid and elec-
trolyte therapy. Most importantly, the clinician
must recognize that hypo- or hypernatremia is
usually a manifestation of impaired water regula-
tion and that therapy must address regulation of
water balance rather than alterations in body
sodium stores.
Water Homeostasis in Acutely Ill
Children or During the Perioperative
Period
In ill children, there are multiple causes of both
physiologic and aberrant vasopressin effect as listed
in Table 4 [23]. As a result, if these children
receive hypotonic intravenous fluids for prolonged
periods of time or in volumes exceeding those
generally recommended, there is the risk of acute
hyponatremia. After volume resuscitation with
isotonic fluids, most hospitalized children were
Table 4 Common causes of vasopressin effect in hospi-
talized children
Category Specific etiology
Physiologic Hyperosmolar state, hypovolemia
Pulmonary Pneumonitis, pneumothorax, asthma,
bronchiolitis, cystic fibrosis
Drug effect Narcotics, barbiturates, carbamazepine,
vincristine, cyclophosphamide
Metabolic Hypothyroidism, hypoadrenalism,
porphyria
CNS Infection (meningitis or encephalitis),
tumor, trauma, hypoxia, shunt
malfunction, nausea, pain, anxiety
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
traditionally provided hypotonic fluids for their
maintenance therapy. Given the tendency for many
of these children to manifest vasopressin effect inde-
pendent of the usual osmotic and volume-related
stimuli, isotonic fluids may be a safer alternative as
the source of maintenance fluid even after acute
volume repletion in this setting [24].
Similarly, some have called for using isotonic
fluids whenever a maintenance infusion is needed
in the perioperative period when oral hydration
has been held and when high ADH levels may be
expected because of pain or anxiety. In these
instances, children could receive intravascular
volume expansion with an initial infusion of
20–40 ml/kg of isotonic fluid over a period of a
few hours and then, rather than a transition to
hypotonic fluid, continued on isotonic fluid as
dictated by clinical circumstance. Proponents
believe it both decreases the number of hospital-
ized children who develop hyponatremia and
prevents hyponatremia-related central nervous
system damage [25].
Others have claimed that maintenance infusion
of isotonic saline to all ill or perioperative
children results in sodium loading in those with-
out any trigger for water retention [8, 26]. In fact,
hypernatremia has been described in some
children receiving even less sodium than that
provided in maintenance isotonic infusions
[27]. Such cases of hypernatremia are often a
result of an underlying renal concentrating defect,
significant free water loss from sources other than
urine, or aggressive fluid restriction [28]. Children
with certain renal or cardiopulmonary problems
may be especially sensitive to such sodium loads
and may more readily develop unintended
sequelae of such sodium provision [29].
Several studies have shown that children with
acute illness requiring emergency department
evaluation or hospitalization do seem to be at
risk for hyponatremia. In one report of 103
children, nearly 80 % had elevated serum ADH
levels and increased plasma renin activity, inde-
pendent of the underlying illness. As expected
with ADH release, plasma osmolality was
reduced significantly [23]. In another report,
fewer than 5 % of children were hyponatremic
at presentation, but nearly 10 % became
369
hyponatremic after hospital admission, most as a
consequence of aggressive intravenous hydration
with hypotonic fluid. These hyponatremic chil-
dren received on average three times more the
volume of electrolyte free water than their
eunatremic peers and were also three times more
likely to receive fluids at rates exceeding
recommended maintenance rates [30].
A retrospective analysis of postoperative
admissions to a pediatric ICU found that 11 % of
children became hyponatremic (serum sodium
<130 mEq/L) [31]. In the hyponatremic children,
there was a trend towards an increased likelihood
of hypotonic fluid provision, although there was
no statistical difference demonstrated in the
incidence of either moderate (<130 mEq/L) or
severe (<125 mEq/L) hyponatremia. None of
these children developed any neurologic sequelae
or other morbidity, but serum electrolytes were
assayed four times daily likely resulting in clinical
interventions to prevent hyponatremia from
exacerbating.
A prospective randomized study of 102 chil-
dren with gastroenteritis demonstrated that the
risk of developing hyponatremia decreased with
provision of isotonic saline [32]. Eunatremic chil-
dren at presentation were most protected from
subsequent falls in serum sodium when provided
isotonic (0.9 % NaCl) versus hypotonic (0.45 %
NaCl) solutions. Urinary biochemistries demon-
strated that hyponatremic children were more
likely to retain sodium and increase serum sodium
levels with isotonic fluids, whereas children with
normal serum sodiums at presentation were
able to excrete excess sodium appropriately and
prevent hypernatremia.
A meta-analysis of six studies comparing
hypotonic and isotonic maintenance intravenous
solutions in children suggested that no single fluid
composition or rate is ideal for all children but that
an isotonic or nearly isotonic solution may prove
less likely to cause symptomatic hyponatremia in
acute illness and the perioperative period [33].
Nonetheless, in many clinical situations where
non-osmotic ADH effect can be expected,
children are still frequently receiving hypotonic
fluid therapy, and such iatrogenic hyponatremia
may not be unusual [34].
370
Derangements in Urinary
Concentrating Ability Affecting Water
Homeostasis
The maximum ability of the kidneys to concen-
trate urine determines the obligatory urine volume
required to excrete the daily solute load. On aver-
age, 600 mOsm are generated daily which would
require approximately 2 L of urine (at a concen-
tration of 300 mOsm/kg) to be excreted. When the
kidneys are faced with a higher solute load, or
the maximum concentrating ability is reduced
despite a stable solute load, the daily urine output
will increase to maintain equilibrium. This clini-
cally manifests as polyuria. Polyuria may be very
difficult to assess in infants and toddlers outside
the hospital setting. However, certain clues
include a history of polyhydramnios in conditions
that are hereditary in nature [35], excessive thirst,
recurrent episodes of dehydration, and in the most
severe cases failure to thrive [36]. In toilet-trained
children and adolescents, nocturia and secondary
enuresis can be the first manifestation of an
acquired urinary concentrating defect.
Polyuria can result from solute (or osmotic)
diuresis, water diuresis, or a combination of
both. Assessment of a random spot urine sample
osmolality should facilitate the distinction
between the two entities in most cases. The classic
case of osmotic diuresis, where urine osmolality is
usually equal to or exceeds the serum osmolality,
can be seen in the child presenting with new-onset
diabetes mellitus. The hyperglycemia produces
a high filtered glucose load that exceeds the
proximal tubule transport maximum for glucose.
Other osmotic agents such as urea and mannitol
can cause a similar picture.
Water diuresis encompasses conditions where
the distal nephron mechanisms for urinary con-
centration are impaired leading to the loss of large
amounts of dilute urine where the osmolality
is typically far less than serum osmolality.
This can be a result of deficient ADH secretion
(central diabetes insipidus), impaired renal ADH
responsiveness (nephrogenic diabetes insipidus),
or reduced renal medullary concentration gradient
required to reabsorb water from the distal nephron
under the influence of ADH. The latter
I.F. Ashoor and M.J.G. Somers
mechanism is seen in conditions with defective
sodium reabsorption from the thick ascending
limb of the loop of Henle (TAL) and distal neph-
ron required to create the medullary osmotic gra-
dient [37], hence the coexistence of a solute
diuresis component to the polyuria in these set-
tings. Chronic polydipsia, either as a primary phe-
nomenon or as a response to polyuria, can lead to
“washout” of the renal medullary concentration
gradient further worsening the polyuria [38].
Nephrogenic Diabetes Insipidus
Nephrogenic diabetes insipidus (NDI) refers to a
state of water diuresis secondary to resistance to the
action of ADH in the distal nephron. Under condi-
tions of volume depletion or hyperosmolality,
ADH acts on arginine vasopressin receptor
2 (AVPR2) on the basolateral membrane of princi-
pal cells leading to insertion of aquaporin 2 (AQP2)
channels in the luminal membrane, thereby increas-
ing water reabsorption [39, 40]. An intact medul-
lary concentration (osmotic) gradient is critical to
fully realize the ADH effect on this segment [41].
In infants and young children, NDI is usually
hereditary, secondary to genetic mutations affecting
the AVPR2 (X linked) [42, 43] or the AQP2 gene
(autosomal) [40, 42, 44, 45]. The condition com-
monly manifests early with failure to thrive, poly-
uria, polydipsia, nocturia, and enuresis [36].
Recurrent episodes of hypernatremic dehydration
are common in the setting of impaired thirst mech-
anism or lack of free access to water, particularly in
infants before their condition is recognized.
The diagnosis of hereditary NDI is typically
suspected on clinical grounds and supported by
high normal serum Na concentration, concomitant
hypoosmotic urine, and lack of response to
desmopressin administration, without resorting to
a water deprivation test. A water deprivation test
should be reserved for older children with equivo-
cal clinical findings and only performed under
close medical supervision. Molecular genetic test-
ing is available to confirm the diagnosis [42].
In older children and adolescents, NDI is usu-
ally acquired secondary to conditions compromis-
ing the renal medullary concentration gradient,
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
which reduces water reabsorption under the influ-
ence of ADH. Such conditions include, among
others, hypercalcemia [46], hypokalemia [47,
48], drug-induced NDI [49–52], and chronic
tubulointerstitial injury seen in some forms of
chronic kidney disease as discussed in the next
section. In addition to nocturia, polyuria, and
polydipsia, children with acquired NDI will also
manifest symptoms of the underlying disorder.
Unlike hereditary NDI, the onset of symptoms is
typically gradual and, oftentimes, at least partially
reversible with correction of the underlying cause.
In the absence of an identifiable acquired
reversible cause for NDI, treatment aims at mini-
mizing polyuria and providing sufficient fluids to
prevent recurrent episodes of hypernatremic
dehydration. This is particularly challenging if
the thirst mechanism is impaired or in infants
and small children who rely on caregivers for
access to water. In some cases, continuous tube
feedings may be required to facilitate normal
growth. Polyuria can be reduced by minimizing
the dietary solute load that allows the kidneys to
excrete it in a smaller volume given the fixed
concentration. This can be accomplished by salt
restriction and in selected cases, where failure to
thrive is not a concern, protein restriction under
close dietary supervision [53]. Another strategy to
reduce the daily urine output involves using
low-dose thiazide diuretics to induce a state of
mild volume depletion which increases sodium
and water reabsorption in the proximal nephron
[54]. The use of nonsteroidal anti-inflammatory
drugs (NSAIDs) to reduce renal prostaglandin
synthesis [55, 56] and ADH analogs (e.g.,
desmopressin) may be effective in selected
patients [57].
Special Considerations in Infants
and Children with Chronic Kidney
Disease
Several mechanisms contribute to impaired
urinary concentrating ability in chronic kidney
disease [58–60]. In vitro studies have suggested
a role for decreased expression of AVPR2 mRNA
in rats with chronic renal failure [61]. Progressive
371
tubulointerstitial injury from chronic pyelonephri-
tis, interstitial nephritis, calcium deposition in
nephrocalcinosis [46], and sickle-cell disease
[62] reduces the medullary concentration gradient
leading to ADH hyporesponsiveness. Children
with congenital or hereditary disorders primarily
affecting the tubulointerstitium and medullary
regions (e.g., nephronophthisis [63] and medul-
lary cystic kidney disease [64]) or solute transport
in the distal nephron required to generate the
medullary concentration gradient (e.g., Bartter
syndrome) [35, 65, 66] will also manifest
impaired urinary concentrating ability. In children
with chronic kidney disease secondary to obstruc-
tive uropathy, a superimposed urinary tract infec-
tion may be followed by a brief period of polyuria
secondary to solute diuresis. The urinary tract
infection is postulated to induce a state of transient
pseudohypoaldosteronism that promotes salt
wasting and resolves with treatment [67, 68].
Also, worsening azotemia from an acute on
chronic obstruction is usually followed by a brief
period of post-obstructive diuresis when the
obstruction is relieved, secondary to the osmotic
diuretic effect of the filtered urea load [69].
The mainstay of treatment in polyuric children
with chronic kidney disease is provision of suffi-
cient free water to prevent dehydration and
hypernatremia. In contrast to children with hered-
itary NDI, they may require salt supplementation
if the salt wasting is the underlying mechanism for
impaired urinary concentration. If these children
are admitted for intravenous fluid therapy, assess-
ment of urine osmolality can be a valuable tool to
guide fluid tonicity choice to correct or maintain
normal serum sodium concentration.
Maintenance Electrolyte Therapy
Estimates for the maintenance requirements of the
major electrolytes sodium, potassium, and chloride
can also be made based on metabolic demands and
daily water needs [6]. For sodium and chloride,
approximately 2–3 mEq/100 ml of daily water
requirement is needed with 1–2 mEq of potassium
for each 100 ml of daily water need. Again, these
estimates require adjustment based on clinical
372
circumstance, but the daily intake of most healthy
individuals contains more than adequate electro-
lytes for maintenance needs. Although at times
there can be significant electrolyte losses through
the skin or the gastrointestinal tract, most electro-
lyte losses are urinary [17, 70]. In the setting of
anuric renal failure and no electrolyte losses from
other sources, lower levels of electrolyte supple-
mentation may be needed, and water supplementa-
tion alone to replace insensible fluid losses may
maintain adequate electrolyte balance.
As with provision of water, electrolyte therapy
should be tailored to individual need. For sodium
and chloride, this requires careful assessment of
the extracellular fluid space, especially the effec-
tive circulating volume. Providing too little
sodium chloride results in volume contraction
and circulatory compromise; providing too much
causes volume overload and sequelae such as
hypertension and edema.
Similarly, inappropriate potassium supplemen-
tation may have significant clinical ramifications.
In children with diminished renal function or who
are at risk of hyperkalemia for other reasons, it
may be appropriate to forego maintenance potas-
sium supplementation. When supplementation is
given, there needs to be timely reassessment of
serum levels. Oral administration of supplemental
potassium is safer than bolus injection and intra-
venous potassium supplements rarely need to
exceed 0.5 mEq/kg/h [20].
Sodium
Alterations in Water Balance, Serum
Sodium, and Cell Volume
Since the regulation of osmolality is achieved by
changes in water intake and excretion, serum
sodium levels can vary with these alterations in
water balance. Generally, serum sodium is regu-
lated between 135 and 145 mEq/L. A serum
sodium concentration below 130 or above
150 mEq/L is out of the range of normal homeo-
stasis and most often indicates a problem with
water balance.
I.F. Ashoor and M.J.G. Somers
Since sodium is the major extracellular osmol,
alterations in serum sodium can result in water
flux between the intracellular and extracellular
spaces. Because significant water movement into
or out of cells could prove deleterious to cell
function, cell volume is closely regulated to min-
imize such shifts [71]. For instance, with
hyponatremia there is decreased effective osmo-
lality in the extracellular space. As a result, water
can shift from the plasma into the intracellular
space and cell swelling will occur. To counterbal-
ance such swelling, the cell acutely regulates its
volume by transporting electrolytes, especially
potassium, from the intracellular space into the
extracellular space, thereby decreasing the
osmotic gradient for water transfer. Over several
days, when faced with chronic hyponatremia or
chronic hypoosmolality, the cells will also achieve
effective volume regulation by losing organic
osmolytes such as taurine and inositol, thereby
further diminishing the osmotic gradient for
water transfer into the cell [72].
With hypernatremia, the osmotic gradient
favors water movement out of the cells and into
the extracellular space, and cells will shrink
without protective mechanisms. Acutely, there is
transport of electrolytes intracellularly, whereas
with chronic hypernatremia production of intra-
cellular organic idiogenic osmoles is enhanced
[72, 73]. Together, these act to blunt the loss
of intracellular volume that would otherwise
occur.
Changes in serum sodium values that evolve
slowly and reflect chronic processes tend to allow
for maximal counter-regulation and fewer clinical
sequelae. On the other hand, sudden profound
alterations – especially with serum sodium values
below 120 mEq/L or above 160 mEq/L – are often
accompanied by neurologic complications
directly related to the acute changes in cell volume
in the central nervous system.
These regulatory mechanisms must also be
kept in mind when formulating specific therapeu-
tic intervention. Acute perturbations can be
corrected more rapidly than chronic conditions
because the full gamut of responses to the imbal-
ance has yet to come into play.
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
Hyponatremia: Initial Approach
Hyponatremia is commonly defined as a serum
sodium concentration less than 135 mEq/L, though
it is rare for there to be any clinical effect in levels
between 130 and 135 mEq/L. Low serum sodium
values are usually the result of persistent ADH
effect and a relative surfeit of water for solute in
the extracellular space; hyponatremia uncommonly
arises secondary to depleted salt stores alone.
Infrequently, pseudohyponatremia may be seen.
Pseudohyponatremia is not a depletion of sodium
stores but a change in the usual makeup of the
extracellular space such that there is a pathologic
elevation of another solute that decreases the rela-
tive concentration of sodium. Common etiologies
of pseudohyponatremia include hypergylcemia,
hyperlipidemia, and hyperproteinemia. In these
clinical conditions, serum sodium values tend to
be only modestly depressed. Since there is no
underlying true anomaly in sodium or water stores
in these conditions, the serum sodium need not be
addressed with any therapeutic maneuvers. With
the introduction of ion-sensitive electrodes for the
measurement of plasma sodium concentration,
pseudohyponatremia related to the presence of
confounding factors in the laboratory assay is
much less commonly encountered.
To clarify the etiology of the hyponatremia, the
clinician should assess the patient’s extracellular
volume status and determine if it is decreased,
normal, or increased. Then, by measuring urine
sodium excretion and determining the renal
response to the hyponatremia, it becomes easier
to determine if the patient should receive sodium
and water or if sodium and water restriction is the
appropriate therapy (Table 5).
Hyponatremia: Decreased Volume Status
and Urine Na < 20 mEq/L
Decreased circulating volume is usually seen with
states of significant sodium loss. Most commonly,
loss is from the gastrointestinal tract as a result of
vomiting, diarrhea, or tube drainage. The renal
response to the decreased effective circulating
volume involves increased activity of the
renin–angiotensin axis and relatively high levels
373
Table 5 Etiology of hyponatremia
Urinary Na (mEq/L)
Circulating
volume 20 20
Decreased Burns Adrenal
insufficiency
Cystic fibrosis Diuretics – early
Diuretics – late Salt wasting
Gastroenteritis
Normal or Cardiac failure Renal failure
increased Hepatic SIADH
cirrhosis
Nephrotic Water
syndrome intoxication
of aldosterone and angiotensin. As a result, urine
sodium values are generally low (<20 mEq/L)
and water reabsorption in the distal nephron is
facilitated by high levels of ADH. In the face of
continuing sodium losses exceeding intake, this
state of vigorous ADH effect leads to a relative
excess of water and concomitant hyponatremia.
A similar clinical state of hyponatremia can
also be seen in cystic fibrosis where there are
increased skin losses of sodium and chloride,
with bleeding, with burns, and with certain losses
of fluid from the intravascular space of the extra-
cellular fluid into the interstitial space as can occur
postoperatively, in conditions of vascular leak
such as sepsis, or with peritonitis. Such
hyponatremia can also be seen following a period
of diuretic therapy. In response to chronic
diuretic-mediated volume contraction, the mech-
anisms outlined above come into play. Thiazide
diuretics, especially in combination with a loop
diuretic such as furosemide, are particularly prone
to inducing such hyponatremia.
The appropriate therapeutic response to these
conditions is the provision of sodium and water
either by use of intravenous or oral electrolyte
solutions. This results in restoration of sodium
balance and volume expansion.
Hyponatremia: Decreased Volume Status
and Urine Na > 20 mEq/L
Decreased circulating volume with a random
urinary sodium excretion >20 mEq/L is
374
indicative of renal salt wasting, either from an
intrinsic tubulopathy or from early diuretic
effect. Less commonly, adrenal insufficiency
can cause sodium wasting from the cells of the
distal nephron. Such a deficiency can arise from
an intrinsic endocrine defect such as congenital
adrenal hyperplasia related to 21-hydroxylase
deficiency, from some secondary impairment of
adrenal function caused by infection, bleeding,
or malignancy or from pharmacologic adrenal
suppression without adequate replacement
therapy. In the setting of adrenal insufficiency,
provision of appropriate adrenal hormone
replacement as well as adequate sodium and
water proves therapeutic. With renal salt
wasting, supplementation with sodium and any
other electrolytes exhibiting impaired renal
reabsorption is useful.
Hyponatremia: Normal or Expanded
Volume and Urine Na < 20 mEq/L
Normal or increased circulating volume and
random urine sodium excretion <20 mEq/L
can be seen in conditions where there is an
excess of both total body water and total body
sodium. The three major disorders that cause
this type of hyponatremia are the nephrotic
syndrome, hepatic failure related to cirrhosis,
and cardiac failure. In all these conditions,
there is a state of sodium and water avidity
related to high levels of ADH and aldosterone.
Most commonly, this is in the setting of
preexisting total body sodium overload as
evidenced by edema. In all of these conditions,
despite the increased extracellular or circulating
volume, the effective circulating volume is
often depressed. As a result of this ineffective
perfusion of the tissues, sodium and water
avidity is only heightened by stimulation of
the renin–aldosterone–angiotensin axis, further
exacerbating the total body excess of salt and
water. Appropriate therapy includes striking a
balance between interventions promoting the
maintenance of effective circulating volume
and restricting the provision of excess water
and sodium which will only contribute to further
total body water and sodium overload.
I.F. Ashoor and M.J.G. Somers
Hyponatremia: Normal or Expanded
Volume and Urine Na > 20 mEq/L
Hyponatremia in the setting of normal or
increased effective circulating volume is always
related to persistent ADH effect [15]. If the ran-
dom urine sodium value is >20 mEq/L, the most
common clinical scenario is the syndrome of inap-
propriate antidiuretic hormone secretion or
SIADH. SIADH can arise from disparate clinical
conditions including the postoperative child, the
child with significant pain, or the child with pul-
monary disease. Ill children may be at risk for
both inappropriate ADH secretion and inappropri-
ate ADH effect [74]. In SIADH, despite a state of
hypoosmolality, the urine is inappropriately con-
centrated as a result of ongoing ADH secretion
and ADH-mediated water reabsorption from the
distal nephron. Appropriate therapy for SIADH
includes restricting water intake and attending to
any underlying clinical factors predisposing to
this syndrome. Provision of fluids containing
higher concentrations of sodium will not neces-
sarily increase serum sodium without attention to
concomitant water restriction.
Normal or increased extracellular volume and
high urine sodium concentration can also be seen
in the setting of renal failure as both glomerular
filtration and water clearance falls, while the frac-
tional excretion of sodium rises. A more unusual
cause of this form of hyponatremia is polydipsia,
usually psychogenic in nature. Such water intox-
ication is rare in children but can occasionally be
seen with emotional or psychiatric illness in older
children or with infants inappropriately provided
with large volumes of water or very hypotonic
fluid in a repetitive fashion by a caretaker. In
both these circumstances, restriction of the vol-
ume of free water ingested on a daily basis may be
beneficial.
Hypernatremia
Hypernatremia is defined as a serum sodium con-
centration greater than 150 mEq/L. Generally,
even higher serum sodium values can be tolerated
with the most significant clinical effects not
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
occurring until levels exceed 160 mEq/L. As with
hyponatremia, hypernatremia is more commonly
a reflection of a problem with water homeostasis
than sodium balance [75]. In most instances, the
patient has a relative deficiency of water for nor-
mal extracellular solute content.
Since sodium is the major determinant of
plasma osmolality, as serum sodium levels rise,
serum osmolality concomitantly increases.
Increases in serum osmolality are sensed by hypo-
thalamic osmoreceptors, triggering ADH release
from the posterior pituitary as serum osmolality
increases over 280 mOsm [76]. In the setting of
high ADH, there can then be increased
reabsorption of free water from filtrate in the cor-
tical collecting duct. Increased serum osmolality
also causes a sensation of thirst and triggers
increased fluid intake. All of these mechanisms
serve to re-equilibrate serum osmolality and
sodium levels before clinically significant
hyperosmolality or hypernatremia occurs.
Outside infancy, hypernatremia as a result of
sodium excess or salt poisoning is infrequent. Its
major cause is improper preparation of powdered
or liquid concentrated formula resulting in a
hypertonic, hypernatremic solution. Since infants
do not have free access to water, they cannot
respond to their increasing sense of thirst as they
develop such hypernatremia. As caregivers con-
tinue to provide the same incorrectly prepared
formula for further feedings, there is further salt
loading. In addition, young infants are unable to
excrete sodium loads as efficiently as older chil-
dren and this limits the intrinsic renal response.
Iatrogenic sodium loading can also be seen in
children who receive large doses of sodium bicar-
bonate during resuscitation, because of persistent
acidosis, or in children who have been given
inappropriate amounts of sodium in peripheral
nutrition. Iatrogenic sodium loading can also be
seen in the child receiving repeated large volumes
of blood products since these are generally iso-
tonic or sodium-rich solutions.
Children who have hypernatremia from
sodium excess should exhibit the physical signs
and symptoms of an expanded extracellular space.
They frequently have peripheral edema and may
375
have hypertension or symptoms of pulmonary
edema. These children can respond to therapy
aimed at augmenting sodium elimination. The
use of diuretics and the provision of adequate
free water decrease the total body sodium burden.
Rarely, dialysis may be necessary when the
hypernatremia must be corrected rapidly [77, 78].
Hypernatremia as a result of salt loading is rare
and the pediatric clinician is much more likely to see
hypernatremia stemming from a free water deficit or
a combined water and sodium deficit where the
water losses exceed the sodium losses.
Hypernatremia secondary to a water deficit arises
in the setting of inadequate access to water or some
impairment in ADH release or response. It is
uncommon to see hypernatremia secondary to
poor water intake except in infants or young chil-
dren who cannot get water for themselves in
response to their sense of thirst [79]. As for
ADH-related anomalies, there are many causes of
central or nephrogenic diabetes insipidus [80].
Again, given normal access to water, it is rare for
the older child to develop hypernatremia even with
impairment in the ADH axis because of the strong
drive to drink in response to thirst [81]. In very
young children with diabetes insipidus, however,
the issue of access to water arises and hypernatremia
may be a concern. Such hypernatremia can also be
seen in the postoperative state in children with con-
centrating defects who are not allowed to drink by
mouth and who are receiving a prescribed volume
of fluid based on a presumed ability to concentrate
urine and conserve water.
The most common etiology of hypernatremia
in children is the loss of hypotonic fluid, which is
fluid with a relative excess of water for its sodium
content. In these situations, the total body water is
decreased more than the total body sodium. The
usual clinical scenario leading to such a condition
is viral diarrheal illness with poor water intake or
persistent vomiting. In this condition, there is loss
of stool with sodium content typically <60
mEq/L. These children tend to excrete small vol-
umes of concentrated urine with urine sodium
content <20 mEq/L, underscoring the fact that
they are conserving both water and sodium.
Their hypernatremia is not a manifestation of a
376 I.F. Ashoor and M.J.G. Somers

total body excess of sodium but a depletion of
sodium that is overshadowed by a larger relative
depletion of body water. Therapy is aimed at
restoring water and sodium balance by providing
back the hypotonic fluid which was initially lost
either by the use of intravenous saline solutions or
with oral electrolyte therapy.
Fluid Replacement Therapy
with marked clinical improvement. In the setting
of more significant compromise of effective vol-
ume such as with loss of vascular tone in sepsis or
a systemic inflammatory response, more than
200 ml/kg may be needed to achieve hemody-
namic stability and effective perfusion. In most
situations other than outright shock, the clinician
can approach fluid resuscitation with either intra-
venous or oral rehydration therapy.

Most commonly, the primary goal of fluid replace-
ment therapy is restoration of an adequate effec-
tive circulating volume. In its absence, significant
metabolic derangements can occur that then exac-
erbate perturbations in fluid and electrolyte
homeostasis. The volume of fluid replacement
required varies with the extent and etiology of
the compromised circulation. In many children
with acute illness, there may be an element of
decreased effective volume that is mild and diffi-
cult to appreciate by clinical examination. Expan-
sion of extracellular volume with infusion or
ingestion of 20–40 ml/kg of fluid over a few
hours often results in better perfusion and
improved clinical appearance from presentation.
Urine output tends to remain normal in these
situations speaking against persistent
non-osmotic ADH activity that would encourage
volume overload [29].
Children with mild dehydration, up to about
5 % weight loss, will usually respond to the pro-
vision of 30–50 ml/kg of fluid over several hours
Assessment of Volume Depletion
In estimating the severity of dehydration, a change
in weight from baseline is the best objective mea-
sure [82]. As rehydration proceeds, following
weights on a serial basis becomes an important
adjunct in assessing the efficacy of fluid repletion.
If no baseline weight is known, most clinicians
use various parameters based on history and phys-
ical examination to judge the severity of dehydra-
tion (Table 6). Children with mild dehydration
will have few clinical signs and only a modest
decline in urine output. As dehydration becomes
more significant, classic findings such as dry
mucous membranes, tenting skin, sunken eyes,
and lethargy become prominent. With profound
dehydration, there is anuria, marked alterations in
consciousness, and hemodynamic instability.
Without access to prior weight values, most
clinicians find it difficult to estimate accurately the
degree of dehydration when it is mild or moderate.
A capillary refill time greater than 2 seconds has

Table 6 Clinical assessment of dehydration

Degree of dehydration
Mild Moderate Severe
Vital signs
Pulse Normal Rapid Rapid and weak
Blood pressure Normal Normal to slightly low Shock
Weight loss
Infant <5 % 10 % >15 %
Older child <3 % 6% >9 %
Mucous membranes Tacky Dry Parched
Skin turgor Slightly decreased Decreased Tenting
Eye appearance Normal tearing Decreased tearing  sunken No tears + very sunken
Capillary refill Normal Delayed (>3 s) Very delayed (>5 s)
Urine output Decreased Minimal Anuric
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
long been touted as a useful physical finding
suggesting effective volume depletion [83]. Unfor-
tunately, delayed capillary refill is neither a sensi-
tive nor specific marker of dehydration [84, 85]. It
may be most useful if normal, as this does seem to
exclude reliably severe dehydration. In a prospec-
tive cohort study of dehydrated children between
3 and 18 months of age, the best correlation
between clinical assessment of degree of dehydra-
tion and actual volume depletion came in children
who had obvious clinical parameters of significant
dehydration such as prolonged skinfold tenting, a
dry mouth, sunken eyes, and altered sensorium
[86]. Similarly, in a review of preschool children
with dehydration, the best clinical indicators of
volume depletion – decreased skin turgor, poor
peripheral perfusion, and Kussmaul breathing –
accompanied more significant dehydration where
there was little clinical question of volume
depletion [87].
A study of 97 American children given intra-
venous fluids for rehydration in an emergency
department underscored the difficulty in assessing
even severe dehydration by standard clinical
approaches [88]. Physicians’ initial estimate of
dehydration compared to the actual percent loss
of body weight varied dramatically, with a sensi-
tivity of 70 % for severe dehydration (>10 % loss)
but only 33 % for moderate dehydration (6–10 %
loss). This study suggested that adding a serum
bicarbonate level to the assessment may be useful,
increasing the sensitivity of the clinical scales to
100 % in severe dehydration and 90 % in moder-
ate dehydration if both clinical features and a
serum bicarbonate <17 mEq/L were found.
Other studies have found that laboratory stud-
ies by themselves are poor indicators of dehydra-
tion. In 40 children receiving intravenous fluids
for dehydration, serum BUN, creatinine, uric acid,
anion gap, venous pH, venous base deficit, uri-
nary specific gravity, urinary anion gap, and frac-
tional excretion of sodium were all assessed from
prehydration blood and urine samples. Only the
serum BUN/Cr ratio and serum uric acid signifi-
cantly correlated with increasing levels of dehy-
dration, but both lacked sensitivity or specificity
for detecting more than 5 % dehydration
[89]. Similarly, in a retrospective review of
377
168 dehydrated children, elevated serum urea
levels and depressed serum bicarbonate
levels were found to be useful adjuncts to clinical
evaluation in accurately assessing the degree of
dehydration but were not by themselves
predictive [90].
In fact, with viral gastroenteritis, the most com-
mon cause of dehydration in children, there rarely
is a significant laboratory anomaly despite clini-
cally detected volume depletion, as underscored
by the report of a cohort of children from the
United Kingdom admitted for rehydration due to
viral gastroenteritis in which only 1 % had an
electrolyte derangement [91–93].
In children with volume depletion accompany-
ing trauma, sepsis, surgery, or underlying renal
dysfunction, it would be more likely to find per-
turbations in electrolyte and acid–base status.
Thus, in the absence of a straightforward case of
mild to moderate diarrheal dehydration, it is gen-
eral consensus that blood should be obtained for
assessment of electrolytes, bicarbonate, and renal
function to help guide specific fluid and electro-
lyte therapy [94, 95].
As the child is volume resuscitated, it is impor-
tant to reassess the child’s clinical status. Initial
estimates of degree of dehydration may need to be
adjusted if the child is not showing progressive
improvement. Most clinicians follow parameters
such as general appearance and sensorium,
change in weight from initiation of rehydration,
and urine output and urine osmolality. In children
with some types of renal dysfunction, there may
often be an underlying chronic urinary concen-
trating defect. In these children, relatively dilute
urine flow may be maintained even in the face of
clinical dehydration and markers other than urine
output and osmolality should be followed.
Oral Rehydration Therapy
Although oral rehydration with electrolyte solu-
tions is a safe, convenient, and effective way
to treat volume depletion, parenteral fluid and
electrolyte therapy has been the mainstay of
treatment for most children presenting with fluid
and electrolyte imbalances [96–99]. Especially
underutilized in North America, oral therapy has
proved successful in clinical settings worldwide
378
in resuscitating children of all ages with profound
fluid and electrolyte anomalies. Short of signifi-
cant circulatory compromise, oral rehydration can
be used as first-line therapy in all fluid and elec-
trolyte aberrations [100, 101]. An example of a
situation calling for oral rehydration is the follow-
ing clinical scenario:
A healthy 4-year-old girl presents to her pedi-
atrician’s office following 3 days of a febrile ill-
ness. Her appetite has been severely depressed
and her parents estimate that she has only had a
few cups of fluid in the last 12 h. She has vomited
once daily. She has continued to urinate, although
less frequently and with smaller volumes. On
physical examination, the girl looks unhappy but
nontoxic and alert. Her pulse is 100 beats per
minute and her sitting blood pressure is 80/50
mmHg. She will not cooperate with attempts at
orthostatic vital signs. Her mucous membranes
are somewhat dry and her weight today is 15 kg,
exactly the same as her weight at a well child
examination 6 months previously. The parents
are concerned that she looks dehydrated.
Primary care and emergency department phy-
sicians face such clinical scenarios daily. Other-
wise healthy children with viral illness causing
mild to moderate dehydration will frequently be
treated by intravenous fluids with the contention
that oral rehydration will be too labor intensive or
will take too much time. In fact, these children are
excellent candidates for oral rehydration, and, in
most developed countries where there is little
concern for cholera-like enteritis, oral solutions
with sodium contents from 30 to 90 mEq/L have
been shown for years to be efficacious for rehy-
dration [102–105].
With this girl, given the history and physical
examination, it is unlikely that any clinically sig-
nificant electrolyte perturbations will be found, so
there is little indication for assaying electrolytes
or renal function prior to starting oral rehydra-
tion [91–93]. The family is given a commercially
available oral rehydration solution containing
75 mEq/L Na, 20 mEq/L K, 30 mEq/L citrate,
and 2.5 % glucose. They are asked to provide 1 l
of fluid (50 ml/kg) to the child over the next
4 h. The child should be offered small aliquots of
fluid very often – 5 mls every 1–2 min at initiation.
I.F. Ashoor and M.J.G. Somers
If this regimen is tolerated with no vomiting, the
aliquots may be gradually increased in volume
and the frequency reduced, aiming to deliver at
least the prescribed total volume over about
4 h. After her rehydration, the child should sub-
sequently continue to be provided free access to
fluid and resume an age-appropriate diet. If, on
the other hand, there are any further episodes of
vomiting, then for each episode of emesis, an
additional 120–240 ml of oral rehydrating solu-
tion should be given, again with the goal to com-
plete rehydration and resume usual fluid intake
and nutrition.
The initial provision of oral fluid given often in
small volumes is far more likely to be well toler-
ated by the dehydrated child than larger aliquots.
If families are unwilling to provide the fluid in this
manner, a nasogastric tube may be placed for
continuous infusion of rehydrating solution.
Although occasional children may fail this
approach and require intravenous rehydration,
most children with mild to moderate rehydration
can be rehydrated orally. A guide for the volumes
of fluid to provide and the duration of rehydration
can be found in Table 7.
The first oral rehydration solutions were devel-
oped in the 1940s at academic medical centers.
Within 10 years, a commercial preparation formu-
lated as a powder to be mixed with water was
available, but its use became associated with an
increased incidence of hypernatremia [106]. Sev-
eral factors contributed to the development of this
problem: the preparation was sometimes incor-
rectly administered as the powder itself or improp-
erly diluted with too little water; when correctly
reconstituted, the solution had a final carbohy-
drate concentration of 8 % predisposing to an
osmotic diarrhea; and it was a common practice
at that time for parents to use high solute fluids
such as boiled skim milk as an adjunctive home
remedy. Taken together, these early experiences
contributed to reluctance by many clinicians to
use oral rehydration solutions, especially since
intravenous rehydration was becoming more stan-
dard in practice.
Over time, there came to be a better under-
standing of the physiology of water and solute
absorption from the gut. Of prime importance
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . . 379

Table 7 Oral rehydration for previously healthy, well-nourished children

Type of Rehydration Replacement of ongoing
dehydration phase Rehydration duration losses Nutrition
Mild (<5 %) 30–50 ml/kg 3–4 h 60–120 ml ORS for each Continue breast
ORT diarrheal stool or episode of milk or resume
emesisa age-appropriate
usual diet
Moderate 50–100 ml/kg 3–4 h As above As above
(5–10 %) ORT
Severe (>10 %) 100–150 3–4 h As above with use of As above
ml/kg ORT nasogastric tube if needed
Evidence of 20 ml/kg 0.9 Repetitive infusions until As above with use of As above
shock % NaCl or perfusion restored then nasogastric tube if needed
lactated transition to ORT 100 ml/kg or consideration of further
Ringer’s IV over 4 h IV therapy
Accompanying Per type of At least 12 h for ORT As above As above
hypernatremia dehydration Monitor fall in serum Na
ORT oral rehydration therapy with fluid containing 45–90 mmol/l Na, 90 mmol/l glucose, 20 mmol/l K, and 10–30 mmol/l
citrate
IV intravenous infusion
a
For children >10 kg, aliquots for replacement of ongoing losses should be doubled to 120–240 ml rehydration solution
for each stool or emesis

was the recognition that many substances actively
transported across intestinal epithelium had
an absolute or partial dependence on sodium
for absorption and that sodium itself was
actually better reabsorbed in their presence
[107–109]. Moreover, it became clear that the
sodium/glucose cotransporter remained intact
not only in the face of enterotoxic gastroenteritis
such as seen with cholera or Escherichia coli but
also in more common viral and bacterial enteriti-
des [100, 101]. This led to the routine introduction
of glucose into oral rehydration solutions in a
fixed molar ratio of no more than 2:1 with sodium.
The World Health Organization (WHO) and
the United Nations Children’s Fund champion
the use of a rehydrating solution that includes Na
90 mmol/L, Cl 80 mmol/L, K 20 mmol/L, base
30 mmol/L, and glucose 111 mmol/L (2 %).
This WHO solution has proved useful in many
pediatric clinical trials and has also been shown to
reduce the morbidity and mortality associated
with diarrheal illness regardless of its etiology
[102, 103], although it may not be readily
commercially available in many locales.
Most commercially available oral rehydration
solutions have somewhat higher carbohydrate
content, a lower sodium content, and a higher
carbohydrate to sodium ratio than WHO solution
(Table 8). Some preparations are available as
powder and other as ready-to-drink formulations.
Some manufacturers have also used rice solids as
a carbohydrate source instead of glucose.
Many of these formulation changes arose from
concerns that using an oral rehydrating solution
with a sodium content >60 mmol/L would prove
problematic in developed countries where most
gastroenteritis is viral in nature and has a lower
sodium content than the secretory diarrheas seen
in less developed areas. Some feared that mini-
mally dehydrated children losing small amounts
of sodium in their stools would become
hypernatremic if exclusively provided WHO
solution and a few studies did document such
iatrogenic hypernatremia [110]. Subsequently,
solutions with sodium content ranging from
30 to 90 mmol/L have proved quite effective in
cases of mild dehydration stemming from causes
other than secretory diarrhea [104, 105, 111].
A meta-analysis of studies focused on the safety
and efficacy of oral rehydration solution in well-
nourished children living in developed countries
documented little evidence that WHO solution
was more likely to cause aberrations in serum
sodium than lower sodium containing oral
380 I.F. Ashoor and M.J.G. Somers

Table 8 Oral rehydration solutions

Concentration, mmol/L
Product Na Sugar K Cl Base Osmolarity (mOsm/L)
WHO ORSa 90 111 20 80 30 311
CeraLyte 90a 90 220b 20 80 30 275
Low-Na ORSa 75 75 20 65 30 245
Rehydralyte 75 140 20 65 30 300
CeraLyte 70a 70 220b 20 60 30 230
CeraLyte 50a 50 220b 20 40 30 200
CeraLyte 50 lemon 50 170b 20 40 30 200
Enfalyte 50 170 25 45 34 167
Pedialyte 45 140 20 35 30 254
a
Provided as powder. Needs to be reconstituted with water
b
Contains rice syrup solids substituted for glucose

Table 9 Composition of common oral fluidsa

Fluid Na (mEq/L) K (mEq/L) Source of base Carbohydrate (g/100 ml)
Apple juice <1 25 Citrate 12
Orange juice <1 55 Citrate 12
Milk 20 40 Lactate 5
Cola 2 <1 Bicarbonate 10
Ginger ale 4 <1 Bicarbonate 8
Kool-Aid <1 <1 Citrate 10
Gatorade 20 2.5 Citrate 6
Powerade 10 2.5 Citrate 8
Jell-O 25 <1 Citrate 14
Coffee <1 15 Citrate <0.5
Tea 2 5 Citrate 10
a
Adapted in part from data found in Feld et al. [113]

rehydration solutions [112]. Why ingestion of
lower tonicity oral rehydration fluids would be
less problematic than infusion of similar tonicity
intravenous fluid is not clear, but does again
underscore the safety of oral rehydration.
Oral Rehydration with Fluids Other
Than ORS
Despite the efficacy and availability of commer-
cial oral rehydration solutions and the ease
with which other electrolyte solutions can be
mixed at home with recipes requiring few ingre-
dients other than water, sugar, and salt, many
children are still given common household bev-
erages for rehydration instead of oral rehydration
solutions. In children with dehydration and elec-
trolyte losses from vomiting or diarrhea, most
common beverages do not contain adequate
sodium or potassium supplementation, and the
base composition and carbohydrate source are
also often suboptimal (Table 9). Similarly, most
sports drinks for “rehydration” following exer-
cise are also depleted of sufficient electrolytes
for gastrointestinal disease given sweat is many
fold lower in electrolyte composition than gas-
trointestinal fluid. In prescribing oral rehydration
to children in an ambulatory setting, the clinician
should specify the appropriate fluid and volume
for the child to ingest, emphasizing the need to
use a fluid with appropriate electrolyte content if
there is concern about evolving imbalances in
sodium, potassium, or bicarbonate homeostasis.
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
Oral Rehydration and Serum Sodium
Abnormalities
Although oral rehydration is often considered for
children with modest dehydration and no presumed
electrolyte anomalies, oral rehydration with WHO
or WHO-like solution has also been used in cases of
dehydration accompanied by hyponatremia or
hypernatremia [114, 115]. Although most children
with severe hypernatremia (>160 mEq/L) can be
successfully rehydrated orally, there have been
reports of seizures, generally as a result of too
rapid correction of serum sodium from the provi-
sion of supplemental water along with the
glucose–electrolyte solution [114, 115]. In those
cases, the average serum sodium fell by 10–15
mEq/L over 6 h rather than over 24 h as advised.
In follow-up studies, no seizure activity was seen in
a similar cohort of hypernatremic children who
received 90 mmol/L Na rehydration solution alone
at a rate calculated to replace the infant’s deficit over
24 h [114]. It is important for the practitioner to
remember that once peripheral perfusion has been
stabilized with initial volume expansion, there is no
benefit to correcting any deficit rapidly and taking
24–48 h may be a more prudent course in the face of
significant electrolyte anomalies.
Oral Rehydration Schemes
Several oral rehydration schemes have been
shown to be quite effective and well tolerated. In
one approach used extensively in developing
countries, the patient’s volume deficit is calcu-
lated on the basis of weight loss and clinical
appearance [116]. The volume deficit is doubled
and this becomes the target rehydration volume to
be given over 6–12 h. Two-thirds of this volume is
given as a glucose–electrolyte solution containing
90 mmol/L Na over 4–8 h; once this has been
ingested, the remaining volume is provided as
water alone over 2–4 h. In cases of suspected or
confirmed hypernatremia with serum sodium
exceeding 160 mEq/L, the volume deficit would
not be doubled and would be administered as
90 mmol/L Na glucose–electrolyte solution
alone over 12–24 h. Patients who refuse to take
381
fluids by mouth have nasogastric tubes placed.
With this approach, successful oral rehydration
is the rule and 95 % of children are fully
rehydrated without the need for intravenous
therapy.
An alternative approach has been to have the
child begin by taking 15 ml/kg/h of a 60–90
mmol/L Na rehydration solution by mouth or
nasogastric tube [117]. The solution is given in
small frequent quantities and increased up to
25 ml/kg/h until hydration has improved at
which point solid feedings are reintroduced and
volumes of 5–15 ml/kg of rehydration solution
offered after feeds until the volume deficit have
been delivered.
Over two decades ago, the American Academy
of Pediatrics issued guidelines for the treatment of
fluid and electrolyte deficits with oral rehydration
solutions [100]. Children with acute dehydration
and extracellular volume contraction were to be
provided 40–50 ml/kg of a glucose–electrolyte
solution containing in each liter 75–90 mmol Na,
110–140 mmol glucose (2–2.5 %), 20 mmol
potassium, and 20–30 mmol base. This volume
was to be administered over 3–4 h and then once
there has been amelioration of the extracellular
volume contraction; the child would be changed
to a maintenance solution with 40–60 mmol/L Na
at half the rate. If the child was still thirsty on
this regimen, there should be free access to
supplemental water or low-solute fluid such as
breast milk.
Based on much of this published clinical expe-
rience, an evidence-based guideline for treating
dehydration in children from industrialized
European countries was created in the late
1990s, recommending oral rehydrating solution
containing 60 mmol/L of sodium, 90 mmol/L of
glucose, 20 mmol/L of potassium, and 10–30
mmol/L of citrate with rehydration occurring
over 3–12 h utilizing from 30 to 150 ml/kg of
fluid depending on the degree and type of
dehydration [82].
In 2004, the American Academy of Pediatrics
updated its oral rehydration recommendations and
endorsed guidelines promulgated by the Center
for Disease Control and Prevention [118,
119]. Minimal dehydration in children weighing
382
less than 10 kg was to be treated with provision of
60–120 ml of oral rehydration fluid for each
watery stool or each episode of vomiting. In larger
children, twice this volume would be provided.
For children with more moderate dehydration,
50–100 ml/kg of oral rehydration solution would
be given over 2 to 4 h to account for estimated
fluid deficit, and ongoing losses would be treated
with the 60–240 ml per stool or emesis depending
on size. Nursing babies would continue to receive
breast milk as desired and formula-fed babies
would be provided age-appropriate diet as soon
as they had been rehydrated. For severely
dehydrated children, a combination of intrave-
nous hydration with isotonic fluids and prompt
transition to oral rehydration solution by mouth
or nasogastric tube was recommended. Overly
restricted diets were to be avoided during gastro-
intestinal illness and attention to adequate caloric
intake emphasized (see Table 7).
Use and Acceptance of Oral
Rehydration Solutions
Despite the availability of guidelines for oral rehy-
dration and their endorsement by professional
organizations, oral rehydration solutions continue
to be underutilized by clinicians in both the devel-
oped and undeveloped world [104, 120]. Even in
Bangladesh where oral rehydration has been
closely studied and championed by both local
and international medical agencies for decades,
its use is still suboptimal [121, 122]. Some studies
do suggest that familiarity with a specific scheme
for oral rehydration increases its use in the emer-
gency department and that younger graduates of
training programs are more likely to use oral rehy-
dration for advanced cases of clinical dehydration
than their older colleagues, even when there was
no generational difference in the baseline knowl-
edge of published data in this field or acceptance
of its validity [123].
When utilized according to recommendation,
oral rehydration has been demonstrated to be
almost universally successful in achieving some
degree of volume repletion [10]. Other advantages
to oral therapy include the stability of the product
I.F. Ashoor and M.J.G. Somers
despite lengthy shelf storage, its ability to be
administered readily by the child’s caretaker in
nearly any locale, and avoidance of the discomfort
and potential complications associated with intra-
venous catheter placement [124]. In developed
countries, there has been the concern that some
powdered ORS formulations may not be looked
upon by parents as convenient since they must be
mixed with water prior to provision, but a ran-
domized controlled trial comparing an urban pedi-
atric clinic and a suburban medical practice found
that parents were equally satisfied with the ease and
effectiveness of a powdered solution as a commer-
cially prepared ready-to-drink solution [125].
Oral rehydration is somewhat less successful in
hospitalized children than in children treated in an
ambulatory setting [10]. This difference may be
directly related to the degree of dehydration or
other complicating clinical issues leading to hos-
pital admission. Moreover, the relatively labor-
intensive slower approach to oral rehydration
may be problematic in medical facilities with
time constraints or space limitations [124, 126].
Frozen-flavored oral rehydration solutions
may be more readily accepted than conventional
unflavored liquid electrolyte solutions. Their use
resulted in higher rates of successful rehydration
in children with mild to moderate dehydration,
even if these children initially failed conventional
oral rehydration [124]. Frozen-flavored rehydra-
tion solution is now commercially available in
many parts of the world, as is a variety of flavored
rehydration solutions.
Another potential issue with oral rehydration is
that its use does not alter the natural course of the
child’s illness. For instance, in gastroenteritis with
dehydration, by far and away the most common
illness requiring rehydration in children, oral rehy-
dration does not lower stool output or change the
duration of diarrheal illness [127]. As a result,
caretakers may abandon oral rehydration because
the child continues to have symptoms, failing to
appreciate the benefits of ongoing hydration. Oral
rehydrating solutions have been formulated with
lower electrolyte composition and different carbo-
hydrate moieties with the goal to reduce the osmo-
larity of solutions and potentially augment
fluid absorption from the small intestine [112].
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
The rice-based oral solutions have been studied
most extensively. In these solutions, glucose is
substituted with 50–80 g/l of rice powder. In a
meta-analysis of 22 randomized clinical trials com-
paring rice-based solution to conventional glucose-
containing solutions, stool output dramatically
decreased in children with cholera given rice-
based hydration but did not change in children
with other bacterial or viral enteritides [128].
There are some reports that suggest that pro-
viding children with non-cholera enteritis with
reduced osmolarity rehydration solution may be
beneficial. A study of 447 boys less than 2 years of
age admitted for oral rehydration compared WHO
solution (osmolarity 311 mmol/l) and a solution
containing less sodium and chloride (osmolarity
224 mmol/l). Children who received the lower
osmolarity solution had reduced stool output,
reduced duration of diarrhea, reduced rehydration
needs, and reduced risk of requiring intravenous
fluid infusion after completion of oral hydration
[129]. A meta-analysis of nine trials comparing
WHO solution to reduced osmolarity rehydration
solution concluded that children admitted for
dehydration had reduced needs for intravenous
fluid infusion, lower stool volumes, and less
vomiting when receiving the reduced osmolarity
solution [130].
Intravenous Therapy
Although absolute indications for parenteral intra-
venous therapy are limited, they do include sig-
nificantly impaired circulation or overt shock. In
addition, there are occasional children who are
truly unable to sustain an adequate rate of oral
fluid intake despite concerted effort or have such
persistent losses that parenteral therapy comes to
be necessary. The mainstays of fluid therapy in
children are saline or buffered saline crystalloid
solutions. Isotonic versions of these crystalloids
are used for volume resuscitation and hypotonic
saline solutions may be used in addition to pro-
vide supplemental maintenance hydration. In
addition to crystalloid solutions, there are several
colloid fluids that are also used by many clini-
cians. Table 10 lists the electrolyte content of
some of the more common intravenous solutions
used for pediatric fluid therapy.
383
Choice and Volume of Parenteral Fluid
Children with significant extracellular volume con-
traction (greater than 10 % acute weight loss in an
infant or 6 % weight loss in an older child) should
receive an isotonic crystalloid solution such as 0.9
% saline (154 mEq/L NaCl) or lactated Ringer’s
(130 mEq/L NaCl) at a rate of 20 ml/kg over 30–60
min. In some children, even more rapid infusions or
serial provision of such aliquots may be necessary
to restore effective volume. Children with less pro-
nounced dehydration may not exhibit signs or
symptoms of volume contraction. In certain situa-
tions, however, it may be clinically warranted to
provide them with an initial rapid intravenous bolus
to initiate rehydration therapy.
Concomitant with the placement of intravenous
access, blood should be obtained for determination
of serum electrolytes, osmolality, and renal func-
tion. Given that dehydrated children often have
high levels of vasoactive hormones and high vaso-
pressin levels, it is most prudent to establish base-
line electrolyte levels since it is possible to alter
electrolyte balance rapidly with intravenous ther-
apy. In the face of inadequate tissue perfusion, a
parenteral fluid infusion should begin immediately
prior to the return of any pertinent laboratory
results. If hemorrhagic shock is suspected, resusci-
tation with packed red blood cells is optimal. In
cases of severe volume depletion, if the child does
not improve with the initial 20-ml/kg crystalloid
bolus, this should be repeated up to two additional
times. In children who have not improved despite
administration of 60 ml/kg of total volume over an
hour or in children in whom underlying cardiac,
pulmonary, or renal disease may make empiric
aggressive rehydration more problematic, consid-
eration should be given to placement of a central
monitoring catheter to more accurately assess intra-
vascular volume and cardiac dynamics [131]. In
some instances of profound ineffective circulating
volume, such as might accompany certain cases of
sepsis, initial volume resuscitation may require
sequential infusions of fluid ultimately exceeding
100 ml/kg.
Within minutes of infusion of a crystalloid
fluid, it becomes distributed throughout the extra-
cellular space. Since this involves equilibration of
384 I.F. Ashoor and M.J.G. Somers

Table 10 Composition of common intravenous fluids

Buffer
Osmolarity Na K Cl (source) Mg Ca Dextrose
Fluid (mOsm/l) (mEq/l) (mEq/l) (mEq/l) (mEq/l) (mEq/l) (mEq/l) (g/l)
Crystalloids
0.9 % saline 308 154 0 154 0 0 0 0
Lactated 275 130 4 109 28 (lactate) 0 3 0
Ringer’s
D5 0.45 % 454 77 0 77 0 0 0 50
saline
D5 0.22 % 377 38 0 38 0 0 0 50
saline
5 % dextrose 252 0 0 0 0 0 0 50
water
Normosol 295 140 5 98 27 (acetate) 3 0 0
23
(gluconate)
Plasma-Lyte 294 140 5 98 27 (acetate) 3 0 0
23
(gluconate)
Colloids
5 % albumin 309 130–160 <1 130–160 0 0 0 0
25 % albumin 312 130–160 <1 130–160 0 0 0 0
Fresh frozen 300 140 4 110 25 0 0 0
plasma (bicarbonate)
3.5 % 301 145 5 145 0 0 6 0
Haemaccel
6% 310 154 0 154 0 0 0 0
hetastarch
Dextran 40 or 310 154 0 154 0 0 0 0
70

the fluid between the two components of the
extracellular space – the intravascular and inter-
stitial spaces – actually only one-third to
one-quarter of infused crystalloid stays in the
blood vessels [132]. This accounts for the need
to give large volumes of crystalloid in the setting
of circulatory collapse and leads some to suggest
that colloid solutions such as 5 % or 10 % albumin
should play a role in resuscitation [133, 134].
Colloid Solutions and Volume
Resuscitation
The use of colloid solutions for volume resuscita-
tion is controversial. Colloids were once included
in a number of widely promulgated guidelines for
the care of patients in emergency facilities and
intensive care units both for hemorrhagic shock
prior to the availability of blood and for
nonhemorrhagic shock as an adjunct to crystalloid
use [135]. Types of colloid utilized included 5 %
albumin, fresh frozen plasma, modified starches,
dextrans, and gelatins. These guidelines, gener-
ally aimed towards the fluid resuscitation of
adults, were composed despite the prior publica-
tion of a systematic review of randomized con-
trolled trials that demonstrated no effect on
mortality rates when colloids were used in prefer-
ence to crystalloids [136]. Moreover, there is a
distinct cost disadvantage to using colloid
solutions.
Subsequent systematic reviews have looked at
this issue anew. In one meta-analysis of 38 trials
comparing colloid to crystalloid for volume
expansion, there was no decrease in the risk of
death for patients receiving colloid [135]. In the
other review, albumin administration was actually
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
shown to increase mortality by 6 % compared to
crystalloid [137]. Proposed mechanisms contrib-
uting to this worse outcome include anticoagulant
properties of albumin [138] and accelerated
capillary leak [139].
A drawback of all these systematic reviews,
however, has been the limited number of studies
that included children other than ill premature
neonates. As a result, generalization of these
results from ill adults may not be germane to all
critically ill volume-depleted children. For
instance, a report of 410 children with meningo-
coccal disease suggests that albumin infusion in
this population may not have been harmful, as
case fatality rates were lower than predicted
[140]. Overall, however, there seems to be no
substantive data to support the routine use of
colloid to complement or replace crystalloid in
fluid resuscitation. Rather, repetitive infusions of
large volumes of crystalloid seem to be well tol-
erated in volume-depleted children, do not seem
to predispose to excessive rates of acute respira-
tory distress syndrome or cerebral edema, and in
some conditions, such as sepsis, play an important
role in improved survival [141]. A recent survey
of pediatric anesthesiologists in Western Europe
reported that colloid solutions are being used less
frequently in infants and older children and
suggested that familiarity with some of the issues
raised in these systematic reviews are affecting
practice patterns [142].
Repetitive infusions of crystalloid may also
prove problematic in some children. Most nota-
bly, if very large volumes of 0.9 % NaCl are used
acutely for volume resuscitation, it is not unusual
for children to develop a hyperchloremic meta-
bolic acidosis. This occurs as acidotic peripheral
tissues begin to reperfuse and already depleted
extracellular bicarbonate stores are diluted by a
solution with an isotonic concentration of chloride
[131, 143]. This acidosis can be ameliorated by
supplemental doses of bicarbonate as well as the
addition of supplemental potassium as needed.
There is sometimes a tendency for clinicians to
react to the hyperchloremic metabolic acidosis
with further saline bolus infusions. In the face of
corrected hypoxia or hypovolemia, however, such
maneuvers may only exacerbate the chloride-
385
driven acidosis [144]. This hyperchloremic
acidosis is seen less frequently when Ringer’s
lactate solution is used as the resuscitation
fluid because of the metabolic conversion of
lactate to bicarbonate. In the setting of significant
preexisting acidosis or underlying hepatic
dysfunction preventing the metabolism of lactate,
infusion of Ringer’s lactate solution may,
however, exacerbate an acidosis.
With the recent suggestion that some children
with acute illness or following surgery may ben-
efit from a prolonged period of isotonic fluid
infusion given high levels of ADH and the possi-
bility for hyponatremia developing with hypo-
tonic fluid therapy, some have expressed concern
that a hyperchloremic acidosis may develop in
these children. In a prospective randomized
study of more than 100 children with gastroenter-
itis given isotonic intravenous rehydration and
maintenance therapy, there was no tendency for
the development of hyperchloremic acidosis even
after a day of isotonic fluid provision. Although
serum chloride levels did tend to increase in these
children, serum bicarbonates also improved,
potentially related to improved effective volume
and subsequent better tissue perfusion [145]. Sim-
ilarly, in children who underwent cardiac surgery
who were given isotonic solutions for their main-
tenance fluid needs, although there was a ten-
dency for a hyperchloremic acidosis to develop,
there seemed to be no significant clinical ramifi-
cations and long-term outcomes were similar to
children who did not develop hyperchloremic
acidosis [146].
Large volume infusion of blood may also pre-
dispose to electrolyte anomalies as well as mani-
festations of citrate toxicity. If aged whole blood is
infused, there is the possibility that a large potas-
sium load will be delivered to the patient as potas-
sium may have moved from less viable
erythrocytes into the plasma. Since most patients
receive packed red blood cells instead of whole
blood, this potential problem is minimized, and
the potassium in the small volume of plasma in a
packed cell transfusion can generally be accom-
modated by intracellular uptake.
Citrate is used as the anticoagulant in stored
blood. Since citrate complexes with calcium, there
386
can be a fall in ionized calcium levels if large
volumes of citrate-containing blood are infused
rapidly or if there are concomitant perturbations
in calcium homeostasis. Similarly, citrate may
complex with magnesium and magnesium deple-
tion may occur. The liver usually metabolizes
infused citrate into bicarbonate and alkalosis can
also occur if large volumes of citrate are delivered.
In the setting of hepatic dysfunction, however,
citrate will not be metabolized and serves as an
acid load and will help create an acidosis or exac-
erbate any underlying acidosis.
Regardless of the initial infusion with either
colloid or crystalloid, once sufficient volume to
restore circulatory integrity has been infused, less
rapid volume expansion is necessary. During this
phase, the rapidity of fluid repletion is most prob-
ably not a concern unless there are severe under-
lying aberrations in the serum sodium or serum
osmolality. In the absence of these derangements
or profound volume deficit, if the child has
improved significantly with the initial parenteral
volume expansion, attempts should be made to
reinstitute oral rehydration. Prolonged intrave-
nous therapy should be rarely necessary.
Rapid Rehydration
In an attempt to minimize the duration of hospital-
based care, a scheme of rapid intravenous resus-
citation and follow-up oral rehydration has been
adopted by many pediatric emergency depart-
ments to treat children with up to 10 % dehydra-
tion secondary to vomiting and gastroenteritis
[147]. After infusion of 20–30 ml/kg of intrave-
nous crystalloid, the child is allowed to take up to
several ounces of a standard oral rehydration fluid,
and if this intake is tolerated without vomiting for
30–60 min, then the child discharged home to
continue rehydration, initially with a prescribed
volume of standard rehydration solution.
If the child does not tolerate oral rehydration or
if there are such significant electrolyte anomalies
that there are concerns regarding potential adverse
CNS sequelae of too rapid rehydration, then intra-
venous rehydration may be the best route for
continued hydration. Children who have had
I.F. Ashoor and M.J.G. Somers
very sudden fluxes in electrolytes may become
symptomatic earlier. On the other hand, children
whose severe sodium abnormalities are thought to
be more chronic in nature must be treated in a
more controlled fashion since they are at higher
risk for developing CNS symptoms during
treatment.
The vast majority of children treated in emer-
gency facilities for volume repletion do well with
such rapid rehydration. These children are gener-
ally healthy with normal cardiac and renal func-
tion and have developed extracellular volume
depletion relatively rapidly. As a result, they suf-
fer no ill effects from rapid rehydration. In fact,
the clinical success of this aggressive restoration
of extracellular volume underlies the calls to
reexamine or abandon the traditional deficit ther-
apy approach to rehydration with its tedious cal-
culations of fluid and electrolytes losses and
requirements, especially in the setting of other-
wise healthy children who are acutely ill [9, 148].
Symptomatic Hyponatremia
In the setting of symptomatic hyponatremia, espe-
cially if the child has seizures, it is important to
raise the serum sodium concentration by approx-
imately 5 mEq/L in an urgent fashion. Generally,
this results in stabilization of the clinical situation
and allows for further more considered evaluation
and treatment of the child. This is one of the few
situations in which hypertonic saline (3 % NaCl,
514 mEq/L) should be utilized.
To calculate the proper volume of 3 % saline to
infuse, the child’s TBW must be multiplied by the
5 mEq/L desired increase in serum sodium to
determine the amount of sodium (in mEq) to
infuse. Since every ml of 3 % saline contains
0.5 mEq of sodium, doubling the number of
mEq of sodium needed results in the proper mil-
liliter volume of 3 % saline to infuse. Thus, in the
20-kg child, the TBW is approximately 12 L (0.6
L/kg  20 kg) and the desired sodium dose
would be 60 mEq (12 L  5 mEq/L). If 120 ml
of 3 % saline were infused, the serum sodium
concentration would be expected to rise by
approximately 5 mEq/L. The infusion should be
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
given at a rate to increase the serum sodium by no
more than 3 mEq/L/h and is often given more
slowly over the course of 3–4 h [149]. If the
child continues to be symptomatic from
hyponatremia after this infusion, additional 3 %
saline may be given until the symptoms improve
or the serum sodium is in the 120–125 mEq/L
range. At that point, further correction of the
hyponatremia should consist of a slower infusion
of more dilute saline to cover the sodium deficit,
the sodium maintenance needs, and any volume
deficit. Consideration of the role of ADH and
prior excess free water provision should also be
considered in determining the volume and tonicity
of fluid to be provided.
Asymptomatic Hyponatremia
If a child has severe hyponatremia but is not
symptomatic, there is no need to administer
hypertonic saline based solely on a laboratory
anomaly. With or without symptoms, in cases of
severe hyponatremia, the child should be carefully
evaluated as to the etiology of the hyponatremia,
keeping in mind that hyponatremia tends to result
from an imbalance of water regulation. If this is
the case, free water should be restricted and appro-
priate supplementation with intravenous saline
solutions begun to provide maintenance sodium
requirements of approximately 2–3 mEq/kg/day
and any ongoing losses of sodium.
Besides these maintenance sodium needs, if
the child has an element of dehydration, every
kilogram of body weight lost from baseline rep-
resents a 1-L deficit of nearly normal saline from
the total body water as well. These losses are often
referred to as isotonic losses. These account for a
sodium deficit of 154 mEq/L that also must be
included in the calculations for sodium
replacement.
In the setting of hyponatremic dehydration,
there have been additional sodium losses as well.
Generally, these occur as viral diarrheal stool
losses with a sodium content 60 mEq/L are
replaced with fluids with lower sodium concen-
tration. To estimate these sodium losses, the dif-
ference between the child’s desired serum sodium
387
and current serum sodium is multiplied by the
child’s estimated TBW. This product represents
the hyponatremic sodium losses that must be
added to the maintenance sodium needs, any
ongoing losses, and the sodium losses that accom-
panied weight loss. An example of the calcula-
tions and therapeutic maneuvers that need to be
considered with significant hyponatremia is
presented in the following case study:
A girl who normally weighs 10 kg suddenly
develops generalized seizures and is brought by
ambulance to the emergency department. She has
had a week of gastroenteritis, has felt warm to
touch, and has been drinking water and apple
juice only, refusing any other liquids or any
solid food for several days. Intravenous access is
placed and lorazepam is administered and the
seizure activity stops. The emergency department
physician orders serum chemistries and the child
is weighed and found to be 8.8 kg. A bolus infu-
sion of 200 ml of 0.9 % NaCl is administered over
the next 30–60 min after which the girl appears
well perfused but she is still lethargic. The serum
sodium is then reported to be 112 mEq/L. While
further evaluation of the child’s overall status is
ongoing, it is important to begin correcting the
symptomatic hyponatremia.
The child has actually already received
approximately 30 mEq of sodium in the 0.9 %
NaCl bolus given because of her dehydration
and poor perfusion. Given her TBW of roughly
5.5 L (wt in kg  0.6 L/kg), this should result in
an increase in her serum sodium by approxi-
mately 5 mEq/L. Since the child has had
hyponatremic seizures and is still exhibiting
some central nervous system effect with her leth-
argy, it is prudent to raise the serum sodium by
5 mEq/L so that it will be in the 120–125 mEq/L
range. Since she is hemodynamically stable, it is
also best not to provide an excess of further vol-
ume until the child undergoes imaging to assess
for cerebral edema, especially given the history of
seizures, lethargy, and hyponatremia. By using a
small volume of hypertonic saline, the serum
sodium can be raised in a controlled manner
while further evaluation of the child continues. It
would take about 28 mEq of sodium (TBW 
desired increase in serum sodium = 5.5 L  5
388
mEq/L) to accomplish the desired elevation. Since
each ml of 3 % saline contains about 0.5 mEq of
sodium, a total of 56 ml of 3 % saline could be
infused over approximately 3–4 h.
In addition to this acute management to restore
initial circulation and perfusion and to raise the
serum sodium to a safer level, plans must be
formulated to attend to the patient’s overall vol-
ume and sodium deficit. To prescribe the proper
follow-up intravenous fluid, the patient’s water
and electrolyte deficits at presentation must be
reconciled with her therapy thus far.
The child’s water deficit is 1.2 l, reflecting the
1.2-kg weight loss. She has “maintenance” water
needs of an additional 1 l/day based on her nor-
mal weight of 10 kg. She is having no other
ongoing water losses and has already received
nearly 250 ml in intravenous fluid in the form of
0.9 % NaCl and 3 % NaCl. Her current water
needs are thus 1,950 ml.
The child’s normal “maintenance” sodium
needs are 30 mEq/day (3 mEq/kg/day). She has
lost 1.2 kg of isotonic fluid in body weight that
represents 185 mEq of sodium. In addition, she
has hyponatremic sodium losses that have arisen
as her diarrheal stool that contained sodium was
replaced with water alone. To calculate these
needs, her normal total body water needs to be
multiplied by the difference in her serum sodium
from a normal value of 135 mEq/L. Her TBW is
6 L (TBW = 0.6 L/kg  10 kg) and the difference
in serum sodium is 23 mEq/L (135 – 112 mEq/L);
her hyponatremic losses are therefore 138 mEq
(6 L  23 mEq/L). Total sodium needs are thus
30 mEq of maintenance, 185 mEq of isotonic
losses, and 138 mEq of hyponatremic losses or a
total of 353 mEq. She has already received
52 mEq of sodium from the 400 ml of 0.9 %
NaCl given in the emergency department. Her
current sodium needs are thus just about
300 mEq.
To choose the proper solution for this child, the
deficit of 1,950 ml of water should contain
300 mEq of sodium. This is best approximated
by 0.9 % NaCl with its NaCl content of
154 mEq/L NaCl. In the past, it has been
suggested that half of the fluid and sodium deficit
be replaced over 8 h and the remainder over the
I.F. Ashoor and M.J.G. Somers
ensuing 16 h. Although such a plan can be
followed, there is little evidence that more rapid
correction of the hyponatremia is harmful except
if the patient has been symptomatic with
hyponatremia or has profound asymptomatic
hyponatremia of chronic duration. In these
cases, it is safest to plan to correct the serum
sodium by no more than 12–15 mEq/L over
24 h. More rapid correction has resulted in
osmotic demyelination injury to the brain with
devastating long-term neurologic outcomes
[149–151].
Severe Hypernatremia
With hypernatremia, therapy is again guided by
the clinical situation and provision of intravenous
fluid is usually reserved for those children with
very elevated serum sodium values who are not
considered candidates for oral rehydration ther-
apy. In cases of hypernatremia due to salt poison-
ing, there should be signs of overhydration and
volume expansion. Excretion of sodium should be
enhanced by using a loop diuretic to augment
urine sodium losses and by replacing urine output
with free water. If the patient has significant
underlying renal or cardiac compromise, dialysis
and ultrafiltration may be necessary to correct the
water and electrolyte imbalance [77, 78]. With
hypernatremia and volume expansion from salt
excess, it will be detrimental to provide further
intravenous saline.
In hypernatremia accompanied by volume
loss, any significant alterations in effective circu-
lation should be addressed with 20 ml/kg bolus
infusions of an isotonic crystalloid solution until
effective peripheral perfusion is restored. Then,
further provision of water and sodium should be
provided based on calculated water and sodium
needs. In the majority of cases, with mild eleva-
tions in serum sodium and minimal degrees of
dehydration, the actual calculation of deficits is
probably unnecessary since the child will be
hemodynamically stable and a candidate for
exclusive oral rehydration. In situations where
there is profound hypernatremia or circulatory
compromise, it remains necessary, however, to
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
be able to calculate a free water deficit to tailor
intravenous rehydration therapy. An example of
such a situation is outlined in the following clin-
ical scenario:
After 2 days of refusing to nurse, a 5-kg infant
boy with a viral syndrome presents to an emer-
gency department in shock, 15 % dehydrated with
a weight of 4.25 kg and a serum sodium of
170 mEq/L. He receives 300 ml of 0.9 % NaCl
urgently and further therapy is now planned.
The child has lost 750 g of weight. Since this is
hypernatremic dehydration, there has been loss of
water in excess to salt. Thus, part of the weight
loss represents isotonic losses but a larger pro-
portion represents free water loss. The child’s free
water deficit can be calculated by the equation
½ðserum Na actualÞ=ðserum Na desiredÞ
 total body water  total body water
Substituting the appropriate data for this baby,
½ð170=145Þ  ð0:6  4:25Þ  ð0:6  4:25Þ
¼ ½1:2  2:55  2:55 ¼ 0:51 L
Thus, of this baby’s 750-ml fluid deficit due to
dehydration, 510 ml is free water and 240 ml is
normal saline.
Too rapid correction of the baby’s serum
sodium with free water could result in cerebral
edema as the water infused into the extracellular
space follows osmotic forces and moves into the
intracellular space. In cases of hypernatremia
where the serum sodium exceeds 160 mEq/L, it
is considered safest to correct the serum sodium
by no more than 15 mEq/day. In this boy’s case,
this would mean that correction to a serum
sodium in the normal range would take about
2 days.
If the fluid and electrolyte therapy must be
given intravenously, the appropriate prescription
again depends on calculation of water and
sodium requirements and deficits. His original
fluid deficit was 750 ml and his maintenance
water needs are estimated at 500 ml/day. Thus,
over the next 2 days, the fluid needed to replace
the deficit and provide maintenance water is
389
about 1,750 ml. Of this volume, the baby has
already received 300 ml of fluid in the emergency
department so a net deficit of 1,450 ml now exists.
The baby has maintenance sodium needs of
15 mEq/day (3 mEq/kg/day). His sodium deficit
reflects only the isotonic fluid losses that have
been estimated above at 240 ml of normal saline
or 37 mEq of sodium. Thus, over the next 2 days,
his sodium needs are 67 mEq of which he has
already received more than 45 mEq in the emer-
gency department due to initial volume
expansion.
Initiating an infusion of 30 ml an hour of free
water should result in the slow and steady correc-
tion of the hypernatremia over 2 days by provid-
ing the nearly 1.5 l of free water that the child
requires to replace losses and provide ongoing
needs. The serum sodium should be monitored
every 4 h initially, and if it is falling faster than
desired (about 0.5 mEq/h), then the sodium
should be added to the rehydration fluid.
Fluid and Electrolyte Therapy
with Renal Dysfunction
Impact of Kidney Disease
Children with compromised renal function often
manifest a reduced tolerance for changes in total
body water as well as changes in the composition
or distribution of volume between the intracellular
and extracellular body spaces. Similarly, alter-
ations in electrolyte balance are more likely prob-
lematic because normal homeostatic mechanisms
are frequently perturbed. Especially in the child
with marked nephrosis or significant impairment
in renal clearance, it becomes vital to approach
the provision of fluids and electrolytes with
great care.
As far as fluid therapy is concerned, of utmost
importance is the recognition that the concept of
maintenance fluids or electrolytes presupposes
normal renal function. Roughly two-thirds of
any daily maintenance fluid prescription is to
replace urinary water losses. Similarly, urinary
electrolyte losses figure prominently in daily elec-
trolyte balance. In the setting of oliguria or anuria,
390
provision of maintenance fluids could contribute
to and, potentially, exacerbate volume overload,
and maintenance electrolyte therapy could result
in electrolyte anomalies.
Fluid and electrolyte needs of the child with
renal dysfunction are better considered in the con-
text of the child’s current volume status and elec-
trolyte needs. For instance, in symptomatic
volume depletion with decreased circulatory per-
fusion, volume expansion would be initiated
regardless of urine output. Once volume is
repleted, the child’s needs could be reassessed
along with his current renal function. The child
who is volume overloaded would best be man-
aged by fluid restriction and provision of only
insensible losses of approximately 300 ml/m2.
Insensible fluid losses should be considered
essentially electrolyte free water. The child who
is volume replete should be kept volume replete.
This is most readily accomplished by providing a
combination of insensible losses as free water and
any other volume losses (urine output, diarrheal
stool, surgical drain output, emesis) on an addi-
tional milliliter-for-milliliter basis. If there are
significant ongoing losses from a single source,
the electrolyte composition of this fluid can be
assayed so that the replacement fluid may more
accurately reflect the electrolyte losses. Other-
wise, a solution of 0.45 % NaCl can be used
initially and altered as the clinical situation con-
tinues to develop and further electrolyte determi-
nations are made.
If the child’s volume status or the adequacy of
renal function is difficult to discern initially, it is
best to provide the child with replacement of both
insensible and ongoing losses. This approach
should maintain the child’s current volume status
and allow for further determination of the appro-
priateness of more vigorous hydration or con-
versely fluid restriction as the clinical situation
clarifies. Monitoring the child’s weight on at
least a daily basis and documenting the child’s
total fluid intake and output will also assist in
arriving at a proper hydration regimen.
Assessing the child’s current electrolyte status
and monitoring the loss of electrolytes in the urine
or in any other source of significant output will
help tailor the daily electrolyte prescription. An
I.F. Ashoor and M.J.G. Somers
understanding of the pathophysiology underlying
the child’s renal dysfunction will also be useful.
The child who has profound tubular electrolyte
losses will require more sodium on a daily basis
than the child who is edematous and total body
salt overloaded from his nephrotic syndrome. The
child with chronic renal insufficiency and hyper-
tension mediated by long-standing salt and water
overload may actually benefit from diuretic ther-
apy to remove salt and water rather than any
further volume expansion with saline.
Certainly the provision of supplemental potas-
sium to the child with renal dysfunction must be
done judiciously. The oliguric or anuric child
should receive no potassium until it is well
documented that serum potassium levels are low
or that there are extrarenal potassium losses (for
instance losses from diarrheal stool). The child
with marginal renal function should receive
small amounts of potassium (approximately
1 mEq/kg/day) with at least daily assessment of
electrolyte balance to determine adequacy and
appropriateness of continued potassium
supplementation.
Fluid and Electrolyte Therapy
in the Pediatric Intensive Care Unit
Critically ill children present a challenge to the
clinician attempting to prescribe appropriate fluid
and electrolyte therapy. Oftentimes, there may be
acute kidney injury or multiorgan failure compli-
cating management decisions. With such children,
rote reliance on standard equations or practice
guidelines to prescribe fluid and electrolyte ther-
apy may create significant fluid and electrolyte
anomalies. Rather than prescribing set mainte-
nance requirement of fluid or electrolytes, the
clinician should assess the patient’s individual
fluid and electrolyte needs in the context of the
underlying pathophysiology, the current volume
status, the efficacy of tissue perfusion, the current
ventilatory requirements, and the current renal
function. Whenever there is concern about incip-
ient or exacerbating fluid overload, it is important
to review the volume and type of fluids being
provided. Maximizing the concentration of
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
continuous medication drips and assessing medi-
cation compatibility for simultaneous infusions
are important steps in limiting total daily fluid
input. Initially, it is crucial in these critically ill
children to ascertain that their intravascular space
is replete to help maintain hemodynamic stability.
Once a patient is felt to be intravascularly replete,
maintaining euvolemia by providing insensible
water losses as well as replacing any ongoing
fluid and electrolyte losses should maintain fluid
and electrolyte balance.
Oftentimes, despite a desire to limit fluids in
the critically ill child, medication requirements,
nutritional needs, and hemodynamic insufficiency
may result in very large daily fluid loads. There
may also be situations in which increased vascular
permeability causes a critically ill child to become
massively volume overloaded but with a
decreased effective circulating volume. In other
words, renal and tissue perfusion may be sluggish
because fluid has moved from the intravascular
space into the interstitial space. In this setting,
there may be a need to continue to administer
large volumes of fluid to maintain circulatory
integrity with the knowledge that such infusions
will only exacerbate the total body fluid overload.
Aggressive diuretic therapy may prove useful
especially if renal function is not compromised.
Combination diuretic therapy utilizing agents that
work at separate sites along the renal tubule may
be necessary. Ultimately, the use of either periodic
or continuous ultrafiltration may be beneficial to
these patients by allowing ongoing fluid adminis-
tration but limiting the daily imbalance between
fluid intake and output. Ultrafiltration may be
accomplished via peritoneal dialysis, by intermit-
tent hemodialysis with ultrafiltration or by utiliz-
ing one of the slow continuous ultrafiltration
techniques now known as continuous renal
replacement therapy (CRRT)). The recognition
that volume overload has a deleterious effect on
many aspects of patient management and seems to
be a strong prognostic indicator of poor ultimate
outcome suggests that early ultrafiltration should
be considered in critically ill patients [152].
If ultrafiltration is initiated, extreme vigilance
is necessary to prevent exacerbation of intravas-
cular depletion and the development of prerenal
391
azotemia or frank renal failure. Special care must
be taken with the continuous modalities to insure
that ultrafiltration rates are periodically reassessed
and readjusted. Furthermore, because the electro-
lyte losses that accompany the ultrafiltration of
fluid are isotonic, the electrolyte content of
infused fluids must be adjusted to match the com-
position of the ultrafiltrate, especially if there is no
component of dialysis ongoing that may blunt the
development of serum electrolyte anomalies. As a
result, serum electrolyte values need to be
followed in a serial fashion with periodic review
and readjustment of the composition of supple-
mental intravenous fluids.
Abnormalities in Serum Sodium
Complicated by Kidney Injury
Because of the important contribution of serum
sodium to serum osmolality, alterations in serum
sodium, especially coupled with alterations in
BUN related to renal failure, can complicate the
usual approach to a child with fluid and electrolyte
anomalies. Generally, there are greater concerns
with hypernatremia and renal failure since the
need to correct the sodium in a slow fashion can
be problematic when renal replacement therapy
needs to be initiated for clearance of urea.
Balancing the correction of sodium and the
hyperosmolar state with the clearance of urea
requires a carefully considered plan that is
grounded in a firm understanding of fluid and
electrolyte homeostasis.
In most cases of hypernatremia related to
severe dehydration, some degree of acute kidney
injury is present. This renal dysfunction is usually
“prerenal” in nature, a result of a decreased effec-
tive circulating volume rather than an intrinsic
glomerular or tubular disorder. Most often, in the
course of rapid restoration of perfusion and early
rehydration, urine output increases and azotemia
begins to resolve.
Alternatively, there are occasional cases in
which due to intrinsic renal dysfunction or acute
tubular necrosis, the renal insufficiency will not
respond to volume infusion, and, in fact, the pro-
vision of excess volume may contribute to
392
significant volume overload. In these cases, there
may be need to consider some form of renal
replacement therapy to assist in the controlled
correction of fluid and electrolyte derangements,
especially if the renal failure is oliguric or anuric
in nature. Such an example is detailed in the
following case study:
A 15-year-old boy presents with several weeks
of polyuria, severe weight loss, fatigue, and poor
oral intake. He is diagnosed as having diabetes
mellitus with ketoacidosis by his pediatrician and
referred to an emergency department for manage-
ment. At this point, his serum sodium is
154 mEq/L, his creatinine is 3.0 mg/dl, and his
BUN is 30 mg/dl. In the emergency department,
the child receives several bolus infusions of nor-
mal saline supplemented with sodium bicarbon-
ate and is started on an insulin drip. He is
admitted and continues to receive brisk intrave-
nous hydration with normal saline with bicarbon-
ate supplementation per a practice guideline for
treating children with diabetic ketoacidosis. He is
noted to be oliguric and this does not improve
with several more hours of hydration with normal
saline following the guideline hydration recom-
mendations. The next morning, laboratory values
reveal a serum sodium of 165 mEq/L, a creatinine
of 4.5 mg/dl, and a BUN of 50 mg/dl. He has made
only 75 ml of urine in the last 8 h and is develop-
ing some mild peripheral edema.
In this case, the renal insufficiency and poor
urine output have complicated the usual manage-
ment of diabetic ketoacidosis and has exacerbated
an underlying hypernatremia. Given the patient’s
evolving renal failure, it is not feasible to provide
the necessary volume of free water to correct the
hypernatremia without contributing to further
volume overload. Because of the apparent pro-
gressive renal failure, it would also be useful to
correct the hypernatremia in case dialysis
becomes necessary for urea clearance. By
performing controlled ultrafiltration on the
patient and replacing back the volume
ultrafiltered with free water, the serum sodium
could be corrected without exacerbating the vol-
ume status.
With a serum sodium of 165 mEq/L and an
estimated TBW of 42 L (70 kg  0.6 L/kg), this
I.F. Ashoor and M.J.G. Somers
boy has free water needs of 7.5 L to lower his
serum sodium to the 140 mEq/L range
([165/140  42]  42). Since the patient is now
significantly hypernatremic and has been subject
to various fluid and electrolyte shifts as his dia-
betic ketoacidosis has been treated, it would be
prudent to correct his serum sodium by no more
than 10–12 mEq/day over the course of 3 days.
Thus, if the boy undergoes ultrafiltration with a
goal of 2.5 l removed daily, and the ultrafiltration
volume each day is replaced back totally as free
water, the serum sodium should be in the normal
range in 3 days time. The ultrafiltration goal
could be achieved over the course of a few hours
each day if the patient were hemodynamically
stable or over a more prolonged period of time
each day if there were concerns regarding hypo-
tension. Thus, either a conventional hemodialysis
setup could be used for relatively rapid ultrafil-
tration only or a continuous filtration circuit for
either rapid or slow filtration.
Since the fluid removed in ultrafiltration is
isonatremic to the serum sodium, the sodium con-
centration of each liter of ultrafiltrate should mir-
ror the serum sodium concentration at the time of
ultrafiltration. Thus, on the initial day of ultrafil-
tration, each liter of ultrafiltrate would contain a
sodium content of 165 mEq/L. By providing back
the volume ultrafiltered each day as free water, the
serum sodium content could be expected to fall, in
this case, by about 8–10 mEq/L/day.
It is important to recognize that free water must
be provided back to the patient to make up for the
ultrafiltration losses. Otherwise, since the ultra-
filtrate is isotonic, there will be no change in the
serum sodium concentration, and the ultrafiltra-
tion may potentially exacerbate the renal failure
by depleting the intravascular space and the effec-
tive circulating volume.
Moreover, it is also important to recognize that
the boy’s overall daily fluid needs will be greater
than the daily ultrafiltration volume alone since
maintenance fluid requirements and any ongoing
fluid losses must also be considered. Since the boy
is in renal failure, his maintenance fluid needs can
be scaled back to insensible losses of 300 ml/m2/
day and, in this case, there are no ongoing losses.
Thus, each day for the next 3 days, this 70-kg
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
patient needs to receive approximately 500 ml/day
of insensible losses and 2,500 ml/day of ultrafil-
tration replacement or a total of 3,000 ml/day. His
maintenance sodium requirements are 3 mEq/kg/
day. Although it may seem counterintuitive to
provide a hypernatremic patient with mainte-
nance sodium, disregarding these requirements
will result in a more rapid correction of the
hypernatremia than desired. If the child were to
receive a saline infusion of 0.45 % NaCl at a rate
of 125 ml/h, this will provide just over 3 mEq/kg/
day of sodium in a total volume of 3 L/day.
If the child with hypernatremia has profound
renal failure and requires dialysis for urea clear-
ance, the dialysis prescription must take into
account the need to correct the serum sodium
slowly. Normally, regardless of the modality of
renal replacement therapy, most dialysate contains
sodium isotonic to the normal serum sodium
range. It may prove detrimental, however, to dia-
lyze a patient who is very hypernatremic against a
dialysate with a sodium concentration that is
30 mEq/L lower than the patient’s serum sodium
concentration. The diffusional gradient during
dialysis would lead to more rapid correction of
the serum sodium than the desired drop of approx-
imately 1 mEq every 2 h.
Although most hemodialysis machines can be
readjusted so that the dialysate produced will have
a sodium content as high as the low to mid-150s,
this still may not reduce the gradient sufficiently
in cases of severe hypernatremia. In those situa-
tions, by maximizing the sodium concentration of
the dialysate and by performing dialysis for lim-
ited amounts of time, one could minimize the drop
in serum sodium. Still, there would need to be
frequent assessments of the serum sodium con-
centration, and overall clearance may need to be
sacrificed to prevent too rapid correction of the
serum sodium and a rapid concomitant decrease in
the serum urea that may increase the chances for
dialysis disequilibrium.
Alternatively, a continuous hemodiafiltration
technique such as continuous venovenous
hemodiafiltration (CVVHDF) could be
performed. By asking the hospital pharmacy to
increase the sodium content of the dialysate and
replacement fluid to within 10–12 mEq/L of the
393
serum sodium concentration, the diffusional gra-
dient for sodium clearance could be minimized.
Then, by making appropriate adjustments in the
sodium content of the dialysate as the serum
sodium falls, the serum sodium levels could be
reduced gradually by 10–12 mEq/L/day, while at
the same time adequate urea clearance and ultra-
filtration for most situations would be achieved.
Peritoneal dialysis has also been used in cases
of severe hypernatremia [153–155]. Again, the
concentration of sodium in the dialysate may
need to be adjusted upwards in severe
hypernatremia to prevent too rapid clearance of
sodium. In addition, since the degree of clearance
and ultrafiltration may not be as precisely con-
trolled as with hemodialysis or hemodiafiltration,
frequent assessment of electrolyte values will be
necessary. Manipulation of dwell volumes and
dwell times will also influence overall clearance
and the use of smaller dwell volumes for longer
periods of time will help to minimize sodium
clearance.
In contradistinction to hypernatremia, since
hyperosmolality is less common with
hyponatremia, in some ways it is easier to employ
renal replacement therapy in the setting of severe
hyponatremia and concomitant renal insuffi-
ciency. Again, the focus needs to be on the rapid-
ity of the correction of the serum sodium. In
conditions of severe but asymptomatic
hyponatremia of some chronicity, the rate of cor-
rection of serum sodium should parallel the rate of
correction recommended in hypernatremia 
approximately 10–12 mEq/L/day. Correction of
chronic hyponatremia at a more rapid rate has
been associated with the development of central
pontine myelinolysis.
All of the manipulations described above for
hypernatremia and renal failure can be utilized
with hyponatremia and renal failure, with the
understanding that the dialysate sodium concen-
tration should now not exceed the serum sodium
value by 10–12 mEq/L. Conventional hemodial-
ysis machines can be adjusted to produce dialy-
sate with a sodium concentration as low as the
mid-120s. In the very rare situation in which a
child with profound hyponatremia (<110 mEq/L)
was being hemodialyzed, brief hemodialysis runs
394
may be necessary initially to prevent too rapid
correction of the serum sodium level and the
attendant risk of central pontine myelinolysis. If
dialysate is being custom prepared for peritoneal
dialysis or hemodiafiltration, precise alterations in
the electrolyte content can be made more readily
to reduce the sodium gradient.
The local resources, the training of ancillary
staff, the unique circumstances of each patient,
and the comfort of the clinician with different
modalities of renal replacement therapy will
guide the choice of therapy when faced with
renal failure and significant serum sodium anom-
alies. The actual modality of renal replacement
therapy utilized is less important than careful
attention to the rate of correction of the electrolyte
anomaly, to the rate of urea clearance being
achieved, and to the clinical response of the
patient to ongoing therapy.
Potassium
There are several homeostatic mechanisms in
place to maintain the usual high concentration of
potassium in the intracellular space. These rely on
the sodium–potassium ATPase found on the cell
membrane, the effects of insulin and adrenergic
hormones on transcellular potassium movement,
and the effects of nephron mass, GFR, hydration,
urinary flow, and especially aldosterone on renal
potassium excretion and net body potassium
balance.
In normal circumstances, potassium distribu-
tion is extremely well regulated since the gradient
between the intracellular and extracellular spaces
plays a crucial role in the process of nerve excita-
tion and myocyte contraction. This explains why
nearly 99 % of total body potassium can be found
intracellularly and why aberrations in potassium
handling that disrupts the usual gradient can be
not only detrimental to growth and development
but also life threatening if cardiac conduction and
contractility or respiratory muscle function is sig-
nificantly affected. The intracellular localization
of most body potassium stores also explains why
serum potassium levels are not a good estimate of
total body potassium stores, and the clinician
I.F. Ashoor and M.J.G. Somers
needs to consider the possibility of transcellular
redistribution when interpreting any aberrant
serum potassium level before making decisions
to supplement or restrict its provision.
In children, abnormalities in potassium homeo-
stasis are uncommon but, as seen with other elec-
trolytes like sodium, physiologic perturbations that
may occur with acute illness or certain chronic
medical conditions can interfere with normal regu-
latory processes and lead to clinically significant
imbalances in potassium. As with other electrolyte
issues, the ramifications of any abnormality gener-
ally reflects on how quickly it has evolved, how
extreme it has become, and whether other medical
conditions are more likely to be adversely affected
or even perpetuate the abnormality.
Hyperkalemia
Hyperkalemia is usually defined as a serum potas-
sium concentration exceeding 5.5 mEq/L. In most
children, intact homeostatic mechanisms keep
serum potassium levels less than 5 mEq/L. With
infants, there can be a higher range of normal
potassium values because of baseline reduced
GFR and some insensitivity to aldosterone, with
levels as high as 6 mEq/L not uncommon. There is
generally little clinical consequence to serum
potassium values up to 6 mEq/L, and most children
rarely manifest any significant symptoms related to
hyperkalemia until serum potassium levels exceed
7 mEq/L. At that level, there may be lethargy and
discernible muscle weakness or even paralysis,
often initially affecting the legs. There can also be
sporadic heart palpitations or arrhythmias that can
progress to asystole. As with any electrolyte aber-
ration, high serum potassium levels should be con-
sidered in the clinical context of the child’s current
medical status, and significantly elevated levels
require urgent assessment and management tai-
lored to take into account any comorbid conditions.
Causes of Hyperkalemia in Children
The causes of hyperkalemia in children can be
grouped into several broad categories based on
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
Table 11 Causes of hyperkalemia in children
Category Cause
Pseudohyperkalemia Technical issue with
phlebotomy
Hemolysis of specimen in
handling
Release of potassium from
leukocytes or platelets in blood
sample
Increased potassium IV fluids, peripheral nutrition,
intake or potassium-containing
medications
Potassium-based table salt
substitutes
Transcellular Conditions with loss of cell
potassium membrane integrity
redistribution Rhabdomyolysis
Tumor lysis
Hemolytic states
Acidosis
Periodic paralysis
Decreased potassium Reduced GFR
excretion Decreased effective circulating
volume
Impaired tubular secretion
Reflux nephropathy
Obstructive uropathy
Type IV renal tubular
acidosis
Sickle cell nephropathy
Impaired
renin–angiotensin–aldosterone
axis
Congenital adrenal
hyperplasia
Pseudohypoaldosteronism
Drug effect (ACE/ARB,
eplerenone, spironolactone,
aliskiren)
underlying pathophysiology (Table 11). In chil-
dren, hyperkalemia may often be spurious and
related to technical issues commonly seen with
pediatric phlebotomy. Such pseudohyperkalemia
needs to be distinguished from true hyperkalemia
that usually arises related to a disturbance in the
renal excretion of potassium or movement of nor-
mally sequestered intracellular potassium into the
extracellular space. Hyperkalemia in children is
rarely due to excess potassium ingestion alone and
sustained high potassium levels usually point
395
towards some impairment in renal potassium
excretion.
Pseudohyperkalemia
In pseudohyperkalemia, the elevated potassium
level does not reflect the child’s true potassium
balance. Especially in small children, there can be
hemolysis of a blood sample due to technical
reasons, with release of erythrocyte potassium
stores [156, 157]. Smaller gauge needles must
often be used or samples withdrawn through
indwelling small gauge intravenous catheters,
with the need to apply significant suction to the
attached syringe to obtain necessary sample vol-
ume. These techniques are up to sixfold more
likely to result in a hemolyzed sample than with
the use of vacuum flow devices [156].
Children are also often less cooperative with
the phlebotomy procedure and may require phys-
ical restraint or may repetitively contract muscles
in an extremity while undergoing phlebotomy,
leading to local potassium release from myocytes
and a hyperkalemic blood sample not reflective of
true systemic potassium levels. Additionally,
hyperventilation seen with intense crying can
mediate a very acute respiratory alkalosis with
potassium shifting out of cells and a rapid
increase in serum potassium by as much as
1 mEq/L [156, 158].
In children with marked leukocytosis or
thrombocytosis such as seen with leukemias or
myeloproliferative disorders, there may also be
potassium egress from white cells or platelets as
the blood sample clots. A plasma potassium assay
decreases the chances of such a confounding
result.
In most cases of pediatric pseudohy-
perkalemia, an elevation in serum potassium
levels is unexpected on clinical grounds, and
details of the phlebotomy procedure or laboratory
confirmation of hemolysis in the sample allow the
result to be put into proper perspective. It does
become more difficult in children with preexisting
alterations in homeostatic mechanisms due to
acute illness or chronic disease to disregard high
potassium levels, and although there can always
be an element of pseudohyperkalemia, it is judi-
cious to repeat another sample to determine that a
396
normal level exists before attributing high levels
to pseudohyperkalemia exclusively.
Increased Potassium Intake
In normal children, even very large dietary potas-
sium loads are unlikely to result in sustained
hyperkalemia. Homeostatic mechanisms that pro-
mote potassium influx into cells and aldosterone-
mediated enhanced kaliuresis tend to prevent
hyperkalemia from intake of most foods. In
small children with smaller volumes of distribu-
tion, exposure to some nutritional supplements
such as salt substitutes that can contain up to
80 mEq potassium chloride/teaspoon can result
in potassium loading that exceeds by many fold
normal dietary potassium intake and can over-
whelm homeostatic mechanisms [159].
Children with impaired GFR may, however,
require dietary potassium restriction, though gut
excretion of potassium increases with chronic
renal dysfunction as a compensatory mechanism,
even in the setting of children maintained chron-
ically normokalemic on dialysis [160].
In hospitalized children, iatrogenic errors in the
concentration of potassium in intravenous fluids,
peripheral nutrition, or certain intravenous medi-
cations can result in the inadvertent provision of
large potassium loads, and hospitalized children
receiving potassium supplementation either orally
or in some intravenous form need to be monitored
for the development of hyperkalemia.
Transcellular Redistribution of Potassium
Breakdown of normal tissue or rapid cell lysis can
result in the release of large amounts of intracel-
lular potassium along with other intracellular elec-
trolytes into the extracellular space and plasma
water. This often occurs in the setting of rhabdo-
myolysis from crush injury sustained in accidents
or natural disasters or after periods of extreme
exercise, in tumor lysis syndrome in leukemia or
lymphoma, or with severe hemolytic processes.
There may often be accompanying acute kidney
injury that exacerbates the hyperkalemia and com-
plicates its management.
Transcellular redistribution may also occur in
the absence of cellular damage. In metabolic aci-
dosis, the movement of hydrogen intracellularly
I.F. Ashoor and M.J.G. Somers
causes extracellular potassium shift to maintain
electroneutrality. In children, metabolic acidosis
is often mediated by decreased effective circulat-
ing volume from either severe dehydration or
infection. This volume imbalance may exacerbate
the hyperkalemia by reducing GFR and reducing
renal potassium excretion. The restoration of ade-
quate intravascular volume and tissue perfusion
generally corrects the acidosis and hyperkalemia.
A similar transcellular shift can be seen with insu-
lin deficiency and diabetic ketoacidosis, though
the hyperkalemia can sometimes be blunted early
in this process by urinary potassium losses from
an osmotic diuresis and excreted keto acids.
In hyperkalemic periodic paralysis,
hyperkalemia is also related to transcellular relo-
cation of potassium. This is an autosomal domi-
nant condition in which a voltage-gated sodium
channel near the neuromuscular junction is
affected and allows for ongoing movement of
potassium ions from the muscle into the blood-
stream in response to stimuli that usually reduce
potassium flux [161]. Hyperkalemic periodic
paralysis may present as early as infancy and
need to be considered in any child with
hyperkalemia and muscle weakness.
Abnormalities in Renal Excretion
Intrinsic or Acquired Glomerular or Tubular
Dysfunction
Intrinsic anomalies in the renal handling and
excretion of potassium or acquired conditions
impairing normal excretion are a common cause
of significant pediatric hyperkalemia. Although
aldosterone effect on the distal nephron is a key
to potassium homeostasis, any renal disease that
impairs effective glomerular filtration will limit
absolute potassium clearance. As GFR falls in a
child with chronic kidney disease, the ability to
excrete potassium tends to stay relatively well
compensated, especially when the GFR exceeds
30 ml/min/1.73 m2. With more profound func-
tional impairment, especially in the setting of
lower urine output, hyperkalemia may be more
commonly encountered.
With children with obstructive uropathy or
reflux nephropathy, there is often a distal tubular
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
resistance to aldosterone that results in a type IV
renal tubular acidosis (RTA) and associated
hyperkalemia, even when the GFR is well pre-
served. In infants, there can also be immature
tubular responsiveness to aldosterone that is
developmental during early life, and this helps to
explain why babies have a tendency towards
higher baseline serum potassium levels than
older children. In children with sickle-cell disease
who have impaired medullary blood flow and
ensuing hypoxic renal tubular injury, there is
also a tendency towards impaired aldosterone sen-
sitivity over time.
One study of children with acute febrile urinary
tract infection but no concomitant urinary tract
obstruction or chronic kidney disease demonstrated
significantly increased rates of hyperkalemia com-
pared to children with other febrile illnesses
[162]. This would suggest that, even in structurally
normal distal renal tubules, conditions with intersti-
tial inflammation such as seen with pyelonephritis
can impact normal potassium handling.
Certain drugs, most notably potassium-sparing
diuretics, can similarly impact normal tubular
renal excretion of potassium. These medications
are often used for disease states such as heart or
liver failure in which effective circulating volume
is also impaired and GFR may be suboptimal,
further exacerbating potential kaliuresis.
Decreased Effective Circulating Volume
A much more common reason to see
hyperkalemia in children is a state of low effective
circulating volume that results in either ineffective
tissue perfusion and the generation of a metabolic
acidosis or sodium and water avidity in the prox-
imal portion of the nephron. As a result, there is
decreased distal flow of filtrate in the nephron and
a decreased concentration of sodium in any filtrate
actually presented to the distal nephron. This leads
to what is often termed a functional renal tubular
acidosis or impaired aldosterone-mediated
kaliuresis and acid excretion that is not related to
any inherent anomaly in the channels or receptors
in the distal nephron. Especially in young chil-
dren, there is an increased tendency to develop
significant dehydration and hemodynamic insta-
bility with acute illness, and it is not uncommon to
397
see significant hyperkalemia related to effective
volume depletion alone and the effects described
above [163].
In some children with chronically marginal
hydration and a diet that is low in sodium content,
this same situation of low distal delivery of
sodium exists and can result in high potassium
levels. This is most often seen in babies with
feeding difficulties since both breast milk and
formula contain little sodium.
Decreased Activity
of Renin–Angiotensin–Aldosterone System
Although infrequently encountered, endocrine
anomalies impacting the RAAS can result in
infants or young children presenting with
hyperkalemia. Congenital adrenal hyperplasia
(CAH), and especially its most common form
21-hydroxylase deficiency, results in impaired
mineralocorticoid production, salt wasting, and
hyperkalemia. CAH is part of the newborn screen
across the entire United States and is included in
newborn screening in many foreign countries,
leading to its identification prior to significant
electrolyte anomalies in many infants. Primary
adrenal insufficiency is a rarer condition than
CAH and affected children may present with
hyperkalemia, though hyponatremia and hypoten-
sion are more common [164].
Pseudohypoaldosteronism, an abnormality of
mineralocorticoid receptor activity, is an even
rarer endocrine condition. Children present char-
acteristically with hyponatremia, hyperkalemia,
and metabolic acidosis, but serum aldosterone
levels are elevated, underscoring that the issue is
in the cell receptor and not in mineralocorticoid
production. An autosomal dominant form only
affects the kidney and, over time, affected chil-
dren may show spontaneous improvement due to
maturational enhancement of tubular sodium
transport. In the autosomal recessive form, there
is generally pronounced distal tubular epithelial
sodium channel dysfunction that presents early in
childhood and requires significant ongoing
sodium supplementation [165].
Children being treated with spironolactone,
eplerenone, aliskiren, or one of the angiotensin-
converting enzyme inhibitors or angiotensin
398 I.F. Ashoor and M.J.G. Somers

receptor blockers will also have decreased miner- Table 12 Diagnostic studies to assess unexplained
alocorticoid production or activity and may hyperkalemia in children
develop hyperkalemia directly related to their Laboratory test Key findings
use. Many of these drugs are used in children Complete blood count, Hemolysis, thrombotic
with medical conditions that already predispose platelet count microangiopathy, blood
dyscrasias
to hyperkalemia due to adverse affects on effec-
Creatinine Reduced GFR
tive volume, GFR, and baseline renal tubular han-
Electrolytes Metabolic acidosis or other
dling of solute. electrolyte aberration
Urine electrolytes and Evidence of inappropriate
urine creatinine (best urinary K excretion
Evaluation of Hyperkalemia in Children done concomitantly with (UK < 20 mEq/L in setting
serum electrolytes and of hyperkalemia)
creatinine) Evidence of reduced
In many children, the clinical presentation or effective volume or
known preexisting medical conditions will point reduced distal tubule
clearly to the underlying etiology of the sodium delivery
hyperkalemia and its proper treatment, and there (UNa < 20 mEq/L)
Lactic dehydrogenase Elevated levels suggesting
is little need for an extensive diagnostic evalua-
hemolysis
tion. For instance, children with tumor lysis, rhab-
Creatine kinase Elevated levels suggesting
domyolysis, structural or functional kidney muscle injury
disease, or markedly decreased effective circulat- Serum aldosterone and Aberrant levels suggest
ing volume all are likely to have correction in their plasma renin activity endocrine abnormality
potassium imbalance as the underlying condition
is treated. In children with no obvious reason for
hyperkalemia and normal renal function and vol-
ume status, a high index of suspicion for potassium in conjunction with reabsorption of
pseudohyperkalemia must exist and potassium sodium, it is often necessary to also measure uri-
values should be confirmed prior to any further nary sodium and urinary osmolality to more
evaluation. completely assess the appropriateness of random
In some children, however, the cause of a per- urine potassium results. Low random urinary
sistently high potassium level will not be obvious, sodium values (<20 mEq/L) demonstrate that
and it is important to assess the renal handling of there is renal sodium avidity, and this may limit
sodium and potassium as well as the renin–angio- the potential secretion of potassium in the distal
tensin–aldosterone system carefully. The direc- nephron even in the face of a normal hormonal
tion of any further evaluation is then guided both milieu and normal renal tubular cell receptors and
by the medical history and initial results from channels. Measures that improve the effective
typical screening laboratories of blood and urine volume and provide more sodium for distal tubu-
(Table 12). lar delivery augment kaliuresis.
In most children, hyperkalemia should result in For some time there was interest in calculation
a random urinary potassium level that exceeds of the transtubular potassium gradient (TTKG) to
20 mEq/L and often exceeds 40 mEq/L. Very help determine if there was appropriate mineralo-
high random urinary potassium values (>80 to corticoid effect in states of hyperkalemia in pedi-
100 mEq/L) almost always point towards intact atric patients [166]. The TTKG estimated the ratio
mechanisms to achieve kaliuresis, and diagnostic of the potassium concentration at the end of the
focus should shift to an issue with potassium intake cortical collecting tubule, the site responsible for
or cellular release rather than renal excretion. most of potassium secretion, to blood potassium
Since random urinary potassium levels may be levels. The TTKG was predicated on the assump-
affected by the degree of urinary concentration tion that changes in the osmolality in the
and by the ability of the distal tubule to secrete collecting duct were only due to water
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . . 399

reabsorption, but there is now evidence of urea Table 13 Treatment of hyperkalemia in children
cycling in the collecting duct that invalidates this Potassium level <6 Limit potassium intake
premise [167]. Accordingly, the TTKG is no lon- mEq/L Optimize effective volume
ger considered a reliable assessment of mineralo- Potassium levels 6–7 Stop potassium intake
corticoid effect and, when there is clinical mEq/L Optimize effective volume
No ECG changes or only Consider loop diuretic
suspicion of an endocrine issue interfering with peaked T waves therapy such as
potassium handling, serum aldosterone and If other ECG changes, furosemide 1 mg/kg q 6 h
plasma renin activity should be measured. consider therapy for Consider sodium
K > 7 mEq/L polystyrene sulfonate
1 g/kg up to 40 g q 4 h
given po, pg, or pr
Treatment of Hyperkalemia in Children Potassium levels >7 Stop potassium intake
mEq/L Optimize effective volume
The degree of potassium elevation and its likeli- Nebulized beta agonist
such as albuterol
hood to cause immediate cardiac arrhythmias
Calcium gluconate (10 %):
guide the urgency of treatment and the therapies 10 mg/kg IV to stabilize
employed. Specific interventions range from lim- myocardium
iting further potassium intake, to facilitating intra- Insulin and glucose:
0.1–0.3 units/kg regular
cellular shifts of potassium from the extracellular
insulin and 2–4 ml/kg D25
space, to augmenting potassium excretion in the Consider sodium
urine or stool, to actually removing potassium bicarbonate (only after
from the extracellular space by dialysis. Outside calcium provision)
1 mEq/kg
of the significant clinical evidence that exists to
Sodium polystyrene
demonstrate that dialysis is an effective way to sulfonate 1 g/kg up to 40 g
correct hyperkalemia and prevent life-threatening q 4 h given po, pg, or pr
complications, there have been few pediatric- Consider loop diuretic
therapy such as
specific studies that have addressed the overall
furosemide 1 mg/kg IV q
efficacy of some of these maneuvers, although 6h
all of them are commonly employed in clinical Urgent dialysis if no
practice [168] (Table 13). improvement or
concomitant significant
renal failure
General Approach to Therapy
In children with potassium levels >6.5 mEq/L or
specific immediate clinical concern for cardiac fibrillation, there may come to be a sinusoidal
effects of hyperkalemia prior to the laboratory pattern as the QRS complex merges with the T
value having returned, an electrocardiogram wave. Not all children with hyperkalemia will
should be obtained to look for abnormal atrial manifest ECG changes and absence of ECG
(P wave) or ventricular (QRS complex) depolari- change does not preclude the need for potential
zation or abnormal repolarization (T wave). In therapy for significant potassium elevation.
children, ECG changes are more likely to be In children with no ECG changes or peaked T
related to serum potassium levels than in adults, waves alone and more moderate levels of
who are more prone to preexisting cardiac con- hyperkalemia (<7 mEq/L), initial therapy
duction abnormalities or cardiovascular disease. includes limiting further potassium intake and
As hyperkalemia develops, initial ECG findings promoting potassium loss in the stool with an
include tall, peaked, symmetric T waves and enteral cation exchange resin, with consideration
shortened QT intervals. With more severe of augmenting urinary potassium losses with the
hyperkalemia, P waves become flattened, PR use of a loop diuretic.
intervals prolonged, and QRS complexes wid- In children with ECG changes other than
ened. Eventually, as a harbinger to ventricular peaked T waves or with more pronounced
400
hyperkalemia (>7 mEq/L), calcium infusion is
used to stabilize the cardiac membrane excitabil-
ity that comes with hyperkalemia. Although its
protective effects begin quickly, its duration may
be short-lived and further infusions may be nec-
essary. Concomitantly, therapies that promote the
shift of potassium into the intracellular space such
as nebulized beta agonists or intravenous insulin
and glucose are begun and enteral cation
exchange resin given.
In children unresponsive to such maneuvers or
with concomitant severe renal dysfunction, dialy-
sis may be necessary. Generally, hemodialysis is
the preferred modality to reduce potassium levels
most quickly and in the most controlled fashion.
In some centers, local resources may make forms
of CRRT a better option than conventional hemo-
dialysis. Although peritoneal dialysis can be used,
potassium loss will be less efficient and less well
controlled.
Therapies Redistributing Potassium
These therapies are rapid in onset but generally
have limited duration since no net potassium is
removed from the child and, as the therapeutic
effect of intervention wanes, potassium can redis-
tribute back to the extracellular space. As a result,
most of these therapies are done along with other
maneuvers to either remove potassium from the
body or to adequately reverse whatever is causing
the hyperkalemia to develop. By themselves, they
are less effective in providing a management
solution.
Since securing intravenous access in young
children can be more problematic than with ado-
lescents or adults, using inhaled beta agonists can
be an attractive initial option to move potassium
intracellularly. This maneuver should be avoided
in children manifesting any preexisting cardiac
arrhythmia and children should be on a cardiac
monitor during its provision. Tachycardia and
tremors are common, but are usually short-lived.
Reductions of serum potassium concentrations by
1.5 mEq/L have been obtained as quickly as
within an hour [169]. Such therapy has been
shown to be safe and effective even in premature
infants, with no differences in heart rate, CNS
symptoms, ECG anomalies, hyperglycemia, or
I.F. Ashoor and M.J.G. Somers
intraventricular hemorrhage when two nebuliza-
tions with albuterol were compared to two saline
nebulization treatments [170].
With intravenous access, other temporizing
maneuvers such as infusion of insulin and glucose
or systemic alkalinization with sodium bicarbonate
can be attempted. Rates of these infusions must be
calculated on a case-by-case basis according to the
child’s weight, given the broad spectrum of body
sizes in pediatric patients. If insulin is provided,
glucose should be given concomitantly to prevent
hypoglycemia, and the potassium level should start
to fall within 15 min of insulin provision with peak
insulin effect by an hour. When sodium bicarbon-
ate is infused repetitively, the risk of developing
hypernatremia exists.
There has been limited experience with a solution
containing fixed concentrations of calcium gluco-
nate, insulin, dextrose, and sodium acetate in treating
hyperkalemic children [171]. An advantage to such a
solution is that it could be readily prepared by hos-
pital pharmacies to be available in hyperkalemic
emergencies and could then be infused continuously
to allow for more sustained effect, although the caus-
tic properties of such a solution may prevent its
provision through peripheral vasculature.
Therapies Removing Potassium from
the Body
Diuretics – Although loop and thiazide diuretics
can promote kaliuresis, given that a large propor-
tion of children with hyperkalemia will have
decreased effective circulating volume or renal
dysfunction, the effectiveness of diuretic therapy
in most hyperkalemic children is limited, espe-
cially in the urgent setting. In children with ade-
quate effective volume, using these diuretics and
maintaining a diuresis with ongoing optimization
of effective volume could be considered as an
adjunct. Such therapy can especially be consid-
ered in children with persistently high but clini-
cally less urgent hyperkalemia (5.5–6.5 mEq/L).
Enteral cation exchange resins – Polystyrene
sulfonates have been used for decades in children
with hyperkalemia. Dissolved in water and taken
orally or given as a retention enema, they work by
exchanging a counterion such as sodium or
calcium for potassium across the large intestine.
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
The potassium polystyrene sulfonate complex that
results cannot be digested and gets excreted in
stool, thereby effectively removing potassium
from the body. Each gram of resin may bind as
much as 1 mEq of potassium. During this process,
the resin releases an equivalent amount of its coun-
terion, and provision of large amounts of these
resins can lead to sodium or calcium imbalances.
To prevent constipation or fecal impaction,
polystyrene sulfonates were mixed for many
years with sorbitol that would serve as an osmotic
laxative. Reports of colonic necrosis and perfora-
tion in patients receiving such mixtures, espe-
cially in postsurgical settings where there was
impaired gastrointestinal mobility, prompted the
FDA to warn that sodium polystyrene sulfonate
should not be administered concomitant with sor-
bitol [172]. To counteract its constipating effects,
polystyrene powder or powder mixed with water
can be given with lactulose or polyethylene glycol
3,350 preparations as laxatives.
Although widely used in children with
hyperkalemia both acutely and chronically, one
comprehensive review found no randomized evi-
dence for the efficacy of these binders in the
emergency situation [173], and most of the limited
studies of its efficacy have been in individuals
with chronic kidney disease or after multiple
doses [173, 174]. Although the only therapy
short of dialysis that can remove potassium from
the body, in most hyperkalemic children, how-
ever, its provision in the setting of significant
hyperkalemia is still regarded as a reasonable
therapy by most pediatric clinicians.
Hypokalemia
Hypokalemia is usually defined as a serum potas-
sium level less than 3.5 mEq/L, although, in most
children and adolescents, levels between 3 and 3.5
mEq/L are asymptomatic, and some reference
laboratories even set the lower limit of normal in
this range. There may also be measurement dif-
ferences between samples processed by conven-
tional laboratory AutoAnalyzers and samples
assayed by point-of-care blood gas analyzers that
are often used in critical care areas for rapid
401
turnaround of results. One study of 60 children
demonstrated that samples done by blood gas
analyzers averaged nearly 0.5 mEq/L lower than
conventional assays [175].
Clinically, hypokalemia is encountered more
commonly than hyperkalemia, especially in hos-
pitalized children. In studies of children in pedi-
atric intensive care unit, up to 20 % had
hypokalemia on admission and up to 40 % ulti-
mately had hypokalemia identified during the
course of their ICU stays [176–178]. Hypokalemia
was more likely seen in children with cardiac or
renal disease or with hemodynamic collapse, as
well as in children with infections or who required
calcium supplementation [176].
Children are often asymptomatic until serum
potassium levels approach 2.5 mEq/L. Since low
extracellular potassium levels alter the usual
transcellular potassium gradient, significant clinical
manifestations of hypokalemia often relate to
changes in muscle contraction or cardiac conduction
[179]. Muscle weakness develops starting in the legs
and then may ascend. With profound hypokalemia,
respiratory muscles can become involved and there
may even be rhabdomyolysis. Smooth muscle dys-
function in the gastrointestinal tract can present as
nausea, constipation, or frank ileus. In terms of
cardiac effects, children with hypokalemia often
manifest characteristic ECG changes with PR inter-
val prolongation, dampening of the T waves, and ST
depression. As hypokalemia exacerbates, there can
be the appearance of U waves as well [179, 180].
With chronic hypokalemia there can also be
direct renal effects leading to polyuria. This poly-
uria arises related to several mechanisms, includ-
ing the stimulating effect of hypokalemia on thirst
and other neuroendocrine factors as well as the
impact of persistent hypokalemia on aquaporin
protein depletion in the cortical collecting duct
limiting the number of effective water channels
[48, 181, 182].
Causes of Hypokalemia in Children
The causes of hypokalemia in children can be
grouped into four broad categories based on
underlying pathophysiology: decreased
402
Table 14 Causes of hypokalemia in children
Decreased potassium intake
Chronic dietary restriction
Malnutrition, eating disorders
Iatrogenic errors in nutritional supplementation
Transcellular redistribution of potassium
Alkalosis
Endocrine effects
Insulin, catecholamines, thyroid hormone
Periodic paralysis
Medications
Beta-adrenergics, chloroquine, antipsychotic agents
Extrarenal potassium losses (usually GI tract)
Diarrhea
Vomiting
Nasogastric tube losses
Renal potassium losses
Increased delivery of sodium and water to distal tubule
Diuretics, mannitol, keto acids
Renal tubular acidosis
Hyperaldosteronism
Related to effective volume depletion
Glucocorticoid-remediable aldosteronism (GRA)
Apparent mineralocorticoid excess (AME)
Congenital adrenal hyperplasia
Tubulopathies
Bartter syndrome
Gitelman syndrome
Medications
Amphotericin B, cisplatin
potassium intake, transcellular redistribution of
potassium, extrarenal potassium losses, and renal
potassium losses (Table 14). Clinical history
makes the etiology apparent and renders extensive
diagnostic evaluation moot in many situations.
Children with a history of recurrent hypokalemia,
especially in the absence of accompanying acute
illnesses, need to be carefully considered for
underlying derangements in renal potassium
handling.
Decreased Potassium Intake
By itself, decreased dietary intake of potassium is
a rare cause of hypokalemia in children. Ongoing
potassium restriction leads to activation of potas-
sium conserving mechanisms, such as a reduction
in the number of excretory potassium channels
I.F. Ashoor and M.J.G. Somers
(ROMK) within the renal tubule and resistance
to the insulin-mediated intracellular flow of potas-
sium [183]. Mild hypokalemia from simple
decreased intake can, however, be exacerbated
by diuretic use, diarrhea, or eating disorders such
as anorexia or bulimia. States of chronic malnu-
trition are especially prone to severe hypokalemia
if potassium loss is then superimposed. For
instance, one report of malnourished children in
Kenya demonstrated a baseline rate of hypokale-
mia of 10 % from decreased intake, but this
increased more than threefold in the setting of
concomitant diarrhea [184]. In hospitalized chil-
dren, inadequate potassium intake may be a reflec-
tion of acute or chronic illness preventing
sufficient provision of general nutrition, or there
can be iatrogenic oversights such as the incorrect
formulation of intravenous peripheral nutrition
solutions. Keeping in mind concomitant clinical
factors that may be influencing potassium balance
should facilitate the provision of proper potassium
supplementation.
Transcellular Redistribution of Potassium
Metabolic Alkalosis
Alkalosis promotes hypokalemia through two
mechanisms. As systemic pH increases, hydrogen
ions in the intracellular space move extracellularly
to help blunt the alkalosis. To maintain
electroneutrality, extracellular potassium then
shifts intracellularly and this will be reflected by
a fall in serum potassium levels.
With alkalosis, there is also increased potas-
sium excretion in the urine. With the increase in
serum bicarbonate, there is an increased load of
bicarbonate filtered and eventually passed in the
urine. Since a cation must accompany the filtered
bicarbonate, this means that there are also
increased urinary loads of potassium and sodium.
Some of this potassium will get directly excreted
along with the bicarbonate. Additionally, the
increased concentration of sodium that is also
presented to the distal nephron allows there to be
more ready exchange of sodium for potassium in
the distal nephron under the influence of aldoste-
rone, with even further urinary potassium losses.
This mechanism is most pronounced when there is
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
concomitant effective volume depletion from gas-
trointestinal losses or diuretic use or if hyperaldos-
teronism exists for other reasons.
Endocrine Effects
Transcellular redistribution of potassium can also
arise from the effects of certain hormones. This is
most commonly seen from the effects of insulin
and from catecholamines with beta-adrenergic
effects, both of which promote cell uptake of
potassium. The ability of these substances to
accomplish effective transcellular shift is
underscored by the common use of both insulin
and nebulized albuterol to treat hyperkalemia.
Insulin-mediated hypokalemia can also be seen
in children with refeeding syndrome or with eat-
ing disorders where insulin release may exacer-
bate a preexisting tendency towards hypokalemia
from long-term poor nutritional intake. Children
receiving intermittent nebulized albuterol are
unlikely to present with significant alterations in
serum potassium levels, but with continuous neb-
ulization there can be a tendency for lower potas-
sium levels to develop [185]. In critically ill
children receiving intravenous catecholamines,
either for bronchodilation or for hemodynamic
reasons, there can be an augmented effect; up to
50 % of children with these treatments have been
reported to develop hypokalemia [186].
In hyperthyroidism, hypokalemia can develop,
mediated in part by increased expression and
activity of the sodium–potassium ATPase
[187]. Thyrotoxic periodic paralysis is a rare con-
dition, almost always seen in adults, where
patients with hyperthyrodism develop profound
hypokalemia and ensuing muscle dysfunction.
This condition is in contradistinction to hypoka-
lemic periodic paralysis caused by autosomal dom-
inant mutations in voltage-gated calcium channels
found in the skeletal muscle and in voltage-gated
sodium channels at the neuromuscular junction.
Affected individuals can present throughout child-
hood but especially in adolescence, have normal
thyroid function, and often have their episodes of
pronounced hypokalemia and paralysis instigated
by an event causing insulin or adrenergic hormone
release to initiate a fall in serum potassium levels
that then gets exacerbated by their channelopathies.
403
Drugs
In addition to beta-adrenergic agents as discussed
above, redistributive hypokalemia has also been
described in chloroquine intoxication and also
with the provision of risperidone and quetiapine
[188, 189]. In children who have inadvertently
ingested barium salts found in certain fireworks
or rodent poisons, hypokalemia has also been
described as a result of inhibition of normal potas-
sium egress from cells [190, 191]. In these cases,
the clinical history and consultation with local
toxicology resources should help guide appropri-
ate diagnosis and management.
Extrarenal Potassium Losses:
Gastrointestinal Tract
Hypokalemia can evolve from upper or lower
gastrointestinal tract losses, and gastrointestinal
illness with unreplaced losses of potassium
accounts for most cases of hypokalemia in other-
wise healthy children. At up to 50 mEq/L,
the potassium content of diarrhea is high
compared to other body fluids, including
vomit or gastric secretions [192]. Although
both types of gastrointestinal loss can cause
hypokalemia, the underlying pathophysiology
differs.
With diarrheal illness, there are not only sig-
nificant potassium losses from the stool, but with
the evolution of concomitant volume depletion,
an aldosterone-mediated kaliuresis can ensue.
This can be exacerbated in the early phases of
rehydration since, with improved effective circu-
lating volume and augmented GFR, there will be
increased delivery of filtered sodium to the distal
nephron and more substrate for aldosterone-
mediated potassium secretion.
In contrast, upper gastrointestinal losses
from vomiting or nasogastic drainage may
contain less than 10 mEq/L of potassium. The
metabolic alkalosis that can develop from these
losses will, however, lead to increased distal tubu-
lar delivery of sodium bicarbonate, and with the
secondary upregulation of aldosterone from vol-
ume losses, the increased availability of sodium in
distal tubular filtrate will allow for increased
exchange for potassium and an effective
kaliuresis.
404
Renal Potassium Losses
Increased Distal Sodium Delivery
Principal cells in the collecting duct contain apical
epithelial cell sodium channels (ENaC) where
sodium is reabsorbed under the influence of
mineralocorticoid (primarily aldosterone) and
exchanged electroneutrally for potassium via
ROMK channels. For potassium excretion to
occur effectively, sodium delivery to these neph-
ron segments must be adequate and mineralocor-
ticoid activity must be present. Increased delivery
of sodium distally augments potassium excretion,
especially in clinical situations where there are
also effective circulating volume depletion and
increased aldosterone activity. This physiology
helps to account for the hypokalemia that can be
seen with diuretic provision, with mannitol provi-
sion, with diabetic ketoacidosis, and, as explained
above, with metabolic alkalosis from vomiting or
nasogastric tube drainage.
Renal Tubular Acidosis
Both type 1 and type 2 RTA are associated with
hypokalemia, although from varying physiologic
mechanisms. With type 1 or distal RTA, the intrin-
sic inability of the distal nephron to acidify the
urine by secreting hydrogen ions leads to potas-
sium secretion to maintain electroneutrality. There
may also be associated tubular cellular membrane
permeability that allows for further potassium
loss. With type 2 or proximal RTA, there is
impaired proximal tubular reabsorption of filtered
bicarbonate, leading to increased urinary losses of
bicarbonate and associated cations. The increased
distal tubular presentation of sodium facilitates
potassium secretion, exacerbated by the aldoste-
rone effect from any effective volume depletion
caused by the augmented sodium and water
losses.
Hyperaldosteronism
Normal physiologic stimuli for aldosterone secre-
tion, most notably volume depletion, commonly
play a contributing role to many of the causes of
pediatric hypokalemia. In comparison, primary
abnormalities in aldosterone secretion are rare in
childhood, though certain inherited conditions
I.F. Ashoor and M.J.G. Somers
can be seen, generally accompanied by hyperten-
sion. In autosomal dominant glucocorticoid-
remediable aldosteronism, there is aldosterone
synthase hyperactivity due to genetic mutations
that allow aldosterone production to become reg-
ulated by ACTH. The provision of glucocorticoid
suppresses the mineralocorticoid excess and ensu-
ing high blood pressure and any associated hypo-
kalemia [193]. In apparent mineralocorticoid
excess (AME), cortisol degradation is impaired
by genetic mutations that alter the efficacy of
11-beta-hydroxysteroid dehydrogenase, and cor-
tisol can then bind to mineralocorticoid receptor
and mediate signs and symptoms mimicking aldo-
sterone excess. This same enzyme can be
inhibited by glycyrrhizic acid that can be found
in natural licorice and this explains why excess
ingestion of natural licorice has been associated
with hypokalemia as well. In forms of congenital
adrenal hyperplasia such as 17-alpha-hydroxylase
deficiency and 11-beta-hydroxylase deficiency,
there can also be excess mineralocorticoid pro-
duction and hypokalemia and hypertension.
Renal Tubulopathies
Certain genetic tubular disorders are characterized
by impaired sodium reabsorption, increased distal
tubular sodium delivery, and ensuing urinary
potassium losses with the development of an asso-
ciated alkalosis. In the various types of Bartter
syndrome, abnormalities in the electrolyte trans-
porters or channels in the thick ascending limb
help mediate a kaliuresis and alkalosis as well as a
tendency towards hypercalciuria. In Gitelman
syndrome, the thiazide-sensitive sodium chloride
symporter in the distal convoluted tubule is
impaired, and there is often concomitant hypo-
magnesemia and normal or lower urinary calcium
excretion. With both syndromes volume depletion
and physiologic aldosterone stimulation will only
serve to aggravate any underlying tendency to
waste potassium in the urine, but provision of
volume will not ameliorate baseline potassium
wasting. In Liddle syndrome, gain-of-function
mutations in ENaC result in dysregulated sodium
reabsorption with ensuing hypertension, hypoka-
lemia, and alkalosis as seen with more classic
cases of mineralocorticoid excess.
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
Any Fanconi syndrome, whether idiopathic in
etiology, related to drug exposure, or related to
renal conditions such as cystinosis, can also result
in hypokalemia as part of the diffuse proximal
tubule solute losses that ensue. Children with
hypokalemia from proximal tubular dysfunction
will also lose large amounts of bicarbonate in their
urine and will be prone to a metabolic acidosis, in
distinction to the metabolic alkalosis that charac-
terizes Bartter and Gitelman syndrome.
Drugs
Diuretics are by far and away the most commonly
prescribed drug that can mediate hypokalemia
through direct urinary potassium losses, by
increasing distal sodium presentation, and by con-
tributing to effective volume depletion and phys-
iologic aldosterone activity. In addition,
amphotericin and some chemotherapy drugs,
most notably cisplatin, can also cause increased
renal potassium wasting. With amphotericin,
there is actual disruption of cellular membrane
integrity and ensuing loss of potassium from the
intracellular space. Some studies have suggested
that up to half of the children receiving
amphotericin will become hypokalemic, though
the provision of liposomal amphotericin seems to
substantially mitigate this problem [194]. With
cisplatin, there can be direct tubular toxicity that
interferes with the handling of potassium as well
as wasting other electrolytes, with effects that can
be quite long lived [195].
Diagnostic Evaluation
History and Physical Examination
The patient’s history is important in guiding the
diagnostic evaluation. An otherwise healthy child
with hypokalemia accompanying an acute gastro-
intestinal illness requires less of an evaluation
than a child with recurrent bouts of unexplained
hypokalemia. Ascertaining any underlying
predisposing medical conditions, medications, or
dietary restrictions may help clarify the cause of
the child’s hypokalemia.
The physical examination should key on find-
ings suggesting significant adverse effects from
405
the low serum potassium such as cardiac arrhyth-
mias or alterations in muscle tone or strength.
There should also be some clinical assessment of
effective circulating volume and respiratory
effort, since severe abnormalities will influence
immediate management decisions and may
impact acid–base balance and, as a result,
transcellular potassium distribution. Blood pres-
sure should also be carefully measured since the
presence or absence of hypertension helps distin-
guish certain of the renal tubulopathies.
Laboratory Testing
In the event that the cause of the hypokalemia is
clear-cut – for instance vomiting with dehydration
or diuretic therapy in the setting of poor potassium
dietary intake – it is probable that follow-up
assessment of serum potassium after proper ther-
apy will show resolution of the problem and no
reason to do further testing. On the other hand, in
cases of less clear etiology, in cases of recurrent
hypokalemia, or when hypokalemia recurs after
initial supplementation is completed, there needs
to be more specific assessment of renal handling
of potassium to determine if there are inappropri-
ate renal losses ongoing.
With profound hypokalemia (<2.5 mEq/L)
and especially when there is evidence or suspicion
of adverse cardiac effect, steps to ameliorate the
hypokalemia should begin immediately and
should not be delayed by further diagnostic eval-
uation. Fortunately, the blood and urine samples
necessary to guide the evaluation can often be
readily collected contemporaneously with initia-
tion of therapy (Table 15).
Urine Potassium Quantification
A 24-h urine collection will give the most accurate
assessment of the degree of urinary potassium
losses since it can be directly compared to potas-
sium intake to assess a net balance. Timed urine
collections are difficult to perform in many chil-
dren, so spot urine chemistries with concomitant
serum chemistries are generally substituted and,
in most cases, will provide enough information to
allow for accurate assessment of renal response.
In the setting of a serum potassium level less
than 3 mEq/L, random urinary potassium values
406 I.F. Ashoor and M.J.G. Somers

Table 15 Initial diagnostic studies to assess unexplained
hypokalemia in children
Laboratory test
Creatinine, electrolytes,
phosphorus, magnesium
Venous pH
Aldosterone and plasma
renin activity
Urinalysis
Urine chemistries and urine
creatinine (best done
concomitantly with serum
electrolytes and creatinine)
Table 16 Hypokalemia and acid–base disorders
Hypokalemia and metabolic acidosis
Key findings Low urinary losses of potassium
Derangement in addition Diarrhea
to potassium High urinary losses of potassium
Systemic acidosis or Renal tubular acidosis
alkalosis
Proximal tubulopathy (Fanconi or Fanconi like)
Variation from normal
Hypokalemia and metabolic alkalosis
levels
Low urinary losses of potassium
Appropriateness of pH
and presence of Diuretics (chronically)
glycosuria High urinary losses of potassium
Urinary K excretion Hyperaldosteronism (often concomitant
Random UK > 20 hypertension)
mEq/L suggests renal Diuretics (acutely)
loss Bartter syndrome (often concomitant
U K/Cr > 15 mEq/g hypercalciuria)
suggests renal loss
Gitelman syndrome (often concomitant
Hypercalciuria
hypomagnesemia)
Urinary Cl excretion

Aldosterone and plasma renin activity will help

usually are less than 20 mEq/L and values signif-
icantly higher suggest renal wasting. Electrolyte
concentration in a random urine void will be
affected by water handling at that point in time,
so spot samples in particularly dilute or concen-
trated samples may provide values that are lower
or higher than if urine output were normal.
Accordingly, urinary potassium-to-creatinine
ratios can be done since urinary creatinine excre-
tion is constant in children with normal renal
function and this will correct for water excretion.
In the setting of hypokalemia, a urine potassium-
to-creatinine ratio <15 mEq/g creatinine (<1.5
mEq/mmol Cr) suggests the hypokalemia is not
due to renal potassium wasting at that time and
focus should shift to GI losses, poor intake, cellu-
lar shift, or prior diuretic use. Ratios >15 mEq/g
creatinine suggest renal wasting.
Further Testing with Renal Potassium
Wasting
In the setting of urinary potassium wasting,
assessment of the child’s blood pressure along
with a few other specific urine and blood tests
helps to clarify the reason for the kaliuresis. Spe-
cifically, a venous pH and serum electrolytes and
serum magnesium level will allow the identifica-
tion of an acid–base disorder, the presence of an
anion gap, and the presence of hypomagnesemia.
determine if there is hyperaldosteronism. Urinary
calcium excretion should also be measured to look
for hypercalciuria. Urinary chloride levels will
help in the differential diagnosis of hypokalemia
with alkalosis (Table 16).
In the hypertensive child with urinary potas-
sium wasting, both low blood aldosterone and
renin levels should make apparent mineralocorti-
coid excess, Liddle syndrome, or a form of adre-
nal hyperplasia the most suspected pathologies.
With high aldosterone but low renin levels,
hyperaldosteronism should be suspected.
In the normotensive child with urinary potas-
sium wasting, the presence of an acidosis suggests
an RTA. With an alkalosis, urinary chloride levels
less than 15 mEq/L speak to renal potassium
wasting from volume depletion in the setting of
a metabolic alkalosis as seen with emesis, naso-
gastric tube losses, or diuretics. With an alkalosis
and higher urinary chloride levels, a renal
tubulopathy such as Bartter or Gitelman syn-
drome is suspected. Hypercalciuria would point
towards Bartter syndrome, and hypomagnesemia
is more frequent with Gitelman, though genetic
testing to clarify specific mutations and which
channels or transporters are affected allows for
there to be a better understanding if the potassium
wasting arises from the thick ascending limb or
the distal convoluted tubule.
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
Treatment of Hypokalemia in Children
Urgency of Potassium Supplementation
In patients with significant cardiovascular or
respiratory compromise related to hypokalemia,
management must proceed emergently and be
focused on acutely increasing the serum potas-
sium level by 0.3–0.5 mEq/L and then
maintaining supplementation until a level of
3 mEq/L is reached. These interventions should
proceed with ongoing cardiac monitoring to
detect new or exacerbating arrhythmia.
Since intravenous potassium can be irritating
to vessels, to prevent phlebitis or ongoing pain,
concentrations exceeding 40 mEq/L are generally
given centrally. When giving intravenous potas-
sium, it is important for patient safety to limit the
absolute amount of potassium available to be
delivered in one single infusion in case the rate
or volume of the infusion is inadvertently altered
from what is intended. Hourly rates of infusion of
0.5–1 mEq/kg will generally result in serum
potassium levels rising by nearly 0.5 mEq/L in
the face of no new increased potassium losses.
Enteral Potassium Supplementation
In children with levels between 2.5 and 3 mEq/L
and no clinical signs or symptoms of hypokale-
mia, potassium supplementation is warranted,
although enteral supplementation is generally pre-
ferred to avoid the complications of intravenous
provision. Although intravenous supplementation
is commonly provided to critically ill children due
to restrictions in enteral intake or other
confounding factors, there is no evidence to sug-
gest that enteral supplementation is inferior to its
intravenous provision. In fact, a study of children
in a pediatric cardiac intensive care unit showed
that the provision of 1 mEq/kg of potassium either
by intravenous or enteral routes resulted in no
difference in efficacy or side effects, with both
groups demonstrating an average improvement
in serum potassium of 0.65 mEq/L [196].
Factors Influencing Efficacy
of Supplementation
Concomitant administration of other medications
that may alter renal potassium handling may affect
407
individual response to potassium supplementa-
tion. For instance, the provision of amphotericin
or furosemide to children receiving potassium
supplementation results in more moderate
increases in serum potassium because of urinary
potassium losses. Conversely, children receiving
ACE inhibitors often exhibit somewhat higher
serum potassium levels after supplementation as
a result of the effect of angiotensin blockade on
GFR [197].
The type of potassium preparation given for
supplementation can be tailored to the need for
other specific electrolyte supplementation. Since
many hypokalemic children have concomitant
alkalosis with hypochloremia, potassium chloride
is most frequently used for both enteral and intra-
venous supplementation. Potassium chloride sup-
plements have also been shown to accomplish
more efficient repletion than potassium phospho-
rus or potassium citrate or bicarbonate, although
with concomitant acidosis or hypophosphatemia,
their provision does make sense [198].
Any supplementation will be less effective if
there are ongoing potassium losses that can con-
tinue to vary independently. In children with
Bartter or Gitelman syndromes, use of
potassium-sparing diuretics may help limit the
extent of renal losses, and in those children with
such tubulopathies and hypomagnesemia, magne-
sium provision can help blunt the attenuating
effects of hypomagnesemia on potassium
reabsorption [199].
Acid–Base Homeostasis
Acid–base homeostasis involves an intricate inter-
play between multiple regulatory mechanisms to
maintain the extracellular arterial pH between
7.35 and 7.45. The typical diet for infants and
children in much of the developed world is rich
in animal proteins and cereal grain resulting in
generation of net acid, as opposed to the genera-
tion of net base with vegetarian diets
[200–202]. When adjusted for body weight,
infants produce about 2–3 mEq/kg/day of nonvol-
atile acid, which is significantly higher than adults
where the net nonvolatile acid production
408
Fig. 2 Acid–base handling in the distal nephron
averages 50–100 mEq/day [203]. The additional
acid load in children is, in part, secondary to rapid
skeletal growth which also produces a net acid
excess as calcium is incorporated into the skeleton
to form hydroxyapatite [204].
This daily acid load is handled by initial buffer-
ing with extracellular buffers (primarily the HCO3
buffer system) followed by intracellular buffers
(proteins and phosphate). The respiratory system
adjusts the ventilation rate to excrete volatile acids
as CO2. The kidneys handle the excretion of non-
volatile acids by secreting H+ in the proximal
tubule, which reclaims filtered HCO3, and by
secreting H+ in the distal nephron, which com-
plexes with phosphate to form titratable acid
(H2PO4) or with ammonia (NH3) to form ammo-
nium (NH4) salts. The H+ secreted distally is
derived from breakdown of water molecules in
the cell, which generates a hydroxyl ion (OH)
that produces new HCO3 in a reaction with CO2
catalyzed by carbonic anhydrase in α -intercalated
cells (Fig. 2). The relationship between pH, serum
HCO3, and PCO2 can be expressed logarithmically
as the Henderson–Hasselbalch equation as follows:
pH ¼ 6:1 þ Log ð½HCO3 =½0:03  PCO2 Þ
where 6.1 is the pKa (log of the dissociation
constant for the reaction) of the CO2-HCO3 buffer
I.F. Ashoor and M.J.G. Somers
system and 0.03 is the solubility constant of CO2
in the extracellular fluid.
Perturbations in Acid–Base Balance
Acid–base disorders result from processes that
lead to changes in the serum bicarbonate (HCO3)
concentration, partial pressure of carbon dioxide
(PCO2), or both. The disorder is considered met-
abolic when the inciting event primarily alters
HCO3 concentration and respiratory if changes
in PCO2 were primarily responsible for altering
systemic pH. Newborns and infants are more sus-
ceptible to develop acid–base disorders given
their baseline higher acid load and the time
required for full maturation of various transporters
involved in acid–base homeostasis in the proxi-
mal and distal nephron [205–208].
Simple Acid–Base Disorders
Under normal conditions, a metabolic acid–base
disorder secondary to a change in serum HCO3
concentration will be associated with a respiratory
compensation that changes the PCO2 in the same
direction as the change in HCO3 concentration to
bring the pH towards normal. For example, a child
with metabolic acidosis secondary to diarrheal
HCO3 losses and resulting lowering serum
HCO3 concentration will hyperventilate to
decrease the PCO2 and raise the depressed sys-
temic pH towards normal. On the other hand, a
child with alkalemia secondary to H+ losses from
vomiting leading to an increase in serum HCO3
concentration will hypoventilate to increase the
PCO2 and lower the systemic pH towards normal.
Similarly, a respiratory acid–base disorder sec-
ondary to a change in PCO2 will be associated
with a metabolic compensation that changes the
HCO3 concentration in the same direction as the
change in PCO2 to bring the pH towards normal.
For example, an infant with respiratory acidosis
secondary to hypercarbia from a process such as
chronic lung disease of prematurity will increase
net renal H+ secretion over time, thereby raising
the serum HCO3 concentration and the systemic
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . . 409

pH towards normal. On the other hand, a child
with respiratory alkalosis secondary to anxiety-
induced hyperventilation lowering PCO2 will
increase the fractional excretion of HCO3 in the
urine, thereby lowering serum HCO3 concentra-
tion and lowering systemic pH towards normal.
The degree by which the body compensates for
a primary metabolic or respiratory process alter-
ing pH can be deduced from various formulas
[209–212]. A simple acid–base disorder exists
when the change in pH can be explained by a
primary metabolic or respiratory disturbance and
an appropriate degree of compensation.
Respiratory Acid–Base Disorders
A respiratory acidosis exists when a low pH is
secondary to a process elevating arterial PCO2,
while respiratory alkalosis refers to an elevated
pH secondary to a process reducing arterial PCO2.
A compensatory metabolic response to a primary
respiratory acid–base disorder will evolve over
time; the full renal compensatory response
requires up to 3–5 days to develop [213]. In the
initial stages of a primary respiratory acid–base
disorder, compensation is accomplished by extra-
cellular and intracellular buffering mechanisms.
As detailed in Table 17, in acute respiratory
acidosis, compensatory response is expected to
raise the serum HCO3 concentration by about
1 mEq/L in the absence of coexisting acid–base
disorders. In comparison, as full compensatory
responses develop in response to a chronic respi-
ratory acidosis, the serum HCO3 concentration
will rise by 4–5 mEq/L [212, 213]. Similarly, in
acute and chronic respiratory alkalosis (Table 18),
the serum HCO3 concentration is expected to fall
by about 2 and 4–5 mEq/L, respectively [213,
214]. The same underlying etiology may cause
respiratory acidosis or alkalosis depending on
the chronicity, degree of ventilation–perfusion
mismatch, and respiratory muscle fatigue.
Metabolic Acidosis
Metabolic acidosis refers to conditions where a
low systemic pH is secondary to a process
decreasing HCO3 concentration. In children, the
clinical presentation of metabolic acidosis is var-
ied and can range from a completely

Table 17 Representative etiologies of respiratory acidosis in children

Mechanism Acute Chronic
Airway obstruction Foreign body aspiration Bronchopulmonary dysplasia
Status asthmaticus Persistent asthma
Epiglottitis Obstructive sleep apnea
Bronchospasm/laryngospasm Hilar lymphadenopathy
Pulmonary – intrinsic Pneumonia Persistent asthma
Respiratory distress syndrome Cystic fibrosis
Pneumothorax/hemothorax Interstitial lung disease
Status asthmaticus Bronchopulmonary dysplasia
Pulmonary – extrinsic Erroneous ventilator settings Chest wall deformity
Diaphragmatic paralysis
Neurologic – central Head injury Congenital CNS malformation
Coma CNS tumor
Stroke Chronic sedative use
Sedative overdose Central sleep apnea
Meningitis
Spinal cord injury
Neurologic – peripheral Guillain–Barre syndrome Muscular dystrophies
Myasthenia gravis Chronic myopathies
Organophosphate poisoning Polio
Familial periodic paralysis
Botulism
410 I.F. Ashoor and M.J.G. Somers

Table 18 Representative etiologies of respiratory alkalo- Table 19 Representative etiologies for metabolic acidosis
sis in children in children
Mechanism Acute Chronic Classification Mechanism Examples
Direct central Head injury Congenital High anion Increased Diabetic
stimulation of the Anxiety CNS gap acidosis endogenous ketoacidosis
respiratory Hyperventilation malformation acid Lactic acidosis
center Meningitis/ CNS tumor production Inborn errors of
encephalitis Meningitis/ metabolism (e.g.,
Drug side effect: encephalitis organic acidemias)
aspirin, nicotine Medication Ingestions
CNS bleed side effect (methanol, ethanol,
Fever ethylene glycol)
Pain Accumulation Advanced renal
Indirect Pulmonary Heart failure of anionic failure
stimulation of the edema Interstitial compounds
respiratory Pulmonary lung disease Normal Loss of Diarrhea
center via embolus anion gap bicarbonate Type 2 (proximal)
pulmonary acidosis renal tubular
stretch receptors acidosis (RTA)
Indirect Hypotension High altitude Carbonic anhydrase
stimulation of the Tissue hypoxia (low FiO2%) inhibitors
respiratory (e.g., asthma, Impaired renal Type I (distal) RTA
center via pneumonia) H+ excretion Type 4 (distal
peripheral hyperkalemic) RTA
chemoreceptors Early CKD

asymptomatic child with incidental findings on
laboratories obtained for another indication to a
critically ill child presenting with circulatory col-
lapse. The history and physical examination find-
ings may point towards the physiologic
perturbation resulting in the metabolic acidosis.
Typically, metabolic acidosis is caused by condi-
tions that can be grouped into three broad catego-
ries: (1) increased endogenous or exogenous acid
generation which produces a high anion gap met-
abolic acidosis, (2) increased HCO3 loss which
produces a normal anion gap metabolic acidosis,
and (3) impaired renal H+ excretion which also
produces a normal anion gap metabolic acidosis
(see below and Table 19).
increasing pH towards normal. In the absence of
severe acidosis (pH < 7.1), Winters’ formula
[210] can predict the expected PCO2 for a given
primary change in HCO3 concentration as
follows:
Expected PCO2 ¼ ð1:5  measured HCO3 Þ
þ 8 ðþ=2Þ mmHg
Alternatively, an appropriate respiratory response
to metabolic acidosis results in a fall in PCO2
concentration by 1.2 mmHg for every 1 mEq/L
fall in HCO3 concentration from a normal baseline
of 24 mEq/L. This relationship tends to be accu-
rate down to a PCO2 of 10–15 mmHg.

Respiratory Compensation Serum Anion Gap

for Metabolic Acidosis
Simple metabolic acidosis exists when there is an
appropriate respiratory compensation for the
degree of reduction in HCO3 concentration. The
respiratory response to a fall in HCO3 concentra-
tion is hyperventilation, lowering PCO2, and
The serum anion gap is a measurement obtained
by subtracting the sum of the major measured
serum anions (chloride and HCO3) from the
major measured serum cation sodium [215] and
is often expressed in the equation form: Serum
AG = Na+  (Cl + HCO3). The serum anion
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
gap reflects the difference between unmeasured
anions and unmeasured cations in the blood. Most
children have an anion gap less than 16 mEq/L
[215]; however, it is prudent to review local lab-
oratory normal values for specific electrolyte
ranges and, in children with an anion gap, to
monitor its trend over time to facilitate accurate
categorization of the acid–base disorder in ques-
tion (Table 3).
Under normal acid–base status, the anion gap
is largely a result of anionic proteins, primarily
albumin. Therefore, the anion gap measurement
has to be adjusted for abnormal albumin concen-
tration, where for every 1 g/dL reduction in serum
albumin the normal anion gap is expected to
decrease by 2.5 mEq/L [216]. This adjustment
becomes particularly important to consider in
children with substantial aberrations in serum
albumin levels, as seen with nephrotic syndrome
or with malnutrition.
Urine Anion Gap
The urine anion gap (UAG) is a useful tool in the
evaluation of metabolic acidosis with a normal
serum anion gap and is often expressed by the
equation: UAG = Urine (Na + K) – Cl. It is a
surrogate measure of ammonium (NH4) excretion
in the urine, as direct urinary NH4 measurement is
not widely available [217]. NH4 is excreted as
NH4 chloride salts in the urine. Chloride is also
excreted along with sodium and potassium in the
urine. Therefore, subtracting the urine chloride
from the sum of urine sodium and urine potassium
concentrations should be a rough estimate of NH4
concentration in the urine assuming that it is
exclusively excreted as NH4 chloride salts.
The ability of the distal nephron to secrete the
excess H+ as NH4+ remains intact in normal AG
metabolic acidosis secondary to gastrointestinal
(e.g., diarrhea) or renal HCO3 losses (e.g., proxi-
mal RTA). The urine Cl concentration will be
high (reflecting high urine NH4+) and the UAG
calculation will yield a negative value. Whereas if
the normal AG metabolic acidosis is secondary to
impaired renal H+ excretion (e.g., distal RTA and
early stages of CKD), the urine Cl concentration
411
will be low (reflecting low urine NH4+) and the
UAG calculation will yield a positive value.
Urine Osmolal Gap
The urine osmolal gap (UOG) is another surrogate
measure of urinary NH4 excretion. It is a better
measure of NH4 excretion when NH4 may be
coupled with other anions besides Cl in the
urine. This can be seen in ketoacidosis and toluene
inhalation where NH4 is excreted in conjunction
with keto acids and hippurate, respectively [218,
219]. The UOG can be calculated by subtracting
the urine-calculated osmolality from the directly
measured urine osmolality as follows:
UOG ¼ urine measuredosmolality
 urinecalculated osmolality ð2  ½Naþ K
þ ½urine ureanitrogen=2:8
þ ½urine glucose=18Þ
Both urine urea nitrogen and urine glucose are
measured in mg/dL. The glucose contribution to
urine osmolality is only relevant in the setting of
glucosuria and in its absence can be ignored.
Similar to the concept of the UAG, a high UOG
(typically greater than 400 mOsm/kg) indicates
increased NH4 excretion by the distal nephron as
would be seen in a child with diarrhea who should
have intact distal tubular acid–base regulatory
mechanisms, whereas a low UOG (typically less
than 150 mOsm/kg) would indicate impaired NH4
excretion in the distal tubule as would be seen in a
child with distal RTA [220].
Metabolic Alkalosis
Metabolic alkalosis refers to conditions where a high
systemic pH is secondary to a process increasing
serum HCO3 concentration. Children with alkalosis
may be asymptomatic or have signs and symptoms
reflecting the underlying etiology of alkalosis such
as vomiting or reflecting other concomitant electro-
lyte abnormalities such as seizures and muscle
cramps from hypomagnesemia and hypokalemia as
seen in Gitelman and Bartter syndrome, respectively.
412 I.F. Ashoor and M.J.G. Somers

Table 20 Representative etiologies for metabolic alkalo- Similarly, dehydration or any state of deceased
sis in children effective circulating volume maintains alkalosis
Mechanism Examples by enhancing proximal tubular HCO3
Excessive loss of H+: Vomiting reabsorption along with the sodium avidity caused
gastrointestinal Nasogastric suction by the ineffective volume, creating what is termed
Excessive loss of H+: Bartter syndrome a “contraction” alkalosis effect.
renal Gitelman syndrome
Primary mineralocorticoid Hypokalemia causes intracellular shift of H+ in
excess exchange for K+ to maintain electroneutrality,
Liddle syndrome effectively adding HCO3 to the ECF
Loop or thiazide diuretic use [229]. Hypochloremia typically accompanies vol-
Excessive gain of Alkali administration
ume depletion; however, it can independently
HCO3 Conversion of potential
bicarbonate precursors to maintain alkalosis by reducing HCO3 secretion
HCO3 (e.g., lactate, citrate, in type B intercalated cells in the cortical
acetate) collecting tubule mediated by the luminal
Intracellular shift of Hypokalemia Cl/HCO3 exchanger, pendrin [230].
H+ Glucose-induced alkalosis in
fasting
Finally, primary activation of the RAS system
Relative increase in Contraction alkalosis contributes to the maintenance of alkalosis by
HCO3 concentration increasing Na reabsorption in the distal nephron
in exchange for H+ and K+ secretion (Fig. 1).
The development of metabolic alkalosis The respiratory compensatory response for
requires a process that generates or adds new metabolic alkalosis is limited to increasing PCO2
HCO3 to the ECF. Most commonly, this is the by about 0.7 mmHg for every 1 mEq/L increase in
result of gastrointestinal H+ loss or excessive HCO3 concentration up to a maximum PCO2 of
renal H+ secretion, which effectively adds HCO3 about 55 mmHg [231, 232]. A higher PCO2 than
to the ECF. Alternatively, intracellular shifting of 55 mmHg in a child with metabolic alkalosis
H+, exogenous alkali administration, or contrac- implies concurrent respiratory acidosis.
tion of the ECF around a relatively fixed HCO3
amount (i.e., contraction alkalosis) will all gener-
ate a metabolic alkalosis [221] (Table 20). Mixed Acid–Base Disorders
The subsequent persistence or maintenance of
metabolic alkalosis will depend on factors A mixed acid–base disorder is typically suspected
impairing net renal HCO3 excretion [222]. Appro- on clinical grounds when the history or physical
priate excretion of excess HCO3 requires normal examination suggests that several physiologic
renal function [223], adequate intravascular vol- processes are perturbed that would alter systemic
ume status [224], normal serum K+ [225] and Cl pH in the same or opposite direction. For example,
concentrations [226], and normal activity of the a child with aspirin toxicity is likely to have a
renin–angiotensin–aldosterone (RAS) combination of both metabolic acidosis and respi-
system [227]. ratory alkalosis and therefore may have a near-
In clinical settings when one of these parame- normal pH. Whereas, a child with renal tubular
ters is altered, there is heightened risk for alkalo- acidosis who suffers from a cardiopulmonary
sis. For example, when a child with chronic arrest has a baseline normal anion gap metabolic
kidney disease stage 4 (GFR 15–30 ml/min/1.73 acidosis that now has a high anion gap metabolic
m2) receives bicarbonate containing intravenous acidosis and respiratory acidosis superimposed,
fluids in an attempt to prevent contrast-induced leading to severe acidemia out of proportion to
nephropathy [228], the low GFR will maintain the what would be expected from each individual
alkalosis that is generated longer compared to a component of his acidosis. Mixed acid–base dis-
child with normal GFR who would readily excrete orders should also be suspected when the
the excess alkali load. observed compensatory response to the primary
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
acid–base disturbance is less than expected or
exceeds what should be expected for compensa-
tion alone.
The D Anion Gap/D HCO3 Ratio
The Δ anion gap (change in AG from patient’s
baseline)/Δ HCO3 (change in HCO3 from
patient’s baseline) ratio is a useful tool to deter-
mine if a mixed acid–base disorder exists. A nor-
mal ratio typically ranges between 1 and 1.6
[233]. A ratio of >1.6 indicates that the HCO3
concentration did not fall as much as would be
expected from the increase in the AG. This can be
seen when metabolic acidosis coexists with met-
abolic alkalosis. On the contrary, a ratio of <1
indicates a coexisting normal AG metabolic aci-
dosis that lowered HCO3 concentration to a
greater degree than would be accounted for by
isolated high AG metabolic acidosis.
Clinical Application and Approach
The following case explores the previously
discussed concepts in interpreting acid–base dis-
orders and underscores how, especially in ill chil-
dren with chronic medical conditions, multiple
physiologic perturbations can arise resulting in
numerous potential acid–base abnormalities:
A newborn boy is delivered prematurely at
34 weeks of gestation secondary to preterm
labor. He does well other than requiring 0.5
L/min of oxygen by nasal cannula. An arterial
blood gas demonstrates a pH of 7.33, PCO2 of
33 mmHg, and HCO3 of 17 mEq/L.
This infant has a simple metabolic acidosis
(low pH and low HCO3) with appropriate
respiratory compensation as expected from
Winters’ formula: expected PCO2 = 1.5  17 +
8+/2 = 33.5+/2 mmHg. The low HCO3 is
most likely secondary to proximal renal tubular
increased fractional excretion of HCO3, an
expected maturational finding at this
gestational age.
The infant starts trials with oral feeds and
experiences an aspiration event. He develops
413
progressive respiratory distress and a chest radio-
graph reveals a right upper lobe infiltrate. A
repeat ABG now demonstrates a pH of 7.2,
PCO2 of 42 mmHg, and HCO3 of 16 mEq/L.
The infant still has metabolic acidosis. How-
ever, Winters’ formula predicts a compensatory
PCO2 of 30–34 mmHg. The PCO2 is much higher,
however, suggesting a concomitant respiratory
acidosis, likely due to hypoventilation in the set-
ting of aspiration pneumonia. In this case, both
acid–base disorders altered the pH in the same
direction, causing a change greater than expected
with either condition operating independently.
The infant’s pneumonia worsens and he is
placed on a ventilator. He remains intubated for
a month and, after extubation, he still has an
oxygen requirement. A blood gas is obtained and
demonstrates a pH of 7.32, PCO2 of 60 mmHg,
and HCO3 of 32 mEq/L.
The infant has now developed chronic lung
disease as indicated by persistent O2 requirement
for >30 days. In such a clinical setting of chronic
respiratory acidosis, the expected full metabolic
compensation would be an increase in normal
serum HCO3 concentration by 4–5 mEq/L for
each 10 mmHg increase in PCO2 from the
expected normal baseline of 40 mmHg, and,
indeed, this child demonstrates such a compensa-
tion. If the infant’s serum HCO3 concentration
differed significantly from expected, this would
raise the specter of a coexisting metabolic alkalo-
sis for higher-than-expected HCO3 values or a
coexisting metabolic acidosis for lower-than-
expected HCO3.
The infant goes on to develop central line-
associated bloodstream infection and hypoten-
sion with poor perfusion. His respiratory status
is unchanged. An arterial blood gas demonstrates
a pH of 7.15, PCO2 of 60 mmHg, and HCO3 of
20 mEq/L. Serum chemistries demonstrate a Na of
145 mEq/L, K of 4 mEq/L, Cl of 95 mEq/L, and
HCO3 of 20 mEq/L.
The infant now has his known chronic respira-
tory acidosis from his baseline lung disease as
well as a superimposed high anion gap
(145 – [95 + 20] = 30) metabolic acidosis
likely secondary to lactic acidosis from
hypoperfusion. The Δ anion gap/Δ HCO3 ratio
414
([30–12]/[30–20] = 1.8) is greater than 1.6,
suggesting a coexisting metabolic process that
raised HCO3 concentration above what otherwise
would be expected, in this case the baby’s baseline
renal compensatory response for his chronic respi-
ratory acidosis.
General Considerations for Therapy of
Acid–Base Disorders
The most effective therapy for any acid–base dis-
order is treatment of the underlying cause or
causes of the disturbance and correction of any
other factors that are helping to maintain the
abnormality. The decision to provide treatment
with exogenous alkali or acid to return the arterial
pH to normal while awaiting response to therapy
generally depends on individual clinical circum-
stance such as disease severity, the duration of the
acid–base abnormality, and whether the underly-
ing perturbation can be effectively reversed with
therapy.
For example, a child with chronic respiratory
acidosis from bronchopulmonary dysplasia who
has normal kidneys will typically develop suffi-
cient renal metabolic compensation to re-establish
a near-normal pH, and the only treatment required
is ongoing optimization of the primary respiratory
condition, whereas a child with metabolic acidosis
from advanced chronic kidney disease will
require exogenous alkali therapy as the underly-
ing cause is irreversible and respiratory compen-
sation for metabolic acid–base disturbances is
limited [209].
In treating metabolic alkalosis, interrupting the
mechanisms responsible for maintaining the alka-
losis is just as important as targeting the underly-
ing cause that generated the alkalosis. In children
with normal renal function, reduced renal excre-
tion of HCO3 which maintains an alkalosis is
typically a result of decreased effective circulating
volume, hypokalemia, chloride depletion, or
excess mineralocorticoid activity [222]. Effective
therapy entails attention to volume expansion,
chloride and potassium replacement, and possible
pharmacologic inhibition of the RAS system if
indicated.
I.F. Ashoor and M.J.G. Somers
In alkalosis, potassium repletion should be
accomplished with potassium chloride salts since
citrate or acetate preparations will generate addi-
tional HCO3. The use of the carbonic anhydrase
inhibitor, acetazolamide, can be a useful transient
adjunctive treatment in selected patients with
sustained metabolic alkalosis [234]. Acetazol-
amide inhibits proximal reabsorption of
NaHCO3 and leads to renal bicarbonate wasting.
The increased Na+delivery to the distal tubule that
ensues can stimulate distal K+ secretion, however,
and potentially induce or exacerbate a preexisting
hypokalemia which could then maintain the alka-
losis. The use of exogenous acid to treat metabolic
alkalosis in children is rarely required and can be
associated with significant morbidity and mortal-
ity if not approached carefully and in the hands of
experienced clinicians [235, 236].
Special Considerations in Infants
and Children with Chronic Kidney
Disease
Children with CKD suffer from metabolic acido-
sis initially secondary to reduced distal H+ excre-
tion and later due to accumulation of anions such
as phosphate, sulfate, and urate from reduced
GFR. As the HCO3 buffer systems become
overwhelmed, excess H+ is buffered by bone,
contributing to bone resorption and CKD-related
metabolic bone disease [237, 238]. Persistent met-
abolic acidosis can also lead to impaired growth in
children outside of the deleterious effects of renal
osteodystrophy [239]. Various mechanisms have
been implicated including blunted growth hor-
mone (GH) release, inhibition of pulsatile GH
secretion, and resistance to insulin-like growth
factor-1 (IGF-1) action in the growth plate
[239–241]. Correction of metabolic acidosis is a
prerequisite for effective response to recombinant
GH therapy in children with CKD and growth
failure.
The 2013 Kidney Disease Improving Global
Outcomes (KDIGO) guidelines recommend
treating metabolic acidosis in CKD with some
sort of alkali therapy to maintain serum HCO3
concentration in the normal range [242]. Treat-
ment can be provided as sodium bicarbonate or
sodium citrate, starting at 1 mEq/kg/day and
13 Physiology of the Developing Kidney: Fluid and Electrolyte Homeostasis and Therapy of. . .
titrated to achieve target serum HCO3 concentra-
tion. Newborns and infants will require higher
alkali doses than older children and adolescents
with the same degree of renal dysfunction due to
their relatively higher acid generation and reduced
acid excretion ability.
Finally, for patients with end-stage renal dis-
ease (ESRD) receiving renal replacement therapy,
metabolic acidosis typically improves with the
additional base load delivered in the dialysate.
Persistent acidosis pre-dialysis may be an indica-
tion of insufficient dialysis dose, higher net acid
generation secondary to high dietary animal pro-
tein intake [243], or some other coexisting etiol-
ogy for metabolic acidosis. On the contrary, a
normal or high serum HCO3 concentration
pre-dialysis may indicate adequate dialysis,
decreased dietary protein intake, or a coexisting
metabolic alkalosis. Correction of abnormal
pre-dialysis serum HCO3 abnormalities entails
addressing the underlying cause and adjusting
the dialysate bicarbonate bath
concentration [244].
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 Part III
Translational Research Methods
 Translational Research Methods:
Basics of Renal Molecular Biology 14
Gian Marco Ghiggeri, Maurizio Bruschi, and
Simone Sanna-Cherchi

Contents Genome-Wide Association in Primary

Glomerulonephritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426 Copy-Number Variations in Congenital
Genomic and Proteomic Technologies Anomalies of the Kidney and Urinary Tract . . . . . . . . 439
and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426 Next-Generation Sequencing in Renal Disease . . . . . 440
Genetics and Genomics for Identification of The Building of the Podocyte Protein Map . . . . . . . . . 440
Susceptibility Alleles for Disease . . . . . . . . . . . . . . . . . . . 426 Proteomic Analysis of Biological Fluids:
DNA Sequencing and Search for Rare Variants . . . . 428 The Normal Urine Map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440
Single Nucleotide Polymorphism (SNP) Peptidome, Degradome, and Metabolomics . . . . . . . . . 441
and Genome-Wide Association Studies (GWAS) . . . 430 Autoantibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441
Structural Variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442
Techniques for Proteome Analysis and Mass
Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431
Bioinformatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434
Bioinformatics Tools Applied to DNA . . . . . . . . . . . . . . 434
Bioinformatics Tools for Proteomics . . . . . . . . . . . . . . . . 436
Protein Interaction, Metabolic Pathways,
and Systems Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437
Application Fields to Renal Diseases . . . . . . . . . . . . . . 437

G.M. Ghiggeri (*) • M. Bruschi

Laboratory of Physiopathology of Uremia, Division of
Nephrology, Dialysis and Transplantation, Istituto
Giannina Gaslini, Genoa, Italy
e-mail: gmarcoghiggeri@ospedale-gaslini.ge.it;
mauriziobruschi@ospedale-gaslini.ge.it
S. Sanna-Cherchi
Division of Nephrology, Columbia University, College of
Physicians and Surgeons, New York, NY, USA
e-mail: ss2517@columbia.edu; ss2517@cumc.columbia.
edu

# Springer-Verlag Berlin Heidelberg 2016 425

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_13
426
Introduction
Molecular biology has developed between years
1940s and 1960s as a branch of science that
spans from genetics to cell biology and biochem-
istry with the goal of understanding the interac-
tions by which DNA, RNA, and protein
synthesis operate into cells [1]. Since then,
numerous technological innovations pushed the
field forward, allowing recombinant DNA tech-
nology to be applied to a wide variety of biologic
problems and to enter the clinical practice. The
discovery of restriction nucleases and DNA
ligases, development of DNA libraries, DNA
cloning procedures, nucleic acid hybridization
techniques, and the polymerase chain reaction
(PCR) together with transgenic animals have
all represented successive steps for a successful
delineation of the role of genes in cellular phys-
iology and pathophysiology. This molecular rev-
olution has affected all of the sciences, including
medical and clinical research, and has culmi-
nated in the completion of the human genome
project [2, 3]. In the past 15 years, we have
witnessed tremendous technological advances
for high-throughput analysis of DNA, RNA,
and protein in a comprehensive and cost-
effective manner, with great promises to trans-
late the analysis of the central dogma of biology
into precision diagnosis and treatment for both
rare and common diseases (Fig. 1). The recent
advances in RNA biology identified numerous
noncoding functional regions of the genome,
which “escape” the central dogma but that
can be analyzed and studied using a similar
framework.
This entry briefly illustrates the basic concepts
of molecular biology that underlie the various
strategies that have been developed for studying
living cells and organisms and summarizes molec-
ular biology techniques that are used in research
and clinical practice. We will show practical appli-
cations of genomic and proteomic tools and strat-
egies applied to kidney disease research in a
concise format. For more information, the reader
can refer to the many specialized reference text-
books that are available.
G.M. Ghiggeri et al.
Genomic and Proteomic Technologies
and Applications
Genetics and Genomics
for Identification of Susceptibility
Alleles for Disease
The type of approach to solve the genetic under-
pinning of a trait largely depends on the epidemi-
ology of the disease, its heritability, race/ethnicity
differences, the underlying genetic model, and the
predicted effect size of the underlying causal var-
iants (Fig. 2). Traditionally, Mendelian diseases
occurring in families have been associated to very
rare variants with large effect on the phenotype
and have been successfully tackled using linkage/
positional cloning strategies, while common traits
have been thought to be mostly caused by com-
mon variants with small-effect size that are ances-
trally inherited in populations (common disease –
common variant hypothesis) and have been the
object of hundreds of genome-wide association
studies.
The recent discovery of the role of rare copy-
number variations in developmental diseases and
the tremendous advances in sequencing technolo-
gies, which lead to the vast use of next-generation
sequencing, boosted the research on rare variants
identification and interpretation in complex dis-
eases. Below, we will briefly review the basic
approaches to gene mapping and identification in
research setting and clinical practice. Examples of
application of different strategies to renal research
will be given in the next section of this chapter.
Mendelian Genetics
Since the landmark paper of Landers in 1986 [4]
and the first discovery of RFLP maps and
then microsatellites, traits with strong genetic
determination, segregating in large pedigrees
following clear Mendelian modes of inheritance
(autosomal dominant, autosomal recessive,
X-linked), were approached by using logarithm
of odds score (LOD score) methods. Using these
methods, if a family is large enough to provide the
statistical evidence that a chromosomal region
co-segregates with the disease trait under the
14 Translational Research Methods: Basics of Renal Molecular Biology 427

Molecular Diagnosis
∼20,000
DNA Counselling
GENES
Personalized Genomics

Gene Expression Regulation Personalized

∼80,000
RNA Pathways Treatment
TRANSCRIPTS
Molecular Signatures Strategies

Protein Networks
>1,000,000
PROTEIN Pathophysiology
PROTEINS
Biomarkers

TRAIT

Fig. 1 Schematic representation of the central dogma of biology

Fig. 2 Graphic
representation of disease 1
models and gene Unlikely
Mendelian
identification strategies to occur
based on penetrance 0.75
Linkage / de novo
Penetrance

(X-axis) and allele

frequency (Y-axis)
0.5

0.25
Difficult
Complex
to identify
0
Next-generation genomics Association

< 0.001 0.001 - 0.01 0.01 - 0.1 > 0.1

Allelic Frequency

hypothesis of linkage versus no-linkage (null Complex Traits: Common Variants

distribution) at a genome-wide significance
level [5], the locus containing the disease-
causing gene is identified. Once the disease
locus is mapped, direct sequencing of the candi-
dates in this region is performed to identify the
gene containing the disruptive segregating muta-
tion. Finally, the proof of genetic causation
requires the identification of independent delete-
rious mutations in additional families or patients
with the same phenotype and functional analyses
in cellular and animal models.
The common variant – common disease theory
stands on the hypothesis that complex traits that
manifest later in life have been less subjected to
natural selection because they are less likely to
impact fitness compared to rare, lethal Mendelian
diseases. With less selection operating, these phe-
notypes are maintained at relatively high fre-
quency in the general population. Given the high
heritability of many of these traits (hypertension,
diabetes mellitus, Alzheimer’s disease, Crohn’s
disease, and many others), it was hypothesized
428
that, similarly to the disease epidemiology, the
underlying disease-causing alleles would be fre-
quent in the general population and have small to
moderate effect size, since purifying selection
would be unlikely to have a major effect on
eliminating such alleles. Therefore, these vari-
ants, once introduced in a population, would be
maintained at relatively high frequency. By the
use of a complete map of SNPs able to effectively
tag the haplotype structure of the human genome
from a specific population, it is then feasible to
identify such variants (or the variants in linkage
disequilibrium with the causal allele). The
HapMap Consortium greatly facilitated the
development and generation of SNP arrays for
genome-wide association studies (GWAS). The
basic hypothesis underlying the GWAS study
design is that variants at the disease locus
(i) would have significantly different allele fre-
quencies between cases and controls compared to
the vast majority of the remaining variants. Usu-
ally, a GWAS is conducted by comparing allele
frequencies for 0.5–1 million markers across the
genome between cases and controls in order to
fully capture the haplotype structure of the
genome. While this approach represents an unbi-
ased, hypothesis-free way to detect association
signal, the large number of pairwise comparisons
mandates rigorous correction for multiple testing
in order to avoid spurious, false-positive results.
In fact, the usual threshold for genome-wide sig-
nificance is set at p < 5  108. While the basic
concept of GWAS is relatively simple and
straightforward, there are numerous technical
and analytical issues to be taken into account,
such as power calculations, population stratifica-
tion, platform and batch effect, and others, that
will be briefly discussed below in the bioinfor-
matics section.
DNA Sequencing and Search for Rare
Variants
DNA sequencing is the backbone of molecular
genetics. Frederick Sanger published his revolu-
tionary paper in 1977, describing a DNA
sequencing technology that dominated the
G.M. Ghiggeri et al.
genetic field for approximately 30 years [6].
This methodology, which is still considered the
gold standard for its accuracy and reproducibil-
ity, relies on enzymatic extension of a DNA
template (usually a PCR amplification product)
using dideoxy terminators initially labeled with
radioactive, subsequently with fluorescent dyes.
The labeled amplicons are then separated on
agarose gel or, currently, through an automated
capillary sequencer. This methodology was the
main force behind the efforts of the Human and
Venter’s Genome Projects [2, 3], which lasted
over a decade and resulted in the first draft of
the human genome at the cost of almost three
billion dollars [7]. After the completion of the
sequence of the human genome, we assisted in
the international private and academic efforts
aimed to bring the whole-genome sequencing
(WGS) to a cost and turnaround that would
allow its implementation into clinical practice.
The recent advent of next-generation technology
has completely revolutionized genetic research,
by increasing sequencing capabilities by five
orders of magnitude and decreasing costs by
nearly six orders of magnitude. It is in fact now
possible to effectively sequence the entire
genome of an individual at approximately
30X coverage in about 2 weeks at a cost of
1,500 dollars using the new Illumina HiSeq X
Ten system. This approaches the goal of the X
Prize Foundation of achieving 100 human
genomes sequenced in less than 30 days at a
cost of $1,000 or less per genome (http://geno
mic.xprize.org/). The basic idea underlying com-
monly used next-generation sequencing (NGS)
technologies is that clonal DNA fragments are
generated and then they are all subjected at once
to massively parallel sequencing using
microfluidics. Sequencing reactions are carried
on by consecutive cycles of base addition by
polymerization or ligation according to the sys-
tem used. Visualization and base calling are then
conducted in different ways: pyrosequencing for
the Roche FLX System, fluorescence for the
Illumina and Life Technologies SOLID instru-
ments, and changes on pH for the Ion Torrent
apparatus. More recent sequencing technologies,
sometimes referred to as third- and
14 Translational Research Methods: Basics of Renal Molecular Biology
fourth-generation sequencing (nanopore, in situ
sequencing, respectively), are still in their exper-
imental phase and will not be discussed here.
Also, since the vast majority of NGS in both
academia and private sector is conducted using
the Illumina system, and since the authors of this
chapter have most of their experience using this
technology, we will describe data generation and
analysis using the Illumina NGS system.
Targeted enrichment systems. During these
past years of NGS, several ways to selectively
enrich portions of the genome have been devel-
oped to capture regions of interest at higher depth
of coverage while attempting to further reduce
costs. Based on the estimation that Mendelian
diseases and traits with stronger genetic determi-
nation are most likely to be caused by protein-
altering variants, exome sequencing has been
developed [8]. By selectively capturing the
exons of the nearly 20,000 human genes, which
constitute about 1–2 % of the euchromatic DNA,
it is possible to sequence at high coverage
(60–90X) the entire protein coding region of the
genome at a cost of $600–800 per sample. This
approach has been tremendously efficient in solv-
ing the molecular diagnosis of a multitude of
Mendelian and complex traits [9–21]. The possi-
bility of performing rapid diagnosis for known
genetic causes for Mendelian diseases led to the
development of array-based targeted gene panels
for diseases (or group of diseases) to use in the
clinical settings. These targeted capture chips,
although of relatively recent introduction, are
already been abandoned due to elevated costs
(higher than whole-exome sequencing) and to
the not comprehensive nature of platform design;
therefore, we will not discuss them in this review
since they are not likely to play a significant part
in the armamentarium of genetic tools in human
disease research and diagnosis.
The availability of individual-level whole
genome or exome data also uncovered that each
individual is a carrier of thousands of single nucle-
otide variants, many of which are very rare or
unique and predicted to be deleterious, thus enor-
mously complicating the attribution of genetic
causality for both Mendelian and complex traits,
especially when no large families or de novo
429
mutations are identified in the discovery studies
(see below). Therefore, for studies that are prom-
inently based on singletons or small families, and
in the setting of high genetic heterogeneity, the
discovery of independent mutations and the
assessment of the mutational load under the null
distribution in large populations rapidly became
imperative. On one side, publicly available
population-based genomes and exomes can be
very helpful in rapidly identifying “intolerant”
genes (genes harboring no or very few loss-of-
function variants). Among such databases, partic-
ularly useful are the 1,000 Genome Project, the
NHLBI Exome Variant Server (http://evs.gs.wash
ington.edu/EVS/; featuring whole exome data
from ~6,500 individuals), and the recently
released Exome Aggregation Consortium (http://
exac.broadinstitute.org/; featuring whole exome
data from ~63,000 individuals). Nevertheless, to
identify rare independent mutations in patients
with the disease object of study, it is becoming
apparent that candidate genes detected in whole
genome or exome studies (usually dozen to a few
hundreds) must be effectively resequenced in
hundreds or thousands of individuals in order to
uncover excess burden of rare variants and, pos-
sibly, establish genetic causation. Obviously,
resequencing studies of this scale cannot be
performed in a time- and cost- effective manner
using Sanger sequencing or targeted NGS chips at
an individual DNA level. Therefore, several
methods have been implemented for multiplex
bar coding DNA capture and massively parallel
resequencing in large cohorts [22]. Among these
methods, the molecular inversion probes (MIP)
and the Fluidigm 48.48 Access Array™ IFC Sys-
tem coupled to NGS have been the most success-
fully used [23–25]. Using these methods, it is
possible to sequence 600–1,000 amplicons
(approximately 40–80 genes) in 1,000–3,000
patients in few months and at a cost of 50–75
cents per gene per sample, corresponding to
approximately $15,000–25,000 to resequence
40 genes in 1,000 patients. As a comparison, the
same undertaking would require approximately
0.8–1.5 million dollars by array-based NGS or
Sanger sequencing and significantly longer
processing time.
430
Single Nucleotide Polymorphism (SNP)
and Genome-Wide Association
Studies (GWAS)
One of the first and most important discoveries
deriving from the completion of the human
genome and the first comprehensive catalog of
single nucleotide variants was that the euchro-
matic DNA is organized in haplotype blocks.
This led to the release of the International
HapMap Project [26] which is an effort to catalog
millions of variants (mostly single nucleotide
polymorphisms) and to describe their pattern of
variations and their correlation (linkage disequi-
librium) in populations from three main ances-
tries: Europeans, Africans, and Asians. The
HapMap work posed the foundation for the devel-
opment of high-density genome-wide SNP arrays
and their application for genome-wide association
study (GWAS) in complex traits. The overarching
hypothesis was in fact that complex traits with
high prevalence in the general population and
high heritability (e.g., diabetes mellitus, hyperten-
sion, Alzheimer’s disease, and many others)
would be caused by common variants with
small-effect size (common disease – common var-
iant hypothesis) [27]. With the description of the
haplotype structure of the human genome, it was
then possible to develop SNP arrays containing
only a reduced fraction of the entire genetic vari-
ation (usually 500,000 to 1 million SNPs) that
would efficiently tag these blocks of disequilib-
rium and therefore enable genome-wide associa-
tion studies. Since the first successful GWAS
reported in 2005 identifying a common variant
in the complement factor H as a strong suscepti-
bility factor for age-related macular degeneration
[28–30], approximately 7,000 variants have been
convincingly identified in complex traits genetics
[31]. While this might seem an enormous success,
these studies and the underlying hypothesis have
also encountered skepticism, especially in regard-
ing the potential for utility and translation in clin-
ical practice [32, 33]. The reason for this
skepticism stands mainly in the fact that most
variations, even in aggregate, account for a small
fraction of the heritability of these traits, thus
hampering a direct prognostication and risk
G.M. Ghiggeri et al.
prediction. Moreover, functional interpretation of
these results is often problematic. Nevertheless, it
is of no doubt that many of such studies have shed
light on disease pathogenesis and treatment and
provided important clues into population struc-
ture, disease epidemiology, and natural selection
[28–31, 34–36]. The challenge still remains now-
adays, and consists in translating each of these
7,000 robust associations into functional assays
to resolve disease pathogenesis and to improve
therapeutic schemes. Finally, despite the success
in the past 10 years of GWAS, a significant frac-
tion of heritability for most complex traits still
remains missing, suggesting that rare or
low-frequency variants with large or moderate
effect size might account for it, hence the interna-
tional efforts for systematic resequencing in large
populations.
Structural Variants
Variations other than single nucleotide variants,
such as change in DNA copy number, inversions,
and translocations are known as structural vari-
ants. Structural variants have traditionally been
implicated in the pathogenesis of rare genomic
disorders [37]. Landmark studies conducted in
healthy population controls in mid-2000s showed
that a significant portion of the euchromatic DNA
is subjected to copy-number variations (CNVs)
[38–41]. The amount of genetic variation derived
from CNVs was estimated to be at least one order
of magnitude larger than the one attributable to
SNPs. Moreover, given the fact that CNVs are
often large and contain multiple genes, it was
suggested that the effect of gene copy number
could be a major driver in the predisposition to
complex traits. While common CNPs (copy-
number polymorphisms) did not seem to explain
much of the missing heritability of complex traits
[42], subsequent studies clearly showed that rare
CNVs are a major source of genetic variation
underlying many developmental traits, from cra-
niofacial defects, cardiovascular and kidney
malformations, and neurodevelopmental
diseases including autism, schizophrenia, epi-
lepsy, intellectual disability, and developmental
14 Translational Research Methods: Basics of Renal Molecular Biology
delay [43–51]. Importantly, the work on
neurodevelopmental diseases also sets the exam-
ple and framework for analysis and interpretation
of rare variants in diseases with extremely high
genetic heterogeneity.
CNVs, usually defined as gain or loss of
genetic material that spans from 1,000 bp to sev-
eral megabases, can be identified using different
technologies. The most used in current research
and in clinical practice are array comparative
genomic hybridization (aCGH), SNP arrays, and
next-generation sequencing.
Array Comparative Genomic Hybridization
(aCGH). One of the first methods for analysis of
CNVs was comparative genomic hybridization,
which was performed by differential labeling of
control and test metaphase chromosomes at low
resolution. Subsequently, BAC clones and then
oligonucleotide probes have been implemented
on a physical support (array) to analyze genomic
DNA at much higher resolution and at a lower
cost. The aCGH is still currently used, often in the
clinical setting, for high-resolution analysis of
CNV, especially in prenatal care and in the
diagnosis of severe developmental diseases
[52, 53]. One major advantage of aCGH is the
very high signal-to-noise ratio, which allows
robust identification of structural variants and
their breakpoints.
Genome-Wide SNP Arrays. While originally
developed for SNP genotyping and GWAS appli-
cation, the observation that raw and allelic inten-
sity data could be used to assess for CNVs made
these platform a valid, and sometimes preferred,
alternative to aCGH. One of the advantages of
aCGH compared to SNP chips for CNV analysis
is the higher signal-to-noise ratio. On the other
side, SNP chips are usually cheaper and allow
better quality control analyses (e.g., identification
of duplicates or hidden relatedness) and correction
for population stratification using genotype infor-
mation, which are critical in large-scale associa-
tion studies. Higher level CNVs (>4 copies) and
other structural variants, such as inversions and
translocations, are very difficult or impossible to
identify by either aCGH or SNP chips.
Next-Generation Sequencing. Genome-wide
analysis of copy-number variation is
431
progressively taking place using whole-exome
and whole-genome sequencing. The observation
that most CNVs implicated in developmental dis-
ease encompass multiple genes, made exome
sequencing a desirable tool for identification of
genic CNVs. Many software packages have been
developed for CNV analysis using exome-
sequencing data (see below), but the capture
design of this technology results in uneven depth
of coverage across the exome, posing serious con-
cern in accuracy and replication of results. Whole-
genome sequencing on the other hand represents a
very powerful way to accurately score CNVs,
identify boundaries, and also to identify other
structural variants such as inversions, transloca-
tions, gene fusions, and complex CNVs by anal-
ysis of atypical reads, thus making it the most
comprehensive and accurate tool for analysis of
structural variations [54]. With the continuous
drop in sequencing costs, WGS will soon repre-
sent the most cost-effective and comprehensive
tool for analysis of rare and common variants,
either SNV or structural variations.
Techniques for Proteome Analysis
and Mass Spectrometry
Principles and techniques. The principle of pro-
tein analysis has considerably evolved over the
years in parallel with key technological advance-
ments, especially in the field of mass spectrome-
try. The basic principle behind any proteomic
approach is that the products of a peptide diges-
tion constitute a fingerprint of the original protein
which can be defined based on adequate strategies
that include peptide mass identification and bio-
informatics analysis. In general, identification of a
protein is preceded by a preparative step.
Two-dimensional (2D) electrophoresis has now
emerged as a powerful tool to accomplish this
task. LC-Mass and MALDI-TOF are commonly
used for the analysis of trypsin digest.
Denaturing and nondenaturing 2D electro-
phoresis. Classic 2D protein electrophoresis is
the technique of choice for the analysis of plasma
proteins or other complex biological samples
(Fig. 3a). High resolution is usually achieved by
432 G.M. Ghiggeri et al.

_
a 3.5 non lianer pH 9 b + Native electrophoresis
250 550

Un-folded and reduced electrophoresis

Albumin 200

IgG heavy chain

Mr Haptoglobin β chain Albumin IgG heavy chain
Mr
kDa
kDa

IgG light chain Haptoglobin β chain

IgG light chain

10
5

414m ∼ 100 fmol

414m ∼ 100 fmol

414m ∼ 200 fmol
414m ∼ 200 fmol
414m ∼ 400 fmol

414m ∼ 400 fmol

c 3.5 non lianer pH 9

250

Ang II _ + + + + + + +

Mr
kDa

5 1 2 3 4 5 6 7 8

Fig. 3 (a) Classical 2D polyacrylamide gel electrophore-
sis (2D-PAGE) and (b) 2D electrophoresis in
nondenaturing condition (Nat/SDS-PAGE) of the same
serum sample. In the former approach, proteins are first
separated according to their charge in the presence of urea
(IEF) and are then run in a polyacrylamide gradient that
separates on the basis of size. In Nat/SDS-PAGE, protein
complexes migrate unresolved in the first dimension and
are then separated in denaturing conditions, in the second
dimension. (c) DIGE systems that utilize thiol-based
reagents (maleimide, iodoacetamide) increase specificity
and reproducibility of protein stainings on a quantitative
basis. Protein mixtures are labeled separately with different
NHS dyes, combined and resolved on a single 2D gel that
is analyzed with different fluorescence excitation wave-
lengths. The differential expression of individual proteins
is by this mean analyzed and quantified. (d) Gel retardation
assay. The figure demonstrates binding of regulatory
nuclear proteins to cis-elements in the COL3A1 promoter
after angiotensin II (Ang II) stimulation. Nuclear extracts
were obtained from Ang II stimulated cells (Ang II +) and
incubated with a 32P-labeled oligonucleotide (414) that
contains sequences +3 to +20 of the COL3A1 promoter.
Ang II stimulates the synthesis of a peptide that binds to the
target DNA and retards its migration in the gel (lanes 2).
This reaction can be competed with increasing amounts of
nonlabeled wild-type oligonucleotide (lanes 3–5) but not
with a cold-mutated analogue sequence (414 m) (lanes
6–8). In the absence of Ang II stimulation (Ang II -), no
DNA-protein complex-generating gel retardation is
observed (lane 1)
14 Translational Research Methods: Basics of Renal Molecular Biology
combining separation by charge and separation by
mass in denaturing conditions (IEF/PAGE)
[55, 56]. This is followed by microscale mass
spectrometry that allows identification of individ-
ual spots, with reliable sensitivity and reproduc-
ibility. The development of softgels is a recent
evolution that improves the resolution of high-
molecular-weight proteins, but requires denatur-
ing conditions, which do not allow to analyze
protein-protein interactions (Fig. 6a) [57]. For
this reason, new techniques that separate protein
mixtures in partially denaturing conditions have
recently been developed. These include Blue-
PAGE, which is performed on membranes, and
Nat/SDS-PAGE, which is performed on a poly-
acrylamide substrate (Fig. 3b) [58–60].
Protein staining. Traditionally, protein staining
after electrophoresis has been performed with
Coomassie R-250 and, from mid-1980s, with sil-
ver ions that allow high sensitivity [61]. Nowa-
days, new dyes allow differential protein
expression analysis on 2D gels (DIGE). This tech-
nique is based on modification of selected amino
acid residues and has become a standard applica-
tion in quantitative proteomics. Peptides are
labeled with matched sets of fluorescent N-
hydroxysuccinimidyl ester cyanines (NHS) that
have different excitation-emission wavelengths
[62]; the use of thiol-based reagents (maleimide,
iodoacetamide) increases specificity and repro-
ducibility (Fig. 3c) [63]. Protein mixtures can be
labeled separately with different NHS/thiol dyes,
combined and resolved on a single 2D gel that is
then analyzed at different fluorescence excitation
wavelengths [64]. This allows analysis and quan-
tification of the differential expression of individ-
ual proteins.
Mass spectrometry. Proteins are usually char-
acterized by mass spectrometry. As ionized mol-
ecules are accelerated through an electric field,
they are separated, reaching the detector at differ-
ent times, depending on their mass and charge.
This “time-of-flight” (TOF) is specific of a given
small solute or for a given peptide, allowing its
identification. Whole proteins are ionized by
electrospray ionization (ESI) or matrix-assisted
laser desorption/ionization (MALDI). The com-
monly used “MALDI-TOF” procedure indicates
433
that the MALDI sample preparation is followed
by TOF mass analysis, while in the surface-
enhanced laser desorption and ionization
(SELDI) procedure, proteins are immobilized on
solid supports or on customized protein chips.
In the so-called top-down strategy, intact pro-
teins are ionized and resolved in the mass ana-
lyzer. Alternatively, proteins are predigested into
smaller peptides before the mass analysis, a pro-
cedure referred to as “bottom-up.” In this case, the
protein is identified by its pattern of digestion that
creates a “peptide mass fingerprinting” (PMF), or
by “de novo sequencing,” tracking back the pro-
tein sequence from the mass-sequence data using
protein databases. Often, both “top-down” and
“bottom-up” strategies are used to optimize pro-
tein identification.
Protein-protein interaction. One important
question in protein analysis is to identify interac-
tions between proteins, which regulate most intra-
cellular signaling and are critical to the assembly
of functional peptides.
Different approaches are possible. In one
approach, proteins are analyzed directly by two-
dimensional electrophoresis in nondenaturing and
in denaturing conditions (Nat/SDS-PAGE) that
allow protein complexes to co-migrate in the
first dimension (nondenaturing) and then dissoci-
ate in the second (Fig. 3b) [60]. Alternatively,
proteins can be extracted from a crude mixture
after binding to target proteins linked to solid
supports or interactions that can be identified
using a yeast two-hybrid system. This methodol-
ogy is based on the modular structure of gene
activation using the GAL4 transcriptional activa-
tor as reporter. This transcription factor has a
DNA-binding domain and an activation domain,
both of which must be in close association to
activate transcription. By DNA recombinant tech-
niques, two protein sequences acting as bait and
target are fused with sequences encoding with one
of the GAL4 domains. When expressed in yeast
cells, the activation domain and the DNA-binding
domain are bridged together when the two pro-
teins interact and promote transcription of a
reporter gene. Similar prokaryotic systems have
been developed based on the modular structure of
bacterial RNA polymerases, in which target and
434
bait cDNAs are fused to either the core enzyme or
a Φ factor.
Protein-DNA interaction. Techniques that ana-
lyze the regulation of DNA expression by tran-
scriptional factors have allowed to identify
consensus DNA-binding regions and their regula-
tory proteins. Most of these studies are based on
the principle that protein-DNA complexes have
high molecular weights and are therefore retarded
when resolved by polyacrylamide gel electropho-
resis. In addition, proteins interacting with DNA
molecules tend to protect the nucleic acid regions
to which they bind from digestion with DNAse,
allowing their identification [65]. In Fig. 3d, for
example, a nuclear extract of proteins obtained
from cells stimulated with angiotensin II was
co-incubated with strings of DNA that encode
for the promoter of type III collagen. As shown,
angiotensin II stimulations promote the synthesis
of a protein that binds to the 32P-labeled DNA
target, forming macromolecular complexes that
are retarded in the gel.
Bioinformatics
Bioinformatics Tools Applied to DNA
In this section of the entry, we will briefly review
and describe the current methodologies and pub-
licly available software packages that are most
commonly used for gene identification in Mende-
lian and complex traits. The scope is to provide a
short, simple, and user-friendly guide for data
analysis. Large-scale genetic data sets are usually
managed using ad hoc scripts and pipelines (Perl,
Python, R, and others), usually in a Unix or Linux
environment, that are generated in-house based on
individual needs, and they will not be discussed in
this chapter.
Traditional Positional Cloning/Linkage
Packages
Linkage analysis refers to the analysis of
co-segregation of chromosomal regions with the
disease trait under a specific mode of inheritance
(X-linked, autosomal dominant, autosomal reces-
sive). The statistical inference underlying linkage
G.M. Ghiggeri et al.
analysis is the logarithm of odds (LOD) score, a
likelihood ratio test, that tests the probability of a
disease gene to be localized in a certain region
(linked to the nearby marker) versus the null
hypothesis or random segregation. The approach
hinges on the availability of single, uniquely large
kindreds or large cohorts of small/medium size.
Since the joint probability across multiple families
is a product of each individual probability, the
LOD scores, which use a logarithmic scale, can
be summed. The first linkage package, Liped, was
described in 1974, and used the Elston-Stewart
algorithm to compute two-point parametric LOD
scores [66]. Subsequent programs, such as LINK-
AGE and FASTLINK, have allowed to calculate
two-point and multipoint LOD scores; while com-
putation time did not increase significantly with
the size of pedigrees, these algorithms, however,
are not efficient enough for large markers datasets
[67, 68]. Efficient multipoint analysis of linkage
for dense marker maps was then developed with
Genehunter [69] and optimized in Allegro and
Merlin software [70, 71]. These linkage packages
allow two-point and multipoint parametric link-
age analysis, nonparametric, model-free analysis,
haplotype reconstruction, and other analyses. The
main limitation of these algorithms is that while
the computation time increases linearly with the
number of markers, it increases exponentially
with pedigree size. Software that can handle larger
genealogies and perform also quantitative traits
linkage analysis include SimWalk [72] and
Loki [73].
Genome-Wide Association Studies
One of the most user-friendly, versatile, complete,
and widely used package for association analysis
is PLINK [74]. This package allows rapid data
management and analysis for hundreds of thou-
sands of markers generated from thousands of
individuals, in a simple research environment
using a Unix or Linux desktop computer. The
main implemented functions include data
management, summary statistics, population
stratification, association analysis, and identity-
by-descent estimation. More recent additions to
this package include analysis or rare variants,
including CNVs, and next-generation sequencing.
14 Translational Research Methods: Basics of Renal Molecular Biology
Fig. 4 Burden analysis
between 1,441 patients with
congenital anomalies of the
kidney and urinary tract
(red) and a control dataset
of 21,610 individuals
Population Frequency
(green), examining the
population frequency of the
largest CNV per genome
using a survival function
(logrank P < 5  108;
Sanna-Cherchi, personal
data)
Correction for population stratification is usually
conducted in PLINK using multidimensional
scaling (MDS) or by principal component analy-
sis as implemented in other packages such as
Spectral-GEM [75, 76].
Often, large studies on complex traits combine
data from multiple cohorts and multiple
genotyping platforms, requiring data harmoniza-
tion prior to performing joint analyses. To uniform
markers map, genotype imputation can be
conducted using Markov chain haplotyping, as
implemented in MACH software [77]. Imputation
of a few million SNPs such has the HapMap panel
is a computational intensive task that usually
requires cluster computing. Joint analyses of
data generated from multiple cohorts can be
performed using an inverse variance-weighted
method (METAL software) [78], and heterogene-
ity across cohorts can be tested by performing
Cochran’s Q test and deriving heterogeneity
index (I2).
Copy-Number Variation Analysis
CNV analysis from SNP chips can be performed
using multiple publicly available software.
Preprocessing and QCs are usually conducted
using PLINK [74]. The most robust algorithms
for CNV calling, such as QuantiSNP and
PennCNV [79, 80], utilize generalized
genotyping methods which incorporate the two
435
100%
75%
50% CAKUT
CONTROL
25%
10%
0%
0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 9,00010,000
Largest CNV Size (kb)
analytic dimensions (genotype and intensity) to
estimate copy-number state. Association tests for
common CNVs can be conducted using the same
framework used for GWAS, with the only caveat
that CNVs have often a higher genotyping error
rate, especially when analyzing data generated
from different platforms. Analysis of rare CNVs
includes burden tests and single variant tests. To
compare the burden of rare CNVs between cases
and controls, the Fisher’s exact test is often used
for testing differences in the type and size distri-
butions of CNV. The average CNV rates per
genome, the mean CNV size per individual, the
largest CNV size per individual, and the total
genomic span per genome are usually calculated.
Given the positively skewed distributions of these
metrics, nonparametric statistics are preferred
(Mann–Whitney U test) and empirical p-values
are calculated using permutation tests. The fre-
quency of the largest CNV per genome in the
population can be analyzed using a survival func-
tion (Fig. 4). In this approach, median sizes are
compared between cases and controls using the
logrank test, while the proportions of cases and
controls with CNVs above a given threshold are
compared using Fisher’s exact test.
Next-Generation Sequencing
The analysis of NGS data varies significantly
based on the application, and, besides standard
436
software for base calling and variant calling, most
of the analyses are conducted using original
scripts and pipelines designed ad hoc for each
study. Usually after completion of a sequencing
run, data are automatically transferred to a cluster
for base calling. Sequence graph files (.sgr) can be
generated to visualize sequencing data. After
quality control assessment, sequence reads are
converted to FASTQ format and mapped to the
reference genome producing BAM files
containing all passed filter reads, alignment infor-
mation, sequence, and quality score information.
BAM files are then converted in VCF files for
downstream analyses [15]. Recalibration and
local realignment around indels are done using
GATK [81]. Standard tools for variants’ annota-
tion include ANNOVAR [82], SNPeff [83], and
SeattleSeq (http://snp.gs.washington.edu/
SeattleSeqAnnotation138/). Usually, allele fre-
quencies are tested against dbSNP, 1,000
Genome, Exome Variant Server (http://evs.gs.
washington.edu/EVS/), and the recently released
Exome Aggregation Consortium (http://exac.
broadinstitute.org/). Metrics for interpretation of
possible functional relevance are computed using
algorithms that take in account DNA and amino
acid conservation across species and chemical
properties of the amino acid substitutions. Some
of the most commonly used include SIFT [84],
PolyPhen-2 [85], GERP [86], and CADD
[87]. Several programs to test for rare variant
association are currently available or underdevel-
opment; one of the most commonly used is
SKAT [88].
Bioinformatics Tools for Proteomics
General considerations. Comparative proteomics
aims at detecting changes in protein expression
profiles between two or more groups of samples
(cell extracts, biological fluids, distinct pharmaco-
logical treatments, etc.). Since an extremely high
number of information derives from mass spec-
trometry, bioinformatics data analysis represents a
crucial aspect. Optimal design of the experiment
is critical to reduce cost and maximize informa-
tion. Quantitative data are traditionally assessed
G.M. Ghiggeri et al.
by univariate statistics, such as ANOVA; multi-
variate analysis, such as principal component
analysis (PCA) and heat maps, allows to better
describe trends and to reduce complexity [89].
A combination of both approaches is often used
and ad hoc software have been developed [90].
Adequate data preprocessing is, however, crucial
to avoid redundancy, data incompleteness, and
errors. Here, we briefly present a statistical
workflow for maximizing the information
obtained by quantitative proteomics. The analyses
are usually conducted with existing statistical
packages; one of the most performing is R [91],
freely available at http://www.r-project.org/. Ana-
lyses of functional protein interaction networks
can be performed with public software; as an
example, cytoscape, an open-source package for
the analysis of protein and genetic interaction
networks, is illustrated.
Sample size. To determine the samples size the
following equation can be utilized:
 2
δ
n ¼ 1 þ 2c
fc
where the quantity c depends on the power of the
test and on the significance level. Power is usually
set at a value between 80 % and 90 %, while the
significance level is set at 0.05 % or 0.01 %; δ is
the standard deviation (SD) of biological replica-
tions, and fc is the difference to be detected that is
usually set at twofold changes. To limit the effect
of systematic variation across different experi-
mental blocks, protein data must be normalized
and expressed as fold changes relative to other
proteins. Normalization of n spot/peptide abun-
dance in each experiment is performed after back-
ground subtraction [92].
Missing values and data filtering. Proteomic
studies are limited by the high frequency of miss-
ing values, which can be determined by using
imputation algorithms such as the k-nearest neigh-
bors [93]. Filtering outlier decreases artifacts, pre-
vents technical biases, and improves the statistical
power of the subsequent analyses. In most cases,
this also reduces sample noise.
Data transformation and testing. Usually, a
small number of replicates produce nonnormal
14 Translational Research Methods: Basics of Renal Molecular Biology
distributions of proteome profiles. In most cases,
this requires log2 transformation of data sets prior
of using classical univariate parametric
approaches, such as the student’s t-test or the
analysis of variance (ANOVA). Often, nonpara-
metric tests are mandatory; these are based on the
analysis of median and are less sensitive than
parametric tests [94]. If multiple hypotheses are
tested simultaneously, a priori control of the error
is required, generally using the family wise error
rate (FWER), the Bonferroni correction, or the
false discovery rate (FDR) procedures
[95]. After an FDR analysis, the statistical perfor-
mance is usually expressed as q-values, which
represent the corrected p-value after FDR and
gives a more accurate indication of the level of
false positivity for a given cutoff value.
Volcano plot. The volcano plot is the most
widely used graphical expression to give a quick
glance on results based on univariate statistical
tests. In this graph representation, the log2 of the
fold change for each spot/peptide is plotted
against the log10 of its p-value (Fig. 5a).
Multivariate statistic: principal component
analysis and heat maps. Multivariate data analy-
sis allows to reduce the complexity of samples
[96]. In the case of biological fluids, it allows to
identify combination of spots/peptides that could
be of value in identifying and characterizing com-
plex mixtures. Principal component analysis
(PCA) generates new variables, termed “compo-
nents,” which condense the variability of the sam-
ples (Fig. 5b). Individual components in PCA do
not correlate with each other are and analyzed
separately to see how much they contribute to
the overall variance of data. Plots of each principal
component provide graphical views of the data
structure and allow defining clusters and outliers.
When specific groups of proteins can be distin-
guished, variables that correlate mostly with the
principal component are analyzed, a process
referred to as loading matrix. Data clustering anal-
ysis is usually performed to identify proteins with
similar behavior. This technique is complemen-
tary to multivariate statistics. Graphical represen-
tation of pathways and visualization of integrated
data sets usually improve data interpretation
and can also be helpful to select candidate
437
variables [89]. Pearson’s or Spearman’s correla-
tion coefficient and the Euclidean distance aggre-
gation method are the most widely used clustering
strategies for proteomics data analysis.
These analyses can also be represented as “heat
maps,” in which the abundance of each variable is
converted in a color scale (Fig. 5c). This should be
best performed after Z-Score standardization.
Frequency tests. These tests are used for nom-
inal or ordinal scales and include the McNemar,
Chi-square, and Fisher’s exact tests. Odds ratio
(OR) can be measured. When the OR is close to
1, the odds of healthy controls and patients are
similar. OR greater than 1 indicates increased risk,
while OR lower than 1 indicates protection against
the risk. Interpretation of these results requires the
determination of the confidence intervals.
Protein Interaction, Metabolic
Pathways, and Systems Biology
Definition of interactive networks further refines
the analysis and is useful to identify pivotal pro-
teins. In Cytoscape, for example, the analysis can
be performed following a simple work flow that
uses plug-in software. The first step consists in
importing data to generate an interactive network
based on univariate/multivariate results. The sec-
ond step consists in matching data with public
database. Together, this generates an interactive
network where proteins that are linked by nodes
and edges represent potential interactions (e.g.,
physical, co-localization, co-expression, common
pathways, etc). Results can be filtered to reduce
complexity. Overall, this approach can provide
insights on cellular pathways and can identify
molecular mechanisms of diseases (Fig. 5d).
Application Fields to Renal Diseases
Genome-Wide Association in Primary
Glomerulonephritis
In recent years, two main glomerular diseases
have been successfully tackled using GWAS
approaches, namely, membranous nephropathy
438
Fig. 5 Analysis of complex sets of data deriving from
proteomics: (a) volcano plot; (b) principal component
analysis (PCA); (c) heat map; (d) example of systems
biology of a complex trait that identifies pathways and
networks associated patterns of protein expression identi-
fied by univariate statistical analysis. The network is
graphically displayed with proteins as nodes and the bio-
logical relationships between the nodes as lines. The color
and IgA nephropathy. By studying only 556
European patients affected by membranous
nephropathy and 1,832 controls, Stanescu and
colleagues identified genome-wide significant
signals in two loci: one on chromosome 2q24
containing the gene encoding M-type
G.M. Ghiggeri et al.
of the node indicates the degree of overexpression (black)
or underexpression (gray) of the proteins in patients com-
pared to controls. Physical interaction between proteins is
represented by solid lines while pathways by broken line.
Different shapes represent proteins with different functions
(diamond = enzymes; triangle = transporters; circle =
other)
phospholipase A2 receptor (PLA2R1) and one
on chromosome 6p21 containing the gene
encoding HLA complex class II HLA-DQ alpha
chain 1 (HLA-DQA1) [36]. Interestingly,
PLA2R1 has been previously shown to be the
target of the autoimmune response for initiation
14 Translational Research Methods: Basics of Renal Molecular Biology
of primary membranous nephropathy [97], thus
providing direct functional support to the genetic
findings. The effect size for both variants was
unusually large for classic GWAS studies, with
an OR of almost 80 when both alleles are present
in homozygosity, supporting a very strong effect
of innate immunity and PLA2R1 in the pathogen-
esis of disease.
IgA nephropathy (IGAN) is the most com-
mon primary glomerulonephritis in the world,
with striking racial differences in prevalence.
Compared to subject of European descent, the
disease is much more common in Asians and
rare in Africans, suggesting a strong genetic
contribution. A GWAS was conducted in 1,194
IGAN cases and 902 controls of Chinese Han
ancestry and was replicated in 1,950 cases and
1,920 controls from Asia and Europe. These
studies identified five novel loci: three indepen-
dent loci were located in the major histocompat-
ibility complex, one tagged a common deletion
of CFHR1 and CFHR3 at chromosome 1q32,
and the last locus was located on chromosome
22q12 in an intergenic region [34]. These five
loci were found to explain 4–7 % of the disease
variance and up to a tenfold variation in
interindividual risk. Interestingly, the IgA
nephropathy risk allele frequencies mirrored
the known variation in disease prevalence
among Asian, European, and African
populations and uncovered a previously
unrecognized north–south gradient in IgAN
prevalence across Europe suggesting complex
selective pressures [34, 98]. Very recently, a
novel GWAS on IGAN studying over 20,000
individuals across three continents identified
six new genome-wide significant loci: four in
ITGAM-ITGAX, VAV3, and CARD9 and two
new independent signals at HLA-DQB1 and
DEFA. This study also replicated previously
reported signals and showed an increased burden
of risk alleles in younger patients. Interestingly,
the genetic risk score computed with all variants
was found to strongly correlate with helminth
diversity, suggesting a role for the interaction
between intestinal pathogens and humans in
driving the disease prevalence across population
of different races and ethnicities [35].
439
Copy-Number Variations in Congenital
Anomalies of the Kidney and Urinary
Tract
Rare CNVs have been associated to multiple
human phenotypes, including neurodeve-
lopmental diseases (intellectual disability, autism,
schizophrenia, epilepsy), cardiac defects, lung
disease, craniofacial malformations, and others
[43–50]. The role of CNVs in renal disease was
unknown until 2012, when a first adequately sized
study was conducted on 522 patients with kidney
malformations and almost 14,000 controls
[51]. Children for this study were recruited in
multiple centers in Europe and at Children’s Hos-
pital, Philadelphia. CNV calls were derived from
genome-wide high-density SNP arrays. Burden
analysis between cases and controls showed a
striking enrichment of large rare CNVs in cases
compared to controls, and these structural variants
were mainly deletions containing one or more
coding element. Per-genome analysis suggested
that kidney malformations could be attributable to
a CNV of 250 kb or larger in at least 17 % of the
cases, indicating that rare, genic submicroscopic
genomic unbalances are a major source of genetic
variation predisposing to renal agenesis and
hypodysplasia. Analysis of individual large rare
CNVs surprisingly identified 34 independent
known genomic disorders in 55 individuals, con-
stituting 10.5 % of the entire CAKUT cohort.
The most frequently identified pathogenic struc-
tural variant was the 1.4 Mb deletion at the
chromosome 17q12 locus associated to the renal
cysts and diabetes (RCAD) syndrome. The other
33 genomic disorders were present in single
individuals or only in few patients (e.g., 1q21
deletion, Wolf–Hirschhorn syndrome,
Potocki–Lupski syndrome, DiGeorge syndrome,
Phelan–McDermid syndrome). Thus, known
copy-number disorders are a frequent cause of
renal malformations. The analysis of cases with-
out known copy-number syndromes identified
38 additional large, rare, genic structural variants
in 32 individuals (6.1 %) representing novel can-
didate genomic disorders. Altogether, these data
show that one in six children with kidney
malformations carries a large structural variant
440
diagnostic of known or novel syndromes. Inter-
estingly, nearly all of these variants have been
previously associated to neurodevelopmental phe-
notypes that develop later in life, with potential
impact on risk stratification and individualization
of care.
Next-Generation Sequencing in Renal
Disease
The establishment of NGS techniques and, in
particular, of exome sequencing has resulted in a
dramatic acceleration in gene identification in
many Mendelian renal phenotypes [10, 14–17,
20, 99, 100]. NGS has been particularly critical
in facilitating gene discovery for traits character-
ized by very high genetic heterogeneity and small
pedigree size. For example, exome sequencing
coupled to linkage analysis in a pedigree segre-
gating with autosomal dominant urinary tract
malformations has recently allowed to identify a
new gene related to these diseases. Specifically,
genome-wide linkage analysis had identified in
this family five candidate regions, corresponding
to ~2 % of the total genome. Subsequent exome
sequencing identified 24 potentially pathogenic
variants shared between two affected individuals,
two of which localized to the regions identified by
linkage analysis; of these, only one variant segre-
gated with the disease in the entire family, namely,
a splice mutation in the DSTYK gene, encoding a
dual specificity serine/threonine and tyrosine
kinase. Resequencing of the DSTYK gene in
311 additional patients with congenital abnormal-
ities of the kidneys and urinary tract (CAKUT)
identified other rare damaging variants of this
gene in 7/311 (2.3 %) unrelated CAKUT patients.
These findings were further supported by experi-
mental data in mice and zebrafish [15].
The Building of the Podocyte
Protein Map
Out of approximately 20,000 genes that are
encoded in the human DNA, only a portion is
ubiquitous. Tissue- and cell-specific cDNA
G.M. Ghiggeri et al.
libraries are available and partially overcome the
limitation due to cell specificity, but they do not
completely reflect the cell protein and peptide
repertoire. Advances in proteomics technology
filled this gap allowing to build cell-specific pro-
tein maps; the podocyte is a relevant example.
Podocytes represent partially differentiated epi-
thelial cells expressing a specialized repertoire of
proteins that play a key role in many renal dis-
eases. The full definition of the podocyte protein
composition is currently in progress and will soon
be available to study pathological conditions
[101]. Currently, most data have been obtained
on podocyte cell lines [102]. Future studies may
directly use podocytes expanded from kidney
biopsies or from urines of patients with specific
glomerular diseases. These studies also allow
defining antigens that are targeted in autoimmune
diseases [97, 103, 104].
Proteomic Analysis of Biological Fluids:
The Normal Urine Map
Biological fluids are potentially an important
source of information in medicine. The plasma
compartment contains important diagnostic
markers or causative molecules of renal diseases.
The expression of most plasma proteins cannot be
assayed by recombinant DNA technologies, as
these are generally not synthesized by circulating
cells. Proteomics represents, therefore, a major
tool to study plasma composition in health and
in diseases. In nephrotic syndrome, for example,
proteomics has been used to search for permeabil-
ity factors and to characterize oxidized protein
products. Using Nat/SDS-PAGE analysis, albu-
min and other proteins, such as α1-anti-trypsine,
have been shown to undergo posttranslational
modifications that include fragmentation, poly-
merization, and formation of adducts [105]. Oxi-
dation end products [106] in specific conditions
may also underscore pathologic processes.
Likewise, proteomic approaches can also be
used to characterize urine samples (Fig. 6). As
the urine proteomic map is nearly completed
[107], researchers are now attempting to use pro-
teomics to identify urinary fingerprints of specific
14 Translational Research Methods: Basics of Renal Molecular Biology 441

Precipitate are not enough to be reproducible. Several, if not

most small urinary peptides, originate ex vivo
85 directly from proteolysis of plasma proteins, and
Untreated it is unclear whether they correlate with significant
22
18 Vesicles biological events. A number of these peptides
4 28
12 42 appear, however, to be biologically active, and
18
12 98 some have been shown to correlate with diseases.
154 744
18 25 Only few studies have attempted to analyze
48 404 small urinary peptide as fingerprints in the diag-
76 36
27 nosis and staging of renal diseases. Most are
41 18
20
derived from digestion of urinary albumin.
16 56 Decramer et al. [111] have utilized capillary elec-
172 51
trophoresis and tandem mass to characterize the
37 29 83
349 346 urine peptide composition in newborns with
hydronephrosis secondary to ureteropelvic junc-
CPLL-beads Unbound
tion: quantitative changes of a type V
preprocollagen α2 chain fragment were found to
Fig. 6 Urine protein composition as a result of a multistep be predictive, with a reasonable sensitivity, of the
procedure [107]. The diagram shows proteins identified by clinical evolution. Similarly, by tandem mass
mass spectrometry during successive fractionation steps
spectrometry, protein chip immunoassay, and
SELDI-TOF, O’Riordan et al. have identified
two peptides of 4.7 and 4.4 kDa derived from
renal diseases. Until now, these studies have been defensin1 and α1antichymotrypsin, respectively,
disappointing and have shown that even distantly and showed that their ratio correlates with epi-
related renal diseases share similar patterns of sodes of acute rejection [112]. Obviously, most
urine protein composition. The level of accuracy of these findings need to be confirmed with large-
of urinary biomarkers allowed to differentiate glo- scale prospective studies. Nonetheless, they rep-
merular from tubular involvement but was insuf- resent one of the forefronts of proteomic research
ficient to characterize glomerular lesions and to in human diseases and may provide in the near
guide treatment [108–110]. Recently, it has been future less invasive diagnosis and monitoring
evaluated that the total number of urinary proteins tools.
is in the range of 3–4.000 [107]; it is reasonable to Recent advances in a new science branch,
expect that in the near future, studies based on a referred to as “metabolomics,” also warrant men-
large number of urinary proteins will expand this tion. Several examples suggest that metabolomics
field and will allow to identify patterns of protein may provide in the future new insights in the
excretion that will have clinical applications. diagnosis and understanding of renal diseases
such as in Fanconi syndrome [113] and/or in
various forms of glomerulonephritis [114].
Peptidome, Degradome,
and Metabolomics
Autoantibodies
Plasma and urine also contain small peptides
(<5 Kda) that are difficult to analyze. Several Analysis of tissue obtained from kidney biopsies
methodological hurdles still need to be overcome currently represents a direct approach to human
before applying the analysis of these small pep- autoimmune diseases. Ultraprecise techniques of
tides in human medicine. Currently, results microdissection, such as laser capture, allow
obtained by different techniques, such as SELDI repetitive analysis of minute amounts of renal
and LC-ESI-MS/MS, are rarely concordant and tissue [115]. In combination with proteomics
442
techniques (i.e., characterization of podocyte pro-
teins recognized by eluted autoantibodies), this
approach has been particularly successful in mem-
branous nephropathy [97, 101, 103] and human
lupus nephritis [104]. In 2009, the identification of
IgG autoantibodies against the M-type-phospho-
lipase A2 receptor (PLA2R) in patients with
membranous nephropathy (MN) has represented
a landmark discovery; subsequent studies have
clearly demonstrated that PLA2R positivity iden-
tifies primary MN patients, which has important
clinical and therapeutic implications [97]. Addi-
tional studies have implicated other proteins that
are normally expressed in podocytes, such as
aldose reductase and superoxide dismutase 2, as
novel targets of autoimmunity [103, 115, 116] and
have also shown that different autoantibodies may
be deposited in the same kidney at the same time
or sequentially [116].
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 Translational Research Methods:
The Value of Animal Models in Renal 15
Research

Jordan Kreidberg

Contents Introduction
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447
Institutional Oversight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447 The use of animal models has been an essential
Animal Models of Kidney Disease . . . . . . . . . . . . . . . . . . 448 aspect of nearly all areas of nephrological
Genetic Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448 research since its earliest days. Research on kid-
ney formation and malformation, physiology
Genetic Approaches with Known Genes:
Genotype to Phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449 and pathophysiology, immunological injury,
Gene Targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449 and tolerance or transplant rejection all depend
Conditional Gene Targeting . . . . . . . . . . . . . . . . . . . . . . . . . 449 on the use of animal experimentation. This chap-
Animal Models Using RNAi Approaches . . . . . . . . . . . 453
ter will emphasize genetic approaches that uti-
Transgenic Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456
Mutagenesis in Zebrafish . . . . . . . . . . . . . . . . . . . . . . . . . . . 458 lize animals, as this area has shown the great
progress in the development of novel technolo-
Gene Identification: Phenotype to Genotype . . . . . 458
Mutational Screens to Obtain New Phenotypes gies that have had great impact in all areas of
and Identify Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458 nephrology.
Gene Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459
Implications of Genome Sequencing . . . . . . . . . . . . . . 461
Institutional Oversight
Other Animals in Nephrology Research . . . . . . . . . . 462
Models of Renal Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463 There is an increasing public awareness of the
Models of Immunological Injury . . . . . . . . . . . . . . . . . . 463 use of animals in research, and with this, comes
Transplant Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463 an increasing concern about the appropriateness
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464 of the use of animals and whether much of the
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464
research that does involve animal models could
be accomplished using nonanimal models.
Therefore, it is important to note that all animal
research in the United States and presumably in
most other countries must be evaluated and
approved by institutional committees, generally
known as the IACUC (Institutional Animal
Care and Use Committee) before any experi-
mentation may commence. These regulatory
J. Kreidberg (*)
Children’s Hospital Boston, Boston, MA, USA committees are charged with evaluating animal
e-mail: jordan.kreidberg@childrens.harvard.edu protocols to make certain of the following
# Springer-Verlag Berlin Heidelberg 2016 447
E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_14
448
criteria: (1) that animals are used in an ethical
manner, with proper use of anesthetics or anal-
gesics to minimize or eliminate any source of
pain during experimentation, (2) that animals
are indeed required for the specific research in
question, (3) that minimal numbers are used,
(4) that large animals are not used when smaller
ones would suffice, (5) and that the investiga-
tors are trained and knowledgeable about proper
use of animals. Furthermore, the US Depart-
ment of Agriculture (USDA) and the National
Institutes of Health Office of Laboratory
Animal Welfare (NIH-OLAW) provide constant
oversight for large animal research, the former
through the use of frequent and usually
unannounced visits to animal facilities with
research institutions and the latter by publishing
detailed regulations concerning animal use that
serve as guidelines for IACUCs and serving as
the NIH agency to whom IACUCs report vio-
lations in NIH-funded use of animals in
research. Additionally, over 900 academic and
industrial animal research facilities in 39 coun-
tries worldwide are accredited by AAALAC
International (Association for Assessment and
Accreditation of Laboratory Animal Care Inter-
national), a private organization that sends
reviewers every 3 years to rigorously inspect
and certify animal research facilities. Despite
these several layers of oversight, in the end it
is up to the principal investigator to be thought-
ful about whether their intended experimental
approach will yield sufficiently important and
worthwhile results to justify the use of labora-
tory animals.
Animal Models of Kidney Disease
The selection of an animal model for some aspect
or type of kidney disease takes several factors into
consideration. Most importantly, the similarity to
human disease that can be observed in a particular
model is taken into account. Other important fac-
tors include the cost of the animals involved: the
cost of maintaining animals larger than rodents
increases dramatically with size, and the numbers
J. Kreidberg
of animals that can be studied consequently
decrease. For this reason, some studies may
begin with a zebrafish, Xenopus, or rodent model
and then progress to a larger model once the
rodent model establishes the feasibility of the
hypothesis under study. The size of an animal
may be important to the extent that it affects the
ability to perform surgical manipulations or phys-
iological measurements. However, since it has
become increasingly desirable to obtain physio-
logical measurements on various strains of knock-
out mice, the equipment available to perform
these measurements has improved and become
commercially available.
Genetic Models
Animal models of disease that have a genetic basis
may either result from spontaneous or induced
mutations. Spontaneous mutations or phenotypes
are those noticed either by chance or through the
directed observation of large numbers of mice that
were not otherwise treated to induce a mutation. In
contrast, induced mutations are those resulting
from the treatment of mice with irradiation or
mutagenic agents known to introduce point muta-
tions or deletions into the genome.
The past 25 years have witnessed an explosion
in the use of genetic approaches to understand
development and physiology, and thus they will
receive appropriate emphasis in this chapter.
Several genetic approaches are available for use
with animal model systems. A gene of interest
may be mutated using gene targeting, or
expressed in transgenic mice in such a way to
interfere with its normal function. On the other
hand, it is possible to start with a phenotype of
interest, which could either be obtained as a
spontaneous mutation or from mice treated with
a mutagen, and an effort is made to identify the
mutated gene responsible for the phenotype.
Genetic approaches using gene-targeted or trans-
genic mice are useful for a wide variety of devel-
opmental and physiological studies in which
there is a need to study the function of a
known gene.
15 Translational Research Methods: The Value of Animal Models in Renal Research
Genetic Approaches with Known
Genes: Genotype to Phenotype
Gene Targeting
Gene targeting was originally used to introduce a
deletion or interruption into a gene of interest,
using the scheme shown in Fig. 1, such that it
could be determined whether mice would be able
to develop in the absence of that gene’s function.
In cases where a gene was shown not to be essen-
tial for development, the homozygous mutant
mouse might serve as a useful model in which to
study the role of a specific gene in a physiological
or disease process. For example, targeted dele-
tions of the Wt1 [1], Pax2 [2], GDNF [3–5],
Wnt4 [6], and BMP7 [7, 8], among others,
showed these genes to be essential for various
aspects of early kidney development. On the
other hand, the absence of many immunology-
related genes does not result in any developmental
impairment, but these mice have served as useful
models to study the role of the immune system in
transplant rejection.
The advent of gene targeting was made possi-
ble through the use of two technologies developed
mainly in the 1980s. The first was the develop-
ment of tissue culture conditions that allowed
embryonic stem (ES) cell lines to be grown indef-
initely in culture while retaining their totipotency
[9]. ES cells grown in culture could then be intro-
duced into mouse preimplantation embryos or
blastocysts and become fully integrated into
those embryos such that their descendant cells
would give rise to all developmental lineages
that are found in adult mice [10]. The second
technology involved the use of homologous
recombination to introduce mutations into mam-
malian genes [11–13]. As shown in Fig. 1, when
long stretches of genomic DNA in recombinant
DNA constructs are introduced into cells in cul-
ture, this DNA will, at variable and often quite low
frequency, recombine into the locus from which
the genomic DNA was originally derived. There-
fore, homologous recombination of the correctly
designed genomic fragment can be used to intro-
duce a deletion or insertion into a genomic locus,
449
which renders the gene unable to be expressed.
This ES cell would in essence be heterozygous for
a mutation in the targeted gene, and heterozygous
ES cells can be isolated and expanded to provide a
population for injection into blastocysts. There-
fore, by combining the ES cell technology and
homologous recombination, it became possible
to target mutations into genes in ES cells and
then introduce ES cells carrying these mutations
into blastocysts, finally obtaining a mutant adult
mouse.
In a typical experiment, gene-targeted ES cells
would contain one mutated allele and one normal
or wild-type allele for the gene under study. The
targeted ES cells would be injected into preimplan-
tation blastocysts, and groups of these blastocysts
would be introduced into female mice that were
previously hormonally primed to allow implanta-
tion of the injected blastocysts into their uteri, to
begin a pregnancy. The resultant mice from these
injections are termed “chimeras,” because any spe-
cific cell is either derived from an ES cells or the
original injected embryo, i.e., the chimeric mouse
essentially has four parents, the male and female
that provided the blastocyst and the male and
female that provided the embryo from which the
ES cell line in use was originally derived. In the
best cases, a chimera might be nearly entirely
derived from the ES cells. Among the tissues that
ES cells contribute to are the germ cells: spermato-
cytes or oocytes. When ES cells heterozygous for a
mutation are used to make a chimera, germ cells
derived from the ES cells have a 50 % chance of
carrying the mutant rather than the wild-type allele.
Therefore, mating chimeric and wild-type mice can
result in some of the offspring being true heterozy-
gotes for the mutated gene. After obtaining both
male and female heterozygotes, they can be mated
to obtain homozygous mutant embryos or mice,
depending on whether or not the gene is essential
for development.
Conditional Gene Targeting
The process described in the preceding section
results in the inactivation of a target gene from
450 J. Kreidberg

Probe
BamH1 BamH1
a Vector
Neo

BamH1 BamH1
Chromosome

BamH1 BamH1 BamH1

Chromosome
Neo

ICM
b Gene-targeted c
ES cells
+/+ +/− −/−

Blastocysts
Reimplantation

Chimeric mouse

d Chimera Wild Type

x
+ / −mix+ / + +/+

Germ-line transmission

x
+/−
+/−

Phenotype of
homozygous mutants ?
−/−

Fig. 1 Gene targeting in mice. (a) The scheme for Restriction sites for restriction enzyme BamH1A are
targeting a deletion of an exon in embryonic stem cells. shown. The replacement vector is constructed such that
Exons are shown as black boxes along a chromosome. the neomycin resistance gene (neo) is shown as an open
15 Translational Research Methods: The Value of Animal Models in Renal Research
the beginning of embryogenesis. In this situation,
an embryo will become nonviable at the first point
at which expression of the inactivated gene
becomes essential for survival. However, it may
be highly desirable to study the function of a gene
product in many later events during development
or adult life. Conditional gene targeting allows the
inactivation of a gene in particular tissues or at
particular times during development or adult life
[14–16]. This technology has been developed
more recently and has proved more difficult to
employ on a widespread basis thus far, for reasons
that will be discussed.
The general approach to conditional gene
targeting is shown in Fig. 2. This is a variation
on traditional gene targeting, in that it also relies
on homologous recombination to introduce a seg-
ment of recombinant DNA into the locus of a gene
in ES cells. However, whereas traditional
gene targeting inactivates the gene, conditional
gene targeting must modify the gene such that it
can be expressed until such time as its inactivation
is desired. The most commonly used approach
involved the insertion of LOX sites, which are
34 base pair sites involved in site-specific recom-
bination by Cre recombinase, and enzyme origi-
nally derived from a bacteriophage [15]. Since
LOX sites are rather small, it is usually possible
to insert them in introns where they have no effect
on gene expression. By placing two LOX sites in a
gene to flank an exon, Cre can be used to inacti-
vate a gene by recombining out the DNA segment
containing the exon that was situated between the
two LOX sites, thus inactivating the gene. There
Fig. 1 (continued) box, in place of one of the exons.
An external probe specifically does not overlap with the
replacement vector. A double homologous recombination
results in the integration of the vector into the chromo-
some, thus replacing the exon with the Neo gene. The
BamH1 site within the neo gene results in a shorter
BamH1 restriction fragment detected by probe after
homologous recombination. (b) ES cells can be injected
through a micropipette into a blastocyst, where they
become part of the inner cell mass. The injected blastocyst
is introduced into the uterus of a hormonally primed mouse
and gives rise to a chimeric mouse, partially derived from
the ES cells and partially from the original inner cell mass
cells. If the ES cells and blastocysts are derived from
451
are experimental approaches for expressing Cre in
temporally and spatially specific manners or both.
Spatial- or lineage-specific expression of Cre is
most often obtained by placing the Cre cDNA
downstream of a known tissue-specific promoter.
Sometimes this is achieved by using homologous
recombination to insert the Cre gene into the
genomic locus of a gene with known tissue-
specific expression, such that Cre replaces the
first exon of that gene. Temporally specific
expression of Cre has proved more difficult to
obtain. One approach is to regulate Cre using the
tetracycline system for inducible gene expression
[17]. The other approach makes use of a fusion
protein consisting of Cre and a portion of the
estrogen receptor that confers steroid-mediated
nuclear localization [18, 19]. The latter is modi-
fied to bind tamoxifen or tamoxifen derivatives
instead of estrogen. The Cre-modified estrogen
receptor fusion protein will remain in the cyto-
plasm and therefore not be able to mediate
site-specific recombination of LOX sites, until
tamoxifen is administered to the mouse to induce
nuclear translocation of the Cre fusion protein.
This system can be used to induce recombination
in embryos, when tamoxifen is administered to
pregnant mice. The major obstacle to employing
conditional gene targeting on a widespread basis
is the availability of promoter/enhancer elements
that are able to confer robust tissue- or cell
lineage-specific expression of Cre recombinase.
However, an increasing number of mouse strains
are available that express Cre recombinase in var-
ious cell lineages within the developing and adult
strains with different coat colors, then the chimeric
mouse will have a variegated coat color pattern on its fur,
providing an indication of its overall extent of chimerism.
In the best cases, the resultant mouse is nearly entirely
derived from ES cells. (c) Shows a possible pattern
obtained in a Southern blot, based on the scheme shown
in (a), using the external probe. A wild-type mouse shows
only the longer band. A heterozygous mouse shows both
the wild-type and gene-targeted band, and the homozygous
mutant shows only the shorter band, due to the presence of
the BamH1 site in the neo gene. (d) The mating involved in
obtaining germ line transmission of the mutation and sub-
sequently obtaining homozygous mutant mice
452
Vector LOX LOX
Chromosome
Homologous recombination in ES cells
Chromosome LOX LOX
1. Inject Es cells into blastocysts to make chimeric mice
2. Mate heterozygous mice with Flp deleter mice to remove neo
Chromosome LOX LOX
X
Fig. 2 Conditional gene targeting. The targeting vector is
different from the previous figure in that LOX sites flank
the exon that will eventually be deleted and the Neo gene is
flanked by Frt sites. The vector is incorporated into the
chromosome through homologous recombination, and ES
cells with this knock-in are used to make chimeric mice,
and germ line transmission is obtained. Although the LOX
sites should not interfere with expression of the gene, the
Neo gene is likely to interfere with normal gene expres-
sion. However, in most cases, mice will tolerate one inac-
tive gene, as long as the other allele is functional. After
obtaining heterozygous mice, they are mated with
Flp-deleter mice that express Flp recombinase in germ
cells. Flp will recombine the FRT sites and eliminate the
J. Kreidberg
FRT FRT
Neo
FRT FRT
Neo
FRT
1. Breed heterozygous mice with
Cre-expressing mice, and breed to
homozygosity.
tissue specific
promoter
Cre
neo gene. Mice without the neo gene, but still containing
the exon flanked by LOX sites, are mated with mice
expressing Cre in a particular tissue or cell type, or
expressing an inducible Cre, to obtain the conditional
knockout. The breeding scheme shown in the figure is
oversimplified. In the actual experiment, a more compli-
cated breeding scheme is required to obtain a mouse that is
homozygous for alleles with LOX sites and that also has
the Cre-expressing transgene. An alternative is to breed
mice with the conditional allele with mice carrying a tra-
ditional knockout. This has the advantage that to obtain the
conditional knockout, Cre must only recombine one, and
not two, pair of LOX sites in each cell
15 Translational Research Methods: The Value of Animal Models in Renal Research
kidney [20]. It is possible to obtain conditional
knockouts restricted to nephron progenitor cells
[21], podocytes [21–24], proximal tubules
[25–27], thick ascending limb [28], ureteric bud
[29], juxtaglomerular cells, and collecting ducts
[30]. It is also possible to obtain vascular knock-
outs [31–33], though not restricted to the kidney.
As more tissue-promoter elements become
available, conditional gene targeting promises to
have a large impact on genetic approaches to kid-
ney disease. As noted above, there are many genes
expressed both in developing and adult kidney,
where the knockout of the gene results in embry-
onic lethality. This precludes study of how that
product of that gene might function in postnatal
kidneys, or why a mutation in that gene leads to
kidney disease in humans. It also raises the question
of why humans carrying such mutations are able
survive, albeit with a genetic disease, when mice
carrying mutations in the same gene do not survive
embryogenesis. Sometimes this is simply because
mice and humans differ in their respective require-
ments for specific genes, but more often, it is
because humans with genetic disease often have
point mutations that lead to partial loss of function,
whereas mouse knockouts often involve complete
loss of function mutations. Conditional gene
targeting can sometimes offer a solution to this
problem, by allowing normal gene expression dur-
ing embryogenesis, and then inactivating a gene in
adult mice. Alternatively, there are variations on the
Cre-LOX approach that allow the introduction of
point mutations into mice. The introduction of
point mutations into mice has been greatly facili-
tated by recent advancements that facilitate homol-
ogous recombination into BACs (bacterial artificial
chromosomes) in E. coli (Fig. 3) [34–36]. BACs
are used as they contain large amounts of genomic
DNA and thus are ideal for use as gene-targeting
vectors. The longer length of BACs compared with
shorter genomic clones should improve the fre-
quency of homologous recombination in ES cells.
Crispr/Cas9 and Talen Knockouts
The zinc finger nuclease (ZFN), TALEN, and
Crispr/Cas9 systems are presently revolutionizing
the ability to place mutations into animal genomes
[37–39] (Fig. 4).
453
ZFNs and TALENs are based on protein back-
bones that can be custom designed to bind specific
nucleotide sequences. In the case of ZFNs (Fig. 4a),
algorithms are used to predict amino acid sequences
that will bind different trinucleotide sequences.
These nucleotide-binding peptides are incorporated
within a zinc finger backbone peptide. With suffi-
cient length, these sequences become unique in the
target genome. For TALENs (Fig. 4b), the specific-
ity is similarly conferred by peptide sequence, but
instead of trinucleotide recognition, “TALE” motifs
recognize single nucleotides. In both systems, these
“designer” peptides are linked to the Fok1 endonu-
clease. As shown in Fig. 4, by designing two ZFNs
or TALENs to flank an intended mutagenesis
site, two Fok1 peptides are brought together, thus
creating an active Fok1 enzyme that produces a
double-stranded break. Repair of this break by
nonhomologous end joining typically introduces a
small deletion into the genome.
The Crispr/Cas9 system (Fig. 4c) is distinct in
using a complex of a “guide” or gRNA (Crispr) and
protein endonuclease (Cas9) to introduce a targeted
cut into a specific sequence within a genome. Thus
far, it appears to be more cost-efficient than the use
of TALENs or ZFNs because it is much easier to
synthesize a custom gRNA to target a specific
genomic sequence than to design and synthesize
sequence-specific peptides as required to use
TALENs or ZFNs. These systems offer the possi-
bility of not only introducing small deletions but
also editing the genome by introducing along with
the TALEN, ZFN, or Crispr/Cas9 reagent, a small
DNAwith flanking sequences homologous to either
side of the cut, and an internal “edited” sequence.
By homologous recombination, this small piece of
DNA can be inserted into the site of the cut, thereby
placing a new sequence into the genome. This tech-
nology offers the ability to repair a mutated
sequence or introduce an entirely new sequence
into a specific site in the genome, giving us unprec-
edented ability to repair or alter genomes [40, 41].
Animal Models Using RNAi Approaches
RNAi technologies have had great impact across
the breadth of molecular biology and the study of
454 J. Kreidberg

SacB Kan DNA fragment with

a b homologous ends
and SacB, Kanamycin
markers

a 2 b 3
1

Select for Kanamycin resistance

X 2 Knock in with
a b mutation in
exon 2.

SacB Kan

a b 3
1

Select against SacB

X

a 2 b 3
1

Additional round of homologous

recombination to place Neo gene
flanked by FRT sites in between two
exons.

1. Linearize BAC and use to target ES cells.

2. Remove Neo gene using Flp deleter mice.
3. Use ES cells to derive chimeric mice.

Fig. 3 Gene targeting using BAC clones. Homologous
recombination is done in E. coli instead of in ES cells. In
the first step, a DNA fragment is prepared that contains the
kanamycin resistance positive selectable marker and the
SacB-negative selectable marker and which also contains
homologous ends (A and B, each about 50–60 bp) is
introduced into E. coli. This fragment can usually be pre-
pared by PCR, using primers that contain the homologies
to the genomic region, and also to a vector containing the
selectable markers. Usually, a strain of E. coli is used that
allows transient activation of the enzymes required for
homologous recombination. Selection for kanamycin
resistance will obtain BAC clones where the selectable
markers have recombined into the BAC. In a second
round of homologous recombination, A DNA fragment
with the same homologous ends but containing a mutated
exon 2, denoted by the “X,” is introduced into the E. coli
containing the BAC. Selection against SacB will obtain
BACs in which the mutated exon 2 has replaced the select-
able markers. A third round of homologous recombination
is used to insert the neo gene, flanked by FRT sites, so that
the BAC can be used for homologous recombination in ES
cells. As shown, this scheme is used to introduce point
mutations or small deletions into a gene. It can also be used
to construct conditional knockout vectors, similar to those
shown in Fig. 2. An additional use is to knock in a green
fluorescent protein (GFP) or β-galactosidase (LacZ)
reporter gene into a locus to obtain information about
patterns of gene expression
15 Translational Research Methods: The Value of Animal Models in Renal Research 455

Fig. 4 Mutagenesis with a ZFNs ZFN_R

Talens and Crispr/Cas9
(Figure from Ref. [39]). (a) kl
ZFNs targeting a specific Fo
genomic sequence to
position Fok1
endonucleases in order to

l
k
Fo
introduce a double-stranded
break. (b) TALENs
similarly targeting a ZFN_L
sequence. The NI peptide
sequence binds adenosine,
b TALENs
HD binds cytosine, NK TALEN_L
binds guanosine, and NG FokI
5’ GCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGTGCATCTG 3’
binds thymidine. By using 3’ CGAAGACTGTGTTGACACAAGTGATCGTTGGAGTTTGTCTGTGGTACCACGTAGAC 5’
specific arrangements of the FokI
FokI
NI, HD, NK, and NG TALEN_R
peptides, a peptide is
constructed to target a RVD: NI HD NK NG
specific sequence. (c) The
Crisrp/Cas9 system uses a Base: A C G T
gRNA designed to target a
c

G
specific sequence CRISPR/CAS9

UCGGUGC
AGCCACGGUGAAAAAAGUUC
(in green). (d) These
technologies can be used to

UAAAAUU CGAU A A
GUUUUAGAGCUA G A
3’-UUUU
disrupt genes by
introducing deletions

GAA
resulting from a double-
stranded break followed by Cas9 Guide
nonhomologous end joining RNA
(NHEJ), genome correction 5’-CNNNNNNNNNNNNNNNNNNN AAGGCUAGUCCGUUAUCAA
by homologous
N N CNNNNNNNNNNNNNNNNNNNNCC N
NN
recombination (HR) to NN NNNNNNNNNNNNNN- 5’
introduce a wild-type (WT) 3’-NNNNNNNN
DNA to repair the double- 5’-NNNNNNNN N N NNNNNNNNNNNNN- 3’
NN NN Chromosome
stranded break, or the N GNNNNNNNNNNNNNNNNNNNNGG N
introduction of a transgene PAM
by homologous
recombination 23 bp genomic target sequence

d Gene targeting by ZFNs, TALENs or CRISPR/Cas9

ZFNs, TALENs or CRISPR/Cas9

targeted DSB

WT donor Transgene donor

DNA DNA

Gene disruption Gene correction Transgene addition

by NHEJ by HR by HR

disease over the past 10 years [42]. RNAi is based complex, hybridizes to a complementary
on a mechanism common to both plant and ani- sequence in a mRNA, causes degradation or inhi-
mals whereby a short strand of RNA, that is asso- bition of translation of the mRNA [43, 44]. In the
ciated with a set of proteins known as the RISC natural setting, these small RNAs are encoded in
456
the genome as microRNAs. The same molecular
machinery used by microRNAs to inhibit expres-
sion of mRNAs can be co-opted for use by
siRNA (silencing RNA) or shRNA (short hairpin
RNA) that are ectopically expressed in cells.
siRNA refers to RNA duplexes that are usually
obtained commercially and transfected into cells.
shRNA refers to small RNAs that are expressed
from plasmids or viral vectors that are introduced
into cells. A major addition to the arsenal of
approaches involving transgenic mice involves
the derivation of transgenic mice that express
shRNA molecules capable of reducing levels of
mRNA for specific genes in specific tissues or
cell lineages [43–46]. shRNAs can be expressed
either constitutively or conditionally using the
Cre-Lox system (Fig. 5). An advantage of
shRNA technology over gene knockouts is that
it is quicker and cheaper, as mice do not need to
be bred to homozygosity and also to carry the Cre
transgene. Instead, a resulting phenotype can be
obtained by mating a Cre-expressing mouse with
a mouse carrying a conditional shRNA transgene
and examining the first-generation offspring car-
rying both transgenes. The major disadvantage in
comparison with gene knockouts is that expres-
sion of the target gene may be variably reduced,
and it may be necessary to examine several
shRNA transgenic lines in order to obtain ones
that demonstrate efficient knockdown of the
desired gene.
Transgenic Mice
As mentioned above, many mutations that result
in human disease are point mutations that result in
“hypomorphic,” or partial loss of function alleles
of a gene. In this case, a disease state may result
from decreased activity of a gene product. In other
cases, a point mutation or deletion mutation may
produce a protein that interferes with the function
of the normal gene; this is referred to as a
dominant-negative effect. This could occur in
instances where a protein requires
homodimerization for activity, and dimerization
of a wild-type and a mutant form of a protein leads
to an inactive complex. Dominant-negative
J. Kreidberg
effects can also be found in cases where two pro-
teins heterodimerize, and an inactive mutant pro-
tein is able to complex with its partner protein, but
as before, the complex is inactive. Dominant-
negative effects can be studied in animal models
using transgenic mice. Although gene-targeted
mice discussed in the previous sections can also
be considered to be transgenic, because foreign
DNA is used to disrupt the endogenous gene, here
the term “transgenic” is reserved for those mice in
which foreign DNA has been inserted into the
murine genome through pronuclear injection
[47–50]. In contrast to gene-targeting schemes in
which genes are modified in ES cells and ES cells
are then used to derive chimeric mice, transgenic
strategies, DNA is directly microinjected into the
pronucleus of a fertilized egg or zygote, and the
injected zygotes are then reimplanted into the
oviduct of a hormonally primed female mouse.
The injected DNA is able to recombine by
nonhomologous or illegitimate recombination
into random locations within the genome and in
variable amounts from zygote to zygote. Once
mice are derived from the injected zygotes, they
are tested to determine if they carry the injected
DNA within their genomes as a transgene, and if
they do, whether the transgene is expressed. By
injecting DNA constructs that contain a tissue-
specific promoter and a mutated gene of interest,
it is possible to study whether expression of the
mutant gene leads to an observable phenotype. In
other instances, the gene to be expressed is not
mutated, and the experiment is designed to deter-
mine if overexpression or de novo expression of
the gene results in an observable phenotype or
disease model. While the original transgenic stud-
ies used relatively short DNA constructs, more
recent studies have used BAC (bacterial artificial
chromosome)- or YAC (yeast artificial
chromosome)-based vectors whose much longer
stretches of DNA containing promoters and other
regulatory regions will hopefully confer more
faithful patterns of expression of the transgene
directed by those regulatory elements
[47, 51]. The great majority of transgenic work
has been done in murine models, but there have
been pioneering efforts in other species such as
pigs and rats [52, 53].
15 Translational Research Methods: The Value of Animal Models in Renal Research 457

a Tissue specific cre transgene shRNA transgene

Promotor Cre U6-Promoter Sense loxP TTTTT loxP Anti-Sense TTTTT

Cre-mediated recombination of loxP sites

U6-Promoter Sense loxP Anti-Sense TTTTT

Sense
loxP
Anti-Sense

Dicer

b Sense
Anti-Sense

Processing to remove hairpin loop

Formation of RISC complex

RISC

Recognition of target mRNA

RISC
5’ 3’
mRNA

Translational repression
mRNA degradation

Fig. 5 Conditional expression of shRNA transgenes. (a)
Mice containing a Cre recombinase-expressing transgene
and a shRNA transgene are crossed to obtain embryos or
offspring mice containing both transgenes. The shRNA
transgene contains a U6 promoter to direct RNA polymer-
ase III-mediated transcription that is terminated by a
poly-T sequence. It also contains a sense and antisense
sequence, designed to target a mRNA, that will become
the double-stranded RNA that associates with the RISC
complex to mediate the inhibitory effect on mRNA stabil-
ity or translation. Therefore, transcription of the shRNA
transgene is interrupted prior to Cre-mediated
recombination of the first poly-T sequence. After removal
of the first poly-T sequence by Cre-mediated recombina-
tion of the two LoxP sites, the transgene is fully transcribed
and terminates at the second poly-T sequence. The resul-
tant shRNA forms a hairpin loop by base pairing of the
sense and antisense sequences. (b) The shRNA associates
with DICER, an RNase III class enzyme that removes the
hairpin loop. The double-stranded RNA then associates
with the RISC complex, the sense strand is removed, and
the RISC complex with the antisense RNA finds its target
mRNA and suppresses translation or decreases mRNA
stability
458
Mutagenesis in Zebrafish
Possibly the single most important advancement in
the development of novel animal models for kidney
disease over the past 20 years is the establishment
of zebrafish (Danio rerio) as a major model for
understanding the genetic and physiological basis
for disease. Zebrafish models of glomerular devel-
opment and disease and polycystic kidney disease
have been particularly useful models for study of
human disease [54–60]. Most recently, the
zebrafish has been more generalized as a model
for exploring genetic variation in human disease
[61, 62]. Zebrafish are much less expensive to
maintain than mice (once a facility has been
constructed), and genetic tools to map zebrafish
mutations nearly equal or in some cases extend
beyond those available for mice [63–65]. The
zebrafish excretory system involves a pronephric
duct and glomus that bears important similarity to
mammalian nephrons and has already been the
subject of many research studies [54, 66–72].
Two great advantages of zebrafish are (1) their
shorter developmental timing and (2) that develop-
ment occurs in nearly transparent embryos that
develop outside the mother, allowing for far greater
observation and intervention than is possible with
rodent embryos. The zebrafish genome is presently
being sequenced at the Sanger Center [73], and
a full set of markers exists for gene mapping
[64, 74, 75]. It is possible to make transgenic
zebrafish [76–78], and gene targeting is now pos-
sible using genome editing approaches that utilize
Crispr/Cas9 or Talens [79–82]. There have
also been large-scale efforts to saturate the
zebrafish genome through insertional mutagenesis
[83–85]. An alternative to gene targeting is the use
of morpholino oligonucleotides that inhibit expres-
sion of specific genes against which the morpholino
is targeted; these can be injected into cells of early
embryos, and phenotypes can be observed at vari-
ous developmental stages thereafter [86, 87]. In one
sense, morpholinos have an advantage over conven-
tional gene knockouts, in that they can be designed
to block expression of specific alternative splice
forms [86], rather than all forms as is often the
case with gene knockouts in mice.
J. Kreidberg
Gene Identification: Phenotype
to Genotype
Mutational Screens to Obtain New
Phenotypes and Identify Genes
ENU mutagenesis: At present, several major
efforts in several countries involve the use of
N-ethyl-N-nitrosourea (ENU) to introduce small
mutations throughout the mouse genome
[88–93]. Similar approaches have been used
with zebrafish, and many interesting phenotypes
have been obtained [94–98]. These large genome-
scale approaches, which can involve very large
mouse or zebrafish colonies, are justified by the
following arguments: (1) Most disease-related
human mutations are caused by point mutations;
therefore, a ENU-mutagenic approach may have a
greater chance of producing a phenotype resem-
bling a human disease than will gene-targeted
mutations that usually completely inactivate a
gene. (2) An ENU-based approach does not rely
on previous identification or cloning of the gene, i.
e., any gene is a theoretical target and can be
studied, to the extent that some degree of compro-
mise in the gene product’s activity will result in an
observable phenotype. The obvious disadvantage
in comparison with gene targeting is that a large
amount of work lies between the observation of a
phenotype and the final identification of the
mutated gene. (3) Given a large enough effort, it
should be possible to eventually “saturate” the
genome with mutations, i.e., examination of sev-
eral hundred thousand mutagen-treated mice or
zebrafish is likely to provide the opportunity to
observe the effects of placing a mutation in every
gene capable of causing an observable phenotype.
However, one important point remains to be men-
tioned that dramatically increases the labor and
expense of an ENU-based effort. Most observable
phenotypes tend to be genetically recessive
instead of dominant, meaning that they are not
apparent in the first-generation offspring of
mutagen-treated animals. Instead, it is necessary
to breed a second generation and then backcross it
to the first-generation mice (or zebrafish),
resulting in a third generation (Fig. 6). Doing
15 Translational Research Methods: The Value of Animal Models in Renal Research
Screen for recessive phenotypes
ENU
Founder
Heterozygote
(with several mutations)
Backcross
to original
heterozygous parent
(with subset of mutations)
Homozygote
(observable recessive
phenotypes)
Fig. 6 ENU mutagenesis. A scheme is depicted for find-
ing recessive phenotypes through ENU mutagenesis.
Dominant phenotypes require a less complicated approach,
as phenotypes will be apparent in the first generation
derived from crossing founders with wild-type mice. In
this scheme, a mutagenized male founder that probably
carries many mutations after mutagenesis is mated with a
wild-type mouse to produce heterozygote offspring that
this on a large scale will result in many third-
generation animals that are now homozygous for
mutations resulting from the original mutagenic
treatment, and some will have observable pheno-
types that can be studied for biological interest
and to map the responsible gene.
These large-scale genetic approaches are not
only suited to study developmental anomalies.
Some of the large-scale efforts on mouse muta-
genesis ongoing around the world will involve
performing basic blood work and a urinalysis on
each mouse from the group being screened for
new phenotypes. Thus, this approach has the
459
Wild type
Wild type
Heterozygote
carry a subset of these mutations. These heterozygotes
are mated with wild-type mice to produce a second gener-
ation, which will carry a smaller subset of the original
mutations. These are then mated to the original heterozy-
gous offspring of the founders, and 25 % of the offspring of
this cross will be homozygous for any particular mutation
that was present in the second heterozygous generation
potential to identify genes involved in disease
progression, as well as those responsible for mor-
phogenetic processes.
Gene Identification
Microsatellite repeats and SNPs: Mapping sites
of induced or spontaneous mutations in mice has
been greatly aided by the development of sets of
microsatellite repeat markers and more recently
by SNP microarray panels. Microsatellite repeats
used in mapping are stretches of CA dinucleotide
460
repeats that are found interspersed throughout
mammalian genomes [99, 100]. Typically, these
CA repeats contain 10–20 CA dinucleotides.
These CA repeats are flanked by unique
sequences, and thus it is possible to design pairs
of PCR primers that correspond to these flanking
sequences that will amplify the (CA)n sequence
between the two primers. Within a genetically
inbred strain of mice each individual mouse will
contain the same number of CA dinucleotides at
each repeat. However, similarly to the variation
observed between human individuals, different
inbred strains may differ in the number of CA
dinucleotides at any particular repeat. By tracking
the segregation of a mutant phenotype with a
particular microsatellite repeat, it was possible to
determine first which chromosome harbored the
mutation and then focus on microsatellite repeats
within a chromosome to further map the mutation
and eventually clone the mutated gene.
SNPs, or single nucleotide polymorphisms, are
single base pair differences found between indi-
viduals within a species. Among the human pop-
ulation, SNPs tend to be found every 100–300
base pairs within the genome and can be used as
a measure of genetic relatedness among, for
example, ethnic groups or people in different geo-
graphic areas [101]. SNPs are also found in com-
paring different inbred strains of mice and
between the commonly used laboratory-inbred
strains that are all derived from Mus musculus
and other mouse strains that are “non-musculus”
that can be intercrossed with Mus musculus strains
to aid in genetic mapping, as described above
[102]. In contrast, within a particular inbred strain
of mice, there is by definition genetic homogene-
ity, and SNPs should not be present between indi-
viduals of the same strain. SNPs are also used to
map genes in zebrafish [75].
It is now possible to detect SNPs using
microarrays [103, 104], such that thousands of
oligonucleotides complementary to sequences
containing known SNPs can be arrayed on a sin-
gle microarray chip, and hybridized to an individ-
ual human or animal’s DNA to determine which
of thousands of particular SNPs that individual
has in their genome [105]. Using comparative
genomic hybridization (CGH) microarrays, this
J. Kreidberg
can also be done quantitatively in humans and
rodents, such that decreased or increased signal
on some SNPS relative to control yields informa-
tion about genomic deletions or amplifications.
SNPs can be used as genetic markers similarly to
the microsatellite repeats described in the previ-
ous section. Therefore, a single microarray can
provide the same information as hundreds of
PCR reactions. To be used in a genetic mapping
experiment, the same type of interspecific cross
would be performed as described in the previous
section, but instead of using PCR reactions to
analyze the segregation of microsatellite repeats,
a microarray is used for each individual to analyze
the segregation of SNPs, to narrow the interval
that contains the gene being mapped.
Using SNP microarray panels and running a
microarray for each of the many mice produced
from a cross between mutant and wild-type mice,
it is now possible to identify SNP polymorphisms
that segregate with the mutant phenotype and
much more quickly identify the genomic locus
containing the mutation.
QTL and GWAS: Almost all human disease
has a genetic component, whether it is the relative
susceptibility to an infectious agent, at one end of
a spectrum, or a disease that is primarily due to a
genetic mutation in a single gene, at the other end.
Between these two extremes lies most human
disease, whose etiology derives from a combina-
tion of genetic and environmental factors. In many
instances, the genetic component is due to the
effects of more than one gene. In other instances,
a single gene might be responsible, but the phe-
notype is not absolute, but rather of variable pen-
etrance or severity. A genetic element that
contributes to a disease phenotype in a quantita-
tive, as opposed to absolute, manner is referred to
as a quantitative trait locus, or QTL [106]. It is
probably not an overstatement to say that most
non-Mendelian genetic components of disease
occur as QTLs, and indeed, there are thousands
of known QTLs that have been reported [107].
A current approach to identifying QTLs in the
human genome that increase (or decrease) risk of
the disease is referred to as GWAS (genome-wide
association studies), whereby the genomes of
many (up to thousands) of individuals are
15 Translational Research Methods: The Value of Animal Models in Renal Research
interrogated for SNP polymorphisms across the
genome to determine the association of specific
SNPs with the presence, absence, or severity of
disease. This approach has found a wide use in the
nephrology community, examples being a search
for risk variants in diabetic nephropathy
[108–110] and search for additional variants that
further increase risk of chronic kidney disease in
individuals with APOL1 risk alleles [111–113].
The disease severity in many animal models of
human disease also appears to be influenced by
QTLs, and the identification of the responsible
gene(s) is a pursuit of many research laboratories
(e.g., [114]). In many examples, the presence of a
trait or disease may be determined in a Mendelian
pattern by a mutation in a single known gene, but
the severity of the trait or disease may be
influenced by so-called “modifier” genes that
show up as QTLs in a GWAS-type screen and
whose identification may be challenging. Impor-
tantly, comparisons of syntenic regions between
human and rodent genomes have aided the search
for modifier genes at QTLs, as it is often apparent
that a QTL identified in humans corresponds to a
QTL for a similar phenotype in rodents
[115]. Comparisons of syntenic regions can also
be complicated, as a region harboring a QTL in a
rodent genome, if not already delimited to a nar-
row interval, may be syntenic with several regions
of the human genome that are found on different
chromosomes. On the other hand, comparisons of
syntenic regions between humans and rodents
may also serve to delimit regions of interest and
facilitate the identification of the gene of interest.
Haplotype analysis: Haplotype analysis has the
potential to make gene identification by SNP map-
ping more efficient [115–117]. Haplotypes are
defined as a group of genetic markers that are
physically linked on chromosomes and that tend
to be inherited together more often than would be
predicted if genetic recombination events were ran-
dom and evenly distributed along chromosomes.
Human haplotype maps now contain over three
million SNPs [118, 119]. Perhaps the best known
examples of haplotypes are the major histocompat-
ibility loci, HLA in humans and H2 in mice, in
which there is considerable polymorphism between
haplotypes that is manifested by transplant
461
incompatibility between individuals with different
HLA haplotypes (in the case of humans) or
between mouse strains of different H2 haplotypes.
It is now known that haplotype units exist through-
out mammalian genomes and that SNPs can be
used as markers to define haplotypes [115–117]. It
then follows that inbred mouse strains that more
closely genetically related are likely to have the
same haplotype at a particular genomic location,
whereas more distantly related strains would be
more likely to have different haplotypes
[115, 116]. Thus, when SNPs are being analyzed
in the progeny of a genetic cross in an attempt to
narrow down the location of a candidate gene,
haplotype analysis provides an alternative to con-
sidering each SNP individually (see Fig. 7). For
example, if strains A and B are being studied with
the aim of identifying a disease-related QTL that
confers greater disease severity in strain A than in
strain B, then genomic areas where strains A and B
share the same haplotype are unlikely to harbor the
disease locus, whereas areas of the genome where
they have differing haplotypes are more likely to
harbor the disease locus. Therefore, consideration
of haplotypes, each of which may contain several to
hundreds of SNPs, may allow a means of
narrowing down the location of candidate genes,
by focusing on areas of the genome where the
stains differ in haplotype. In practice, such an anal-
ysis may involve multiple strains of mice and
crosses between them in an effort to narrow down
a genetic interval and physically locate a QTL
[120, 121]. Indeed, a recent effort known as the
“collaborative cross” involves the intercrosses of
several inbred strains of mice and then genotyping
and phenotyping resultant recombinant inbred
strains [122, 123]. This represents an animal
model version of a GWAS-like approach to identify
human disease genes.
Implications of Genome Sequencing
Sequencing of the entire human genome was com-
pleted in 2001 [124, 125] and of the mouse genome
in 2002 [126]. These continue to be refined, with
more detailed reports and annotations of the
sequence of specific human chromosomes
462 J. Kreidberg

Strain published from 2003 to the present. In the 2008

a version of this chapter, it was noted that the geno-
1 2 3 4
mic sequences of approximately 180 organisms,
including bacteria, plants, and animals, have been
A A G G SNP1 reported. This number is now in the tens of thou-
Haplotype A T T T T sands, and the new frontier in human biology lies in
assembling complete genome sequences of thou-
C C C C sands of individuals. This rapidly expanding data-
base made possible by high-throughput sequencing
G G G G (also referred to as “next-generation” or “massively
parallel” sequencing) has transformed modern biol-
Haplotype B C C A A SNP2
ogy and the study of disease [127–129]. For exam-
G G G G ple, in disease gene identification studies described
above, it is no longer the situation that a disease
gene might be mapped to an area within a chromo-
A A A A
some that had never been sequenced, and the hunt
T T T C SNP3 for the gene becomes a large-scale sequencing pro-
Haplotype C
ject. Now, once an area is delimited, the candidate
G G G G
genes in that area are largely known from prior
C C C C genome sequencing, allowing much more directed
sequencing to be done in attempts to find disease-
b causing mutations in affected individuals. The
Strains Strains
Haplotype A
1, 2 3, 4
genome sequences for most, if not all, animals
used as disease models is also known, greatly
Strains Strains accelerating studies such as those involving
Haplotype B
1, 2 3, 4 disease-related gene identification in animal
models. In many of these accelerated efforts, only
Strains Strain coding exons are sequenced (“whole exome
Haplotype C
1, 2, 3 4
sequencing”), rather than the entire genome. The
advantage of whole exome sequencing is that it
Fig. 7 SNPs and haplotypes. (a) A highly simplified
schematic of SNPs and haplotypes is depicted. Four greatly reduces the amount of sequencing per indi-
mouse strains, labeled at top, are compared. A three vidual, and the corresponding disadvantage is that
nucleotide stretch is shown for haplotypes A and B and mutations outside coding regions will be missed.
four nucleotides for C, though in reality a haplotype may Thus, a major frontier in genome sequencing
span megabases and be defined by hundreds or more
SNPs. The sequences in haplotypes A, B, and C are not related to animal disease models is not so much
necessarily contiguous and might even be on different the sequencing of additional species as it is the
chromosomes. In the haplotype at locus A, SNP1 is pre- sequencing of multiple genomes among individ-
sent at the first nucleotide, in the haplotype at locus B, uals that vary in their severity of, or susceptibility
SNP2 is present at the second nucleotide, and in the
haplotype at locus C, SNP3 is present at the second nucle- to, a disease, so that we can increase our under-
otide. (b) Haplotype grouping defined by the SNPs. For standing of inter- and intraspecies genetic variation
haplotypes at loci A and B, strains 1 and 2 are the same and how it relates to disease susceptibility [130].
haplotype, and 3 and 4 define a different haplotype. For
haplotype at locus C, strains 1, 2, and 3 are the same
haplotype, and strain 4 is a different haplotype, suggesting
that strain 3 might be more closely related to strains Other Animals in Nephrology Research
1 and 2 than is strain 4. If strains 1 and 2 differ from
3 and 4 for a disease phenotype, it is more likely that the The emphasis on mice and zebrafish thus far in
gene is within the area covered by haplotypes A and B
than within haplotype C that is shared between strains this chapter is not meant to overlook the enormous
1, 2, and 3 benefit that has derived from the use of other
15 Translational Research Methods: The Value of Animal Models in Renal Research
species. Since the previous edition of this text-
book, gene targeting has been extended to rats
and rabbits, and the advent of TALENs and the
Crispr/CAS9 system will accelerate efforts in
other species. ENU mutagenesis has also been
used to obtain rat mutant models of disease and
gene mapping of disease phenotypes in rats has
yielded important insights [131–135]. Rats have
also been widely used in studies of nutrition as it
related to kidney development and disease
[136]. Furthermore, their larger size makes rats
more amenable to studies that require precise
physiological monitoring or imaging, though the
technology to perform such studies in murine
models, to take advantage of knockout mouse
models, has also improved dramatically
[137–144].
Large animal research has also had an impor-
tant legacy in nephrology that continues to this
day. Historically, several animal models have
been used to study renal physiology, including
swine, sheep, guinea pigs, rabbits, rats, and
mice. Fetal lambs have been a particularly impor-
tant model in which developmental aspects of
physiology have been studied, particularly relat-
ing to obstructive uropathy [145–157]. Studies in
large animals are also vital in efforts to use tissue
engineering to develop artificial tissues or in
models of tissue regeneration [158–160]. As in
other situations, there is a constant need to balance
the advantages of a large animal model with the
lower costs of smaller animal models.
Models of Renal Failure
Approaches to the study of renal failure include
acute and chronic models. Acute renal failure has
been induced using pharmacological agents,
antisera against kidney tissue, or other antigens
in which immune complex formation leads to
glomerular disease [161–167]. Ischemia-
reperfusion models of acute renal failure are
achieved by temporarily ligating a renal artery
allowing study of the pathological processes
involved in tubular damage, as well as the effect
of various pharmacological treatments on the
pathologic process (e.g., [168–188]). An alternate
463
form of tubular injury involves temporary
obstruction of a ureter [189–191]. Chronic kidney
disease has also been studied using a variety of
mouse and rat models, including Adriamycin-
induced podocyte injury [192–194] and
glomerular injury from nephrotoxic serum
[161, 164, 195]. There are also several mouse,
rat, and other mammalian models for dominant
and recessive forms of polycystic kidney disease
that will be discussed in the respective chapters on
those diseases [196–198]. Combining these injury
models with genetic models is an emerging fron-
tier in nephrology research.
Models of Immunological Injury
There are many models of autoimmune injury to
the kidney. A traditional model for a lupus-like
autoimmune disease is the NZB mouse that has
been studied for many years [199–203]. These
mice develop autoantibodies similar to those
observed in humans with systemic lupus
erythematosus and other related autoimmune dis-
orders. More recently, many strains of mice car-
rying mutations in genes involved in the
regulation of the immune response have been
used to increase our understanding of the role
the immune system plays in the onset and pro-
gression of kidney disease [204–214]. These
knockout strains have allowed investigators to
begin a genetic dissection of genes involved in
autoimmune and other disorders. Transgenic tech-
nologies have also had an important impact in the
development of new immunological models. One
particular contribution is the use of live-cell imag-
ing that exploits fluorescent transgenic reporter
genes which mimic the expression of genes
expressed in specific immunological cell types
[215, 216]. These can be used either to trace the
location of these cells in animals or to show evi-
dence of gene expression in an in vivo setting.
Transplant Models
Animal models have been used extensively to
study transplant rejection and in efforts to
464
understand how tolerance to transplanted tissue
may be improved. Over the past 20 years, there
has been an extraordinary advancement in our
mechanistic understanding of the immune func-
tion, and this has been brought to bear on the
study of transplant rejection and tolerance
[217–223]. Important models under study include
skin and heart transplants in mice and kidney
transplants in rats [224–231]. Additionally, it is
possible to produce “humanized” mice by
transplanting human tissue into immunodeficient
or irradiated mice whose immune system has been
reconstituted with human lymphocytes, thus
allowing the study of human immune function in
an animal model [232–235]. One area of research
that remains controversial is that involving xeno-
grafts [236, 237]. Since the supply of human
kidneys and other organs for transplants continues
to fall far short of the demand, there is a desirabil-
ity of determining whether nonhuman animals
provide an alternative source of organs for trans-
plantation. The major concerns here include the
strong immunological rejection to a xenograft that
must be overcome and the danger that xenografts
might serve as vectors for the introduction of
novel infectious agents into the human
population.
Summary
Animal models are of increasing importance in
the study of kidney disease. An important shift
over the past 20 or more years is the use of rodent
models and the use of genetic models. Large ani-
mal use remains an important aspect of these
studies. The use of large versus small animals
must take into account cost and ethical
considerations.
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2. Torres M, Gomez PE, Dressler GR, Gruss P. Pax-2
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3. Moore MW, Klein RD, Farinas I, Sauer H, Armani M,
Philips H, et al. Renal and neuronal abnormalities in
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 Basics of Clinical Investigation
16
Susan L. Furth and Jeffrey J. Fadrowski

Contents Sources of Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487

Clinical Research Question . . . . . . . . . . . . . . . . . . . . . . . . 474
Hallmarks of a Good Research Question . . . . . . . . . . . . 474
Steps in Refining a Good Research Question . . . . . . . 474
Scientific Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Inference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Validity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Reliability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Study Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477
Observational Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477
Experimental Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 482
Important Issues in Carrying Out a
Research Plan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484
Selection of Subjects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484
Attrition of Study Subjects . . . . . . . . . . . . . . . . . . . . . . . . . . 486
Data Collection: Measurement . . . . . . . . . . . . . . . . . . . . 487
Identifying the Variables to Be Measured . . . . . . . . . . . 487
Types of Variables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487
Assessing Data Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488
Analytic Plan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488
Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488
Statistical Significance and Confidence
475 Intervals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491
475
476 Topics Related to Clinical Decision Making . . . . . . 492
477 The Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
The Diagnostic Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
Screening Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495
Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495
Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495
Person/Population . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495
Ethics in Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495
Evaluating the Literature: Rating the
Strength of Scientific Evidence . . . . . . . . . . . . . . . . . . . . 496
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497

S.L. Furth (*)

Departments of Pediatrics and Epidemiology, Perelman
School of Medicine at the University of Pennsylvania,
Philadelphia, PA, USA
e-mail: furths@email.chop.edu
J.J. Fadrowski
Department of Pediatrics, John Hopkins University School
of Medicine, Baltimore, MD, USA
e-mail: fadrowsj@jhmi.edu

# Springer-Verlag Berlin Heidelberg 2016 473

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_15
474
Clinical Research Question
How can we best evaluate, treat, and assess long-
term risks for children with kidney disease? Who
is at risk of developing end-stage renal disease
(ESRD) in childhood or young adulthood? Clini-
cians are often faced with questions such as these
with uncertain answers in the practice of pediatric
nephrology. Parents ask, “Why did my child get
this disease?” “What is the most effective method
to treat this condition?” “What’s the prognosis of
this condition in my child?” Frequently, these
answers are not known, and these questions are
the inspiration for high-quality clinical research.
The first step in developing a valuable clinical
study is determining whether the initial query
can be translated into a good research question.
Hallmarks of a Good Research Question
A good research question gives useful informa-
tion, is interesting to the researcher, builds on
what is known, and can be answered with avail-
able resources. Research is a labor of love,
demanding attention to detail, perseverance, hon-
esty, and imagination. Developing a good
research question is an iterative process. One
needs input from knowledgeable colleagues and
collaborators. The researcher must become thor-
oughly familiar with what is already known about
the topic by reviewing the literature and consult-
ing with experts in the area. Investigating what is
already known has several benefits. First, it can
reveal that the candidate research question has
already been answered adequately. Second, learn-
ing what is already known provides insight into
potentially useful methods for addressing a
research question. For example, previous studies
may demonstrate good ways to measure a variable
of interest or provide background information for
determining sample size. Third, a literature review
may suggest ways to frame the research question
at hand. For example, a literature review may
reveal that particular risk factors are consistently
associated with a disease process, and an interven-
tion to modify these risk factors may form a sound
basis for a clinical trial.
S.L. Furth and J.J. Fadrowski
Finally, a good research question needs to be
answerable with available resources. These
include subjects available for study, technical
expertise of the research staff, and the time
and money that can be devoted to the
project. Once a question is framed, the researcher
needs to outline the study protocol or
methods, which include specifying the recruit-
ment method, number of subjects and how they
will be recruited, how each variable will be
defined, and the plan for data analysis. A poorly
designed study is worse than no study at all
because, like imprecise measurements and an
improper analytic plan, it can also lead to false
conclusions.
Steps in Refining a Good Research
Question
A good research question usually begins with a
broadly stated concept. The initial question is then
made more specific by identifying independent
and dependent variables. Often, research
questions are concerned with causal relationships.
Independent variables are those conceptualized to
be causes; dependent variables are those
conceptualized to be effects. The research ques-
tion can be modified to ask about the role of
multiple potential causes in leading to the specific
outcome. A simple research question asks
whether x (independent variable) causes
y (dependent variable). More complex research
questions could assess the relative importance of
x and other variables (e.g., a and b) as causes of y.
A different research question might ask how
strongly x predicts y in one population versus
another.
The next step is to translate a research
question into a hypothesis. In our simple
example, the researcher may hypothesize that
x causes y. In the actual research project, the
information collected is examined to
determine whether it is reasonable to conclude
that x does cause y. In examining the data, the
investigator tests the null hypothesis that x does
not cause y versus the alternative hypothesis that
it does.
16 Basics of Clinical Investigation
Scientific Method
A study’s potential value is determined by the
relevance of the research question. Its ultimate
worth is determined by the study methods.
Methodologic issues concern the study design,
subject selection, data collection techniques, and
the analytic plan. Subsequent sections in this
chapter discuss each of these aspects. As a foun-
dation, this section describes the concepts of infer-
ence, generalizability, and validity.
Inference
As Fig. 1 shows, scientific research begins with
the research question. It then moves (clockwise in
the figure) to the controlled arena of the study
design and then through the implementation of
the actual study and findings. Inferences from
the findings in the study approximate the “truth
in the study.” From these “truths,” we attempt to
infer the applicability of the findings to general
clinical practice. Researchers describe and explain
reality by sampling a portion of it, measuring
characteristics of the sample, analyzing the mea-
surements, and interpreting the results. Errors in
the design or implementation of the study can lead
to false conclusions. The strength of inference
depends largely on the research methods used in
the recruitment of study subjects (sampling) and
in the choice and integrity of the study design.
At any stage in study design and implementation,
bias can occur. Bias is the result of any process
that tends to produce results that depart
Fig. 1 The role of
inference in drawing
conclusions from clinical
research studies
(generalizability)
475
systematically from the truth. Three broad catego-
ries of bias include selection bias, measurement
bias, and confounding bias. Selection bias arises if
the manner in which subjects with the outcome of
interest and the comparison group were selected
yields an apparent association, when, in reality,
exposure and disease are not associated. Measure-
ment bias occurs when the methods of measure-
ment systematically differ between groups.
Confounding occurs when a factor is associated
both with the exposure and the outcome and the
effect of the main exposure under study is con-
fused or distorted by the confounder.
Statistical inference depends on the methods
used to define and sample the population. The
researcher uses inferential statistics to extrapolate
the sample findings to the larger population of
children from which the sample was drawn. Infer-
ential statistics assume that the studied sample is
drawn by probability methods and can be used to
make inferences about the larger population. The
size of a probability sample determines the cer-
tainty of inferences from it. All other things being
equal, the larger the sample size, the greater the
certainty of inferences to the population.
The researcher’s ability to make a causal infer-
ence from study results depends largely on issues
of study design. Study designs are observational
when the investigator does not manipulate the risk
factor but merely selects children with and with-
out disease and compares them in terms of the risk
factor(s). Study designs are experimental when
the investigator not only observes but actually
manipulates the relationship between two vari-
ables. Observational designs provide somewhat
476
weaker evidence of causation, because they fail to
rule out explanations other than association
between the variables studied. Experimental
designs can provide much stronger evidence for
causation. In an experimental study, the investi-
gator controls the independent variable, which is
the factor hypothesized to produce change in a
dependent variable. In an experimental study, sub-
jects are randomized to receive or not receive the
independent variable. The goal of the process of
randomization is to produce study groups that are
“balanced” in terms of other factors that could
influence the dependent variable. Unfortunately,
experimental designs often are not feasible, ethi-
cal, or desirable. Epidemiologic studies of disease
preclude manipulation of risk factors in humans.
Health services researchers studying the public
health impact of changes in health policy rarely
can control these changes.
Within the broad categories of observational
and experimental designs, there are many varia-
tions. These variations, distinguishing character-
istics, primary uses, strengths, and weaknesses,
are discussed, in the section Study Design. Study
design also influences the validity of study results.
Validity
Validity is the extent to which study findings cor-
rectly reflect and explain reality (Fig. 2). Validity
Fig. 2 External and
internal validity in
experimental designs (From
Fletcher RH, Fletcher SW,
Wagner EH. Clinical
epidemiology: the
essentials, 3rd
ed. Baltimore: Williams &
Wilkins, 1996:12, with
permission)
S.L. Furth and J.J. Fadrowski
can also be thought of as accuracy. Systematic
bias undermines validity. As a research question’s
relevance increases, so does the need for validity.
The clarity and rigor of study design as well as the
careful implementation of the research plan
increase the likelihood that inferences from the
study are valid.
Cook and Campbell [1] define several aspects
of validity. Statistical validity is the correctness of
study conclusions regarding the existence of a rela-
tionship between two variables. A study lacks sta-
tistical validity when it concludes there is no
relationship between variables when in fact there
is one or when it concludes there is a relationship
when in fact there is none. Statistical validity is
jeopardized most often by inadequate sample size
and by improper use of statistical tests.
If there is a relationship between two variables,
internal validity is the correctness of conclusions
about whether the relationship between the inde-
pendent and dependent variables is causal. Inter-
nal validity is jeopardized when a study design
fails to control for factors that could confound the
hypothesized causal relationship.
Finally, given a causal relationship between the
independent and dependent variables, external
validity (Fig. 2) is the correctness of generalizing
to other persons, settings, and times. Poor choice
of a study population and inadequate research
procedures are common challenges to external
validity.
16 Basics of Clinical Investigation
Reliability
While validity is the degree to which data measure
what they were intended to measure, reliability is
the extent to which repeated measurements at
different times and places, or by different people,
are reproducible. An instrument measuring an
assay may be accurate if, on average, the measures
vary around the true value, but may not be reli-
able, because the measures are widely scattered
around the true value. Reliability and precision are
related concepts. A precise measure is one that has
nearly the same value each time it is measured. It
is very reliable. Precision can be described statis-
tically using the standard deviation of repeated
measures. The standard deviation divided by the
mean is the coefficient of variation. Small coeffi-
cients of variation imply precise measurements.
In summary, the scientific method involves
extrapolating inferences from a study situation to
the larger world. The value of research depends on
the validity of such inferences, which in turn is
determined by the researcher’s choice of methods.
The following sections explain the strengths and
weaknesses of the methodologic choices
available.
Study Design
Observational Studies
There are four major types of observational stud-
ies: case series, cross-sectional, case–control, and
cohort. Observational designs are weaker than
experimental designs in establishing causation,
but they are useful when it is not feasible to
manipulate the independent variable. Studies of
disease etiology usually are observational. In
these types of observational studies, risk factors
or exposures are the independent variables, and
disease is the dependent variable.
Case Series
In a case series study, a sample of patients with
common characteristics are selected to describe a
clinical or pathophysiological aspect of a disease,
treatment, or diagnostic procedure. A case series
477
study is easy to conduct and is useful as a prelim-
inary study to reinforce anecdotal evidence, to
generate hypotheses, or to establish variable dis-
tributions in planning future research. In a field
like pediatric nephrology which deals with many
rare diseases, this study design can be quite useful.
However, because there is no comparison group,
the case series design cannot provide an estimate
of risk. An example of a case series is the report by
Porras et al. examining 53 children presenting to a
single hospital with midaortic syndrome over the
past 53 years [2]. The aim of this study was to
better understand the outcomes associated with a
multidisciplinary management approach to
patients with this diagnosis. The authors described
clinical characteristics at presentation, cardiac and
kidney function parameters at follow-up, and
interventions such a number of blood pressure
medications used in treatment, and number of
percutaneous and surgical procedures.
Cross-Sectional Design
A cross-sectional study is one in which the disease
and risk factors are measured at the same time in a
sample of subjects. Subjects can be categorized as
either having or not having the risk factor. Within
each group, the presence of the disease can be
determined. Analytically, the association between
a particular risk factor and the disease is measured
as the relative prevalence of the disease among
those with, versus those without, the risk factor.
The cross-sectional study design is superior to
the case series in that it provides a means for
comparison. Cross-sectional studies are relatively
economic, are easy to conduct, and allow simul-
taneous examination of multiple risk factors.
Cross-sectional studies have a number of lim-
itations. Whether the results of such studies gen-
eralize to the population of interest is significantly
impacted by the population from which a cross-
sectional slice is taken. The simultaneous exami-
nation of exposure and disease in the study popu-
lation can lead to the introduction of significant
biases that may directly impact results of the
study. Perhaps the most important limitation of a
cross-sectional study that must be recognized is
the inability to infer causality. If an exposure and
disease are assessed simultaneously, then one
478 S.L. Furth and J.J. Fadrowski

Fig. 3 Cystatin
C-estimated glomerular 130
filtration rate (GFR) by
blood lead levels. The thick
line represents estimates
based on restricted
quadratic spline

Cystatin C–Based GFR, mL/min/1.73 m2

transformation for
log-transformed blood lead
levels with knots at the 110
10th, 50th, and 90th
percentiles. Thin lines
represent corresponding
95% confidence intervals.
Data points for the
scatterplot represent
adjusted blood lead and 90
estimated GFR values. The
horizontal axis is in the log
scale (From Fadrowski and
Navas-Acien [3])

70

0.7 1.7 4.1 10.0

Blood Lead Level, μg/dL

cannot be sure the exposure preceded the disease, Case–Control Studies

which is critical to the inference of causality.
In the United States, the National Health and
Nutrition Examination Survey (NHANES) con-
tinually collects data on various exposures and
outcomes simultaneously in a representative sam-
ple of the general population. This data lends itself
well to cross-sectional studies. The association
between lead levels in children and estimated
glomerular filtration rate (GFR) was examined
among 769 children who participated in the sur-
vey from 1988 to 1994 [3]. As can be observed in
Fig. 3, higher blood lead levels were associated
with lower estimated GFR. The authors hypothe-
sized that the toxicity of higher lead levels may be
causing the lower GFR, but acknowledged that
the lower GFR, possibly caused by some other
factor, could be causing the decreased excretion of
lead thus leading to higher lead levels. Given the
simultaneous examination of lead and GFR in this
study, it is impossible to know if lead was causal
of the lower GFR observed with higher lead
levels.
Case–control studies are also known as
case–referent, compeer, retrospective, case his-
tory, and cohort studies. A case–control study
starts with the identification of persons with the
disease or other outcome variables of interest in
the population at risk (Fig. 4). A suitable control
group of persons without the disease or outcome
is also selected from the population at risk. This is
pictured on the right side of Fig. 4. To examine the
possible relation of one or more exposures to the
given disease or outcome, the researcher then
looks back in time to compare the proportions of
the cases and controls exposed and not exposed to
the risk factor in question.
The case–control design has several advan-
tages. It provides stronger evidence of causation
than the cross-sectional design. In a cross-
sectional study, outcomes and exposures are
assessed simultaneously, and the investigator
must infer cause and effect relationships because
the temporal sequence cannot be established. In a
case–control design, an attempt to establish a
16 Basics of Clinical Investigation
Fig. 4 Design of a
case–control study (From
Fletcher RH, Fletcher SW,
Wagner EH. Clinical
epidemiology: the
essentials, 3rd
ed. Baltimore: Williams &
Wilkins, 1996:213, with
permission)
temporal relationship between the outcome and
exposure is made by starting with a population
of persons with and without the outcome and then
working backward to examine suspected expo-
sures. Thus, compared to the cross-sectional
design, the investigator is more confident that
the exposure of interest came before the outcome,
not as a result of the outcome. As compared to
other study designs, case–control studies are effi-
cient in the study of rare diseases or those with
long latent periods between exposure and out-
come. Whereas cross-sectional or cohort designs
would require a large number of subjects and time
to identify risk factors for a rare disease, a well-
designed case–control study can identify similar
risk factors with comparatively fewer subjects and
much less time and expense. Also adding to the
efficiency of the case–control design, several
potential risk factors for a disease or outcome
can be examined simultaneously.
A recent case–control study by Bettinelli
et al. [4] examined phosphate homeostasis in
Bartter syndrome. The authors noted that
hypercalciuria and nephrocalcinosis were well
described in patients with this rare disease, but
characterization of phosphate and calciotropic
hormones in this disorder was quite limited. To
expand knowledge in this area, the authors com-
pared plasma phosphate, calcium, PTH,
25-hydroxyvitamin D, alkaline phosphatase, and
osteocalcin levels between 15 patients with
Bartter syndrome and 15 healthy subjects. It was
found that patients with Bartter syndrome had a
tendency toward renal phosphate wasting and
479
elevated circulating PTH levels. The authors
suggested these findings could help motivate a
larger multicenter study in which the mechanisms
underlying these abnormalities could be more
fully elucidated.
Case–control studies yield an odds ratio as an
estimate of relative risk. This measure is calcu-
lated by dividing the odds that a patient was
exposed to a given risk factor by the odds that a
control was exposed to the risk factor. It can also
be obtained from logistic regression analysis.
Logistic regression allows the investigator to
obtain the odds ratio for a given risk factor inde-
pendent of other potential risk factors or con-
founders using the technique of adjustment.
Odds ratios are generally a good approximation
of relative risk if the outcome is rare.
As with any study design, the case–control
method has limitations. Case–control studies
allow for the study of only one disease at a time,
as opposed to cross-sectional or cohort studies.
This design does not allow for the measurement
of incidence, prevalence, or excess risk.
Case–control studies are also subject to error, or
bias, which can threaten the validity of the study.
Selection and information biases, the two major
categories of bias, are possible in the case–control
design. Selection bias arises if the manner in
which cases and controls were selected yields an
apparent association when, in reality, exposure
and disease are not associated. For example,
cases, by definition, include only individuals
who have been identified as having the disease
and who are available for study. Those who have
480
not been diagnosed, have been misdiagnosed, or
have died are excluded. If diagnosis or availability
is related to the exposure being studied, the sam-
ple of cases will be biased.
Avoiding selection bias can be even more chal-
lenging in the selection of controls. The control
group must be comparable to the cases. They
should not be chosen in such a way that important
differences between cases and controls exist that
might influence exposure history and thus limit
the inferences derived from the study. A number
of strategies exist to select a control group that is
at risk for the disease and otherwise representative
of the same population as the cases. These include
sampling cases and controls in the same way (e.g.,
from the same clinical setting), matching controls
to cases on key variables related to the disease
(e.g., age), using multiple control groups, and
using population-based samples of both cases
and controls (e.g., using disease registries).
Information bias occurs when the case and
control groups differ in terms of the quality of
the data collected to measure risk factors. The
retrospective approach to measuring an exposure
in the case–control design introduces the possibil-
ity of differential recall between the cases and
controls. Cases may have been asked more often
about the presence of a given exposure and/or
may be more circumspect in their recall of such
exposures. This introduces recall bias, a form of
information bias because of better, and sometimes
exaggerated, recollection of exposures by cases as
compared to controls.
It can be difficult for an investigator to remain
objective in collecting exposure information. In
interviewing subjects and in reviewing records,
there may be a tendency to look more carefully
or evaluate evidence differently for cases than for
controls. Strategies for dealing with this problem
include the use of objective measures and to
ensure that the individuals collecting data are
unaware of the subject’s group status (blinding/
masking). The more subjective the method for
measuring the exposure, the more important it is
to mask the observer. Blinding as to the specific
exposure being studied or study hypothesis is
useful and also can be used to attempt to control
recall bias.
S.L. Furth and J.J. Fadrowski
Nested case–control studies and nested
case–cohort studies are alternative case-based
hybrid designs that have many advantages. A
nested case–control study involves selecting all
cases and control subjects from a known cohort.
In this design, the controls are free of the outcome
or disease. Nested case–control studies eliminate
the problem of recall bias, because the exposure
information is obtained before the outcome has
developed (cohort design). Also, the temporal
sequence between exposure and outcome is
defined. This design is also much more econom-
ical and efficient; the entire cohort need not be
analyzed for a given exposure (e.g., via a labora-
tory specimen). Nested case–cohort studies also
use the selection of cases and controls from a
known cohort. However, in this design, controls
are randomly selected from the initial cohort
irrespective of outcome. This design permits the
delineation of relative risk for an exposure.
Cohort Design
Various names, including prospective, follow-up,
and longitudinal, have been used to label cohort
studies in the past, reflecting the temporal
sequence of exposure and disease in this category
of observational studies (Fig. 5). The word cohort
originated from the Latin word cohors, describing
a group of warriors that marched together. Clinical
investigators have adapted this term to a specific
type of research study: a group of individuals free
of the disease(s) of interest is assembled, their risk
status is determined, and the group is followed
over time to measure the incidence of disease.
Comparison of the incidence of disease (or rate
of death from disease) between those with and
without the exposure of interest permits measure-
ment of the association between the risk factor and
the disease.
The significance of the cohort design has been
emphasized by the wealth of scientific data
obtained from famous cohorts such as the Fra-
mingham Heart Study or the Physicians Health
Study. Pediatric nephrology has also benefited
from studies using the cohort design, including
the ongoing Chronic Kidney Disease in Children
study (CKiD), which is a multicenter, prospective
cohort study of greater than 500 children with
16 Basics of Clinical Investigation
Fig. 5 Design of a cohort
study (From Fletcher RH,
Fletcher SW, Wagner
EH. Clinical epidemiology:
the essentials, 3rd
ed. Baltimore: Williams
&Wilkins, 1996:102, with
permission)
mild to moderate CKD from 48 pediatric nephrol-
ogy centers in North America.
The cohort design has an obvious niche in
clinical research. Ethical and practical consider-
ations often do not allow for randomization of
individuals to an exposure of interest. Cohort
designs allow for the examination of exposure
and disease associations under such
circumstances.
Cohort studies can be classified as concurrent
or nonconcurrent. In a concurrent cohort study
(also referred to as a prospective or longitudinal
study), the clinical investigator identifies the pop-
ulation and collects extant exposure information
and then follows the cohort to a designated point
in the future. Nonconcurrent cohort studies (i.e.,
retrospective, historical, and nonconcurrent pro-
spective) require the investigator to identify a
cohort that has been delineated in the past, along
with information regarding the exposure(s) of
interest. This population can then be followed
for the development of a given disease in the
more recent past, the present, or into the future.
Traditionally, the outcome of interest in cohort
studies is the ratio of the incidence of disease in
those with the exposure divided by the incidence
of disease in those without the exposure. This can
be interpreted as the relative risk for disease in
many cases. When calculating and interpreting
risks in the cohort design, the absence of random-
ization must be taken into account. Because the
investigator is merely observing the exposure and
not controlling for it via randomization, subjects
with and without the risk factor might differ in
terms of other characteristics that are related to the
disease. If the characteristic is related to both the
exposure being evaluated and the disease, it can
481
lead to a misleading association between the
exposure and the disease. Such a characteristic
would be a confounder.
To avoid misinterpreting such an association,
the investigator must measure potential con-
founders and adjust for them in the analysis. Mul-
tivariable analyses are examples of statistical tools
used to adjust for confounders. However,
unsuspected confounders might still jeopardize
the validity of conclusions.
The relationship between blood pressure con-
trol and left ventricular hypertrophy was recently
examined in children in the CKiD cohort
[5]. Using a linear mixed statistical model, the
study looked at the exposure of casual blood pres-
sure measurement z scores on the outcome of left
ventricular mass index (LVMI). In acknowledg-
ment of their potential impact on the exposure
and/or outcome, additional variables were
included in the statistical model including sex,
race, type of CKD diagnosis, duration of CKD
diagnosis, age, height at each visit, GFR, use of
angiotensin-converting enzyme inhibitor (ACEi)
or angiotensin receptor blocker (ARB) medica-
tion, use of other antihypertensive medications,
and anemia status. In addition to systolic blood
pressure, predictors of LVMI in this study
included anemia and use of antihypertensive med-
ications other than ACEi’s or ARBs.
Because risk factors are measured before out-
come or disease, the temporal sequence of risk
and disease is established, and the potential for
biased risk measurement is avoided. Several dis-
eases or outcomes can be measured, and disease
occurrence can be measured in terms of incidence,
not just prevalence. Cohort studies are an ineffi-
cient way to study rare outcomes because in this
482
Fig. 6 Design of a
randomized trial (From
Fletcher RH, Fletcher SW,
Wagner EH. Clinical
epidemiology: the
essentials 3rd
ed. Baltimore: Williams
&Wilkins, 1996:139, with
permission)
scenario, large sample size and long follow-up are
needed, which significantly increases the cost and
effort associated with the study. A nonconcurrent
cohort design can reduce cost, but it decreases the
investigator’s control over subject selection and
risk factor measurement.
Experimental Studies
In an experimental study, the investigator controls
the independent variable, also known as the inter-
vention or treatment, and then observes the effect
on an outcome or series of outcomes. The classic
form of experimental design is the clinical trial.
Randomized Controlled Clinical Trials
Most investigators, clinicians, and patients are
familiar with the “gold standard” of experimental
designs, the randomized blinded controlled clini-
cal trial. Such trials are considered “gold stan-
dard” because the rigid design helps minimize
the influence of confounding variables and bias,
allowing the true effect of the intervention to be
elucidated.
The Effect of Strict Blood Control and ACE
Inhibition on the Progression of Chronic Renal
Failure in Pediatric Patients (ESCAPE) trial is an
example of a randomized controlled clinical trial
in children with kidney disease [6]. In this study,
children with CKD received an ACEi at a stan-
dardized dose for a 6-month period. They were
then randomly assigned to intensified blood pres-
sure control (target 24-h mean ambulatory blood
pressure below the 50th percentile) or “conven-
tional” (at that time) blood pressure control (mean
arterial blood pressure in the 50th to 95th percen-
tile). The primary end point in this study was time
S.L. Furth and J.J. Fadrowski
to a decline of 50 % in the GFR or progression to
end-stage renal disease. In this and other con-
trolled clinical trials, randomization is the key
feature of its experimental nature. Participants
are randomly assigned to the group that will
receive the intervention (the intervention, treat-
ment, study or experimental group) or the group
that will not receive the intervention (the control
or placebo group) (Fig. 6). Through randomiza-
tion, all potential confounders, both those recog-
nized by the investigator and those that are not
suspected, are likely to be balanced between the
study groups. In other words, the groups in this
study are considered to be the same except for the
treatment they receive. Differences in rates of
progression of kidney disease in the groups are
attributable to the intervention, because the effect
of confounders has been ruled out by the balance
achieved by randomization. Therefore, experi-
mental designs offer stronger evidence of causal-
ity than do observational designs.
In an experimental study, it is important to
ensure that subject assignment is truly random.
This can be achieved by using random numbers,
either through computer-assisted assignment or
manually, with a table of random numbers. Some-
times blocking is used in conjunction with random
assignment. A block of subjects is simply a set
number of consecutive study enrollees. Within
each block, a predetermined number of subjects
are randomly assigned to each study group. For
example, if the block size is set at six and two
study groups of equal size are desired, then three
subjects in each block of six are randomly
assigned to one group and three to the other.
Blocking is useful when study enrollment is
expected to be prolonged. Over extended periods,
both study procedures and outside conditions can
16 Basics of Clinical Investigation
change. Blocking ensures that the study groups
will be balanced with regard to such changes.
A block randomization scheme, with a block
size of four, was used in the ESCAPE trial.
Experimental studies, like observational stud-
ies, are subject to measurement bias. Research
staff should be masked or blinded to the subject’s
group assignment during data collection, espe-
cially if any outcome measures are not strictly
objective. If outcomes are measured, without sub-
ject or staff knowledge of the subject’s group
status, they are less likely to be influenced by
expectations about potential differences between
treatment and control group outcomes. Studies in
which both the participant and the staff are
unaware of the treatment group status are known
as “double blinded.” There may be situations in
which blinding the clinical staff and patients is
difficult or impossible. Clinical trials that are not
blinded are known as open-label trials. Such
designs are common in cancer clinical trials, as
treatments being compared are often complex,
with different side effect profiles and delivery
protocols, and thus masking is not feasible. The
medical staff in the ESCAPE trial had to be aware
of the assigned blood pressure target as they were
integral in titrating medications to achieve this
target. Thus, the ESCAPE trial was not a double-
blinded study.
Clinical trials typically provide the necessary
medical evidence to bring a new treatment into
use, and for drugs, such trials are referred to as
“phases.” A Phase I trial is most often the first
stage in testing a new drug in humans and may
include healthy participants and/or patients. In
these trials, information on the distribution,
metabolism, excretion, and side effects of the
drug is investigated. The optimal dose of the
drug to deliver is also investigated. Such studies
are typically not randomized or blinded. Phase II
clinical trials are designed to evaluate the effec-
tiveness and short-term safety and side effects
associated with the drug. These trials are generally
carried out in persons having the disease of inter-
est. Phase III trials are expanded trials to prove the
effectiveness of a drug and provide the informa-
tion necessary for physician labeling of the drug.
These studies are most often randomized and
483
blinded. Once completed, the pharmaceutical
manufacturer may apply to market the drug for
the indication(s) studied in the phase III trial.
Phase IV clinical trials are post-marketing studies
(post-licensure) designed to delineate more infor-
mation about the drug’s risks, benefits, and opti-
mal use in circumstances approximating “real-
world” conditions.
Alternative Designs
It is not always feasible to conduct a traditional
randomized controlled clinical trial for every
treatment. In such scenarios, multiple alternative
experimental study designs are available and may
provide useful information regarding the treat-
ment under consideration. A few examples of
such alternative designs follow. Crossover trials
randomly assign half of the study population to
start with the control period and then subsequently
switch to active treatment; the other half are on the
opposite schedule. Such trials allow each partici-
pant to serve as their own control, allowing for
increased statistical power and thus fewer partic-
ipants. However, these studies generally take a
longer period of time. The analysis and
interpretation of the results may also be
complicated if the treatment effects are thought
to persist for a period of time even after the
intervention has been ceased. A before–after trial
compares the outcomes of different types of treat-
ments in a group or groups of interest by taking
advantage of calendar time. In such a study, out-
comes in individuals receiving one type of treat-
ment during a given period are compared with
individuals at a subsequent time, who have
received a different treatment. Although econom-
ical to perform, the results of such designs may be
more prone to error as it may be difficult to know
and/or control factors independent of the treat-
ment that may have also influenced the outcome
of interest.
Issues with Analysis in Experimental
Designs
It is not uncommon in clinical trials to have
patients who were assigned to one group switch
to a different group. For example, a patient
assigned to receive the active treatment under
484
study may discontinue the treatment. Alterna-
tively, a patient assigned to the control group
may end up receiving active treatment. To avoid
introducing bias in the results, it is common prac-
tice in clinical trials to analyze the results
by intention-to-treat. In such conservative
analyses, every patient is grouped according to
his/her original randomization assignment when
analyzing the results, regardless of whether the
patient actually received the assigned treatment
or not. Intention-to-treat analyses may dilute the
effect of the treatment of interest, but more impor-
tantly, likely minimize the introduction of bias
into the study which may lead to erroneous
conclusions.
To maximize the yield of arduous and costly
clinical trials, investigators may wish to perform
subgroup analyses, defined as an evaluation for
treatment effects within a subset of patients.
Related to subgroup analyses, post hoc analysis
refers to examining the data after the study has
concluded for associations that were not specified
a priori. Although subgroup analyses and post hoc
analyses may provide additional useful informa-
tion, due to analytic challenges, particularly in the
area of sample size, results may be misleading.
Given these statistical limitations, it is
recommended that all hypotheses and intended
analyses be stated prior to the initiation of the
study, which may help in planning the design of
the study, including the sample size. In the
event that post hoc analyses are performed,
they should be clearly labeled as such so that the
reader is able to identify the potential limitations
to any conclusions derived from such analyses.
A report in the New England Journal of Medicine
reviews the challenges of subgroup analyses and
provides guidelines for their use within the
journal [7].
In summary, many research designs are avail-
able to the investigator. No single design is best
for all research questions. Although experimental
designs are superior to observational designs in
addressing threats to internal validity, they are not
always feasible or ethical. The most appropriate
design for a given research question is the design
that maximizes internal validity within the con-
straints of the research environment.
S.L. Furth and J.J. Fadrowski
Important Issues in Carrying Out a
Research Plan
Selection of Subjects
In any research study, one would like to extrapo-
late the findings to all patients with the condition
of interest (Fig. 2). The study population is the
group that is meant to represent the target popula-
tion from which a sample is drawn. Sampling
decisions involve defining the study population
and sample.
Defining the Target Population
Although there is no one single ideal target popu-
lation, the investigator needs to consider the ram-
ifications of one definition versus another. If the
investigator were interested in studying risk fac-
tors for a specific disease, the target population
could be defined as all children with this disease
or a subset of them (e.g., children of a certain age).
The broader the target population, the greater the
generalizability of the study findings. On the other
hand, the increased heterogeneity of a broadly
defined target population could introduce variabil-
ity among subgroups in terms of the importance of
risk factors. For example, a particular characteris-
tic could be a major risk factor in some population
subgroups but not in others. Assessing the impor-
tance of risk factors within subgroups requires a
larger study sample and perhaps a more complex
sampling design.
Defining the Study Population
A practical consideration in defining the target
population is availability of the population for
study. The investigator could have all children
available seen in a particular clinical setting. Inso-
far as children seen in this setting are representa-
tive of the target population, the clinical site
would be a good choice for study; the experience
of its enrollees could be considered generalizable
to the target population. If children enrolled in the
clinical setting differ systematically from the tar-
get population, sampling bias is introduced. For
example, tertiary care pediatric nephrology cen-
ters might be more likely to serve children with
advanced stages of the kidney disease or more
16 Basics of Clinical Investigation
severe or complicated cases. Studying only these
cases may introduce bias toward only studying the
most complex forms of a particular disease. Sam-
pling bias impairs the generalizability of study
findings. Representativeness, therefore, is a
prime consideration in defining a study popula-
tion. Investigators should evaluate the representa-
tiveness of candidate settings and the likely
implications of potential biases. One possible
approach to this in studies of patients with kidney
disease is to compare the characteristics of partic-
ipants in a study to known characteristics of the
larger population to whom one would like to
generalize the results.
Defining the Sampling Scheme
Just as we generalize from the study population to
the target population, we generalize from the
study sample to the study population. Sample
statistics are measures that pertain to the samples
that are studied. A sample mean, for example, is
the sample’s average score on a particular mea-
sure, and a sample standard deviation expresses
the variability of the sample scores. The sample
statistics are the investigator’s best estimates of
the population parameters. The sample mean is
the best estimate of the population mean; the
sample standard deviation is the best estimate of
the population standard deviation.
Extending beyond inference to hypothesis test-
ing, sample statistics of the association between
variables are the best estimates of these associa-
tions in the target population. The association
between a hypothesized risk factor and the occur-
rence of disease in the study sample (perhaps
measured by odds ratios or relative risks) is the
investigator’s best estimate of the association
between the risk factor and disease in the target
population.
Probability theory is the rationale for extrapo-
lating inferences from a study sample to the refer-
ence population. A probability sample is one in
which every subject, or element, in the study pop-
ulation has a known probability of being selected.
A nonprobability sample is one in which the prob-
abilities of selection are unknown. It is legitimate to
extrapolate from a sample to its population only if
probability sampling has been used.
485
There are several types of probability sam-
pling. In simple random sampling, each element
has an equal chance of being selected. In system-
atic sampling, each element in the population is
assigned a consecutive number, and every nth
element is sampled. Systematic sampling is easy
to use, but it will generate a biased sample if the
sampling fraction (e.g., every tenth case) is the
same as some periodicity in the ordering of cases
in the population. For example, if every tenth
patient is sampled in a clinic where ten patients
are seen each session and the most complex cases
are scheduled first, then the sample will contain
either all complex cases or no complex cases,
depending on the first element drawn. Stratified
random sampling is useful when one believes that
population subgroups differ in important ways.
The population is divided into the subgroups, or
strata, of interest. Simple or systematic random
samples are then drawn from each stratum. Clus-
ter sampling is useful when it is difficult or costly
to sample elements in a population individually.
Instead of elements, groups of elements are sam-
pled. For example, in a study of school children,
the investigator could take a probability sample of
classrooms and then study all the students within
each selected classroom. The selected classrooms,
in combination, must be representative of the
overall population. As with stratified sampling,
formulas for calculating variance must be modi-
fied, and consultation with a statistician is
recommended.
Nonprobability sampling techniques include
convenience sampling, quota sampling, and pur-
posive sampling. A convenience sample is one
that is most readily obtained without die use of
random sampling. A quota sample is a conve-
nience sample drawn to assure specified numbers
of subjects in specified strata, without the use of
random sampling. A purposive sample is one in
which subjects are selected because they are
judged to be representative of the population of
interest.
Probability sampling is preferred but not
always possible. In clinical research, the investi-
gator is often limited to a particular clinic popula-
tion. If a clinic population is believed to be
representative and if it is larger than the number
486
of subjects needed for study, the investigator
should use a probability sampling technique to
draw the study sample.
An example of probability sampling using
stratified sampling techniques can be seen in a
survey study of adult and pediatric nephrologists
[8]. The authors created a survey containing ten
case vignettes to assess whether increased experi-
ence with pediatric patients influenced nephrolo-
gists’ recommendations for peritoneal or
hemodialysis in otherwise identical patients with
ESRD described in the vignettes. Because the
authors wanted the survey respondents to repre-
sent the population of US adult and pediatric
nephrologists, they randomly selected a represen-
tative sample of nephrologists in five geographic
regions of the United States. Each randomly
selected nephrologist was mailed a survey
containing ten case vignettes to assess what fac-
tors affected the nephrologists’ dialysis
recommendations.
Determining Sample Size
In any study, several factors determine the
required sample size. This section describes
those that come into play in several common
types of investigations. Detailed sample size for-
mulas and tables are beyond the scope of this
chapter, but several excellent references are listed
in Suggested Reading. Briefly, to estimate sample
size, the researcher needs to set the acceptable
level of α (probability of type I error) and β (prob-
ability of type II error) and determine the effect
size that one is likely to see. In determining sam-
ple size, the probability of making a type I error, α,
is usually set at 0.05. This is the probability of
concluding that an association between two vari-
ables exists when it does not. β error is the prob-
ability of concluding that no association exists,
when in fact it does. The reader will be more
familiar with β error in terms of its relationship
to “power.” The power of a study is equal to 1  β.
In many studies, β is customarily set at 0.2 for a
power of 80 %. If β is set to 0.1, the power of the
study is 90 %.
In addition to specifying α and β, the researcher
must also determine an estimate of the response to
treatment in one of the groups (in a clinical trial)
S.L. Furth and J.J. Fadrowski
Table 1 Example illustrating how a, b, and effect size
affect sample size
Confidence Power
Total
1  α (%) 1  β (%) Effect size (%) sample no.
95 80 10 ! 40 76
95 90 10 ! 40 96
95 90 10 ! 20 572
95 99 10 ! 40 156
Adapted from sample size calculator (Statcalc) in Epi-info
Stat Calc. Available at http://www.cdc.gov/epiinfo
or the rate of occurrence of disease (in a cohort
study). The effect size is an estimate of how much
better than the comparison group you expect a
treatment group to be in a clinical trial or how
increased the risk of a particular disease is in the
setting of a particular risk factor (in a cohort
study). An illustration of estimated sample sizes
for given α, β, and effect sizes is shown in Table 1
for a study comparing differences in proportions
in two groups.
Table 1 illustrates how varying the acceptable
levels of α, β, and effect size influences sample
size. For example, if we designed a study to deter-
mine whether a new drug could “cure” 40 % of
patients compared to an old drug that “cured”
10 % of patients, we would have 90 % power to
see such an effect with 95 % confidence in a total
sample of 96 patients (see row 2 in Table 1). In
contrast, to obtain a significant result
documenting a smaller effect size from the old
drug cure rate of 10 % to a new drug cure rate of
20 % with the same α = 0.05 and 90 % power, we
would need to study 572 patients.
Attrition of Study Subjects
Sample size calculations determine the number of
subjects needed at the study’s conclusion. In
determining the number of subjects to enroll, the
investigator must estimate attrition rates and
enroll a sufficiently large sample to compensate
for study dropout.
Even if probability sampling is used to define
the study, subject attrition could produce a biased
16 Basics of Clinical Investigation
sample at the study’s conclusion. An attrition rate
of more than 25 % is cause for concern. In data
analysis, subjects completing the study should be
compared with those who drop out to determine
whether the two groups differ in a clinically sig-
nificant way. Such differences must be considered
in interpreting the study findings.
Data Collection: Measurement
Decisions on what data to collect and how to do so
begin with specifying the variables that need to be
measured and operationally defining each. The
investigator will need to evaluate the suitability
of existing measures and determine whether to use
an existing measure or develop a new one. The
data sources for each variable must also be iden-
tified. Finally, the investigator should specify the
level of measurement of each variable. An effi-
cient way to document the data collection plan is
to make a table with columns listing the variables
to be measured, their operational definitions, the
data source(s) for each variable, and level of mea-
surement for each. This section describes issues
pertaining to each of these tasks.
Identifying the Variables to Be
Measured
Researchers are often tempted to collect as much
information as possible. This can be costly, in
terms of time, money, and data quality. The inves-
tigator should be able to justify each variable to be
measured. Most important are the hypothesized
independent and dependent variables. In addition,
identified potential confounders should be mea-
sured. Finally, data characterizing the study pop-
ulation and sample will be needed to describe the
study’s generalizability.
Types of Variables
Variables can be continuous, such as age, height,
and weight, or categorical. Categorical variables
can be binary, with two possible outcomes such as
487
male or female gender; nominal, i.e., non-ordered
categories such as race: White, African American,
Asian, Native American, or Pacific Islander; or
ordinal, i.e., ordinal categories such as a pain
rating scale: none, mild, moderate, and severe.
Sources of Data
Study data can be collected from existing sources
or can be generated specifically for the specific
research hypothesis being tested, using surveys,
interviews, or observations. Most studies combine
both strategies.
An enormous variety of existing data sources is
available, including medical records, vital
records, national and local health surveys, and
census data. Health programs often keep records
of services provided, and billing records can be
especially helpful. Examples of existing data
sources in pediatric kidney disease include the
US Renal Data System (USRDS) (www.usrds.
org) and data collected by the North American
Pediatric Renal Trials and Collaborative Studies
(NAPRTCS) (web.emmes.com/study/ped/).
Existing sources can provide data for time periods
and individuals otherwise unavailable to the
investigator. The number of studies published by
the NAPRTCS and their tremendous contribution
to our understanding of clinical outcomes in pedi-
atric kidney transplantation and dialysis illustrate
this point. The chief disadvantage of existing
sources gleaned from registry data is that the
data are often not collected as systematically as
in a prospective research study. Important data
elements are sometimes missing. Incomplete
data and inaccuracies are also possible with
registry data.
Primary data collection is expensive and is
limited to subjects available to the investigator.
On the other hand, the investigator’s control over
data collection makes data quality more certain.
Many primary data collection strategies are avail-
able, including mail surveys, mass-administered
questionnaires, telephone and in-person struc-
tured and unstructured interviews, direct observa-
tion, and videotaping and audiotaping. Choosing
a strategy should be based on the research
488
question, the sensitivity of the data to be collected,
the literacy of the population to be studied, and the
resources available for the study. The key factor
should be data quality – that is, which method will
provide the most complete and accurate informa-
tion within budgetary constraints.
Assessing Data Quality
One strategy to enhance data quality is to train
research staff thoroughly. Often, data collection
staff works independently. To ensure that they
follow study procedures, the protocol for data
collection should be detailed in a study manual.
Training sessions should be held to explain the
study’s aim to staff, as well as how each one’s role
fits into the big picture. Staff should be given
ample time to practice their data collection skills.
Once the fieldwork of the main study begins, staff
should be encouraged to bring problems to the
attention of supervisory staff. Such problems
should be resolved in a timely fashion, and the
resolution should be documented and added to the
study manual. In this way, research staff are kept
apprised of changes in the study protocol and are
impressed with the importance of adhering to it.
After the instrument pretesting and staff train-
ing, the investigator should pilot test the data
collection activities. A pilot test is a dress
rehearsal of the activities for selecting study sub-
jects, contacting them, securing informed consent,
and collecting and processing data. Activities that
do not work as planned should be modified and
the pilot testing continued until the fieldwork pro-
cedures run smoothly.
Data quality should be monitored during the
main study. Interviews and questionnaires should
be reviewed as they are completed to allow
recontacting subjects to correct errors. The reliabil-
ity of subjective measures and those requiring spe-
cial technical skill should be assessed. For studies
with unsupervised interviewing of subjects, it is
wise to validate a portion of the completed inter-
views handed in by fieldworkers. This can be done
by recontacting a random sample of subjects and
then asking them to verify their responses to a
subsample of the interview questions.
S.L. Furth and J.J. Fadrowski
Analytic Plan
Data Analysis
Investigators often defer considering data analysis
until the data have been collected. This is a serious
mistake. Study planning should include an ana-
lytic plan of the steps needed to answer the study
questions once the fieldwork is completed.
Use of Statistical Tests
As noted earlier, statistical validity is the correct-
ness of study conclusions regarding group differ-
ences and variable relationships. A key threat to
statistical validity is the use of inappropriate sta-
tistical tests. Variable types and variable distribu-
tion, as well as the hypothesis being tested,
determine the correct type of statistical test. The
important properties of a variable’s distribution
are its location, spread, and shape. Measures of
location include the median (middle observation),
the mean (arithmetic mean or average), and the
mode (most frequent value). Measures of spread
of a distribution include the range, which equals
the maximum minus the minimum value; the
interquartile range, which equals the 75th minus
the 25th percentile; and the variance, which
equals the average squared deviation from the
mean and the standard deviation. The shape of a
distribution can be described by its skewness, i.e.,
symmetry, and its kurtosis, i.e., “peakedness.”
Parametric statistical tests are based on assump-
tions about parameters of the population and are
the most powerful tests available in situations in
which these assumptions are met. Nonparametric
statistical tests are based on fewer assumptions
about the population, so they are appropriate in
situations in which the assumptions underlying
parametric statistics are not met.
Assumptions vary by statistical test. If a test is
used in a situation that violates its assumptions, it
will be inaccurate, leading to a misleading mea-
sure of statistical significance. This, in turn, will
lead to an incorrect estimate of the likelihood of a
type I error.
In developing the analytic plan, the investiga-
tor should consider the assumptions of
candidate tests in determining which ones to use.
16 Basics of Clinical Investigation
A discussion of specific statistical tests is beyond
the scope of this chapter, but a framework for
deciding which tests to use can be given. In this
framework, three factors determine the type of test
to use: the major analytic question to be answered,
the levels of measurement used, and the number
and independence of comparison groups.
In preparing the analytic plan, the investigator
needs to translate the research question into ana-
lytic terms. Three major analytic approaches are
to describe characteristics of the sampled popula-
tion, to compare groups of subjects, and to mea-
sure associations among variables.
Where group comparisons are to be made, the
appropriate statistical test is also determined by
the number of groups to be compared (two
vs. three or more) and by whether comparison
groups are independent or matched. Thus, in a
study of cases matched with sibling controls, it
would be inappropriate to use a statistical test for
independent groups.
As decisions about level of measurement and
the selection of study groups are part of study
planning, it is easy to see how these decisions
are better informed if their ramifications for data
analysts are considered. Level of measurement,
study group formation, and data analysis are all
interrelated, and all should be considered part of
study planning.
When the study’s purpose is to assess the rela-
tionship between two continuous variables, the
degree of concordance can be expressed as a
simple correlation coefficient. A related approach
Table 2 Bivariates Statistical Tests
Two groups
Level of Independent
measurement (unpaired groups) Paired groups
Nominal Chi-square of McNemar’s test
dichotomy Fisher’s exact test
More than Chi-square McNemar’s test
two categories
Ordinal Mann-Whitney Sign test
test
Wilcoxon matched-
pairs signed-ranks test
Interval t Test for groups t Test for pairs
489
is the use of a kappa statistic which expresses the
degree of concordance beyond that due to chance.
Cronbach’s alpha expresses internal consistency
among three or more variables that measure the
same general characteristic.
When the study’s purpose is to describe a pop-
ulation, the investigator makes inferences from
sample statistics to population parameters. Sam-
ple proportions and measures of central tendency
(mean, median, and mode) and dispersion (stan-
dard deviation, range) are used to estimate these
parameters in the population. Confidence inter-
vals can be constructed around proportions and
means to express the certainty of the sample-
based population estimates. When the study’s
objective is to compare two or more groups, sam-
ple group differences in proportions and means
are used to estimate such differences in the popu-
lation. Statistical tests of significance can be used
to assess the certainty of sample-based inferences
about group differences in the population. The
appropriate statistical test depends on the number
of groups compared, whether subjects in the
groups are matched, and the level of measurement
of the variable on which the groups are being
compared. Table 2 displays bivariate statistical
tests commonly used in assessing the significance
of group differences. For a variable with a normal
distribution, a t-test compares two means, while
ANOVA can compare means in three or more
groups. Chi-square compares proportions.
When normality assumptions cannot be made,
the sign test can be used for a single sample or
Three or more groups
Independent (unmatched
groups) Matched groups
Chi-square Cochran’s test
Chi-square Cochran’s test
Kruskal-Wallis one-way Friedman two-
analysis of variance ANOVA way ANOVA
One-way ANOVA ANOVA for
repeated
measures
490
paired sample to assess whether the medians of
the sample and a reference, or two samples being
compared differ. The Wilcoxon signed rank test
can be used, when the data are on an interval scale,
and makes use of the magnitudes of the differ-
ences between measurements and a hypothesized
location parameter. If the variable of interest is
measured on an ordinal scale, the Mann–Whitney
test can be used to assess whether the two
populations have different median values.
When the research aim is to measure the asso-
ciation between variables, sample statistics again
are used to estimate population parameters. The
variables’ levels of measurement determine the
appropriate statistical measure of the strength of
their association, the appropriate test of the statis-
tical significance of the association, and the cer-
tainty of estimates of its strength. For continuous
data, Pearson’s correlation coefficient is used to
measure association. For dichotomous variables,
the odds ratio and relative risk (RR) are measures
of the degree of association between two factors.
Analytic studies in the medical literature often are
designed to determine whether there is an associ-
ation between exposure to a factor and develop-
ment of disease. If there is an association, the
question is how strong the association is. To
assess the strength of the association, we measure
the ratio of the incidence of disease in exposed
individuals to the incidence of disease in
nonexposed individuals. This ratio is called the
relative risk. If the risk in exposed individuals is
equal to the risk in unexposed individuals, the
relative risk is 1.0, and there is no association. If
the risk in exposed individuals is greater than in
unexposed (RR > 1.0), then there is an associa-
tion that may suggest that the exposure confers
risk. If the risk in exposed is less than in
unexposed (RR < 1.0), the exposure may be
protective against risk. The relative risk can only
be calculated in a prospective study, as it requires
incidence of disease. In a case–control study,
since we do not know incidence, we cannot cal-
culate the RR directly. Instead of the proportion of
the exposed population who develop disease com-
pared to the proportion of the unexposed
S.L. Furth and J.J. Fadrowski
population who develop disease, in case–control
studies, we have the proportion of the cases who
were exposed and the proportion of the controls
who were exposed. In case–control studies we
utilize the concept of odds to define the odds
ratio, which approximates the relative risk if the
incidence of disease is low. We compare the odds
of a case having been exposed to a particular
factor to the odds of a control being exposed to
that factor. As in the case of relative risk, if the
exposure is not related to the disease, the odds
ratio will equal 1.0. If the exposure is positively
related to the disease, the odds ratio will be greater
than one.
Studies of the combined and relative impacts
of multiple independent variables, or the effect of
an independent variable after controlling for other
factors, will require multivariable analytic tests.
The appropriateness of a multivariable technique
is determined by the levels of measurement of the
independent and dependent variables. Multiple
linear regression analysis can be used to assess
association between a putative exposure and a
continuous outcome while adjusting for other pos-
sibly confounding factors. Multiple logistic
regression analysis can be used to assess the asso-
ciation between a putative risk factor and a binary
outcome measure while adjusting for other poten-
tial confounding factors. These measures can be
used to calculate an adjusted relative risk, or an
adjusted odds ratio. The references cited at the end
of this chapter describe the applicability and inter-
pretation of the most commonly used multivari-
able statistical tests.
In addition to describing association between
two variables, and assessing the risk associated
with an exposure and an outcome, another com-
mon research question in the medical literature
includes assessing the time from a particular expo-
sure until an outcome such as death, or hospitali-
zation or transplantation. A commonly used
statistical tool to assess the time to an event is
survival analysis, or Kaplan–Meier analysis.
Kaplan–Meier analyses can be used to compare
survival between two treatment modalities. When
adjusting for other potential confounding
16 Basics of Clinical Investigation
variables in a survival or time to event analysis,
Cox proportional hazards methods can be used,
yielding a hazard ratio which can be thought of as
comparable to a relative risk.
Statistical Significance and Confidence
Intervals
Most readers of the medical literature will be
familiar with the term statistical significance,
which is most often referred to in clinical reports
as a “p value <.05.” This highly sought after
result of a statistical test refers to the probability
of α, or a type I error. A p value of .05 in a study
means that there is a 5 % chance that the results
seen in the study could have occurred by chance.
However, the authors have concluded that this
probability is low enough for them to accept the
alternative hypothesis (that there is a real differ-
ence between groups) and to reject the null
hypothesis (there is no difference between
groups). It is important to note that the p value in
a study result depends on the size of the observed
difference between the groups in question and the
size of the sample of patients studied. Standing
alone, the p value does not convey any sense of
the magnitude of the treatment effect seen in the
study or the precision of the estimate of the size of
the treatment effect. Confidence intervals, in con-
trast to p values, can convey this information in a
more meaningful way.
For any estimated value, it is useful to have an
idea of the uncertainty of the estimate in relation
to the true value it is trying to approximate. For
example, if we designed a study to estimate the
beneficial effect of a new lipid-lowering medica-
tion in chronic renal failure in adolescents, we
would try to recruit a large representative sample
of hyperlipidemic adolescents and randomize
them to treatment with a new lipid-lowering med-
ication. From our study, we might want to esti-
mate the magnitude of lipid level reduction
associated with the new medicine. We might also
want to use this estimate as an approximation for
the “true” reduction in lipid levels that would be
491
seen in the “universe” of pediatric patients with
hyperlipidemia and chronic renal failure. To esti-
mate the “true” reduction in lipid levels (which
can never be directly measured), we can generate
a confidence interval around our estimate.
In any study, construction of a confidence
interval around the point estimate gives us a
range of values in which we can be confident
that the true value resides. A confidence interval
gives a sense of the estimates precision; it extends
evenly on either side of the estimate by a multiple
of the standard error (SE) of the estimate. In our
example, our study might yield an estimate of the
drop in serum low-density lipoprotein cholesterol
levels of the treatment group of 30 % with a
standard deviation of that estimate of 20 %. One
could then use this estimate to generate a confi-
dence interval around this estimate. In the medical
literature, one will most often see references to
95 % confidence intervals. The general equation
for a 95 % confidence interval is equal to the
estimate 1.96 times the SE of the estimate. The
factor 1.96 comes from the standard normal dis-
tribution, in which 95 % of estimates would fall
within 1.96 SEs of the mean. If one wanted to
increase the probability of including the true esti-
mate in the confidence interval, one could gener-
ate a 99 % confidence interval, which would equal
the estimate 2.56 times the SE of the estimate.
Because the SE of an estimate is equal to the
standard deviation of the population divided by
the square root of the sample size, N, one can see
that a larger sample size is needed in a study to
generate a precise estimate of a treatment’s effect.
Given the same standard deviation, the SE in our
study would be smaller if we studied 100 children
compared to 20 children. The larger study would
generate a narrower 95 % confidence interval. The
strict interpretation of a 95 % confidence interval
is that this is the range of values for the true
population estimate that is consistent with the
data observed in the study. In our hypothetical
example, the smaller study might give us the
opportunity to conclude that the new lipid lower-
ing is associated with a 30 % reduction in
low-density lipoprotein cholesterol with a
492 S.L. Furth and J.J. Fadrowski

relatively broad 95 % confidence interval of
21–39 %, whereas the larger study yields a more
precise estimate. The 95 % confidence interval
around the same point estimate of a 30 % reduc-
tion is 26–34 % in the study with the larger
sample size.
million population. This compared to 217 in 1991.
The prevalence of ESRD in 2011 (also referred to
as point prevalence) was 1,924 cases per million
population. Based on these definitions, we know
that 362 (19 %) of the 1,924 prevalent cases of
ESRD per million population in 2005 were new
cases.

Topics Related to Clinical Decision

Making The Diagnostic Test

Clinicians are routinely faced with patients with When utilizing a diagnostic or screening test to aid
unknown diagnoses, and patients expect that the in the care of a patient, a clinician must know
clinician will know how to efficiently and accu- “how good” or valid is the test. Terms used to
rately diagnose the problem with which they are describe “how good” a test is include sensitivity
presenting. Diagnostic acumen relies in large part and specificity. The sensitivity of a test describes
in understanding the “epidemiology” of the pos- the ability of the test to correctly identify those
sible diagnoses, as well as the strengths and lim- that have the disease. A sensitive test has a low
itations of the various tests that might be false-negative rate, meaning the test result will
performed to diagnose such diseases. The follow- not frequently be negative in those who have the
ing section provides an introduction to concepts disease. The specificity of a test describes the
and terms that are related to the science of clinical ability of the test to correctly identify those who
decision making. do not have the disease. A specific test has a low
false-positive rate, meaning the test result will not
frequently be positive in those who do not have
The Disease the disease.
Table 3 from the American Academy of Pedi-
When describing the burden of a disease in a atrics’ Practice Parameter for the diagnosis of
population, the terms incidence and prevalence initial urinary tract infections in children exam-
are often used. Incidence is defined as the number ines the sensitivity and specificity of various com-
of new cases of a disease that occur in a popula- ponents of the urinalysis [10]. For example, the
tion at risk for developing the disease during a sensitivity of nitrite is reported as 53 %. Thus,
specified period of time. Prevalence is defined as among children with a urinary tract infection,
the proportion of persons present in the population
currently affected by the disease at a specified
point in time. Distinguishing between these
Table 3 Sensitivity and specificity of urinalysis compo-
terms is important when considering the burden nents (13)
of given disease. When prevalence is reported,
Sensitivity % Specificity
people affected by the disease for varying Test (range) % (range)
amounts of time are included; these are not nec- Microscopy: WBCs 73 (32–100) 81 (45–98)
essarily all new cases. Those with severe forms of Microscopy: bacteria 81 (16–99) 83 (11–100)
the disease may have died and thus, depending on Leukocyte esterase 83 (67–94) 78 (64–92)
the time specified in the definition, may not be Nitrite 53 (15–82) 98 (90–100)
included in the prevalence. The incidence and Leukocyte esterase or 93 (90–100) 72 (58–91)
prevalence of end-stage renal disease (ESRD) nitrite positive
are routinely reported by the US Renal Data Sys- Leukocyte esterase or 99.9 (99–100) 70 (60–92)
tem (USRDS) ([9] Annual Data Report). In 2011, nitrite or microscopy
positive
the incidence was 362 (new) cases of ESRD per
16 Basics of Clinical Investigation
this test will be positive approximately 53 % of the
time. Knowing the relatively low sensitivity of the
nitrite test, a clinician would not be reassured that
a urinary tract infection does not exist if the result
is negative. The specificity of the nitrite test is
98 %. Thus, among children without a urinary
tract infection, this test is negative approximately
98 % of the time. The high specificity of the nitrite
test informs the clinician that a low false-positive
rate exists. As can be observed in the figure, and as
often happens in clinical practice, using a combi-
nation of diagnostic tests can significantly
increase the overall sensitivity and specificity,
and thus the accuracy of the diagnosis.
Often in clinical medicine, clinicians are faced
with a positive test result, and the next question
asked is, “among patients with a positive test
result, what proportion will actually have the dis-
ease?” This is known as the positive predictive
value of the test. The negative predictive value
of a test relays the probability that if the test is
negative, the patient does not have the disease. It
is important to remember that the predictive value
of a test is affected by both the prevalence of the
disease in the population being considered and, if
the disease in uncommon, the specificity of the
test being used. Higher disease prevalence gener-
ally leads to an increase in the positive predictive
value of a test. However, as most diseases are rare,
tests with higher specificity likely have a greater
impact on increasing the positive predictive value
for a given test. These values can be easily calcu-
lated as demonstrated in Fig. 7.
Fig. 7 Positive and
negative predictive values
493
Likelihood ratios (LR) are another tool to help
the clinician utilize the results of a given test
diagnostically. When presented with a patient
with particular signs and symptoms, the clinician
has an initial assessment of the likelihood (pretest
probability) of a particular disease. A diagnostic
test ordered to help determine the presence of a
disease may be positive or negative. LRs help
inform the clinician as to how much the test result
should shift his/her initial assessment of the prob-
ability of the disease being present or absent (post-
test probability). Strong, conclusive tests yield
very big or very small LRs. Weak, inconclusive
tests yield modest LRs, close to 1.0. For example,
following the results of a positive test result, if the
positive LR is found to equal 1, then there is no
change in the likelihood of the disease. If the LR
was found to be 10 after the positive test result,
then the posttest odds of the disease is equal to the
likelihood ratio multiplied by the pretest odds.
The odds of the disease can be calculated from
the probability as probability/1-probability. The
higher the LR, the better the test is for ruling in a
diagnosis. These ratios depend upon the validity
of the test being ordered in distinguishing who has
the disease in question from who does not; hence,
these ratios may be derived from sensitivity and
specificity of the test (see below). LRs may be
more useful than sensitivity and specificity in
certain situations. LRs can be calculated for tests
without dichotomous results such as those that are
resulted as “positive, intermediate, or negative.”
The results of several diagnostic tests may be
494
combined to provide a single LR. Finally, with
some relatively simple calculations, the posttest
probability of a disease can be calculated using the
LR and the pretest odds of the disease. Formulas
for calculating LRs follow.
The Positive LR is the probability of an individual
with the disease having a positive test/proba-
bility of an individual without the disease hav-
ing a positive test.
The Negative LR is the Probability of an individ-
ual with the disease having a negative test/the
probability of an individual without the disease
having a negative test
For dichotomous tests:
Positive LR: sensitivity/(1  specificity)
Negative LR: (1  sensitivity)/specificity
Receiver operating characteristic (ROC)
curves, originally developed in the field of elec-
tronics, allow for a graphical display of the trade-
off between sensitivity and specificity for diag-
nostic tests with ordinal or continuous results, in
which several values of sensitivity and specificity
are possible. Several cutoff points are determined,
and the sensitivity and specificity are determined
at each point. The sensitivity (or true-positive
rate) is graphed on the Y-axis as a function of
1-specificity (the false-positive rate) on the
X-axis. Tests with values falling in the upper left
corner of the graph are considered ideal (100 %
true positives and no false positives). If a test
followed the diagonal line from the lower left
corner to the upper right corner, it would be con-
sidered useless – on this diagonal line the true-
positive rate equals the false-positive rate. The
area under the ROC curve can range from 0.5 for
a worthless test to 1.0 for a perfect test.
Filler et al. made use of both an ROC curve and
LRs in a study examining non-minimal change
nephrotic syndrome in children referred to a ter-
tiary care medical center in Canada, as can be seen
in Fig. 8 included from this study [11]. The
authors noted an increase in the incidence in
focal segmental glomerulosclerosis (FSGS) over
a 17-year study period. Based on the International
S.L. Furth and J.J. Fadrowski
Fig. 8 ROC curve for the detection of non-MCNS in
relation to remission time (14). The graph plots the true-
positive rate expressed as sensitivity (%) as a function of
the falsepositive rate (100-specificity [%]) at different cut-
off points. Area under the ROC CURVE = 0.859; SE =
0.057; 95% confidence interval, 0.793 to 0.911. Data in the
table present selected cutoff points derived from the graph
as mean and 95% CI
Study of Kidney Disease in Children (ISKDC),
the approach to a child with new-onset nephrotic
syndrome has been to perform a kidney biopsy if
the disease was unresponsive to a standard dose of
corticosteroid therapy of at least 28 days in dura-
tion [12]. Given the increased incidence of FSGS
observed in their study, Filler et al. considered that
kidney biopsies to distinguish FSGS from mini-
mal change disease may need to be performed
sooner after presentation with the nephrotic syn-
drome, and based on their data, investigated the
ideal time to perform a kidney biopsy for the
detection of non-minimal change nephrotic syn-
drome (i.e., FSGS). The clinical feature
(as opposed to a “diagnostic” test) represented in
the ROC curve is “time to remission after starting
corticosteroid therapy.” By plotting and compar-
ing the various sensitivities and specificities of
values for “time to remission,” the authors con-
cluded that the cutoff of 28 days was statistically
the best point when a biopsy should be consid-
ered. At this cutoff point, the detection of “true
positives” (non-minimal change nephrotic syn-
drome) was maximized, and the detection of
16 Basics of Clinical Investigation
“false positives” (minimal change nephrotic syn-
drome) was minimized. The LRs for “time to
remission” are also listed. The longer a patient
took to enter remission, the higher the positive
LR for the diagnosis of a non-minimal change
pathology underlying the nephrotic syndrome.
Screening Programs
Screening programs are designed to detect or diag-
nose a disease as early as possible, in hopes of
improving the prognosis. A common screening
program is the use of mammography for the earlier
detection of breast cancer. Detecting breast cancer
early, before it is “symptomatic” or advanced, has
been proven to improve outcomes and survival in
older women. A sampling of the many factors that
must be considered when evaluating the feasibility
and effectiveness of a screening program follows.
Disease
Is there a preclinical phase of the disease – a time
when the disease is present but clinical symptoms
have not yet manifested? Does intervening earlier
in the natural history of the disease make treat-
ment easier and/or improve morbidity or mortal-
ity? Is the disease prevalence high enough to make
a screening program cost-effective?
Test
Does a screening test exist with acceptable sensi-
tivity and specificity for a screening program – are
the false-positive and false-negative rates accept-
able? Is the test acceptable to the population – will
they consent to the test? Do the benefits gained
from early diagnosis of the disease outweigh the
cost of the test?
Person/Population
Does screening/early diagnosis improve out-
comes for an individual, and the population?
495
Do those with earlier diagnosis of the disease
comply with treatment recommendations and reg-
imens – how many of those who screen positive
receive a final diagnosis and treatment? Is there an
improvement in the quality of life in those
screened?
Ethics in Research
The foundation of medical research is to help
improve the lives of patients. In keeping with
this, it is important to be familiar with the various
terms and concepts related to the responsible and
ethical conduct of medical research.
Conflicts of interest may occur between a
researcher’s interest in advancing medical knowl-
edge and his/her self-interest in fame, prestige,
academic, or financial advancement. Transpar-
ency of contractual obligations and relationships
is mandatory, and in some circumstances, these
must be dissolved, or the researcher should not
participate in a related project. For example, a
conflict of interest would exist if a physician was
paid by a pharmaceutical company as a medical
consultant, and this physician also served as a
primary investigator in a clinical trial of a medi-
cation produced by the same drug company. Con-
flicts of interest may also occur in the
clinician–investigator role when a researcher is
also the clinician for the research subject. In
such situations, what may be best for the research
project may not be best for the individual patient.
In such conflicts, the physician is expected to do
what is best for the patient.
Several types of scientific misconduct have
been described. Scientists have a responsibility
to report misconduct, and institutions have the
responsibility to investigate the misconduct and
protect the person alleging the misconduct. Fab-
rication/forgery is the invention and reporting of
data that does not exist from an experiment that
was not performed. Falsification/fraud is the
manipulation of research data such that what is
reported misrepresents the actual findings. Pla-
giarism is the presentation of another person’s
words or ideas as one’s own or without giving
appropriate credit to the original author(s).
496
Research on human subjects requires special
ethical considerations and protections. Respect
for research participants requires investigators to
obtain informed consent or assent, maintain pri-
vacy and confidentiality, and protect the vulnera-
ble. Informed consent involves relaying an
unambiguous description of the research project
and allowing the subject to make an informed
decision regarding participation. It must be clearly
stated that the patient will be involved in a
research project and participation is entirely vol-
untary. A clear description of the potential risks
and benefits, and any compensation, should be
provided. For pediatric patients participating in
research, assent is also required. Children cannot
legally give permission to participate in a research
study, nor can they give “consent” as consent
implies full understanding. However, ethicists
and medical and legal professionals have agreed
that children should be routinely asked if they
agree (assent) to participate in a research project,
and their wishes should be respected. Other vul-
nerable populations that require special attention
to ensure their safety include prisoners, pregnant
women and their fetuses and embryos, and people
with impaired capacity to make decisions, such as
the mentally ill. During the consent process, con-
fidentiality and privacy procedures utilized by the
study should be outlined. The extent of confiden-
tiality should be disclosed – the subject should
understand who will and who will not have access
to the data.
Evaluating the Literature: Rating the
Strength of Scientific Evidence
Health-care decisions should be based on
research-based evidence. Whether the individual
nephrologist is making a clinical decision or a
national organization is developing clinical prac-
tice guidelines, efforts should be made to system-
atically assess the strengths of scientific evidence
related to a particular clinical diagnostic or treat-
ment plan. Guidelines first developed more than
20 years ago at the Department of Clinical Epide-
miology and Biostatistics at McMaster University
first introduced tools to allow clinicians to
S.L. Furth and J.J. Fadrowski
critically review original articles on etiology,
diagnosis, prognosis, and therapy [13]. In the
following decade, the series was widely read and
cited, was modified for use by the general public,
and was published in clinical epidemiology texts
[14]. At the same time, clinicians at McMaster
University and across North America continued
to expand and improve the guidelines. Their focus
has expanded to include clinicians’ ability to
access, summarize, and apply information from
the literature to everyday clinical problems,
transforming the Readers’ Guides to Users’
Guides [15–38].
Such systematic approaches have also been
adapted to assess entire bodies of research on
particular subjects. In 1999, the US Congress
directed the Agency for Health Care Research
and Quality to identify methods to assess health-
care research results. The results of that effort
were published in a report entitled “Systems to
rate the strength of scientific evidence.” The goals
of this project were to describe systems to rate the
strength of scientific evidence, including evaluat-
ing the quality of individual articles that make up a
body of medical evidence related to a particular
disease, allowing for the most informed medical
assessments and decision making. The report pro-
vides the framework for the clinician regarding
the evaluation of various types of study design as
described in this chapter [39].
In summary, the busy clinician can afford to be
selective in reviewing the literature. In rating the
strength of scientific evidence in evaluating a spe-
cific clinical problem or treatment, one needs to
be selective. The simplest criteria for choosing
which studies to read in detail or which to weigh
heavily in evidence are clinical relevance and
methodologic soundness. This chapter has intro-
duced a simple framework for evaluating such
features in the context of sound clinical research
methodology and has outlined the most recent
guidelines for assessing the strength of scientific
evidence for making decisions in clinical care.
These tools for systematic assessment of existing
research can also guide the clinical investigator
toward areas that require further study in which
current evidence for treatment or outcomes is
scant.
16 Basics of Clinical Investigation
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 Genomic Methods in the Diagnosis
and Treatment of Pediatric Kidney 17
Disease

Karen Maresso and Ulrich Broeckel

Contents The completion of the Human Genome Project

The Genomics Revolution . . . . . . . . . . . . . . . . . . . . . . . . . . 500 (HGP) in 2003 has laid the foundation and driven
the technological advancements necessary for
The Application of Genomic Methods
to Pediatric Kidney Diseases . . . . . . . . . . . . . . . . . . . . . . . 504
the study of the genetics of complex, multifacto-
Acute Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504 rial diseases, such as those affecting the kidney.
Hemolytic Uremic Syndrome (HUS) . . . . . . . . . . . . . . . . 506 The International HapMap Project has built upon
Chronic Kidney Diseases and End-Stage Renal the HGP through the systematic identification
Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
The Impact of Novel Sequencing Technologies
and cataloging of genetic variation across
on Gene Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512 human populations. Translating the mass of
Next-Generation Sequencing . . . . . . . . . . . . . . . . . . . . . . . . 512 data generated by these studies into useful clini-
Data-Analytic Techniques in NGS . . . . . . . . . . . . . . . . . . 513 cal knowledge is now a major undertaking in
Data Analysis in NGS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514
NGS in Pediatric Kidney Disease . . . . . . . . . . . . . . . . . . . 514
nearly all areas of medicine, including the field
The Past, Present, and Future Application of pediatric nephrology. Much of this work will
of Genomic Methods in Pediatric Nephrology . . . . . . 516 revolve around linking particular patient pheno-
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517 types to genomic and proteomic data, such as
genotype, expression profile, and protein bio-
markers. As evidenced by the etiological
advances made in various kidney disorders
resulting from the application of genome-wide
linkage analyses in the 1990s, there are a number
of unique aspects of pediatric nephrology that
make it an area particularly suitable to genomic
exploration with such novel technologies such as
genome-wide association and next-generation
sequencing. These aspects relate both to the clin-
ical characteristics and to public health and epi-
K. Maresso demiological concerns of pediatric kidney
Section of Genomic Pediatrics, Medical College of diseases.
Wisconsin, Milwaukee, WI, USA
A number of pediatric kidney diseases are
U. Broeckel (*) considered to have a multifactorial pathogenesis,
Department of Pediatrics, Medical College of Wisconsin
often reflected in their variable clinical presenta-
and Children’s Hospital of Wisconsin, Milwaukee,
WI, USA tions. Such diseases are often difficult to diag-
e-mail: ubroeckel@mcw.edu nosis; and invasive biopsies are often required.
# Springer-Verlag Berlin Heidelberg 2016 499
E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_77
500
Genotype sequence and/or expression informa-
tion obtained from DNA analysis and profiling
studies may refine the diagnosis of a number of
kidney diseases and even allow for individual-
ized disease management and therapy. Addition-
ally, there remains an urgent need for clinically
useful biomarkers, which can assist in the
prevention and early diagnosis of both acute
and chronic kidney diseases in children. Novel
serum and/or urine biomarkers identified
through the application of high-throughput pro-
teomics offer another possibility for enhanced
diagnosis and treatment. Moreover, from a pub-
lic health standpoint, proper management of
renal disease is expensive, particularly renal
replacement and dialysis. The treatment of kid-
ney patients based on genomic and proteomic
data may help lessen this burden by enhancing
disease risk prediction and creating disease sub-
sets with associated prognostic and therapeutic
implications. While these issues are relevant to
all of nephrology in general, as well as to many
other areas of medicine, they are particularly
germane to the study of childhood kidney dis-
eases where there can be high mortality, the need
for expensive lifetime treatment, as well as seri-
ous growth and nutrition implications. The
proper application of genomics in pediatric
nephrology is well suited to address the unique
challenges posed by this field.
This chapter aims to provide an overview of
the application of genomic technologies to the
study of pediatric kidney diseases. We will begin
with a discussion of the polygenic nature of com-
plex diseases and the recent genomic advances
enabling their study. This will include outcomes
of the HGP and International HapMap Project. We
will then discuss the application of various geno-
mic methods to the more common pediatric kid-
ney diseases, covering the range of both chronic
and acute, with a focus on the DNA level. With
the recent developments in novel sequencing
technologies, the field is poised to capitalize on
these recent advances as demonstrated by most
recent publications. We will end with a discussion
of the future prospects and recommendations for
applying genomics to pediatric nephrology.
K. Maresso and U. Broeckel
The Genomics Revolution
In 2003, the completion of the Human Genome
Project brought the scientific community one step
closer to identifying the genes underlying com-
mon, polygenic diseases. Prior to this achieve-
ment, the goal of identifying the genetic factors
responsible for diseases presenting substantial
public health burdens was elusive. Research
efforts focused on monogenic disorders following
Mendelian patterns of inheritance. Linkage anal-
ysis was the main tool for the identification of
genes behind these monogenic diseases. Although
linkage analysis proved to be an effective method
for the mapping of numerous genes in monogenic
disorders during the 1980s and 1990s, including
for familiar forms of various kidney diseases
[1–5], it’s subsequent application to complex dis-
eases did not meet with the same success. As
linkage analysis is more suitable for the identifi-
cation of genes with relatively large effect sizes,
its application to polygenic diseases, where the
contribution of each locus or allele is believed to
be relatively small, resulted in less than significant
findings and results that could not be replicated.
Only a handful of genes contributing to common
diseases have been identified to date through link-
age and subsequent positional cloning methods
[6–9].
In 1996, Risch and Merikangas demonstrated
the power of genome-wide association analysis
(GWA) over linkage methods for the identification
of the genes behind complex diseases [10]. Associ-
ation analysis relied upon the availability of a stan-
dardized dense set of markers covering the genome
that could be easily and inexpensively typed, as
well as sample sizes of thousands of cases and
controls. Until the technology existed to identify,
accurately map, and then genotype such large num-
bers of markers in such great numbers of individ-
uals, linkage analysis followed by fine mapping
remained the best option at that time for uncovering
complex disease genes. However, the work of the
HGP began to drive the technological break-
throughs needed to map polygenic diseases.
Analysis of the human genome sequence has
demonstrated the abundance and uniformity of
17 Genomic Methods in the Diagnosis and Treatment of Pediatric Kidney Disease
single nucleotide polymorphisms (SNPs), single
base changes in DNA that occur in at least 1 % of
the population. (This is in contrast to a mutation,
which occurs in <1 % of the population.) It is
some of these common DNA variants that are
believed to be responsible for at least some of
the phenotypic variation observed within and
between populations, including various diseases.
The HGP contributed to developing technologies
for the rapid, large-scale identification and scoring
of SNPs and creating the intellectual resources
needed to study sequence variation. The Interna-
tional HapMap Project built upon these discovery
efforts by characterizing the patterns of linkage
disequilibrium (LD) and haplotype structure
across the human genome. LD occurs when two
or more markers segregate together with signifi-
cantly different frequencies than would be
expected if they segregated independently from
each other. LD is generally greater for SNPs in
close physical proximity. As LD tends to reduce
the number of possible haplotypes, or set of
alleles, present in a population, it is useful for
association mapping, in that knowing the geno-
type of one marker can predict the genotype of
another marker in LD with it (Fig. 1). The work of
the HapMap Project, along with others, has
resulted in the identification, mapping, character-
ization, and the public availability of over four
million validated SNPs to date (www.hapmap.
org). The identification, mapping, and cataloging
of these SNPs in various human populations have
helped make association studies a reality. In con-
junction with the advent of mass-throughput
genotyping platforms, such data has potential to
contribute extensively to our basic understanding
of human biology and disease.
The two types of association studies that are
commonly used are the candidate-gene approach
and the genome-wide approach (Table 1, Fig. 2).
Candidate-gene association studies rely upon a
specific hypothesis and are often limited by our
current knowledge of biological and/or disease
mechanisms. The strategy has been a frequently
employed method in the study of numerous traits
and diseases, including many related to nephrol-
ogy. Nevertheless, replicable results have been
501
Possible Allelic Combinations of Two SNPs in the Absence of LD
SNP1(T/C) SNP2(G/A)
AC T GGTACGTACCC A ATGTTGCATACGTT
AC T GGTACGTACCC G ATGTTGCATACGTT
AC C GGTACGTACCC A ATGTTGCATACGTT
AC C GGTACGTACCC G ATGTTGCATACGTT
Possible Allelic Combinations of Two SNPs in Presence of LD
SNP1(T/C) SNP2(G/A)
AC T GGTACGTACCC A ATGTTGCATACGTT
AC C GGTACGTACCC G ATGTTGCATACGTT
Fig. 1 The principle of linkage disequilibrium. A set of
DNA sequences illustrating two SNPs (indicated in red and
blue). In the top half, the red and blue SNP are not in LD
with each other and segregate independently. With two
SNPs in linkage equilibrium, four possible allelic combi-
nations are possible. In the bottom half, two SNPs in the
presence of LD are illustrated. Notice that LD reduces the
number of possible allelic combinations, as the SNPs no
longer segregate independently. Here, the “T” allele of
SNP1 segregates only with the “A” allele of SNP2, while
the “C” allele of SNP1 segregates only with the “G” allele
of SNP2. In such a case, only one of these two SNPs would
need to be genotyped, as the genotype at one SNP will
provide the genotype information for the other
difficult to achieve for various reasons, including
the small, underpowered sample sizes character-
istic of many of these types of studies [11].
In the second type of association study, the
genome-wide strategy is hypothesis-free and
does not make any assumptions regarding the
mechanisms behind the phenotype being studied.
Genome-wide association studies are ideally
suited for common complex diseases, where the
physiological mechanisms and genetic factors that
contribute to them are often not well understood.
GWA studies are based on hundreds of thousands
to millions of SNPs spread across the genome,
each one tested for association with a particular
outcome. There are currently two main
approaches to this type of study. One type
employs an LD-based selection strategy, as exem-
plified by the Illumina HumanHap550 and
Human1M sets of variants. The LD-based strat-
egy exploits patterns of genomic LD to select a
minimum number of SNPs (tagSNPs) to be
assayed, which captures the maximum amount
of information across the genome. SNPs on
502 K. Maresso and U. Broeckel

Table 1 Comparison of genome-wide (GWA) and candidate-gene association studies

Advantages Disadvantages
GWA Hypothesis-free Genotyping more technically
May be more economical and efficient when large numbers demanding
of loci need to be worked up May be costly and time-consuming
Ideal in cases where little is known regarding disease Requires sophisticated bioinformatic
mechanisms approaches to data analysis and storage
Often allow for collection of data relating to copy number
polymorphisms (CNPs) in addition to SNPs – two genetic
variants for the price of one
Candidate Less costly and time-consuming if large numbers of loci do Candidate-gene selection constrained
gene not need to be analyzed by known disease biology
Ideal in situations where evidence implicates particular Less economical and efficient when
disease mechanisms and related genes or when following-up testing great numbers of loci
a QTL containing likely functional candidates
Straightforward data-handling and analytical approaches
Various methods of genotyping available; less technically
demanding

Select Study Design: Extract DNA from

Collect Clinical Data:
1) GWA v. Candidate-gene all study participants
1) Determine phenotypes of interest
2) Case-control v.Families
2) Standardized phenotyping
3) Collect data on confounders
Data Analysis:
1) Perform Association Analysis Determine method of Genotyping:
2) Control for Multiple Testing 1) GWA:
3) Determine best p-values a) TagSNPs (Illumina)
4) Replication / Finemapping b) Random selection of SNPs
5) Functional Analysis (Affymetrix)

6) Combine with Transcriptomic/Proteomic Data 2) Candidate-gene: numerous methods

Fig. 2 Workflow of a genetic association study

these arrays serve as proxies for those not assayed.
The second approach ignores patterns of LD and
relies on the random selection of markers. This
approach is the basis for the Affymetrix 500 K and
1 million SNP array products. A number of GWA
reports have recently been published identifying
novel genes for various diseases in adult
populations, including many outcomes relevant
to kidney disease, such as obesity and type II
diabetes [12–14], as well as quantitative traits
like urinary albumin excretion [15].
Within the field of pediatric nephrology, many
of the known disease genes were initially identi-
fied through the study of familiar forms of a
17 Genomic Methods in the Diagnosis and Treatment of Pediatric Kidney Disease
disease using linkage analysis followed by posi-
tional cloning methods [16–19]. These genes have
become obvious candidates to examine in the
context of seemingly sporadic occurrence, where
a patient has no prior family history of the disease
and which constitutes the majority of many
childhood-onset kidney diseases. This is often
done through mutation scanning in case series.
While linkage analysis proved to be a useful tool
for identifying the first disease genes in pediatric
kidney disease, allowing for some of the first
insights into the molecular pathogenesis of many
of these diseases, these genes generally only
account for a minority of cases. New methods
which result in the identification of novel genes
can assist in making further advances. While
GWA studies are still relatively new, candidate-
gene studies have been an actively pursued
approach in nephrology to novel gene identifica-
tion. The popularity of this approach is illustrated
by a review of the 2004 and 2005 abstracts of the
American Society of Nephrology (ASN) and the
European Renal Association/European Dialysis
and Transplantation Association (ERA/EDTA),
where over 180 studies encompassing 205 poly-
morphisms in 92 different genes were submitted
[20]. The genes most frequently studied were
those of the renin–angiotensin system (RAS) and
those related to inflammation [20]. The best evi-
dence to date indicates minor statistical associa-
tions and a negligible clinical relevance of the
most frequently studied RAS polymorphism, the
insertion/deletion (I/D) variant of the angiotensin
I-converting enzyme (ACE) gene [21–23].
Despite their frequent study, a genetic association
has yet to be convincingly demonstrated for any
of the RAS genes. (These genes will not be further
discussed in the chapter.) Of particular note, the
authors highlight the extremely small sample sizes
of the vast majority of these studies. In 2004, the
average study size was 185 (cases and controls
combined), with a slight increase in 2005 to 276.
In addition, the authors found that while only
16 % of these studies met the newly adopted
criteria for publishing candidate-gene association
studies in the Journal of the American Society of
Nephrology (JASN) in 2004, this number jumped
to nearly half in 2005 [20]. These data highlight
503
two important points. First, it is unlikely that the
often thousands of subjects needed for adequately
powered studies can be collected at a single site;
and consequently, there is a need for multisite
collaborative studies. This reflects the fact that
large-scale gene association studies of novel
genes in adult or pediatric populations are criti-
cally required. Second, by adopting strict publish-
ing guidelines, journals can help assure the proper
design and analysis of association studies. To
address the first point, European investigators
interested in nephrology and genetics established
the Renal Genome Network (ReGeNet; www.
regenet.eu) in 2003 to encourage large, collabora-
tive clinical studies focused on the genetic epide-
miology of renal disease [24].
When many genes are to be tested, or when one
desires a hypothesis-free approach, it may be
more efficient and economical to perform a
GWA study. While these studies may assist in
the identification of novel genes in acute kidney
disorders, where currently half or less of all cases
are explained by known mutations, GWA
approaches in pediatric nephrology are likely to
be most beneficial when applied to chronic kidney
disease (CKD) and end-stage renal disease
(ESRD) resulting from the increasing rate of
hypertension, diabetes, and obesity in children.
The mechanisms leading to CKD and ESRD in
these settings are likely to be polygenic in nature
and the current lack of understanding regarding all
disease mechanisms leading to these outcomes
precludes a selection of appropriate candidate
genes. In addition, the mechanisms of these dis-
orders and their precipitating causes may be dif-
ferent from those occurring in adults. While some
of the GWA findings from adult populations may
be relevant to pediatric populations as well, more
work will be required to determine this. A more
in-depth discussion on the application of
candidate-gene association and GWA approaches
to the more common pediatric kidney diseases
will be presented in the following section.
While much of this discussion has focused
exclusively on the role of DNA sequence varia-
tion in disease outcomes, there are also extensive
research efforts dedicated to the role of mRNA
and proteins in the prevention, diagnosis, and
504
treatment of kidney disease. Expression profiling,
as with high-throughput SNP genotyping, has
benefited substantially from recent advances in
microarray technology, which are allowing for
the measurement of genome-wide expression
levels, also called “transcriptomics,” and their
correlation with various disease states. As with
cancer, the identification of certain common
expression “signatures” through the simultaneous
profiling of thousands of genes from kidney
biopsy samples may help determine treatment
course and patient prognosis in various renal dis-
eases. The genome-wide study of proteins, or
proteomics, has also benefited from recent tech-
nological breakthroughs in mass spectrometry,
allowing for a greater number of proteins to be
simultaneously analyzed. There is currently an
urgent need in pediatric nephrology for clinically
relevant biomarkers which can be obtained non-
invasively and which will eventually allow for
earlier disease detection [25, 26]. Because these
two fields are closely aligned with the study of
DNA sequence variation, in that changes affecting
gene sequence can ultimately affect gene expres-
sion and protein production, we focus in the fol-
lowing section on the application of the study of
DNA sequence variation to the various forms of
pediatric renal disease. The application of all three
fields to the study of pediatric nephrology can
ultimately lead to enhanced prevention efforts,
diagnoses, and treatment options through the
identification of novel genes and genetic markers,
gene expression signatures, and biomarkers asso-
ciated with various renal disease states and out-
comes in pediatric populations.
The Application of Genomic Methods
to Pediatric Kidney Diseases
Acute Diseases
Table 2 provides a summary of the genes and loci
implicated in the following conditions, as well as
information on known genotype–phenotype
correlations.
Nephrotic syndrome (NS) NS as a heteroge-
neous collection of disorders represents an excellent
K. Maresso and U. Broeckel
example of where the application of genetic and
genomic methods can assist in a greater understand-
ing of disease pathogenesis and treatment. See also
▶ Chap. 27, “Idiopathic Nephrotic Syndrome in
Children: Genetic Aspects” in this text.
In children, minimal change nephropathy
(MCN) is the overwhelming cause of NS, such
that renal biopsies are often not performed before
beginning treatment with potentially harmful
immunosuppressive therapies [27]. Corticoste-
roids are often prescribed first. The majority of
children respond well to this treatment and they
are said to have “steroid-sensitive nephrotic syn-
drome” (SSNS). However, there is a subset of
children who do not respond to this therapy and
they are said to have “steroid-resistant nephrotic
syndrome” (SRNS). In such cases, a renal biopsy
may be indicated and the underlying cause may be
focal segmental glomerularsclerosis (FSGS).
Current treatment strategies for MCN and FSGS
are similar; however, they are generally less
effective for FSGS; and children with the latter
may go on to develop ESRD. Despite the exis-
tence of these therapeutic strategies, the underly-
ing pathological mechanisms of these two
histological classifications are poorly understood.
It is a subject of debate in the literature as to
whether or not these classifications represent dis-
tinct diseases or subtypes of a single condition
[27]. In addition to a need for a greater under-
standing of the disease processes involved in NS,
less toxic therapies are also needed for treatment
in children. In the last decade, much progress has
been made in the area of NS genetics and
more can be expected in the coming years with
novel genomic technologies. These advances
should help to clarify NS-related pathologies and
offer a pharmacogenetic approach to the treatment
of NS.
In the late 1990s, genome-wide linkage analy-
sis followed by positional cloning was a particu-
larly successful method for the identification of
the genes behind the familial form of NS,
although sporadic cases, those without any previ-
ous family history, account for the majority of
pediatric NS cases. In 1998, Kestila
et al. identified the first NS-related gene, NPHS1
(nephrin), to be the cause of an autosomal
17 Genomic Methods in the Diagnosis and Treatment of Pediatric Kidney Disease 505

Table 2 Genes and genotype–phenotype correlations in selected pediatric renal diseases

Genotype–phenotype
Disease Gene(s)/loci correlations Comments
Nephrotic NPHS1, NPHS2, LAMB2, NPHS1, NPHS2, LAMB2, Additional susceptibility genes
syndrome WT1, PLCE1 WT1 commonly mutated in and modifier genes likely.
(steroid- congenital onset and resistant GWA studies may assist with
resistant) to steroid treatment; type and their discovery
number of NPHS2 mutations
correlate with age of onset
Atypical CFH, CFI, CFB, MCP CFH-severe clinical course, Known complement genes
hemolytic (complement regulatory genes) rapid progression to ESRD, account for ~50 % of cases.
uremic earlier age at onset, disease Additional susceptibility genes
syndrome recurrence after transplant; likely. Modifier genes likely
MCP, most favorable course, due to high incomplete
no recurrence post transplant; penetrance. SNPs may also
CFI, intermediate course play a role along with known
mutations
Autosomal PKD1, PKD2 PKD1 – more severe course PKD1 accounts for ~85 % of
dominant and earlier onset; 50 mutations families. Most mutations in
polycystic may progress to ESRD earlier these genes are unique to single
kidney and be more susceptible to family. Genetic background
disease ICAs may account for variability
within a family. Medical
resequencing can assist in
increasing the numbers of
particular mutational profiles.
GWA studies may help to
uncover modifier genes
Autosomal PKHD1 Presence of two truncating ~33 % of mutations are unique
recessive mutations results in neonatal to a single family. Medical
polycystic death, regardless of position resequencing can assist in
kidney Missense mutations associated increasing the numbers of
disease with milder phenotype particular mutational profiles
IgA 6q22-23; 4q26-31; 17q12-22; Gene verification and No gene(s) yet identified in
nephropathy 2q36; megsin gene, discovery and families. Many results from
uteroglobin, C1GALT1, E- and genotype–phenotype case–control studies need
L-selectins, PIGR, IGHMBP2, correlations still needed verification. GWA studies
TNFα, TGFβ, IL-4, IFγ, and needed
HLA-DRA
End-stage 10p; 18q; see Iyengar et.al. for Gene verification and Genes identified in adult
renal disease additional loci; D10S558, discovery and populations and many in
D10S1435, D6S281, genotype–phenotype setting of DN. Verification in
D4S2937, D2S291, D17S515, correlations in pediatric cohorts adults and children required.
CNDP1, ELMO1, PVT1 still needed GWA studies needed

recessive form of congenital NS in the Finnish
population [16, 17]. This was a particularly severe
form of NS, characterized by heavy proteinuria in
utero, lesions of the glomerular filtration barrier,
and often death within the first 2 years of life
[17]. These findings were quickly followed by
the identification of the NPHS2 (podocin) gene
and its mutations as the cause of autosomal reces-
sive steroid-resistant NS in older children from
families of Northern European and North African
descent [18, 19]. This form presented between
3 months and 5 years of age and was characterized
by minimal glomerular changes on early biopsy
samples, FSGS present at later stages, and rapid
progression to ESRD. Both nephrin and podocin
localize to the slit diaphragm and are key glomer-
ular filtration barrier proteins.
Since the identification of these genes, numer-
ous studies have investigated their role in all
forms of NS. It is now well established that
506
10–30 % of sporadic NS cases with FSGS are due
to mutations within NPHS2 [28]. Mutations
within the laminin-β-2 (LAMB2), Wilms’ tumor
1 (WT1), and phospholipase C-ε (PLCE1) genes
have also been reported in pediatric NS, although
they are far less frequent [29–31]. Hinkes
et al. reported that in 89 patients from 80 families
manifesting NS within the first year of life, muta-
tions in one of four genes (NPHS1, NPHS2, WT1,
and LAMB2) were identified in 66 % of the cases,
with nearly 38 % and 23 % due to NPHS2 and
NPHS1 mutations, respectively [32]. When
examined by age of onset, the proportion of con-
genital cases explained by mutations in one of the
four genes was nearly twice that of infantile-onset
cases. Also of note, the study reported that chil-
dren with mutations in any one of the four genes
did not respond to steroid treatment. The authors
concluded that children with onset of NS during
the first year of life should be screened for muta-
tions within these four genes in order to guide
therapeutic course and that there are likely addi-
tional unknown genes playing a role in early-
onset NS.
Previous studies have suggested both allelic and
locus heterogeneity of NS. While nephrin muta-
tions have been found only in patients with con-
genital onset, podocin mutations have been found
in cases presenting at varying ages [32, 33]. The
reasons for this phenotypic variability are not yet
completely understood; however, there is data
supporting that the specific type or number of
NPHS2 mutations may play a role [34, 35].
Perhaps the largest study to date on podocin
mutations in sporadic cases best demonstrates the
significant locus and allelic heterogeneity of
NS. Using direct sequencing in 430 patients
from 404 families from around the world, Hinkes
et al. was the first to document that patients with a
specific type and number of mutations presented
with symptoms at a significantly earlier age than
patients without such mutations [33]. Neverthe-
less, the authors did observe that identical muta-
tions did result in different ages of onset within
their study. Consequently, Hinkes et al. state that
there are likely to be additional modifying factors
affecting the phenotype. It must also be noted that
nearly 300 individuals in this study did not carry
K. Maresso and U. Broeckel
any mutation within the podocin gene, highlight-
ing the likelihood of as yet undiscovered genes.
This is the largest report to date and the first to
establish statistical significance of an important
genotype–phenotype correlation. It serves as an
example of the advantages of multinational col-
laborations and demonstrates that further such
studies are required in order to identify additional
correlations with therapeutic and prognostic
implications in the treatment of NS.
Currently, genetic screening is recommended
for those children presenting with NS during their
first year of life, as this could allow carriers of
mutations in the NPHS1, NPHS2, LAMB2, and
WT1 genes to be spared the potentially harmful
treatments to which they have been shown not to
respond [32, 36]. Genetic testing may eventually
become more widespread in children presenting
with NS, regardless of age, as further studies estab-
lish links between currently known mutations and
clinically relevant phenotypic outcomes, as well as
with the discovery of novel genes and variants. It
should be noted that a different set of genetic var-
iants may predispose to the steroid-sensitive form
of NS [37]; however, it has not been as intensively
studied as SRNS due to its relatively benign clinical
course. While there is currently much research
focused on the genes and proteins of the slit dia-
phragm complex, further inroads may be made in
both SRNS and SSNS with the application of
genome-wide association and expression studies
to uncover novel susceptibility and modifying
genetic variants. Such studies will require large-
scale cohorts resulting from multi-institution
and/or multinational collaborations in order to fur-
ther elucidate the pathogenetic mechanisms behind
this rare and heterogeneous syndrome. Most
recently, next-generation sequencing (NGS) was
applied to study NS. As will be discussed below
in the section on the application of NGS, this
approach can offer additional novel insights into
the genetic basis of this disease.
Hemolytic Uremic Syndrome (HUS)
Hemolytic uremic syndrome (HUS) is similar to
NS in that there are different manifestations of the
17 Genomic Methods in the Diagnosis and Treatment of Pediatric Kidney Disease
disease and it appears to be genetically heteroge-
neous. See also ▶ Chap. 47, “Renal Involvement
in Children with HUS” in this text. HUS occurs in
its typical form following an infection associated
with diarrhea, often from the E. coli serotype
0157:H7. This form is relatively benign, and
once resolved, complete renal function is restored.
However, approximately 5–10 % of HUS patients
present with no previous infection. This atypical
form, aHUS, can occur at any age, can be
familiar or sporadic in nature, and is associated
with a poor clinical outcome, including a 25–30 %
mortality rate [38]. While a number of environ-
mental factors triggering aHUS have been
reported [39–45], a genetic basis for this form
has been suggested by familial cases [46–48]. In
1998, the first aHUS-associated gene, comple-
ment factor H (CFH), was localized [49]. Since
then, aHUS has become largely recognized as a
disease of complement alternative pathway
dysregulation.
As with NS, some of the initial advances in
understanding the etiology of aHUS were the
result of genetic linkage and subsequent associa-
tion studies. Using a candidate-gene linkage
approach in three families, Warwicker et al. first
demonstrated linkage of aHUS to a region on chr.
1 containing a number of genes involved in com-
plement activation regulation [49]. Subsequent
mutational screening of the CFH gene identified
two different mutations in one of the three fami-
lies and in a sporadic aHUS patient. Since this
first identification, numerous studies have
reported various mutations within CFH associ-
ated with aHUS [49–54]. Most mutations are
missense mutations occurring in the C-terminal
region of the protein, and functional analysis of
CFH mutations shows that they result in a
reduced ability of CFH to protect cells from com-
plement lysis [55]. Additional mutations within
the complement regulatory genes of membrane
cofactor protein (MCP) [56–58] and factor I
(CFI) [59, 60], as well as within complement-
activating components factor B (CFB) [61] and
C3 genes [62], have also been associated with
aHUS. Together, these genes are estimated to
account for approximately 50 % of aHUS cases
[39, 63–65]. There is now an online database,
507
www.fh-hus.org, which catalogs the various
complement activation gene mutations associ-
ated with aHUS and provides a host of informa-
tion regarding this disease and its genetic risk
factors.
In order for complement gene mutation status
to be translated into clinically useful information,
a few reports have examined genotype–phenotype
correlations in aHUS. Although each report is
based on a small number of children, the results
are consistent across studies. Carriers of CFH
mutations have the most severe clinical course,
demonstrating earlier age of onset, rapid progres-
sion to ESRD, and disease recurrence after
kidney transplantation [63, 66]. Those with MCP
mutations show the most favorable prognosis,
with no disease recurrence post-transplantation
[39, 63, 66]. CFI mutation carriers have an
intermediate course, with 50 % quickly
progressing to ESRD, 50 % recovering, and a
majority with disease recurrence post-
transplantation [63, 66]. These data demonstrate
that aHUS presentation, response to treatment,
and long-term outcome are closely linked to geno-
type status. This should allow for more tailored
management of children with aHUS and under-
scores the urgent need for new and improved
therapies for those with CFH and IFH mutations.
An interesting feature of aHUS is its high rate
of incomplete penetrance, approaching 50 %,
where disease-causing mutations are seen in
those who do not present with the disease. It has
been speculated that additional genetic factors
may be required in addition to the known causa-
tive mutations in order for the disease to manifest
itself. In addition, approximately half of aHUS
patients do not carry a known causative mutation,
suggesting as yet unidentified genetic variants. In
this regard, a handful of polymorphisms, or SNPs,
have also been associated with aHUS [50, 56, 65,
67, 68]. Caprioli et al. documented association of
three SNPs in CFH with aHUS, concluding that
they may predispose to the disease in those with-
out CFH mutations, as well as contributing to the
full manifestation of aHUS in CFH mutation car-
riers [50]. Findings from Fremeaux-Bacchi
et al. support these data [56]. A report by
Esparza-Gordillo identified a SNP haplotype
508
block spanning the complement activation gene
cluster or chr. 1q32 which was more frequent in
aHUS patients, particularly those with CFH,
MCP, or F1 mutations, and which associated
strongly with the severity of the disease
[68]. This same group later reported on a pedigree
where three independent aHUS genetic risk fac-
tors were segregating, but only those members
who inherited all three manifested aHUS
[69]. The authors hypothesize that multiple
“hits” are required in complement activation
genes in order to induce the aHUS phenotype.
These data demonstrate the allelic heterogeneity
and polygenic nature of aHUS.
Clearly, more work remains to be done, as
only 50 % of cases are explained by currently
known mutations. It is likely that more genes and
mutations await discovery. Moreover, the role of
SNPs and their functional impact alongside the
currently known mutations need to be better elu-
cidated. As with NS, large-scale genomic associ-
ation and expression studies may be helpful in
this regard.
Chronic Kidney Diseases and End-Stage
Renal Failure
As with the acute disorders, Table 2 provides a
summary of the genes, loci, and genotype–phenotype
correlations relevant to the below conditions.
Polycystic Kidney Disease (PKD)
PKD represents a diverse collection of disorders
of the tubules of the kidney, where healthy renal
tissue is progressively replaced by fluid-filled
cysts. See also ▶ Chap. 36, “Childhood Polycys-
tic Kidney Disease” in this text. Extrarenal man-
ifestations are also seen in PKD, including hepatic
cysts, abnormal heart valves, and intracranial
aneurysms. Autosomal dominant PKD
(ADPKD) is the most common form, with symp-
tom onset usually during the third and fourth
decades of life, although childhood onset has
been documented [70, 71]. Autosomal recessive
PKD (ARPKD) is rarer, with symptoms often
beginning in utero or during the neonatal period.
Aside from palliative care, there are currently no
K. Maresso and U. Broeckel
known treatments for either form. While the
genetics of both ADPKD and ARPKD are well
established, additional advancements await
through medical resequencing and the identifica-
tion of genetic disease modifiers.
As with the aforementioned acute conditions,
the genes for ADPKD, polycystin-1 (PKD1) and
polycystin-2 (PKD2)/transient receptor potential
channel (TRPP2), and the gene for ARPKD,
fibrocystin (PKHD1), were identified through
linkage analyses and subsequent positional clon-
ing efforts [1–5]. In ADPKD, the PKD1 gene
accounts for approximately 85 % of all families
[72–74] and is associated with both a more
severe clinical course and earlier onset
[74–77]. There is substantial allelic heterogene-
ity of both PKD1 and PKD2, with most muta-
tions unique to a family. The increased pace of
genomic diagnostics is evidenced by evaluation
of the ADPKD database (http://pkdb.mayo.edu)
at two points in time. The ADPKD database
identified an increase from 314 to 929 and
91 to 167 likely pathogenic germline mutations
within PKD1 and PKD2, respectively, between
mid-2008 and 2014.
As most mutations are private,
genotype–phenotype studies must generally clas-
sify mutations in some way, usually based on type
(missense v. truncating) and position within the
protein. However, no strong correlations have yet
been established between either type or position
and phenotypic outcome for either gene. There is
some evidence to suggest that patients with muta-
tions in the 50 region of PKD1 progress to ESRD
earlier and may be more susceptible to intracranial
aneurysms [78, 79]. While these two genes
account for nearly all cases, the disease often pre-
sents with significant variability within families.
While some of this variability is likely due to
environmental factors, additional genetic factors
are also suspected in modifying disease outcomes.
Studies have estimated that 18–78 % of the phe-
notypic variance in PKD1 and PKD2 populations
may be attributable to genetic background
[80–82]. Many candidate-gene studies have been
performed in an attempt to identify some of these
modifying factors; however, the results to date
have been inconsistent [23, 83, 84]. Because of
17 Genomic Methods in the Diagnosis and Treatment of Pediatric Kidney Disease
the diversity of disease mutations and a lack of
phenotypic correlations, the usefulness of
genetic testing in ADPKD is limited, although it
may be helpful in childhood cases with no previ-
ous family history. This may change as
genotype–phenotype correlations and new thera-
pies are eventually established.
In ARPKD, allelic effects play a greater role
than in ADPKD. The ARPKD database provides
an up-to-date infrastructure for the annotation of
disease variants (http://www.humgen.rwth-
aachen.de/) and noted 748 distinct pathogenic
germline mutations (accessed 15 April 2015).
Although genotype–phenotype studies are limited
by the substantial amount of allelic heterogeneity
present within PKHD1, some conclusions can be
drawn from these reports. For example, the pres-
ence of two truncating mutations has been shown
to result in neonatal death, regardless of their
position [85–87]. All ARPKD patients which sur-
vive the neonatal period have been shown to carry
at least one amino-acid substitution, as opposed to
all chain-terminating mutations [85]. While trun-
cating mutations in general are associated with
more severe disease, missense mutations have
been shown to result in a milder phenotype
[85]. Larger cohorts of patients will be required
to establish consistent and more detailed
genotype–phenotype correlations that can be
used to improve management of the disease and,
in particular, to assist in the development of novel
therapeutic approaches. As ARPKD is quite rare,
and thus the numbers small, no candidate-gene
studies have been carried out to identify genetic
modifiers, although some have been mapped in
mouse models [74, 88].
In both ADPKD and ARPKD, the availability
of fast, high-throughput medical resequencing,
where patients are resequenced in depth over
long genomic regions in a matter of hours or
days, offers a new opportunity for mutation detec-
tion, with a consequent increase in numbers of
patients with specific mutational profiles. This
should eventually aid in establishing clinically
useful genotype–phenotype correlations. More-
over, the use of GWA studies in ADPKD, where
the numbers are larger, may allow for the identifi-
cation of novel genetic modifiers.
509
Primary IgA Nephropathy (IgAN)
Primary IgAN is the most common form of glo-
merulonephritis in children worldwide, although
its prevalence varies according to country
[89–91]. See also ▶ Chap. 32, “Immunoglobulin
A Nephropathies in Children (Includes HSP)” in
this text. Some of this variance is attributable to
geographic differences in the policies affecting
renal biopsy in the young [92], while some may
also be due to geographic differences in genetic
and environmental factors [93, 94]. Its incidence
has been estimated as high as 25–30 % in some
Asian countries where children routinely undergo
annual urinary screening; however, lower rates
have been reported in Europe and the United
States [95–98]. IgAN is characterized by glomer-
ular mesangial deposits of IgA and a widely vary-
ing presentation [99]. IgAN often presents with
gross hematuria attendant to an upper respiratory
tract infection [95, 98, 100, 101]. However, it is
estimated that in as many as half of all cases, there
is only microscopic hematuria, which can con-
tinue for years before the development of protein-
uria [95, 98, 101, 102]. Because symptoms can be
intermittent, and because in most countries there
are no routine urinary screening programs, a diag-
nosis can be missed.
IgAN was previously considered a benign dis-
ease; however, this outlook is changing based on
the recognition of increased morbidity and mortal-
ity after long-term follow-up [95, 100, 103, 104],
including the finding that approximately 10 % and
20 % of children progress to ESRD after 10 and
20 years, respectively [95, 100, 105–108]. The dis-
ease progresses slowly over decades, and with clin-
ical symptoms often not presenting until years after
onset, it is possible that many adult IgAN cases
actually had a pediatric onset. Consequently, child-
hood IgAN may be considered an early stage of
adult IgAN with lifelong follow-up and evaluation
required to detect the signs of disease progression
[95]. Enhanced detection of the disease in child-
hood may allow for its earlier treatment and more
aggressive management, offering a reduced risk of
chronic kidney disease and ESRD at its later stages.
A complete understanding of the pathogenesis
of IgAN is still lacking, although abnormally low
glycosylation of IgAI molecules is a consistent
510
finding in patients [109–111]. Treatment of the
disease is based upon symptoms, but ACE inhib-
itors are commonly prescribed due to their reno-
protective effects. Some new drugs are currently
under investigation, but more specific novel treat-
ments are needed which can be used safely over
the long term in children. Obtaining a better
understanding of this disease from a genetic
standpoint should allow for insights into IgAN
pathogenesis, as well as offering options for its
improved diagnosis and treatment.
As with the diseases previously discussed,
IgAN is genetically heterogeneous, with familial
aggregation identified in a subset of cases, while
the bulk of cases present with seemingly sporadic
onset. Currently, it is estimated that 15 % of cases
are “familial”; however, it may actually be higher
due to the difficulty in diagnosing the disease.
Within families, IgAN appears to follow an
autosomal dominant inheritance pattern with
incomplete penetrance. The most likely genetic
model for IgAN resembles that of NS or aHUS,
where there are a handful of primary susceptibility
genes with additional modifying genetic and envi-
ronmental factors also required. Yet the identifi-
cation of genes for IgAN lags behind that of the
acute kidney disorders, with no gene yet identified
in familial cases. However, three genome-wide
linkage studies have established a number of chro-
mosomal regions linked to the disease. Gharavi
et al. identified a locus, termed IGAN1, on chro-
mosome 6q22-23 as being linked to IgAN in 60 %
of the families from a Caucasian cohort
[112]. Using 22 informative Italian families from
the IgAN Biobank [113], Bisceglia et al. reported
linkage to IGAN1 and also identified two sugges-
tive loci on chromosomes 4q26-31 and 17q12-22
[114]. A genome-wide linkage scan identified a
locus on chromosome 2q36 in a Canadian family
of Austrian-German descent with 14 affected sub-
jects [115]. These studies support the likely
genetic heterogeneity of the disease. Review of
the genes within these loci does not reveal likely
candidates; however, this is not unexpected given
the lack of understanding surrounding IgAN path-
ological mechanisms [116].
Not unlike the previous diseases, numerous
smaller-scale case–control and family-based
K. Maresso and U. Broeckel
association studies have been undertaken for
IgAN. Some of the genes studied to date, and for
which significant associations have been reported,
include the megsin gene, found in glomerular
mesangium and upregulated in IgAN [117, 118];
the uteroglobin gene, a multifunctional anti-
inflammatory protein [119–121]; the C1GALT1
gene, an enzyme involved in the O-glycosylation
process [122]; and the E- and L-selectin genes,
involved in endothelial and leukocyte cell–cell
interactions, respectively [123]. Associations
have also been reported for a number of inflam-
matory candidates as well, including TNFα,
TGFα, IL-4, IFγ, and HLA-DRA [124–127]. In a
partial genome-wide screen, a Japanese group has
reported significant associations of the polymeric
immunoglobulin receptor (PIGR) gene [128] and
the immunoglobulin mu-binding protein 2 gene
[129] using over 80,000 gene-based SNPs in a
stepwise association design. Finally, while not
associated with IgAN onset, a 32 bp deletion in
the chemokine receptor 5 (CCR5) gene has been
associated with increased renal survival in IgAN
patients [130, 131]. Results for many of the above
genes require confirmation in larger, independent
cohorts.
High-throughput genotyping of dense sets of
SNPs within the currently known linkage peaks,
now made possible through the HapMap Project
and the commercial availability of mass-
throughput SNP genotyping platforms, may assist
in the identification of the genes responsible for
the signals. GWA studies may also be useful and
more economical in this regard. Once susceptibil-
ity genes are firmly established, the IgAN field
can progress to identifying genotype–phenotype
correlations to aid in diagnosis and treatment.
End-Stage Renal Disease (ESRD)
While many chronic kidney diseases can lead to
ESRD, the causes of it differ between children and
adults. See also ▶ Chap. 66, “Management of
Chronic Kidney Disease in Children” in this text.
Although hypertension and diabetes are the pri-
mary causes of ESRD in adult populations, FSGS
and congenital structural kidney abnormalities are
the primary causes in children. However, the well-
documented worldwide increase in rates of
17 Genomic Methods in the Diagnosis and Treatment of Pediatric Kidney Disease
childhood obesity, and its related complications of
diabetes and hypertension, has the potential to sig-
nificantly impact the rate of pediatric renal failure
[132–137]. The prevalence of ESRD in pediatric
cohorts is generally less than that in adults; never-
theless, these children experience significant mor-
bidity and mortality [138–140]. In addition, ESRD
disproportionately affects minorities, with African-
American children having nearly double to triple
the rates of Caucasian children within the same age
categories [140].
Due to well-established statistics indicating a
potentially catastrophic increasing public health bur-
den of CKD and ESRD overall, the identification of
genes related to these two outcomes represents a
substantial opportunity for improvement in under-
standing the complex pathological mechanisms
behind CKD progression. A vast majority of the
work in this area has been conducted in adult
populations due to the higher prevalence of these
outcomes, as well as to the availability of large
cohorts of adults well phenotyped for the more com-
mon complex diseases which result in CKD and
ESRD within this population. We will briefly review
findings from the adult studies here; however, as the
precipitating causes differ between adults and chil-
dren, disease mechanisms leading to the end point of
ESRD may also differ. Therefore, genes identified in
adults need to be tested for relevance in pediatric
patients. Large, multi-institutional/multinational
cohorts of CKD and ESRD pediatric patients
phenotyped in a standardized manner will be required
to dissect the genetics of these complex and critical
outcomes of various pediatric kidney diseases.
A number of groups have performed linkage
analyses for ESRD in the context of one of its
major precipitating causes in adults, diabetic
nephropathy (DN) [141–153]. Results from
these linkage studies have identified numerous
distinct loci; however, the best evidence supports
DN susceptibility loci on chromosomes 10p and
18q [154]. A susceptibility gene, carnosinase-1
(CNDP1), has been identified from the 18q
locus. Two independent groups have demon-
strated diabetic patients harboring a particular leu-
cine repeat polymorphism of CNDP1 to be at a
significantly reduced risk of developing nephrop-
athy and ESRD [155, 156].
511
Although numerous association studies examin-
ing single genes have been published for ESRD, as
in other cases, the small sample sizes and limited
coverage of the candidate genes make results diffi-
cult to interpret [154]. However, some larger-scale
and more in-depth association scans, including a
GWA study, have been published in this area and
warrant discussion [15, 157–160]. In the first com-
prehensive, family-based screen of candidate genes
for ESRD, Ewens identified twenty nominally sig-
nificant genes, including twelve novel ones, in
72 type I diabetic trios [160]. McKnight
et al. used 6,000 microsatellites in 400 Irish patients
with and without type I DN to identify two signif-
icant makers on chromosome 10 and four addi-
tional markers worthy of investigation on
chromosomes 2, 4, 6, and 17 [159]. A Japanese
scan using over 80,000 SNPs in 87 type II diabetics
with nephropathy and 92 diabetic controls without
nephropathy identified the engulfment and cell
motility 1 (ELMO1) gene as a DN susceptibility
gene [158]. In a recent GWA SNP association
study, Hanson et al. found a significant association
with the plasmacytoma variant translocation
(PVT1) gene with ESRD in 207 type II diabetics
with and without DN [157]. Unfortunately, many
of the above results still await verification [154].
It is noteworthy that the above studies all
examined a qualitative outcome and were
conducted in the setting of DN. However, there
have been a number of both large-scale linkage
and association studies published examining
quantitative kidney function traits, such as glo-
merular filtration rate and urinary albumin excre-
tion, in various populations [15, 141,
161, 162]. Results from most of these studies
require verification. A number of advantages
have been suggested for the use of quantitative
traits in the genetic dissection of complex diseases
[163–165]. The continued use of large and com-
prehensive linkage and association scans should
assist in the further identification and replication
of ESRD susceptibility loci. However, their rele-
vance to children will have to be tested. The
establishment of cohorts of children with ESRD
is challenging given the small numbers; neverthe-
less, such cohorts can be collected through long-
term collaborative efforts. Such cohorts are
512
required to map genetic variation, which may
ultimately allow for more aggressive and tailored
prevention and treatment efforts in this life-
threatening disorder.
The Impact of Novel Sequencing
Technologies on Gene Identification
Significant resources have been invested in iden-
tifying the genetic variants underlying common,
complex human diseases and traits. If we look at
the genetics field in general, large-scale GWASs
have been performed in populations around the
world for almost every common disorder and
numerous complex traits. To date, these studies
have yielded thousands of genomic loci associ-
ated with human diseases and traits, and the list
will continue to grow (http://gwas.nih.gov/index.
html). However, despite this tremendous success,
population genetics analysis clearly demonstrates
that only a portion of the overall genetic risk has
been identified using the GWAS approach [166]
of the most widely discussed reasons for this
might lie on the fact that GWASs focus toward
common DNA variants and one apparent source
of this missing heritability can be hidden in less
frequent variants. This notion is now leading to
the next wave of comprehensive genome analysis
allowing for novel ways to sequence genomes.
Recent advances in the area of next-generation
sequencing (NGS) offer the opportunity to build
and expand upon those findings by creating again
a paradigm shift in the way we approach studying
disease at a genetic and genomic level.
The area of NGS is rapidly evolving, with con-
stant improvements both in instrumentation and in
downstream data-analytic techniques [167]. While
the cost of whole-genome sequencing (WGS) con-
tinues to decrease, it can still be associated with a
substantial cost. Nevertheless, it is now well in
reach that a human genome can be analyzed for
around one thousand dollars [168]. One should
note however that this cost estimate often refers
only to the actual sequencing component and the
downstream bioinformatics analysis and interpre-
tation will require resources beyond this estimate.
Whole-exome sequencing, where only the coding
K. Maresso and U. Broeckel
portion of the genome is sequenced, offers a less
expensive alternative to WGS and has been suc-
cessfully applied to the identification of unknown
genetic defects in various Mendelian disorders.
However, the application of either whole-genome
or whole-exome sequencing to common, complex
diseases, as opposed to Mendelian, is far from
straightforward and many challenges remain. Cur-
rently, significant efforts are being devoted toward
developing the overall infrastructure to process the
wealth of DNA information generated by these
technologies [169]. Ultimately the challenge will
be to interpret each individual’s cumulative collec-
tion of polymorphisms and more specifically iden-
tify disease-causing or disease risk variants.
A comprehensive analysis needs to differentiate
the functional or causative variants from the large
amount of variations that are identified using these
NGS techniques. As a first step, bioinformatics
analyses are a first step to describe the frequency
of variants as well as modeling-based approaches
to predict the functional effects and implications.
As these methods will provide a first statistical
attempt of categorizing each individual’s genome
information, bioinformatics is limited in the ability
to describe functional effects in a complex biolog-
ical system. Consequently, the challenge will be to
more comprehensively assess the impact of vari-
ants in a biological system, which captures each
individual’s unique genome and at the same time
allows for a comprehensive functional analysis on a
biological level.
While it is apparent that the field is very much
dominated by the technological advancements,
understanding the current state of sequencing tech-
nology, the necessary steps involved in genome
analysis, and the associated limitations will be crit-
ical for applying these methods for disease identi-
fication. Consequently we will discuss examples of
the application of both whole-genome and whole-
exome sequencing and their challenges and limita-
tions for complex disease in general.
Next-Generation Sequencing
Using any of the available next-generation
sequencing (NGS) platforms which yield millions
17 Genomic Methods in the Diagnosis and Treatment of Pediatric Kidney Disease
of DNA sequence reads in a single run is now a
reality [170]. While in the beginning these tech-
nologies have been limited to larger genome cen-
ters or sequencing groups due to the substantial
cost of the equipment, the technology has
advanced and the cost for the equipment has
become in reach for more laboratories. Conse-
quently NGS has been spreading rapidly over the
past years and many laboratories around the world
are now using the technology to sequence cohorts
and individuals in the hopes of identifying novel
variants responsible for common diseases and
traits. Data from such studies is revealing a vast
amount of rare DNA variation [171, 172], which
presents specific analytical challenges to our cur-
rent methodologies but also poses a significantly
greater challenge to the overall field of genetics
and personalized medicine: exactly how “per-
sonal” does personalized medicine need to be? In
other words, the extent to which risk prediction,
diagnosis, and treatment needs to be tailored is
reflective of the extent to which rare variation
plays a role in common disease. If, for example,
each individual and their risks of the different
common diseases are a compilation of rare, very
rare, and even unique/private mutations, then risk
prediction, diagnosis, and treatment will need to
be much more “individualized,” approaching that
seen in cases of Mendelian disease, than if more
common variation were the culprit and risk pre-
diction, diagnosis, and treatment could be based
much more upon overall population inferences.
With NGS, the field of genomics is now poised
to answer important questions related to the
genetic architecture of disease since in the near
future it will be possible to comprehensively
describe the overall polymorphic structure of
each individual’s genome.
Data-Analytic Techniques in NGS
With the development of every new technology,
significant challenges must be addressed if we
want to capitalize upon it. A number of key tech-
nologies, which enable the generation of large
amounts of sequence data, have emerged over a
short period of time. The improvement in
513
sequencing technology, the increase in through-
put, and, at the same time, the reduction in cost
have been truly breathtaking. While sequencing
technologies from companies such as
454, Illumina, SOLiD, and other more recent plat-
forms such as PacBio and Ion Torrent are cur-
rently marketed and have captured significant
market share in both research and diagnostic
areas, all methods still present significant chal-
lenges to sequence and data analysis. Despite
different approaches to obtaining the sequence
data, all current methods have a few characteris-
tics in common which influence the downstream
analysis as well as data interpretation [173]. Cur-
rent methods utilize a shotgun sequencing
approach as the target DNA is prepared as a
sequencing library which will be more or less
randomly analyzed and numerous reads for each
base will be obtained. The sequence analysis will
assemble the obtained reads and various analyti-
cal approaches are utilized to determine the actual
base call in addition to the determination of the
genotype. The difference between the
abovementioned methods is determined by the
length of a single read which can be obtained, as
well as the method to obtain or read the sequence
itself. Improvement in sequencing technologies
now allows for increased read lengths, which rep-
resent substantial improvements over the initial
very short reads obtained just a short time ago.
The methods also differ in their detection
approach. More established light-based detection
is the underlying approach for SOLiD or Illumina,
while newer technologies such as Ion Torrent rely
on the detection of electrical signals as this method
is based on the use of computer chip technology.
Numerous other yet less publicized methods are
currently under development, and, therefore, at
this time it is hard to predict which method or
platform will be the most widely used in the future.
In fact, with the anticipated broad applications
which can be perceived for sequencing, it appears
likely that a number of robust and cost-effective
approaches will be utilized, rather than just one
single method. Different methods will address dif-
ferent needs and applications driven by require-
ments related to throughput, accuracy, ease of
sample processing, turnaround time, and cost.
514
Data Analysis in NGS
A critical next step in next-generation sequencing
represents the actual data analysis, sequence
assembly, and the identification and annotation
of DNA variants. While this field is rapidly devel-
oping and novel analysis algorithms are con-
stantly being explored and tailored for the
specific NGS methods, a few general issues have
emerged. A number of manufacturers of sequenc-
ing equipment provide software as part of the
analysis related to their platform; other software
packages have been developed and can integrate
the output from the various sequence platforms
[174]. In general, the programs conduct the
assembly of the reads in relation to a
predetermined and assembled human reference
sequence. Most commonly, the assembly for the
genome sequence will be conducted against the
whole human reference genome sequence, even
for an exome sequencing approach. Variant call-
ing will be conducted in reference to the baseline
sequence and as a starting point for more compre-
hensive annotation of downstream functional
effects. An initial analysis might focus on coding
sequence, the identification of synonymous and
non-synonymous variants, as well as the estima-
tion of their likely impact on gene function. More
comprehensive approaches focus also on annotat-
ing noncoding sequence and the prediction of the
impact of noncoding and copy number variation.
In addition, an assessment of the observed popu-
lation frequency is often used as a means to cate-
gorize an individual’s variants as common, in
comparison to rare, disease-causing variants.
This approach has proved particularly helpful in
studies focusing on rare diseases. Thus far, many
important and unexpected insights into the poly-
morphic structure of the genome have been
obtained. First and foremost, initial results of the
1,000 Genomes Project identified a vast number
of rare DNA variants in addition to the previously
known common variants. This substantially
expands the overall catalog of known human
genome variation. Beyond the basic annotation
with regard to frequency, the functional categori-
zation has provided some interesting insights.
A publication by McArthur et al. comprehensively
K. Maresso and U. Broeckel
assessed the frequency of loss of function variants
in the human genome. The authors estimate that a
human genome may contain up to approximately
100 loss of function variants, in addition to about
20 genes which are completely inactivated
[175]. These findings in healthy individuals sug-
gest a surprising tolerance to a reduced function in
genes. While the authors perform a sophisticated
and complex analysis to determine which variants
are most likely of greater functional importance,
this clearly demonstrates some of the major chal-
lenges: With a large number of rare variants, and an
unexpectedly high rate of variants with potentially
significant functional effects, it will be challenging
to evaluate these variants and their downstream
effect solely based on sequence data. This also
demonstrates the difficulties related to diagnostic
sequencing. Comprehensive whole-genome
sequencing will identify a number of significant
gene variants with significant effects when ana-
lyzed solely based on sequence prediction. While
the functional effects for some of these variants
might be well compensated for due to functional
redundancy, it will be challenging to differentiate
potential disease-causing variants from this loss of
function background.
NGS in Pediatric Kidney Disease
As the NGS methodology matures, a number of
recent publications showcase the power of NGS in
the field of pediatric nephrology. Since NGS can
lend itself both to targeted candidate-gene
sequencing and whole-genome approaches, the
published literature reflects all these approaches.
Using NGS, Otto et al. performed targeted
sequencing in a cohort of 120 patients
with nephronophthisis-associated ciliopathies
[176]. For this group of diseases with a recessive
mode of inheritance, causative mutations have
been previously identified in 18 different genes.
Consequently, clinical mutation analysis for test-
ing all genes in patients with a suspected disease
will be expensive and time-consuming. The
authors developed a targeted sequencing
approach using PCR amplification covering all
exomes in the described genes. Subsequent
17 Genomic Methods in the Diagnosis and Treatment of Pediatric Kidney Disease
pooling of amplified PCR products and NGS
allowed for a rapid, cost-effective, and highly
sensitive analysis of the samples. Interestingly in
75 % of the analyzed cases, this approach did not
identify a causal variant. Expanding the sample
size in a follow-up publication, the sequence anal-
ysis in 1,056 patients established the molecular
diagnosis in 127/1,056 independent individuals
(12.0 %) and identified a single heterozygous
truncating mutation in an additional 31 individuals
(2.9 %) [177]. Taken together these reports dem-
onstrate the power of targeted sequencing for a
comprehensive analysis of the mutational spec-
trum in selected candidate genes and demonstrate
its power for diagnostics. Nevertheless, these
results also demonstrate the substantial heteroge-
neity of the disease and further sequence analysis
in order to identify causal variants in patients
without an established genetic diagnosis is
warranted.
Again using a targeted candidate-gene
approach, Saisawat et al. analyzed a subset of
genes for congenital abnormalities of the kidney
and urinary tract (CAKUT) [178]. Five heterozy-
gous missense mutations were detected in
two candidate genes (FRAS1 and FREM2),
which have not been previously linked to
non-syndromic CAKUT.
In order to further understand the genetic basis
of steroid-resistant nephrotic syndrome,
36 patients identified through the UK Renal Reg-
istry were chosen and next-generation-sequenced
to screen 446 genes, including 24 genes known to
be associated with hereditary steroid-resistant
nephrotic syndrome. The subsequent mutation
analysis revealed known and novel disease-
associated variations in expected genes such as
NPHS1, NPHS2, and PLCe1 in 19 % of patients.
Interestingly, mutations were also detected in
COQ2 and COL4A4 in two patients with isolated
nephropathy and associated sensorineural deaf-
ness. Taken together, these studies demonstrate
the power of targeted NGS sequencing for the
analysis of previously identified disease and can-
didate genes. However, it also showcases that a
candidate-gene approach might be limited in that
only a small portion of the disease variants might
be located in previously identified genes. While
515
targeted sequencing can represent a rapid and
cost-effective approach to test known genes, in
case where this methodology does not yield suf-
ficient clarification, expanding to whole exome or
genome will likely provide important additional
information.
Expanding the targeted sequencing results for
CAKUT in including families with VACTERL
association, which represents a rare disorder that
involves congenital abnormalities in multiple
organs including the kidney and urinary tract,
Saisawat et al. demonstrated that combined
homozygosity mapping and whole-exome
resequencing was able to identify recessive muta-
tions in the TNF receptor-associated protein
1 gene (TRAP1) in two families with isolated
CAKUT and three families with VACTERL asso-
ciation. TRAP1 is a heat-shock protein 90-related
mitochondrial chaperone, expressed in renal epi-
thelia of mouse kidney and in the kidney of adult
rats [179]. Using a similar approach for nephrotic
syndrome (NS), Gee at al. aimed to identify
genetic factors, which might influence the differ-
ence between steroid-sensitive (SSNS) and
steroid-resistant forms (SRNS). The authors
detected biallelic mutations in epithelial mem-
brane protein 2 (EMP2) in four individuals from
three unrelated families affected by SRNS or
SSNS. EMP2 is exclusively localized to glomeruli
in the kidney and subsequent knockdown of emp2
in the zebra fish resulted in pericardial effusion,
supporting the pathogenic role of mutated EMP2
in human NS. At the cellular level the authors also
showed that knockdown of EMP2 in podocytes
and endothelial cells resulted in an increased
amount of CAVEOLIN-1 and decreased cell pro-
liferation [180]. To further expand on NGS in
kidney disease, Malone et al. report using WES
and targeted sequencing in families with focal
segmental glomerulosclerosis [181]. In seven
families they identified rare or novel variants in
COL4A3 or COL4A4. The predominant clinical
finding at diagnosis was proteinuria associated
with hematuria. In all seven families, there were
individuals with nephrotic-range proteinuria with
histologic features of FSGS by light microscopy.
In one family, electron microscopy showed thin
GBM, but four other families had variable
516
findings inconsistent with classical Alport nephri-
tis. Families with COL4A3 and COL4A4 variants
that segregated with disease represent 10 % of the
cohort. Consequently COL4A3 and COL4A4 var-
iants can be considered in the interpretation of
next-generation sequencing data from such
patients.
Taken together, these NGS studies demonstrate
the beginning of what one might anticipate as an
avalanche of results from large-scale sequencing
studies. The studies demonstrate a number of
notable findings. First and foremost, NGS and
targeted NGS can identify and confirm the results
from previous studies using standard sequencing
technologies. NGS is likely to emerge as a fast and
cost-effective methodology not only for research
but also for comprehensive, cost-effective, and
rapid screening of candidate genes. Depending
on the genetic architecture, phenotypic disease,
and genetic heterogeneity, a targeted approach
might identify genetic disease variants in a limited
subset of patients. Consequently, expanding to an
unbiased, whole-exome, or whole-genome
approach is likely to identify novel, potentially
rare, or fully private mutations, which contribute
to the disease phenotype. The interpretation of
rare variants in the context of the overall genome
variability will emerge as the main challenge in
the years to come. This will be of particular impor-
tance as we utilize NGS for clinical applications.
The Past, Present, and Future
Application of Genomic Methods
in Pediatric Nephrology
With the application of genome-wide linkage ana-
lyses as one of the first genomic technologies,
pediatric nephrology witnessed some of the first
fundamental advances in the understanding of
many chronic and acute kidney diseases. With
the completion of the HGP and the availability
of genome-wide SNP markers that were easily
and inexpensively typed, the field witnessed the
candidate-gene era, where one or a few genes
were genotyped for a handful of SNPs in usually
small- to moderate-size samples with inadequate
statistical power for detecting true disease
K. Maresso and U. Broeckel
associations. The popularity of this method was
due in large part to its ease of application; how-
ever, the current and widespread use of candidate-
gene studies has limited ability to result in the
novel breakthroughs required to enable paradigm
changes in the prevention, diagnosis, and treat-
ment of these often life-threatening conditions.
Another major step in the field was accomplished
through the use of high-throughput genotyping
methods including GWA studies with hundreds
of thousands to millions of SNPs. Expanding on
the tremendous pace of technological develop-
ments in DNA analysis, NGS represents another
technical revolution, which is likely to change the
landscape of genetic research in fundamental
ways again. Beyond all technological advances,
a few major requirements and concepts remain for
the field to move forward. In order for these
genetic studies to become a reality, large, well-
phenotyped cohorts of children are necessary. The
sample sizes needed for GWA studies to be suc-
cessful, those samples approaching 1,000 or
more, cannot be collected at a single center due
to the often modest number of cases of any par-
ticular kidney disorder. For whole-genome
sequencing, sample size requirements might be
different. While theoretically for rare diseases,
one single sample might be sufficient to identify
potentially underlying disease variants, this needs
to be analyzed on the background of many rare
variants. For this, family and pedigree information
will be helpful in addition to larger numbers of
normal samples in order to estimate whether a rare
variant is potentially rare and disease relevant or
just simply rare.
In any case, it is apparent that investigators
need to form interinstitutional and international
collaborations with standardized methods of
phenotyping. This is now being pursued for
many diseases including adult renal patients as
well as pediatric diseases. More collaborations
can be expected as the importance of genomic
technologies in pediatric nephrology is increas-
ingly recognized, as such technologies become
more readily available to investigators and as the
infrastructure required for such studies becomes
more firmly established. GWA and NGS studies
offer a hypothesis-free design to gene discovery
17 Genomic Methods in the Diagnosis and Treatment of Pediatric Kidney Disease
and are therefore ideal for many complex kidney
diseases where there is limited knowledge of dis-
ease mechanisms. Being able to analyze a genome
in a short time is likely to create a vast amount of
genetics information. This poses the obvious
questions of data sharing and dissemination. The
amount of data generated through NGS exceeds
by far the amount of data, which was generated as
part of the GWAS approach. Novel standards for
data sharing and guidelines for depositing large
amounts of sequence information are developing.
Finally, information on potential disease-causing
variants needs to be put in context with the poten-
tial impact and the functional relevance. Just a
short time ago it was hard to imagine that identi-
fying a comprehensive set of potential disease
mutations becomes simply a technical question,
which is solvable in a short time. Consequently
the field of genetics will need to expand from
DNA analysis and mapping to the functional anal-
ysis of genome variation. At this time, genetics is
likely to incorporate functional genome annota-
tion, high-throughput phenotyping of targeted cell
systems, and complex tissue models. Animal
studies and model organisms will gain impor-
tance, and model systems, which comprehen-
sively assess the complete human genome, will
be important. Historically, animal models have
served as a main platform. Novel technologies
such as induced pluripotent stem cells hold a
tremendous potential for disease modeling. This
field is rapidly developing and the combination of
genome analysis, together with the other genome-
wide technologies of transcriptomics and proteo-
mics, offers the best hope for future fundamental
advances in the field of pediatric nephrology.
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 Translational Research Methods:
Renal Stem Cells 18
Kenji Osafune

Contents Clinical Application and Practical Use

of Renal Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 526
Renal Stem Cell Research: Future Directions . . . . 558
Embryonic Renal Stem/Progenitor Cells . . . . . . . . . 527
Kidney Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 527 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559
Identification of Embryonic Renal
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560
Stem/Progenitor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 530
Embryonic Renal Stem/Progenitor Cells
Differentiated from Pluripotent Stem Cells . . . . . . 533
Pluripotent Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533
Mechanisms of Cues for Directed
Differentiation of Pluripotent Stem Cells . . . . . . . . . . . 534
Directed Differentiation of Mouse ESCs/iPSCs
into Embryonic Renal Stem/Progenitor Cells . . . . . . . 535
Directed Differentiation of Human ESCs/iPSCs
into Embryonic Renal Stem/Progenitor Cells . . . . . . . 540
Embryonic Renal Stem/Progenitor Cells
Reprogrammed from Adult Renal Cells . . . . . . . . . . . . . 545
Adult Renal Stem/Progenitor Cells . . . . . . . . . . . . . . . 545
Stem Cells of Extra-Renal Sources . . . . . . . . . . . . . . . . . . 545
Sources of Adult Renal Cells Responsible for
In Vivo Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 546
Adult Renal Stem/Progenitor Cells Expanded
Ex Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547
Stem Cell Niche in Adult Kidney . . . . . . . . . . . . . . . . . . . 554
Cancer Stem Cells in Kidney . . . . . . . . . . . . . . . . . . . . . . 555
Cancer Stem Cells in Adult Renal Cell
Carcinoma (RCC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 555
Cancer Stem Cells in Wilms’ Tumor . . . . . . . . . . . . . . . . 556

K. Osafune (*)
Center for iPS Cell Research and Application (CiRA),
Kyoto University, Sakyo-ku, Kyoto, Japan
e-mail: osafu@cira.kyoto-u.ac.jp

# Springer-Verlag Berlin Heidelberg 2016 525

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_16
526
Introduction
The kidney is the main excretory organ in the
body and performs essential functions, such as
the excretion of waste products from the blood,
the regulation of water volume, electrolyte bal-
ance and pH levels, and the maintenance of blood
pressure, erythropoiesis and bone metabolism. In
humans, these functions are carried out by the
combined action of approximately two million
nephrons, which are the functional units of the
kidney. Any pathologic process that leads to a
loss of nephrons may eventually cause decreased
renal function and perturbs the physiological
homeostasis maintained by the kidney, which is
a process collectively termed chronic kidney dis-
ease (CKD) [1–3]. CKD gradually progress to end
stage renal disease (ESRD). At present, ESRD
patients have only two therapeutic options: renal
transplantation and dialysis therapy. Renal trans-
plantation is a radical treatment, but hampered by
serious problems, including the shortage of donor
organs and chronic use of immunosuppressive
drugs. Dialysis therapy can substitute for a part
of renal function, but it eventually causes systemic
complications in ESRD patients [3–6]. The prev-
alence of CKD is increasing worldwide, mainly
due to the increased incidence of diabetes mellitus
and hypertension. Moreover, CKD is associated
with elevated morbidity and mortality due to the
increased risk of cardiovascular diseases, which
gives growing economic burden to health care
systems [7]. Since CKD represents a gradual
loss of nephron number, substantial efforts have
sought cell sources that can compensate the loss.
One option might be the supplementation of renal
stem cells [8].
Stem cells are defined as cells that have self-
renewal capacity and can give rise to multiple
terminally-differentiated cell types constituting the
body (Fig. 1a). Progenitor cells that differentiate
from stem cells are more developmentally commit-
ted to some cell lineages than stem cells, but are
nevertheless undifferentiated or immature com-
pared with fully-differentiated and matured cells
(Fig. 1b). In fact, it is sometimes difficult to clearly
distinguish progenitor cells from stem cells, and
both cell types are often equated. Therefore, the
K. Osafune
two cell types specific for kidneys will be described
as one entity, “renal stem/progenitor cells” or “renal
progenitors,” in this chapter.
With regard to usage for regenerative medicine,
stem cells are largely classified into two major
categories: tissue-specific or somatic stem cells
and pluripotent stem cells (Fig. 1c, d). Tissue-
specific stem cells, such as hematopoietic stem
cells in bone marrow [9] and neural stem cells in
nervous systems [10], have been identified in most
organs or tissues of our body, including renal stem
cells. Tissue-specific stem cells generally show
relatively limited proliferation capability on culture
dishes and limited differentiation potential into a
restricted number of cell types constituting the
original organ or tissue, while pluripotent stem
cells, such as embryonic stem cells (ESCs)
[11–14] and induced pluripotent stem cells
(iPSCs) [15–17], have virtually unlimited replica-
tive capacity on culture dishes and are theoretically
able to give rise to any cell type in the body. These
stem cells have been increasingly used as sources
for cell replacement therapy. Like the generation of
other clinically useful cell types, vigorous efforts to
differentiate renal lineage cells from ESCs/iPSCs
have already been carried out [18–24].
Recently, there have been an increasing num-
ber of reports that describe the successful genera-
tion of the desired cell types by manipulating core
transcriptional factors to convert fully-
differentiated cells to a pluripotent state or from
one fully-differentiated cell type to another
[25–27]. This experimental strategy, cellular
reprogramming, has already been utilized to gen-
erate renal progenitors [28].
To date, a growing body of evidence has
supported the idea that malignant tumors are initi-
ated and maintained by a population of tumor cells
that share similar biologic properties with normal
stem cells [29, 30]. This cancer stem cell model is
based on the observation that tumors arise from
stem cells that show the ability to self-renew as
well as give rise to differentiated tissue cells. Can-
cer stem cells are usually thought to occur by the
transformation of normal embryonic or adult stem
cells in their own tissues. While most knowledge
on cancer stem cells had initially come from leuke-
mia stem cells that were prospectively isolated by
18 Translational Research Methods: Renal Stem Cells 527

a Stem cell b

Unlimited Progenitor cell

self-renewal
capacity

Limited
Limited differentiation
Multipotency
self-renewal potential
capacity

ESC
HSC

c d

Red blood Platelet White blood

cell cell Neuron Blood cell Hepatocyte

Fig. 1 Stem cell and progenitor cell. (a, b) Stem cells (a)
show unlimited self-renewal capacity and multipotent dif-
ferentiation potentials, while progenitor cells (b) exhibit
more restricted proliferation and differentiation capability.
(c, d) Tissue-specific stem cells, such as hematopoietic
stem cells (HSCs; c) differentiate into cell types constitut-
ing their original tissue, while pluripotent stem cells, such
as embryonic stem cells (ESCs; d) differentiate into any
cell type in the body

flow cytometry sorting and transplanted into immu-
nocompromised mice to examine their tumorigenic
potentials, similar techniques have recently been
used to identify cancer stem cells from solid
tumors, such as breast carcinomas and brain tumors
[31, 32]. The elucidation of the biological proper-
ties of cancer stem cells would allow the develop-
ment of therapies that target the stem cell
populations. However, due to differences between
tumors derived from other organs, only a small
number of studies have focused on the identifica-
tion of cancer stem cells in kidney [33].
This chapter will summarize the current status
of research on the identification and isolation of
renal stem/progenitor cells from embryonic or
adult kidneys and on the generation of embryonic
renal stem/progenitor cells by the directed differ-
entiation of pluripotent stem cells or by the cellu-
lar reprogramming of other cell types. Present
knowledge on cancer stem cells of kidney-related
malignancies will also be described (Fig. 2).
Embryonic Renal Stem/Progenitor
Cells
Kidney Development
To understand the entity of embryonic renal stem/
progenitor cells, this section starts by briefly sum-
marizing mammalian kidney development (Fig. 3;
[34–39]). See also ▶ Chap. 1, “Embryonic
528
Embryo Cancer
patient
Isolation
Transformation?
Embryonic
stem cell
Cancer
stem cell
Directed
differentiation Cellular
reprogramming
ESC/iPSC Fully differentiated
renal cell
Fig. 2 Renal stem cell. Three types of renal stem cells
have been reported: embryonic renal stem cells, adult renal
stem cells and renal cancer stem cells. Embryonic renal
stem cells can be isolated from embryonic kidney, or
generated from pluripotent stem cells by directed differen-
tiation or from adult renal cells by cellular reprogramming.
Development of the Kidney” in this text. The
kidneys are derived from an early embryonic
germ layer, the intermediate mesoderm (IM);
[40–42], which is located between the lateral and
paraxial mesoderms and derived from the primi-
tive streak, which is the progenitor population for
both the mesoderm and endoderm [43, 44]. A
number of factors, such as Wnt, bone morphoge-
netic proteins (BMPs), fibroblast growth factors
(FGFs), Nodal/activin and retinoic acid
(RA) signals, contribute to the cell fate commit-
ment of primitive streak or mesodermal lineage,
including the IM [40, 45–50]. Especially, BMP
signalling is the key morphogen regulating
lateral-medial patterning of early mesoderm
development. Low BMP4 induces IM, while
K. Osafune
Adult
Isolation
Isolation
Transformation?
Adult
stem cell
Adult renal stem cells can be isolated from adult kidneys
by various methods, but are not necessarily equivalent to
embryonic renal stem cells. Renal cancer stem cells are
isolated from tumor tissues and may arise from the trans-
formation of embryonic or adult renal stem cell
populations
high BMP4 induce lateral plate mesoderm, and
the antagonism of BMP signals is required for
paraxial mesoderm differentiation [40].
In vertebrates, the IM successively develops
three types of kidneys: the pronephros, mesoneph-
ros and metanephros. The three kidneys are similar
in that they consist of a basic functional unit, the
nephron, although the number of nephrons differs
among the three. Mesonephros is the adult kidney
of fish and amphibians, while metanephros is the
functional kidney of adult amniotes, reptiles, birds
and mammals. The mammalian adult kidney, the
metanephros, is formed by reciprocally inductive
interactions between two embryonic precursor tis-
sues derived from the IM: the metanephric mesen-
chyme (MM) and the ureteric bud (UB), a
18 Translational Research Methods: Renal Stem Cells
Metanephric
mesenchyme
Posterior (Hox11, Wt1,
intermediate Six1, Eya1)
Primitive mesoderm
streak (Osr1, Eya1)
Fertilized (Brachyury,
egg Mixl1)
Anterior
intermediate
mesoderm
Ureteric bud
(Osr1, Lhx1,
(Ret, Hoxb7,
Pax2)
Spry1)
Fig. 3 Developmental process and lineage commitment
of kidney. Each developmental stage and its specific
marker gene are shown. Three kinds of embryonic renal
derivative of the nephric (Wolffian) duct. The UB
induces the MM to differentiate into epithelialized
nephrons and interstitium, while the MM directs
the UB to elaborate the lower urinary tract from the
collecting ducts through the renal pelvis and ureter
to a part of the urinary bladder [51]. Recently,
Taguchi et al. redefined the origin of the MM and
elucidated the lineage commitment from the
nascent mesoderm through the IM into either the
MM or UB [52]. They demonstrated that the MM is
generated from the posterior IM, which is distinct
from the anterior UB precursor IM population.
A portion of MM cells condenses around the tips
of the UB and differentiates into structures termed
cap mesenchyme (CM). CM cells self-renew at the
UB tips and give rise to various cell types comprising
the nephron. Thus, the CM has been considered
multipotent nephron stem/progenitor cells, which
can self-renew and differentiate into different types
of nephron epithelia [53, 54]. Additionally, CM cells
express a unique combination of transcription factors,
such as the Hox11 paralogs, Osr1, Pax2, Eya1, Wt1,
Six2, Sall1 and Cited1, before UB induction [55].
The MM produces the secreted neurotrophin,
glial cell line-derived neurotrophic factor (GDNF;
529
Nephron Podocyte
progenitor
(Six2, Sall1)
Proximal
tubule
Distal
tubule
Stromal
progenitor
(Foxd1, Pbx1) Interstitium
?
Mesangium
Vascular progenitor (Flk1)
Collecting
duct
Lower urinary
tract
progenitor cells, nephron progenitors, stromal precursors
and ureteric bud cells, are highlighted by boxes
[56, 57], which is a chemotactic factor for
UB. GDNF drives the outgrowth of the UB from
within the Wolffian duct and the extension of the
UB toward the MM. The attracted UB in turn
secretes Wnt9b, which is the primary inducer for
the MM [58] to induce the adjacent MM to con-
dense around the UB tip, thereby forming the
CM. Thereafter, Wnt4 secreted from the induced
CM epithelializes the CM itself in an autocrine
fashion [59]. During the process of mesenchymal-
epithelial transition (MET), the CM cells sequen-
tially go through specific developmental stages,
such as renal vesicles, pre-tubular aggregates, and
C- and S-shaped bodies, to eventually differenti-
ate into all nephron segments ranging from glo-
merular podocytes to distal renal tubules.
Among the key molecules involved in the pro-
cess of kidney development, a homeodomain
transcriptional factor, Six2, which serves as the
most specific marker of CM cells, has been
shown to play a central role in the regulation of
renal progenitor CM cells, particularly on the
ability of CM cells to continuously self-renew
throughout nephrogenesis [53, 54]. It was
reported that FGF9 and FGF20 are required for
530
the formation and maintenance of the CM pro-
genitors, respectively [60]. MM survival in vitro
also depends on FGF2 and BMP7 [61]. Urbach
et al. have recently demonstrated that a
RNA-binding protein, Lin28, sustains early renal
progenitors by preventing the final wave of
nephrogenesis during kidney development in
mice [62]. Interestingly, the overexpression of
Lin28 markedly expands nephrogenic progeni-
tors, resulting in a pathology highly reminiscent
of Wilms’ tumor, a pediatric kidney cancer that
evolves from the failure of terminal differentiation
of the embryonic kidney.
Upon completion of the kidney development,
transcription factors expressed in the CM, includ-
ing Six2, are no longer expressed and the embry-
onic nephron progenitors disappear, which leads to
no formation of new nephrons. Embryonic nephron
progenitors do not exist in adult mice even after
acute kidney injury (AKI) in ischemia-reperfusion
(I/R) models, which was elegantly demonstrated by
a lineage tracing study for Six2 [63]. In contrast to
mammals, which lose the renal progenitor pool
around birth, a self-renewing nephron stem cell
population has been shown to exist in the adult
kidneys of lower organisms, such as zebrafish and
drosophila, and to form new nephrons throughout
adult life, contributing to regenerating nephrons de
novo after injury [64, 65].
In addition to the Six2-expressing CM nephron
progenitors that give rise to several epithelial cell
types constituting nephrons, such as glomerular
podocytes and renal tubular epithelia, the devel-
oping kidney also contains progenitor cell types of
other lineages. These include Foxd1(+) stromal
progenitors in the MM, which differentiate into
renal interstitial cells, mesangial cells and vascu-
lar smooth muscle cells and pericytes in adult
kidney [66–72]. Kobayashi et al. have recently
demonstrated by performing lineage tracing
experiments that the Foxd1-expressing cortical
stroma represents a distinct multipotent self-
renewing progenitor population that gives rise to
the adult renal cell types described above through-
out kidney organogenesis [73]. Interestingly, it
was demonstrated that a small subset of
Foxd1(+) stromal cells contributes to Six2(+)
nephron progenitors at early developmental
K. Osafune
stages, but thereafter a strict nephron and stromal
lineage boundary are formed between the nephron
and stromal progenitor compartments.
Despite the evidence described above, the origin
of renal stromal cells remains unknown. A fate
mapping study by Guillaume et al. demonstrated
that large populations of renal stromal cells origi-
nate from a different part of mesoderm, the paraxial
mesoderm, which gives rise to mesenchymal tis-
sues, such as bones, skeletal muscle and tendon
[74]. Moreover, some studies have indicated that
an embryonic ectodermal tissue, the neural crest,
may contribute to renal stroma [75–79]. In addition
to stromal progenitors, embryonic kidneys have
been shown to harbor resident Flk1(+) vascular
progenitors, which give rise to renal vessels
[80–82], although cross-species transplantation
studies have suggested that extrarenal cells also
contribute to the glomerular vasculature [83]. There-
fore, details of the developmental origin of stroma
and vasculatures in kidney remain to be elucidated.
Most knowledge on kidney development has
been obtained from genetic approaches on experi-
mental animal models (see also ▶ Chap. 15, Trans-
lational Research Methods: The Value of Animal
Models in Renal Research in this text), including
fish, frog, chick and mice [34–39]. Previous works
have also examined embryonic kidneys in human
and nonhuman primates, such as rhesus monkey
[84–86]. Humans, nonhuman primates and mice
share a number of molecules and corresponding
spatial expression patterns that act as developmen-
tal markers, although major differences have
been found in the temporal patterns of kidney
development. Nephrogenesis starts in human and
nonhuman primates at the end of the first trimester
and continues throughout the mid-third trimester
with a term of 165  10 days, while mouse
nephrogenesis begins in mid-gestation and com-
pletes postnatally with a term of 20 days [85, 87].
Identification of Embryonic Renal
Stem/Progenitor Cells
As described above, the metanephric kidney con-
tains several stem/progenitor cell populations
required for the formation of a fully functional
18 Translational Research Methods: Renal Stem Cells
kidney. This property has also been demonstrated
by the transplantation of whole embryonic kid-
ney rudiments into extrarenal space of host ani-
mals. Hammerman et al. [88, 89] and Dekel
et al. [90, 91] demonstrated the formation of
vascularized renal tissues from transplanted
metanephroi inside the rodent body. Notably,
when mouse, rat, porcine or even human early-
gestational metanephroi are transplanted into the
kidney subcapsule or the abdominal cavity of
immunodeficient mice, they survive, grow,
undergo complete nephrogenesis and form func-
tional organs that are able to produce diluted
urine by integrating with the host vasculature
and forming vascularized glomeruli [91].
In addition to the transplantation of whole
metanephric rudiments, Kim et al. reported that
transplantation of a heterogeneous population of
dissociated embryonic day (E) 14.5 and E17.5 rat
fetal kidney cells under the renal capsule of a 5/6
nephrectomy kidney injury model resulted in the
formation of renal structures and improved renal
functions [92]. However, the research group later
demonstrated that rat fetal kidney cells experi-
enced proliferation arrest and lost in vivo regen-
erative potential after repeated passages of in vitro
expansion culture [93].
The selection or isolation of specific lineage of
progenitor cells included in embryonic kidneys
have also been performed either by sorting based
on the expression of specific markers or by selec-
tive in vitro assays. By creating a clonogenic
assay system which uses stimulation of Wnt4, an
epithelializing factor for MM in embryonic devel-
opment, Osafune et al. previously demonstrated
that the MM contains multipotent nephron pro-
genitors that can give rise to several kinds of
epithelial cells, such as glomerular podocytes
and the epithelia of the proximal and distal renal
tubules and Henle’s loop [94]. The progenitor
cells are contained only in the cell population
that strongly expresses Sall1, a zinc finger tran-
scription factor that is essential for kidney devel-
opment [95], and can reconstitute three-
dimensional kidney structures in an organ culture
setting that contain glomeruli- and tubule-like
components (Fig. 4). This clonogenic assay, how-
ever, has not yet been applied to human fetal
531
kidney and cannot be used to expand undiffer-
entiated nephron progenitors.
Other approaches to isolate progenitor cells
include the formation of spheres based on the
ability of progenitor cells grown in
low-attachment conditions. Lusis et al.
established nephrospheres from murine embry-
onic kidneys, which, although robustly propa-
gated in vitro, displayed limited nephrogenic
capacity [96]. To date, expansion culture of these
murine renal progenitors in vitro has not been
successful, underscoring the major role of the
in vivo stem cell niche in supporting the growth
of these cells. Therefore, the detailed mechanisms
regulating progenitor cell proliferation by the
corresponding niche need to be elucidated.
Methods to prospectively isolate fetal kidney
progenitor cells using surrogate surface markers
of intra-cellular renal transcription factor expres-
sion have also been examined to supply an unlim-
ited source of nephron progenitors, which in turn
can be used to regenerate damaged epithelial cells
within the nephron by combining culture condi-
tions for expanding nephron progenitors.
By using a previously-reported isolation
method for the putative progenitor population in
adult kidney [97], Lazzeri et al. isolated a similar
CD24(+)CD133(+) cell population from human
fetal kidney [98]. They reported that the CD24
(+)CD133(+) population represents 35–50 % of
embryonic kidney cells, but gradually decreases in
proportion and becomes restricted to the urinary
pole of the Bowman’s capsule. This population
may possibly persist into adulthood as progeni-
tors. Importantly, when isolated and injected into
immunodeficient mice with AKI by glycerol-
induced rhabdomyolysis, CD24(+)CD133(+)
cells regenerated different portions of the nephron,
reduced tissue necrosis and fibrosis, and signifi-
cantly improved renal function without tumor for-
mation [98]. The authors concluded that CD24(+)
CD133(+) cells represent a subset of multipotent
embryonic progenitors that persist in human kid-
neys from the early stages of nephrogenesis.
A group headed by Benjamin Dekel employed
a unique stepwise strategy in order to identify cell
surface markers for isolation of human embryonic
renal stem/progenitor cells [27]. To identify
532
a b
Day3 Day20
c d
Day3 Day7
f Renal Colony
progenitors formation
Fig. 4 Multipotent nephron progenitors in mouse meta-
nephric kidney (Ref. [94]). (a) In vitro colony formation
from a single progenitor cell within metanephric mesen-
chyme on NIH3T3 feeder cells expressing Wnt4. (b) A
single progenitor cell is seen to divide and differentiate into
two renal lineages: (1) podocytes (red: peanut agglutinin;
PNA) and proximal tubular epithelia (green: Lotus
Tetragonobulus lectin; LTL) in the left panel, and (2) prox-
imal (green: LTL) and distal tubular epithelia (red:
E-cadherin) in the right panel. (c) A cell population
strongly expressing Sall1 (Sall1high) and containing neph-
ron progenitors differentiates into renal structures in an
candidate markers that are highly expressed in
renal progenitor-rich tissues compared with dif-
ferentiated renal tissues, the group compared the
global gene expressions of human embryonic kid-
ney and Wilms’ tumor to human adult kidney by
oligonucleotide microarray [99]. Since Wilms’
tumor specimens frequently harbor cells that have
differentiated into renal tubular epithelia or stroma,
in addition to uninduced metanephric blastema, the
group serially propagated Wilms’ tumor xenografts
in mice to purify blastema tissues at the expense of
differentiated elements for the analysis. This anal-
ysis uncovered a large number of renal “stemness”
genes, including nephron progenitor markers
K. Osafune
PNA LTL E-cad LTL
e
Wt1 LTL
g
t t
g
Renal Glomerulus
progenitors
Proximal tubule
Henle`s loop
Distal tubule
organ culture setting. In contrast, metanephric cells weakly
expressing Sall1 (Sall1low) or not expressing Sall1 (Sall1)
disappear without differentiation (data not shown). (d)
Hematoxylin-eosin staining of sections of Sall1high aggre-
gates at day 10 of the organ culture shown in (c). Tubule-
(t) and glomerulus-like structures (g) are seen. (e) Double
staining of a renal tissues section formed from Sall1high
cells with the podocyte marker Wt1 (red) and the proximal
tubule marker LTL (green). Bars: (a, b) 50 μm, (c) 500 μm,
(d, e) 25 μm. (f) Schematic drawing of multipotent nephron
progenitors in a colony-forming assay. Several kinds of
epithelia constituting nephron arise from the progenitors
(e.g., SIX2, OSR1 and PAX2) and surface markers
(NCAM1, PSA-NCAM, FZD7, FZD2, DLK1,
ACVRIIb and NTRK2) [100]. The group narrowed
the candidate surface molecules by observing
exclusive expression in undifferentiated cortical
areas of embryonic kidneys and the possession of
characteristic traits of tissue-specific stem cells
determined by a series of assays, such as over-
expression of renal progenitor genes, self-renewal
capacity, colony formation capability and renal dif-
ferentiation potential [101]. They eventually iden-
tified NCAM1 (CD56) as a potential marker for
human embryonic renal progenitors that fulfils all
these criteria [100, 102].
18 Translational Research Methods: Renal Stem Cells
NCAM1(+) cells isolated from human embry-
onic kidney cells and cultured in serum-free
medium showed significant enrichment of nephron
progenitor genes, such as SIX2, OSR1, PAX2,
SALL1, CITED1 and WT1, compared to NCAM1
() cells [102]. The renal differentiation potential of
NCAM1(+) cells was demonstrated using a chorio-
allantoic membrane (CAM) transplantation model,
in which cells are grafted onto a highly vascularized
niche, rich in developmental cues [103]. The model
showed that NCAM1(+) cells generated well-
developed epithelial tubular structures expressing
markers for the proximal tubule, distal tubule and
loop of Henle. Harari-Steinberg et al. evaluated the
regenerative potential of the NCAM1(+) population
in 5/6 nephrectomy CKD models. They demon-
strated that NCAM1(+) nephron progenitors from
human embryos engrafted and integrated into dis-
eased murine kidneys after transplantation, and that
consecutive intra-parenchymal administration
halted the progression of renal disease, as
manifested by the increased creatinine clearance
throughout 12 weeks of treatment [102]. Therefore,
it was concluded that NCAM1(+) human nephron
progenitors are a pool of mitotically competent yet
strongly nephron-generating-biased cells.
Although the embryonic kidney progenitors
represent a promising cell source for renal regen-
eration, the ethical problems arising from the use
of human embryos as well as the allogeneic nature
of the human cells limit the use of these cells.
Therefore, efforts to identify a nephrogenic cell
population in adult kidneys or to generate the
embryonic renal progenitors from autologous
stem cells, such as iPSCs, are required to acceler-
ate research towards the clinical application of
transplantation therapy for kidney diseases.
Embryonic Renal Stem/Progenitor
Cells Differentiated from Pluripotent
Stem Cells
Pluripotent Stem Cells
Pluripotent stem cells are characterized by their
high proliferation potential and multipotent differ-
entiation ability into any cell type of the body. They
533
can be broken down into various types, including
the aforementioned ESCs [11–14] and iPSCs
[15–17], embryonal carcinoma (EC) cells [104],
embryonic germ (EG) cells [105, 106] and
multipotent germ stem (mGS) cells [107]. EC
cells are the first derived pluripotent stem cells
and come from teratocarcinomas, a type of tumor
that consists of three germ layer components
[104]. EG cells are generated from primordial
germ cells, which are embryonic cells that give
rise to germ cells, sperm and oocytes [105,
106]. Finally, mGS cells occur as a rare cell popu-
lation in the culture of sperm stem cells from mouse
testis [107]. However, when it comes to directed
differentiation strategies for regenerative medicine,
ESCs and iPSCs receive the most attention.
ESCs are pluripotent stem cells derived from
the inner cell mass of fertilized eggs in mammals
([11–14]; Fig. 5a–c), while iPSCs are generated
from somatic cells by the transduction of
reprogramming factors, such as most famously
the combination of Oct3/4, Sox2, Klf4 and
c-Myc, and closely resemble ESCs ([15–17];
Fig. 5d–f). The successful derivation of human
ESCs has stimulated regenerative medicine strat-
egies that aim to recover dysfunctional organs by
transplanting organ-specific cell types generated
from ESCs in vitro. iPSCs are appealing because
they offer the same attractive features of ESCs
while potentially overcoming two drawbacks
associated with the use of human ESCs: ethical
problems related to the use of human fertilized
eggs and the potential for immune rejection after
transplantation due to antigenic differences
between the ESCs and patients.
Although iPSCs were originally derived from
fibroblasts, the stem cell lines have since been
generated from terminally-differentiated somatic
cells, such as adult liver and stomach cells [108],
keratinocytes [109], hematopoietic cells [110,
111] and pancreatic β cells [112]. In fact, it
appears that reprogramming potential exists in
almost any cell type, including the cells that con-
stitute adult kidneys [113–116], although the effi-
ciency of the iPSC derivation differs among
original cell types.
Despite the excitement and potential of iPSCs
for medicine, they have the risk of cancer
534
a b
Inner cell mass
Fertilized egg ESC
d e
Oct4
Somatic Sox2
cells Klf4
c-Myc
iPSC
Fig. 5 Embryonic stem cell (ESC) and induced pluripo-
tent stem cell (iPSC). (a) ESCs are generated from the inner
cell mass of fertilized eggs. (b) Mouse ESCs (D3). (c)
Human ESCs (khES-3). (d) iPSCs are derived from
development. iPSCs were originally derived using
genome-integrating vectors, such as retroviral and
lentiviral vectors, to introduce the reprogramming
factors into somatic cells [15–17]. To solve the
mutagenesis problem, integration-free and safer
methods to generate iPSCs have been reported,
including (1) non-genome integrating vectors,
such as adenoviruses, Sendai viruses, plasmids
and episomal plasmid vectors, (2) genetic manip-
ulation, such as transposons and Cre-loxP sys-
tems, (3) the introduction of mRNA and proteins
of reprogramming transcription factors and
(4) treatment with chemical compounds [19, 20].
For clinical application, xeno-free and defined
culture conditions have been developed to main-
tain and differentiate ESC/iPSC cultures
[117]. Clinical trials using human ESC-derived
somatic cell types have been already performed
to treat spinal cord injury with oligodendrocytes
[118] and retinal degenerative disorders with ret-
inal pigmented epithelial cells [119]. Recently, the
first clinical trial using human iPSC-derived reti-
nal pigmented epithelial sheets was performed for
a patient suffering from age-related macular
degeneration [120].
Already, many cell types have been generated
from ESCs/iPSCs, including neural cells, cardiac
K. Osafune
c
f
somatic cells by introducing reprogramming transcrip-
tional factors. (e) Mouse iPSCs (178B5). (f) Human
iPSCs (201B7). Bars: 100 μm
and vascular cells, hematopoietic cells, pancreatic
β cell-like cells and hepatocyte-like cells
[18–21]. However, methods for the directed dif-
ferentiation of ESCs/iPSCs into renal lineage cells
have not been fully established. Most reports have
described the differentiation of mouse ESCs into
renal lineages, but few have examined human
ESCs or iPSCs. However, several research groups
have more recently published methods to induce
human ESCs/iPSCs into kidney lineages [22–24].
Mechanisms of Cues for Directed
Differentiation of Pluripotent Stem
Cells
There are two common differentiation formats of
pluripotent stem cells, EB formation and mono-
layer or adherent cultures. Conventional differen-
tiation protocols developed by Doetschman
et al. have used multicellular aggregations, termed
embryoid bodies (EBs), which constitute three-
dimensional cultures mimicking developing
embryos, although the three body axes of the
embryo, i.e., anterior-posterior, dorsal-ventral
and left-right, are randomly formed [121]. In
these systems, complex intercellular signals
18 Translational Research Methods: Renal Stem Cells
within three-dimensional structures that resemble
those in developing embryos can be reproduced to
generate target cell types from ESCs/iPSCs. On
the other hand, Nishikawa et al. established
two-dimensional monolayer differentiation of
mouse ESCs into vascular endothelia
[122]. Here, exogenous inducing factors can uni-
formly affect almost all cells to result in a higher
induction rate of the target cell types than EB
formation does. Two-dimensional cultures also
have the advantage of simplicity and are easy to
use for mechanistic analyses. Therefore, an
increasing number of studies for the directed dif-
ferentiation of ESCs/iPSCs have adopted mono-
layer culture formats [123, 124].
Regarding the inducers used to direct the dif-
ferentiation of ESCs/iPSCs into specific lineages
by mimicking the signals involved in normal
development, combinatorial treatments have
been developed. These treatments combine dif-
fusible factors (growth factors, cytokines and
chemical compounds), cell-cell interactions
(co-culture with cell lines) and positional cues
(extracellular matrix) that ultimately regulate the
gene expression, which together confer a specific
cellular identity and function. Genetic and epige-
netic manipulations using the overexpression of
cDNA, small interfering RNA (siRNA), short
hairpin RNA (shRNA) and microRNA (miRNA)
have also been utilized to induce specific cell fates
[19, 20, 22–24].
Some reports have demonstrated that high-
throughput screening (HTS) of low-molecular-
weight chemical compounds, including synthetic
and natural compounds, can be used to identify
small molecules that have the potential to induce
the differentiation of ESCs/iPSCs into specific
lineages, such as cardiomyocytes, pancreatic pro-
genitor cells, definitive endoderm and pancreatic
endocrine cells [125–129]. Araoka et al. has used
this approach to investigate directed differentia-
tion into kidney lineages and recently identified
two retinoids, AM580 and TTNPB, that effi-
ciently induce human ESCs/iPSCs into a
nephrogenic IM [130]. These IM cells induced
with chemicals give rise to renal lineage cells
that have the developmental potential to form
renal tubular structures. The appeal of HTS is
535
that it can help identify small molecule inducers
for renal lineage differentiation, which will also
help elucidate the mechanisms underlying kidney
development and differentiation.
Substantial differences exist in the differentia-
tion potential among human ESC/iPSC lines,
although only marginal differences have been
observed in the undifferentiated state
[131–133]. Some human ESC/iPSC lines even
have a propensity to differentiate into certain lin-
eages or cell types. Moreover, it has been shown
that iPSCs harbor residual DNA methylation sig-
natures characteristic of their original somatic
cells. Such “epigenetic memory” of the parental
tissue may predispose the differentiation into lin-
eages related to the parental cells, a property
observed in multiple iPSC lines including those
from mouse blood cells [134, 135] and human
pancreatic β cells [112]. These findings indicate
that iPSCs derived from renal lineage cells might
favour differentiation into kidney lineages.
Indeed, human iPSCs have been derived from
mesangial cells [113] and renal tubular cells
dropped into urine [114, 115]. In addition,
mouse iPSCs were also generated from renal
tubular epithelial cells [116]. These findings
underscore the importance of using suitable cell
lines for renal lineage differentiation.
Directed Differentiation of Mouse
ESCs/iPSCs into Embryonic Renal Stem/
Progenitor Cells
By recapitulating the signals involved in the dif-
ferentiation steps during normal embryonic devel-
opment, step-by-step protocols have been
explored to directly differentiate ESCs/PSCs into
the specific cell types included in adult organs,
such as pancreas and liver [123, 124]. For exam-
ple, pancreatic β cells have been induced by dif-
ferentiating ESCs/iPSCs into the definitive
endoderm, then the foregut endoderm, followed
by pancreatic progenitors and eventually into
insulin-producing cells [123]. Most studies
aiming to generate renal cells from ESCs/iPSCs
have so far adopted a similar strategy of recapitu-
lating kidney development [22–24].
536
The in vitro injection of ESCs into immunode-
ficient mice can produce a teratoma, which is a
tumor that contains the derivatives of all three
embryonic germ layers: the ectoderm, mesoderm
and endoderm. Yamamoto et al. reported that glo-
merulus- and renal tubule-like structures can be
formed in teratomas generated from mouse ESCs,
indicating that mouse ESCs have the developmen-
tal potential to differentiate into renal cells
[136]. Steenhard et al. demonstrated that undiffer-
entiated mouse ESCs incorporate into the
renal tubules and glomeruli of host kidneys by
responding to developmental signals within
the host kidney microenvironment when
microinjected into E12.5-13.5 mouse kidneys in
organ culture settings [137].
Moreover, substantial efforts have been made
to selectively or specifically induce renal progen-
itors and fully-differentiated renal cell types
in vitro from mouse ESCs (Table 1). Kramer
et al. reported that the differentiation cultures of
mouse ESCs with EB formation in the presence of
serum alone produced cells expressing renal line-
age markers [140].
Kobayashi et al. generated mouse ESC lines
that stably express Wnt4, a renal-epithelializing
factor [138]. They induced Wnt4-transformed
mouse ESCs into renal tubular cells that expressed
Aquaporin-2 (AQP2) and three-dimensional tubu-
lar structures in vitro by EB formation and treat-
ment with hepatocyte growth factor (HGF) and
activin A in addition to Wnt4. The transplantation
of the treated EBs into mice renal cortices also
resulted in the formation of teratomas, some of
which contained AQP2(+) tubular structures.
Kim and Dressler reported that EBs formed
from mouse ESCs can be induced into the IM
in vitro by a combinatorial treatment with
nephrogenic growth factors, such as RA, activin
A and BMP7 [139]. These EBs developed renal
tubule-like structures by co-culturing with embry-
onic spinal cord, which is a classical inducer for the
MM, and the induced cells were integrated into the
proximal tubules when microinjected into E12.5
mouse kidney rudiments in organ culture settings.
Vigneau et al. generated mouse ESC lines with
green fluorescent protein (GFP) knocked into the
Brachyury (T) locus as well as lacZ into the
K. Osafune
ROSA26 locus (LacZ/T/GFP) for cell selection
and lineage tracing [141]. They further optimized
the differentiation conditions using only activin A
treatment for renal progenitors and used the
expression of Cadherin11, Wt1, Pax2 and Wnt4
as markers of differentiation. The renal progeni-
tors, isolated via flow cytometry, were injected
into developing live-newborn mouse kidneys
and stably integrated into the proximal tubules
for 7 months without teratoma formation. It was
concluded that the defined differentiation of
mouse ESCs via EB formation and with activin
A treatment generated proximal renal tubular pro-
genitors that may have adequate safety for regen-
erative therapies.
Bruce et al. induced mouse EBs into the meso-
derm by culturing with serum or serum-free cul-
tures supplemented with BMP4 and detected the
gene expression of kidney lineage markers by
real-time RT-PCR analysis [142]. The generation
of urogenital Pax2(+) cells was demonstrated by
immunostaining and flow cytometry using
reporter mouse ESC lines containing either a
modified Pax2 promoter-lacZ or bacterial artificial
chromosome (BAC)-GFP transgene. This
approach enabled direct monitoring of renal rather
than the mid-hindbrain Pax2 expression. In addi-
tion, three BMPs, BMP2, BMP4 and BMP7, were
compared in EB systems to evaluate the specifi-
cation of the mesoderm. It was found that a low
concentration of BMP4 activates ventral (blood)
and intermediate (kidney) mesoderm gene expres-
sion, while BMP2 and BMP7 promote only kid-
ney lineage.
Nakane et al. generated mouse ESC lines using
a tetracycline-inducible gene expression of Pax2
and examined the effects of Pax2 overexpression
on renal lineage differentiation, since Pax2
expression was shown to be sufficient to induce
the ectopic formation of nephric structures in
chick [143]. They found that Pax2 overexpression
in undifferentiated ESCs failed to induce the
expression of renal lineage markers, while its
overexpression in EBs led to the expression of
two renal lineage markers, integrin α8 and Aqp1,
suggesting that along with Pax2, additional fac-
tors induced in EBs are required for renal lineage
differentiation.
 18

Table 1 Summary of reports on the directed differentiation of mouse ESCs/iPSCs into kidney lineages
Primary
Author Cell line Format Medium Inducer target cell Functional examination References
Kobayashi T BL6 EB IMEM/15 % Wnt4 overexpression, 10 ng/mL HGF NP (MM) Tubule formation by three- [138]
et al. 2005 FCS and 10 ng/mL activin A for 5–20 days dimensional culture in Matrigel.
Formation of teratoma that contain
tubules by injection into renal
cortex of immunodeficient mice
Kim D R26 EB DMEM/10 % 0.1 μM RA, 10 ng/mL activin A, NP (MM) Tubule formation by co-culture [139]
et al. 2005 FCS 50 ng/mL BMP4 or BMP7 for 5–7 with embryonic mouse spinal cord.
days Integration into proximal tubules
by injection into mouse
metanephric rudiments
Kramer J D3 EB DMEM/20 % Spontaneous differentiation: 20 % Renal Formation of ring-like glomerular [140]
et al. 2006 FCS FCS for up to 40 days. Induction using lineage structures consisting of podocyte-
recombinant proteins: 100 ng/mL cell like cells in EB outgrowths
FGF2 for 5 days followed by
Translational Research Methods: Renal Stem Cells

100 ng/mL FGF2 in combination with

50 ng/mL LIF for an additional 7 days
Vigneau C E14.1,129/ EB 50 % 2–100 ng/mL activin A for 1–8 days NP (MM) Integration into nephrogenic zone [141]
et al. 2007 Ola Neurobasal/50 by injection into mouse
% DMEM/F12 metanephric rudiments.
Integration into proximal tubule by
injection into live newborn mice
kidneys
Bruce SJ W9.5 EB DMEM/10 % Spontaneous differentiation: 10 % Renal NA [142]
et al. 2007 FCS or serum- FCS or 2 ng/mL BMP4 and 1 U/mL lineage
free chemically LIF for 19 days. Induction using cell
defined media recombinant proteins: 2 ng/mL BMP4
and 1 U/mL LIF for 4 days followed
by 7 days with BMP2, 4 or 7
Nakane A MGZRTcH2 EB GMEM/10 % 15 % FCS for 5 days followed by Pax2 Renal NA [143]
et al. 2009 cell FCS overexpression lineage
(MG1.19) cell
Morizane R EB3, EB DMEM/10 % 10 ng/mL activin A for 4 days Renal NA [144]
et al. 2009 MEF-Nanog- FCS followed by 150 ng/mL GDNF and lineage
38C (iPSC) 15 ng/mL BMP7 for an additional cell
537

14 days
(continued)
 538

Table 1 (continued)
Primary
Author Cell line Format Medium Inducer target cell Functional examination References
Ren X R1 EB DMEM/15 % 10 ng/mL activin A and 0.1 μM RA Renal NA [145]
et al. 2010 FCS for 8 days followed by 10 days with lineage
conditioned medium from E13.5 mice cell
ureteric bud
Mae S 129/sv Monolayer DMEM/F12/2 1 μM JAK inhibitor, 5 μM LY294002, IM NA [146]
et al. 2010 % FCS 1 μM CCG1423 and 0.1 μM RA for
8 days
Nishikawa M J1 Monolayer DMEM/10 % Stage 1: 10 ng/mL activin A NP NA [147]
et al. 2012 FCS (A) during days 0–2, A plus 50 ng/mL (MM) and
BMP4 during days 2–4, A4 plus UB
10 mM lithium chloride (L) during
days 4–6; Stage 2: A4L plus 0.1 μM
RA during days 6–8; Stage 3:
conditioned media from UB or MM
Oeda S D3 Monolayer ESF medium/ 10 ng/mL activin A and 0.1 μM RA IM Integration into renal tubules by [148]
et al. 2013 0.5 mg/mL BSA for 6 days injection into mouse metanephric
rudiments
Taguchi A E14.1 (Osr1- EB 75 % IMDM/25 Stage 1: 0.5–1 ng/mL activin A for NP Formation of glomeruli and renal [52]
et al. 2014 GFP), % Ham’s F12 1 day; Stage 2: 1 ng/mL BMP4, 10 μM tubules by co-culture with
EB3-DsRed) with N2 and CHIR99021 for 1.5 days; Stage 3: embryonic mice spinal cord
B27 1 ng/mL BMP4, 10 μM CHIR99021
supplements for 1 day; Stage 4: 10 ng/mL
and 0.05 % BSA activin A, 3 ng/mL BMP4, 3 μM
CHIR99021, 0.1 μM RA, 10 μM
Y27632 for 1 day; Stage 5: 1 μM
CHIR99021, 5 ng/mL FGF9, 10 μM
Y27632 for 2 days
EB embryoid body, NP nephron progenitor, MM metanephric mesenchyme, NA not applicable, IM intermediate mesoderm, UB ureteric bud
K. Osafune
18 Translational Research Methods: Renal Stem Cells
Ren et al. used the factors secreted from
embryonic renal cells to differentiate mouse
ESCs into renal precursor cells [145]. They first
generated mesodermal populations expressing
Pax2 and Brachyury by treating mice EB with
activin A and RA. The mesodermal cells were
then treated with conditioned medium collected
from the cultures of UB cells from E13.5 mice
embryos, which resulted in the generation of dif-
ferentiated cells expressing marker genes charac-
teristic of the initiation of nephrogenesis (Wt1 and
Pax2) and terminally-differentiated renal cell
types (POD1 and E-cadherin).
Mae et al. reported the differentiation of mouse
ESCs into IM cells in monolayer culture with
combinatorial treatments of small molecules
[146]. They established the requirement for a
combination of four small molecules: (1) Janus-
associated tyrosine kinase (JAK) inhibitor 1, an
inducer of differentiation from ESCs into epi-
blasts by inhibiting leukemia inhibitory factor
(LIF) signals, to maintain an undifferentiated
state of mouse ESCs [149]; (2) LY294002, an
inhibitor of phosphatidylinositol 3-kinase (PI3K)
to promote differentiation into the mesendoderm
[150]; (3) CCG1423, an inhibitor of RhoA tran-
scriptional signalling to promote the epithelial-
mesenchymal transition (EMT) during gastrula-
tion by breaking down the basement membrane
[151]; and (4) RA, to upregulate the expression of
IM marker genes in mouse ESC differentiation
when combined with activin A treatment
[139]. The combination of JAK inhibitor
1, LY294002 and CCG1423 resulted in the gen-
eration of BMP7(+) mesoderm cells, and treat-
ment with all four factors efficiently induced
mouse ESCs into IM cells.
Nishikawa et al. reported a stepwise renal lin-
eage differentiation from mouse ESCs by tracing
in vivo development, which consisted of three
stages performed in monolayer cultures: the
induction of a mesoderm that expresses
Brachyury (Stage 1), an IM that expresses Pax2
and Lim1 (Stage 2) and MM cells that express
Wt1, Gdnf and Cadherin 11 along with UB cells
that express Hoxb7, Ret and Wnt11 (Stage 3;
[147]). For Stage 1, three factors were sequen-
tially added: activin A (A) during days 0–2, A
539
plus BMP4 during days 2–4 and A4 plus lithium
(L), a surrogate for Wnt signalling, during days
4–6. For Stage 2, RA was added to A4L during
days 6–8. For Stage 3, conditioned media derived
from a mouse UB cell line, CMUB-1 [152], and a
mouse undifferentiated MM cell line, MK3 [153],
were used to induce MM and UB, respectively.
The group also found a gradual increase in the cell
population expressing CD24 from Stages 1–3 and
accordingly proposed CD24 as a cell surface
marker that can be used for the purification of
IM and MM cells in the culture when combined
with other surface markers.
Oeda et al. established a differentiation proto-
col from mouse ESCs into IM cells by treatment
with activin A and RA under defined, serum-free,
adherent, monolayer culture conditions
[148]. These mesoderm cells integrated into lam-
inin(+) tubular cells and Pax2(+) renal cells when
co-cultured with embryonic kidneys in organ cul-
tures. The group also demonstrated that signals
initiated through RA receptors (RARs), but not
retinoid X receptors (RXRs), are primary in the
differentiation of the IM.
Viñas et al. used microarray analysis to show
that miRNA let-7e was the most upregulated
miRNA in mouse EBs treated with activin A and
RA [154]. Furthermore, the expression of several
marker genes for IM and MM, such as Pax2, Wt1,
Wnt9b, Wnt4 and Notch2, had increased. The
group demonstrated that miRNA let-7e is
involved in renal lineage differentiation by stim-
ulating the Wnt pathway, which suppresses the
phosphorylation of glycogen synthase kinase
(GSK)3β and subsequently stabilizes β-catenin
by inhibiting protein kinase C (PKC) β.
Morizane et al. examined the renal lineage
differentiation from both mouse ESCs and iPSCs
by combinatorial treatment of EBs with activin A,
BMP7 and GDNF [144]. Using the expression
analysis of several renal marker genes, including
Pax2, Wt1 and Kidney-specific protein (Ksp),
both mouse ESCs and iPSCs could be differenti-
ated into renal lineages, although differences in
the gene expression levels between the two stem
cells were found.
In some reports using mouse ESCs or iPSCs,
the induced renal lineage cells were shown to
540
form tubule-like structures in organ cultures [139]
or to be integrated into developing renal tubules of
mouse kidneys [141]. Notably, Taguchi
et al. recently established a multistep differentia-
tion protocol from mouse ESCs into nephron pro-
genitors that could form glomerulus-like
structures in addition to renal tubules [52]. As
described above, the group demonstrated that
nephron progenitors are derived from the poste-
rior mesoderm and identified the optimal combi-
nations of inducers that promote differentiation
from the posterior mesoderm into nephron pro-
genitors. They established a stepwise differentia-
tion protocol from mouse ESCs through the
epiblast, nascent mesoderm, posterior nascent
mesoderm and posterior IM into nephron progen-
itors. The induced nephron progenitors could
form three-dimensional kidney structures
containing both proximal and distal renal tubules
as well as numerous glomeruli by co-culturing
with embryonic spinal cords. The researchers
even developed vascularized glomeruli when
integrated with the host vasculature after trans-
plantation under the kidney capsule of
immunodeficient mice.
In summary, nephron progenitors can be gen-
erated from mouse ESCs/iPSCs through the IM,
and renal tissues containing glomeruli and renal
tubules can be also induced in vitro from the
induced nephron progenitors. Moreover,
transplanted mouse ESC/iPSC-derived nephron
progenitors are capable of integrating into the
proximal renal tubules of developing mouse
kidney.
Directed Differentiation of Human
ESCs/iPSCs into Embryonic Renal Stem/
Progenitor Cells
In contrast to the works using mouse ESCs/iPSCs,
a small number of reports have described the
directed differentiation of the renal lineages from
human ESCs or iPSCs [22–24]. Like mouse
ESCs, the presence of renal cells and tissues was
demonstrated in teratomas generated from human
ESCs, which indicated that human ESCs also
have the potential to differentiate into kidney
K. Osafune
lineages [13, 155]. Schuldiner et al. examined
the effects of eight growth factors (nerve growth
factor (NGF), HGF, epidermal growth factor
(EGF), RA, bFGF, BMP4, transforming growth
factor (TGF)-β1 and activin A) on the differenti-
ation of human ESCs, showing that six (RA, NGF,
bFGF, activin A, HGF and EGF) could induce the
expression of two kidney markers, RENIN and
KALLIKREIN, according to RT-PCR
analysis [156].
That report was followed by studies on selec-
tive differentiation methods for kidney lineages
from human ESCs/iPSCs (Table 2). Batchelder
et al. spontaneously differentiated human ESCs
via EB formation with 10 % serum and used real-
time RT-PCR to detect the expression of several
marker genes for the IM (OSR1, PAX2, SIX2 and
WT1), early kidney precursors (CD24, CDH11,
EYA1, LIM1 and SIX1), the developing kidney
(GDNF, RET and WNT4) and more mature renal
cells (AQP2, CDH16, CLCN5, CYP27, NPHS1,
NPHS2, PODXL and SLC12A1; [157]). Directed
differentiation of these ESCs using activin A, RA,
BMP4 and BMP7, which are the same
nephrogenic factors used in mouse ESCs [139],
showed that marker gene expression for kidney
lineages was highest when human EBs attached to
gelatin-coated plates [157]. Additionally, the
immunostaining patterns of PAX2 and vimentin
in human ESC differentiation cultures were simi-
lar to those in human fetal kidneys and embryonic
rhesus monkey kidneys.
Lin et al. differentiated human ESCs with
reduced serum concentration and a reduced den-
sity of feeder cells [155]. MM cells were then
selected via flow cytometry with two surface
markers, CD24 and PODOCALYXIN [36], but
lacking the well-characterized marker for
undifferentiated ESCs, GCTM2 [13]. Microarray
analyses of the purified CD24(+)
PODOCALYXIN(+)GCTM2() cell population
and comparison with GUDMAP, a transcriptome
profile database for urogenital development [166],
showed that the top 200 differentially
up-regulated genes in the cell fraction clustered
into a group of genes associated with the MM at
E11.5 and the cortico-nephrogenic interstitium at
E15.5 of murine kidney development [155].
 18

Table 2 Summary of reports on the directed differentiation of human ESCs/iPSCs into kidney lineages
Primary
Author Cell line Format Medium Inducer target cell Functional examination References
Batchelder HSF-6 EB αMEM/10 % Spontaneous differentiation: 10 % FCS for Renal NA [157]
CA FCS, High- up to 22 days. Directed differentiation: 0.1 lineage
et al. 2009 glucose DMEM/ μM RA, 10 ng/mL activin A, 50 ng/mL cell
10 % FCS BMP4 or BMP7 for 6–8 days
Lin SA HES-2, Monolayer DMEM/20 % FCS Reduced density (2  104 cells/cm2) of Renal NA [155]
et al. 2010 HES-3, HES-4 feeder cells with 20 % FCS for 2 days and lineage
with 5 % FCS for an additional 12 days cell
Song B iPSCs derived EB DMEM/F12/2.5 10 ng/mL activin A, 15 ng/mL BMP7 and 0.1 Podocyte Podocytes showing a contractile response to [158]
et al. 2012 from normal % FCS μM RA for 10 days. the addition of angiotensin II (AII) and the
human kidney uptake of albumin. Integration into glomeruli
mesangial by co-culture with mouse metanephric cells
cells (NKMC)
Mae S hiPSCs Monolayer DMEM/F12/2 % Stage 1: 100 ng/mL activin A and 3 μM IM Formation of renal tubular structures by [159]
et al. 2013 (201B7, FCS or B27 for CHIR99021 for 2 days; Stage 2: 100 ng/mL co-culture with mouse metanephric cells
OSR1-GFP) Stage 1, DMEM/ BMP7 and 3 μM CHIR99021 for 4–20 days
F12/10 % KSR or
Translational Research Methods: Renal Stem Cells

B27 for Stage 2

Narayanan K HUES7 Monolayer REGM medium/ 10 ng/mL activin A, 0.1 μM RA, 10 ng/mL Proximal Proximal tubular epithelia showing increase [160]
et al. 2013 0.5 % FCS BMP2 and 2.5 ng/mL BMP7 for 20 days renal in cAMP levels in response to PTH, GGT
tubular activity, ammonia production and water
epithelial transport. Integration into tubules by
cell injection into newborn mice kidneys.
Formation of tubular structures by
subcutaneous injection into immunodeficient
mice
Xia Y H1, human Monolayer DMEM/F12/17.5 Stage 1: 30 ng/mL BMP4 and 50 ng/mL UB Integration into ureteric bud structures by [161]
et al. 2013 fibroblast mg/mL BSA bFGF for 2 days; Stage 2: 1 μM RA, co-culture with mouse metanephric cells
episomal- 10 ng/mL activin A and 100 ng/mL BMP2
derived iPSCs for 2 days
Taguchi A 201B7 EB DMEM/F12/B27 Stage 0: 0.5 ng/mL BMP4, 10 μM Y27632 NP Formation of glomeruli and renal tubules by [52]
et al. 2014 for 1 day; Stage 1: 1 ng/mL activin A, co-culture with embryonic spinal cord
20 ng/mL FGF2 for 2 days; Stage 2: 1 ng/mL
BMP4, 10 μM CHIR99021 for 2 days; Stage
3: 1 ng/mL BMP4, 10 μM CHIR99021 for
4 days; Stage 4: 10 ng/mL activin A, 3 ng/mL
BMP4, 3 μM CHIR99021, 0.1 μM RA,
10 μM Y27632 for 2 days; Stage 5: 1 μM
CHIR99021, 5 ng/mL FGF9, 10 μM Y27632
for 3 days
541

(continued)
 542

Table 2 (continued)
Primary
Author Cell line Format Medium Inducer target cell Functional examination References
Takasato M HES3, H9, Monolayer Serum-free APEL Stage 1: 30 ng/mL BMP4 and 10 ng/mL MM and Integration into ureteric epithelia, renal [162]
et al. 2014 CRL2429C11- mediuma activin A or 8 μM CHIR99021 only for UB vesicles, and nephron progenitors by
hiPSC 2 days; Stage 2: 200 ng/mL FGF9 for 4 days; co-culture with mouse metanephric cells.
Stage 3: 200 ng/mL FGF9, 50 ng/mL BMP7 Formation of ureteric epithelia, renal vesicles,
and 0.1 μM RA and nephron progenitors in an organ culture
Lam AQ H1,H9,CHB8- Monolayer Advaced RPMI Stage 1: 5 μM CHIR99021 for 24–48 h; IM and Integration into renal tubules by co-culture [163]
et al. 2014 H2B-GFP Stage 2: 100 ng/mL bFGF, 1 μM RA for NP with mouse metanephric cells. In vitro
hESCs, dermal 36 h–3 days; Stage 3: 100 ng/mL FGF9 and formation of renal tubular structures
fibroblast- 10 ng/mL activin A for 3 days
derived
hiPSCs
Araoka T 201B7 Monolayer DMEM/F12/2 % Stage 1: 3 μM CHIR99021 and 1 μM AM580 IM Formation of renal tubular structures by [130]
et al. 2014 (OSR1-GFP) FCS or B27 for or TTNPB for 2 days; Stage 2: 1 μM AM580 co-culture with mouse metanephric cells
Stage 1, DMEM/ or TTNPB for 3–12 days
F12/10 % KSR or
B27 for Stage 2
Kang M H9, Monolayer RPMI/B27 Stage 1: 100 ng/mL activin A and 100 ng/mL NP In vitro differentiation into renal tubular [164]
et al. 2014 CRL-iPSCs Wnt3a for 1 day; Stage 2: 20 ng/mL BMP4 epithelia and glomerular podocytes. Tubule
and 10 ng/mL bFGF for 2 days; Stage 3: formation in collagen type I gel
10 μM RA, 50 ng/mL BMP7 and 10 ng/mL
bFGF for 6 (hESCs) and 8 days (hiPSCs);
Stage 4: 150 ng/mL BMP7 and 50 ng/mL
bFGF for 15 days
EB embryoid body, NA not applicable, IM intermediate mesoderm, cAMP cyclic adenosine monophosphate, PTH parathyroid hormone, GGT γ-glutamyl transferase, UB ureteric bud, NP nephron
progenitor, MM metanephric mesenchyme
a
See Ref. [165]
K. Osafune
18 Translational Research Methods: Renal Stem Cells
a b
PNA Nuclei LTL Nuclei
c d
AQP1 HuNu DBA Nuclei
Fig. 6 Renal cells and tissues differentiated from human
iPSC (Ref. [159]). (a, b) Adult renal cells differentiated
from human iPSC-derived intermediate mesoderm (IM)
cells in vitro. Glomerular podocytes (a) and proximal
tubular cells (b). (c, d) Adult renal cells differentiated
from human iPSC-derived IM cells in vivo. Proximal tubu-
lar cells (c) and collecting duct cells (d). Peanut agglutinin
(PNA): a marker for glomerular podocytes; Lotus
More recent research has established differen-
tiation methods from human ESCs/iPSCs into
renal lineage cells that indicate developmental
potential as well as marker gene expression
[167]. Mae et al. used BAC-based targeting vec-
tors to induce homologous recombination in
human iPSCs [159]. This approach successfully
generated reporter human iPSC lines in which the
GFP reporter gene was knocked into the locus of
an IM marker, OSR1. These cell lines allow for
quantitative evaluation of the IM cell induction
rate. The researchers found that combinatorial
treatment with activin A, BMP7 and
CHIR99021 in two-dimensional monolayer cul-
tures can achieve an induction rate of over 90 %
for OSR1(+) cells. These human mesoderm cells
have the developmental potential to further differ-
entiate into renal lineage cells both in vitro and
in vivo, such as glomerular podocytes and renal
tubular cells (Fig. 6a–d). Furthermore, the cells
can form three-dimensional renal tubular
543
e
hMito LTL Nuclei
hMito LAMININ Nuclei
tetragonolobus lectin (LTL) and AQUAPORIN (AQP) 1:
markers for the proximal renal tubule; Dolichos biflorus
aggulutinin (DBA): a marker for the collecting duct; HuNu:
human nuclear antigen. (e) Three-dimensional renal tubu-
lar structure formed from human iPSC-derived IM cells by
co-culturing with mouse metanephric cells in an organ
culture setting. hMito human mitochondria. Bars: 50 μm
structures in organ culture settings when com-
bined with dissociated mouse E11.5 metanephric
cells (Fig. 6e).
In another study, Xia et al. published the gen-
eration of UB-like progenitors from human ESCs/
iPSCs [161]. They established a 4-day monolayer
culture protocol for human ESCs/iPSCs that
included a combinatorial treatment with BMP4
and FGF2 for the first 2 days in order to induce
mesoderm-committed cells, followed by an addi-
tional 2-day treatment with RA, activin A and
BMP2 to generate the IM. They found that marker
genes for UB, including HOXB7, RET and
GFRA1, were expressed in the culture rather than
marker genes for the MM. When human IM cells
were co-cultured with dissociated mouse E11.5
metanephric cells in organ cultures, the differenti-
ated cells were integrated only into UB structures
and no chimaeric MM structures were formed.
These observations suggest the generation of UB
progenitor-like cells from human ESCs/iPSCs.
544
Taguchi et al. applied the differentiation proto-
col that induces mouse ESCs into nephron pro-
genitors described above to human iPSC
differentiation cultures by modifying the early
steps for mesoderm induction and adjusting the
culture periods of the later steps [52]. The induced
human cells expressed several nephron progenitor
markers, including WT1, PAX2, SALL1 and SIX2,
and formed three-dimensional kidney structures
that consisted of WT1(+)NEPHRIN(+) glomeruli,
CADHERIN6(+) proximal tubules and
E-CADHERIN(+) distal tubules when
co-cultured with mouse embryonic spinal cords.
The report was the first to show that kidney tissues
containing glomerular structures can be generated
from mouse ESCs and human iPSCs.
Takasato et al. reported the generation of “self-
organizing kidneys” from human ESCs that were
synchronously formed from derivative tissues of
both the MM and UB [162]. They treated human
ESCs in monolayer cultures with a combination of
high BMP4 and low activin A or with only high
CHIR99021 to induce the posterior primitive
streak and then included FGF9 treatment to
induce the IM. The resulting IM cells were treated
with a combination of FGF9, BMP7 and RA,
which caused the expression of marker genes for
the MM, UB and several adult renal cell types,
including glomerular podocytes and epithelial
cells in the proximal renal tubule and collecting
duct. Additionally, the formation of
E-CADHERIN(+)PAX2(+) ureteric epithelia
surrounded by WT1(+)SIX2(+) MM cells and
even luminal connections between ureteric epithe-
lia and renal vesicles were observed in these cul-
tures. When co-cultured with mouse E12.5
metanephric cells in organ cultures, the induced
cells were integrated into ureteric epithelia, early
nephron/renal vesicles and clusters of nephron
progenitors. Furthermore, when the induced cells
were reaggregated to pellets and cultured in organ
cultures with 10 % serum, the resulting cellular
organoids spontaneously developed into renal
structures, such as E-CADHERIN(+)PAX2(+)
AQP2(+) ureteric epithelia that were surrounded
by WT1(+)PAX2(+) MM, AQP1(+)SLC3A1(+)
proximal tubules and JAG1(+)E-CADHERIN
(+) renal vesicles.
K. Osafune
Lam et al. established a simple, rapid and effi-
cient differentiation protocol from human iPSCs/
ESCs into the IM [163]. They first induced human
ESCs/iPSCs into a BRACHYURY(+)MIXL1(+)
mesendoderm with CHIR99021 and then into a
PAX2(+)LHX1(+) IM with bFGF and RA with
70–80 % efficiency. These IM cells gave rise to
apically ciliated tubular structures expressing
proximal tubule markers, such as Lotus
tetragonolobus lectin (LTL), N-CADHERIN and
KSP, and partially integrated into embryonic kid-
ney explant cultures. In addition, the IM cells
were further differentiated into cells expressing
markers for nephron progenitors following addi-
tional treatment with FGF9 and activin A.
Araoka et al. aimed to replace growth factors
with low-molecular-weight compounds for the
induction of the IM [130]. High-throughput chem-
ical screening identified two retinoids, AM580 and
TTNPB, as potent inducers for the IM from human
iPSCs/ESCs. The combination of either AM580 or
TTNPB with CHIR99021 efficiently induced
OSR1(+) IM cells that have the developmental
potential to differentiate into renal lineage cells
and form three-dimensional renal tubular structures.
Kang and Han published a stepwise differenti-
ation method from human ESCs/iPSCs into neph-
ron progenitors through the primitive streak and
IM in a serum- and feeder-free two-dimensional
culture with the combination of bFGF, activin A,
Wnt3a, BMP4, RA and BMP7 [164]. These
human nephron progenitors further differentiated
into cells that expressed markers for podocytes
(SYNAPTOPODIN and PODOCALYXIN) and
renal tubules (AQP1, CD13 and MUC1) and
formed three-dimensional tubule-like structures.
Selective differentiation methods for the gener-
ation of adult renal cell types from human iPSCs
into glomerular podocytes were reported by Song
et al. [158]. They formed EB-like cellular aggre-
gates from human iPSCs in suspension cultures
treated with activin A, BMP7 and RA, and attached
to gelatin-coated plates. After 10 days, the cultures
gave rise to podocytes that showed typical mor-
phology, marker expression (SYNAPTOPODIN,
NEPHRIN and PODOCIN) and function, includ-
ing a contractile response to angiotensin II (AII)
and the endocytic uptake of albumin.
18 Translational Research Methods: Renal Stem Cells
A specific differentiation protocol from human
ESCs into proximal renal tubular epithelia was
also reported by Narayanan et al. in which cultur-
ing undifferentiated human ESCs in renal epithe-
lial growth medium (REGM) with a combination
of activin A, RA, BMP2 and BMP7 generated
AQP1-expressing cells at an induction rate of
around 40 % [160]. In addition to the expected
marker gene expression and morphology of prox-
imal renal tubular epithelia, these cells showed
physiological functions, such as an increase in
the intracellular levels of cyclic adenosine
monophosphate (cAMP) in response to parathy-
roid hormone (PTH), γ-glutamyl transferase
(GGT) activity, ammonia production and water
transport. Moreover, these cells retained their
functional characteristics under conditions in
bioartificial kidneys.
In summary, human ESCs/iPSCs can be differ-
entiated into both nephron progenitors and ure-
teric bud cells through the IM, and nephron
structures containing glomeruli and renal tubules
and ureteric bud-like tubular structures can be
formed in vitro from induced human embryonic
renal progenitors. Selective differentiation into
glomerular podocytes and proximal renal tubular
epithelia from human ESCs/iPSCs has also been
achieved.
Embryonic Renal Stem/Progenitor Cells
Reprogrammed from Adult Renal Cells
The success of direct reprogramming from one
terminally-differentiated cell type into another
has been shown in several cell types including
pancreatic exocrine cells into β cells [25] and
fibroblasts into neurons [26]. In these reports, the
transduction of a combination of three or more
transcription factors essential for the development
or differentiation of the target cell types were
utilized. This strategy was successfully applied
to the artificial generation of embryonic nephron
progenitors from fully-differentiated adult renal
cell types [28]. Hendry et al. reported that the
forced expression of six transcription factors
(SIX1, SIX2, OSR1, EYA1, HOXA11 and
SNAI2) in an adult proximal tubule cell line
545
resulted in the activation of a network of genes
consistent with nephron progenitors and that the
induced cells showed the ability to contribute to
the Six2(+) nephron progenitor fields of an
embryonic kidney explant.
In addition, Omer et al. showed that treating
mature human adult kidney epithelial cells with
valproic acid, a histone deacetylase (HDAC)
inhibitor, results in significant activation or
embryonic renal progenitor genes, such as SIX2,
OSR1, SALL1, NCAM, and PSA-NCAM
[168]. However, the same treatment inhibits cell
de-differentiation towards a more replicative mes-
enchymal state at the same time, which is required
for the clonogenic expansion and acquisition of
stem cell characteristics.
Research on direct cellular reprogramming
into renal cell types is still nascent, and further
studies will be required to establish methods for
generating renal stem/progenitor cells. For exam-
ple, the in vitro reprogramming of somatic cell
types other than renal cells, such as fibroblasts,
into renal progenitors remains to be achieved.
Adult Renal Stem/Progenitor Cells
Stem Cells of Extra-Renal Sources
Two types of tissue-specific stem cells identified
in bone marrow have been reported to contribute
to renal tissues: hematopoietic stem cells [9] and
mesenchymal stem cells or multipotent marrow
stromal cells (MSCs: [169]). MSCs were hypoth-
esized to be circulating from bone marrow and
responsible for the homeostasis, regeneration and
repair of adult mesenchymal tissues [170], but are
now considered a subpopulation of perivascular
cells or pericytes that reside in almost all tissues of
our body [171, 172]. MSCs are currently thought
to be recruited from their perivascular niche in
each tissue to sites of tissue injury, where they
establish a regenerative microenvironment to
facilitate tissue regeneration by secreting various
bioactive molecules [173].
Early reports described that bone marrow-
derived cells can differentiate into multiple
somatic cell types including renal tubular cells,
546
indicating the “plasticity” of stem cells to transdif-
ferentiate into cells belonging to other organs,
including organs derived from other germ layers
[174–179]. However, since those reports, sponta-
neous cell fusion was demonstrated between cells
from mouse brain and mouse ESCs and between
mouse bone marrow cells and mouse ESCs [180,
181]. These findings suggested that at least part of
the transdifferentiation of bone marrow stem cells
could be explained by spontaneous cell fusion
between transplanted bone marrow cells and
recipient host tissue cells.
It has also been reported that bone marrow-
derived cells contribute to renal regeneration
after injury by transdifferentiating into tubular
cells, suggesting the renal regeneration potential
of exogenously administered bone marrow-
derived cells [182–187]. Other reports showed
that hematopoietic stem cells and MSCs can dif-
ferentiate into glomerular mesangial cells in vivo
[184, 188]. Yokoo et al. demonstrated the ability
of human MSCs to differentiate and contribute to
nephrogenesis during development in whole
rodent embryo cultures [189]. The group injected
human MSCs that were engineered to produce
GDNF and lacZ using a replication-defective ade-
noviral construct into the nephrogenic IM region
of E9.5 and 11.5 rodent embryos, which gives rise
to the developing metanephros. After ex vivo
whole-embryo culture for up to 48 h, these cells
showed a contribution to the developing rodent
kidneys.
In addition, the injection of bone marrow-
derived cells, especially MSCs, has repeatedly
been shown to improve renal function in AKI
induced by toxins (cisplatin and glycerol) or I/R
injury [190–194]. Similar beneficial effects on
renal function may be obtained by administering
growth factors, such as granulocyte colony-
forming factor (G-CSF), granulocyte/monocyte
colony-forming factor (GM-CSF), monocyte
colony-forming factor (M-CSF), or stem cell fac-
tor, to mobilize a patient’s bone marrow cells.
Several studies have indicated such administra-
tion improves renal function [195–198], but a
counter observation was also reported [199].
Current data suggest that any contribution
made by non-renal stem cells, including MSCs,
K. Osafune
to the regeneration of renal tissues is not because
of their differentiation into tubular cells in vivo
Humphreys et al. elegantly demonstrated this con-
cept by using mice lineage tracing experiments
[63]. They demonstrated that the cells responsible
for tubular regeneration after AKI induced by I/R
injury originate from remaining renal tubular
cells, thereby excluding any possible contribution
of extra-renal cells to regenerating kidney.
Instead, MSCs seem to contribute to kidney repair
after damage by secreting trophic factors, such as
vascular endothelial growth factor (VEGF),
bFGF, monocyte chemoattractant protein-1
(MCP-1), HGF and insulin-like growth factor
1 (IGF1), and immunomodulators, such as
TGF-β and prostaglandin E2 (PGE2)
[200]. While MSCs have consistently been
shown to exert renoprotective effects in animal
models of AKI [190–194], the therapeutic effec-
tiveness of MSCs on CKD is controversial
[201–203]. Indeed, phase-I clinical trials testing
MSC transplantation in patients at high risk of
developing AKI following cardiac surgery are
currently being carried out [204].
Sources of Adult Renal Cells
Responsible for In Vivo Regeneration
As mentioned above, the lineage tracing experi-
ments done by Humphreys et al. demonstrated
that the cells responsible for tubular regeneration
after I/R injury are of tubular origin, not extra-
renal cells [63]. The study also demonstrated that
adult kidneys do not contain embryonic nephron
progenitor-like cells expressing Six2, and that the
reactivation of this cell population does not occur
even after ischemia damage. It was therefore indi-
cated that the regeneration of new tubular cells
originates from intrinsic epithelial cells in adult
kidneys. However, it remains unclear whether
these regenerating cells are epithelial progenitors
with more restricted differentiation potential than
Six2(+) embryonic progenitors or are fully-
differentiated tubular cells that can expand after
ischemia damage.
Kusaba et al. answered this question by genet-
ically labelling fully-differentiated proximal
18 Translational Research Methods: Renal Stem Cells
tubule cells using mice in which a CreER
(T2) cassette was knocked into the sodium-
dependent inorganic phosphate transporter
SLC34a1 locus, which is expressed only in
fully-differentiated proximal tubules, and exam-
ined the cells’ contribution to regenerating renal
epithelia after ischemia injury [205]. After injury,
all tubular cells remained labelled, indicating that
new regenerating tubular cells could originate
from the proliferation of surviving differentiated
tubular epithelia. Interestingly, injury to labelled
proximal tubule epithelia induced a certain degree
of de-differentiation as manifested by the marker
expression of putative epithelial stem cells, such
as CD24, CD133, vimentin, and kidney-injury
molecule-1 (KIM-1). Their results provided no
evidence for an intratubular stem cell population,
but rather indicated that terminally-differentiated
tubular epithelia de-differentiate to some extent
by re-expressing stem cell markers and repair the
damaged tubules [205].
Consistent with these results, Vogetseder et al.
investigated the contribution of stem cells to the
growth of proximal tubular S3 segments in healthy
rats by examining cell cycle status with the applica-
tion of thymidine analog and bromo-deoxyuridine
(BrdU) and the expression of cell cycle markers,
such as the G(1)-phase marker cyclin D1
[206, 207]. It was shown that cycling and
non-cycling tubular cells have the same degree of
differentiation, that the transit amplifying cells, usu-
ally thought to be the descendent of stem cells, were
absent and that most tubular cells in the S3 segment
divided or entered the cell cycle in response to a
proliferative stimulus. These results indicate that
tubular cells originate from differentiated tubular
cells, not from stem cells [206]. The group also
demonstrated that around 40 % of S3 tubular cells
are in G(1) phase and that a strong mitotic stimulus
shortens the period of quiescence following cell
division, suggesting that the capacity of the proxi-
mal tubule to rapidly recruit cells into division
depends on a large reserve pool of G(1) cells and
on the shortening of the quiescence period [207].
Similar results were demonstrated by Berger
et al. using the transgenic PEC (parietal epithelial
cell)-reverse-tetracycline trans-activator (rtTA)
mouse, which allowed cell fate tracing of
547
regenerating proximal tubular cells [208]. They
found that tubular regeneration after I/R injury
occurred from any surviving tubular cells rather
than from fixed progenitor cells.
Rinkevich et al. examined the mechanisms and
magnitude by which the mammalian kidney gen-
erates and maintains tubular cells by using multi-
colored “Rainbow” mice, in which every cell is
randomly marked by one of four fluorescent
colors, allowing long term lineage tracing and
clonal analysis of new tubular cells [209]. It was
demonstrated that the adult mammalian kidney
undergoes tubulogenesis during homeostasis and
after damage by clonal expansion of lineage- or
fate-restricted precursor cells that give rise only to
cells of the same nephron segment, and that
the adult renal clones are derived from
Wnt-responsive precursors. They concluded that
fate-restricted precursors functioning as unipotent
progenitors continuously maintain and self-
preserve the mouse kidney throughout life.
In summary, the origin of regenerating tubular
cells is fully-differentiated tubular epithelia, but at
the same time these cells may acquire some features
of unipotent epithelial stem-like cells that can clon-
ally expand under specific proliferation stimuli.
However, most studies examining the origin of
tubular regeneration have been performed in AKI
mice models induced by I/R injury. It is known that
different regenerative mechanisms can take place
in different disease status in other organ systems.
For example, liver normally regenerates from
injury, such as partial hepatectomy, by simply
expanding the remaining hepatocytes, whereas
some stem/progenitor populations, known as oval
cells, are responsible for the regeneration after
specific types of insults, such as toxic drugs
[210, 211]. These oval cells are termed facultative
stem/progenitor cells. It is therefore possible that
different regenerative mechanisms may be
involved in other kidney injury models too.
Adult Renal Stem/Progenitor Cells
Expanded Ex Vivo
Many studies have already characterized adult
renal stem/progenitor cells by mainly using
548 K. Osafune

Glomerulus Juxtaglomerular
Proximal compartment
tubule ・Sagrinati et al., ・Bruno et al.,
2006, CD24(+) 2009, resident ・Pippin et al., 2013,
CD133(+) cell MSC Renin(+) cell
・Maeshima et al.,
2003, LRC
・Kitamura et al., Distal
2005, Cloned cell tubule
・Challen et al., 2006,
SP cell
・Gupta et al., 2006,
MRPC
・Buzhor et al., 2013,
NCAM1(+) cell

Interstitium

・Oliver et al.,2004,
LRC
・Bussolati et al.,
2005, CD133(+) cell
・Hishikawa et al., Henle’s
2005, SP cell loop
Collecting
・Dekel et al., 2006,
duct
Sca1(+)Lin(-) cell
・Lee et al., 2010, ・Li et al., 2014,
MKPC MSC-like cell

Fig. 7 Adult renal stem/progenitor cells isolated from various locations by different methods

in vitro cultures. These cell populations might
result from cell-culture–induced changes in mor-
phology, behavior, and marker expression, but
still show the possibility of clinical usefulness.
The establishment of methods to expand
nephron-forming cells from adult kidney while
preserving their stem/progenitor function would
supply a source for cell therapy.
Renal stem/progenitor cells have been reported
to exist in or were isolated from various locations
in adult kidneys [212, 213], including the renal
papilla [214, 215], interstitium [216–218], tubular
epithelial cells [219–223], Bowman’s capsule
[97, 224, 225], decapsulated glomeruli [226],
juxtaglomerular apparatus [227] and collecting
ducts [228], by various identification methods,
such as label-retaining assays [214, 219–221],
clonogenic assays [222, 223, 229], sphere or
spheroid assays [230, 231], flow cytometry using
cell surface marker [97, 224, 225], lineage tracing
[227] and side population (SP) assays ([216,
232–234]; Fig. 7, Table 3).
The label-retaining cells (LRCs) examined by
in vivo BrdU labeling exhibited a characteristic
slow cycling of tissue-specific stem cells.
Maeshima et al. showed that LRCs were found
in renal epithelial tubular cells of rat kidney and
that they reentered mitosis and differentiated into
tubular epithelia in response to renal ischemia,
thus contributing to tubular regeneration after
damage [219]. The group also reported that these
tubular LRCs proliferate, migrate and transdif-
ferentiate into fibroblast-like cells during renal
fibrosis [220]. In addition, tubular LRCs isolated
 18

Table 3 Summary of reports on the isolation or identification of adult renal stem/progenitor cells
Location or
Author Assay Species origin in kidney Medium and main factor Functional examination References
Maeshima A LRC Rat Proximal tubule DMEM/F12/5 % FCS with 200 ng/mL EGF, Formation of tubular or tubulocystic structures [219–221]
et al. 2003 200 ng/mL TGF-α, 200 ng/mL IGF-I, or in collagen gel. Integration into proximal
100 ng/mL HGF tubules and collecting ducts by injection into
metanephros in an organ culture
Oliver JA LRC Rat, Interstitium DMEM/F12/10 % FCS with 5 % chicken In vitro differentiation into mesenchymal, [214]
et al. 2004 mouse (Papilla) embryo extract and 5 % rat serum epithelial and neural cells and myofibroblasts,
and sphere formation. Integration into renal
parenchyma by injection into the renal
subcapsule of transient ischemic rat
Bussolati B Surface Human Intestitium 60 % LG-DMEM/40 % MCDB-201 with In vitro differentiation into tubular epithelia [217]
et al. 2005 markers (Cortex) 10 ng/mL EGF, 10 ng/mL PDGF-BB and 2 % and endothelia. Formation of tubules and
CD133(+) FCS vessels by subcutaneous injection into
immunodeficient mouse and integration into
Translational Research Methods: Renal Stem Cells

tubules of AKI models by glycerol-induced

rhabdomyolysis
Hishikawa K SP Mouse Intestitium NA Integration into interstitium and therapeutic [216]
et al. 2005 effects by injection into cisplatin-induced AKI
models
Kitamura S Clonogenic Rat Proximal tubule 1:1 mixture of culture supernatant of mouse In vitro differentiation into proximal and distal [222, 223]
et al. 2005 culture (S3 segment) mesenchymal cells in DMEM/10 % FCS and renal tubular cells and epithelia of Henle’s
modified K1 medium (1:1 DMEM and Ham’s loop. Formation of tubular structures and
F12)/10 % FCS with 25 ng/mL HGF integration into tubules and therapeutic effects
on cisplatin-induced and I/R injury AKI
models. Responsiveness to PTH and AVP
Challen GA SP Mouse Mainly proximal DMEM/10 % FCS In vitro differentiation into osteocytes and [234]
et al. 2006 tubule adipocytes and integration into metanephric
mesenchyme- and ureteric bud-derived
structures by injection into mouse
metanephros. In vivo integration into
glomeruli, proximal and distal tubules and
collecting ducts at low frequency. Therapeutic
effects on adriamycin-induced acute
glomerular injury models
549

(continued)
 550

Table 3 (continued)
Location or
Author Assay Species origin in kidney Medium and main factor Functional examination References
Sagrinati C Surface Human Bowman’s Endothelial growth medium-microvascular In vitro differentiation into proximal and distal [97]
et al. 2006 markers capsule (EGM-MV)/20 % FCS tubular epithelia, osteocytes, adipocytes and
CD24(+) neural cells. Formation of tubular structures of
CD133(+) different nephron segments with therapeutic
effects by intravenous injection into AKI
models by glycerol-induced rhabdomyolysis
Gupta S Low Rat Proximal tubule 60 % DMEM-LG/40 % MCDB-201 with In vitro differentiation into cells expressing [235]
et al. 2006 density 1 mg/mL LA-BSA, 2 % FCS, 10 ng/mL EGF, endothelial, hepatocyte and neural markers.
culture 10 ng/mL PDGF-BB and 10 ng/mL LIF Integration into proximal and distal tubules by
injection into I/R injury AKI models without
therapeutic benefits
Dekel B Surface Mouse Intestitium DMEM 1.0 g/mL glucose/10 % FCS In vitro differentiation into myocytes, [215]
et al. 2006 markers (Papilla) osteocytes, adipocytes and neural cells.
Sca1(+)Lin Integration into tubules by injection into I/R
() injury AKI models
Bruno S Surface Human Decapsulated RPMI/10 % FCS In vitro differentiation into osteocytes, [226]
et al. 2009 markers glomerulus adipocytes, chondrocytes, endothelia,
CD133() podocytes and mesangial cells
CD146(+)
Lee PT Myh9(+) Mouse Interstitium LG-DMEM/10 % charcoal-stripped calf In vitro differentiation into endothelia and [218]
et al. 2010 cells from (Medulla and serum (CCS) osteoblasts. Integration into endothelia and
Myh9-GFP papilla) tubular epithelia and formation of blood
mice vessels by injection into normal mice.
Therapeutic effects on I/R injury AKI models
with improved mortality
K. Osafune
 18

Buzhor E Spheroid Human Proximal and Serum-containing medium (SCM): IMDM/10 In vitro differentiation into tubular structures [229]
et al. 2011 culture distal renal % FCS with 50 ng/mL bFGF, 50 ng/mL EGF of different nephron segments by grafting onto
tubules and and 5 ng/mL SCF. Serum-free medium the chorioallantoic membrane (CAM) of chick
collecting duct (SFM): DMEM/F12 with B27 and N2 embryo
supplements, 10 ng/mL bFGF and 20 ng/mL
EGF
Pippin JW Lineage Mouse Juxtaglomerular NA In vivo differentiation into glomerular [227]
et al. 2013 tracing compartment podocytes and parietal epithelial cells
(Renin(+) cell)
Bombelli S Sphere Human ND DMEM/F12 with B27 supplement, 20 ng/mL In vitro and in vivo formation of 3D tubular [231]
et al. 2013 EGF and 20 ng/mL bFGF structures. In vitro differentiation into
proximal and distal tubular epithelia,
podocytes and endothelia
Buzhor E Clonogenic Human Proximal tubule Serum-containing medium (SCM): IMDM/10 In vitro differentiation into osteoblasts and [230]
et al. 2013 culture (NCAM1(+) % FCS with 50 ng/mL bFGF, 50 ng/mL EGF adipocytes, and formation of 3D kidney
cell) and 5 ng/mL SCF. Serum-free medium spheroid and tubular structures by grafting
(SFM): DMEM/F12 with B27 and N2 onto CAM of chick embryo. Formation of
supplements, 10 ng/mL bFGF and 20 ng/mL tubular structures by injection into
Translational Research Methods: Renal Stem Cells

EGF subcutaneous space of mice

Li J Clonogenic Mouse Collecting duct α-MEM/20 % FCS In vitro differentiation into adipocytes, [228]
et al. 2014 culture chondrocytes and osteocytes and formation of
tubular structures in collagen gel. Integration
into medullary collecting ducts by injection
into live newborn mice kidneys
LRC label-retaining cell, SP side population, NA not applicable, I/R ischemia/reperfusion, PTH parathyroid hormone, AVP arginine vasopressin, ND not determined
551
552
by flowcytometry sorting formed three-
dimensional tubular structures in vitro by cultur-
ing in collagen gel and integrated into proximal
tubules and collecting ducts when transplanted
into the metanephric kidney [221].
Oliver et al. identified a population of LRCs in
the interstitium or upper papilla of adult rodent
kidneys [214]. These LRCs can be cultured under
hypoxic conditions to form aggregates of cells
expressing Nestin, a marker in other stem cell
types. Single cell-derived clones of these cells
co-expressed mesenchymal and epithelial
markers, and gave rise to myofibroblasts and
cells expressing neuronal markers. They also
formed a compartment of rapidly proliferating
cells in response to ischemic insult, suggesting
that they may play a role after acute injury.
However, the LRCs from these reports might
represent transiently amplifying cells rather than
stem cells. Proof of the existence of a pluripotent
adult renal stem cell population with long-term
self-renewal capacity and clonogenicity remains
lacking.
Kitamura et al. screened for stem cells in var-
ious regions of the postnatal rodent kidney by the
dissection of various nephron segments and cul-
ture after dissociation to single cells [222]. They
established clonally expanded cells from the S3
segment of rat proximal renal tubules, which are
known to be most susceptible to injury and to have
the highest proliferation rate in nephron segments.
The established cell line showed features of stem
cells, could be maintained long term without
transformation, and expressed Pax2, Wnt4 and
Wt1. The transplantation of these progenitors
resulted in the contribution to renal tubules and
significantly improved the renal function in both
cisplatin-induced and I/R injury AKI
models [223].
Buzhor et al. established three-dimensional
spheroid cultures of human kidney epithelial
cells retrieved from human nephrectomy tissue
samples by suspension cultures in
low-attachment conditions [229]. The human kid-
ney spheroids upregulated certain renal develop-
mental and “stemness” markers, including PAX2,
SALL1, SIX2, WT1, NANOG and GPC3, and indi-
cated mostly an EpCAM(+)CD24(+)CD133(+)
K. Osafune
CD44(+) phenotype. When grafted in low cell
numbers onto the CAM of chick embryos, the
spheroid cells exclusively reconstituted renal
tubular epithelia of different nephron segments.
Bombelli et al. reported a modified sphere-
formation protocol to generate nephrospheres
from normal human adult kidneys [231]. The cul-
tured nephrospheres activated stem cell pathways,
contained cells at different levels of maturation
and generated three-dimensional tubular struc-
tures both in vitro and in vivo. The group identi-
fied a quiescent PKHhigh stem cell population
within the spheres that demonstrated in vitro
self-renewal capacity and differentiation potential
into epithelial, endothelial and podocytic
progenies.
Regarding isolation using the combination of
cell surface markers, Bussolati et al. first used this
methodology to isolate tubule cells expressing a
hematopoietic stem cell marker, CD133, from
human renal cortex [217]. These cells expanded,
differentiated in vitro into epithelial or endothelial
cells and showed developmental potential to form
tubule-like structures and functional vessels. They
were also incorporated into tubules when intrave-
nously injected into immunodeficient mice with
AKI induced by glycerol-induced rhabdomyolysis.
Dekel et al. used a stem cell marker, Sca1, and
identified non-tubular Sca1(+) progenitor cells in
the papillary interstitial space of the adult mouse
kidney [215]. The cloned renal Sca1(+)Lin()
cells showed in vitro differentiation potential for
myogenic, osteogenic, adipogenic and neural lin-
eages, and newly isolated Sca1(+)Lin() cells
adopted a tubular phenotype when injected
directly into the renal parenchyma of AKI mice
by I/R injury. These cells displayed features
overlapping those of the papillary LRCs described
above [214].
A group headed by Paola Romagnani has
reported that a population of CD24(+)CD133(+)
cells localized at the urinary pole of the Bowman’s
capsule in adult human, mouse or rat kidneys
represents renal progenitors that give rise to both
glomerular podocytes and renal tubular epithelia.
Clonally-expanded CD24(+)CD133(+) cells also
contributed to the regeneration of tubular struc-
tures and significantly ameliorated functional
18 Translational Research Methods: Renal Stem Cells
kidney damage after their intravenous injection
into immunodeficient mice with AKI induced by
rhabdomyolysis [97, 224]. It has also been
reported that these progenitor cells compose the
hyperplastic glomerular lesion in several human
proliferative glomerulonephritis [225].
Bruno et al. found that human glomeruli
deprived of Bowman’s capsule contain a popula-
tion of CD133()CD146(+) cells that express
MSC markers and renal stem cell markers,
CD24 and PAX2 [226]. These MSCs exhibit
self-renewal capability, clonogenicity and
multipotency capable of osteogenic, adipogenic
and chondrogenic differentiation. In addition,
they are able to differentiate into endothelia, epi-
thelial cells expressing podocyte markers and
mesangial marker-expressing cells. The expres-
sion of the kidney-specific PAX2 gene and deter-
mination of donor sex identity when the MSCs
were isolated from glomeruli of a renal allograft
suggested these cells are a tissue-resident
population.
Buzhor et al. established a two-dimensional and
low-density clonal culture of human kidney epithe-
lial cells from adult kidney tissue of
nephrectomized patients, in which the reactivation
of gene expression of an embryonic mesenchymal
marker, NCAM1, was observed in a specific cell
population that attained a stem/progenitor state
[230]. The isolated NCAM1(+) cells showed prox-
imal tubular origin, expressed early nephron pro-
genitor markers, such as PAX2, SALL1, SIX2 and
WT1, and acquired a mesenchymal fate, indicated
by high VIMENTIN and reduced E-CADHERIN
expression levels. The cell population showed
clonogenic potential by single cell cultures, gener-
ated three-dimensional kidney spheroids, and dif-
ferentiated into tubular structures when grafted
onto the CAM of chick or into the subcutaneous
space of immunodeficient mice.
Gupta et al. reported the isolation of
multipotent renal progenitor cells (MRPCs) from
rat kidneys by modifying the culture conditions of
renal cells to preserve only progenitor cells
[235]. MRPCs were isolated from adult rat kid-
neys using low density culture conditions for 4–6
weeks that were similar to those used for the
culture of bone marrow-derived multipotent
553
adult progenitor cells [236]. MRPCs expressed
Oct4 and Pax2 and differentiated into tubular-
structures in vitro and in vivo. However, these
cells displayed spindle-shaped morphology,
suggesting non-tubular and possibly extra-renal
origin.
Pippin et al. demonstrated by performing line-
age tracing experiments that cells of renin lineage
in the juxtaglomerular compartment serve as pro-
genitors of glomerular podocytes and parietal epi-
thelial cells in experimental glomerular disease
[227]. Renal cells expressing renin were geneti-
cally fate-mapped in adult Ren1cCreER x
Rs-tdTomato-R, Ren1cCre x Rs-ZsGreen-R- and
Ren1dCre x Z/EG reporter mice, and podocyte
depletion was induced in these mice by cytotoxic
anti-podocyte antibody. After a decrease in
podocyte number, a significant increase in the
number of labelled cells of renin lineage was
observed in glomeruli in a focal distribution
along Bowman’s capsule, within the glomerular
tuft, or in both locations, and the labelled cells
expressed markers for glomerular parietal cells or
podocytes.
SP cells are defined as a cell population that
can rapidly efflux DNA-binding dyes, such as
Hoechst 33342 and Rhodamine 123, and may
exhibit stem cell-like characteristics, including
the expression of specific membrane transporters
associated with stemness [237]. SP cells in the
bone marrow overlap hematopoietic stem cells,
and the cell type has also been isolated from
other tissues, including the adult kidney, to exam-
ine their characteristics as tissue-specific stem
cells. Several groups have reported the existence
of SP cells in adult rodent kidney [216,
232–234]. However, the relative size, origin and
differentiation ability of these cells have not been
consistent. Iwatani et al. showed no evidence for a
capacity to transdifferentiate into renal cells
in vivo [233]. Hishikawa et al. reported that
Musculin/MyoR was a marker of renal SP cells,
and these cells were located in the interstitium of
the recipient kidney when administered into a
cisplatin-induced AKI mouse model, but showed
little evidence for transdifferentiation [216].
Challen et al. using more stringent isolation pro-
cedures, demonstrated that renal SP cells
554
represent 0.1 % of the kidney [234]. These cells
showed multilineage differentiation potential into
osteocytes and adipocytes in vitro, and into sev-
eral nephron segments in vivo at low efficiency
after their injection into an adriamycin-induced
acute glomerular injury model. However, despite
stringent selection, the cell population remains
heterogeneous, including a monocytic fraction,
which may result in the apparent phenotypic plas-
ticity. In both studies, the injection of adult kidney
SP cells did not show significant tubular integra-
tion in acute renal damage models, although the
treatment reduced renal damage. While SP cells in
bone marrow overlap the hematopoietic stem cells
isolated by flowcytometry sorting using cell sur-
face markers, the population derived from other
organs does not always show characteristics of
tissue-specific stem cells. This finding may also
apply to renal SP cells. Nevertheless, these results
suggested that humoral factors from SP cells may
directly ameliorate renal injury rather than con-
tributing to the host tissues. Isolation techniques
for renal SP cells with high reproducibility could
solve the problem of heterogeneity and facilitate
further analyses.
Lee et al. isolated mouse kidney progenitor
cells (MKPCs) with MSC properties that reside
in the mouse kidney interstitium of the medulla
and papilla [218]. MKPCs displayed features
including spindle-shaped morphology, self-
renewal of more than 100 passages without evi-
dence of senescence, and expression of Oct4,
Pax2, Wnt4, Wt1, vimentin, α-smooth muscle
actin, CD29 and S100A4. MKPC exhibited plas-
ticity and multipotent differentiation potential, as
demonstrated by the ability to differentiate into
endothelial cells and osteoblasts in vitro and endo-
thelial and tubular epithelial cells in vivo. Impor-
tantly, intrarenal injection of MKPCs in
immunodeficient mice with I/R injury rescued
renal damage both histologically and functionally,
and some MKPCs formed vessels with red blood
cells inside and incorporated into renal tubules. In
addition, MKPC treatment reduced the mortality
of mice after I/R injury.
Li et al. has recently identified a MSC-like
population from the collecting duct within adult
mouse kidney that undergoes an epithelial-to-
K. Osafune
mesenchymal transition to form clonogenic,
long-term self-renewal and retains a capacity to
form epithelial structures in vitro and in vivo
[228]. After extensive passage, these kidney
MSC-like cells selectively integrated into the
AQP2(+) medullary collecting duct when
microinjected into the kidneys of neonatal mice.
Interestingly, examination of the origin of kidney
MSC-like cells revealed that a Wnt4-expressing
interstitial population intercalates into the
collecting duct in neonatal kidney, giving rise to
the MSC-like cells.
Various types of adult renal stem/progenitor
cells have been identified by different methods.
Although these adult stem/progenitor cells have
been reported to contribute to the epithelia of
glomeruli and renal tubules like embryonic pro-
genitors, they may represent a different cell pop-
ulation from embryonic nephron progenitors and
possess different gene expression profiles and cel-
lular origins. Detailed characterization of these
cells and examination of the therapeutic potential
of each stem/progenitor cell population are there-
fore required.
Stem Cell Niche in Adult Kidney
Stem cells are usually located in specific anatomic
locations termed “niches,” which regulate how
stem cells participate in tissue generation, main-
tenance and repair [238]. Stem cells are located in
deep, narrow tissue niches, while transiently
amplifying precursor cells and differentiated
cells at various stages of maturation lie in the
progressively more distal cell layers. In addition
to heterologous cellular components, any given
niche is thought to consist of an extracellular
matrix and other non-cellular constituents. Both
paracrine and metabolic factors could also serve
as a niche, since they respond to exogenous sig-
nals via a sensing system consisting of the ner-
vous and/or circulatory system. Accordingly, the
perivascular site is considered a stem cell niche
[171, 239, 240].
Other studies have indicated that the MSC
compartment, which resides in the connective tis-
sue of most organs and is capable of
18 Translational Research Methods: Renal Stem Cells
differentiating into osteoblasts, adipocytes and
chondrocytes, extends throughout the body,
including the kidneys, as a result of its
perivascular location. Based on similar marker
genes, such as CD146, 3G5 [241], and differenti-
ation potential into osteoblasts, adipocytes and
chondrocytes, it has been hypothesized that vas-
cular pericytes may represent the MSC population
within any given organ. Indeed, pericytes are qui-
escent, slow-cycling cells, but in the angiogenic
stage they show highly-proliferative potential,
capacity for self-renewal, and the ability to gener-
ate osteoblasts, chondroblasts, fibroblasts and
pre-adipocytes in vitro, indicating adult stem cell
characters [240].
The microvasculature of the renal papilla is
particularly rich in pericytes that regulate micro-
vascular integrity in the perivascular capillary net-
work and give the descending vasa recta, the
arteriolar segments supplying blood to the
medulla, their contractile function. Therefore,
the MSC compartment residing in the renal papilla
might be linked to the perivascular niche. Consis-
tent with this hypothesis, Lee et al. [218] identified
MKPCs with MSC properties that reside in the
mouse kidney interstitium of the medulla and
papilla in close association with endothelial cells.
Also consistent are reports by Oliver et al. [214],
who showed LRCs with several characteristics of
adult stem cells in the papillary interstitium and
collecting duct, and Dekel et al. [215], who dem-
onstrated that non-tubular Sca1(+) multipotent pro-
genitor cells are found mainly in the papillary
interstitial space of adult mouse kidney.
To date, the renal papilla has been a candidate
stem cell niche in kidney. However, there are
relatively little data on kidney stem cell niches
compared with other organs, meaning there is
still much to learn about the identify of these
niches and their mechanisms for regulating renal
stem cells.
Cancer Stem Cells in Kidney
Cancer stem cells drive tumor growth by their
self-renewal, as well as giving rise to a large
population of differentiated progeny that make
555
up the majority of cells in a tumor
[29, 30]. Their ability to initiate tumor growth
when grafted into immunocompromised mice
demonstrates that cancer stem cells exist in a
variety of human tumors [31]. Specific signalling
pathways play a functional role in self-renewal
and/or differentiation of cancer stem cells,
suggesting targeted therapies are possible. In
addition, the identification of markers that enable
the prospective isolation of cancer stem cells,
which are a minority population of tumor-
constituting cells, from whole tumor tissues
should help clarify the biological properties of
cancer stem cells [32]. To date, only a small num-
ber of studies have reported the identification of
renal cancer stem cells [33].
Cancer Stem Cells in Adult Renal Cell
Carcinoma (RCC)
Renal cell carcinoma (RCC) is a common form of
urologic tumors, accounting for approximately
3.8 % of total adult human malignancies with an
annual increasing incidence [242]. Histologic
subtypes of RCC include the clear cell carcinoma,
which is the most common form of RCC, papil-
lary carcinoma, chromophobe carcinoma,
collecting duct carcinoma, and unclassified carci-
nomas [243]. Although the prognosis of RCC has
recently improved due to earlier detection and
increased treatment options, including tyrosine
kinase inhibitors and mTor inhibitors, RCC
remains a tumor of unpredictable presentation and
poor clinical outcome [244]. RCC is considered to
originate from a mutated tubular renal population
with stem/progenitor cell properties, much like in
other organs [245]. Renal cancer stem cells have
been isolated from RCC using different stem cell
markers or by functional approaches of stem cell
isolation by sphere-formation or SP assays.
Bussolati et al. identified a population of
tumor-initiating cells from renal clear cell and
undifferentiated carcinomas by means of cell
sorting with a mesenchymal marker, CD105
[246]. These cells were cloned in stringent culture
conditions and displayed tumor-initiating ability,
generating serially transplantable carcinomas.
556
Functionally, CD105(+) cells are clonogenic and
able to generate spheres. Moreover, these cells
differentiate into epithelial cells in vitro and
in vivo to recapitulate the histological pattern of
the tumor. Interestingly, CD105(+) cells do not
express CD133, which is a candidate marker for
adult renal progenitors and also expressed in can-
cer stem cells of tumors in other organs, such as
the nervous system, colon, prostate, pancreas and
lungs [32].
Other functional assays commonly used to iden-
tify stem/progenitor cells have been used to isolate
renal cancer stem cells. Zhong et al. selected cells
from a renal cancer cell line, SK-RC-42, that have
the ability to form spheres in serum-free medium
supplemented with EGF and bFGF [247]. The cell
population expressed several stemness genes,
including Oct4, BMI, β-catenin and Nanog,
showed resistance to chemotherapeutic agents and
irradiations and have higher in vivo tumorigenicity
than monolayer adherent cells.
Addla et al. used the SP assay to identify renal
cancer stem cells [248]. SP cells isolated from
renal carcinomas expressed mesenchymal
markers including CD105, but not CD133, and
showed high proliferative potential and an ability
to give rise to differentiated spheroids. However,
the characteristics and roles of this cell population
in carcinogenesis are unknown.
Cancer Stem Cells in Wilms’ Tumor
Among pediatric malignancies, Wilms’ tumor
(nephroblastoma) is the most common and is
rated fourth in overall incidence among childhood
cancers [249]. Wilms’ tumor is typically com-
posed of three elements: undifferentiated blas-
tema, stroma and tubular structures at varing
levels of differentiation, which combined resem-
ble the nephrogenic zone of developing kidney
[33, 250]. Therefore, it is widely accepted that
Wilms’ tumor arises from transformed renal
stem cells, which maintain themselves as the
undifferentiated blastemal component of the
tumor as well as maldifferentiate to give rise to
tubular elements [250]. Thus, while embryonic
nephrogenesis is driven by normal renal stem
K. Osafune
cells, Wilms’ tumor is propagated by an equiva-
lent transformed population, termed Wilms’
tumor cancer stem cells [250]. It was reported
that the nephrogenic process in the tumor was
deranged, as the transcription factors that specify
renal stem/progenitor cells (Six2, Wt1 and Pax2)
were epigenetically dysregulated in the Wilms’
tumor blastema with aberrant DNA methylation
at the promoter regions [251] and chromatin mod-
ifications [252]. These changes may lead to an
enhanced activation and accumulation of early
stem/progenitor cells instead of undergoing the
kidney-specific differentiation program.
Pode-Shakked et al. reported that NCAM1, a
marker for normal embryonic renal progenitors, is
also a putative cancer stem cell marker in Wilms’
tumor based on microarray data [253]. It was
further found that NCAM1(+) cells showed the
expression of a “Wilms’ tumor-stem” signature
gene set and clonogenicity. The group also
performed serial transplantation of a NCAM1(+)
cell fraction within Wilms’ tumor into immuno-
deficient mice, demonstrating that NCAM1(+)
cells give rise to new tumors as well as
NCAM1() differentiated cells. Therefore, the
NCAM1(+) population fits within the criteria of
cancer stem cells, as it can self-renew and has
multipotency in vivo [254].
Shukrun et al. screened to purify the cancer stem
cell population in Wilms’ tumor and revealed
that NCAM1(+) aldehyde dehydrogenase 1
(ALDH1)(+) cells exclusively localized within the
blastemal compartment of the tumor, but that
NCAM1(+)ALDH() or NCAM1()ALDH1(+)
cells did not, and that the population had tumori-
genic potential [255]. Surprisingly, they showed
that ALDH1(+) Wilms’ tumor cancer stem cells
are not the most primitive cell population in
Wilms’ tumor blastema, but correspond to a more
committed epithelial progenitor that is manifested
by a lower expression of several renal progenitor
markers including SIX2, OSR1, PAX2, CITED1,
WT1 and SALL1, and a relatively high expression
of epithelial markers. Thus, it can be deduced that
Wilms’ tumor cancer stem cells, which sustain
tumor growth, both de-differentiated toward less
differentiated SIX2high blastemal cells and differ-
entiated into mature epithelial structures.
18 Translational Research Methods: Renal Stem Cells
In summary, both RCC and Wilms’ tumor pos-
sess cancer stem cells with high tumorigenic abil-
ity and resistance to chemo and radio therapy, thus
providing a rationale for new therapies targeting
these stem cell populations.
Clinical Application and Practical Use
of Renal Stem Cells
In order to develop regenerative therapies for kid-
ney diseases, substantial efforts have already been
made to examine the therapeutic effectiveness of
transplanting renal stem/progenitor cells. Most
studies have examined stem/progenitor cells
from adult kidneys, such as LRCs, cloned cell
lines, isolated cells with surface markers and SP
cells, by using acute renal damage models, includ-
ing I/R injury-, rhabdomyolysis- and cisplatin-
induced tubular injury models, and an
adriamycin-induced glomerular injury model
(Table 3). On the other hand, few studies have
used embryonic renal stem/progenitor cells or
chronic renal injury models. Lazzeri et al. reported
that CD24(+)CD133(+) cell populations isolated
from human embryonic kidney regenerated differ-
ent portions of the nephron, reduced tissue necro-
sis and fibrosis, and significantly improved renal
function when injected into AKI mice by
glycerol-induced rhabdomyolysis [98]. Of note,
Harari-Steinberg et al. demonstrated that the
transplantation of NCAM1(+) nephron progeni-
tors derived from aborted human embryos have
therapeutic benefits when transplanted into a 5/6
resection mice model for chronic renal
failure [102].
These reports suggest the possibility that
embryonic nephron progenitors differentiated
from human ESCs/iPSCs or reprogrammed from
adult renal cells could be used to develop cell
therapies for kidney diseases. However, no reports
describe the transplantation of renal lineage cells,
including embryonic renal progenitors, generated
from human ESCs/iPSCs or reprogrammed from
adult renal cells, into renal disease models. Since
it is difficult to obtain a sufficient number of renal
cell samples from embryonic or adult kidneys,
especially human embryos, or to expand the
557
human renal cells in vitro, renal lineage cells
generated from human ESCs/iPSCs might
become an important source for the clinical appli-
cation of cell therapy approaches.
Other research areas that utilize ESC/iPSC-
derived renal lineage cells for clinical application
or practical use include disease modelling,
drug discovery and toxicology screening
[19, 20]. iPSC technology enables the creation
of patient- or disease-specific iPSC lines that
carry disease genotypes. Differentiation of these
iPSCs into the cell types injured by the disorder
can be used as in vitro models for the study of
disease mechanisms and for the identification of
new drug compounds [256–258]. As an example,
Xia et al. reported that human iPSCs generated
from somatic cells of a patient with autosomal
dominant polycystic kidney disease (ADPKD)
could be differentiated into collecting duct cells,
one of the renal cell types injured by ADPKD,
using their differentiation method, but no results
on the mechanistic analysis of the disorder were
shown [161]. Regarding toxicology screening, Li
et al. reported that renal proximal tubule-like cells
differentiated from human ESCs could be used to
examine the nephrotoxicity of drug
compounds [259].
Research aiming to generate entire kidneys has
been conducted using ESCs/iPSCs or renal lineage
cells derived from ESCs/iPSCs [260]. One
approach to generate entire kidneys would be the
use of a biological scaffold that forms a three-
dimensional architecture. Ross et al. decellularized
rat kidneys by detergent perfusion and reseeded
them with undifferentiated mouse ESCs, resulting
in the differentiation of mouse ESCs into renal cells
in the scaffold [261]. Song et al. published a report
showing that decellularized rat kidneys were
repopulated with rat neonatal kidney cells and
HUVECs, and that the regenerated kidneys showed
urine production both in vitro and after transplan-
tation in vivo [262]. Decellularizing patient-
derived kidneys and refilling them with the renal
precursors and vascular progenitors differentiated
from the patient’s own iPSCs might lead to the
reconstruction of three-dimensional whole kidneys
that could be used for autologous renal
transplantation.
558
On the other hand, Kobayashi et al. demon-
strated that entire organs can be generated using a
method involving interspecific blastocyst comple-
mentation [263]. They injected mouse and rat
ESCs/iPSCs into Pdx1 (/) (pancreatogenesis-
disabled) rat and mouse blastocysts, respectively,
and generated an almost entirely ESC/iPSC-
derived pancreas. Similar experiments have
already been performed to generate entire kidneys
by injecting mouse ESCs/iPSCs into the blasto-
cysts of Sall1(/) mice, which lose the parts of
their renal tissues derived from the metanephric
mesenchyme and die of renal insufficiency after
birth [264]. The resultant kidneys showed a chi-
merism of donor-derived mesenchyme- and
stroma-lineage renal cells and host-derived vascu-
lar and ureteric bud-lineage cells. These
approaches hold tremendous promise for generat-
ing functional whole kidneys. The generation of
genetically engineered host animals that lack all
the components of kidneys, including the vascu-
lature, is required for further analyses.
Renal Stem Cell Research: Future
Directions
Regarding kidney development, further efforts to
identify specific marker genes for the IM and renal
lineage cells and to elucidate the developmental
mechanisms of mesoderm formation and the cell
fate commitment into renal lineages and fully-
differentiated adult renal cell types, including
those that regulate how nephron progenitors give
rise to several types of nephron-forming epithelia,
such as glomerular podocytes and renal tubular
epithelia, should be made. These investigations
are necessary to establish methods that can manip-
ulate isolated embryonic renal stem/progenitor
cells, including a method for their in vitro expan-
sion, and methods that can induce the population
by the directed differentiation of pluripotent stem
cells or by cellular reprogramming from other
adult cell types. Furthermore, investigations on
directed differentiation or cellular reprogramming
to generate renal progenitors as a model for kid-
ney development will facilitate our understanding
of kidney lineage specification. Regarding
K. Osafune
embryonic renal progenitors, while knowledge
on nephron progenitors has been increasing, a
relatively small number of studies have examined
other precursor populations, such as ureteric bud
and stromal progenitors.
In addition to the works using mouse ESCs,
recent advances in the directed differentiation of
human ESCs/iPSCs into kidney lineages make it
possible to generate embryonic renal progenitors
and immature kidney tissues in vitro. However,
further improvement in the differentiation methods
as well as more detailed characterization of the
induced renal cells and tissues based on compari-
sons with their in vivo counterparts are needed. It
remains to be answered whether the renal tissues
formed from ESC/iPSC-derived renal progenitors
can exert proper renal physiological functions, such
as glomerular filtration, tubular reabsorption and
secretion, and hormone synthesis and secretion.
There are currently few functional assay systems,
however, that can evaluate the developmental or
physiological functions of induced renal lineage
cells. Further studies will also be required to estab-
lish selective and efficient differentiation methods
for adult renal cell types from ESCs/iPSCs through
embryonic renal progenitors, such as glomerular
podocytes, renal tubular epithelia and collecting
duct cells. A stable supply of these cell types will
accelerate research for disease modelling, drug dis-
covery and toxicology screening.
Various types of adult renal progenitors have
been identified by different methods, some of
which have been shown to have therapeutic ben-
efits on renal disease models. The detailed char-
acterization of these progenitor cells and the
comparison of the therapeutic potential among
different progenitor populations are required. To
conduct these characterizations will require the
development of in vitro expansion methods and
therapeutically effective transplantation methods.
Regarding renal cancer stem cells, a definitive
selective marker for the isolation and targeting of
stem cells is still lacking. Future studies to identify
these selection markers and to understand the
intracellular pathways that regulate renal cancer
stem cell maintenance and differentiation should
contribute to the development of new therapeutic
strategies.
18 Translational Research Methods: Renal Stem Cells
Conclusion
Because of the recent advances in elucidating the
mechanisms of kidney development, the biology
of embryonic renal stem/progenitor cells has been
increasingly clarified. The CM population in
metanephros, which is marked by Six2 expres-
sion, represents a pool of embryonic renal pro-
genitors, and it is currently accepted that the
post-natal mammalian kidney does not harbor
a progenitor population equivalent to the
CM. NCAM1(+) progenitors in human embryonic
kidney might also be nephron-forming and clini-
cally useful cells, although ethical issues and the
allogenetic nature may limit the use of these cells.
The generation of embryonic renal progenitors
from ESCs/iPSCs by directed differentiation or
from adult renal cells by cellular reprogramming
would also provide a potential source for the
clinical application of renal progenitors. Directed
differentiation methods from mouse and human
ESCs/iPSCs into embryonic renal progenitors,
such as nephron progenitors and ureteric bud,
have been increasingly reported, and renal struc-
tures, including glomeruli, tubules and collecting
ducts, can be generated in vitro from the progen-
itor cells, although the differentiation methods
needed for such strategies have not yet been
fully established.
Several recent studies have revealed the source
of the newly-formed regenerating renal tubular
epithelia after acute injury, demonstrating that
the repair of injured renal epithelia starts from
mature tubular cells, rather than extra-renal or
resident stem cells. In this process, mature tubular
cells show a marked plasticity, entering into an
activated proliferative state characterized by the
reactivation of mesenchymal markers detectable
during nephrogenesis.
Adult renal stem/progenitor cells contain sev-
eral different cell populations, although they may
not contribute to in vivo tubular regeneration
after acute injury. These adult progenitor cells
may not be equivalent to those in embryonic
kidneys. The renal papilla contains a population
of adult stem cells with MSC-like characters and
might represent the perivascular renal stem cell
niche. Several studies have shown that the
559
administration of embryonic or adult stem/pro-
genitor cells can be therapeutically beneficial in
kidney injury models, suggesting this strategy
should be further examined for clinical
application.
Cancer stem cells, which exhibit tumor-
initiating capability and pluripotency, have been
isolated in renal carcinomas, although no specific
markers for the cell population have been identi-
fied and thus their characterization is mainly
based on functional assays. As in other tumors,
however, renal cancer stem cells show a high
tumorigenic ability and are resistant to chemo
and radio therapy, providing a rationale for
targeting these stem cell populations with new
therapies. A pediatric malignancy, Wilms’ tumor,
has been thought to arise from a transformed
embryonic renal progenitor. Cancer stem cells
have been isolated using several selection
markers, including NCAM1 and ALDH. Since
there exists significant relevance between kidney
development and Wilms’ tumor formation, a
tumor formation model could contribute to the
understanding of kidney development in addition
to establishing anti-cancer therapeutic strategies.
Continued efforts to isolate and characterize
the renal stem/progenitor cells in embryonic or
adult kidneys as well as in renal tumors, and to
generate renal progenitors from ESCs/iPSCs by
directed differentiation or from other cell types by
cellular reprogramming, are required. Because of
the annual increase in the number of patients with
CKD around the world, regenerative medicine
strategies using renal stem/progenitor cells are
being aggressively sought.
Acknowledgements The author would like to thank all
the members of CiRA, Kyoto University, especially
Dr. Peter Karagiannis for critically reading and revising
the manuscript, Dr. Kazutoshi Takahashi for providing the
photos of iPSCs, and apologize to authors whose studies
could not be cited owing to space limitations. The research
of the author is supported by the Japan Society for the
Promotion of Science (JSPS) through its “Funding Pro-
gram for World-Leading Innovative R&D on Science and
Technology (FIRST Program),” and by the Japan Science
and Technology Agency (JST) through its research grant
“Core Center for iPS Cell Research and Technological
Development, Research Center Network for Realization
of Regenerative Medicine.”
560
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 Translational Research Methods:
Tissue Engineering of the Kidney 19
and Urinary Tract

Austin G. Hester and Anthony Atala

Contents Introduction
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571
Patients suffering from diseased or injured geni-
The Basic Components of Tissue Engineering . . . 572
Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572
tourinary organs are often treated with reconstruc-
Biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576 tive surgery or transplants, but there is a severe
shortage of donor tissue and organs. This shortage
Engineering Specific Tissues and Organs . . . . . . . . 578
Urethra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578 worsens yearly as modern medicine increases the
Bladder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579 human lifespan. The aging population grows, and
Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581 the need for organs grows with it. Physicians and
Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . 588 scientists have begun to look to the fields of
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588
regenerative medicine and tissue engineering to
provide new options for these patients. These
fields apply the principles of cell transplantation,
material science, and bioengineering to construct
biological substitutes that can significantly
improve the quality of life of the urologic patient
by eliminating the need for intensive grafting pro-
cedures or transplant surgery.
Tissue engineering, one of the major compo-
nents of regenerative medicine, follows the prin-
ciples of cell transplantation, materials science,
and engineering to develop biological substitutes
that can restore and maintain normal organ func-
tion. Tissue engineering strategies generally fall
into two categories: the use of acellular matrices
designed to direct the body’s natural ability to use
its own cells to regenerate damaged tissue and the
use of matrices seeded with cells in the laboratory
to produce novel tissues and organs. Acellular
A.G. Hester • A. Atala (*) tissue matrices are usually prepared by
School of Medicine, Department of Urology, Wake Forest
manufacturing artificial scaffolds or by removing
Institute for Regenerative Medicine, Wake Forest
University, Winston Salem, NC, USA cellular components from donor tissues via
e-mail: ahester@wakehealth.edu; aatala@wfubmc.edu mechanical and chemical manipulation to
# Springer-Verlag Berlin Heidelberg 2016 571
E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_17
572
produce collagen-rich matrices [1–4]. These
matrices slowly degrade after implantation and
are replaced by the extracellular matrix (ECM)
proteins secreted by the ingrowing cells. Cells
themselves can also be used for therapy via injec-
tion, either with carriers such as hydrogels or
alone.
The most common way to use cells in tissue
engineering is to obtain a small piece of donor tissue
and dissociate it into individual cells in the labora-
tory. These cells are either implanted directly into
the host or are expanded in culture and attached to a
support matrix. The cell/matrix construct is then
reimplanted into the host. The source of the donor
tissue can be heterologous (such as bovine), alloge-
neic (same species, different individual), or autolo-
gous. Ideally, autologous cells are used, because in
this case, both structural and functional tissue
replacement will usually occur with minimal com-
plications. To accomplish this, a biopsy of tissue is
obtained from a host, the cells are dissociated and
expanded in culture, and the expanded cells are
implanted back into the same host [2, 5–12]. The
use of autologous cells, although it may cause an
inflammatory response, avoids rejection, and thus,
the deleterious side effects of lifelong immunosup-
pression can be avoided.
However, for many patients with extensive
end-stage organ failure, a tissue biopsy may not
yield enough normal cells for expansion and
transplantation. In other instances, primary autol-
ogous cells cannot be expanded from a particular
organ, such as the pancreas. In these situations,
stem cells are envisioned as an alternative source
of cells from which the desired tissue can be
derived. Stem cells can be derived from discarded
human embryos (human embryonic stem cells),
from fetal tissue, or from adult sources (bone
marrow, fat, skin). However, there are ethical
issues involved in the use of embryonic stem
cells, and most human applications are currently
banned in the United States. Despite this, the field
of stem cell biology is advancing rapidly, and
cutting-edge techniques such as therapeutic clon-
ing and somatic cell reprogramming circumvent
some of the ethical questions and offer a poten-
tially limitless source of these cells for tissue
engineering applications.
A.G. Hester and A. Atala
This chapter will review the major components
of most tissue engineering techniques and will
describe how these techniques are being applied
to the reconstruction and regeneration of the gen-
itourinary system.
The Basic Components of Tissue
Engineering
Cells
Native Cells
In the past, one of the limitations of applying cell-
based regenerative medicine techniques to organ
replacement was the inherent difficulty of grow-
ing certain cell types in large quantities. Even
when some organs, such as the liver, have a high
regenerative capacity in vivo, cell growth and
expansion in vitro can be difficult. By studying
the privileged sites for committed precursor cells
in these organs, as well as by exploring the con-
ditions that promote differentiation and/or self-
renewal of these cells, it has been possible to
overcome some of the obstacles that limit cell
expansion in vitro. One example is the urothelial
cell. Typically, in the past, intestinal segments
have been used to replace urinary structures in
patients with congenital or acquired defects. How-
ever, this comes with a series of complications,
including metabolic imbalances, urinary stone
formation, hypocontractility, and neoplasia, to
name a few [13]. Several protocols have been
developed that identify the undifferentiated cells
in a mixed culture of cells isolated from the uri-
nary tract and keep them undifferentiated during
their growth phase, which now allows expansion
of a urothelial culture that initially covered a sur-
face area of 1 cm2 to one covering a surface area
of 4202 m2 (the equivalent of one football field)
within 8 weeks [11, 14–17]. More recent studies
have developed methods of isolating these cells
from urine samples and bladder washings rather
than from more invasive endoscopic and surgical
techniques [18, 19]. More studies have indicated
that it is possible to collect autologous bladder
cells from human patients, expand them in cul-
ture, and return them to the donor in sufficient
19 Translational Research Methods: Tissue Engineering of the Kidney and Urinary Tract
quantities for reconstructive purposes [11, 15–17,
20–22]. In fact, seven pediatric patients requiring
cystoplasty secondary to a myelomeningocele
were implanted with bladders grown from autol-
ogous urothelial and muscle cells [23]. Major
advances in cell culture techniques have been
made within the past decade, and these techniques
make use of autologous cells possible for clinical
application.
Embryonic Stem Cells
In 1981, pluripotent cells were found in the inner
cell mass of the human embryo, and the term
“human embryonic stem cell” was coined
[24]. These cells are able to differentiate into all
cells of the human body, excluding placental cells
(only cells from the morula are totipotent, i.e.,
able to develop into all cells of the human body).
These cells have great therapeutic potential, but
their use is limited by both biological and ethical
factors.
The political controversy surrounding stem
cells began in 1998 with the creation of human
embryonic stem (hES) cells derived from
discarded embryos. hES were isolated from the
inner cell mass of a blastocyst (an embryo 5 days
postfertilization) using an immunosurgical tech-
nique. Given that some cells cannot be expanded
ex vivo, ES cells could be the ideal resource for
tissue engineering because of their fundamental
properties: the ability to self-renew indefinitely
and the ability to differentiate into cells from all
three embryonic germ layers. Skin and neurons
have been formed, indicating ectodermal
differentiation [25–28]. Blood, cardiac cells, car-
tilage, endothelial cells, and muscle have been
formed, indicating mesodermal differentiation
[29–31]. Pancreatic cells have been formed, indi-
cating endodermal differentiation [32]. In addi-
tion, as further evidence of their pluripotency,
embryonic stem cells can form embryoid bodies,
which are cell aggregations that contain all three
embryonic germ layers while in culture, and can
form teratomas in vivo [33]. These cells have
demonstrated longevity in culture and can main-
tain their undifferentiated state for at least 80 pas-
sages when grown using current published
protocols [34, 35]. Recent studies have
573
demonstrated the ability to differentiate bone mar-
row mesenchymal stem cells into smooth muscle
and urothelium-like cells, resulting in another
potential cellular basis for bladder tissue engineer-
ing [36]. Additionally, another study indicated
that urine-derived stem cells demonstrate even
more growth potential than bone marrow mesen-
chymal stem cells, thus producing yet another
cellular background. These cells have demon-
strated the ability to expand in culture and to
differentiate into multiple cell lines [37].
However, in addition to the ethical dilemma
surrounding the use of embryonic stem cells,
their clinical application is limited because they
represent an allogeneic resource and thus have the
potential to evoke an immune response. New stem
cell technologies (such as somatic cell nuclear
transfer and reprogramming) promise to over-
come this limitation.
Therapeutic Cloning (Somatic Cell
Nuclear Transfer)
Somatic cell nuclear transfer (SCNT), or thera-
peutic cloning, entails the removal of an oocyte
nucleus in culture, followed by its replacement
with a nucleus derived from a somatic cell
obtained from a patient. Activation with
chemicals or electricity stimulates cell division
up to the blastocyst stage.
At this point, it is extremely important to dif-
ferentiate between the two types of cloning that
exist – reproductive cloning and therapeutic clon-
ing. Both involve the insertion of donor DNA into
an enucleated oocyte to generate an embryo that
has identical genetic material to its DNA source.
However, the similarities end there. In reproduc-
tive cloning, the embryo is then implanted into the
uterus of a pseudopregnant female to produce an
infant that is a clone of the donor. A world-famous
example of this type of cloning resulted in the
birth of a sheep named Dolly in 1997 [38]. How-
ever, there are many ethical concerns surrounding
such practices, and as a result, reproductive clon-
ing has been banned in most countries.
While therapeutic cloning also produces an
embryo that is genetically identical to the donor,
this process is used to generate blastocysts that are
explanted and grown in culture, rather than in
574
Fig. 1 Strategies for therapeutic cloning in regenerative medicine
utero. Embryonic stem cell lines can then be
derived from these blastocysts, which are only
allowed to grow up to a 100-cell stage. At this
time, the inner cell mass is isolated and cultured,
resulting in ES cells that are genetically identical
to the patient. This process is detailed in Fig. 1.
It has been shown that nuclear-transferred ES cells
derived from fibroblasts, lymphocytes, and olfac-
tory neurons are pluripotent and can generate live
pups after injection into blastocysts. This shows
that cells generated by SCNT have the same
developmental potential as blastocysts that are
fertilized and produced naturally [39–42]. In addi-
tion, the ES cells generated by SCNT are perfectly
matched to the patient’s immune system and no
immunosuppressants would be required to pre-
vent rejection should these cells be used in tissue
engineering applications.
Although ES cells derived from SCNT contain
the nuclear genome of the donor cells, mitochon-
drial DNA (mtDNA) contained in the
oocyte could lead to immunogenicity after trans-
plantation. To assess the histocompatibility of
tissue generated using SCNT, Lanza
et al. microinjected the nucleus of a bovine skin
A.G. Hester and A. Atala
fibroblast into an enucleated oocyte [43].
Although the blastocyst was implanted (reproduc-
tive cloning), the purpose was to generate renal,
cardiac, and skeletal muscle cells, which were
then harvested, expanded in vitro, and seeded
onto biodegradable scaffolds. These scaffolds
were then implanted into the donor steer from
whom the cells were cloned to determine if cells
were histocompatible. Analysis revealed that
cloned renal cells showed no evidence of T-cell
response, suggesting that rejection will not neces-
sarily occur in the presence of oocyte-derived
mtDNA. This finding represents a step forward
in overcoming the histocompatibility problem of
stem cell therapy.
Although promising, SCNT has certain limita-
tions that require further improvement before its
clinical application, in addition to the ethical con-
siderations regarding the potential of the resulting
embryos to develop into cloned embryos if
implanted into a uterus. In addition, this technique
has not been shown to work in humans. The initial
failures and fraudulent reports of nuclear transfer
in humans reduced enthusiasm for human appli-
cations [44–46], although it was recently reported
19 Translational Research Methods: Tissue Engineering of the Kidney and Urinary Tract
that nonhuman primate ES cell lines were gener-
ated by SCNT of nuclei from adult skin fibroblasts
[47, 48].
Before SCNT-derived ES cells can be used as
clinical therapy, careful assessment of quality of
the lines must be determined. For example, some
cell lines generated by SCNT have contained
chromosomal translocations, and it is not known
whether these abnormalities originated from
aneuploid embryos or if they occurred during ES
cell isolation and culture. In addition, the low
efficiency of SNCT (0.7 %) and the inadequate
supply of human oocytes further hinder the ther-
apeutic potential of this technique, as well as the
continued need for cell cycle synchronization
between the donor cells and recipient oocytes
[49, 50]. Still, these studies renew the hope that
ES cell lines could one day be generated from
human cells to produce patient-specific stem
cells with the potential to cure many human dis-
eases that are currently untreatable.
Reprogrammed Somatic Cells
Recently, exciting reports of the successful trans-
formation of adult cells into pluripotent stem cells
through a type of genetic “reprogramming” have
been published. Reprogramming is a technique
that involves dedifferentiation of adult somatic
cells to produce patient-specific pluripotent stem
cells, eliminating the need to create embryos.
Cells generated by reprogramming would be
genetically identical to the somatic cells (and
thus, the patient who donated these cells) and
would not be rejected. Yamanaka was the first to
discover that mouse embryonic fibroblasts
(MEFs) and adult mouse fibroblasts could be
reprogrammed into an “induced pluripotent state
(iPS)” [51]. These iPS cells possessed the immor-
tal growth characteristics of self-renewing ES
cells, expressed genes specific for ES cells, and
generated embryoid bodies in vitro and teratomas
in vivo. When iPS cells were injected into mouse
blastocysts, they contributed to a variety of cell
types. However, although iPS cells selected in this
way were pluripotent, they were not identical to
ES cells. Unlike ES cells, chimeras made from iPS
cells did not result in full-term pregnancies. Gene
expression profiles of the iPS cells showed that
575
they possessed a distinct gene expression signa-
ture that was different from that of ES cells.
In addition, the epigenetic state of the iPS cells
was somewhere between that found in somatic
cells and that found in ES cells, suggesting that
the reprogramming was incomplete.
These results were improved significantly by
Wernig and Jaenisch in July 2007 [52]. In this
study, DNA methylation, gene expression pro-
files, and the chromatin state of the reprogrammed
cells were similar to those of ES cells. Teratomas
induced by these cells contained differentiated
cell types representing all three embryonic germ
layers. Most importantly, the reprogrammed cells
from this experiment were able to form viable
chimeras and contribute to the germ line like ES
cells, suggesting that these iPS cells were
completely reprogrammed.
It has recently been shown that reprogramming
of human cells is possible [53, 54]. Yamanaka
generated human iPS cells that are similar to
hES cells in terms of morphology, proliferation,
gene expression, surface markers, and teratoma
formation. Thompson’s group showed that retro-
viral transduction of the stem cell markers OCT4,
SOX2, NANOG, and LIN28 could generate plu-
ripotent stem cells. However, in both studies, the
human iPS cells were similar but not identical to
hES cells. The major concern with this technique
is that these retroviral genes could increase tumor-
igenesis [49]. A study by Okita in 2007 demon-
strated 20 % of subjects generated from Nanog-
iPS cells developed tumors secondary to c-myc
retroviral expression [49, 55]. However, this study
along with another study has indicated that by
using transient expression, there is silencing of
the viral transcripts and thus can be used only
for induction of pluripotency rather than mainte-
nance [49, 55, 56]. The study by Meissner has also
indicated that iPS cells do not necessarily require
transgenic donor cells but can in fact be formed
from non-transgenic donor cells [56].
Even more recently, studies are now indicating
there is a more effective manner to reprogram
somatic cells using a technique called stimulus-
triggered acquisition of pluripotency (STAP),
which uses external stimuli to induce pluripotency
in somatic cells as opposed to nuclear
576
manipulation [57, 58]. In particular, Obokata
noted that cells treated in a low-pH environment
(pH 5.4–5.8) were able to be reprogrammed to
express the gene Oct4-GFP, a marker of
pluripotency, with considerable decreases in the
methylation patterns in the regulatory regions.
In addition, cells produced via the STAP method
have been shown to contain both embryonic and
placental tissues [57, 58]. While this technique
still requires further exploration and many ques-
tions remain, including why this mechanism
exists, this is certainly a promising path for tissue
engineering.
Placental and Amniotic Fluid Stem Cells
Recently, it has been shown that pluripotent cells
may be derived from the amniotic fluid and pla-
centa. Both amniotic fluid and placenta are known
to contain multiple partially differentiated cell
types derived from the developing fetus. Stem
cell populations have been isolated from these
sources. Called amniotic fluid and placental stem
cells (AFPSC), they express embryonic and adult
stem cell markers [59]. The undifferentiated stem
cells expand extensively without a feeder cell
layer and double every 36 h. Unlike human
embryonic stem cells, the AFPSC do not form
tumors in vivo. Lines maintained for over
250 population doublings retained long telomeres
and a normal complement of chromosomes. AFS
cells are broadly multipotent, and human lines can
be induced to differentiate into cell types
representing each embryonic germ layer, includ-
ing cells of adipogenic, osteogenic, myogenic,
endothelial, neuronal, and hepatic lineages.
Examples of differentiated cells derived from
AFS cells and displaying specialized functions
include neuronal lineage secreting the neurotrans-
mitter L-glutamate or expressing G-protein-gated
inwardly rectifying potassium (GIRK) channels,
hepatic lineage cells producing urea, and osteo-
genic lineage cells forming tissue-engineered
bone. In this respect, they meet a commonly
accepted criterion for pluripotent stem cells, with-
out implying that they can generate every adult
tissue. The cells could be obtained either from
amniocentesis or chorionic villous sampling in
the developing fetus or from the placenta or
A.G. Hester and A. Atala
human fetal somatic cells from the chorion at the
time of birth and thus stir less of an ethical debate
[60, 61]. They could be preserved for self-use and
used without rejection, or they could be banked.
A bank of 100,000 specimens could potentially
supply 99 % of the US population with a perfect
genetic match for transplantation. Such a bank
may be easier to create than with other cell
sources, since there are approximately 4.5 million
births per year in the United States [59].
Biomaterials
In the most common tissue engineering proce-
dures, isolated cells are seeded onto a scaffold
composed of an appropriate biomaterial. These
biomaterials replicate the biologic and mechanical
function of the native extracellular matrix (ECM)
found in tissues in the body by serving as an
artificial ECM. Biomaterials provide a three-
dimensional space for the cells to develop into
new tissues with appropriate structure and func-
tion. They can also allow delivery of appropriate
bioactive factors (e.g., cell adhesion peptides,
growth factors) to the developing tissue [62] to
help regulate cellular function. As the majority of
mammalian cell types are anchorage-dependent
and will die if no cell adhesion substrate is avail-
able, biomaterials provide this substrate that can
deliver cells to specific sites in the body with high
loading efficiency. Biomaterials can also provide
mechanical support against in vivo forces so that
the predefined three-dimensional structure of the
engineered implant is maintained during tissue
development.
The ideal biomaterial should be biodegradable
and bioresorbable and support the replacement of
normal tissue without inducing inflammation.
Incompatible materials are destined for an inflam-
matory or foreign-body response that eventually
leads to rejection and/or necrosis. Degradation
products, if produced, should be removed from
the body via metabolic pathways at an adequate
rate so that the concentration of these degradation
products in the tissues remains at a tolerable level
[63]. The biomaterial should also provide an envi-
ronment in which appropriate regulation of cell
19 Translational Research Methods: Tissue Engineering of the Kidney and Urinary Tract
behavior (adhesion, proliferation, migration, and
differentiation) can occur. Cell behavior in the
newly formed tissue has been shown to be regu-
lated by multiple interactions of the cells with
their microenvironment, including interactions
with cell-adhesion ligands [64] and with soluble
growth factors. Since biomaterials provide tem-
porary mechanical support while the cells
undergo spatial reorganization into the tissue, the
properly chosen biomaterial should allow the
engineered tissue to maintain sufficient mechani-
cal integrity to support itself in early development,
while in late development, it should begin to
degrade so that it does not hinder further tissue
growth [62].
Generally, three classes of biomaterials have
been utilized for engineering tissues: naturally
derived materials (e.g., collagen and alginate),
acellular tissue matrices (e.g., bladder submucosa
and small intestinal submucosa), and synthetic
polymers such as polyglycolic acid (PGA),
polylactic acid (PLA), and poly(lactic-co-glycolic
acid) (PLGA). These classes of biomaterials have
been tested in respect to their biocompatibility
[65, 66]. Naturally derived materials and acellular
tissue matrices have the potential advantage of
biological recognition. However, synthetic poly-
mers can be produced reproducibly on a large
scale with controlled properties such as strength,
degradation rate, and microstructure, which
would aid in the preparation of easily used, “off-
the-shelf” scaffold material.
Naturally Derived Materials
Collagen is the most abundant and ubiquitous
structural protein in the body and may be readily
purified from both animal and human tissues with
an enzyme treatment and salt/acid extraction
[67]. Collagen implants, under normal conditions,
degrade through a process involving phagocytosis
of collagen fibrils by fibroblasts [68]. This is
followed by sequential attack by lysosomal
enzymes including cathepsins B1 and D. Under
inflammatory conditions, the implants can be rap-
idly degraded largely by matrix metalloproteins
(MMPs) and collagenases [68]. However, the
in vivo resorption rate of a collagen implant can
be regulated by controlling the density of the
577
implant and the extent of intermolecular cross-
linking – the lower the density, the greater the
space between collagen fibers and the larger the
pores for cell infiltration, leading to a higher rate
of implant degradation. Collagen contains cell
adhesion domain sequences (e.g., RGD) that
may assist to retain the phenotype and activity of
many types of cells, including fibroblasts [69] and
chondrocytes [70]. Additionally, recent studies
have employed the usage of chitin as an adhesion
molecule in the collagen-based scaffolds [71].
Alginate, a polysaccharide isolated from sea-
weed, has been used as an injectable cell delivery
vehicle [72] and a cell immobilization matrix [73]
owing to its gentle gelling properties in the pres-
ence of divalent ions such as calcium. Alginate is
relatively biocompatible and is approved by the
Food and Drug Administration (FDA) for human
use as a wound dressing material. Alginate is a
family of copolymers of D-mannuronate and
L-guluronate. The physical and mechanical prop-
erties of alginate gel are strongly correlated with
the proportion and length of polyglucuronic block
in the alginate chains [72].
Acellular Tissue Matrices
Acellular tissue matrices are collagen-rich matri-
ces prepared by removing cellular components
from tissues. The matrices are often prepared by
mechanical and chemical manipulation of a seg-
ment of tissue [1–4]. These matrices slowly
degrade upon implantation and are replaced and
remodeled by ECM proteins synthesized and
secreted by transplanted or in growing cells. The
most commonly used acellular tissue matrices are
small intestinal submucosa (SIS) and bladder sub-
mucosa (BSM) [49].
Synthetic Polymers
Polyesters of naturally occurring a-hydroxy acids,
including PGA, PLA, and PLGA, are widely used
in tissue engineering. These polymers are
FDA-approved for a variety of applications,
including sutures [74]. The ester bonds in these
polymers are hydrolytically labile, and they
degrade by nonenzymatic hydrolysis. The degra-
dation products of PGA, PLA, and PLGA are
nontoxic natural metabolites and are eventually
578
eliminated from the body in the form of carbon
dioxide and water [74]. The degradation rate of
these polymers can be tailored to the application
by altering crystallinity, initial molecular weight,
and the copolymer ratio of lactic to glycolic acid.
Generally, the optimal degradation time ranges
from several weeks to several years. Since these
polymers are thermoplastics, they can be easily
formed into a three-dimensional scaffold with a
desired microstructure, gross shape, and dimen-
sion by various techniques, including molding,
extrusion, solvent casting [75], phase separation
techniques, and gas-foaming techniques
[76]. Many applications in tissue engineering
often require a scaffold with high porosity and
ratio of surface area to volume. Other biodegrad-
able synthetic polymers, including poly(anhy-
drides) and poly(ortho esters), can also be used
to fabricate scaffolds for tissue engineering with
controlled properties [77].
Engineering Specific Tissues
and Organs
Investigators around the world, including our lab-
oratory, have been working toward the develop-
ment of several cell types, tissues, and organs for
clinical application. The following sections will
describe this research in detail.
Urethra
Various biomaterials without cells, such as PGA
and acellular collagen-based matrices derived
from decellularized small intestine and bladder,
have been used in animal models for the regener-
ation of urethral tissue [4, 78–82]. Some of these
biomaterials, like acellular collagen matrices
derived from bladder submucosa, have also been
seeded with autologous cells for urethral recon-
struction. Our laboratory has been able to replace
tubularized urethral segments with cell-seeded
collagen matrices [83, 84].
Acellular collagen matrices derived from blad-
der submucosa by our laboratory have been used
A.G. Hester and A. Atala
experimentally and clinically. In animal studies,
segments of the urethra were resected and
replaced with acellular matrix grafts in an onlay
fashion. Histological examination showed com-
plete epithelialization and progressive vessel and
muscle infiltration, and the animals were able to
void through the neourethras [4]. These results
were confirmed in a clinical study of patients
with hypospadias and urethral stricture disease
[85] (Fig. 2). Decellularized cadaveric bladder
submucosa was used as an onlay matrix for ure-
thral repair in patients with stricture disease and
hypospadias. Patent, functional neourethras were
noted in these patients with up to a 7-year follow-
up. The use of an off-the-shelf matrix appears to
be beneficial for patients with abnormal urethral
conditions and obviates the need for obtaining
autologous grafts, thus decreasing operative time
and eliminating donor site morbidity.
Unfortunately, the above techniques are not
applicable for tubularized urethral repairs. The
collagen matrices are able to replace urethral seg-
ments only when used in an onlay fashion. How-
ever, if a tubularized repair is needed, the collagen
matrices should be seeded with autologous cells to
avoid the risk of stricture formation and poor
tissue development [83]. Therefore, tubularized
collagen matrices seeded with autologous cells
can be used successfully for total penile urethra
replacement. An initial study in 2008 with 5 adult
patients requiring tubularized repairs secondary to
lichen sclerosis was somewhat successful. These
patients had grafts engineered from the buccal
mucosa, but 2 of the 5 patients required excision
secondary to fibrosis and contraction and the
remaining 3 required some form of instrumenta-
tion [86]. We performed a study in the past few
years where five boys were successfully
implanted with urethras engineered from autolo-
gous cells for posterior urethral defects. Upon
biopsy following only 3 months from implanta-
tion, these grafts were found to contain normal
urethral architecture, and at a median follow-up of
71 months, these patients all remained patent and
functional [87]. Another study performed with
children, this time with hypospadias, demon-
strated similar results. These children had
19 Translational Research Methods: Tissue Engineering of the Kidney and Urinary Tract
Fig. 2 Urethral repair using a collagen matrix. (a) Repre-
sentative case of a patient with a bulbar stricture. (b)
During surgery, strictured tissue is excised, preserving the
urethral plate on the left side, and the matrix is anasto-
mosed to the urethral plate in an onlay fashion on the right.
The boxes in both photos indicate the area of interest,
including the urethra, which appears white in the left
photograph. In the left photograph, the arrow indicates
autologous urothelial cells isolated from bladder
washes, and urethral implants were engineered.
Most of these children had minor complications
over the following 3–5 years, but only one
required a urethrotomy for an obstruction. The
remaining children were able to void in a standing
position [13, 88].
Despite the success of these techniques, there
are still the continued complications of synthetic
materials used and implanted into a human subject
serving as a nidus for fibrosis and stricture forma-
tion. A more recent technique has been more
successful in animal models [13]. In this model,
the graft and scaffolding are created from human
cells without relying on any exogenous materials
and by using a self-assembly method [89]. In fact,
an additional study has shown how using endo-
thelial cells in the process of growing these grafts
produces earlier vascularization of the
material [90].
579
the area of stricture in the urethra. On the right, the arrow
indicates the repaired stricture. Note that the engineered
tissue now obscures the native white urethral tissue in an
onlay fashion in the right photograph. (c) Urethrogram
6 months after repair. (d) Cystoscopic view of urethra
before surgery on the left side and 4 months after repair
on the right side
Bladder
Currently, gastrointestinal segments are com-
monly used as tissues for bladder replacement or
repair. However, gastrointestinal tissues are
designed to absorb specific solutes, whereas blad-
der tissue is designed for the excretion of solutes.
Due to the problems encountered with the use of
gastrointestinal segments, numerous investigators
have attempted alternative materials and tissues
for bladder replacement or repair.
The success of cell transplantation strategies
for bladder reconstruction depends on the ability
to use donor tissue efficiently and to provide the
right conditions for long-term survival, differenti-
ation, and growth. Urothelial and muscle cells can
be expanded in vitro, seeded onto polymer scaf-
folds, and allowed to attach and form sheets of
cells [91]. These principles were applied in the
creation of tissue-engineered bladders in an
580
Fig. 3 Construction of engineered bladder. (a) Scaffold
material seeded with cells for use in bladder repair. (b) The
seeded scaffold is anastomosed to native bladder with
animal model that required a subtotal cystectomy
with subsequent replacement with a tissue-
engineered organ in beagle dogs [12]. Urothelial
and muscle cells were separately expanded from
an autologous bladder biopsy and seeded onto a
bladder-shaped biodegradable polymer scaffold.
The results from this study showed that it is pos-
sible to tissue engineer bladders that are anatom-
ically and functionally normal. In subsequent
years, these findings have been replicated, and
the subjects have been followed for longer periods
of time, indicating native histology, good capac-
ity, and excellent compliance [92, 93]. Clinical
trials for the application of this technology are
currently being conducted.
Despite the success of the previously men-
tioned studies for subtotal cystectomy, there
have been subsequent reports of difficulties
while attempting to engineer larger sections of
the bladder. In 2008, a study, again in canines,
compared the results of seeded versus
unseeded small intestinal segments for large-
portion (90 %) bladder replacements. Many of
the subjects in this study demonstrated graft
shrinkage and adhesions [94]. Additionally, there
has been some recent interest in using bioreactors
to mechanically stimulate the engineered
bladder. Theoretically, the mechanical stimulation
provided by the bioreactor allows for functional
filling and emptying of the tissue which
would provide an increase in cell-scaffold
A.G. Hester and A. Atala
running 4–0 polyglycolic sutures. (c) Implant covered
with fibrin glue and omentum
interactions and potentially allow studying of the
cell growth and development process in these
engineered organs [93, 95].
A clinical experience involving engineered
bladder tissue for cystoplasty reconstruction was
conducted starting in 1999. A small pilot study of
seven patients was reported, using a collagen
scaffold seeded with cells either with or without
omentum coverage or a combined PGA-collagen
scaffold seeded with cells and omental coverage
(Fig. 3). The patients reconstructed with the
engineered bladder tissue created with the
PGA-collagen cell-seeded scaffolds showed
increased compliance, decreased end-filling pres-
sures, increased capacities, and longer dry periods
[23] (Fig. 4). Although the experience is promis-
ing in terms of showing that engineered tissues
can be implanted safely, it is just a start in terms of
accomplishing the goal of engineering fully func-
tional bladders. Since this time, two multicenter
Phase II clinical trials have been performed, one in
pediatric patients and one in adult patients, in
order to further test the safety of engineered
Neo-Bladder Augments™, produced by Tengion,
Inc. These studies had good results but did find
that the engineered bladder augments were more
likely to succeed in patients who still retained
natural bladder cycling function, further indica-
tion to pursue bioreactors for mechanical stimula-
tion [13, 93]. Further experimental and clinical
work is being conducted.
19 Translational Research Methods: Tissue Engineering of the Kidney and Urinary Tract
Fig. 4 Cystograms and urodynamic studies of a patient
before and after implantation of the tissue-engineered blad-
der. (a) Preoperative results indicate an irregular bladder in
Kidney
Renal tissue is arguably one of the most difficult
tissues to replicate in the laboratory. The kidney is
a complex organ and the unique structural and
cellular heterogeneity present within this
organ creates many challenges. The system of
nephrons and collecting ducts within the kidney
is composed of multiple functionally and
morphologically distinct segments. For this
reason, appropriate conditions must be provided
to ensure the long-term survival, differentiation,
and growth of many types of cells. Recent efforts
in the area of kidney tissue regeneration have
focused on the development of a reliable cell
source [96–101]. Moreover, optimal growth con-
ditions have been extensively investigated to pro-
vide adequate enrichment to achieve stable renal
cell expansion systems [102–106].
Isolation of particular cell types that produce
specific factors may be a good approach for selec-
tive cell therapies. For example, cells that produce
erythropoietin could be isolated in culture, and
these cells could eventually be used to treat ane-
mia that results from end-stage renal failure. How-
ever, total renal function would not be achieved
using this approach. To create kidney tissue that
would deliver full renal function, a culture
containing all of the cell types comprising the
581
the cystogram and abnormal bladder pressures as the blad-
der is filled via urodynamic study. (b) Postoperatively,
findings are significantly improved
functional nephron units should be used. Optimal
culture conditions to nurture renal cells have been
extensively studied and cells grown under these
conditions have been reported to maintain their
cellular characteristics [107]. Moreover, renal
cells placed in a three-dimensional culture envi-
ronment are able to reconstitute into renal
structures.
Recent investigative efforts in the search for a
reliable cell source have been expanded to stem
and progenitor cells. The use of these cells for
tissue regeneration is attractive due to their ability
to differentiate and mature into specific cell types
needed. This is particularly useful in instances
where primary renal cells are unavailable due to
extensive tissue damage. Bone marrow-derived
human mesenchymal stem cells have been
shown to be a potential source due to their
ability to differentiate into several cell lineages
[96, 97, 100]. These cells have been shown to
participate in the kidney development when they
are placed in a rat embryonic niche that allows for
continued exposure to a repertoire of nephrogenic
signals [101]. These cells, however, were found to
contribute mainly to regeneration of damaged
glomerular endothelial cells after injury. In addi-
tion, the major cell source of kidney regeneration
was found to originate from intrarenal cells in an
ischemic renal injury model [96, 99]. Another
582
potential cell source for kidney regeneration is
circulating stem cells, which have been shown to
transform into tubular and glomerular epithelial
cells, podocytes, mesangial cells, and interstitial
cells after renal injury [97, 98, 108–112]. These
observations suggest that controlling stem and
progenitor cell differentiation may lead to suc-
cessful regeneration of kidney tissues.
Although isolated renal cells are able to retain
their phenotypic and functional characteristics in
culture, transplantation of these cells in vivo may
not result in structural remodeling. In addition,
cell or tissue components cannot be implanted in
large volumes due to limited diffusion of
oxygen and nutrients [113]. Thus, a cell-
support matrix, preferably one that encourages
angiogenesis, is necessary to allow diffusion
across the entire implant. A variety of synthetic
and naturally derived materials has been exam-
ined in order to determine the ideal support
structures for the regeneration [12, 23, 85,
114, 115]. Biodegradable synthetic materials,
such as polylactic and polyglycolic acid poly-
mers, have been used to provide structural support
for cells. Synthetic materials can be easily fabri-
cated and configured in a controlled manner,
which makes them attractive options for tissue
engineering. However, naturally derived mate-
rials, such as collagen, laminin, and fibronectin,
are much more biocompatible and provide a sim-
ilar extracellular matrix environment to normal
tissue. For this reason, collagen-based scaffolds
have been used increasingly in many applications
[116–119].
Over the years, the engineered scaffoldings
have continued to have only partial success with
angiogenesis. However, in recent years, the con-
cept of whole-organ decellularization has come to
fruition. By using a natural acellular organ bed,
the internal vascular system and microarchitecture
can be maintained while generating simple tissue
matrices for production of heart valves and intes-
tinal segments [120]. However, this technology
suggests that more complex organ structures can
be produced in a similar manner. These scaffolds
can then be recellularized and have been studied
in animal models [121–123], with the potential for
future clinical studies [120].
A.G. Hester and A. Atala
Developmental Approaches to Kidney
Regeneration
Transplantation of a kidney precursor, such as the
metanephros, into a diseased kidney has been
proposed as a possible method for functional res-
toration. In an animal study, human embryonic
metanephroi, transplanted into the kidneys of an
immune-deficient mouse model, has developed
into mature kidneys [124]. The transplanted meta-
nephroi which produced urine-like fluid, how-
ever, failed to develop ureters. This study
suggests that development of an in vitro system
in which metanephroi could be grown may lead to
transplant techniques that could produce a small
replacement kidney within the host. In another
study, the metanephros was divided into mesen-
chymal tissue and ureteral buds, and each of the
tissue segments was cultured in vitro [125]. After
8 days in culture, each portion of the mesenchy-
mal tissues had grown to the original size.
A similar method was used for ureteral buds,
which also propagated. These studies indicate
that if the mesenchyme and ureteral buds were
placed together and cultured in vitro, a
metanephros-like structure would develop and
suggest that the metanephros could be propagated
under an optimal condition.
In another study, transplantation of metaneph-
roi into a non-immunosuppressed rat omentum
showed that the implanted metanephroi are able
to undergo differentiation and growth that is not
confined by a tight organ capsule [126]. When the
metanephroi with an intact ureteric bud were
implanted, the metanephroi are able to enlarge
and become a kidney-shaped tissue within
3 weeks. The metanephroi transplanted into the
omentum were able to develop into a kidney tis-
sue structure with a well-defined cortex and
medulla. Mature nephrons and collecting system
structures are shown to be indistinguishable from
those of normal kidneys by light or electron
microscopy [127, 128]. Moreover, these struc-
tures become vascularized via arteries that origi-
nate at the superior mesenteric artery of the host
[127, 128]. It has been demonstrated that the
metanephroi transplanted into the omentum sur-
vive for up to 32 weeks postimplantation
[129]. These studies show that developmental
19 Translational Research Methods: Tissue Engineering of the Kidney and Urinary Tract
approach may be a viable option for regenerating
renal tissue for functional restoration.
Currently, there is a lot of interest in branching
morphogenesis and its impact on tissue regenera-
tion for the kidney. A recent review discussed how
the epithelial buds in certain tissues, including the
kidney, form into iterative tip-stalk generators
(ITSG) which are able to form the necessary
ducts and tubules needed for tissue development.
Recent studies have demonstrated how this
branching strategy can be used to form
3-dimensional scaffolds for tissue engineering
[130–132].
Tissue Engineering Approaches
to Kidney Regeneration
The ability to grow and expand renal cells is one
of the essential requirements in engineering tis-
sues. The feasibility of achieving renal cell
growth, expansion, and in vivo reconstitution
using tissue engineering techniques was investi-
gated [114]. Donor rabbit kidneys were removed
and perfused with a non-oxide solution which
promoted iron particle entrapment in the glomer-
uli. Homogenization of the renal cortex and frac-
tionation in 83–210 μm sieves with subsequent
magnetic extraction yielded three separate puri-
fied suspensions of distal tubules, glomeruli, and
proximal tubules. The cells were plated separately
in vitro and after expansion, were seeded onto
biodegradable polyglycolic acid scaffolds and
implanted subcutaneously into host athymic
mice. This included implants of proximal tubular
cells, glomeruli, distal tubular cells, and a mixture
of all three cell types. Animals were sacrificed at
one week, two weeks, and one month after
implantation, and the retrieved implants were ana-
lyzed. An acute inflammatory phase and a chronic
foreign-body reaction were seen, accompanied by
vascular ingrowth by 7 days after implantation.
Histological examination demonstrated progres-
sive formation and organization of the nephron
segments within the polymer fibers with time.
Renal cell proliferation in the cell-polymer scaf-
folds was detected by in vivo labeling of replicat-
ing cells with the thymidine analog
bromodeoxyuridine [99]. BrdU incorporation
into renal cell DNA was confirmed using
583
monoclonal anti-BrdU antibodies. These results
demonstrated that renal-specific cells can be suc-
cessfully harvested and cultured and can subse-
quently attach to artificial biodegradable
polymers. The renal cell-polymer scaffolds can
be implanted into host animals where the cells
replicate and organize into nephron segments, as
the polymer, which serves as a cell delivery vehi-
cle, undergoes biodegradation.
Initial experiments showed that implanted cell-
polymer scaffolds gave rise to renal tubular struc-
tures. However, it was unclear whether the tubular
structures reconstituted de novo from dispersed
renal elements, or if they merely represented frag-
ments of donor tubules which survived the origi-
nal dissociation and culture processes intact.
Further investigation was conducted in order to
examine the process [133]. Mouse renal cells were
harvested and expanded in culture. Subsequently,
single isolated cells were seeded on biodegradable
polymers and implanted into immune-competent
syngeneic hosts. Renal epithelial cells were
observed to reconstitute into tubular structures
in vivo. Sequential analyses of the retrieved
implants over time demonstrated that renal epithe-
lial cells first organized into a cord-like structure
with a solid center. Subsequent canalization into a
hollow tube could be seen by 2 weeks. Histolog-
ical examination with nephron segment-specific
lactins showed successful reconstitution of prox-
imal tubules, distal tubules, loop of Henle,
collecting tubules, and collecting ducts. These
results showed that single suspended cells are
capable of reconstituting into tubular structures,
with homogeneous cell types within each tubule.
Although these studies demonstrated that renal
cells seeded on biodegradable polymer scaffolds
are able to form some renal structures in vivo,
complete renal function could not be achieved in
these studies. In a subsequent study, we sought to
create a functional artificial renal unit which could
produce urine [134]. Mouse renal cells were
harvested, expanded in culture, and seeded onto
a tubular device constructed from polycarbonate
[119]. The tubular device was connected at one
end to a Silastic catheter which terminated into a
reservoir. The device was implanted subcutane-
ously in athymic mice. The implanted devices
584
Fig. 5 Combining therapeutic cloning and tissue engi-
neering to produce kidney tissue. (a) Illustration of the
tissue-engineered renal unit. (b) Renal unit seeded with
cloned cells, 3 months after implantation, showing the
accumulation of urine-like fluid. (c) Clear unidirectional
continuity between the mature glomeruli, their tubules, and
Silastic catheter. (d) ELISPOT analyses of the frequencies
were retrieved and examined histologically and
immunocytochemically at 1, 2, 3, 4, and 8 weeks
after implantation. Fluid was collected from
inside the implant, and uric acid and creatinine
levels were determined.
Histological examination of the implanted
device demonstrated extensive vascularization as
well as formation of glomeruli and highly orga-
nized tubule-like structures. Immunocytochemi-
cal staining with anti-osteopontin antibody,
which is secreted by proximal and distal tubular
cells and the cells of the thin ascending loop of
Henle, stained the tubular sections. Immunohisto-
chemical staining for alkaline phosphatase stained
proximal tubule-like structures. Uniform staining
for fibronectin in the extracellular matrix of newly
formed tubes was observed. The fluid collected
from the reservoir was yellow and contained
66 mg/dl uric acid (as compared to 2 mg/dl in
plasma) suggesting that these tubules are capable
of unidirectional secretion and concentration of
uric acid. The creatinine assay performed on the
A.G. Hester and A. Atala
of T cells that secrete IFNγ after stimulation with alloge-
neic renal cells, cloned renal cells, or nuclear donor fibro-
blasts. Cloned renal cells produce fewer IFNγ spots than
the allogeneic cells, indicating that the rejection response
to cloned cells is diminished. The presented wells are
single representatives of duplicate wells
collected fluid showed an 8.2-fold increase in
concentration, as compared to serum. These
results demonstrated that single cells form
multicellular structures can become organized
into functional renal units that are able to excrete
high levels of solutes through a urine-like
fluid [134].
To determine whether renal tissue could be
formed using an alternative cell source, nuclear
transplantation (therapeutic cloning) was
performed to generate histocompatible tissues,
and the feasibility of engineering syngeneic renal
tissues in vivo using these cloned cells was
investigated [107]. Nuclear material from bovine
dermal fibroblasts was transferred into
unfertilized enucleated donor bovine eggs. Renal
cells from the cloned embryos were harvested,
expanded in vitro, and seeded onto three-
dimensional renal devices (Fig. 5a). The devices
were implanted into the back of the same steer
from which the cells were cloned and were
retrieved 12 weeks later.
19 Translational Research Methods: Tissue Engineering of the Kidney and Urinary Tract
This process produced functioning renal
units. Urine production and viability were
demonstrated after transplantation back into the
nuclear donor animal (Fig. 5b). Chemical
analysis suggested unidirectional secretion and
concentration of urea nitrogen and creatinine.
Microscopic analysis revealed formation of orga-
nized glomeruli and tubular structures (Fig. 5c).
Immunohistochemical and RT-PCR analysis con-
firmed the expression of renal mRNA and
proteins.
Since previous studies have shown that bovine
clones harbor the oocyte mitochondrial DNA
[135–137], the donor egg’s mitochondrial DNA
(mtDNA) was thought to be a potential source of
immunologic incompatibility. Differences in
mtDNA-encoded proteins expressed by cloned
cells could stimulate a T-cell response specific
for mtDNA-encoded minor histocompatibility
antigens when the cloned cells are implanted
back into the original nuclear donor [138]. Mater-
nally transmitted minor histocompatibility anti-
gens in mice have been shown to stimulate both
skin allograft rejection in vivo and cytotoxic
T-lymphocytes expansion in vitro [138] that
could prevent the use of these cloned constructs
in patients with chronic rejection of major histo-
compatibility matched human renal transplants
[139, 140]. We tested for a possible T-cell
response to the cloned renal devices using
delayed-type hypersensitivity testing in vivo and
ELISPOT analysis of interferon-γ-secreting T
cells in vitro. Both analyses revealed that the
cloned renal cells showed no evidence of a T-cell
response, suggesting that rejection will not neces-
sarily occur in the presence of oocyte-derived
mtDNA (Fig. 5d). This finding may represent a
step forward in overcoming the histocompatibility
problem of stem cell therapy [140].
These studies demonstrated that cells derived
from nuclear transfer can be successfully
harvested, expanded in culture, and transplanted
in vivo with the use of biodegradable scaffolds on
which the single suspended cells can organize into
tissue structures that are genetically identical to
that of the host. These studies were the first dem-
onstration of the use of therapeutic cloning for
regeneration of tissues in vivo.
585
However, a naturally derived tissue matrix
with existing three-dimensional kidney architec-
ture would be preferable to the artificial matrix
used in these experiments, because it would allow
for transplantation of a larger number of cells,
resulting in greater renal tissue volumes. Thus,
we developed an acellular collagen-based kidney
matrix, which is identical to the native renal archi-
tecture. In a subsequent study, we investigated
whether these collagen-based matrices could
accommodate large volumes of renal cells and
form kidney structures in vivo [141].
Acellular collagen matrices, derived from por-
cine kidneys, were obtained through a multistep
decellularization process. During this process,
serial evaluation of the matrix for cellular rem-
nants was performed using histochemistry, scan-
ning electron microscopy (SEM), and RT-PCR.
Mouse renal cells were harvested, grown, and
seeded on 80 of the decellularized collagen matri-
ces at a concentration of 30  106 cells/ml. Forty
cell-matrix constructs grown in vitro were ana-
lyzed 3 days and 1, 2, 4, and 6 weeks after
seeding. The remaining 40 cell-containing matri-
ces were implanted in the subcutaneous space of
20 athymic mice. The animals were sacrificed
3 days and 1, 2, 4, 8, and 24 weeks after implan-
tation for analyses. Gross, SEM, histochemical,
immunocytochemical, and biochemical analyses
were performed.
Scanning electron microscopy and histological
examination confirmed the acellularity of the
processed matrix. RT-PCR performed on the kid-
ney matrices demonstrated the absence of any
RNA residues. Renal cells seeded on the matrix
adhered to the inner surface and proliferated to
confluency 7 days after seeding, as demonstrated
by SEM. Histochemical and immunocytochemi-
cal analyses performed using H&E, periodic acid-
Schiff, alkaline phosphatase, anti-osteopontin,
and anti-CD-31 identified stromal, endothelial,
and tubular epithelial cell phenotypes within the
matrix. Renal tubular and glomerulus-like struc-
tures were observed 8 weeks after implantation.
MTT proliferation and titrated thymidine incorpo-
ration assays performed 6 weeks after cell seeding
demonstrated a population increase of 116 % and
92 %, respectively, as compared to the 2-week
586
time points. This study demonstrates that renal
cells are able to adhere to and proliferate on the
collagen-based kidney matrices. The renal cells
reconstitute renal tubular and glomeruli-like
structures in the kidney-shaped matrix. The
collagen-based kidney matrix system seeded
with renal cells may be useful in the future for
augmenting renal function.
We also investigated the feasibility of creating
three-dimensional renal structures for in situ
implantation within the native kidney tissue. Pri-
mary renal cells from 4-week-old mice were
grown and expanded in culture. These renal cells
were labeled with fluorescent markers and
injected into mouse kidneys in a collagen gel for
in vivo formation of renal tissues. Collagen injec-
tion without cells and sham-operated animals
served as controls. In vitro reconstituted renal
structures and in vivo implanted cells were
retrieved and analyzed.
The implanted renal cells formed tubular and
glomerular structures within the kidney tissue, as
confirmed by the fluorescent markers. There was
no evidence of renal tissue formation in the con-
trol and the sham-operated groups. These results
demonstrate that single renal cells are able to
reconstitute kidney structures when placed in a
collagen-based scaffolding system. The implanted
renal cells are able to self-assemble into tubular
and glomerular structures within the kidney tis-
sue. These findings suggest that this system may
be the preferred approach to engineer functional
kidney tissues for the treatment of end-stage renal
disease.
More recent studies have explored ways to
optimize the decellularization process as well as
the recellularization process, with the intention of
decreasing processing times and increasing vas-
cular perfusion within the grafts [142]. Bonandrini
and colleagues successfully decellularized rat kid-
neys following a 17 h perfusion of sodium
dodecyl sulfate while still maintaining the basic
microarchitecture, including glomeruli and vascu-
lar structures. The scaffold was recellularized with
murine embryonic stem cells through the renal
artery while using pressure-controlled perfusion
for 24–72 h, and initial cellular differentiation was
noted at 24 h and marked at 72 h. The major
A.G. Hester and A. Atala
implication of this study is that under the correct
conditions, it is possible to rapidly decellularize
and recellularize a 3-dimensional scaffold while
maintaining vascular integrity and differentiation
of the cells.
Genital Tissues
Reconstructive surgery is required for a wide vari-
ety of pathologic penile conditions, such as penile
carcinoma, trauma, severe erectile dysfunction,
and congenital conditions such as ambiguous gen-
italia, hypospadias, and epispadias. One of the
major limitations of phallic reconstructive surgery
is the scarcity of sufficient autologous tissue.
The major components of the phallus are cor-
poral smooth muscle and endothelial cells. The
creation of autologous functional and structural
corporal tissue de novo would be beneficial.
Autologous cavernosal smooth muscle and endo-
thelial cells were harvested, expanded, and seeded
on acellular collagen matrices and implanted in a
rabbit model [143, 144]. Histological examination
confirmed the appropriate organization of
penile tissue phenotypes, and structural and
functional studies, including cavernosography,
cavernosometry, and mating studies, demon-
strated that it is possible to engineer autologous
functional penile tissue. Corporal body grafts
have been successfully formed using small intes-
tinal submucosa (SIS), but results do seem to vary,
with a single layer SIS resulting in fewer compli-
cations than the four-layer SIS [145]. Multiple
studies have reiterated this point and have
established this technique as the standard for cor-
poral body grafting [146–149]. Congenital
malformations of the uterus may have profound
implications clinically. Patients with cloacal
exstrophy and intersex disorders may not have
sufficient uterine tissue present for future repro-
duction. We investigated the possibility of engi-
neering functional uterine tissue using autologous
cells [150]. Autologous rabbit uterine smooth
muscle and epithelial cells were harvested and
then grown and expanded in culture. These cells
were seeded onto preconfigured uterine-shaped
biodegradable polymer scaffolds, which were
then used for subtotal uterine tissue replacement
in the corresponding autologous animals.
19 Translational Research Methods: Tissue Engineering of the Kidney and Urinary Tract
Upon retrieval 6 months after implantation, histo-
logical, immunocytochemical, and Western blot
analyses confirmed the presence of normal uterine
tissue components. Biomechanical analyses and
organ bath studies showed that the functional
characteristics of these tissues were similar to
those of normal uterine tissue. Breeding studies
using these engineered uteri are currently being
performed. A recent study of regenerated endo-
metrial tissue in mice has demonstrated the ability
to implant functional endometrial tissue in vivo
[151]. Additionally, in vitro studies have demon-
strated the ability of tissue-engineered endome-
trial tissues to respond to cues allowing for normal
uterine activity such as menstruation [152]
(Fig. 6).
Similarly, several pathologic conditions,
including congenital malformations and malig-
nancy, can adversely affect normal vaginal devel-
opment or anatomy. Vaginal reconstruction has
traditionally been challenging due to the paucity
of available native tissue. The feasibility of
engineering vaginal tissue in vivo was investi-
gated [153]. Vaginal epithelial and smooth muscle
Fig. 6 Autologous chondrocytes for the treatment of
vesicoureteral reflux. (a) Preoperative voiding cystoure-
throgram of a patient with bilateral reflux. A catheter was
inserted into the bladder via the urethra, and contrast mate-
rial was instilled intravesically. Here, contrast material can
be seen within both ureters and within the kidneys, indi-
cating reflux is present. (b) Postoperative radionuclide
cystogram of the same patient 6 months after injection of
587
cells of female rabbits were harvested, grown, and
expanded in culture. These cells were seeded onto
biodegradable polymer scaffolds, and the cell-
seeded constructs were then implanted into nude
mice for up to 6 weeks. Immunocytochemical,
histological, and Western blot analyses confirmed
the presence of vaginal tissue phenotypes. Electri-
cal field stimulation studies in the tissue-
engineered constructs showed similar functional
properties to those of normal vaginal tissue.
When these constructs were used for autologous
total vaginal replacement, patent vaginal structures
were noted in the tissue-engineered specimens,
while the non-cell-seeded structures were noted to
be stenotic [153]. A prospective study performed in
2013 in patients with Mayer-Rokitansky-K€uster-
Hauser syndrome undergoing vaginoplasty dem-
onstrated positive results. These patients received
tissue-engineered acellular dermal matrix and were
advised to dilate the vagina postoperatively to
avoid contraction of the graft. At a mean follow-
up of 21.1 months, all patients had patent vaginal
grafts with a similar Female Sexual Function Index
to their age-matched controls [154].
autologous chondrocytes. A catheter was inserted into the
bladder via the urethra, and a radioactive solution was
inserted into the bladder. The bladder was scanned during
filling and emptying phases. This panel includes sequential
images of the bladder as it was filled and emptied. This
shows a normal, round bladder that fills and empties prop-
erly. If reflux had been present, the ureters would have been
visible in the scan above the round bladder
588
Summary and Conclusions
Regenerative medicine efforts are currently
underway experimentally for virtually every type
of tissue and organ within the human body.
As regenerative medicine incorporates the fields
of tissue engineering, cell biology, nuclear trans-
fer, and materials science, personnel who have
mastered the techniques of cell harvest, culture,
expansion, transplantation, and polymer design
are essential for the successful application of
these technologies to extend human life. Various
tissues are at different stages of development, with
some already being used clinically, a few in pre-
clinical trials, and some in the discovery stage.
Recent progress suggests that engineered tissues
may have an expanded clinical applicability in the
future and may represent a viable therapeutic
option for those who would benefit from the life-
extending benefits of tissue replacement or repair.
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 Part IV
Clinical Approach to the Child with
Suspected Renal Disease
 Clinical Evaluation of the Child with
Suspected Renal Disease 20
Mohan A. Shenoy and Nicholas J. A. Webb

Contents Clinical Examination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602

General Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602
Presentation of Renal Disease in Children . . . . . . . 596 Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596 Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604
Antenatal Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596 Hydration/Volume Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604
Abnormalities of Appearance of Urine . . . . . . . . . . . . . . 596 Cardiovascular System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604
Abnormalities of Volume of Urine . . . . . . . . . . . . . . . . . . 598 Respiratory System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605
Wetting and Abnormalities of the Passage of Urine 598 Abdomen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605
Edema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599 Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606
Asymptomatic Presentation Following Screening or Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606
Routine Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599 Face . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607
Urinary Tract Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600 Eyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607
Clinical History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600 Ears . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607
Antenatal History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600 Musculoskeletal System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608
Birth History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601 Examination of the Urine . . . . . . . . . . . . . . . . . . . . . . . . . . 608
Family History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601 Macroscopic Examination . . . . . . . . . . . . . . . . . . . . . . . . . . . 608
General Medical History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601 Dipstick Examination of Urine . . . . . . . . . . . . . . . . . . . . . . 608
Urine and Micturition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602
Dietary History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
Psychosocial History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602

M.A. Shenoy • N.J.A. Webb (*)

Department of Pediatric Nephrology, Royal Manchester
Children’s Hospital, Manchester, UK
e-mail: nicholas.webb@cmft.nhs.uk

# Springer-Verlag Berlin Heidelberg 2016 595

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_18
596
Presentation of Renal Disease
in Children
Introduction
Renal disease in children may present with overt
symptoms clearly associated with the urinary
tract, such as the development of macroscopic
hematuria or profound oliguria. However, in
many instances, symptoms may be very
nonspecific or seemingly mild. Children with
chronic kidney disease present with a wide variety
of symptoms including enuresis, failure to thrive,
short stature, lethargy, and pallor. The onset may
be silent and the progress insidious, with symp-
toms only developing late in the disease course.
Urinary tract infection in infants and small chil-
dren may, in contrast to older children, present
with nonspecific manifestations including poor
feeding, vomiting, irritability, abdominal pain,
failure to thrive, lethargy, and restlessness. The
possibility of renal disease should therefore be
considered in the differential diagnosis of any
child presenting to the hospital with acute or
chronic symptoms.
Antenatal Imaging
Since the first report of renal abnormalities being
detected by antenatal ultrasonography in the
1970s, there have been major developments in
the antenatal assessment of the urinary tract
[1–4]. In most countries, pregnant women
undergo routine ultrasound assessment of the
fetus at various stages throughout pregnancy,
including a detailed scan performed at around
20 weeks of gestation. Ultrasound is particularly
informative with regard to renal abnormalities,
which account for around 20 % of all significant
fetal abnormalities detected during gestation
[5]. Recent developments including 3D ultra-
sound and magnetic resonance imaging of the
fetus allow these anomalies to be studied in fur-
ther detail. This has resulted in many significant
congenital and inherited abnormalities of the uri-
nary tract being detected antenatally, notably pos-
terior urethral valves, the multicystic dysplastic
M.A. Shenoy and N.J.A. Webb
kidney (MCDK), pelvi-ureteric junction obstruc-
tion, and the polycystic kidney diseases. In the
case of posterior urethral valves, this has resulted
in the possibility of antenatal therapy, though the
efficacy and safety of this require further confir-
mation in clinical trials. These may be difficult to
recruit to: the UK PLUTO clinical trial investigat-
ing vesico-amniotic shunting had to close early
because of suboptimal recruitment [6]. Large kid-
neys on antenatal imaging may suggest
hydronephrosis, polycystic kidney disease
(PKD), MCDK, congenital nephrotic syndrome,
or rarely a renal tumor, whereas small kidneys
may point to the presence of renal dysplasia or
hypoplasia. PKD, cystic dysplasia, and
glomerulocystic disease are common causes of
echobright kidneys. TCF2 gene mutations are
found in almost one third of the children with
antenatally diagnosed bilateral echogenic kidneys
[7]; however, counseling families with this muta-
tion is difficult as the phenotype is extremely
variable, even within a single family. The pres-
ence of renal macrocysts in the antenatal period
should alert the physician towards a diagnosis of
autosomal dominant PKD, PKD associated with
tuberous sclerosis, MCDK, or cystic dysplasia.
The sensitivity of ultrasound imaging does,
however, result in the detection of many
minor abnormalities, particularly unilateral renal
pelvic dilatation, which appear to resolve
spontaneously in later pregnancy or after birth
and are of no long-term significance. Their detec-
tion may be a source of significant parental anxi-
ety and multiple invasive investigations in the
neonatal period where appropriate protocols are
not in operation.
Abnormalities of Appearance of Urine
Red or Dark Urine
The presence of blood in the urine causes it to
develop a pink to red color. Only a relatively small
amount of blood is necessary to produce discolor-
ation. Prolonged contact between blood in the
urinary tract and acidic urine causes the heme
pigment to become oxidized to a methaem deriv-
ative, giving the urine a brown color. In general,
20 Clinical Evaluation of the Child with Suspected Renal Disease
Table 1 False-positive hematuria; alternative causes of
red urine
Foods, e.g., beetroot, berries containing anthocyanins,
and food dyes
Hemoglobinuria, e.g., in intravascular hemolysis
Myoglobinuria, e.g., in rhabdomyolysis
Urate crystals (a cause of pink discoloration of nappies)
Drugs, e.g., rifampicin, phenothiazines, desferrioxamine,
phenindione
Inborn errors of metabolism, e.g., porphyria and
alkaptonuria
the longer the contact and the more acidic the
urine, the darker brown the urine becomes.
Contamination with blood from menstruation
in older girls needs ruling out. There are a number
of causes of false-positive hematuria, where alter-
native substances produce red discoloration
(Table 1). One of the most common of these is
the pink discoloration seen in nappies caused by
the precipitation of urate crystals. Urine micros-
copy is therefore mandatory following the detec-
tion of red or dark urine. It is important that this is
performed on a fresh sample as red cell lysis
occurs where samples are allowed to stand for
prolonged periods prior to examination.
The passage of fresh red blood in the urine,
with or without clots, is most likely to originate
from the lower urinary tract, particularly where
this is most marked at the beginning or the end of
the urinary stream. This highlights the importance
of obtaining a comprehensive clinical history in
such children. Causes include urethritis, trauma,
bladder calculus, and schistosomiasis. Urological
assessment and cystoscopy are indicated in
most such cases. More uncommon causes of
bleeding from the upper tracts include trauma,
arteriovenous malformations, tumors, and
angiomyolipomas associated with tuberous
sclerosis.
Fabricated and induced illness by proxy, pre-
viously termed Munchausen syndrome by proxy,
is a very rare disorder, though falsification of urine
samples by contamination with maternal or other
blood is one of the more common modes of pre-
sentation [8]. A high index of suspicion needs to
be maintained to detect such cases. Absolute con-
firmation of contamination requires analysis of
597
urinary red cells for differences in blood group
type or, if required, DNA profiling.
Dark urine associated with jaundice in the neo-
nate or older child should alert the physician to the
presence of conjugated hyperbilirubinemia, indic-
ative of significant liver or biliary tract disease.
Bilirubin and urobilinogen will be detected on
dipstick testing the urine in such cases.
Cloudy Urine
The urine may become cloudy secondary to the
presence of white blood cells (pyuria) associated
with urinary infection, calcium phosphate crys-
tals, or a combination of calcium salts, uric acid,
oxalate, cystine, or struvite. These substances are
normally present in the urine, though may be
present in excess in various disease states. The
precipitation of phosphates and urates is enhanced
by refrigeration of the urine sample.
Frothy Urine
The presence of significant quantities of protein in
the urine will result in it becoming frothy. Presen-
tation with frothy urine should therefore prompt
the physician to perform dipstick analysis
followed by formal quantification of protein
content.
Offensive or Unusual-Smelling Urine
A number of factors may result in an alteration in
the smell of urine. Infection with urea-splitting
organisms including Ureaplasma urealyticum, Pro-
teus, Staphylococcus, Klebsiella, Providentia, and
Pseudomonas species may generate an ammonia-
like smell, though the positive predictive value of
offensive urine for bona fide urinary infection is
very low. A number of the inborn errors of metab-
olism, including maple syrup urine disease, phenyl-
ketonuria, and isovaleric acidemia, also produce
characteristic odors. The change in urine odor fol-
lowing the ingestion of asparagus was first reported
in the eighteenth century by a physician of the
French royal family [9]. This is thought to be due
to the presence of S-methyl-thioacrylate and
S-methyl-3-(methylthio)thiopropionate [10],
though a combination of methyl mercaptan,
dimethyl sulfide, and small amounts of sulfur-
oxidized compounds could also be responsible [11].
598
Passage of Gravel or Stones
The passage of gravel or stones in the urine is an
unusual though recognized mode of presentation
of urinary tract calculi. Stones are either metabolic
or infective in origin.
Abnormalities of Volume of Urine
Oliguria
The otherwise healthy neonate is oliguric for the
first 2–3 days of life until the onset of the postnatal
diuresis. Ninety-two percent of neonates will pass
urine within the first 24 h of life, and almost all
newborns will do so in the first 48 h [12]. Beyond
the immediate neonatal period, oliguria is defined
as urine output of less than 500 ml/24 h/1.73 m2,
which is sufficient to maintain homeostasis. The
most common cause of oliguria in children is
intravascular volume depletion. Where this has
been excluded or adequately treated and volume
depletion persists, a search for intrinsic or obstruc-
tive renal disease should be commenced.
Polyuria and Polydipsia
Of those children presenting to medical practi-
tioners with symptoms of polyuria, only a very
small proportion will have a significant renal or
other pathology. A strict definition of polyuria
does not exist, though a daily urine output exceed-
ing 2 l in school-aged children is unusual. It is
important to distinguish polyuria from frequency
of micturition where small volumes of urine are
passed frequently though the 24 h urine output is
within normal limits.
Significant pathologies which result in poly-
uria are shown in Table 2 and include excessive
fluid intake, increased osmotic load, failure to
produce or release antidiuretic hormone (ADH),
and resistance to the actions of ADH in the kidney.
The distinction between primary polydipsia and
primary polyuria may be inferred from the relative
osmolality of plasma and urine and the response to
controlled water deprivation. The distinction
between pituitary and renal causes of polyuria
may be made by the plasma ADH concentration
and the urinary response to D-amino-arginine
vasopressin.
M.A. Shenoy and N.J.A. Webb
Table 2 Causes of polyuria in children
Increased fluid intake
Psychogenic/behavioral polydipsia
Hypothalamic polydipsia
Hyperreninemia including Wilms tumor
Increased osmotic load
Recovery from acute kidney injury
Diabetes mellitus and other causes of hyperglycemia
Chronic kidney disease
Following mannitol infusion
Failure of ADH production
Cranial diabetes insipidus
Basal skull fracture
Cranial tumors
Post hypophysectomy
Infection; encephalitis, meningitis, tuberculosis
Vascular aneurysm or thrombosis
Failure of renal response to ADH
Nephrogenic diabetes insipidus (X-linked)
Acquired unresponsiveness to ADH
Obstructive uropathy
Hypokalemia
Hypercalcemia
Sickle cell disease
Drugs, e.g., amphotericin, methoxyflurane,
tetracycline
Wetting and Abnormalities
of the Passage of Urine
Wetting is the most common urinary tract disorder
of childhood, though in most cases, there is no
organic pathology. The majority of children will
achieve daytime bladder control between 3 and
4 years of age [13]. The prevalence of nocturnal
enuresis is 15–20 % at age 5 years and up to 3 % in
young adults [14]. Nocturnal enuresis has a strong
genetic predisposition and on its own, is a benign
self-limiting condition. Daytime wetting, which
can occur as a result of a number of disorders
including detrusor overactivity, dysfunctional
voiding, and, rarely, giggle incontinence, which
is more common in girls and usually resolves by
the age of 9 years. Secondary enuresis, which is
defined as enuresis that develops after at least
6 months of dryness, raises the possibility of an
organic cause (e.g., neuropathic bladder) espe-
cially in boys; however, it is commonly associated
20 Clinical Evaluation of the Child with Suspected Renal Disease
with psychosocial disturbances such as parental
separation, the birth of a new child, or a death in
the family. Diabetes mellitus should always be
excluded. A careful voiding and wetting history
along with a focused physical examination will
help to identify any organic pathology. The pres-
ence of dysuria and frequency might suggest a
diagnosis of urinary tract infection, whereas poor
urinary stream and an enlarged bladder would
point to a diagnosis of a neuropathic bladder or
an obstructive pathology such as posterior ure-
thral valves. Examination of the urine for infec-
tion is essential in all children with wetting. In a
child with persistent daytime wetting, a pre- and
post-void ultrasound of the renal tract is useful to
screen for urinary tract anomalies and to assess
bladder emptying. Urodynamic assessment to
identify abnormalities of bladder and sphincter
function is indicated in children with a neuro-
pathic bladder and in some children with persis-
tent and troublesome wetting who do not
demonstrate a good response to conservative
management.
Pollakiuria, a benign, self-limiting condition,
is characterized by the very frequent (every 5–20
min) passage of urine during the daytime hours. It
most commonly presents in preschool-age chil-
dren and is not associated with wetting.
Edema
Children with renal disease may present with
edema, a major clinical manifestation of ECF
volume expansion. Edema may occur in the
acute nephritic syndrome and other causes of
acute renal impairment as a result of failure of
salt and water excretion. Here, peripheral edema
is accompanied by intravascular volume expan-
sion with hypertension and pulmonary edema. In
the nephrotic syndrome, loss of plasma oncotic
pressure secondary to hypoproteinemia and
increased vascular permeability result in the loss
of fluid from the intravascular into the extravas-
cular space. Here, edema is initially first evident as
swelling of the periorbital region, which is often
most marked in the morning. With progressive
fluid retention, more generalized edema develops
599
in a gravity-dependent distribution, the ankle and
sacral areas being the most severely affected. This
may be associated with abdominal distension sec-
ondary to ascites.
Asymptomatic Presentation Following
Screening or Routine Assessment
While the universal routine testing of children’s
urine by dipstick examination is not performed in
the large majority of countries, this has been
undertaken in Japan, having been introduced by
the Ministry of Education in 1973 with the aim of
the early detection of asymptomatic renal disease
[15]. Outside of universal screening programs,
children may have their urine tested at the time
of attendance at hospital emergency departments
and outpatient clinics or as part of a routine med-
ical assessment for insurance or immigration pur-
poses. Such testing may detect abnormalities in
the urine (commonly microscopic hematuria),
which results in referral for further assessment.
It is estimated that 5 % of children have hyper-
tension [16]. Children may have hypertension
detected following a routine medical examination
performed at school or for participation in partic-
ular sporting activities. In such instances, it is
firstly essential to rule out erroneously high
blood pressure, which has occurred as a result of
the use of incorrect measuring equipment or white
coat hypertension. While renal disease is widely
reported to be the commonest cause of hyperten-
sion in children, the rising tide of childhood obe-
sity may soon see this become the predominant
cause [17]. There is an ongoing debate as to
whether children ought to be screened for hyper-
tension, especially since up to 40 % of children
will have end-organ damage at the time of diag-
nosis of hypertension [18].
The screening of asymptomatic siblings of
index cases of renal disease is a difficult area.
Clearly where the detection of significant pathol-
ogy necessitating the commencement of specific
therapy is possible, e.g., cystinosis, hemolytic
uremic syndrome associated with complement
abnormalities, and Alport syndrome, then appro-
priate testing should be performed. Siblings and
600
close contacts of children presenting with STEC-
associated hemolytic uremic syndrome are not
infrequently found to have evidence of varying
degrees of renal impairment and anemia in the
absence of any significant symptoms, and such
detection will allow the commencement of
appropriate therapy and follow-up. In contrast,
there is no consensus as yet regarding screening
of asymptomatic siblings of children with
autosomal dominant PKD. Many authorities
would recommend that ultrasound or genetic
screening does not take place in these children as
there is no proven treatment as yet. Potentially
affected individuals could undergo an annual
check of blood pressure and urinalysis until they
are able to make a fully informed decision about
the relative merits of presymptomatic detection,
bearing in mind the implications that this may
have. However, preventative treatment for
ADPKD may not be far off, and there may be
advantages in early identification of the condition.
There are a number of situations where medical
opinion is divided, for instance, whether
newborn siblings of children with vesicoureteric
reflux should undergo radiological assessment;
definitive answers to these questions are only
likely to be obtained through the enrolment of
such children in prospective randomized con-
trolled trials.
Urinary Tract Infection
Urinary tract infection is common. Swedish data
report a cumulative incidence by 2 years of age of
2.2 % in boys and 2.1 % in girls [19]. In the
northern United Kingdom, cumulative referral
rates by 7 years of age are as high as 2.8 % for
boys and 8.2 % for girls, rising to 3.6 and 11.3 %,
respectively, by 16 years of age [20]. While older
children may present with classical symptoms of
either lower urinary tract infection (dysuria, fre-
quency, wetting) or upper urinary tract infection
(systemic upset, fever, loin pain), these features
are much less pronounced in the younger child;
here, nonspecific manifestations may include poor
feeding, vomiting, irritability, failure to thrive,
lethargy, and abdominal pain.
M.A. Shenoy and N.J.A. Webb
Diagnosis of urinary tract infection is impor-
tant; as aside from the acute morbidity associated
with infection, the development of infection is an
important marker of underlying congenital abnor-
malities of the kidney and urinary tract (CAKUT),
in particular, vesicoureteric reflux. Furthermore,
infection itself is an important cause of renal cor-
tical scarring in those predisposed to this. There-
fore, a diagnosis of urinary tract infection should
always result in consideration being given to
radiological assessment of the urinary tract. Ultra-
sonography of the kidneys and bladder should be
performed to detect anatomic abnormalities. Pre-
vious guidelines recommended that all children be
investigated after a single infection, though more
recent guidelines, for instance, those produced by
the UK National Institute for Health and Clinical
Excellence and the American Academy of Pedi-
atrics, have recommended that further radiologi-
cal investigations are only performed on the very
young and those with recurrent or difficult-to-treat
infections (http://www.nice.org.uk/guidance/
index.jsp?action=byID&o=11819, http://pediat
rics.aappublications.org/content/early/2011/08/
24/peds.2011-1330).
Clinical History
Antenatal History
A record should be made of the results of antenatal
imaging studies, particularly the detailed scan
performed at around 20 weeks of gestation.
Abnormalities of liquor volume should be
recorded. In the absence of rupture of the amniotic
membranes, oligohydramnios or anhydramnios
after 16 weeks of gestation represents failure of
urine excretion due to obstruction to urine flow or
inability of the kidneys to produce urine. Polyuric
states including neonatal Bartter syndrome and
congenital nephrotic syndrome are associated
with polyhydramnios; it should be remembered,
however, that only a small proportion of cases of
polyhydramnios are caused by renal disease.
A raised maternal serum alpha-fetoprotein
level between 15 and 20 weeks of gestation is
associated with spinal cord defects and the
20 Clinical Evaluation of the Child with Suspected Renal Disease
congenital nephrotic syndromes and is an indica-
tion for a detailed antenatal ultrasound scan and
amniocentesis.
Close attention should be paid to drug history.
Maternal intake of medications such as COX-2
inhibitors, angiotensin-converting enzyme inhibi-
tors (ACEis), or angiotensin receptor blockers
(ARBs) in the second and third trimester is asso-
ciated with renal failure in the neonatal period.
First-trimester exposure to ACEis or ARBs can
lead to major cardiovascular, central nervous sys-
tem, and renal malformations [21].
Other aspects of maternal medical history
should be noted. Previous recurrent miscarriages
should alert the physician to the possibility of a
chromosomal abnormality. Maternal gestational
diabetes and obesity are associated with increased
risk of CAKUT. Maternal obesity is also associ-
ated with increased risk of neural tube
defects [22].
Birth History
An enlarged placenta (greater than 25 % of the
infant’s birth weight) is a feature of congenital
nephrotic syndrome. The presence of hematuria
following perinatal asphyxia may be due to renal
venous thrombosis. Umbilical arterial catheteriza-
tion is associated with vascular damage and is the
commonest cause of hypertension in the neonatal
period [23]. The presence of a single umbilical
artery in an infant is associated with an increased
risk of a variety of renal anomalies including
vesicoureteric reflux, MCDK, and renal aplasia
and dysplasia [24]. It is debatable as to whether
these infants merit further imaging unless the
antenatal scan has demonstrated any abnormality;
in such cases, an ultrasound scan of the renal tract
will provide useful initial information and guide
further evaluation.
Family History
A detailed family history forms an essential com-
ponent of the clinical evaluation and may often
provide important clues to the diagnosis. It is
601
considered good practice to document the
genogram in the case records. A variety of renal
diseases including PKD, familial glomerulone-
phritis, and tubular disorders, e.g., Bartter and
Gitelman syndromes, are inherited. A family his-
tory of epilepsy or learning difficulties should
alert the clinician to the possibility of tuberous
sclerosis in a child with polycystic kidneys. An
increasing number of hereditary renal disorders
can now be diagnosed by DNA analysis, and it
is therefore essential that the family is provided
with detailed counseling with regard to the poten-
tial future implications.
Parental consanguinity is common in a number
of societies including the Muslim Middle Eastern
countries and the UK Pakistani community, where
up to 55 % are married to a first cousin [25]. This
should raise the possibility of conditions which
are transmitted in an autosomal recessive manner
such as autosomal recessive PKD, juvenile
nephronophthisis, and cystinosis. End-stage
renal failure of all causes is also more prevalent
in this population [26].
Deafness and renal failure in a male relative
may suggest a diagnosis of Alport syndrome,
which is most commonly inherited in an
X-linked manner. The inheritance of isolated
vesicoureteric reflux is thought to be consistent
with multifactorial or autosomal dominant with
reduced penetrance and has a sibling recurrence
rate of 30–50 % [27].
General Medical History
Renal disease may be a feature of a number of
childhood disorders. A pediatric nephrologist
working in a tertiary center will often be asked
to see children under the care of other specialists
and must therefore be familiar with the renal man-
ifestations of systemic disorders and iatrogenic
problems. A number of drugs including nonste-
roidal anti-inflammatory drugs (NSAID),
aminoglycosides, and acicyclovir are associated
with AKI. Renal involvement may be a part of a
multisystem disorder such as Henoch-Schönlein
purpura or systemic lupus erythematosus.
Hyponatremia due to cerebral salt wasting or
602
syndrome of inappropriate ADH secretion is a
common problem on the neurosurgical ward.
A diagnosis of retinitis pigmentosa should alert
to the possibility of juvenile nephronophthisis.
Tubulopathy is often seen in children on the pedi-
atric oncology ward following chemotherapy with
agents such as adriamycin or ifosfamide. Atypical
hemolytic uremic syndrome is known to be asso-
ciated with cisplatin, the oral contraceptive pill,
and tacrolimus. Asymptomatic renal calculi are
known to occur after a short course of ceftriaxone,
and nephrocalcinosis is associated with the
prolonged use of furosemide.
Urine and Micturition
The clinical history should record information
regarding daytime and nighttime wetting (see
above) and abnormalities of micturition, includ-
ing the nature of the urinary stream (e.g., hesi-
tancy, staccato micturition, terminal dribbling, the
need to sit to void, etc.). Poor urinary stream
should raise the possibility of posterior urethral
valves in a male infant, though following the
introduction of routine ultrasound scanning, the
majority of such cases would be detected in the
antenatal period. By 3 years of age, most children
have some conscious control over micturition and
achieve daytime control, but enuresis and daytime
accidents can continue to occur. In an older child,
the voiding frequency and an estimate of the uri-
nary volume should be documented [28].
Dietary History
The fluid and dietary restrictions in renal disease
depend on the type and stage of disease. For
instance, while salt and fluid restriction is not
necessary in polyuric states such as proximal
renal tubular disorders and Bartter syndrome,
anephric children on dialysis will require tight
control of their salt and fluid intake. Most children
with advanced renal failure will be on a restricted
potassium, phosphorus, and protein diet.
Anorexia associated with renal failure means
M.A. Shenoy and N.J.A. Webb
that these children are often malnourished, and
input from an experienced pediatric dietician is
mandatory.
One of the consequences of the obesity epi-
demic has been the increasing numbers of over-
weight children with hypertension, disturbed
glucose metabolism, and hyperlipidemia
[29]. Excessive salt intake may be associated
with hypercalciuria.
Psychosocial History
The impact of the disease on the child, for exam-
ple, the amount of school missed, body image,
limitations on lifestyle, self-esteem, and peer reac-
tions, should be assessed. There is a wealth of
evidence supporting the view that chronic physi-
cal illness and disability are risk factors for mental
health problems in children. Social disadvantage,
poverty, poor housing, educational failure, and
family instability are other risk factors. Caring
for a chronically ill child is a huge challenge for
any family, and parents often have to give up
employment in order to achieve this. Impact on
siblings should be given consideration. A skilled
social worker and child psychiatrist or psycholo-
gist are therefore essential members of a multipro-
fessional pediatric nephrology team. The impact
of the disease on the child’s intellectual, emo-
tional, and social progress must be assessed. An
awareness of the risk factors will help the physi-
cian identify those children who are at particularly
high risk of developing mental health problems.
Adherence with prescribed medications is a par-
ticular problem during adolescence and is the
commonest cause of rejection and graft loss in
this age group [30].
Clinical Examination
General Assessment
An initial rapid assessment of the level of con-
sciousness, airway, breathing, and circulation
should be performed to determine how ill the
20 Clinical Evaluation of the Child with Suspected Renal Disease 603

1. Airway 6. Abdomen
Breathing Scars, PD catheter
Circulation Distension, peritonitis
III or well child Renal mass
Bowel sounds
2. General assessment
Transplant kidney-
Hydration
tendemess/bruit
Growth - centile charts
Genitalia, urethral opening
Nutrition
Anus
Dysmorphic features
7. Central nervous system
Tanner staging
Mental status
3. Skin Cranial nerves
Sallow Tone, power, reflexes
Pallor Gait, coordination
Jaundice
Oedema 8. Musculoskeletal system
Cushingold features Muscle wasting, weakness
Scratch marks Hemihypertrophy
Rash Joint swelling
Central venous/ Spine, sacrum
haemodialysis catheter Linb deformity
Wrist swelling
Eyes 9. Urine analysis
Face and oral cavity Microscopy
Ear deformity Dipstick for blood, protein,
nitrite leucocytes and glucose
4. Cardiovascular system
Pulse including femorals
BP
JVP
AV fistula
Cardiomegaly
Murmur
Bruits
5. Chest
Kussmaul breathing
Rib rosary
Harrison’s sulcus
Pleural effusion/pulmonary oedema

Fig. 1 Assessment of the child with renal disease

child is (Fig. 1). Any obvious dysmorphic features Growth

should be recorded. Pain associated with renal
disease is often confused with back pain and
other causes of abdominal pain in childhood.
The state of the child (clothing and hygiene)
should be noted. A record of the mood and
demeanor of the child together with the family
interaction is informative.
Growth is an invaluable measure of health during
childhood, and accurate measurement of height
and weight along with the head circumference in
younger children is therefore essential. Obtaining
parental height is important to calculate the target
height centile for the child. Most growth charts
604
express anthropometric measures in terms of
percentiles; however, not infrequently children
with renal disease are significantly below the
lowest percentile lines, and therefore, expressing
height, weight, and body mass index in terms of
standard deviation score (SDS or z-score) is
sometimes more informative. Pubertal develop-
ment (Tanner staging) should also be formally
assessed.
Nutrition
Assessment of nutrition in children with renal
disease can be a challenge; fluid overload and
abnormalities in the distribution of fat and lean
body tissue can lead to misinterpretation of the
nutritional anthropometric measures [31]. The
commonly used nutritional markers are dietary
assessment, height, estimated dry weight, body
mass index, serum albumin, and skin fold thick-
ness. Tools for assessing body composition such
as dual energy x-ray absorptiometry and bioelec-
trical impedance analysis can sometimes provide
useful information.
Hydration/Volume Status
Assessment of the state of hydration and circula-
tory status is an important skill for all physicians
dealing with children with kidney disease. An
understanding of total body water and its distribu-
tion between the intravascular (plasma) and extra-
cellular fluid (interstitial) compartments is
essential. A child with nephrotic syndrome usu-
ally presents with a recent increase in weight and
peripheral edema, reflecting an increase in ECF
volume, though at the same time may exhibit
signs of intravascular volume depletion such as
reduced capillary refill time, cold extremities,
tachycardia, and oliguria. Occasionally, clinical
signs may be subtle, and measurement of urinary
sodium, fractional excretion of sodium (FeNa),
and urine osmolality may provide additional
information. In contrast, expansion of the intra-
vascular volume, such as in nephritic states,
results in hypertension, raised jugular venous
M.A. Shenoy and N.J.A. Webb
pulse, hepatomegaly, and the development of pul-
monary edema. Sometimes, assessment of hydra-
tion state may be difficult. In this situation, rather
than administering a large-volume fluid bolus
such as 20 ml/kg, it might be more appropriate
to administer a smaller volume such as 5–10
ml/kg and then to re-evaluate the child.
Cardiovascular System
Pulse
The rate, rhythm, and volume of the pulse should
be recorded. Tachycardia with a low-volume
pulse is a feature of intravascular volume
depletion. The presence of a gallop rhythm sug-
gests congestive cardiac failure. The femoral
pulses should always be examined in a child
with hypertension; coarctation of the aorta may
present with hypertension and cardiac failure. The
presence of bruits on auscultation over major
arteries such as the carotid or renal arteries sug-
gests extensive vascular disease or renal artery
stenosis.
Blood Pressure
Measurement of the blood pressure is an integral
part of the assessment of the child with renal
disease. It is important to ensure that the right
equipment and standards are used to ensure that
hypertension is correctly diagnosed; a wrong
diagnosis due to incorrect measurement can lead
to a battery of unnecessary invasive tests. There is
an increasing use of ambulatory blood pressure
monitoring in children, especially to diagnose
essential and white coat hypertension [32].
The investigation of hypertension is discussed
in ▶ Chap. 61, “Evaluation of Hypertension in
Childhood Diseases”.
Precordium
Fluid overload, long-standing hypertension, and
cardiomyopathy will give rise to cardiomegaly.
Pericarditis with a rub may be a feature of severe
uremia. A heart murmur may suggest a structural
anomaly but can also be a feature of infective
endocarditis or a hyperdynamic state as occurs in
anemia.
20 Clinical Evaluation of the Child with Suspected Renal Disease 605

Table 3 Causes of an abdominal mass of renal origin

Abnormality Causes if unilateral Causes if bilateral
Hydronephrosis (obstructive and PUJ obstruction Causes of bladder outlet obstruction including
nonobstructive) posterior urethral valves
VUJ obstruction Eagle-Barrett syndrome
Primary megaureter
Cystic mass Multicystic dysplastic PKD (dominant and recessive)
kidney
Simple cyst Cystic disease associated with syndromal
diagnosis
Cystic dysplasia Cystic dysplasia
Infection Acute pyelonephritis
Xanthogranulomatous
pyelonephritis
Perinephric abscess
Vascular Renal venous thrombosis
Arterial thrombosis
Acute cortical necrosis
Tumors Wilms tumor
Mesoblastic nephroma
Leukemic infiltrate
Miscellaneous Compensatory Beckwith-Wiedemann syndrome
hypertrophy
Duplex kidney Acute renal failure
Fused crossed ectopia Storage disorders
Congenital nephrotic syndrome

Respiratory System aware of the possibility of adhesions and obstruc-

Hyperventilation with Kussmaul respiration sug-
gests the presence of metabolic acidosis. Rachitic
deformity of the ribs can be seen in long-standing
chronic kidney disease. Dullness on percussion
over the lung fields might suggest pleural effusion
in a child with nephrotic syndrome or a peritino-
pleural leak in a patient on peritoneal dialysis.
Fine crepitations over the lung bases may indicate
the presence of pulmonary edema.
Abdomen
Abdominal Distension
Abdominal distension in renal disease may be due
to ascites, bladder enlargement due to urinary
retention and mass (see below), and the presence
of peritoneal dialysis fluid. In children who have
undergone previous abdominal surgery, for exam-
ple, urinary diversion procedures, one should be
tion presenting as an acute surgical abdomen.
Tenderness with guarding and rigidity is highly
suggestive of peritonitis, and in these circum-
stances, the opinion of a pediatric surgeon should
be sought.
Abdominal Mass
Prior to the onset of routine antenatal imaging, the
detection of an abdominal mass was a reasonably
frequent mode of presentation of a variety of renal
and urological disorders, including the MCDK,
pelvi-ureteric junction obstruction, and other
causes of hydronephrosis. In the neonatal period,
55 % of abdominal masses are renal in origin
(hydronephrosis 25 % and MCDK 15 %). In
later infancy and childhood, the proportion of
abdominal masses which are renal in origin
increases, largely due to the increased rate of
malignant tumors in the age group [33].
Renal causes of an abdominal mass are shown
in Table 3.
606
Features in the clinical history may help in
ascertaining the likely cause of the mass. In the
newborn period, the large majority of these
lesions will have been identified antenatally,
though additional clinical information may
inform subsequent investigation and likely diag-
nosis, i.e., the presence of oligohydramnios or
polyhydramnios. Beyond the newborn period, a
family history of severe hypertension may point to
a diagnosis of PKD. Presentation with symptoms
and signs of urinary tract infection may point to
many of the anomalies associated with urinary
obstruction or stasis. Children on peritoneal dial-
ysis are at increased risk of developing inguinal
and abdominal herniae.
Hepatosplenomegaly
Liver enlargement in association with renal dis-
ease can be seen in a number of conditions includ-
ing glycogen storage disease type 1, tyrosinemia,
and Fanconi-Bickel syndrome. Splenomegaly in a
child with hematuria might suggest infective
endocarditis. Autosomal recessive PKD may
cause hypersplenism as a consequence of hepatic
fibrosis. Hepatosplenomegaly in association with
renal disease reflects a multisystem disorder,
which can be inflammatory such as systemic
lupus erythematosus or a neoplastic process, for
instance, lymphoma presenting with AKI second-
ary to uric acid nephropathy.
Urinary Bladder
An enlarged urinary bladder can be detected by
palpation and percussion and is characteristically
associated with suprapubic pain. Gross constipa-
tion and local genital inflammation are the com-
moner causes of acute urinary retention in
children and rarely require urethral catheteriza-
tion. Lower urinary tract obstruction due to uro-
logical causes such as posterior urethral valves,
pelvic tumors, and the neuropathic bladder should
be considered an emergency, immediate decom-
pression of the urinary tract being indicated.
Genitalia
Clinical evaluation of the renal patient is incom-
plete without examination of the genitalia. The
presence of vulvovaginitis, a common
M.A. Shenoy and N.J.A. Webb
gynecological problem in prepubertal girls caus-
ing dysuria, should be noted. In male children, the
state of the foreskin and the position of the ure-
thral meatus should be recorded. Hypospadias is a
common problem with an incidence of up to 1:130
live births; parents should be made aware that
circumcision is contraindicated where this is pre-
sent. Eagle-Barrett syndrome is associated with
bilateral cryptorchidism. The presence of male
pseudohermaphroditism in association with pro-
teinuria and renal impairment in an infant is sug-
gestive of Denys-Drash syndrome.
Anus
The presence of an imperforate anus in a neonate
should alert the physician towards other associ-
ated anomalies such as sacral dysplasia, spinal
dysraphism, and vesicoureteric reflux.
Nervous System
Whilst a detailed neurological examination is
often not required, it is important to record the
level of consciousness and mental status and
examine the cranial nerves and muscle tone,
power, and reflexes along with the gait and coor-
dination. Hypertension can present with
papilledema and is the commonest cause of bilat-
eral Bell’s palsy. Seizures are associated with
electrolyte abnormalities, uremia, hypertensive
encephalopathy, vasculitis, and hemolytic uremic
syndrome. Blindness can result from rapid lower-
ing of blood pressure in severe hypertension. Pos-
terior reversible encephalopathy syndrome is
known to occur in association with the use of
immunosuppressive agents including methylpred-
nisolone [34] and tacrolimus following renal
transplantation [35]. In a child with continence
issues, careful examination of the peripheral sen-
sory and motor functions together with the assess-
ment of the anal tone is essential.
Skin
Itching is a feature of severe uremia. SLE can
present with a characteristic fixed erythematosus
20 Clinical Evaluation of the Child with Suspected Renal Disease
malar rash. The presence of a palpable purpura
over the extensor surfaces and buttocks suggests
Henoch-Schönlein purpura. Multiple café au lait
spots are seen in neurofibromatosis, which can
give rise to hypertension secondary to renal artery
stenosis. Tuberous sclerosis is characterized by
typical dermatological lesions including facial
adenofibromas (adenoma sebaceum), shagreen
patches over the lower back, and hypopigmented
ash leaf macules which may be present anywhere
in the body. Ichthyosis is a recognized feature of
the arthrogryposis, renal Fanconi syndrome, and
cholestasis (ARC) syndrome. Dystrophic nails
should raise the possibility of nail patella
syndrome.
Face
Examination of the face can provide valuable
clues to the underlying clinical problem. Any
obvious dysmorphic features such as Down or
William syndrome should be noted. Long-term
ciclosporin use is associated with hypertrichosis,
particularly in the Asian population [36]. Hirsut-
ism is a well-recognized feature of steroid toxicity
and Cushing syndrome. Nephrotic syndrome
often presents with early morning periorbital
swelling and is frequently misdiagnosed as aller-
gic rhinitis. Examination of the oral cavity is
important and may reveal gingival overgrowth as
a consequence of ciclosporin therapy. Asking the
child to smile may reveal a facial expression
resembling crying in the child with Ochoa syn-
drome, which is associated with the presence of a
neuropathic bladder.
Eyes
The eyes are affected in a number of renal dis-
eases, and therefore input from an experienced
pediatric ophthalmologist is invaluable.
Coloboma of the eyelid is seen in Goldenhar
syndrome. Uncontrolled renal osteodystrophy
can lead to scleral calcification. The presence of
hypercalcemia may cause redness of the eyes sec-
ondary to the metastatic crystallization of calcium,
607
phosphate, and hydroxyapatite in the conjunctiva
[37]. Slit lamp examination may reveal character-
istic crystal deposits in the cornea in cystinosis. In
a child with unexplained acute kidney injury,
examination of the eyes may clinch the diagnosis,
for example, in tubulointerstitial nephritis and
uveitis (TINU) syndrome. Uveitis is also a feature
of systemic diseases such as juvenile rheumatoid
arthritis. Aniridia is associated with an increased
risk of Wilms tumor. Presence of anterior
lenticonus, which is a conical protrusion in the
anterior aspect of the lens (seen almost exclu-
sively in males) along with macular changes, is a
characteristic feature of Alport syndrome. Cata-
racts are seen in Lowe syndrome and galacto-
semia but can also be a consequence of
corticosteroid therapy.
Not infrequently, a child with severe hyperten-
sion is referred by an optician with papilledema
and retinal hemorrhages and exudates. Diabetic
retinopathy, which parallels nephropathy, is rare
in childhood and correlates with the duration and
control of disease. Nephronophthisis is associated
with several characteristic eye lesions: retinitis
pigmentosa in Senior-Loken syndrome,
tapetoretinal degeneration in juvenile
nephronophthisis, and oculomotor apraxia in chil-
dren with Cogan syndrome. Retinitis pigmentosa
is a feature of Bardet-Biedl syndrome.
Ears
Minor external ear malformations such as
preauricular skin tags and pits are found in
0.5–1 % of newborns and do not warrant renal
evaluation unless accompanied by other systemic
malformations [38]. Branchial fistulae are a feature
of branchio-oto-renal syndrome. Alport syndrome
is characterized by the insidious onset of high-tone
sensorineural deafness. Sensorineural deafness is
also a feature of type 4 Bartter syndrome which is
the most severe phenotype presenting in the neo-
natal period with life-threatening volume depletion
and chronic kidney disease. Deafness can be a
consequence of aminoglycoside toxicity and is
also seen following rapid administration of a large
dose of furosemide.
608
Musculoskeletal System
Muscles
Muscle wasting is a feature of chronic uremia.
Myopathy can also be secondary to rickets and
renal osteodystrophy, which may improve follow-
ing vitamin D therapy. One of the recognized side
effects of corticosteroid therapy is proximal
myopathy, demonstrated by eliciting Gower’s
maneuver. Up to half of children with mitochon-
drial disorders will have renal involvement with
tubular dysfunction being the commonest
pathology.
Skeletal System
Florid skeletal deformities of the weight-bearing
limbs, such as genu varum or valgum, can be seen
in infants with rickets. Rachitic rosary and swell-
ing of the wrists and ankles due to epiphyseal
swelling are other well-recognized features of
rickets. Slipped femoral capital epiphysis is a
feature of renal osteodystrophy, and avascular
necrosis of the head of the femur may complicate
corticosteroid therapy, particularly following
renal transplantation.
Whilst polydactyly is a recognized feature of
disorders such as Bardet-Biedl and Meckel-
Gruber syndrome, isolated polydactyly is usually
associated with a favorable outcome.
Hemihypertrophy and Beckwith-Wiedemann
syndrome are associated with an increased risk
of Wilms tumor. Spinal dysraphism should be
suspected in infants with a lower midline back
lesion such as a subcutaneous mass, dermal vas-
cular malformation, hypertrichosis, a midline
dimple or sinus tract, a skin tag, or an asymmetric
gluteal cleft. Sacral agenesis is seen in infants of
insulin-dependent diabetic mothers but may also
be part of the familial Currarino triad syndrome
(presacral mass, sacral agenesis, and anorectal
malformation) [39].
Joints
Arthropathy is a characteristic feature of Henoch-
Schönlein purpura and SLE. It is also seen in
systemic onset juvenile idiopathic arthritis,
which if poorly controlled can lead to renal amy-
loidosis. Arthralgia and arthritis affect almost one
M.A. Shenoy and N.J.A. Webb
half of children with sarcoidosis. Metabolic disor-
ders such as Lowe syndrome and Lesch-Nyhan
syndrome may present with arthropathy.
Although hyperuricemia is often seen in children
with HNF1 beta mutation and pediatric transplant
recipients, gouty arthritis is rare.
Examination of the Urine
Macroscopic Examination
The usual yellow color of urine is due to the
presence of a number of pigments, some of
which are derived from food (i.e., riboflavin) and
others produced endogenously. The intensity of
the coloration depends upon the urinary flow rate;
where large quantities of urine are produced, e.g.,
following high fluid intake, the urine is paler in
color though darker in color at times of reduced
urine production.
Where the urine is abnormally red or dark in
color, dipstick testing and microscopy examina-
tion of the sample should be performed.
Dipstick Examination of Urine
Dipsticks
It is important that urine dipsticks are kept dry in
their container with the lid on and that the manu-
facturer’s expiry date is adhered to. A number of
automated devices are available. These ensure
that the stick is read at the correct time, thus
reducing inter-observer variability. Most will pro-
duce a printout, which can be attached to the
patient’s medical record.
Specific Gravity
Most modern urine dipsticks measure urinary spe-
cific gravity, though this information is rarely
utilized in clinical practice. Specific gravity is a
measure of the mass of individual solutes present
per unit volume of urine, as opposed to osmolal-
ity, which is the measure of the total particle
concentrations, irrespective of the mass of the
individual particles. Low values of specific grav-
ity (less than 1.010) are found at times of maximal
20 Clinical Evaluation of the Child with Suspected Renal Disease
water excretion and high values (greater than
1.025) at time of maximal urinary concentration.
pH
The pH of healthy urine varies between 4.5 and
8.0. Fasting produces low values, and the highest
pH measurements are seen following meals. pH
values are low where acidemia is present, except
for where this is secondary to renal tubular
acidosis.
Blood
Urine dipsticks detect the presence of hemoglobin
in the urine through its ability to catalyze a reac-
tion between hydrogen peroxide and o-tolidine.
Spotted positivity indicates intact red cells,
whereas uniform positivity indicates free hemo-
globin (e.g., in intravascular hemolysis or red cell
lysis in the urinary tract).
There are a number of causes of false positiv-
ity, including the presence of myoglobinuria,
oxidizing agents in the urine, and heavy bacterial
contamination. The presence of reducing agents,
such as ascorbic acid in the urine, may cause a
false-negative result. These highlight the
importance of confirmation of the presence of
red blood cells by microscopy of a fresh sample
of urine.
Protein
Urine dipsticks undergo color change from yellow
to green following binding with proteins. Albu-
min is better detected than other urinary proteins
including globulins, tubular proteins, etc. There
are a number of causes of false-negative and false-
positive results (Table 4). Dipstick analysis is not
a good quantitative test because of the effect of
urinary concentration; more concentrated urine
will show higher protein content, and where pro-
teinuria is detected, formal quantification with a
urinary protein/creatinine or albumin/creatinine
ratio is indicated.
It is well recognized that urinary protein excre-
tion increases throughout the daytime with
prolonged duration in the upright position, and
first morning samples should be assessed to rule
out any element of orthostatic proteinuria. Tran-
sient proteinuria may occur following exertion,
609
Table 4 Causes of false-positive and false-negative pro-
teinuria on dipstick testing
False-negative
False-positive proteinuria proteinuria
Concentrated urine Dilute urine
Alkaline urine Acidic urine
Gross hematuria, pyuria,
bacteriuria
Dipstick left in urine too long or
delay in reading
Contamination and drugs
Antiseptics: chlorhexidine,
benzalkonium
fever, or acute illness and is of no significance
with regard to long-term renal outcome.
Glucose
Glucose is a small molecule, which is freely fil-
tered in the glomerulus. In health, the proximal
tubule reabsorbs all of the filtered glucose by an
active process, which has an upper rate limit or
transport maximum (TM). Certain normal individ-
uals have a lower TM for glucose and will have
glycosuria at normal or only slightly increased
plasma glucose concentrations (so-called renal
glycosuria). Glycosuria also occurs where overt
hyperglycemia is present, e.g., in diabetes mellitus
and in a number of tubular abnormalities, includ-
ing Fanconi syndrome. The lower limit of detec-
tion for glucose in the urine is 0.5 mmol/l.
Leukocytes
A number of routinely available urine testing
sticks will detect the presence of leukocyte ester-
ase, indicating the presence of pyuria. Where test-
ing is positive, urine microscopy should be used to
confirm this finding.
Nitrites
A number of routinely available urine testing
sticks will also detect the presence of nitrites.
These are produced by the majority of pathogenic
bacteria, and their detection provides further
screening data to assist in the diagnosis of urinary
tract infection. The test has a high specificity but a
low sensitivity for the diagnosis of UTI. As such,
as a standalone test, it is of limited usefulness.
610
Where UTI is suspected or needs to be excluded, a
urine culture is necessary to determine the bacte-
riological cause and antibiotic sensitivities or to
confidently rule out UTI.
Microscopy of Urine
There are many who advocate the routine bedside
microscopy of urine in all children who present
with suspected renal disease or urinary tract infec-
tion. This is not, however, widely performed for a
variety of reasons including unfamiliarity with the
technique and availability of appropriate micro-
scopes in clinical areas.
Casts
Casts are produced by the aggregation of Tamm-
Horsfall protein with cells or cellular debris in the
renal tubule. They are best seen in unspun urine as
centrifugation may damage casts. Where urine has
been centrifuged for other reasons, casts are most
frequently seen at the edge of the coverslip.
Hyaline casts are present in proteinuric states,
though may be found in concentrated specimens
of urine from normal individuals. These may
appear waxy if lipid droplets are present.
Cellular casts contain cellular material, the
source of which may provide some clues regard-
ing the underlying pathology. Red blood cell casts
are always pathological and indicate glomerular
bleeding. White blood cell casts indicate renal
inflammation secondary to pyelonephritis or
immunologically mediated disease. Epithelial
cell casts (often present with red and white blood
cell casts) are produced from shed tubular epithe-
lial cells and may be seen during recovery from
acute tubular necrosis.
Red Blood Cells
The excretion of a small number of red cells is a
normal phenomenon, which increases with
increasing age and following vigorous exercise.
Children with febrile illness may develop a tran-
sient increase in red cell excretion. However, the
persistent presence of greater than 5 red cells/mm3
in uncentrifuged urine is abnormal. Urine micros-
copy can distinguish anatomically normal RBCs
M.A. Shenoy and N.J.A. Webb
of lower urinary tract origin from dysmorphic
RBCs of glomerular origin, which have been
distorted during their passage through the filtra-
tion barrier. This is best performed using phase
contract microscopy, though possible with ordi-
nary light microscopy. In its purest form, the dis-
tinction is useful, though often a mixture of
abnormal and normal cells is present and interpre-
tation is difficult. Acanthocytes are red blood cells
with thorn-like projections of varying lengths dis-
tributed over the surface due to an increase in
membrane cholesterol:lethicin ratio. Where
acanthocytes constitute greater than 5 % of the
urinary red cell population, this may indicate the
presence of a glomerulonephritis.
White Blood Cells
The presence of greater than 10 white cells/mm3
in a midstream sample of urine is considered
abnormal. Neutrophils are detected in urinary
tract infection but are also seen in contaminated
urine samples, proliferative glomerulonephritis,
and interstitial nephritis. The presence of eosino-
phils in the urine is a sensitive and specific sign of
acute interstitial nephritis.
Bacteria and Other Organisms
Urinary bacteria may be clearly visible without
Gram staining. Their detection may be enhanced
by the use of phase contrast microscopy. Fungi, e.
g., Candida and Schistosoma species (a rare cause
of hematuria), may also be detected.
Epithelial Cells
The presence of epithelial cells in the urine
may represent desquamation from the urinary
tract. Tubular cells may be seen following
tubular injury (acute tubular necrosis, acute trans-
plant rejection). Squamous cells are commonly
exfoliated from the urethra and are a normal
finding.
Crystals
Normal urine contains crystals of calcium phos-
phate and oxalate. Other crystals may be cystine,
uric acid, or dihydroxyadenine.
20 Clinical Evaluation of the Child with Suspected Renal Disease
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 Laboratory Investigation of the Child
with Suspected Renal Disease 21
George van der Watt, Fierdoz Omar, Anita Brink, and
Mignon McCulloch

Contents Calcium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626

Magnesium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614 Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 627
Glomerular Function and Injury . . . . . . . . . . . . . . . . . . 614 Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 628
Use of Endogenous Substrates in Clinical Urine Amino Acid Analysis . . . . . . . . . . . . . . . . . . . . . . . . . 628
Medicine for the Estimation of GFR . . . . . . . . . . . . . . . . 615 Potassium and Transtubular Gradient of
Use of Exogenous Substrates for Measurement Potassium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 628
of GFR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 618 Assessment of Renal Acid/Base Homeostasis . . . . . . 629
Urate, Oxalate, Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 630
Proteinuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 621 Renal Tubular Concentrating Ability . . . . . . . . . . . . . . . . 631
Measurement of Urinary Protein . . . . . . . . . . . . . . . . . . . . 622
Renal Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624 Parathyroid Hormone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 632

Urinalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633

Tubular Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625

Fractional Excretion (FE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626
Sodium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626
Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626

G.Watt (*) • F. Omar

Chemical Pathology, University of Cape Town and
National Health Laboratory Service, Red Cross Children’s
Hospital and Groote Schuur Hospital, Cape Town,
South Africa
e-mail: George.vanderwatt@uct.ac.za;
Fierdoz.omar@uct.ac.za
A. Brink
Department of Pediatrics and Child Health (Nuclear
Medicine), University of Cape Town, Red Cross War
Memorial Children’s Hospital, Cape Town, South Africa
e-mail: anita.brink@uct.ac.za
M. McCulloch
Department of Paediatric Intensive Care/Nephrology,
University of Cape Town, Red Cross War Memorial
Children’s Hospital, Cape Town, Western Cape,
South Africa
e-mail: mignon.mcculloch@uct.ac.za;
mignonmcculloch@yahoo.co.uk

# Springer-Verlag Berlin Heidelberg 2016 613

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_19
614
Introduction
The kidney can be injured by a variety of different
mechanisms. Investigating the type and assessing
the degree of injury and its progression involves
laboratory assessment and often tissue sampling.
This chapter will discuss laboratory assessment and
investigation with emphasis on the use of blood and
urine samples to investigate renal function.
There are a number of terms frequently used in
laboratory assessment that are now more precisely
defined and help us think about how we use labora-
tory assessment. In general, laboratory studies are
used to help with diagnosis, prognosis, follow-up,
or effect of therapy. Usually, the specific laboratory
study performed fits the definition of a biomarker – a
characteristic that is measured and evaluated objec-
tively as an indicator of normal biologic or patho-
genic processes or pharmacologic responses to a
therapeutic intervention. This chapter will discuss
those measurements used primarily in patient care.
Glomerular Function and Injury
The glomerular filtration rate (GFR) is considered
to be the most accurate measure of renal func-
tional capacity and reliably reflects the number
of functional nephrons. As a physiological mea-
surement, it has high sensitivity and specificity for
changes in renal function and by convention is
expressed in mL/min/1.73 m2. The GFR is a
dynamic measurement and is defined as the vol-
ume of plasma that can be cleared of an ideal
substance per unit of time, corrected to body sur-
face area. Clearance of an ideal substance is there-
fore considered to be equal to the GFR where
clearance = [U]/[P]  V where [U] is the urine
concentration of the substance, [P] the plasma
concentration, and [V] the urine flow rate in
mL/min. Body surface area is usually calculated
according to the formula: BSA (m2) = 0.024265
 weight (kg)0.5378  height (cm)0.3964 [1].
For a clearance measurement to be equal to the
GFR, the substance to be measured should fulfill
the so-called “ideal” criteria, namely, be present at
a stable plasma concentration, be freely filterable
through the glomerulus, not be reabsorbed or
G. Watt et al.
secreted by the tubules, and not have inherent
toxicity or affect renal function. Thus, the sub-
stance is neither added to urine nor removed from
urine during its path through the tubule.
Compounds used to measure clearance are
either exogenous or endogenous. Exogenous
compounds are nonphysiological substances that
are infused and measured in plasma and/or urine
over time to calculate GFR, while endogenous
compounds are produced by the body at a rela-
tively constant rate and maintain a stable plasma
concentration at a specific GFR.
Creatinine clearance is arguably still consid-
ered the most convenient endogenous estimate
of GFR despite the fact that it does not fulfill all
the criteria for an ideal substance, whereas inulin
clearance (a fructose polymer) is considered the
“gold exogenous standard” as it fulfills all the
ideal criteria. In clinical practice, however, less
ideal radiolabeled agents such as technetium-
99m-labeled diethylenetriamine pentaacetic acid
(99mTc-DTPA), chromium-51-labeled ethylene-
diaminetetraacetic acid (51Cr-EDTA), and
iodine-125-labeled iothalamate (125I-iothalamate)
clearances are usually employed due to the fact
that routine measurement is less onerous than
inulin. Briefly stated, 51Cr-EDTA and 99mTc-
DTPA clearances correlate well with inulin clear-
ance [2, 3], whereas 125I-iothalamate’s correlation
to inulin clearance is only fair [4]. In general,
99m
Tc-DTPA and 51Cr-EDTA do not perform
well in neonates, 51Cr-EDTA appears to have an
extrarenal source of elimination, and 99mTc may
dissociate from DTPA during clearance studies,
whereas iothalamate appears to undergo some
tubular secretion and reabsorption [2]. Further-
more, poor correlation exists between techniques
and the various radionuclide agents, and as such,
it is recommended that technique-specific refer-
ence intervals are used [5].
Iohexol is increasingly being used as an alterna-
tive to inulin. Iohexol is a nonionic, nonradioactive,
X-ray contrast medium of low molecular weight
that is eliminated from plasma solely by glomerular
filtration [6]. It is easily measured by HPLC using
very small sample volumes, increasing its useful-
ness in children, and clearance correlates well with
inulin clearance [7, 8].
21 Laboratory Investigation of the Child with Suspected Renal Disease 615

Table 1 Plasma creatinine reference intervals (2.5–97.5th percentiles) [9]

Enzymatic creatinine Jaffe creatinine
Age group mg/dL umol/L Age group mg/dL umol/L
0–14 days 0.32–0.92 28–81 0–14 days 0.42–1.05 37–93
15 days to <2 years 0.1–0.36 9–32 15 days to <1 year 0.31–0.53 27–47
2 to <5 years 0.2–0.43 18–38 1 to <4 years 0.39–0.55 34–49
5 to <12 years 0.31–0.61 27–54 4 to <7 years 0.44–0.65 39–57
12 to <15 years 0.45–0.81 40–72 7 to <12 years 0.52–0.69 46–61
15–19 years male 0.62–1.08 55–95 12 to 15 years 0.57–0.80 50–71
15 to <19 years female 0.49–0.84 43.3–74 15 to <17 years male 0.65–1.04 57–92
15 to <17 years female 0.59–0.86 52–76
17 to <19 years male 0.69–1.10 61–97
17 to <19 years female 0.60–0.88 53–78

Use of Endogenous Substrates
in Clinical Medicine for the Estimation
of GFR
Creatinine
Creatinine, a waste product, is the nitrogen-
containing cyclic anhydride of phosphocreatine,
an intracellular component of the skeletal muscle,
involved in the temporary storage and transfer of
high-energy phosphate moieties required for mus-
cle contraction (Table 1). Creatine itself is synthe-
sized in the liver, pancreas, and kidneys prior to
transport to the muscle tissue where it is phos-
phorylated and utilized in a cyclical manner. Syn-
thesis is however self-limiting to a certain extent,
and production decreases at very high plasma
creatinine levels. Approximately 1–2 % per day
of total muscle creatine spontaneously converts to
creatinine, giving rise to a stable production rate.
The stable plasma concentration of creatinine is
therefore a function of muscle mass relative to the
body surface area and the GFR. Creatinine clear-
ance tends to overestimate the true GFR because
although primarily filtered at the glomerulus, cre-
atinine is also secreted into urine by tubular epi-
thelial cells and is also reabsorbed to a certain
extent [10, 11]; these effects are more pronounced
at high levels of plasma creatinine. Because of the
combination of effects of relative muscle mass
and evolving GFR, the plasma concentration of
creatinine in childhood changes over time; fur-
thermore, with the onset of puberty, creatinine
levels are higher in males compared females, and
sex-specific reference intervals are required in this
age group. At birth, the plasma creatinine value
reflects maternal concentrations, giving rise to
higher creatinine values in the first days of extra-
uterine life. The GFR itself at term birth is only
approximately 30 mL/min/1.73 m2 and lower in
premature neonates but increases rapidly in the
first 18 months of life to stabilize at normal adult
values of approximately 104 mL/min/1.73 m2
from where it remains relatively constant
(Fig. 1). Best available normative data for GFR
in premature neonates and children < 15 years of
age are presented in Tables 2 and 3 respectively.
As can be appreciated from the presented data,
both plasma creatinine and GFR are subject to
wide interindividual variation, and patients with
relatively low muscle mass and significant renal
dysfunction may still have a plasma creatinine
level within reference intervals, making it difficult
to identify early stages of renal dysfunction with-
out the hindsight of serial measurements or clear-
ance studies. With the loss of nephrons, the GFR
falls, and plasma creatinine tends to increasingly
overestimate the GFR [14, 15]. This is due, in
part, to the fact that the tubular secretion of creat-
inine contributes an increasing fraction of total
creatinine excreted, leading to a clearance that is
increasingly greater than actual GFR.
Creatinine is routinely measured in plasma and
urine using either the chemical Jaffe method or
enzymatically by means of either creatininase or
creatininase-/creatinase-based assays. A major
limitation of the Jaffe reaction is the fact that the
reagent (picric acid) cross-reacts with protein,
glucose, ascorbic acid, ketones, and
616 G. Watt et al.

Fig. 1 GFR values derived 160

from 51Cr-EDTA clearance,
corrected for body surface
area in children under 140
2 years. The 10th, 25th,
50th, 75th, and 90th 120
percentiles are represented

GFR ml/min / 1.73 m2

[13]
100

80

60

40

20

0
0 0.5 1 1.5 2
age (years)

Table 2 Reference intervals for GFR (mL/min per 1.73 m2) in very preterm infants during the first month of life based on
creatinine clearance (3rd to 97th percentiles) [12]
Day 27 weeks GA 28 weeks GA 29 weeks GA 30 weeks GA 31 weeks GA
7 7.9–18.9 10.7–21.7 13.6–24.6 16.4–27.4 19.3–30.3
14 10.7–21.7 13.5–24.6 16.4–27.4 19.3–30.3 22.1–33.1
21 12.5–23.5 15.3–26.3 18.2–29.2 21–32 23.9–34.9
28 15.5–26.5 18.3–29.4 21.2–32.2 24–35 26.9–37.9

Table 3 Reference intervals for GFR (mL/min per aligned to those originally used to derive creati-
1.73 m2) in children <0.1 to >2 years of age based on nine clearance, GFR, and estimated GFR (eGFR)
51
Cr-EDTA clearance (mean 2 SD) [13]
51
reference intervals. To correct this discrepancy,
Age in years Cr-EDTA clearance (mL/min/1.73 m2) the Creatinine Standardization Program was initi-
0.1 34–70
ated by the National Kidney Disease Education
0.1–0.3 33.1–90.3
Program’s laboratory working group in collabora-
0.3–0.66 43.9–99.5
tion with the International Federation of Clinical
0.66–1 48–117.2
Chemistry and the European Federation of Clini-
1–1.5 55.9–127.1
1.5–2 58.3–130.7
cal Chemistry and Laboratory Medicine to reduce
2 63.6–145.2 interlaboratory variation in creatinine assay cali-
bration and to enable more accurate estimates of
glomerular filtration rate [16]. As of 2011, all
cephalosporins, among others, and although these major Jaffe and enzymatic creatinine assays
limitations are corrected for in modern assays by worldwide were standardized to a reference mate-
employing kinetic assays that measure the rate of rial, NIST SRM 967, traceable to an accurate
chromogen formation during a creatinine-specific isotope dilution mass spectrometric (IDMS)
window, assay manufacturers until recently still method. This standardization has also resulted in
calibrated their assays to overestimate creatinine the need for reformulation of reference intervals
by a factor of 20–30 % so as to yield results for GFR and creatinine, and eGFR equations
21 Laboratory Investigation of the Child with Suspected Renal Disease 617

Table 4 Recommended equations for estimating GFR (mL/min/1.73 m2) based on plasma creatinine values and
demographic characteristics
Age Recommended equation Mass units – mg/dL, height in cm SI units – creat in μmol/L, height in cm
<18 Bedside IDMS-traceable GFR = (0.413  height)/creat GFR = (36.20  height)/creat
years Schwartz eGFR for children
<18 years old [19]
18 IDMS-traceable MDRD GFR = 175  (creatinine)–1.154 GFR = 175  (creatinine /88.4)1.154
years Study Equation eGFR for  (age)0.203  (0.742 if  (age)0.203  (0.742 if female) 
Adults [17] female)  (1.212 if African (1.212 if African American)
American)
CKD-EPI (2009) (where GFR = 141  min(creat/κ, 1)α GFR = 141  min((creat/88.4)/κ, 1)α
κ = 0.7 for females and 0.9  max (creat/κ, 1)1.209  0.993  max (creat/κ, 1)1.209  0.993 Age
for males, α is 0.329 for Age
[1.018 if female] [1.159 if [1.018 if female] [1.159 if black]
females and 0.411 for black]
males, min indicates the
minimum of creat/κ or 1, and
max indicates the maximum
of creat/κ or 1) [18]

based on plasma creatinine. Currently, the best
performing eGFR equations based on plasma cre-
atinine values in adults are the IDMS-traceable
Modification of Diet in Renal Disease (MDRD)
Study equation and the Chronic Kidney Disease
Epidemiology Collaboration (CKD-EPI) equa-
tions [17, 18] and the Bedside IDMS-traceable
Schwartz equation for children under the age of
18 years [19]; the latter equation was derived
using an enzymatic creatinine method. It is impor-
tant to realize that all eGFR equations derived
prior to the IDMS standardization of creatinine
such as the Cockroft-Gault and original Schwartz
formula are no longer valid and should not be used
under any circumstances with standardized creat-
inine methods as they will overestimate the GFR.
Despite standardization of creatinine measure-
ments internationally, evaluations have demon-
strated a small positive bias, most prominent in
pediatric samples at low levels of creatinine
between Jaffe and enzymatic methods, and as
such, it is recommended that only enzymatic cre-
atinine methods be used when estimating GFR in
children with the Bedside IDMS-traceable
Schwartz equation [20]. As a general rule, eGFR
equations are superior to creatinine clearance-
derived GFRs in the majority of patients with
normal relative muscle mass but should not be
used in patients with unstable creatinine levels
(acutely ill patients, acute renal failure) or those
with extremes of relative muscle mass or creatine
intake (amputees, paraplegics, obese patients;
muscle-wasting disease, neuromuscular disor-
ders; malnutrition or patients on a vegetarian or
low-meat diet). In these patients, formal creatinine
clearance with timed urine collection is still
recommended. Estimated GFR equations are sub-
ject to higher variability at low plasma creatinine
levels, limiting their reliability in identifying early
stages of renal dysfunction. Most laboratories
only report numerical eGFR results below a
threshold of 60 mL/min/1.73 m2. Estimated GFR
equations are however very useful in monitoring
progression of renal dysfunction over time in an
individual patient. The recommended creatinine-
based equations are presented in Table 4.
To effectively calculate creatine clearance,
urine collections must be accurately timed to
avoid erroneous results. Ideally, a 24-h (1,440
min) urine collection is required to calculate
urine flow rate in mL/min which may be difficult
to achieve in small children. Under-collection will
report a falsely decreased GFR and over-
collection the opposite. To complete the clearance
calculation, plasma creatinine should ideally be
measured simultaneously with the urine collection
using the same assay methodology, and as with
eGFR calculations, clearance studies should not
be performed in patients with unstable creatinine
levels.
618 G. Watt et al.

Table 5 Equations for estimating GFR (mL/min/1.73 m2) based on plasma creatinine  urea and cystatin C values and
demographic characteristics
Mass units – creat in mg/dL, cys C
in mg/L, urea in mg/dL, height SI units – creat in μmol/L, cys C in mg/L,
Age Recommended equation in m urea in mmol/L, height in m
<18 IDMS-traceable Schwartz GFR = 39.1  (height/ GFR = 39.1  (height/(creat/
years eGFR-creat-cys for creat)0.516  (1.8/cystatin C) 0.294 88.4))0.516  (1.8/cys C) 0.294 
children <18 years old  (30/urea)0.169  (1.099)male  (30/(urea/0.357)0.169  (1.099)male 
[19] (height/1.4)0.188 (height/1.4)0.188
Adults CKD-EPI creatinine- GFR = 135  min(creat/κ, 1)α GFR = 135  min((creat/88.4)/κ, 1)α 
cystatin C equation (2012)  max (creat/κ, 1)0.601  min max ((creat/88.4)/κ, 1)0.601  min (cys
(where κ = 0.7 for (cys C/0.8, 1)0.375  max(cys C/0.8, 1)0.375  max(cys C/0.8, 1)0.711
females and 0.9 for males, C/0.8, 1)0.711  0.995 Age  0.995 age [0.969 if female] [1.08 if
α is 0.248 for females [0.969 if female] [1.08 if black]
and 0.207 for males, min black]
indicates the minimum of
creat/κ or 1, and max
indicates the maximum of
creat/κ or 1) [23]

Cystatin C reference material, ERM-DA471/IFCC across

Cystatin C can also be used as an endogenous
marker of renal function and to estimate GFR.
Cystatin C is a nonglycosylated low molecular
weight protein of 13.3 kd produced in all cells
with relative constancy. Plasma levels are less
significantly influenced by age, gender, body hab-
itus, or composition compared to creatinine
[21]. Renal handling of cystatin C is by complete
filtration with near total reabsorption and catabo-
lism in the proximal tubule and little or no urinary
excretion [22]. Reference intervals for plasma
cystatin C are presented in Table 5. Contrary to
earlier meta-analyses, the best available current
data indicates that plasma cystatin C-based
eGFR equations (eGFR-cys) do not more accu-
rately predict GFR than the creatinine-based equa-
tions (eGFR-creat) and that equations derived
from both analytes (eGFR-creat-cys) such as the
CKD-EPI creatinine-cystatin C 2012 are more
precise with less bias at the critical threshold of
60 mL/min/m2 [23–26]. Equations based on cre-
atinine and cystatin C for both adult and pediatric
populations are presented in Table 5. Presently, a
widespread implementation of eGFR-creat-cys
equations in favor of eGFR-creat is limited by
the fact that cystatin C is far more expensive
than creatinine to assay, utilizing nephelometric
or immunoturbidimetric methodologies, and has
only recently been standardized to an acceptable
assay platforms. As with creatinine, standardiza-
tion has led to the need for reevaluation or
reformulation of eGFR-creat-cys equations, and
current recommendations are that equations
should only be implemented in those populations
from which they were developed [27].
Use of Exogenous Substrates
for Measurement of GFR
Inulin
This is the classic marker for measuring GFR. The
test requires a constant infusion of inulin and
urinary catheter placement, and it is not routinely
used in clinical practice [28]. Reference values for
inulin clearance are demonstrated in Fig. 2.
51
Cr-EDTA
Availability of this radiopharmaceutical is not
universal, but 51Cr-EDTA is regarded as the best
radiopharmaceutical tracer for the measurement
of kidney function because it is eliminated exclu-
sively by glomerular filtration ([19, 99]). The ref-
erence single-injection plasma disappearance
curve method consists of an injection of 51Cr-
EDTA and blood samples drawn at 2 and
4 h. There is no collection of urine. Measuring
the decrease in activity in the blood allows one to
21 Laboratory Investigation of the Child with Suspected Renal Disease 619

Fig. 2 Inulin clearance as a 250

Inulin clearance, ml/min per 1.73 m2

function of age X  SD

200

150
120
100

50

0
Neonate 2–8 9–22 1–5.9 6–11.9 12–19 2–12 Adult Adult
days days months months months years

calculate the GFR [99]. It is recommended that the absolute differential renal function [33]. It should
two-sample method is used because improving however be noted that when combining imaging
the accuracy over the two-sample method would and GFR determination a larger dose of radiophar-
require at least 13 samples drawn between 2 and maceutical is administered. In addition, 99mTc-
4 h [29]. Correction factors are employed to cor- DTPA image quality is poor in patients with
rect for the initial fast clearance of activity from poor renal function and immature kidneys
the blood. The reproducibility of 51Cr EDTA GFR [33]. If there is reason to suspect a decrease in
measurement differs in patients with normal and renal function imaging with 99mTc MAG3 in com-
poor renal function. The day-to-day variation in bination with a separate GFR test, using a
measurement of GFR using this method is 4.1 % low-dose 99mTc-DTPA or 51Cr-EDTA is advised.
in patients with a GFR of greater than 30 mL/min
and 11.5 % in patients with a GFR less than The Two-Sample Method
30 mL/min [30]). This method is valid in patients The early exponential is neglected in this simpli-
down to a GFR of 15 mL/min/1.73 m2 [13, 19]. fied method.
The effective radiation dose that the patient Clearance is expressed as
receives from this investigation is approximately
D
0.011 mSv per examination [99]. This compares Cl ¼
favorably with the average radiation dose of A
where
0.1 mSv received by an anterior/posterior and
lateral chest X-ray [31].
Cl = clearance
D = dose
99m
Tc-DTPA A = the estimated area under the plasma curve
This radiopharmaceutical is readily available.
Variations in protein binding have been described The dose (D) is calculated from the following
as a problem in the past. This problem is rarely measurements:
encountered with the newer DTPA preparations
available commercially [32]. ðDm  RÞ  ADS  DS
D¼
When using the slope intercept method to per- AS
form this test, the same method is used as the one
described for 51Cr-EDTA [99]. This agent is rou-
tinely used as an imaging agent. This gives the Dm = dose measured before intravenous injec-
advantage of combining a renogram with the GFR tion: if the weighing technique is used, this is
determination and allows the clinician to calculate the weight of the dose.
620
R = residual activity in the syringe after injec-
tion: for the weighing technique, this is the
weight of the empty syringe after injection.
ADS = activity in 1 mL of the dilution of the
standard.
DS = the dilution factor of the standard, for
example, the drop is diluted in a 100 or
500 mL flask of water.
AS = the activity of the standard: if the weighing
technique is used, this is the weight of the
aliquot used to make the standard.
The estimated area under the curve (A), is
obtained from the following equation:
y0
A¼
b
where
Y0 is the intercept of the exponential of the late
plasma sample (2 and 4 h) at time 0 and b is the
slope of the exponential.
Using only the plasma sample activities and
plasma times, the equation can then be expressed
as
 
p
D  In 1
p2
Cl ¼ exp ððt1  lnp2 Þ  ðt2  lnp1 ÞÞ=ðt2  t1 Þ Where FBSA ¼ 0:00185:BSA0:3
ðt 2  t 1 Þ
where
D = administered activity in counts per minute
P1 = plasma activity in counts per minute/mL at
time 1
P2 = plasma activity in counts per minute/mL at
time 2
The clearance calculated from this equation is
then corrected for body surface area.
Clearance corrected for body surface area
(BSA) = Cl1
Cl1 = Cl  1.73/the patient’s body surface
area in mL/min/1.73 m2
The omission of early blood samples used to
measure the initial fast clearance of activity from
G. Watt et al.
blood leads to an overestimation of GFR, and a
correction factor should be employed. Currently,
the correction factors by Chantler and Brøchner-
Mortensen are routinely employed [99]. The
Brøchner-Mortensen correction factor results in
underestimation of renal clearance if the clearance
values are higher than 130 mL/min [34]. The
Chantler correction factor underestimates low
clearance values [99].
The Chantler method:
GFR ¼ 0:87  Cl1 in ml=min=1:73m2
The Brøchner-Mortensen method:
GFR ¼ 1:01  Cl1  0:0017
 Cl21 in ml=min=1:73m2
Newer correction factors by Fleming and
Brøchner-Mortensen and Jødal employ correction
factors for body surface areas [35].
Fleming’s method:
GFR ¼ ððCl1 Þ=ð1 þ FÞÞ  ðCl1 Þ in ml=min=1:73m2
Where F ¼ 0:00170 min=ml
Brøchner-Mortensen and Jødal:
GFR ¼ ððCl1 Þ=ð1 þ FBSA ÞÞ  ðCl1 Þ in ml=min=1:73m2
These newer correction factors do not have the
problems of overestimations seen with the
Chantler method and underestimation of the
Brøchner-Mortensen method [34, 35].
The Distribution Volume Method or
One-Sample Method
This method has been validated for both
51
Cr-EDTA and 99mTc-DTPA. In this method, a
single blood sample is drawn at 110 to 130 min
after the injection of the tracer. The volume
of distribution in this method is calculated and
not measured [36]. It is important to note
that this method is not accurate in patients with
clearance values below 30–35 mL/min/1.73 m2
[19, 37].
21 Laboratory Investigation of the Child with Suspected Renal Disease
When using the one-sample method, the GFR
is calculated as follows:
Cl ¼ ð2:602  V120Þ  0:273
V120 is the “virtual distribution of volume,”
where the dose injected is divided by the
120-min plasma concentration. It is rare that a
plasma sample is taken at exactly 120 min after
injection and a correction factor for the blood
sample time is employed. This correction factor
is not valid if the blood sample is taken outside the
allowed time window of 110–130 min:
P120 ¼ Pt  e0:008ðt120Þ
The final result is then corrected for body surface
area [36, 99].
125
I Iothalamate
At the time of this publication, no standardized
guideline for the determination of GFR using 125I
iothalamate in children is available. Protocols vary
from single injection and subcutaneous injection to
constant infusion techniques, with or without the
addition of urinary clearance measurements [28].
Nonradioactive Iothalamate
Nonradioactive iothalamate can be measured using
high-performance liquid chromatography. A prim-
ing dose of iothalamate is given to achieve a
plasma serum level of 1 mg/dL. The prime is
followed by a constant infusion to maintain a con-
sistent plasma level. Due to differences in volumes
of distribution, children less than three years of age
are infused over 8 h, and children older than three
years are infused over 6 h. Blood samples are then
drawn at 120 min before, 60 min before, and at the
end of the infusion. If the final three samples varied
by less than 10 %, the patients have achieved
steady state, and the calculation can then be
performed using the following equation:
I1
Iocl ¼  infusate
S1
where
Iocl = iothalamate clearance in mL/min
I1 = concentration of the iothalamate infusion
in mg/mL
621
S1 = mean iothalamate concentration of the
three serum samples also in mg/mL
Infusate = the mL/min of the infusion
This result is then corrected for surface
area [38].
Iohexol
This nonionic low osmolar contrast agent is rou-
tinely used in high doses for intravenous-
contrasted radiological examinations. Low doses
of iohexol are used for the measurement of GFR.
Iohexol is excreted completely unmetabolized in
the urine; is not reabsorbed, metabolized, or
secreted by the kidney; and has a low
toxicity [19].
The HPLC method is used to assay the sera for
the iohexol concentration. Different methods have
been described with a four-sample method being
used in routine clinical practice with success. The
dose is given over 1 min. Blood samples are then
taken at approximately 5, 120, 180, and 240 min
after dosing with the times of blood samples accu-
rately recorded. The 5-min sample is taken to
check that the dose has been administered intra-
venously and not, inadvertently, subcutaneously.
The GFR is calculated using a single compart-
mental model based on the iohexol clearance
rate between 120 and 240 min. Results are then
corrected using the formula proposed by
Brøchner-Mortensen. The GFR is then corrected
for body surface area.
A two-sample method was also recently
described by Ng et al.; this method employs a
correction factor similar to the newer correction
factors by Fleming and Brøchner-Mortensen and
Jødal [39]. In terms of clinical application, the
two-sample method of iohexol with low toxicity
and only two blood samples required is gaining
popularity.
Proteinuria
Normal values for urinary total protein excretion
are <240 mg/m2/day in children <6 months of
age and <150 mg/m2/day in older children,
622
whereas excretion >3 g/1.73 m2/day is classified
as nephrotic range proteinuria in all age groups
[40, 41]. Approximately half of normal urine pro-
tein is secreted by the tubular epithelium in the
form of Tamm-Horsfall protein or uromodulin, a
glycoprotein. The balance is made up of albumin,
accounting for approximately 40 % of the total
and other low molecular weight plasma proteins,
such as beta-2-microglobulin. The normal glo-
merulus retards the passage of proteins into the
Bowman space by charge-repelling negatively
charged proteins such as albumin and by size
exclusion of larger proteins. Freely filtered low
molecular weight (LMW) proteins less than
25,000 Da are normally reabsorbed by the proxi-
mal tubule through endocytosis at the luminal
membrane. Excessive proteinuria can therefore
be classified as:
• Glomerular proteinuria – due to increased fil-
tration of protein across the glomerular mem-
brane. This is the most common cause of
proteinuria in children and is seen with glomer-
ular disease and other non-pathologic condi-
tions such as orthostatic proteinuria, fever, and
excessive exercise. Albumin is the major urine
protein present, but as disease severity pro-
gresses and glomerular selectivity decreases,
larger proteins such as immunoglobulins
appear in urine. Orthostatic proteinuria is
quite common during adolescence, affecting
2–5 % of the adolescent population [42].
• Tubular proteinuria – due to reduced
reabsorption of freely filtered low molecular
weight proteins such as alpha-1-
microglobulin, beta-2-microglobulin, and
retinol-binding protein. This form of protein-
uria is seen in a variety of tubulointerstitial
diseases and is often associated with other
defects of proximal tubular function such as
glycosuria, aminoaciduria, phosphaturia, and
proximal renal tubular acidosis, known as the
Fanconi syndrome.
• Overflow proteinuria – due to increased levels
of low molecular weight proteins in the plasma
that overwhelm tubular reabsorptive capacity.
This form of proteinuria is rarely seen in chil-
dren and usually associated with
G. Watt et al.
immunoglobulin light-chain production in the
plasma cell dyscrasias (multiple myeloma).
Measurement of Urinary Protein
Two screening tests are initially used to quantita-
tively assess urine protein excretion, namely, uri-
nary dipstick analysis and the urine total protein
assay using a colorimetric automated assay such
as sulfosalicylic acid. It is important to realize that
extremes of urine dilution and concentration can
significantly affect protein concentration in urine
and a dilute urine may underestimate the degree of
pathological proteinuria, whereas highly concen-
trated but normal urine may have a protein con-
centration of up to 1 g/L. For accurate assessment,
protein should be measured quantitatively and
expressed as a protein/creatinine ratio or based
on a 24-h urine collection and expressed as
mg/m2/day. Normal values as a function of age
are presented in Table 6.
Urinary dipstick estimates albumin concentra-
tion by a specific colorimetric reaction with
tetrabromophenol blue, yielding a green color.
For this reason, it is unsuitable as a screening
test for detecting tubular or overflow proteinuria.
False-positive results can occur with highly alka-
line urine or in urine contaminated with antiseptic
agents such as benzalkonium chloride and chlor-
hexidine and/or containing iodinated contrast
media [43]. Typical dipstick results are expressed
semiquantitatively as follows:
• Trace: – <0.3 g/L
• 1+:0.3 to <1 g/L
Table 6 Normal values of protein excretion as a function
of age
Age g/g creatinine g/mol creatinine
0–6 months 0.70 80
6–12 months 0.55 60
1–2 years 0.40 45
2–3 years 0.30 30
3–5 years 0.20 20
5–7 years 0.15 19
7–17 years 0.15 18
21 Laboratory Investigation of the Child with Suspected Renal Disease
• 2+:1 to 3 g/L
• 3+: 3 to <20 g/L
• 4+: >20 g/L
Routine laboratory colorimetric total protein
assays however detect all proteins present in
urine and can be used to assess all forms of
proteinuria.
Albumin is accurately quantified in urine using
albumin-specific immunoassays with high sensi-
tivities that enable the recognition of subtle
glomerular injury such as that associated with
early diabetic nephropathy and hypertensive
renal disease. Although variable and influenced
by age, albuminuria at a level of <30 mg/g
Cr (<3 mg/mmol) is considered normal, whereas
levels between 30 and 300 mg/g Cr (3–30
mg/mmol) are considered moderately increased
and levels >300 mg/g Cr (>30 mg/mmol) are
considered severely increased. Albuminuria can
be used in this way along with GFR to screen for
and classify renal disease severity, although
current data suggest that total protein should
preferentially used in children despite the fact
that acceptable guidelines and cutoffs have not
been formulated yet [44]. In addition to the
above, albumin is also measured quantitatively
together with a larger protein such as IgG as a
measure of glomerular selectivity in glomerular
disease. Whereas loss of anionic charge alone
results in increased albumin excretion (selective
proteinuria), as glomerular injury worsens,
Fig. 3 Urine protein
electrophoresis on agarose
gel: lanes 1 and 2 – tubular
proteinuria, showing Albumin
albumin band and alpha-2-
globulin doublet; lane 3 –
selective glomerular
proteinuria with albumin
and transferrin and some α-2 doublet
gamma globulins; lanes Transferrin
4 and 5 – moderate and
severe nonselective Immunoglobulins
glomerular proteinuria; lane
6 - normal serum sample
623
protein loss becomes nonselective. Highly
selective proteinuria will have an IgG/albumin
ratio of <0.1.
Tubular proteinuria can be differentiated from
glomerular proteinuria by the selective
immunonephelometric measurement of specific
low molecular weight proteins such as beta-2-
microglobulin, alpha-1-microglobulin, lysozyme,
and retinol-binding protein. In tubular proteinuria,
these levels are typically increased 10- to 100-fold
above normal. A relatively simple and
underutilized method to asses and differentiate
variable degrees of glomerular from tubular pro-
teinuria is by analysis of urinary protein by gel or
capillary electrophoresis, where the ratios of high
to low molecular weight plasma proteins present
in urine are visualized. This test is performed by
most laboratories, usually for the diagnosis and
monitoring of myeloma.
While normal urine contains very little protein,
a tubular proteinuria pattern consists primarily of
LMW proteins, the hallmark being an alpha-2-
globulin doublet consisting of alpha-2-
microglobulins, with some albumin. In more
severe tubular proteinuria, beta-2-microglobulin
is also present, just cathodal to the normal location
of transferrin. In contrast, urine with selective and
nonselective glomerular proteinuria demonstrates
larger amounts of albumin along with varying
degrees of higher molecular weight proteins,
resembling the protein pattern present in serum;
see Fig. 3 [98].
URINE AGAROSE
PROTEIN ELECTROPHORESIS +
1 2 3 4 5 6 -
624
Renal Biomarkers
Recent efforts have been made over the last few
years to identify novel urinary biomarkers to iden-
tify early acute kidney injury (AKI) allowing early
identification and management before AKI is
established. A need for precise assessment is
important as there is an independent association
between AKI and mortality rate of critically ill
patients of up to 60 %.
Until now, reliance has been placed on reduced
urine output as well as rising serum creatinine to
make the diagnosis of AKI [45]. This is a concern,
as serum creatinine changes can occur without
kidney damage and similarly, functional changes
only occur after significant damage has occurred.
An ideal biomarker should be accurate, reliable,
and easy to measure, which makes urine the ideal
fluid. Biomarkers for AKI that have been
suggested include: neutrophil gelatinase-
associated lipocalin (NGAL), interleukin 18, kid-
ney injury molecule-1, and liver fatty acid-
binding protein [46]. The most promising of
these markers is NGAL, a protein that mediates
innate immunity by inhibiting bacterial growth
through iron sequestration. It is expressed princi-
pally in neutrophils but also in renal tubular cells
and increases in urine and plasma within hours
after AKI. Due to its low molecular mass and
protease resistance, it is stable and easily mea-
sured in urine [47]. Plasma and urinary NGAL
elevations are able to detect AKI far sooner than
plasma creatinine, with higher sensitivity and
specificity and have also been shown to have
good prognostic value in predicting outcome in
many clinical scenarios associated with AKI. Fur-
ther clinical trials on outcomes and cost/benefits
comparing the use of NGAL and other novel bio-
markers to standard clinical practices are however
still required to demonstrate the clinical utility of
these biomarkers. Development of specific refer-
ence intervals, action cutoffs, and intervention
protocols are also required before NGAL can be
implemented into widespread clinical
practice [46].
Ultimately, there is a search for the renal equiv-
alent of troponin T or I that would allow early
recognition and therapeutic intervention in
G. Watt et al.
patients at risk of AKI. In all likelihood, a panel
of biomarkers will be developed and used in a
similar manner to a urine dipstick [48].
Patients with chronic kidney disease (CKD)
have traditional risk factors such as hypertension,
hypercholesterolemia, and obesity as well as
nontraditional risks such as inflammation, ane-
mia, and bone and mineral abnormalities. Fibro-
blast growth factor-23 (FGF-23) has recently
emerged as powerful predictor of adverse out-
comes in patients with CKD. Elevated FGF-23
exerts its negative impact through mechanisms
of action independent of role as regulator of phos-
phorus hemostasis [49]. These FGF-23 levels can
be measured in the laboratory setting.
Urinalysis
Likely the most frequently requested but least under-
stood laboratory test for examining renal function or
injury to the kidney or urinary tract, the urinalysis, is
easy to perform and is used as a screening test, a
diagnostic test, and as a follow-up examination to
test the efficacy of therapy. Its utility is at times
overestimated, and the risk of either false positives
or false negatives should be understood [50].
Typical urinalysis usually characterizes three
aspects of urine:
• Physical character: color, clarity, specific grav-
ity, and/or osmolality
• Chemical character: pH, glucose, protein,
blood, ketones, urobilinogen, conjugated bili-
rubin, leukocyte esterases, and nitrites
• Microscopic character: red cells (RBC), white
cells (WBC), tubular casts, organisms, and
crystals
Normal urine should be clear. Urine may be
turbid or cloudy due to the presence of crystals
(e.g., calcium phosphate in alkaline urine) or cel-
lular components – WBC, RBC, or both. Urine
may also have unusual color or odors; examples
include a grey or black discoloration in urine that
has been alkalinized or exposed to air in patients
with alkaptonuria, development of a wine red
21 Laboratory Investigation of the Child with Suspected Renal Disease
color in urine exposed to air and light in
patients with some porphyrias, a distinct pleasant
caramelized sugar odor with maple syrup urine
disease, and a noxious cheesy odor in patients
with glutaric aciduria type 2 or isovaleric
acidemia.
The most commonly used method for some
physical, chemical, and even cellular elements
present in urine is a dipstick test which employs
a colorimetric reaction to identify abnormalities.
These impregnated sticks should be used appro-
priately. Certain rules pertain:
• Urine tested should be around body or even
room temperature. Refrigeration will affect the
test results.
• The observations of change should be made
after 15 s and up to 1 min after dipping the
reagent stick in urine. Too rapid or too late, an
observation of color change can give a false
reading.
• Moisture, sunlight, cold, heat, or time (strips
have expiration dates) will result in false
readings.
• A number of substances present in urine may
yield false-positive or false-negative results on
the dipstick test; examples include:
– False-positive protein readings with high
levels of ascorbic acid, iodinated contrast
agents, some antiseptic agents, and highly
alkaline urine.
– False-negative glucose readings with high
levels of ascorbic acid.
– False-positive urobilinogen readings in
patients with acute porphyrias and elevated
urinary porphobilinogen.
– False-positive hemoglobin reading can be
obtained with myoglobinuria.
When leukocytes, especially neutrophils, are
present, leukocyte esterase may be positive.
Nitrite is found in urine as a result of metabolic
release by bacteria. Unfortunately, neither is sen-
sitive or specific enough to be able to definitively
diagnose urinary tract infections even when used
together [51]. Routine urinalysis as a screening
tool in the general population remains
controversial [52].
625
Hematuria may be due to renal trauma, glo-
merular injury, interstitial or tubular injury, stone
disease, cystic disease, malignancy, or trauma to
the urinary tract. Red cells can appear in urine
even though they derive from outside the urinary
tract. Two examples include menses or excoria-
tion of the epidermis at the urethral meatus. Nor-
mal urine has few, if any, intact red cells, generally
<2 red blood cell/high-powered field on micros-
copy on a urine specimen, which has been
centrifuged. This small number will not result in a
positive result on dipstick. At the other extreme,
urine will appear red with red cells, hemoglobin,
or myoglobin in urine. Because hematuria is a
nonspecific finding, the finding usually indicates
some injury but usually does not provide more
information. There are two exceptions. First, dys-
morphic red cells seen using phase contrast micros-
copy on microscopic examination of the urine may
help determine the site of injury along the urinary
tract. If 75 % or greater of red cells seen are dys-
morphic, the kidney, and most likely the glomeru-
lus, is the site of injury. If <25 % of RBC are
dysmorphic, the injury site is the urinary tract –
renal pelvis or below [53]. Second, if a dipstick is
positive for RBCs but no RBCs are noted on
microscopy, then hemoglobinuria or myoglobinuria
should be suspected, and direct measurement of
hemoglobin or myoglobin should be undertaken.
Tubular Function
The renal tubules are responsible for modification
of the glomerular ultrafiltrate by reabsorption and,
to a lesser extent, secretion of solutes and water in
order to maintain an optimal internal milieu, in the
process, producing a concentrated urine. When
assessing tubular function, therefore, the
reabsorptive and/or secretory capacity, as well as
tubular ability to concentrate urine, is assessed.
Commonly measured urinary solutes for the
assessment of reabsorptive (particularly proximal
tubular) function are sodium, phosphate, glucose,
bicarbonate, calcium, and amino acids. Further
testing involves assessing distal tubular capacity
for secreting potassium and hydrogen ions and for
water reabsorption or excretion [54, 55].
626
Fractional Excretion (FE)
Reabsorptive capacity can be assessed by calcu-
lating the FE of the solute. This refers to the
fraction of filtered solute that is eventually
excreted in urine, normalized to the relative excre-
tion of creatinine. The FE can be calculated as
follows, where U represents concentration in
urine, P concentration in plasma, and X the solute
of interest,  100 to obtain the result as a
percentage:
UX  PCreatinine
FEX ¼  100
PX  UCreatinine
This calculation is most accurate when performed
on a 24-h urine collection, with a blood sample
taken during this collection.
Sodium
The FE of sodium (FE-Na) is often used to differ-
entiate prerenal azotemia, due to decreased renal
perfusion, from intrinsic renal failure. As sodium is
almost completely reabsorbed in the functional but
underperfused kidney, an FE-Na <1 % implies
appropriate tubular reabsorption and therefore
prerenal disease. It is not valid in patients receiving
diuretics where it can be falsely elevated. It may
also be falsely depressed and therefore inaccurate
with certain intrinsic causes of kidney injury such
as acute glomerulonephritis, acute interstitial
nephritis, radiocontrast-induced nephropathy, and
rhabdomyolysis with myoglobinuria and in
patients with chronic low-flow states such as con-
gestive cardiac failure. The FE of urea may be a
better discriminator in these patients, with an
FE-urea <35 % supporting the diagnosis of
prerenal failure and >50 % suggestive of an intrin-
sic renal problem although there are differing opin-
ions on these cutoffs [55–57].
Chloride
Urinary chloride excretion is tied to urinary
sodium excretion. Urinary chloride depends on
G. Watt et al.
diet and extracellular volume status. With extra-
cellular volume contraction, enhanced sodium
reabsorption results in enhanced chloride
reabsorption. During metabolic alkalosis, the uri-
nary chloride concentration can be useful diag-
nostically. A urinary chloride concentration >10
mmol/L is consistent with diuretic-induced renal
loss, whereas urine chloride concentration under
10 mmol/L is consistent with gastric loss and
volume contraction.
Calcium
Urine calcium measurement is used in the inves-
tigation of hyper- and hypocalcemia,
normocalcemic hypercalciuria, parathyroid func-
tion, and renal calcium-sensing receptor function.
In children, hypercalciuria is defined as calciuria
>4 mg/kg/day (0.1mmol/kg/day) based on a 24-h
urine sample collection [58]. The urine calcium/
creatinine ratio is commonly used to screen for
hypercalciuria defined as >0.2 mg/mg (0.6 mmol/
mmol) [59]. A fasting random calcium/creatinine
ratio on the second morning voided urine sample
correlates best with a 24-h urine calcium measure-
ment [60]. In infants, values vary with age: 0–6
months <0.8 mg/mg (2.3 mmol/mmol), 6–12
months <0.6 mg/mg (1.7 mmol/mmol), and 1–2
years <0.5 mg/mg (1.4 mmol/mmol) [61]. In chil-
dren with decreased muscle mass, and therefore
decreased creatinine excretion, the calcium/osmo-
lality ratio is a comparable alternative [62]. The
FE of calcium, based on a 24-h urine collection
and measurement of calcium and creatinine con-
centrations in plasma and urine, is another alter-
native and is particularly useful in assessing
calcium-sensing receptor function or parathyroid
hormone function.
Magnesium
Magnesium in the ultrafiltrate is reabsorbed pri-
marily in the loop of Henle. The FE of magnesium
(FE-Mg) is typically performed to determine ade-
quacy of renal handling of magnesium. Normally,
21 Laboratory Investigation of the Child with Suspected Renal Disease
Fig. 4 Determining the
Tmp/GFR: a straight line
drawn through the plasma
phosphate concentration
and the TRP will pass
through the TmP/GFR [65]
approximately 5 % of filtered magnesium is
excreted. With hypomagnesemia, the FE-Mg can
fall to 1 %. In the presence of hypomagnesemia,
an FE-Mg 5 % suggests renal wasting of mag-
nesium [63, 64].
Phosphate
Renal phosphate handling may be abnormal even
in the presence of a normal serum phosphate level.
The tubular phosphate reabsorptive capacity is
therefore more effectively assessed by calculating
the tubular reabsorption of phosphate (TRP)
which can be considered the converse of the FE
and is calculated as follows:
TRP ¼ 1  FEphosphate
The best indicator of renal tubular handling of
phosphate, however, is the tubular maximum
627
reabsorption threshold of phosphate per glomeru-
lar filtration rate (TmP/GFR). The TmP/GFR rep-
resents the serum phosphate concentration at
which phosphate begins to appear in urine [65]
and can either be calculated or determined from a
nomogram (Fig. 4), using the TRP and the serum
phosphate concentration. This calculation
requires fasting urine and plasma phosphate and
creatinine measurements. Results should be com-
pared with age-appropriate reference intervals. A
typical reference interval in children aged 2–15
years is 3.6–7.6 mg/dL (1.15–2.44 mmol/L) [66].
Decreased TmP/GFR is associated with vitamin D
deficiency and hyperparathyroidism, while
increased TmP/GFR is seen with hypo- and
pseudohypoparathyroidism:
TmP=GFR ¼ TRPserumphosphate ðmg=dL or mmol=LÞ
It can be calculated directly using the following
equation:
628
TmP=GFR ðin mg=dL or mmol=LÞ
Pphosphate  Uphosphate  Pcreatinine
¼ transporter defect leading to increased excretion
Ucreatinine
Glucose
Glucose is reabsorbed by the proximal tubule. The
renal threshold for reabsorption is a plasma con-
centration of 9.9 (180 mg/dL) mmol/L, implying
that at normal plasma glucose concentrations,
glycosuria is absent. When the plasma glucose
concentration exceeds this threshold as in diabetes
mellitus, the proximal tubule’s capacity to reab-
sorb glucose is exceeded and glycosuria ensues.
Familial renal glycosuria is a rare benign condi-
tion due to a defective renal glucose transport and
is associated with isolated glycosuria and normal
plasma glucose concentration [67]. Here, the renal
threshold is lowered such that glycosuria occurs at
a normal plasma glucose concentration. Glycos-
uria may also occur with other tubular disorders
such as in the Fanconi syndrome, cystinosis, and
hereditary tyrosinemia. In these cases, it is usually
associated with other tubular losses of phosphate,
uric acid, amino acids, and bicarbonate [68, 69].
Urine Amino Acid Analysis
Amino acids are almost entirely reabsorbed in the
proximal tubule. As with glucose and phosphate,
there is a maximum capacity of reabsorption for
each amino acid. Any condition leading to
increased plasma concentrations, such that this
maximum capacity is exceeded, will lead to
increased excretion in the urine. Generalized ami-
noaciduria, in the presence of normal plasma con-
centrations, indicates proximal tubular damage or
dysfunction (e.g., the Fanconi syndrome,
cystinosis, and Lowe syndrome), while defects
of individual amino acid transporters are associ-
ated with excretion of specific amino acids or
groups of amino acids. These include cystinuria
(dibasic transporter defect with elevated cystine,
arginine, ornithine, and lysine excretion), dicar-
boxylic aciduria (dicarboxylic acid transporter
G. Watt et al.
defect, with increased glutamate and aspartate
excretion), Hartnup’s disease (neutral amino acid
of the neutral amino acids), lysinuric protein intol-
erance (cationic amino acid transporter leading to
increased excretion of lysine, ornithine, and argi-
nine), and iminoglycinuria (increased proline,
hydroxyproline, and glycine excretion) [68, 70].
Urine collection should preferably be accom-
panied by plasma analysis for comparison. Urine
and plasma samples must be frozen immediately
after collection to maintain analyte integrity.
Urine amino acid concentrations are reported as
a ratio to creatinine concentration to accommo-
date for varying degrees of dilution.
Potassium and Transtubular Gradient
of Potassium
In contrast to the previous analytes discussed,
urine potassium concentration is determined by
renal secretion in the distal tubule, which, in
turn, is associated with sodium uptake, via miner-
alocorticoid action. Urine potassium however is
not an accurate indication of adequate potassium
handling as it does not take into account the effect
of water movement across the distal tubule. Renal
handling of potassium is therefore assessed by
calculating the transtubular potassium gradient
(TTKG) which uses the serum/urine osmolality
ratio to adjust for the concentrating effects occur-
ring in the medullary collecting duct, thereby esti-
mating the potassium concentration at the point in
the collecting duct where tubular fluid is isotonic
with that of plasma [71, 72]:
UKþ  Posmolality
TTKG ¼
PKþ  U osmolality
A high TTKG is suggestive of renal potassium
wasting, which would be appropriate in the pres-
ence of hyperkalemia but inappropriate in hypo-
kalemia. Causes of a high TTKG in the setting of
hypokalemia include increased mineralocorticoid
activity due to hyperaldosteronism or pseudohy-
peraldosteronism (Cushing syndrome or Liddle
syndrome). A low TTKG suggests impaired distal
21 Laboratory Investigation of the Child with Suspected Renal Disease
secretion of potassium which may occur in min-
eralocorticoid deficiency such as type IV renal
tubular acidosis. In adults, a TTKG >5 means
aldosterone is active, while <3 suggests limited
or no mineralocorticoid activity. In children with
hyperkalemia, a TTKG <4.1 (or 4.9 in infants)
suggests decreased mineralocorticoid activity.
With hypokalemia, a TTKG >2 is indicative of
ongoing aldosterone secretion. It is important to
note that the TTKG can only be used in the
presence of sufficient distal delivery of sodium
(urine sodium >25 mmol/L) so that sodium
delivery is not rate limiting for potassium secre-
tion and a urine osmolality equal to or greater than
that of serum, implying adequate vasopressin
levels – required for optimal distal tubular secre-
tion of potassium [71].
Assessment of Renal Acid/Base
Homeostasis
The following tests are useful in ascertaining the
site of tubular dysfunction in the presence of a
suspected tubular cause for metabolic acidosis –
renal tubular acidosis (RTA). Proximal and distal
RTA differ in the mechanism by which the acido-
sis occurs. In the former, there is a lowered thresh-
old for the loss of bicarbonate, while in the latter,
the problem is an inability to excrete protons. The
following tests are useful to distinguish the types
of RTA.
Urine pH
In the normal kidney, the introduction of an acid
load leads to acidification of the urine, with
decreased excretion of bicarbonate and increased
buffering of acid in the form of ammonium chlo-
ride and sodium or potassium phosphates, yield-
ing a urine pH of <5.5. With both forms of RTA, a
paradoxically, relatively alkaline urine is often
found in the setting of a normal anion gap meta-
bolic acidosis. In proximal RTA, however, the
distal tubule is still able to excrete protons, and
therefore the urine pH can still achieve a pH of
<5.5 provided the plasma bicarbonate concentra-
tion is below the renal threshold [73]. In contrast,
with distal RTA, the urine pH is always >6.0 [61].
629
Urine pH should be measured immediately after
collection or kept refrigerated to prevent bacterial
action which may alter the pH (typically
increases, but may decrease in the presence of
glucose) [74].
Fractional Excretion of Bicarbonate
This test requires the patient’s serum bicarbonate
(HCO3) concentration to be restored to normal
levels via intravenous administration prior to mea-
suring HCO3 in urine and plasma along with
creatinine to calculate the FE-HCO3. An
FE-HCO3 >15 % confirms the diagnosis of
proximal RTA [73]. Again urine and plasma sam-
ples must be collected anaerobically and prefera-
bly measured without delay.
Estimating Urine Ammonium Production
The normal renal response to acidosis is to
increase the excretion of ammonium (NH4+) in
order to efficiently buffer excreted protons,
together with an equal increase in Cl ions to
maintain electroneutrality. With distal RTA, there
is decreased excretion of ammonium ions, and
therefore concomitantly, Cl excretion is not
appropriately increased. The urine ammonium
concentration is therefore an indicator of appro-
priate hydrogen excretion by the distal tubule. As
this analyte is not routinely measured, two surro-
gate methods for estimating ammonium excretion
can be used:
(a) Urine anion gap:
Urine anion gap ¼ ðNaþ þ Kþ Þ
 Cl ðin mmol=LÞ
Bicarbonate levels are usually insignificant in
acidic urine (pH < 6) and can therefore be
ignored in the calculation. The normal renal
response to acidosis results in a negative gap
due to the increase in Cl, while in distal RTA,
this gap remains positive, implying defective
ammonium production [61]. The urinary
anion gap has also been used in neonates,
but is more difficult to interpret especially in
the premature [75].
630
(b) Estimated ammonium concentration using
the urine osmolar gap
This method makes the same assumptions
as the urine anion gap that the gap is generally
caused by an increase in ammonium chloride.
It has been shown to correlate well with net
acid excretion and ammonium levels and is
the best indirect test for assessment of ammo-
nium excretion [76, 77]:
Estimated urine ammoniumðmmol=LÞ
¼ urine
 osmolar gap
2 
¼ U osmolality  U osmolarity
2
Urine osmolarity (mOsm/L) can be calcu-
lated using the following formula (all analytes
in mmol/L):
Urineosmolarity ¼ 2½Naþ þ Kþ  þ Urea þ glucose
To convert from urea nitrogen in mg/dL to
urea in mmol/L, divide by 2.8. For glucose,
convert from mg/dL to mmol/L by dividing
by 18.
In the event that distal tubular dysfunction is
still not demonstrable, other assessments of uri-
nary acidification may be required. Three methods
are employed to stimulate and therefore assess
distal hydrogen ion secretion:
(a) Acid loading by administration of ammonium
chloride (NH4Cl) and measuring the change
in urine pH: pH <5.5 is normal, whereas pH
remains above 6.5 in distal RTA.
(b) Increasing sodium delivery to the distal tubule
by administration of furosemide in combina-
tion with mineralocorticoid stimulation
(fludrocortisone) and measuring the urine pH
response.
(c) Increasing the delivery of HCO3 to the distal
tubule and measuring the increase in urine
PCO2.
While the urine acidification response to
NH4Cl is commonly described, this test is not
the preferred method in children, and the
G. Watt et al.
alternative methods are recommended. The urine
maximum PCO2 is measured after collection
under oil and in glass to prevent air contamina-
tion, following intravenous sodium bicarbonate
administration with a urine pH >7. This test can
be performed using either sodium bicarbonate or
acetazolamide or both [78–81]. Bicarbonate
administration results in a large delivery of
HCO 3 to the distal tubule, and HCO3 now

becomes almost the only acceptor of excreted
protons in the lumen, resulting in the production
of carbonic acid which, in turn, dissociates into
carbon dioxide and water. Therefore, with normal
distal hydrogen ion secretion, the urine PCO2 is
expected to rise. With defective distal hydrogen
ion secretion, the PCO2 will be similar to that in
blood. Acetazolamide, a carbonic anhydrase
inhibitor, enhances this effect. These tests need
not be performed if urine pH is <5.5 which
implies adequate distal acidification.
Urate, Oxalate, Citrate
Urate, derived from purine catabolism, is excreted
predominantly by the proximal tubules but also
filtered and reabsorbed. Abnormal serum urate
levels in children should always be investigated
as a number of rare disorders may otherwise be
missed; in addition, elevated levels carry the risk
of nephrolithiasis and AKI. Increased serum
levels are ascribed to either systemic
overproduction or undersecretion in the kidney.
Examples of overproduction in children include
the tumor lysis syndrome and rare metabolic dis-
orders such as Lesch-Nyhan syndrome and glyco-
gen storage disease type 1. Idiopathic gout is rare
in children. Undersecretion in the kidney may be
due to metabolic abnormalities or competition
with other chronically elevated anions such as
lactate, salicylate, and ketones. Decreased serum
urate levels may be associated with hepatocellular
disease, increased renal losses in the Fanconi syn-
drome, deficiency of xanthine oxidase activity
(molybdenum cofactor deficiency), and purine
nucleoside phosphorylase deficiency [82]. Frac-
tional excretion of uric acid (Fe-UA) is useful for
differentiating the causes of both hypouricemia
21 Laboratory Investigation of the Child with Suspected Renal Disease
and hyperuricemia in children; Fe-UA is very
high in young infants at approximately 28 %,
dropping rapidly in the first months of life to an
average of 8 % in childhood, with normal adult
values of approximately 5 % attained in adoles-
cence [83]. In children over 2 years of age, uric
acid excretion is approximately (520  145
mg/1.73 m2/24 h) 3,093  875 μmol/1.73
m2/24 h [84].
Oxalate is usually only measured in urine to
diagnose hyperoxaluria as it forms highly insolu-
ble crystals in urine and, like urate, may precipi-
tate nephrolithiasis and/or AKI. Normal oxalate
excretion is in the range of 20–50 mg/1.73 m2/
24 h (0.3–0.55 mmol/1.73 m2/24 h). In infants, the
value may be slightly higher [85]. Above this
range is suspicious for hyperoxaluria. The pri-
mary hyperoxalurias constitute a number of met-
abolic disorders of oxalate metabolism, whereas
secondary hyperoxaluria may be idiopathic or
associated with ethylene glycol poisoning and
fat malabsorption syndromes where oxalate is
preferentially absorbed from the gut.
Citrate excretion in urine is important in reduc-
ing the risk of stone formation especially calcium-
containing stones. Hypocitraturia contributes to
stone formation. Citrate excretion below
300 mg/g creatinine (0.176 mol/mol) in girls and
125 mg/g/ creatinine (0.074 mol/mol) in boys
suggests hypocitraturia [86].
Renal Tubular Concentrating Ability
The concentrating ability of the kidney relies on
the action of vasopressin on vasopressin receptors
in the renal collecting ducts, resulting in insertion
of aquaporin-2 channels into the apical membrane
facilitating the reabsorption of water. Defective
concentration of urine with associated polyuria
and polydipsia can therefore be ascribed to either
vasopressin deficiency (central diabetes
insipidus), loss of vasopressin action
(nephrogenic diabetes insipidus), or primary poly-
dipsia, defined as water intake of more than
2 L/m2/day [87].
When a diagnosis of diabetes insipidus is
suspected, polyuria must first be confirmed by a
631
24-h urine collection, and other causes excluded;
these include hyperglycemia, hypokalemia, and
hypercalcemia. Polyuria is defined as >4 mL/kg/
h in infants and children and >6 mL/kg/h in the
newborn [88]. An early morning urine osmolality
>800 mOsm/kg H2O implies normal concentrat-
ing ability with no further testing required.
Water Deprivation Test
A water deprivation test is performed to establish
renal ability to concentrate urine. The water dep-
rivation test should only be performed if the base-
line serum sodium and osmolality are within
reference intervals and urine osmolality is below
300 mosm/kg [88]. The test involves fluid restric-
tion for a number of hours until significant dehy-
dration has occurred, accompanied by hourly
measurements of osmolality and sodium in
serum and urine, as well as weight. The test is
stopped when either 2–5 % body weight loss has
been reached, when the upper limit of the refer-
ence interval has been exceeded for serum osmo-
lality or sodium, or when a normal urine response
to dehydration has occurred, typically urine osmo-
lality >800 mosm/kg. The test is usually limited
to 4 h in infants and 7 h in children. If the urine
osmolality exceeds >800 mosm/kg, both central
and nephrogenic diabetes can be excluded, and a
tentative diagnosis of primary polydipsia made
[88, 89]. In children where the urine osmolality
has not risen above 300 mosm/kg, vasopressin
(1-desamino-8-argininovasopressin) is adminis-
tered intranasally. Partial fluid restriction is
recommended by some (50–75 % of usual fluid
intake in infants), to avoid water overload. Intake
and output are strictly monitored. Urine is col-
lected after 4 h (2 h in infants) for osmolality
measurement. If urine osmolality then increases
by >50 %, a diagnosis of central diabetes
insipidus can be inferred, whereas an increase of
<50 % implies a nephrogenic cause for diabetes
insipidus.
In those cases where the urine osmolality fol-
lowing water deprivation falls between 300 and
800 mosm/kg, further investigation is required to
differentiate between primary polydipsia, partial
central, and partial nephrogenic diabetes
insipidus [89].
632
It is important to realize that current methods
for serum osmolality measurement may not
perform at the level of accuracy required for the
water deprivation test and therefore serum
sodium is a better measure of dehydration.
A basal sodium concentration above the reference
interval in the presence of a urine osmolality of
<300 mosm/kg makes the water deprivation
test unnecessary and potentially dangerous.
Vasopressin can be administered immediately.
The water deprivation test must not be performed
in patients with renal insufficiency, hypovolemia,
uncorrected hypothyroidism, and uncontrolled
diabetes insipidus. Dehydration may develop
rapidly; therefore, careful monitoring of
water balance must occur via hourly body
weight and serum sodium and osmolality
measurements.
Direct Vasopressin Measurement
Urine osmolality measurement following the
water deprivation test is an indirect measurement
of vasopressin activity. In locations where vaso-
pressin measurement is readily available, proto-
cols include direct vasopressin measurement as
part of the hourly biochemical measurements.
Vasopressin and urinary osmolality increase in
normal subjects and with primary polydipsia,
while neither increases with central diabetes
insipidus. In nephrogenic diabetes insipidus,
vasopressin levels increase as for normal subjects,
while urine osmolality does not increase
[90]. Vasopressin measurement is useful in differ-
entiating partial diabetes insipidus. Unfortunately,
vasopressin assays are not readily available, and
vasopressin is a highly unstable analyte with a
short half-life.
Co-peptin (C-Terminal ProAVP) Direct
Assay
Co-peptin, the C-terminal part of the vasopressin
precursor, is co-secreted with vasopressin from
the neurohypophysis. It is superior to vasopressin
in that is more stable ex vivo and is easier to
measure [91]. It has been shown to be a good
differentiator in partial diabetes insipidus when
G. Watt et al.
used in conjunction with post-osmotic stimulus
sodium concentration [92].
Parathyroid Hormone
Circulating parathyroid hormone (PTH) consists
of a range of immunoreactive fragments. Biolog-
ically active PTH 1–84 is the principle hormone
making up approximately 15–20 % of circulating
levels. It exerts its effects via the first 34 amino
acids on the amino terminal of the molecule,
interacting with the type 1 PTH receptor, and has
a very short half-life of approximately 3 min. Fur-
ther degradation in the circulation and in the para-
thyroid gland results in additional PTH fragments
containing carboxyl (C-terminal) or amino
(N-terminal) terminal portions of the peptide.
These fragments have much longer half-lives
and so make up the majority of PTH type peptides
in plasma. In addition, some of the N-terminally
truncated fragment PTH(7–84) interact with other
PTH receptors such as the C-PTH receptor and
may play important roles in the regulation of bone
resorption and serum calcium concentration [93].
Automated routine laboratory PTH immunoas-
says rely on antibodies directed at different epitopes
on the PTH molecule. The first widely used immu-
noassay called the intact PTH assay employs a cap-
ture antibody directed toward C-terminal epitopes
39–84 and a second detection antibody directed
toward N-terminal epitopes 13–34. This assay mea-
sures biologically active PTH 1–84 together with
large C-terminal fragments lacking portions of the
N-terminus and can therefore overestimate PTH
levels in uremic patients [94]. To address this issue,
a more specific bioactive PTH 1–84 assay was devel-
oped, utilizing a similar capture antibody to the intact
PTH assays but with a detection antibody
directed against the extreme N-terminal epitopes
1–4 [95]. Both assays are adequate for making a
diagnosis of primary hyperparathyroidism, but evi-
dence suggests that bioactive PTH 1–84 assays have
higher diagnostic accuracy in patients with renal
failure and primary hypoparathyroidism and in
patients with PTH-producing carcinoma [96, 100].
21 Laboratory Investigation of the Child with Suspected Renal Disease
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 Growth and Development of the Child
with Renal Disease 22
Bethany Foster

Contents Introduction
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 637
Normal growth and development is a central goal
Normal Growth and Development . . . . . . . . . . . . . . . . 637
Bone Age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 646
in the management of children with chronic kid-
ney disease (CKD). This chapter will review the
Growth and Development in Chronic Kidney
growth patterns observed in children with CKD as
Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 647
Protein Energy Wasting in CKD . . . . . . . . . . . . . . . . . . . . 649 well as the causes of growth impairment and the
signs and causes of protein energy wasting (PEW)
Evaluation of Growth and Development
in CKD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 652 in pediatric CKD. The methods for assessing
growth and nutritional status, along with the lim-
Optimizing Growth in Children with CKD . . . . . . 655
Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 656
itations of these measures in CKD, will also be
Fluid and Sodium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658 reviewed. Finally, management strategies to opti-
Acidosis Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658 mize growth among children with CKD will be
Control of Hyperparathyroidism and Metabolic outlined. However, before considering the growth
Bone Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658
Recombinant Human Growth Hormone
abnormalities associated with childhood CKD,
Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 659 normal growth and development and the tools
Enhanced Dialytic Clearance . . . . . . . . . . . . . . . . . . . . . . . . 660 available to assess the adequacy of the growth of
Minimized Exposure to Corticosteroids . . . . . . . . . . . . . 660 an individual child will be reviewed.
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660
Normal Growth and Development

Normal phases of growth: Normal growth can be

divided into three phases – infancy, childhood,
and puberty. Each phase is primarily influenced
by a different factor [1–3]. The infancy phase of
growth is essentially a continuation of fetal
growth and, like fetal growth, is driven primarily
by nutrition. Nutritional intake stimulates insulin
and insulin-like growth factors, the major regula-
B. Foster (*)
tors of growth in this phase. Growth is most rapid
The Research Institute of the McGill University Health
Centre, Montreal, QC, Canada in the first 2–3 months of life, with weight gains
e-mail: bethany.foster@mcgill.ca of ~30 g per day in the first 2 months and
# Springer-Verlag Berlin Heidelberg 2016 637
E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_20
638
20–25 g/day in the third month. Linear growth is
similarly brisk in the first 3 months of life, but
shows rapid deceleration thereafter [2]. At 6–12
months of age, the early, nutrition-dominated
infancy phase of growth is replaced by the child-
hood phase of growth. Growth hormone is the
major driver of growth in the childhood phase;
during this phase, growth velocity is greater than
it is at the end of the infancy phase and decelerates
slowly over time [3]. In the childhood phase,
growth rates of ~7–8 cm/year are typical at
3 years of age and of ~6–7 cm/year at 10 years
of age. Finally, the onset of puberty marks the
beginning of the pubertal phase of growth,
influenced mainly by sex hormones [1–4]. Puber-
tal growth patterns differ for girls and boys. Girls
experience a major increase in growth velocity
during the first 2 years of puberty, followed by
an additional 2–3 years of slower growth. In con-
trast, boys show a smaller increase in growth
velocity in early puberty. In fact some boys expe-
rience a decline in growth velocity for 12–18
months in early puberty before the growth spurt
in mid-puberty. Like in girls, the growth spurt
lasts about 2 years and is followed by 2–3 addi-
tional years of gradual growth.
Normal puberty: There is substantial varia-
tion in the age of onset of puberty in healthy
children. Puberty typically begins earlier in girls
than in boys, and sexual development is also
completed at a younger age in girls than boys.
Sexual development is assessed using Tanner
staging (Fig. 1) [5]. For girls, breast and pubic
hair development are staged separately. For boys,
genital (testicular volume and penile size) and
pubic hair development are staged separately.
Pubic hair and breast (girls)/genital (boys) staging
may be discordant. In boys, testicular volume
should be measured using a Prader orchiometer;
a testicular volume of 4 ml signals the onset of
puberty [6].
Sexual development is considered to be
delayed in a girl if there is no breast development
by 14 years, or if there is more than a 5-year
interval between breast development and menar-
che, or no menarche by 16 years. In boys, the
absence of testicular enlargement by 14 years or
an interval of more than 5 years between onset and
B. Foster
completion of genital development indicated
delayed sexual maturation [7].
Growth measurements: Linear growth in
children is assessed by measuring recumbent
length (in children 24 months of age or younger)
or standing height (children >24 months old) and
weight. These measures are then compared with
the distributions among healthy children of the
same age to determine the adequacy of growth.
Similarly, weight is compared with the distribu-
tion of weights among healthy children of the
same age. For children under 3 years of age,
head circumference is also assessed.
Body mass index (BMI): BMI is calculated by
dividing weight (kg) by height (m) squared and
provides a means of evaluating the appropriate-
ness of a child’s weight relative to height, at a
specific age. Pediatric BMI reference values
have been available for children over 2 years of
age since 2000 (US Centers for Disease Control
(CDC) [8]) and from birth since 2006 (World
Health Organization (WHO) [9]). These reference
curves are necessary because, unlike in adults,
raw BMI is not meaningful in children. Normal
BMI changes with age-related changes in body
proportions and body composition. Therefore,
BMI is expressed as a percentile or standard devi-
ation score relative to age in healthy children [10],
allowing meaningful and consistent interpretation
of BMI regardless of age. The BMI percentile is
used to determine whether a child’s weight-for-
height can be classified into a specific “at risk”
category, including underweight, overweight, or
obese. However, the cutoffs defining these cate-
gories vary somewhat depending on the source of
the reference data. A BMI-for-age less than the
fifth percentile represents underweight according
to the US CDC [11, 12]. The WHO defines under-
weight or “thinness” as a BMI-for-age less than
the third percentile (standard deviation score less
than 2.0). It is important to recognize that these
are both distributional cutoffs; no evidence-based
definitions exist linking underweight or thinness
to poor outcomes in the general population. The
CDC and the WHO agree on the definition of
overweight (BMI-for-age greater than the 85th
percentile or more than about 1 SD above the
average), but definitions of obesity differ [13].
22 Growth and Development of the Child with Renal Disease
Fig. 1 Stages of pubertal development for boys and girls
(a–c). Stages of genital development for boys (a). (1) Pre-
adolescent – testes, scrotum, and penis are of about the
same size and proportions as in early childhood.
(2) Enlargement of scrotum and of testes – the skin of the
scrotum reddens and changes in texture; little or no
enlargement of penis at this stage. (3) Enlargement of
penis, which occurs at first mainly in length – further
growth of testes and scrotum. Increased size of penis with
growth in breadth and development of glans, further
639
enlargement of testes and scrotum, increasing darkening
of scrotal skin. Stages of breast development for girls (b).
(1) Preadolescent – elevation of papilla only. (2) Breast bud
stage – elevation of papilla as a small mound, enlargement
of areolar diameter. (3) Further enlargement and elevation
of breast and areola, with no separation of their contours.
(4) Projection of areola and papilla to form a secondary
mound; the development of the areolar mound does not
occur in all girls. (5) Mature stage – projection of papilla
only, caused by recession of the areola to the general
640
The CDC defines obesity as a BMI-for-age greater
than the 95th percentile (>1.64 SD above the
average), whereas the WHO defines obesity as a
BMI-for-age >2 SD above the average (> ~ 97th
percentile).
Growth reference values: In 2006, the WHO
released growth reference standards indicating the
typical distribution of length/height, weight, and
BMI relative to age for children from birth to
5 years of age [9]; growth velocity standards are
also available for infants up to 24 months [14]. All
WHO growth charts can be found at http://www.
who.int/childgrowth/en/. These growth charts are
referred to as growth standards because the chil-
dren contributing measurements were specifically
selected to represent children growing under ideal
conditions: they had nonsmoking mothers, were
from areas of high socioeconomic status, and
received regular pediatric healthcare, including
immunizations. Longitudinal growth data, from
birth to 24 months, were collected in a subset of
882 infants, all of whom were breastfed for at least
4 months. These longitudinal data were used to
generate the growth velocity standards. The refer-
ence population used to generate the WHO
Growth Standards was of broad ethnic diversity,
including children from Brazil, Ghana, India,
Norway, Oman, and the USA. Although there
may be some theoretical advantages to country-
specific growth charts, it is notable that ethnicity
had very little impact on length in the WHO
population; only 3 % of the variability in length
within the population could be attributed to the
country of origin [9]. This suggests that the
growth standards reflect a reasonable expectation
for growth regardless of ethnicity.
The growth of children 5–19 years old is
represented in the WHO Growth Reference 2007
[15]. These curves, showing the typical
Fig. 1 (continued) contour of the breast. Stages of pubic
hair development for boys and girls (a–c). (1) Preadoles-
cent – the vellus over the pubes is not developed further
than over the abdominal wall. (2) Sparse growth of long,
slightly pigmented downy hair, or only slightly curled,
appearing chiefly at the penis or along the labia. (3) Con-
siderably darker, coarser, and more curled; the hair spreads
sparsely over the junction of the pubes. (4) Hair now
B. Foster
distributions of height, weight, and BMI relative
to age, were created by merging data from the
1977 American National Center for Health Statis-
tics (NCHS)/WHO growth reference (1–24 years)
with data from the WHO <5-year old growth
standards’ cross-sectional sample (18–71 months)
to smooth out the transition between the two
samples.
The American CDC published revised Ameri-
can growth reference charts for infants and chil-
dren from birth to 20 years of age in 2000 [8]. The
CDC reference curves and the WHO Growth
Standards differ very little after 2 years of age.
Because the WHO Growth Standards represent
ideal growth, the WHO Growth Standards should
be used as the reference for children from birth to
2 years. The recommendations as to which growth
reference should be used after 2 years of age differ
by country, with some countries recommending
country-specific reference curves and other coun-
tries recommending the WHO Growth Standards
to age 5 years and the WHO Growth Reference
2007 after 5 years.
Growth velocity: Growth velocity, measured
in cm/year, may be calculated to determine if the
growth rate is appropriate for age. The WHO
growth velocity standards represent the best avail-
able reference values for children younger than
2 years; reference values are available for growth
in 2-, 3-, 4-, and 6-month increments. Reference
values are also available for older children
[16, 17]. These were determined by taking serial
measurements of healthy, growing children to
determine growth rates. It is important to remem-
ber that the Tanner growth velocity reference
values (Fig. 2) are based on measurements taken
at 1-year intervals. The Baumgartner tables
(Table 1) provide growth data at 6-month intervals
to allow determination of growth velocity based
resembles adult in type, but the area covered is still con-
siderably smaller than in the adult; no spread to the medial
surface of the thighs. (5) Adult in quantity and type with
distribution of the horizontal (classically feminine) pattern;
spread to the medial surface of thighs but not up to linea
alba or elsewhere above the base of the inverse triangle
(From (39a) Tanner J. Growth at adolescence. Oxford:
Blackwell, 1962, with permission)
22 Growth and Development of the Child with Renal Disease 641

Fig. 2 (continued)
642 B. Foster

Fig. 2 (a) Growth velocity for age in boys 2–19 years (From [17]). (b) Growth velocity for age in girls 2–19 years (From
Ref. [17])
Table 1 Growth velocity tables in 6-month increments (Reproduced from Baumgartner et al. 1986 [16])
22

Percentiles of 6-mo increments

Age at end of interval 3 (2 SD) 5 10 (1 SD) 25 50 (mean) 75 90 (+ 1SD) 95 97 (+ 2SD) n
mo cm/6 mo
Recumbent length of boys: birth to 36 mo of age in 6-mo increment (Fels Longitudinal Study)
6 12.22 (12.00) 12.96 13.71 (14.49) 15.33 17.09 (16.98) 18.57 19.91 (19.47) 20.79 21.70 (21.96) 271
9 8.39 (819) 8.90 9.39 (9.72) 10.22 11.08 (11.25) 12.14 13.26 (12.77) 13.93 14.51 (14.30) 265
12 5.89 (5.64) 6.20 6.69 (7.06) 7.55 8.51 (8.48) 9.29 10.14 (9.90) 10.71 10.91 (11.32) 286
18 4.24 (4.03) 4.57 4.99 (5.29) 5.82 6.51 (6.56) 7.31 8.05 (7.82) 8.67 9.02 (9.08) 286
24 3.26 (2.94) 3.58 3.89 (4.17) 4.57 5.40 (5.41) 6.13 6.85 (6.64) 7.39 7.57 (7.88) 280
30 2.84 (2.73) 3.00 3.49 (3.70) 4.00 4.63 (4.67) 5.29 5.77 (5.63) 6.10 6.63 (6.60) 270
36 2.33 (2.23) 2.47 3.02 (3.18) 3.58 4.05 (4.12) 4.66 5.38 (5.07) 5.75 6.01 (6.01) 266
Recumbent length of girls: birth to 36 mo of age in 6-mo increment (Fels Longitudinal Study)
6 12.00 (11.55) 12.36 13.26 (13.84) 14.51 16.06 (16.13) 17.66 18.85 (18.42) 19.76 20.34 (20.71) 254
9 8.18 (7.92) 8.40 9.10 (9.38) 9.78 10.82 (10.83) 11.77 12.58 (12.29) 13.22 13.53 (13.74) 248
12 5.94 (5.43) 6.20 6.76 (6.99) 7.41 8.49 (8.56) 9.43 10.34 (10.12) 10.87 11.32 (11.69) 263
18 3.94 (4.17) 4.61 5.19 (5.46) 6.00 6.79 (6.76) 7.50 8.15 (8.05) 8.81 9.17 (9.35) 262
24 3.72 (3.41) 4.00 4.40 (4.57) 4.95 5.59 (5.73) 6.45 7.09 (6.89) 7.60 7.87 (8.04) 259
Growth and Development of the Child with Renal Disease

30 2.90 (2.70) 3.19 3.54 (3.71) 4.02 4.66 (4.72) 5.23 6.00 (5.74) 6.39 6.77 (6.75) 245
36 2.54 (2.29) 2.72 2.99 (3.22) 3.62 4.13 (4.16) 4.67 5.25 (5.10) 5.78 6.16 (6.04) 246
Stature of boys: 3 to 18 yr of age in 6-mo increment (Fels Longitudinal Study)
3.5 2.54 (2.22) 2.64 2.84 (3.03) 3.36 3.81 (3.84) 4.33 4.72 (4.65) 5.00 5.32 (3.46) 208
4.0 2.14 (2.09) 2.49 2.69 (2.84) 3.11 3.59 (3.60) 4.02 4.41 (4.35) 4.77 4.86 (5.11) 233
4.5 2.27 (2.21) 2.52 2.73 (2.88) 3.05 3.53 (3.55) 4.03 4.40 (4.23) 4.59 4.77 (4.89) 244
5.0 2.08 (2.03) 2.34 2.67 (2.77) 3.05 3.53 (3.50) 3.92 4.41 (4.24) 4.66 4.93 (4.97) 262
5.5 2.25 (2.15) 2.33 2.67 (2.79) 3.02 3.45 (3.43) 3.79 4.20 (4.07) 4.51 4.62 (4.71) 241
6.0 1.99 (2.00) 2.07 2.43 (2.63) 2.93 3.35 (3.30) 3.67 4.10 (3.90) 4.23 4.48 (4.53) 240
6.5 2.00 (1.92) 2.07 2.31 (2.55) 2.76 3.21 (3.19) 3.60 3.96 (3.82) 4.22 4.39 (4.45) 233
7.0 1.99 (1.99) 2.24 2.45 (2.58) 2.70 3.19 (3.17) 3.53 3.93 (3.75) 4.11 4.26 (4.34) 235
7.5 1.83 (1.72) 2.01 2.20 (2.38) 2.62 2.99 (3.03) 3.45 3.88 (3.69) 4.11 4.29 (4.34) 229
8.0 1.69 (1.69) 1.86 2.18 (2.34) 2.51 3.02 (2.98) 3.45 3.75 (3.62) 3.90 4.18 (4.27) 226
8.5 1.80 (1.82) 2.01 2.23 (2.36) 2.59 2.96 (2.90) 3.21 3.56 (3.44) 3.79 3.86 (3.98) 214
9.0 1.77 (1.73) 1.89 2.11 (2.28) 2.45 2.84 (2.83) 3.21 3.52 (3.37) 3.67 3.70 (3.92) 212
9.5 1.46 (1.43) 1.61 1.83 (2.02) 2.28 2.67 (2.62) 2.96 3.36 (3.22) 3.64 3.75 (3.82) 204
643

(continued)
 644

Table 1 (continued)
Percentiles of 6-mo increments
Age at end of interval 3 (2 SD) 5 10 (1 SD) 25 50 (mean) 75 90 (+ 1SD) 95 97 (+ 2SD) n
mo cm/6 mo
10.0 1.80 (1.56) 1.89 2.06 (2.15) 2.35 2.70 (2.73) 3.06 3.44 (3.31) 3.69 3.91 (3.89) 202
10.5 1.44 (1.30) 1.56 1.83 (1.94) 2.14 2.56 (2.57) 2.93 3.38 (3.20) 3.67 3.94 (3.84) 208
11.0 1.49 (1.31) 1.68 1.87 (1.96) 2.22 2.52 (2.61) 2.96 3.43 (3.26) 3.74 4.11 (3.91) 204
11.5 1.58 (1.22) 1.69 1.86 (1.96) 2.20 2.57 (2.70) 3.06 3.70 (3.44) 4.00 4.18 (4.18) 198
12.0 1.49 (0.99) 1.60 1.86 (1.93) 2.24 2.73 (2.86) 3.27 4.07 (3.79) 4.68 5.01 (4.72) 196
12.5 1.31 (0.67) 1.49 1.83 (1.85) 2.20 2.77 (3.03) 3.53 4.93 (4.20) 5.44 5.83 (5.38) 195
13.0 1.61 (1.05) 1.87 2.10 (2.28) 2.55 3.29 (3.52) 4.45 5.22 (4.75) 5.75 6.06 (5.98) 191
13.5 1.27 (1.26) 1.55 2.19 (2.42) 2.75 3.49 (3.58) 4.41 5.03 (4.74) 5.31 5.55 (5.89) 188
14.0 1.53 (1.49) 1.84 2.15 (2.65) 2.93 4.01 (3.81) 4.64 5.21 (4.97) 5.49 5.71 (6.13) 190
14.5 1.40 (1.00) 1.51 1.75 (2.28) 2.60 3.59 (3.57) 4.54 5.26 (4.85) 5.48 5.63 (6.13) 186
15.0 0.65 (0.67) 0.90 1.42 (1.90) 2.26 3.19 (3.13) 4.01 4.66 (4.36) 5.11 5.22 (5.59) 179
15.5 0.04 (0.54) 0.32 0.66 (0.87) 1.24 2.05 (2.29) 3.26 4.28 (3.70) 4.66 4.95 (5.12) 178
16.0 0.28 (0.62) 0.12 0.60 (0.58) 0.94 1.56 (1.78) 2.48 3.57 (2.98) 3.91 4.21 (4.18) 176
16 5 0.67 (1.07) 0.50 0.03 (0.01) 0.44 0.94 (1.08) 1.50 2.44 (2.16) 2.98 3.81 (3.23) 155
17.0 0.78 (0.98) 0.55 0.16 (0.04) 0.25 0.83 (0.89) 1.33 1.94 (1.83) 2.85 3.24 (2.76) 153
17.5 0.84 (1.03) 0.66 0.37 (0.23) 0.12 0.41 (0.57) 1.00 1.43 (1.37) 1.73 2.26 (2.17) 137
18.0 0.87 (0.83) 0.55 0.33 (0.24) 0.03 0.32 (0.36) 0.72 1.01 (0.96) 1.45 1.61 (1.56) 137
Stature of girls: 3 to 18 yr of age in 6-mo increment (Fels Longitudinal Study)
3.5 2.55 (2.31) 2.84 2.98 (3.09) 3.34 3.79 (3.87) 4.33 4.84 (4.65) 5.24 5.52 (5.43) 195
4.0 2.50 (2.37) 2.72 2.91 (3.04) 3.25 3.65 (3.71) 4.09 4.49 (4.38) 4.97 5.21 (5.05) 211
4.5 2.09 (2.07) 2.23 2.71 (2.82) 3.06 3.54 (3.57) 4.05 4.46 (4.31) 4.81 4.99 (5.06) 230
5.0 2.20 (2.06) 2.33 2.52 (2.80) 3.02 3.52 (3.54) 4.03 4.36 (4.27) 4.84 4.89 (5.01) 240
5.5 2.24 (2.08) 2.42 2.57 (2.74) 2.98 3.39 (3.39) 3.79 4.16 (4.05) 4.48 4.55 (4.71) 226
6.0 2.07 (1.99) 2.23 2.46 (2.64) 2.84 3.30 (3.29) 3.75 4.10 (3.95) 4.49 4.62 (4.60) 227
B. Foster
 22

6.5 1.85 (1.76) 1.93 2.20 (2.42) 2.60 3.11 (3.08) 3.49 3.94 (3.74) 4.29 4.35 (4.41) 223
7.0 1.96 (1.86) 2.11 2.36 (2.48) 2.66 3.08 (3.09) 3.50 3.81 (3.71) 3.98 4.17 (4.33) 223
7.5 1.86 (1.81) 1.98 2.23 (2.42) 2.64 3.07 (3.02) 3.39 3.67 (3.63) 4.03 4.14 (4.23) 221
8.0 1.62 (1.64) 1.82 2.17 (2.31) 2.56 3.01 (2.98) 3.35 3.85 (3.65) 4.07 4.20 (4.32) 222
8.5 1.77 (1.58) 1.86 1.99 (2.22) 2.41 2.88 (2.86) 3.27 3.68 (3.50) 3.90 4.09 (4.14) 222
9.0 1.81 (1.70) 1.93 2.13 (2.28) 2.47 2.84 (2.85) 3.22 3.53 (3.43) 3.77 4.08 (4.01) 216
9.5 1.64 (1.50) 1.78 1.95 (2.16) 2.36 2.80 (2.83) 3.29 3.58 (3.49) 3.92 4.09 (4.16) 219
10.0 1.63 (1.31) 1.70 1.99 (2.11) 2.38 2.91 (2.90) 3.28 3.83 (3.70) 4.38 4.61 (4.49) 220
10.5 1.57 (1.13) 1.64 1.99 (2.07) 2.37 2.82 (3.01) 3.56 4.21 (3.95) 4.70 5.27 (4.90) 212
11.0 1.67 (1.38) 1.87 2.08 (2.27) 2.53 3.06 (3.16) 3.74 4.33 (4.05) 4.76 4.92 (4.94) 203
11.5 1.61 (1.41) 1.84 2.09 (2.38) 2.54 3.34 (3.35) 4.02 4.63 (4.32) 4.84 5.17 (5.29) 202
12.0 1.67 (1.22) 1.60 1.98 (2.25) 2.65 3.32 (3.28) 3.98 4.52 (4.31) 4.86 4.98 (5.34) 196
12.5 0.79 (1.04) 1.11 1.61 (2.07) 2.41 3.19 (3.10) 3.87 4.38 (4.13) 4.58 4.72 (5.16) 186
13.0 0.36 (0.37) 0.52 1.05 (1.52) 1.99 2.76 (2.67) 3.46 4.09 (3.82) 4.21 4.43 (4.97) 185
13.5 0.25 (0.21) 0.35 0.57 (0.92) 1.20 1.98 (2.05) 2.90 3.56 (3.19) 3.92 4.24 (4.32) 184
14.0 0.28 (0.71) 0.09 0.28 (0.43) 0.78 1.33 (1.58) 2.35 3.11 (2.72) 3.66 4.09 (3.86) 179
14.5 0.32 (0.79) 0.21 0.04 (0.16) 0.47 0.88 (1.11) 1.65 2.77 (2.06) 2.97 3.32 (3.01) 154
15.0 0.62 (0.87) 0.47 0.16 (0.04) 0.24 0.68 (0.78) 1.24 1.75 (1.60) 2.33 2.51 (2.43) 150
Growth and Development of the Child with Renal Disease

15.5 0.88 (0.88) 0.67 0.43 (0.20) 0.07 0.49 (0.49) 0.83 1.27 (1.17) 1.62 1.91 (1.85) 143
16.0 0.53 (0.68) 0.48 0.33 (0.13) 0.04 0.46 (0.42) 0.75 1.08 (0.97) 1.42 1.54 (1.52) 139
16.5 0.66 (0.70) 0.61 0.37 (0.21) 0.05 0.23 (0.28) 0.63 0.91 (0.77) 1.02 1.19 (1.25) 132
I7.0 1.14 (0.94) 0.84 0.65 (0.38) 0.13 0.19 (0.17) 0.56 0.81 (0.73) 0.94 1.08 (1.29) 133
17.5 0.81 (0.97) 0.74 0.51 (0.41) 0.21 0.04 (0.14) 0.38 0.97 (0.70) 1.25 1.42 (1.26) 126
18.0 0.80 (0.91) 0.78 0.64 (0.39) 0.15 0.09 (0.13) 0.46 0.83 (0.65) 1.05 1.08 (1.17) 123
645
646
on shorter intervals. Although it is common prac-
tice to measure height at even shorter intervals and
extrapolate, this approach should be used cau-
tiously in children over 2 years old. Growth rates
are not constant even over a 6-month interval;
briefer intervals may capture periods of slower
or more rapid growth than may be observed over
a longer period. Nevertheless, height may be mea-
sured more frequently and plotted on the growth
curves to give a general impression of growth
adequacy.
Special populations: Special growth curves
are available for a variety of special populations,
including Trisomy 21, Turner syndrome, and pre-
mature infants [18, 19]. Diagnosis-specific curves
were constructed from fairly small reference
populations, but provide useful guidance on
expected growth in these unique groups. Prema-
ture infants should have growth assessed relative
to their corrected age rather than chronological
age. Corrected age is determined by subtracting
the number of weeks early that the child was born
from the chronological age. Separate curves are
available for low birth weight (1,500–2,500 g)
and very low birth weight (<1,500 g) infants
[20, 21].
Frequency of growth assessment: Among
healthy children, growth should be assessed at
each well-child health visit. In the first year of
life, these are suggested to occur at birth, 1–2
weeks of age, and at 1, 2, 4, 6, 9, and 12 months.
Between 1 and 3 years old, assessments are
suggested every 6 months, at 18, 24, 30, and
36 months. After 3 years, annual assessments are
sufficient.
Estimating height potential: When assessing
the adequacy of growth, it is important to consider
the child’s height not only relative to the appro-
priate reference group but also relative to his or
her genetic potential. Although many factors
influence growth in addition to genetic factors, a
child’s adult height potential may be estimated
from the sex-adjusted midparental height.
Midparental height is calculated from the heights
of the biological parents. For girls, 13 cm is
subtracted from the father’s height and averaged
with the mother’s height. For boys, 13 cm is added
B. Foster
to the mother’s height and averaged with the
father’s height. This average of parental heights
is then plotted at age 20 years on the growth chart
relevant for the sex of the child; the heights 8.5 cm
above and below the calculated midparental
height represent two standard deviations around
this estimate [22].
Bone Age
An assessment of skeletal development will give
an idea as to the remaining growth potential of the
long bones. By convention, bone age is evaluated
by obtaining a left hand and wrist radiograph. This
is compared with the images of “standard” hand/
wrist radiographs from children of different ages
in the Greulich and Pyle skeletal age atlas
[23]. The age of the child in the atlas’ standard
image that is closest in appearance to the child’s
X-ray represents the bone age. This provides a
fairly rough estimate of bone age, since bone age
images are available at 1-year intervals. In addi-
tion, discrepancies between the maturity of bones
in the hand and the wrist may be difficult to
reconcile, and interobserver variability is com-
mon. Furthermore, the Greulich and Pyle atlas
was generated with images from white children
of high socioeconomic status living in the USA in
the 1930s; the applicability of these standards to
an ethnically diverse, contemporary population is
debatable [24–26].
Tanner and Whitehouse developed another
method of estimating skeletal maturation, in
which 20 bones of the hand are scored into
one of eight categories; the scores are translated
into a bone age [27]. This method has superior
reproducibility to the Greulich and Pyle method
and allows assessment of bone age at 0.1-year
increments. However, it is significantly more
time consuming and does not show major
advantages over the Greulich and Pyle atlas in
most populations. Bone age should be recognized
as an estimate of skeletal maturity, rather than
a precise measure, and should be interpreted in
the context of the sexual maturity and overall
health.
22 Growth and Development of the Child with Renal Disease 647

Growth and Development in Chronic

Kidney Disease

Growth disturbance is one of the hallmarks of

CKD in childhood [28–32]. Short stature is com-
mon among children with CKD, with younger age
of onset, and more advanced CKD associated
with larger height deficits. Short stature has long
been recognized as a complication of CKD, with
30–50 % of individuals with childhood onset
CKD being reported as having height deficits as
adults [33, 34]. CKD affects each of the phases of
growth in a slightly different way.
Disruption of normal phases of growth in
CKD: During the infancy phase of growth, poor
nutritional intake may result in growth restriction.
In addition, the childhood phase of growth may be
delayed in onset until 2–3 years of age in about
40 % of boys and 27 % of girls with CKD; 60 % of
both boys and girls may show a transient resump-
tion of the infancy pattern of growth after starting
the childhood phase [4]. Because linear growth is
quite slow at the end of the infancy phase and Fig. 3 Typical growth pattern in patients with congenital
chronic kidney failure is shown in red. Loss of statural
faster at the onset of the childhood phase, delayed height predominates in infancy and adolescence, with
onset of the childhood phase or return to the growth parallel to the normal curves in mid-childhood.
infancy phase will result in overall slower growth The shaded area represents the normal range (3–97 %)
than normal. (Reproduced with permission from Mehls and Schaefer
[150])
Children with CKD growing in the childhood
phase may have normal growth velocity. How-
ever, children with lower glomerular filtration pituitary gland. In CKD, GnRH secretion may be
rate (GFR) have been shown to have significantly blunted, resulting in a decreased frequency of LH
lower growth velocity than children with higher pulses [37]. Although LH levels may be increased
GFR [29, 35]. In addition, the onset of pubertal in CKD due to decreased renal clearance, pubertal
growth is delayed among children with CKD, and patients receiving maintenance dialysis produce
the pubertal growth spurt is shorter [36]. The typ- substantially less LH overnight than healthy peers
ical growth pattern for a child with CKD is illus- [38, 39]. As a result of these abnormalities, the
trated in Fig. 3. onset of puberty may be delayed in children with
Together, the disturbances in the normal pat- CKD. However, both boys and girls with CKD do
tern of growth may lead to severe short stature. eventually progress through the normal stages of
Children with CKD from a very young age are puberty and attain full sexual maturation. Because
particularly severely affected; failure to grow at a growth and pubertal development are so tightly
normal rate during critical periods when growth is linked, it is important that pubertal development
normally rapid is very difficult to catch up later. be carefully monitored in conjunction with
Pubertal development in CKD: In healthy growth assessment.
children, gonadotropin-releasing hormone Etiology of growth impairment in CKD:
(GnRH) stimulates the release of luteinizing hor- There are a variety of reasons that children with
mone (LH) in pulsatile nocturnal bursts from the CKD experience impaired growth. As noted
648
above, poor caloric intake – common among
infants with CKD – has the most important effect
on growth in the first 1–3 years of life. Anorexia
appears to be the primary reason for growth failure
in infants and very young children with CKD
[40–42]. Anorexia has been observed in infants
with glomerular filtration rates as high as
70 ml/min/1.73 m2 [43]. A variety of factors may
cause anorexia in CKD, including disturbances in
the appetite-regulating hormones leptin and
ghrelin [44, 45], altered taste [46], gastroesopha-
geal reflux, delayed gastric emptying, and ele-
vated levels of several cytokines such as IL-1,
IL-6, and tumor necrosis factor (TNF)-α
[47]. Although some older children may have
poor energy intake, this is not common; most
older children have normal energy intake relative
to body size [31]. Developmental abnormalities
and/or psychological factors may also contribute
to poor intake. Children with a history of anorexia
may also have poor oral-motor skills, resulting in
very slow eating and low intake. The struggles
around eating between parents and anorectic chil-
dren, and the use of nasogastric tubes, may also
result in negative psychological reactions to food
and further food refusal [48, 49]. Caloric intake
may also be limited by frequent vomiting in CKD.
A large proportion of children with CKD have
poor urinary concentrating ability and therefore
high urine volumes. Such children often feel
thirsty and prefer to drink water over milk or
other fluids. The volume of water taken may be
large enough to diminish their appetite for milk or
food. Children with CKD may also experience
salt wasting, resulting in chronic volume deple-
tion; this will also limit growth. Chronic meta-
bolic acidosis may also contribute to poor
growth [50]. The mechanisms by which acidosis
impairs growth include decreased thyroid hor-
mone levels, disturbances in the growth hormone
IGF axis and response to IGF-1, and promoting
catabolism [22, 51, 52].
The role of CKD-related anemia in growth
impairment is not clear. Although some evidence
suggests an association between hemoglobin level
and growth [53], this relation may be confounded by
other factors. No studies have demonstrated a clear
growth benefit with erythropoietin therapy [54].
B. Foster
The interplay between CKD-related metabolic
bone disease and growth is complex; the effects of
calcium and phosphate metabolism and parathy-
roid hormone (PTH) levels on growth have not yet
been elucidated. Whereas some small studies
showed a positive association between PTH levels
and growth, others found that high PTH levels
were associated with poor growth [55]. Normal
growth has been demonstrated in children with
CKD and PTH at ~1.5 times the upper limit of
normal [56], children with PTH <2 times the
upper limit of normal [57], and even among chil-
dren with adynamic bone disease [58]. Contro-
versy remains regarding the optimal target PTH
level to best support growth and healthy bones.
The very therapies given to control PTH levels –
cholecalciferol and active vitamin D – have direct
inhibitory effects on the chondrocytes of the
growth plate; both dose and dosing schedule of
these medications may influence growth [55].
Disturbances in the growth hormone (GH) axis
also play an important role in the growth retarda-
tion associated with CKD [59]. The nature of the
disturbance depends somewhat on age. In puber-
tal children GH release is reduced, possibly due to
diminished stimulation by sex hormones, whereas
in prepubertal children GH release is normal or
even increased [38, 60, 61]. Poor growth in the
face of normal GH levels suggests a resistance to
the actions of GH. GH resistance may be due to
either reduced GH receptor density or a defect in
signal transduction following GH binding to the
receptor; either of these would result in decreased
insulin-like growth factor-1 (IGF-1) production –
which is normally stimulated by GH. Signal trans-
duction following GH binding to its receptor
involves the activation of Janus kinase 2 (JAK2),
which then activates signal transducer and activa-
tor of transcription (STAT) proteins [62]. Acti-
vated STAT proteins upregulate transcription of
GH-regulated genes. The activation of JAK2 and
STAT proteins appears to be impaired in uremia
[62, 63]. Another family of proteins known as
SOCS (suppressor of cytokine signaling) also
plays a role in control of GH signaling. SOCS
are induced by GH and provide negative feedback
by inhibiting activation of JAK2 and STAT pro-
teins [62, 64]. SOCS are also upregulated in
22 Growth and Development of the Child with Renal Disease 649

response to inflammation and may in this way
play a role in PEW [65].
Individuals with CKD also show low levels of
circulating growth hormone-binding protein
(GHBP), a product of proteolytic cleavage of the
extracellular domain of the GH receptor.
Although GHBP levels are associated with
growth rate and with response to recombinant
human GH therapy in children with CKD
[66, 67], it is not clear whether GHBP levels
are a reliable marker of GH receptor
expression [68].
As noted above, one of the major effects of GH
is to stimulate IGF-1 production. However,
despite GH resistance, IGF-1 levels are normal
in children with CKD and only slightly decreased
in ESRD [67]. There is evidence of IGF-1 resis-
tance in CKD. This appears to be mediated in two
separate ways. First, increased levels of
IGF-binding proteins in CKD will lead to a reduc-
tion in free (active) IGF-1. Second, signal trans-
duction following binding of IGF-1 to its receptor
may be impaired [69, 70].
Protein Energy Wasting in CKD
The term “protein energy wasting” (PEW) is used
to describe the wasting syndrome seen in some
patients with CKD [71]. The term “cachexia” is
also used to describe this syndrome; however, in
the setting of CKD, PEW is the more widely used
term. PEW is distinct from simple malnutrition.
Malnutrition is caused by inadequate intake and
accompanied by adaptive responses, including
hunger, a protective decrease in energy expendi-
ture, and preferential use of fat stores for energy
with preservation of lean body mass. In contrast,
maladaptive responses prevail in PEW. PEW is
characterized by anorexia and increased meta-
bolic rate; fat mass is preserved, or even increased,
while muscle mass is reduced [72]. Although poor
nutritional intake may contribute, other factors
may be more important in the pathogenesis of
PEW in CKD; systemic inflammation and meta-
bolic acidosis have been shown to play a role
[73–78]. Figure 4 summarizes the purported
causes and consequences of PEW in CKD. Ure-
mic toxins are suspected as the root cause of
endocrine disturbances such as growth hormone
resistance and abnormal neuropeptide signaling –
both of which are also believed to contribute to
PEW [72, 78]. Interestingly, current growth hor-
mone use was found to be protective against skel-
etal muscle wasting in a study of children with
CKD [79]. Importantly, unlike malnutrition, in
which increased food intake or altered dietary
composition usually leads to rapid weight gain
and restoration of normal body composition, cal-
orie supplements or other dietary manipulations
are less successful in treating patients with PEW
[75, 76, 80–82].

Fig. 4 Schematic Causes of PEW Consequences of PEW

illustrating the causes and
consequences of PEW in ↓ Dietary intake
pediatric CKD. Double- Loss of kidney function
headed arrows indicate that Uremic toxins
the factor may be both a
cause and a consequence of Infection
PEW (Adapted, with
permission, from Ikizler Comorbid conditions
et al. KI 2013 [78]) Protein
Cardiovascular disease
energy
wasting
Dialysis-associated
catabolism

Metabolic derangements Poor growth (?)

(hyperparathyroidism,
metabolic acidosis,
hypogonadism, GH resistance) Inflammation
650 B. Foster

In adults with CKD, at least one of the criteria Table 2 Proposed criteria for protein energy wasting in
in each of the following categories must be met to adults and children with CKD
make a diagnosis PEW: (1) biochemical distur- Criteria for PEW in adults Modified criteria for PEW
bances, including serum albumin <38 g/L, with CKD in children with CKD
prealbumin <30 g/L (dialysis patients only), and Serum chemistry
total cholesterol <100 mg/dl; (2) low body Serum albumin <3.8 g per Serum albumin <3.8 g per
100 ml 100 ml
weight, reduced body fat, or weight loss,
Serum prealbumin Serum transferrin <140
defined by body mass index <23, unintentional (transthyretin) <30 mg per mg/dl
weight loss of 5 % over 3 months or 10 % over 100 ml (for maintenance
6 months, or total body fat percentage <10 %; dialysis patients only;
levels may vary according
(3) decreased muscle mass, defined as loss of
to GFR level for patients
muscle mass of 5 % over 3 months or 10 % with CKD stages 2–5)
over 6 months or mid-arm muscle circumference Serum cholesterol Total cholesterol <100
>10 % below the reference average, or low uri- <100 mg per 100 ml mg/dl
nary creatinine appearance [83]; and (4) low pro- C-reactive protein >3
tein or energy intake, defined as protein intake mg/L
Body mass
<0.6 g/day for at least 2 months for dialysis
BMI <23 BMI-for-height-age and
patients and <0.8 g/day for at least 2 months for
sex <5th percentile
patients with CKD stage 2–5. Unintentional weight loss Unintentional decrease in
In pediatric CKD, there is no consensus on the over time: 5 % over BMI-for-height-age and
criteria that should be used to define PEW. There 3 months or 10 % over sex percentile
are significant challenges in adapting the adult 6 months
criteria to growing children. For example, weight Total body fat percentage –
<10 %
loss is insensitive as a criterion in children who are
Muscle mass
expected to be gaining weight with growth.
Muscle wasting: reduced Mid-upper arm
Efforts have been made to adapt the adult criteria muscle mass 5 % over circumference for height-
to children, with limited success [72, 84]. The 3 months or 10 % over age and sex <5th
criteria for PEW in adults with CKD and proposed 6 months percentile
modified criteria for children are summarized in Reduced mid-arm muscle Decrease in mid-upper
circumference area arm circumference for
Table 2. (reduction >10 % in height-age and sex
There should be a sound rationale for each of relation to 50th percentile percentile
the criteria accepted as important in defining of reference population)
PEW. Ideally, the abnormalities making up the Creatinine appearance –
syndrome of PEW should predict poor clinical Dietary intake
outcomes. A clear outcome-based definition has Unintentional low DPI Self-reported poor appetite
<0.80 g kg1 day1 for at
yet to be determined for either adults or children least 2 months for dialysis
with CKD. The evidence that the proposed criteria patients or <0.6 g kg1
for PEW predict adverse outcomes is reviewed day1 for patients with
below. CKD stages 2–5
Biochemical disturbances: Hypoalbuminemia Unintentional low DEI
<25 kcal kg1 day1 for
has been consistently demonstrated to be a pre- at least 2 months
dictor of mortality in both adults [85] and children Growth
[86] treated with dialysis. However, N/A Height-for-age and sex
hypoalbuminemia is a nonspecific predictor of <3rd percentile
PEW in CKD [87–94]. Low serum albumin may Low height velocity or a
be a consequence of both volume overload and decrease in height-for-age
and sex percentile
systemic inflammation [90, 91] – both of which
are independently associated with adverse
22 Growth and Development of the Child with Renal Disease
outcomes. In the absence of inflammatory
markers, hypoalbuminemia is not predictive of
increased mortality [94]. Although it is agreed
that hypoalbuminemia is neither necessary nor
sufficient for a diagnosis of PEW, it is considered
an important biochemical indicator of PEW given
its strong association with mortality.
The limitations of serum prealbumin (also
known as transthyretin) are similar to those of
albumin. However, adult hemodialysis patients
with prealbumin concentrations <20 mg/dl were
demonstrated to have an elevated death risk even
when albumin levels were normal [4]. Low serum
transferrin level [95] and low serum cholesterol
have also been suggested as possible indicators of
PEW; however, their ability to predict poor out-
comes is not established. There are other circulat-
ing inflammatory markers that are commonly
elevated in PEW and may predict mortality, such
as C-reactive protein, and proinflammatory cyto-
kines such as IL-6 [96, 97]; however, these were
not included as part of the adult diagnostic criteria
for PEW.
Low body weight, weight loss, or reduced
body fat: In adults, stable body weight, within a
certain range relative to height, is expected in
healthy individuals. Therefore, low body weight
or weight loss may be important indicators of
PEW in adults. Despite the fact that it provides
little information about body composition, BMI
may be a particularly useful method of evaluation
for PEW because BMI is strongly correlated with
lean body mass at the low end of the BMI spec-
trum [98]. PEW is characterized by loss of lean
mass; therefore low BMI may be a reasonable way
to identify lean mass deficits. In addition, low
BMI is associated with an elevated mortality risk
in both adults [99, 100] and children [101] on
maintenance dialysis.
BMI is expected to be fairly stable in adults;
therefore the PEW criterion of BMI < 23 is log-
ical in adults. In children, however, BMI must be
expressed as a percentile or Z-score relative to age
and sex due to the expected changes in BMI with
growth and development. The BMI percentile
cutoff below which a diagnosis of PEW should
be considered in children with CKD is not clear.
As noted above, the WHO and the American CDC
651
use different cutoffs to define underweight, and
neither definition has been shown to indepen-
dently predict adverse outcomes in children
with CKD.
Unintentional weight loss, independent of
absolute BMI, is a reasonable criterion for PEW
in adults; weight loss is associated with adverse
outcomes [71]. However, while weight loss would
certainly be cause for concern, weight loss would
be an insensitive indication of PEW in growing
children; weight must always be assessed relative
to height in children. A crossing down of BMI
percentiles is a more appropriate diagnostic crite-
rion for pediatric PEW [72, 84].
Linear growth failure has also been suggested
as an indicator or PEW in children [84, 102] and
has been associated with a greater mortality risk in
CKD [103, 104]. However, poor growth may have
many causes distinct from PEW in the setting of
CKD, including inadequate caloric intake,
delayed sexual maturation, and growth hormone
insensitivity. Importantly, some of the causes of
PEW may independently cause growth
failure, even if PEW is absent. Therefore, growth
failure may have poor specificity as a criterion
for PEW.
Decreased muscle mass: Given that muscle
wasting is a central feature of PEW, reduced mus-
cle mass is probably the single most valid criterion
for the presence of PEW in CKD [71]. However,
although muscle mass can be estimated
using techniques such as dual-energy X-ray
absorptiometry (DXA), there is currently no prac-
tical method of measuring muscle mass in the
clinical setting. Mid-arm muscle circumference
has been suggested as a surrogate for
muscle mass, but has not been validated for this
purpose. While indirect measures, such as
creatinine appearance (estimated by the
quantification of creatinine in a 24-h urine collec-
tion and in the collected spent dialysate), may
have some value in adults with CKD [71], refer-
ence values for creatinine appearance are not
available for children, making interpretation
impossible.
Low protein or energy intake: Poor appetite is
associated with an increased mortality risk in adult
dialysis patients [105]. While anorexia leading to
652
poor energy intake is common among infants with
CKD, both calorie and protein intake are usually
appropriate for body size in older children with
CKD [31]. Nevertheless, the presence of anorexia
may be a sign of PEW in some children.
A study of children with CKD stages 2–3 in the
Chronic Kidney Disease in Children (CKiD)
cohort tested a variety of different pediatric PEW
definitions in predicting hospitalization rates
[84]. The criteria for PEW in that study included
the following: (1) biochemical, serum albumin
<38 g/L, transferrin <140 mg/dl, total cholesterol
<100 mg/dl, and C-reactive protein >3 mg/L;
(2) low body mass, BMI-for-height-age and sex
<5th percentile, or a decrease in BMI-for-height-
age and sex percentile of 10 % over 12 months
from an initial BMI <80th percentile;
(3) decreased muscle mass, mid-arm muscle cir-
cumference for height-age and sex <5th percen-
tile or a decrease in mid-arm muscle
circumference for height-age and sex percentile
of 10 % over 12 months; (4) decreased appe-
tite, as a surrogate for low protein or energy
intake; and (5) poor growth, height-for-age and
sex <3rd percentile or a decrease in height-for-
age and sex percentile of 10 % over 12 months.
PEW was alternately defined as having at least
one criterion in two of categories 1–4, in three of
categories 1–4, or in three of categories 1–4 plus a
growth criterion. Although there was a trend
toward a higher risk of hospitalization in children
with PEW regardless of the definition used, the
increased risk was only statistically significant
when poor growth was included in the PEW def-
inition. Because PEW is not yet clearly defined in
children with CKD, it is not possible to estimate
the prevalence. However, the study mentioned
above noted a prevalence of 7–20 % depending
on the definition used; there was no clear relation-
ship between the stage of CKD and prevalence
of PEW.
Further research is needed to better understand
the pathogenesis and manifestations of PEW in
pediatric CKD. However, the concept is a useful
one because it has highlighted the distinctions
between simple intake-related malnutrition and
more complex wasting syndromes, with multiple
causes.
B. Foster
Evaluation of Growth
and Development in CKD
Because of the high risk for impaired growth, and
the unquantified (but at least theoretical) risk for
protein energy wasting in children with CKD, it is
recommended that growth and nutritional status
be evaluated at regular intervals. The National
Kidney Foundation Kidney Disease Outcomes
Quality Initiative 2008 update to the Clinical
Practice Guideline for Nutrition in Children with
CKD provided guidance on which measures to
follow and at what intervals [22]. These guide-
lines were developed by a working group with
international representation. Table 3 summarizes
the recommendations. Recommendations differ
slightly for children under 3 years old versus
older children, based on the different factors
influencing growth among infants compared
with older children with CKD. Methods of
assessing each suggested parameter, and the ratio-
nale for assessing it, are detailed below.
Dietary intake: A skilled registered dietician is
an important member of the care team for children
with CKD. Dieticians are able to assess dietary
intake using a variety of methods. The guidelines
suggest that dietary intake be assessed regularly
using a 3-day diet diary, but acknowledge that
three 24-h recalls may be better suited to adoles-
cents. Dietary records allow the dietician to esti-
mate daily intake of calories, macronutrients
(carbohydrates, protein, and fat), vitamins, and
minerals. Prospective 3-day dietary diaries have
been shown to give unbiased estimates of energy
intake in normal weight children under 10 years
old; however, underreporting is common among
adolescents [106, 107], making 24-h recalls a
potentially more suitable option in adolescents.
The 24-h recall method is limited by its inability
to capture the day-to-day variability in dietary
intake. A more complete evaluation of intake
can be made by examining three 24-h recalls,
including 1 weekend day and 2 weekdays.
Information on dietary intake allows the
treating team to evaluate the adequacy of a
patient’s intake before significant adverse changes
in body composition result. This is particularly
important in infants, in whom poor intake is
22 Growth and Development of the Child with Renal Disease 653

Table 3 Recommended parameters and frequency of assessment for children with CKD stages 2–5 (including dialysis).
a
Applies only to adolescents receiving hemodialysis (Reproduced from K/DOQI [22])
Minimum interval
Age 0– < 1 year Age 1–3 years Age >3 years
CKD CKD CKD CKD CKD CKD CKD CKD CKD
Measure 2–3 4–5 5D 2–3 4–5 5D CKD2 3 4–5 5D
Dietary intake 0.5–3 0.5–3 0.5–2 1–3 1–3 1–3 6–12 6 3–4 3–4
Height- or 0.5–1.5 0.5–1.5 0.5–1 1–3 1–2 1 3–6 3–6 1–3 1–3
length-for-age
percentile or SD
score
Height- or 0.5–2 0.5–2 0.5–1 1–6 1–3 1–2 6 6 6 6
length velocity-
for-age
percentile or SD
score
Estimated dry 0.5–1.5 0.5–1.5 0.25–1 1–3 1–2 0.5–1 3–6 3–6 1–3 1–3
weight and
weight-for-age
percentile or SD
score
BMI-for-height- 0.5–1.5 0.5–1.5 0.5–1 1–3 1–2 1 3–6 3–6 1–3 1–3
age percentile or
SD score
Head 0.5–1.5 0.5–1.5 0.5–1 1–3 1–2 1–2 N/A N/A N/A N/A
circumference-
for-age
percentile or SD
score
nPCR N/A N/A N/A N/A N/A N/A N/A N/A N/A 1a

expected. Nutritional supplements should be ini-
tiated without delay in infants with poor intake if
there is any evidence of inadequate weight gain or
poor growth. Dietary intake information is also
useful to determine intake of calcium, phospho-
rus, sodium, and potassium. These minerals and
electrolytes must be carefully controlled in indi-
viduals with CKD.
Height- or Length-for-Age Percentile or
Standard Deviation Score: Length should be
measured in infants younger than 2 years using a
length board, and height should be measured in
older children using a wall-mounted stadiometer.
Ideally the same well-trained person should take
the measurement at each assessment. Stature mea-
surements should then be plotted on the growth
curves recommended for use in the country where
the child is being treated. Alternatively, or in
addition to the visual assessment provided by the
curves, reference data for healthy children can be
used to calculate the standard deviation score (cal-
culated by subtracting the child’s height from the
mean height for children of the same age and sex
and dividing by the standard deviation for chil-
dren of the same age and sex). A standard devia-
tion score of zero represents an average height; a
positive standard deviation score indicates above
average height, and a negative standard deviation
score indicates below average height.
Height- or Length Velocity-for-Age Percentile
or Standard Deviation Score: The growth veloc-
ity (change in height per unit time) can be deter-
mined by recording serial height measurements.
As noted above, it is also possible to calculate
growth velocity standard deviation scores using
WHO Reference Standards in infants under
2 years or using reference data from the Fels
Longitudinal Study [16] in children over 2 years
old. A negative height velocity standard deviation
score indicates that the child is growing at a lower
654
than average rate, resulting in a crossing down of
percentiles. A positive height velocity standard
deviation score may indicate catch-up growth.
The determination of height velocity standard
deviation score or percentile provides a quantifi-
able measure of relative growth velocity and can
be a useful adjunct to plotting heights on the
growth curves.
Estimated dry weight and weight-for-age and
body mass index-for-age percentiles or standard
deviation scores: In children with predialysis
CKD without edema, measuring weight and plot-
ting it on weight-for-age curves are fairly simple.
In children being treated with dialysis, or children
with edema related to nephrotic syndrome, weight
assessment is more challenging. The first step is to
estimate the euvolemic weight. This is generally
referred to as the “target dry weight,” since vol-
ume overload is the more common abnormality in
CKD. However, some children with CKD may
also be at risk for volume depletion due to high
urine volumes (e.g., renal dysplasia, cystinosis).
Physical findings such as edema and jugular
venous distention indicate volume overload.
Hypertension is also commonly a sign of fluid
overload in patients on maintenance dialysis
[108]. Hypoalbuminemia may also be a marker
for fluid overload. In contrast, thirst, tachycardia,
decreased skin turgor, and sunken eyes and/or
fontanelle signify volume depletion. In mainte-
nance dialysis patients, response to dialytic fluid
removal (hypotension, cramping) may also give
clues as to the volume status of the patient. Esti-
mation of target dry weight is difficult, particu-
larly in growing children, in whom weight is
expected to increase over time. Errors in the esti-
mation of target dry weight may have an impor-
tant impact on the assessment of the
appropriateness of body weight relative to height.
For example, an overestimation of target dry
weight representing 10 % of the body weight
will result in a BMI standard deviation score
about 0.5–1.0 SD units above what it would be
at true target dry weight.
Once the target dry weight has been estimated,
weight should be plotted on the weight-for-age
curves and the standard deviation score calcu-
lated. Weight-for-age cannot be interpreted in
B. Foster
isolation because it will be low in children who
are short for age. Therefore, weight-for-age
should be interpreted in the context of the height-
for-age percentile or standard deviation score.
Appropriateness of weight relative to height can
be assessed using weight-for-length percentiles in
younger children or BMI in children of all ages.
There are some caveats to the assessment of
BMI in children with CKD due to the high prev-
alence of growth retardation and delayed matura-
tion. If BMI is expressed relative to chronological
age in a child with growth and/or maturational
delay, this will result in inappropriate underesti-
mation of his/her BMI compared to peers of sim-
ilar height and developmental age. It has been
shown that this problem can be avoided by
expressing BMI relative to height-age in children
with CKD [109]. Height-age is the age at which
the child’s actual height would be on the 50th
percentile. Although this approach works well
for most children with CKD, it may not be appro-
priate for all children. For example, when sexual
maturation is complete, it may be more appropri-
ate to express BMI relative to chronological age.
As in healthy children, the BMI percentile can
be used to classify children with CKD as under-
weight, normal weight, overweight, or obese.
However, it must be recognized that the risk asso-
ciated with any of these categories in a CKD
population remains controversial. Although obe-
sity and overweight have been associated with
adverse outcomes in otherwise healthy adults,
the ability of these categories to predict poor out-
comes in the CKD population is unknown. Large
studies of adult hemodialysis patients showed an
inverse relationship between BMI and mortality
risk. These studies found no clear BMI threshold
above which the mortality risk stabilized or
began to increase, even as BMI increased above
30 [100, 110]. A smaller study, also of adult
hemodialysis patients, found a U-shaped relation-
ship between BMI and mortality risk, with higher
mortality risk among those with BMI <17 or >23
compared to those with BMI between 17.0 and
18.9 [111]. A similar U-shaped association has
been seen in children with stage 5 CKD: mortality
risk was lowest among children with a BMI SD
score around +0.5. A 1.0 SD unit difference in
22 Growth and Development of the Child with Renal Disease
BMI SD score in either direction was associated
with a 6 % higher risk of mortality [101] – which
represents a fairly small effect. All of these studies
can only conclude an association between BMI
and outcome; a causal relationship has not been
established. Nonetheless, BMI, interpreted in the
overall clinical context, can provide useful infor-
mation to clinicians caring for children with CKD.
Head circumference-for-age percentile or SD
score: Like for healthy children 3 years of age,
head circumference should be measured regularly
and plotted on the head circumference-for-age
curves in children 3 years old with CKD. The
WHO Growth Standards are the most appropriate
reference curves [112]. Infants with CKD have
been shown to be at risk for poor head growth –
which reflects poor brain growth [113, 114]. Head
circumference assessment should be coupled with
intermittent developmental assessments in this
population.
Normalized protein catabolic rate (nPCR):
The protein catabolic rate (PCR) provides an
objective measure of dietary protein intake in
patients receiving maintenance hemodialysis.
When PCR is expressed normalized for the
patient’s weight, it is referred to as the normalized
PCR (nPCR). Although nPCR was originally cal-
culated using formal urea kinetic modeling [115],
simpler algebraic formulas appear to provide
almost identical results to more complex model-
ing [116]. The nPCR can be calculated using the
modified Borah equation, where G represents the
urea generation rate in mg/min and V1 represents
the total body water at the end of dialysis in liters
(0.58  weight in kg) [117]:
nPCR ¼ 5:43  G=V1 þ 0:17
The urea generation rate is determined from the
rise in blood urea nitrogen (BUN) from the end of
one HD treatment to the beginning of the next
treatment and is calculated as
Gðmg=minÞ ¼ ½ðC2  V2 Þ  ðC1  V1 Þ=t
where C1 = postdialysis BUN (mg/dl), C2 =
predialysis BUN (mg/dl), V1 = postdialysis total
body water in deciliters (5.8  postdialysis weight
655
in kg), V2 = predialysis total body water in deci-
liters (5.8  predialysis weight in kg), and t = time
(min) from the end of the dialysis treatment to the
beginning of the following treatment. Adult stud-
ies suggest that pre- and postdialysis BUN levels
from the same treatment can be used to calculate
nPCR [118].
The value of nPCR in children with CKD –
beyond as a means of estimating protein intake
– is not clear. Higher nPCR was shown to be
associated with subsequent weight gain and
lower nPCR with future weight loss among ado-
lescents treated with dialysis [119, 120]. It is
suggested that nPCR level be targeted to the
age-specific recommended protein intake.
Frequency of growth and nutritional assess-
ment: Given the high risk among children with
CKD for disturbances in growth and nutritional
status, it is suggested that these parameters be
assessed at least twice as frequently as in healthy
children and more often with severe CKD and/or
when there is an ongoing concern.
Optimizing Growth in Children
with CKD
Why is growth important? Growth may be one
of the most important outcomes in pediatric CKD.
There are two main reasons that growth is so
important. First, growth is an excellent index of
overall health. Second, stature has important
effects on self-esteem and perceived quality
of life.
Large retrospective cohort studies of children
with ESRD showed a strong association between
stature and risk for morbidity and mortality [103,
104]. A study of children recorded in the United
States Renal Data Systems (USRDS) Pediatric
Growth and Development Study compared hospi-
talization rates and mortality rates over 5 years of
follow-up among children with moderate or
severe growth failure, defined as a height velocity
SD score calculated over a 1-year interval of 2.0
to 3.0 (moderate) or < 3.0 (severe)
[103]. Compared with children with height veloc-
ity SD scores of > 2.0, those with moderate
growth failure had a relative risk (RR) for
656
hospitalization over the next 5 years of 1.24 (95 %
confidence interval (CI) 1.2, 1.3) and those with
severe growth failure a RR of 1.14 (95 % CI 1.1,
1.2), after adjustment for age, gender, race, cause
and duration of ESRD, and treatment modality
(dialysis or transplant). Similarly, moderate and
severe growth failure were associated with a sig-
nificantly higher risk of death (RR 2.01 (95 % CI
1.1, 3.6) for moderate and 2.9 (95 % CI 1.6, 5.3)
for severe) [77]. A study from the North American
Pediatric Renal Trials and Collaborative Studies
registry had similar findings [104]. Children with
a height-for-age SD scores of < 2.5 at the time
of dialysis initiation had significantly more hospi-
tal days per month of dialysis than patients with
height-for-age SD score > 2.5 [78]. Height-for-
age SD score < 2.5 was also associated with a
two times higher risk of death, after adjustment for
age, sex, race, primary renal disease, dialysis
modality, and whether the patient was listed for
kidney transplant. These studies should not be
interpreted as showing that short stature causes
morbidity or death. Rather, they show that growth
failure is a marker for illness severity. Efforts
should be directed toward the root causes of
growth failure in order to improve outcomes.
Short stature is highly visible and may be one
of the most obvious features distinguishing indi-
viduals with childhood onset CKD from their
healthy peers. Although existing studies of adult
survivors of pediatric CKD cannot draw causal
links between stature and quality of life, associa-
tions were quite strong [121–123]. Among
244 adults who had received a kidney transplant
in childhood, greater final adult height was asso-
ciated with a significantly higher likelihood of
being married or living outside of the parents
home [121]. Short stature, defined as a final
adult height >2.5 standard deviations below the
mean, was also associated with a lower likelihood
of full-time employment [121]. In a small study of
39 adults with childhood onset ESRD, 36 %
reported dissatisfaction with their height com-
pared with 4 % of healthy age-matched controls;
greater satisfaction with adult height was signifi-
cantly positively correlated with a better per-
ceived quality of life [123]. A study of
433 children with CKD found significant
B. Foster
associations between better physical and social
functioning and both of catch-up growth and treat-
ment with growth hormone [124].
Because of the important associations between
growth and morbidity, mortality, and quality of
life, efforts should be made to optimize growth in
children with CKD. Optimal growth may also be
especially important in infants with CKD who
must reach a specific body size before being eli-
gible for a kidney transplant. Growth can be opti-
mized by systematically addressing each of the
potential contributors to growth failure in the
pediatric CKD population.
Nutrition
Adequate energy intake is necessary for normal
growth. Assessment of dietary intake combined
with examination of weight and height trajectories
will help determine whether poor energy intake
may be contributing to poor growth. Children
with CKD should take in 100 % of the estimated
energy requirement based on sex, age, weight,
height, and physical activity level [22]. The equa-
tions used to calculate estimated energy require-
ment are provided in Table 4 and physical activity
coefficients in Table 5. Children experiencing
inadequate growth who have insufficient energy
intake will need dietary supplements. The vast
majority of infants <2 years old with moderate
to severe CKD will be unable to take sufficient
calories by mouth and will need tube feeding. It is
important to intervene preemptively, before
stunting is evident; height potential lost in infancy
is very difficult to catch up later. Infants in whom
tube feedings are started before major height def-
icits are noted have superior final adult height
[125]. Supplemental tube feedings should be con-
sidered for any infant failing to meet the energy or
fluid requirements for normal growth [22].
Tube feedings can be delivered either via a
nasogastric tube or via a gastrostomy tube.
Gastrostomy feeding has cosmetic advantages
(hidden under clothing) and avoids negative oro-
pharyngeal stimulation associated with chronic
presence of and frequent replacement of an NG
tube [126]. The unpleasant stimulation of the
22 Growth and Development of the Child with Renal Disease 657

oropharynx associated with nasogastric tube use
may exacerbate oral feeding aversion and contrib-
ute to poor development of oral-motor skills
[126]. In addition, a gastrostomy avoids the poten-
tial complications associated with nasogastric
tubes such as otitis, sinusitis, pulmonary aspira-
tion, exacerbation of gastroesophageal reflux, and
nasoseptal erosion [126]. Despite the advantages
of gastrostomy feeding, nasogastric tube feeding
still has a place in the nutritional care of children
with CKD, especially when tube feedings are
expected to be needed for only a short time.
Table 4 Equations to estimate energy requirements for
children (Reproduced from K/DOQI [22]). PA represents
the physical activity coefficient
Estimated energy requirement (EER)
(Kcal/day) = total energy expenditure +
Age energy deposition
0–3 EER = [89 X weight (kg) 100] + 175
months
4–6 EER = [89 X weight (kg) 100] + 56
months
7–12 EER = [89 X weight (kg) 100] + 22
months
13–35 EER = [89 X weight (kg) 100] + 20
months
3–8 years Boys: EER = 88.5–61.9 X age (y) + PA X
[26.7 X weight (kg) +903 X height (m)] +
20
Girls: EER = 135.3–30.8 X age (y) + PA X
[10 X weight (kg) +934 X height (m)] + 20
9–19 Boys: EER = 88.5–61.9 X age (y) + PA X
years [26.7 X weight (kg) + 903 X height (m)] +
25
Girls: EER = 135.3–30.8 X age (y) + PA X
[10 X weight (kg) + 934 X height (m)] + 25
In infants weighing less than 4 kg, nasogastric
feeding is the route of choice [126].
In contrast to infants and very young children,
in whom the growth benefits of tube feeding are
well established [42, 127, 128], there is little evi-
dence supporting the use of supplemental tube
feeding to promote growth in older children with
CKD. In fact, there is some evidence that poor
intake in older children with CKD may be a con-
sequence of poor growth, rather than the cause:
spontaneous calorie intake increased by almost
12 % during treatment with recombinant human
growth hormone in a study of 33 children with
CKD [129]. Nevertheless, selected older children
may need, and benefit from, tube feeding. Older
children treated with nutritional supplements
should be observed closely for evidence of a
response in the form of improved growth. Thera-
pies of proven benefit, such as growth hormone,
should not be unduly delayed by prolonged trials
of calorie supplementation [59].
In addition to energy intake, the protein intake
of children with CKD should also be monitored
and controlled. While children with CKD tend to
spontaneously consume less protein than their
healthy counterparts, it is rare that spontaneous
protein intake does not meet requirements. Rather,
protein intake among children with CKD is often
in the range of 1.5–2 times the recommended
dietary allowance [130–132]. In view of the
important correlations between protein intake
and phosphorus intake, and the negative effects
of phosphorus overload on cardiovascular health,
the current recommendations are to maintain

Table 5 Physical activity coefficients for the determination of energy requirements in children 3–18 years of age (From
Health Canada: http://www.hc-sc.gc.ca/fn-an/alt_formats/hpfb-dgpsa/pdf/nutrition/dri_tables-eng.pdf)
Level of physical activity
Sedentary Low active Active Very active
ADL +
Typical ADL +30–60 min of 60 min of ADL + 60 min of daily moderate
activities of daily moderate activity daily activity + an additional 60 min of
daily living (e.g., walking at 5–7 moderate vigorous activity or 120 min of moderate
Gender (ADL) only km/h) activity activity
Boys 1.0 1.13 1.26 1.42
Girls 1.0 1.16 1.31 1.56
658 B. Foster

Table 6 Recommended dietary protein intake in children with CKD stages 3–5 and 5D (dialysis) (Reproduced from
K/DOQI [22])
Dietary reference intake (DRI)
DRI Recommended for CKD Recommended for CKD Recommended Recommended for
(g/kg/ stage 3 (g/kg/day) stages 4–5 (g/kg/day) for hemodialysis peritoneal dialysis
Age day) (100–140 % DRI) (100–120 % DRI) (g/kg/day)a (g/kg/day)b
0–6 1.5 1.5–2.1 1.5–1.8 1.6 1.8
months
7–12 1.2 1.2–1.7 1.2–1.5 1.3 1.5
months
1–3 1.05 1.05–1.5 1.05–1.25 1.15 1.3
years
4–13 0.95 0.95–1.35 0.95–1.15 1.05 1.1
years
14–19 0.85 0.85–1.2 0.85–1.05 0.95 1.0
years
a
DRI + 0.1 g/kg/day to compensate for dialytic losses
b
DRI + 0.15–0.3 g/kg/day depending on patient age to compensate for peritoneal losses

protein intake at 100–140 % of the dietary refer-
ence intake for children with CKD stage 3 and at
100–120 % for those with CKD stages 4–5
(including 5D – dialysis) [22]. Low protein
diets are not recommended. The recommended
dietary protein intake by age is summarized in
Table 6.
Fluid and Sodium
Many children with CKD, including those with
renal dysplasia, obstructive uropathy,
nephronophthisis, and cystinosis, are polyuric.
When urine volumes are high, there may also be
substantial sodium losses. Infants on peritoneal
dialysis may also experience large sodium losses
through the dialysate [133]. Chronic sodium and
water losses may result in significant volume
depletion, which will impair linear growth [134]
and may even result in neurologic injury
[22]. When sodium losses are significant, sodium
supplements are required to support optimal
growth [40, 41, 125, 135]. In infants with polyuric
CKD, the volume of fluid and doses of sodium
needed to promote adequate growth can rarely be
taken orally. Tube feeding facilitates delivery of
adequate sodium and fluid, as well as calories
[125]. Fluid volumes of 180–240 ml/kg/day and
sodium intakes of 2–4 mEq per 100 ml of fluid are
suggested for infants with polyuric CKD [125].
Acidosis Control
Current recommendations suggest that the bicar-
bonate level be corrected to at least 22 mmol/L
[22]. This can be achieved using oral supplements
of sodium bicarbonate or sodium citrate. Citrate
should be avoided in the rare situation where
aluminum-based phosphate binders are being
used due to enhanced aluminum absorption in
the presence of citrate. For children being treated
with hemodialysis, acidosis may also be corrected
by a higher bicarbonate concentration in the bath
or by an increased dose of dialysis.
Control of Hyperparathyroidism
and Metabolic Bone Disease
The metabolic bone disease associated with CKD
(formerly referred to as renal osteodystrophy) has
a negative impact on growth. Management of
CKD-related metabolic bone disease is dealt
with in ▶ Chap. 66, “Management of Chronic
Kidney Disease in Children” of this book. The
main components of therapy include dietary
22 Growth and Development of the Child with Renal Disease 659

phosphate restriction, the use of intestinal phos- SDS; the most pronounced effects are seen in the
phate binders to limit phosphate absorption, and first year of treatment [139]. Importantly, the fre-
active vitamin D therapy. quency of adverse effects did not differ between
rhGH-treated subjects and the control group
[139]. Although prepubertal children with CKD
Recombinant Human Growth Hormone treated with rhGH have been shown to achieve
Therapy normal final adult height [59], the impact of rhGH
treatment on final height in pubertal patients is
Recombinant human growth hormone (rhGH) less clear. While some studies showed a good
therapy has become the standard of care for chil- growth response to rhGH in pubertal patients,
dren with CKD with evidence of growth failure. without advancing bone maturation beyond that
Numerous studies have demonstrated its safety expected [136], most studies suggested a more
and effectiveness [136–138]. A Cochrane review vigorous response in prepubertal than pubertal
including 15 randomized control trials of rhGH children [59, 139–141]. Most agree that existing
treatment in children with predialysis CKD, those evidence supports early initiation of rhGH to opti-
on dialysis, and with kidney transplants con- mize final height. Unfortunately rhGH remains an
cluded that rhGH treatment significantly improves underutilized therapy [142]. A consensus state-
height SDS, height velocity, and height velocity ment, published in 2006, provided guidance on

Consider GH therapy in patients with CKD stage 2-5 and 5D AND height SDS <-
1.88 or height-for-age <3rd percentile

Assess and treat complicating factors for poor growth including: acidosis, inadequate calorie intake, salt-
wasting, metabolic bone disease, and hypothyroidism

No Is growth velocity improved? Yes

Perform baseline assessments for GH therapy:

Continue current therapy
(a) Recalculate height SDS, height velocity SDS, and height velocity
(b) Assess pubertal stage, bone age, hip and knee X-rays, fundoscopic
exam, chemistries, PTH, and thyroid studies

Start GH therapy:
0.05 mg/kg/day SC (0.35 mg/kg/week)

Monitor during GH therapy:

(a) Every 3-4 months: height, weight, head
circumference (until 3 years old), pubertal stage,
nutritional evaluation, fundoscopic exam,
chemistrics, PTH, and toxicity
(b) Every year: Bone age
(c) For symptoms: hip and knee X-rays

Is growth adequate? (i.e. growth Yes

No
velocity 2cm/yr> baseline)

Assess and correct: Is growth Continue GH therapy and adjust dose

adequate? every 3-4 months based on weight
(a) Dose for weight Discontinue GH therapy in the following circumstances:
(b) Metabolic status (a) Achieved height goal based on mid-parental
(c) Nutrition height or 50th percentile for age

(d) Adherence (b) Closed epiphyses

(c) Active neoplasia
(d) Slipped femoral epiphyses
(e) Intracranial hypertension
If height velocity remains <2 cm/yr
Consider pediatric (f) Nonadherence over baseline and reason for
No
endocrine consult discontinuation resolved, consider
(g) Severe hyperparathyroidism per CKD stage:
reinitiation of GH therapy
Stage 2-4: PTH>400 pg/ml
Stage 5: PTH>900 pg/ml

Fig. 5 Proposed algorithm for evaluation and treatment of growth failure in children with CKD (Modified with
permission from Mahan et al. 2006 [137])
660
the evaluation and treatment of growth failure in
children with CKD in the form of a practical
algorithm [137] (Fig. 5).
Enhanced Dialytic Clearance
The first evidence that enhanced dialytic clearance
may promote better growth in children was a 1999
study of 12 children 7 months to 14 years of age
being treated with hemodialysis [143]. By provid-
ing enhanced clearance (average Kt/V of 2.0 per
treatment; average of 14.8 h per week of dialysis),
these children were able to achieve catch-up
growth, without growth hormone therapy. Fol-
lowing that study, others have also demonstrated
improved growth with intensified dialysis therapy
[144]. A study of 15 children (median age 8 years;
interquartile range 6–10 years) treated with
hemodiafiltration 3 h per day, 6 days per week,
achieving an average single treatment Kt/Vof 1.4,
showed an increase in height velocity to 14.3 
3.8 cm/year during intensified therapy compared
with 3.8  1.1 cm/year with conventional dialysis
doses [145]. There are no randomized trials of the
impact of enhanced clearance on growth. How-
ever, the existing evidence is quite compelling.
Intensified dialysis should be considered if growth
cannot be optimized using other means of improv-
ing growth.
Minimized Exposure to Corticosteroids
Many children with CKD are treated with cortico-
steroids, either for an underlying condition or fol-
lowing a kidney transplant. The growth
suppressing effects of corticosteroids are well
known [146, 147]. When growth is inadequate in
a child with CKD who is being treated with corti-
costeroids, efforts should be made to reduce the
dose or discontinue corticosteroid therapy if possi-
ble. An observational study of steroid minimization
after renal transplantation, by early withdrawal,
appeared to result in improved growth outcomes
in pediatric transplant recipients [148]. However, a
randomized trial of steroid-based immunosuppres-
sion versus complete steroid avoidance showed no
B. Foster
difference in growth outcomes at 3 years
posttransplant [149]. Therefore, other reasons for
growth failure besides steroid use should be
sought, and treated, before considering reduction
or discontinuation of steroids.
Summary
Kidney disease can have a profound effect on
growth and development. In the past, a normal
final adult height was out of reach for the majority
of children with CKD. However, much has been
learned about the causes of growth failure in pedi-
atric CKD, and effective treatments are readily
available. It is now a realistic goal to have children
with CKD reach (or at least come close to) their
full height potential. This requires close monitor-
ing by a multidisciplinary team, a strong commit-
ment by the family, and early intervention to
prevent growth deficits.
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 Diagnostic Imaging of the Child with
Suspected Renal Disease 23
Jonathan Loewen and Larry A. Greenbaum

Contents Diagnostic Procedures

Diagnostic Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
Ultrasound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667 Imaging is essential for the diagnosis and man-
Voiding Cystourethrography . . . . . . . . . . . . . . . . . . . . . . . . . 670 agement of many disorders of the urinary tract
Intravenous Pyelography . . . . . . . . . . . . . . . . . . . . . . . . . . . . 671 (UT). Clinicians should be familiar with the avail-
Computed Tomography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 672
Nuclear Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 673 able techniques, including their indications, limi-
Magnetic Resonance Imaging . . . . . . . . . . . . . . . . . . . . . . . 677 tations, and potential complications.
Congenital Kidney and Urological
Ultrasound (US) is the most widely used
Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 678 modality because of the information provided,
Fetal Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 678 safety, and low cost. Intravenous pyelography
Hereditary Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 685 (IVP) is currently seldom utilized, but voiding
Infections of the Urinary Tract . . . . . . . . . . . . . . . . . . . . 686 cystourethrography (VCUG) and nuclear medi-
Renal Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 690 cine studies continue to have important roles.
Renal Transplant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 690
Computed tomography (CT) is readily available
Urolithiasis and Nephrocalcinosis . . . . . . . . . . . . . . . . . 691 and can acquire images rapidly, generally obviat-
Urolithiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691 ing the need for sedation. Magnetic resonance
Nephrocalcinosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691
imaging (MRI) generates anatomically precise
Trauma to the Urinary Tract . . . . . . . . . . . . . . . . . . . . . . 691 images without radiation exposure, but at high
Renal and Urinary Tract Tumors . . . . . . . . . . . . . . . . . 692 direct financial cost and with the need for sedation
Role of Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 692 in young children. Imaging studies should be
Imaging of Specific Renal Tumors . . . . . . . . . . . . . . . . . . 692 carefully selected, with consideration of diagnos-
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 696 tic utility, cost, and potential complications such
as contrast nephropathy and radiation exposure.

Ultrasound

US is the principal imaging modality for visuali-

zation of the UT. US provides a rapid assessment
of kidney shape and size, and it is especially good
J. Loewen (*) • L.A. Greenbaum
at identifying hydronephrosis. Studies are com-
Department of Pediatric Radiology, Emory University
School of Medicine, Atlanta, GA, USA pleted without discomfort or need for sedation,
e-mail: jonathan.loewen@choa.org; lgreen6@emory.edu and there are no known complications or side
# Springer-Verlag Berlin Heidelberg 2016 667
E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_21
668 J. Loewen and L.A. Greenbaum

effects. In addition, US is relatively inexpensive and evaluation of the renal pelvis, ureters, and
and readily available. The skill of the examiner bladder. The ureters are not usually visible, but
can significantly influence the information may be seen when dilated due to underlying
obtained via US. Lack of patient cooperation and pathology.
large body habitus may also limit image quality. There are published standards for US determi-
Because of its low cost and the absence of side nation of renal length for fetuses, preterm infants,
effects, US is the optimal modality for clinical full-term neonates, infants, and older children
situations requiring repeated evaluations. For ([2–7]; Table 1). Length measurements should
example, US can assess hydronephrosis before be made with the patient supine or in the contra-
and after an intervention such as a catheter, stent, lateral decubitus position; length may be
or nephrostomy tube placement. US is also ideal underestimated with the patient prone [8, 9].
as a screening examination for children who need Gestational age and birth weight correlate with
surveillance for kidney stones or malignancy. kidney length in newborns [2, 5]. In older chil-
Advances in sonographic technology including dren, age is a convenient parameter, but length or
compound imaging, harmonic imaging, pano- height is a better predictor of normal kidney
ramic imaging, and 3D imaging have improved length [4, 10, 11]. A multivariable approach
image quality [1]. allows for improved prediction equations for
renal length [12], but such equations are not
Technique widely used in clinical practice.
Images are generally obtained with the patient In adults and children, the average left kidney
supine, which optimizes visualization of the is slightly longer than the average right kidney
upper poles, using the spleen and liver as acoustic [4, 5, 10], although this is only observed in
windows. The contralateral decubitus position
may permit better visualization in some patients.
In certain situations, placing the patient in the Table 1 Renal length
prone position may allow for better visualization Range (2 SD
of the lower pole if subtle pathology is Age Mean length (cm) in cm)
present. Ideally, the bladder should be visualized Term newborn 4.48 3.86–5.10
while filled with urine. A cooperative child 2 months 5.28 3.96–6.60
should be asked not to void for a few hours prior 6 months 6.15 4.81–7.49
to the exam. In a patient who is not toilet 1.5 years 6.65 5.57–7.73
trained, the bladder should be visualized first 2.5 years 7.36 6.28–8.44
to allow imaging of the bladder before the 3.5 years 7.36 6.18–8.54
4.5 years 7.87 6.87–8.87
patient voids.
5.5 years 8.09 7.01–9.17
US transducers produce sound waves of
6.5 years 7.83 6.39–9.27
varying frequencies. High-frequency sound
7.5 years 8.33 7.31–9.35
waves do not penetrate tissue as deeply, but pro-
8.5 years 8.90 7.14–10.66
vide greater resolution. In most situations, the 9.5 years 9.20 7.40–11.00
sonographer should use the highest frequency 10.5 years 9.17 7.53–10.81
that penetrates to the desired depth. Hence, high- 11.5 years 9.60 8.32–10.88
frequency US sound permits excellent resolution 12.5 years 10.42 8.68–12.16
in infants and small children. Conversely, resolu- 13.5 years 9.79 8.29–11.29
tion is decreased in very large or obese 14.5 years 10.05 8.81–11.29
individuals. 15.5 years 10.93 9.41–12.45
US evaluation of the UT should include mea- 16.5 years 10.04 8.32–11.76
surement of renal lengths and assessment of renal 17.5 years 10.53 9.95–11.11
shape, thickness and echogenicity of the cortex, 18.5 years 10.81 8.55–13.07
the presence of corticomedullary differentiation, Adapted from Rosenbaum et al. [7]
23 Diagnostic Imaging of the Child with Suspected Renal Disease
Fig. 1 (a) Normal ultrasound of a neonatal kidney. Med-
ullary pyramids are hypoechoic with a relatively thin cor-
tex. (b) Normal 2-year-old kidney. (c) Normal 16-year-old
slightly more than 50 % of measurements of indi-
vidual patients (the remainder have kidneys of
equal length or a longer right kidney) [11]. In
infants and adults, the median left renal length is
about 3 mm longer than the median right renal
length [4, 13]. In adults and older teenagers, males
have longer kidneys, which is explained by
their increased height compared to females.
Black children have shorter kidneys than white
children [12].
Measurement of renal length in children has a
fairly high rate of intraobserver and interobserver
variation [14]; hence, US measurement of renal
length lacks the accuracy to allow assessment of
renal growth over limited time periods. Measure-
ment of the left kidney length is less accurate,
probably because the spleen provides a smaller
acoustic window than the liver. Nevertheless,
renal length discrepancy in children can be
used to predict abnormalities on technetium
99m-dimercaptosuccinic acid (99mTc-DMSA)
renal scintigraphy [15].
669
kidney. Note decreasing cortical echogenicity, decreased
conspicuity of medullary pyramids, and increased renal
sinus fat echoes with increasing age
The appearance of the kidneys changes with
age. The relative volume of medullary tissue com-
pared to cortical tissue is increased in neonates
and infants (Fig. 1a; [16]). Cortical echogenicity
is increased in the term newborn kidney, with the
majority having the same echogenicity as the liver
or spleen [6]. Renal cortical echogenicity is fur-
ther increased in premature infants, with some
kidneys having cortical echogenicity greater than
the echogenicity of the liver or spleen [17]. Corti-
cal echogenicity decreases after birth, and only a
small percentage of normal kidneys of term
infants have cortical echogenicity equivalent to
the liver or spleen by 6 months. Cortical
echogenicity is compared to the liver or spleen,
but this comparison may not be valid if there is
underlying liver disease. Transient increased cor-
tical echogenicity of the kidneys may occur in
children with a variety of nonrenal illnesses [18].
The kidneys of term newborns have prominent
medullary pyramids that are relatively hypoechoic
[16]. This results in marked corticomedullary
670
Table 2 Society of fetal urology grading system for
hydronephrosis
Grade Ultrasound
0 No hydronephrosis
1 Only renal pelvis is seen
2 Renal pelvis and a few calices are seen
3 Virtually all calices are seen
4 Similar to grade 3, but parenchymal thinning
present
differentiation. The echogenicity of the medullary
pyramids decreases after birth, and prominent
medullary pyramids are not normally present by
1 year. In contrast, the central sinus echo is min-
imal at birth and gradually increases during the
first 10 years of childhood [6] (Fig. 1b, c).
Hydronephrosis may indicate obstruction or
vesicoureteral reflux (VUR), or it may be a normal
variant, especially when mild. The Society for
Fetal Urology grading system for hydronephrosis
is predictive of the presence or absence of obstruc-
tion (Table 2; [19, 20]). Mild hydronephrosis is
common in newborns and occurs in a higher per-
centage of males than females [21].
An increase in bladder wall thickness may be
an indication of pathology, especially chronic
increased bladder pressure. Measurement of blad-
der wall thickness is highly dependent on the
degree of bladder filling, but does not appear to
be dependent on patient age or gender [22],
although male neonates may have slightly greater
bladder wall thickness than females [21]. The
upper limit of normal is 5 mm for an empty
bladder and 3 mm for a well-distended bladder
[22]. US can be used to accurately estimate blad-
der volume [23, 24]. US measurement of bladder
volume after voiding is a useful parameter for
assessing bladder function. US may detect
ureteroceles or bladder masses.
Doppler US permits evaluation of blood flow
and has been utilized to identify renal artery dis-
ease, renal vein thrombosis, tumor thrombosis in
the renal vein and inferior vena cava, and arterio-
venous fistulas [25–27]. Resistance to blood flow
can be quantified by measuring the resistive index
(RI), which is calculated from the formula: RI =
(peak systolic velocity-end diastolic velocity)/
peak systolic velocity. A normal RI is below 0.7
J. Loewen and L.A. Greenbaum
in adults and children who are 6 years of age or
older, but higher values are observed in children
less than 6 years [28].
Power Doppler mode allows increased sensi-
tivity for blood flow detection, but does not pro-
vide information on flow direction or velocity.
Power Doppler US is superior to color Doppler
US in detecting intrarenal blood flow [29] and in
identifying areas of decreased perfusion within
the kidney [30]. Power Doppler increases the sen-
sitivity of US for detecting acute pyelonephritis
[31]. Power Doppler is very sensitive to motion
artifact and hence requires a very cooperative
patient, limiting its utility in young children. 3D
US of the kidneys can also be performed, with
subtraction of a dilated collected system in order
to calculate renal parenchymal volumes [32].
Voiding urosonography (VUS) is an alternative
technique for detecting VUR that uses intravesical
instillation of a US contrast agent [33, 34]. The
principal advantage is the elimination of radiation
exposure, but bladder catheterization is still nec-
essary. VUS does not provide adequate imaging
of the bladder and urethra and thus should only be
used for the assessment of VUR. The results with
VUS correlate well with conventional VCUG [33,
35–38]. It is not yet clear if operator skill or other
barriers such as availability of US contrast agents
may limit the widespread use of this
technique [32].
Application
US is the initial imaging modality in a variety of
clinical situations, including evaluation of renal
failure, kidney transplant dysfunction, hematuria,
screening for renal malignancy, abdominal
mass, and follow-up of abnormal prenatal US.
Procedural applications of US in pediatric
nephrology include pre-biopsy or real-time local-
ization of native and transplant kidneys as well as
venous localization prior to hemodialysis catheter
placement [39].
Voiding Cystourethrography
VCUG is the most frequently performed fluoro-
scopic examination and the gold standard for the
23 Diagnostic Imaging of the Child with Suspected Renal Disease 671

diagnosis of VUR [1]. Most clinical studies of

VUR have utilized VCUG. Moreover, VCUG is
the only modality that detects VUR and provides
detailed information about the bladder and
urethra.
Radiation exposure is the most important side
effect of VCUG. The radiation dose has been
reduced through technological innovations, and
a variety of additional approaches are available
to further reduce radiation exposure [40, 41].
A known allergy to contrast material is a contra-
indication to the study [42]. Because of the need
for catheterization, VCUG creates a small risk of
iatrogenic infection and trauma to the urethra,
although these risks are low [43]. In addition,
many children and families find catheterization
frightening and emotionally traumatic. Sedation
with midazolam appears safe and effective
[44–46].

Technique
VCUG is done using fluoroscopy. An initial film
prior to bladder catheterization detects calcifica-
tions or bony abnormalities; an abnormal bowel
gas pattern may suggest a mass. The bladder is
sterilely catheterized using a feeding tube (5 F in
young infants; 8 F in older children), and urine can
be obtained for culture. After emptying the blad-
der of urine, contrast material is infused to fill the
bladder based on estimates of bladder capacity,
but using gravity to avoid overfilling. The bladder
must be filled to capacity to insure an adequate
study.
An early film during filling of the bladder is
useful for identifying filling defects, such as
caused by a ureterocele or bladder tumor. Films
are taken before and after voiding, with VUR
graded based on the International Reflux Study
in Children [47]. In young children, who may not
allow their bladder to fill to capacity, the sensitiv-
ity of VCUG for detecting VUR improves with
multiple cycles of bladder filling and voiding
[48, 49], but this increases the length and radiation
exposure of the procedure. A film during voiding
permits visualization of the urethra and is essential
in male children (Figs. 2 and 3). At the end of
voiding, a final film identifies residual urine and
previously unidentified VUR. Technological
Fig. 2 Voiding cystourethrogram. Frontal voiding image
of normal 5-year-old girl
advances including digital fluoroscopy with
pulsed fluoroscopy and last-image-hold options
have made it possible to diminish radiation
dosage [1].
Intravenous Pyelography
The availability of US and other imaging modal-
ities such as MR urography has dramatically
decreased the use of intravenous pyelography
(IVP) [50]. The risks of IVP include radiation
and contrast exposure; intravenous access is
necessary.
Technique
An initial supine film is performed prior to con-
trast injection. Approximately 5–7 min after con-
trast, a film centered on the kidneys allows
visualization of the nephrogram and the calices.
Subsequent films are full length and taken 15 and
30 min after injection. Delayed films are useful in
cases of urinary obstruction. Bowel preparation
does not improve image quality [51, 52], although
672
Fig. 3 Voiding
cystourethrogram. Oblique
voiding image of a normal
1-year-old boy
a short fasting period is usually recommended.
The radiation exposure of IVP can be reduced by
adjusting the number of films based on the clinical
question and the results of initial films. Nonionic
contrast medium improves image quality and
decreases the risk of contrast nephropathy.
Application
Poor renal function limits the utility of IVP due to
inadequate contrast accumulation in the kidneys.
Moreover, poor renal function increases the risk
of contrast nephropathy. IVP is currently rarely
used in the evaluation of obstructive disease; US
and nuclear medicine studies are the preferred
imaging modalities. 99mTc-DMSA has replaced
IVP for detection of renal scarring, but IVP pro-
vides excellent detail and is certainly an appropri-
ate substitute [53, 54]. IVP is useful for evaluation
of ectopic ureters, ureteroceles, and fusion anom-
alies, although alternative imaging (US, MRI, CT)
is generally utilized when available.
Computed Tomography
CT provides excellent anatomic resolution of the
UT (Fig. 4), and CT has many applications. Radi-
ation exposure is an important risk of CT and
should limit its use to cases where the detail pro-
vided outweighs the risk of the study [55, 56].
IV contrast is necessary for optimal studies in
J. Loewen and L.A. Greenbaum
most situations. Multiple strategies and innova-
tions have reduced the radiation dose with CT
[55, 57], but it remains an important concern
[58]. Sedation is currently rarely necessary due
to rapid image acquisition [59, 60], which also
decreases motion artifact [57].
Technique
Thin-section helical imaging, now performed on
all CT scanners, allows for multiplanar
reformatted images to be obtained quickly and
displays the kidneys, ureters, and bladder in
three planes. Oral contrast, while often unneces-
sary for evaluation of the UT, is useful for exam-
inations where differentiating bowel from
pathology is necessary. Time constraints (acute
trauma) or risk of aspiration may prevent the use
of oral contrast. For younger children, excessive
movement can be prevented by using blankets or
adhesive tape.
Intravenous contrast is usually indicated, with
the notable exception of nephrolithiasis evalua-
tion. Low osmolality nonionic contrast is
preferred; contrast is dosed based on weight
[57, 61]. Neonates and infants require a longer
delay from contrast injection to imaging.
Application
CT is the preferred modality for initial evaluation
of possible symptomatic nephrolithiasis, although
US is generally utilized for surveillance to
23 Diagnostic Imaging of the Child with Suspected Renal Disease
Fig. 4 CT of a normal 11-year-old boy. (a) Axial image through the mid-pole of both kidneys. (b) Coronal image. (c)
Sagittal image
minimize radiation exposure. CT is used for renal
tumor staging and is the optimal modality when
trauma of the UT is suspected. CT provides excel-
lent resolution of renal scarring, but 99mTc-DMSA
is more commonly used. CT angiography is useful
in assessing for renal artery stenosis [62, 63].
Diminishing radiation exposure by using age-/
weight-based exposure parameters and by limit-
ing the field of view is suggested. Efforts should
be made to achieve the lowest radiation dose
needed in order to generate a diagnostic exam
[64, 65].
Nuclear Medicine
A variety of radiotracers are utilized for imaging
of the UT [66, 67]. The radiotracers emit photons,
which are detected by a scintillation detector.
Nuclear medicine imaging does not provide ana-
tomic detail; it provides functional data that is
673
typically utilized to answer a specific question.
Nuclear medicine is often a complementary tech-
nique. While there is radiation exposure, it is less
than other modalities such as VCUG, IVP, or CT.
Technique
Dynamic Renal Scintigraphy
Technetium 99m-diethylene triamine pentaacetic
acid (99mTc-DTPA) and technetium
99m-mercaptoacetyltriglycine (99mTc-MAG3)
are the radiotracers currently utilized for dynamic
renal scintigraphy. 99mTc-DTPA is primarily
excreted via glomerular filtration; it is not
reabsorbed or secreted by the tubules; its clear-
ance is slightly lower than inulin. 99mTc-MAG3 is
predominantly excreted via tubular secretion. For
most indications, 99mTc-MAG3 is superior to
99m
Tc-DTPA because 99mTc-MAG3 is more rap-
idly excreted and thus provides a superior renal/
background ratio. The nuclear radiotracers have a
674
Fig. 5 Posterior image of a normal 99mTc-MAG3 renal scan in an 8-year-old with hydronephrosis by ultrasound
lower allergic reaction profile than radiographic
contrast media [68].
Patients are encouraged to consume liquids
immediately prior to the procedure as this facili-
tates exam interpretation, but also decreases the
radiation dose to the urinary bladder [68]. Patients
should also ideally void before injection of iso-
tope. The uncooperative child should be
immobilized, but sedation is rarely necessary.
Images are obtained with the patient supine; the
scintillation detector is below the patient for
native kidneys and above the patient for renal
transplants. After bolus injection of radiotracer,
dynamic images are recorded for 30 min. The
initial images assess renal perfusion, and later
images evaluate excretion. Differential renal func-
tion is calculated using the early images, typically
during the second minute, prior to radiotracer
appearance in the collecting system.
Diuresis scintigraphy is performed to evaluate
a patient for renal obstruction [69, 70]; use of a
diuretic is only necessary if radiotracer does not
rapidly drain from the renal pelvis during initial
imaging. A dilated renal pelvis that is not
obstructed may have slow drainage of radiotracer
due to the increased pelvic volume. Administra-
tion of diuretic allows differentiation of an
obstructed kidney from a non-obstructed dilated
renal pelvis. Furosemide is given 15–20 min after
administration of radiotracer.
J. Loewen and L.A. Greenbaum
Following administration of a diuretic, radio-
tracer drains rapidly from a non-obstructed renal
pelvis (Figs. 5 and 6); there is limited washout of
radiotracer if obstruction is present. A half-life of
radiotracer disappearance greater than 20 min
indicates obstruction [71]. There is a good corre-
lation between the test results and histological
changes [72], and the test is sensitive and specific
[71, 73]. Diuresis scintigraphy is accurate in new-
borns, despite their lower glomerular filtration rate
[73, 74], although follow-up imaging of normal
results is necessary to ensure that obstruction does
not subsequently develop [75]. Potential causes of
false-positive results are a massively dilated renal
pelvis or a kidney with very poor function [76],
but technical modifications may allow for more
accurate assessment of a poorly functioning kid-
ney [77–79]. False-positive results can also be
decreased by obtaining images after the patient
voids to avoid bladder pressure effects and after
placing the child in the erect position to maximize
gravitational drainage [80–82].
Cortical Scintigraphy
99m
Tc-DMSA is the preferred radiotracer for
imaging of the renal cortex [83, 84]. 99mTc-
DMSA is taken up by the cells of the proximal
tubule, where it binds to sulfhydryl groups
(Fig. 7). The accumulation of 99mTc-DMSA in
the kidney is relatively slow; hence, imaging is
23 Diagnostic Imaging of the Child with Suspected Renal Disease 675

Fig. 6 Time-activity curve

from 99mTc-MAG3 renal
scan showing symmetric
prompt excretion of
radiotracer, indicating no
obstruction

Fig. 7 (a) Posterior image and coned oblique images of normal kidneys on DSMA scan in a 3-year-old with a urinary
tract infection. (b) Regions of interest drawn for split-function calculation

typically performed 1.5–3 h after injection,
although diagnostic accuracy may improve with
an increased delay in imaging if renal function is
significantly impaired. Posterior and left and right
posterior oblique views should be obtained
[83, 84], but there is minimal loss in diagnostic
accuracy if only a posterior view is possible in a
restless child [85]. Anterior imaging is preferred
for pelvic or horseshoe kidneys. Additional pin-
hole images are useful for improving resolution
when imaging infants.
A variety of factors may complicate interpreta-
tion of 99mTc-DMSA imaging. Fetal lobulations
may give the false impression that there are areas
of decreased radiotracer uptake. The splenic
impression explains flattening of the superolateral
surface of the left kidney [84]. The poles of nor-
mal kidneys may have relatively decreased uptake
of radiotracer. Nevertheless, there is good
interobserver agreement in the interpretation of
99m
Tc-DMSA scans for scarring and acute pyelo-
nephritis [86, 87].
99m
Tc-DMSA is not specific for scars or acute
pyelonephritis. Alternative explanations for
defects such as cysts, hydronephrosis, infarcts,
masses, or a dysplastic half of a duplex kidney
can only be identified using alternative imaging
strategies (e.g., US). Children with proximal
tubulopathies have decreased uptake of 99mTc-
DMSA and increased urinary excretion [88].
676
Single-photon emission computed tomogra-
phy (SPECT) is an alternative to planar scintigra-
phy. SPECT appears to offer superior sensitivity
for detecting renal scars [89, 90], although with a
higher rate of false-positive results [91, 92]. The
clinical relevance of the increased detection of
small scars via SPECT is not clear. An
intrarenicular septum, a common normal variant,
may lead to a photopenic area with SPECT, but
can be differentiated from a scar because of pre-
served cortical uptake [93]. The increased imag-
ing time required with SPECT may necessitate
sedation in young children.
Split function is determined by outlining the
appropriate regions of the kidneys and subtracting
the activity of an appropriate background area in
the posterior image. Normal split function is 50 %
+/ 6 %. Estimates of split function must be
corrected for attenuation if there is an anteriorly
placed ectopic kidney. Such correction is not
accurate with a pelvic kidney due to the effect of
the pelvic bone. Imaging may need to be delayed
for up to 24 h in an obstructed kidney because
isotope accumulation in the renal pelvis may
cause an error in estimating split function, with
an overestimation of the function of the obstructed
kidney if the image is obtained prematurely. A
similar approach may be necessary for a
non-obstructed, severely dilated renal pelvis,
although injection of furosemide, with the subse-
quent diuresis clearing radiotracer from the
dilated pelvis, is an alternative strategy. Estimates
of split function are reliable, with good
interobserver agreement, except in cases where
renal function is significantly impaired [94].
Adrenal Gland Scintigraphy
Metaiodobenzylguanidine (MIBG), when labeled
with either 131I or 123I, is used for the detection of
catecholamine-secreting tumors, including pheo-
chromocytomas and neuroblastomas [95–97].
MIBG, a guanethidine analog that structurally
resembles norepinephrine, is taken up by sympa-
thetic nerve cells. The patient should receive oral
nonradioactive iodine before and after administra-
tion of radiolabeled MIBG to compete with free
radioactive iodine, minimizing radioactive expo-
sure to the thyroid gland. A variety of medications
J. Loewen and L.A. Greenbaum
Table 3 Drugs that interfere with uptake of MIBG
Propranolol
Labetalol
Calcium channel blockers
Phenylephrine
Pseudoephedrine
Tricyclic antidepressants
Phenothiazines
Monoamine oxidase inhibitors
interfere with MIBG uptake into tissue and should
be discontinued prior to MIBG imaging (Table 3).
MIBG radiotracer is injected, with images
obtained at various times depending on the iso-
tope. MIBG accumulates in a variety of organs,
including the liver, heart, and salivary glands;
isotope is detected in the kidney and bladder due
to renal excretion. Tumors can be identified
because of the stronger radiotracer uptake, which
is typically focal. 123I has a much lower radiation
burden than 131I and results in superior
images [98].
Radionuclide Cystography
Radionuclide cystography (RNC) is an alternative
to VCUG for the detection of VUR (Fig. 8;
[99–101]). In RNC, a catheter is placed into the
bladder, and a solution containing a radioisotope
is infused, with complete filling of the bladder.
RNC, because it allows for continuous monitor-
ing, is more sensitive at detecting VUR than
VCUG, which cannot utilize continuous monitor-
ing due to radiation concerns. Furthermore, the
radiation burden is considerably less than VCUG.
However, radionuclide cystography does not per-
mit evaluation of the bladder or the urethra.
Applications
99m
Tc-DMSA is the preferred radiopharmaceuti-
cal for evaluation of the renal cortex and the gold
standard for the identification of pyelonephritis
and renal scars [102]. 99mTc-DMSA can be uti-
lized to determine split renal function and to iden-
tify and characterize renal infarcts or anatomically
abnormal kidneys such as horseshoe kidneys, pel-
vic kidneys, and crossed fused ectopia [103, 104].
Dynamic renal imaging with 99mTc-MAG3 or
99m
Tc-DTPA permits evaluation of renal blood
23 Diagnostic Imaging of the Child with Suspected Renal Disease
flow, split renal function, kidney location, and
urinary drainage. Dynamic renal imaging allows
determination of whether hydronephrosis is sec-
ondary to renal obstruction or an alternative
Fig. 8 Radionuclide cystogram. Posterior image from a
nuclear cystogram in a 4-year-old demonstrating left
vesicoureteral reflux
Fig. 9 Renal MRI of a normal 5-year-old. (a) Axial T2-weighted image through the mid-pole of the kidneys. (b) Coronal
T2-weighted image
677
etiology such as VUR, high urine flow, or relieved
obstruction [105]. Dynamic renal imaging can be
utilized to determine split renal function and to
identify renal artery stenosis when used with
captopril [106].
Magnetic Resonance Imaging
MRI, with its ability to image the abdomen in
multiple planes and with sequences showing dif-
ferent signal characteristics, provides excellent
resolution of the UT without radiation exposure
(Figs. 9 and 10) [107]. Faster MR sequences
allowing for rapid image acquisition and the con-
tinued trend in reducing radiation exposure in the
pediatric population have also increased imaging
of the urinary tract by MRI [1].
Gadolinium-based contrast at standard doses is
significantly less nephrotoxic than iodinated con-
trast [108, 109], but gadolinium-based contrast is
nephrotoxic when used at radiographic doses for
angiographic procedures [110–112]. Gadolinium-
based contrast is the dominant risk factor for the
development of nephrogenic systemic fibrosis
(NSF) [113], an entity that occurs almost exclu-
sively in patients with end-stage renal disease or
severe renal impairment. Most cases of NSF have
been associated with gadodiamide, although other
gadolinium-based contrast agents may lead to
NSF [114]. Gadolinium-based contrast should be
678
Fig. 10 Renal MRI. 3D image of the kidneys and ureters
in a normal 12-year-old
used cautiously in patients with moderate to
severe renal impairment [115].
Sedation is typically necessary for infants and
young children. MRI is the most expensive imag-
ing modality, but it may replace multiple other
studies and provide superior resolution in certain
clinical situations.
Technique
Magnetic resonance urography (MRU) provides
superb anatomic detail combined with functional
information [116]. In this technique, a gadolinium
chelate (Gd-DTPA) is injected, and the kidneys
are imaged over time. In addition to anatomic
assessment of renal parenchyma and collecting
system anatomy, post-processing of MRU data
derived from the study allows for determination
of split differential renal function, transit time of
the contrast agent through the kidney, and an
estimate of glomerular filtration rate. MRU has
J. Loewen and L.A. Greenbaum
superior contrast and temporal and spatial resolu-
tion when compared to renal scintigraphy [1].
Application
Conventional MRI is useful in characterization of
solid and cystic renal masses, renal vascular dis-
ease, and congenital abnormalities including
lower urogenital tract malformations and complex
post-traumatic conditions [64, 117, 118]. The
most common indication for MRU is
hydronephrosis. This technique allows for simul-
taneous anatomic and physiologic assessment of
the parenchyma, collecting system, and surround-
ing tissues [1, 119]. The goal of the exam is to
determine whether obstruction of a
hydronephrotic system is present based on its
response to a fluid/diuretic challenge [119, 120].
Congenital Kidney and Urological
Disorders
Fetal Imaging
The widespread use of fetal US has led to a dra-
matic increase in postnatal imaging of the
UT. Most congenital abnormalities of the UT are
now detected prenatally, and this has led to a
decrease in the diagnosis of obstructive uropathies
as a consequence of urinary tract infection (UTI),
potentially diminishing the role of renal US in
children with UTI [121, 122].
Fetal kidney length relates to gestational age,
and normal values are available (Table 4; [3]).
Fetal US is excellent at identifying
hydronephrosis, and this may identify infants
with UT obstruction or VUR. Oligohydramnios,
which may indicate poor urine output due to renal
insufficiency, is easily quantified and followed.
Persistent oligohydramnios is associated with pul-
monary hypoplasia. The fetal bladder, which may
be distended with impaired bladder emptying, and
the echogenicity of the renal parenchyma are also
visualized via fetal US.
Hydronephrosis is the most common renal
abnormality on fetal US, and ureteropelvic junc-
tion (UPJ) obstruction is the most common path-
ologic condition, although a high percentage of
23 Diagnostic Imaging of the Child with Suspected Renal Disease 679

Table 4 Mean renal lengths for various gestational ages

Gestational age Mean 95 %
(Weeks) length (cm) SD CI n
18 2.2 0.3 1.6–2.8 14
19 2.3 0.4 1.5–3.1 23
20 2.6 0.4 1.8–3.4 22
21 2.7 0.3 2.1–3.2 20
22 2.7 0.3 2.0–3.4 18
23 3.0 0.4 2.2–3.7 13
24 3.1 0.6 1.9–4.4 13
25 3.3 0.4 2.5–4.2 9
26 3.4 0.4 2.4–4.4 9
27 3.5 0.4 2.7–4.4 15
28 3.4 0.4 2.6–4.2 19
29 3.6 0.7 2.3–4.8 12
30 3.8 0.4 2.9–4.6 24
31 3.7 0.5 2.8–4.6 23
32 4.1 0.5 3.1–5.1 23 Fig. 11 Sagittal T2-weighted fetal MR image of a
33 4.0 0.3 3.3–4.7 28 28-week fetus with dilated bladder and posterior urethra
34 4.2 0.4 3.3–5.0 36 (arrow) in the setting of posterior urethral valves
35 4.2 0.5 3.2–5.2 17
36 4.2 0.4 3.3–5.0 36 evaluation [125]. The severity of hydronephrosis
37 4.2 0.4 3.3–5.1 40 is determined by ultrasonography after 48 h of life
38 4.4 0.6 3.2–5.6 32 by the measurement of the anterior posterior
39 4.2 0.3 3.5–4.8 17 diameter of the pelvis. A diameter less than
40 4.3 0.5 3.2–5.3 10 7 mm is considered as normal, 7–8 mm is a mild
41 4.5 0.3 3.9–5.1 4 hydronephrosis, 9–16 mm is a moderate
Gestational age is an average of the gestational ages in hydronephrosis, and more than 16 mm is a severe
weeks determined on the basis of biparietal diameter, fem-
oral length, and abdominal circumference. SD standard
hydronephrosis which requires a voiding
deviation; 95 % confidence interval; n number of fetuses. cystourethrogram and a functional renal scan
A t distribution was used when n < 30 (From Cohen diuretic renography [126].
et al. [3]) Prenatal US can detect children with a variety
of other pathologic conditions, including
posterior urethral valves, multicystic dysplastic
kidneys are ultimately diagnosed as kidney, VUR, megaureter, duplex anomalies
normal [123]. There is a high rate of false- with upper pole hydronephrosis, single kidney,
negative results when a postnatal US to follow- polycystic kidney disease, and Eagle-Barrett
up on prenatal hydronephrosis is done in the first syndrome [127].
few days of life, presumably because of low urine While fetal US remains the primary means of
output due to physiologic dehydration in utero imaging, fetal MRI has a role in further
[124]. Hence, the initial postnatal US should characterization of complex fetal genitourinary
either be done at least 4 weeks after birth or the abnormalities. The pregnant patient is most fre-
child should have a second US. Deferral of the quently scanned in the supine position during the
initial US is inappropriate if the prenatal US second or third trimester. Intravenous contrast
suggests the presence of disease that would material is not used for the exam. The most com-
require immediate intervention (e.g., bilateral monly encountered GU abnormalities on fetal
hydronephrosis or oligohydramnios). The degree MRI include both upper urinary tract obstruction
of prenatal hydronephrosis is highly predictive of (UPJ stenosis) and lower urinary tract obstruction
the likelihood of pathology on postnatal such as posterior urethral valves (Fig. 11).
680
Fig. 12 Ectopic ureterocele with vesicoureteral reflux. (a)
Voiding cystourethrogram shows rounded filling defect in
bladder base from ureterocele. (b) Voiding cystoure-
throgram shows reflux into the lower pole moiety of the
More complex abnormalities such as cloacal
malformations and bladder exstrophy can also be
imaged [128].
Megaureter
Megaureter may be primary or secondary to VUR
or obstruction. VCUG permits identification of
VUR, and diuretic scintigraphy establishes the
presence of obstruction. MRU provides excellent
anatomic detail, often allowing identification of
the site of obstruction [129].
Ureterocele
Ureteroceles may be demonstrated by US,
VCUG, IVP, or MRU (Fig. 12). An everting
ureterocele may be confused with a bladder diver-
ticulum on VCUG [130]. Ureteroceles may
obstruct the ureter during voiding.
Ureteropelvic Junction Obstruction
UPJ obstruction is one of the most common con-
genital kidney disorders, and most cases are now
diagnosed by fetal US. Dynamic renal scintigra-
phy remains the gold standard imaging technique
for diagnosing UPJ obstruction (Fig. 13). MRI is
an alternative approach that provides functional
J. Loewen and L.A. Greenbaum
left duplex system with eversion of the ureterocele
(asteric). Opacification of the kidney indicates intrarenal
reflux
information and superior anatomic detail without
exposure to radiation, albeit with the need for
sedation (Fig. 14; [131, 132]).
A second alternative technique for diagnosing
obstruction is the measurement of the resistive
index (RI) by Doppler US, with an elevated RI
suggesting obstruction. The sensitivity and spec-
ificity of this approach is improved by adminis-
tering normal saline and furosemide prior to US
[133]. The principal advantage is the elimination
of radiation exposure, but this approach is not yet
widely utilized.
Abnormal Kidney Number or Location
Unilateral renal agenesis must be distinguished
from an ectopic kidney, which can be located
anywhere along the course of renal ascent. Such
kidneys are often smaller in size. Detection by US
is possible, but MRI, IVP, or DMSA is often
necessary (Fig. 15). IVP may have limited sensi-
tivity if the ectopic kidney has poor renal function.
Horseshoe Kidney and Crossed Fused
Ectopia
Horseshoe kidney, with an incidence of 1 of
400 births, is the most common fusion anomaly.
23 Diagnostic Imaging of the Child with Suspected Renal Disease 681

99m
Fig. 13 Left ureteropelvic junction obstruction. (a) US Tc-MAG3 scan shows a normal right kidney. On the
image of the left kidney shows dilatation of the renal pelvis left, little drainage occurs in response to furosemide. T ½
and calices. (b) 99mTc-MAG3 renal scan shows retention of exceeds 20 min
radiotracer in the left kidney. (c) Time-activity curve from
682
Fig. 14 Left ureteropelvic junction obstruction. MR
urogram in patient with UPJ obstruction shows dilatation
of the left renal pelvis and calices
Fig. 15 Crossed fused ectopia. DMSA renal scan anterior
image demonstrates a single fused renal moiety on the right
J. Loewen and L.A. Greenbaum
Crossed ectopia occurs in an estimated 1 of
1,000–2,000 births. While US can be diagnostic,
additional imaging, including IVP, MRI, CT, or
DMSA, is often necessary to establish the diag-
nosis (Fig. 16).
Renal Dysplasia
Renal dysplasia is a histological diagnosis char-
acterized by primitive ducts and cartilage. US
evaluation demonstrates increased echogenicity;
most dysplastic kidneys are decreased in size. US
may detect cysts, which may vary in size and
number.
The incidence of multicystic dysplastic kidney
(MCDK) is estimated at 1 in 2,500 newborn
[134]; it is one of the common prenatally detected
fetal anomalies. Postnatal US shows cysts of vary-
ing size that do not appear to communicate with
each other or the collecting system and a small
amount of abnormal-appearing renal parenchyma.
It may be difficult to differentiate MCDK from
severe hydronephrosis [135]. In MCDK, there is
typically no communication between cysts, and
the larger cysts are not medial (Fig. 17). In
hydronephrosis, the calices extend outward from
the dilated renal pelvis, and there is functional
renal parenchyma surrounding the central cystic
structure. If the diagnosis is unclear, a DMSA
shows uptake of tracer if hydronephrosis is pre-
sent, but usually no uptake with MCDK (Fig. 18).
Alternatively, MRI can distinguish these entities.
Duplex Collecting System
A duplex collecting system is a common inciden-
tal finding and usually of no clinical significance
unless hydronephrosis is also present. Associated
anomalies include ureterocele, VUR, ectopic ure-
ters, renal dysplasia, and UPJ obstruction
(Figs. 19 and 20; [136]). The upper pole is more
likely to be associated with obstruction at the
distal ureter, while the lower pole is associated
with VUR or UPJ obstruction. US can usually
demonstrate division of the kidney, but is not
reliable in distinguishing between bifid ureter
and complete ureteral duplication. IVP is being
supplanted by MRU for delineating the anatomy
of the ureteral duplication when such information
is necessary for planning surgery [129]. MRU is
23 Diagnostic Imaging of the Child with Suspected Renal Disease 683

Fig. 16 Horseshoe kidney.

CT scan of abdomen shows
a horseshoe kidney with a
bridge of renal tissue fused
across the midline
(arrowhead)

Fig. 17 Left multicystic

dysplastic kidney. Renal US
in an infant shows multiple
noncommunication cysts in
the left kidney

especially useful for characterization of the distal
ureter [137]. VCUG may demonstrate VUR or
ureterocele.
Ectopic Ureters
Ectopic ureters are three times more common in
girls, and approximately 80 % are associated with
a duplicated collecting system and ectopic inser-
tion of the upper pole ureter. Greater distance from
the normal ureter insertion site is associated with
more severe ipsilateral renal abnormalities
[138]. US can demonstrate duplication,
hydronephrosis, megaureter, and abnormal renal
parenchyma. IVP is useful for delineating the
course of the ectopic ureter, although lack of con-
trast may limit the study if the associated renal
unit is poorly functioning. MRU may allow detec-
tion of the ectopic ureter if other studies are unsuc-
cessful [137] and has largely replaced IVP in the
evaluation of ectopic ureteral insertion. VCUG is
useful for showing reflux and may indicate the
insertion point of the ureter if it is proximal to
the external sphincter. DMSA is useful for identi-
fying an ectopic kidney that is the source of an
ectopic ureter in a patient with unexplained incon-
tinence [103, 104]. By indicating relative renal
function, DMSA may also help with surgical
planning.
Bladder Abnormalities
US and VCUG are the standard imaging modalities
for patients with a neurogenic bladder (Fig. 21).
684 J. Loewen and L.A. Greenbaum

VCUG readily identifies bladder diverticula, which Urethral Abnormalities

are easily missed by US. Bladder wall thickening,
consistent with high bladder pressures, can be
quantified by US.
VCUG is the definitive test for diagnosing
posterior urethral valves (Fig. 22). The prostatic
urethra is dilated, the membranous urethra is
stenotic, and the distal urethra appears
normal. Secondary VUR, hydronephrosis,
and bladder abnormalities are often
present (Fig. 23). Other urethral abnormalities
(anterior urethral valves, urethral duplication,
Cowper’s duct cyst, urethral diverticulum,
megalourethra) are also best visualized via
VCUG.

Fig. 18 Left multicystic dysplastic kidney. DMSA renal
scan (posterior image) shows no uptake in the left
multicystic dysplastic kidney
Vesicoureteral Reflux
VCUG is the gold standard for diagnosing VUR,
with VUR graded based on the International
Reflux Study in Children (Fig. 24; [47]). RNC
and VUS, because of decreased or no
radiation burden, respectively, are appropriate
substitutes for VCUG in clinical situations
where the purpose of the study is solely to assess
the patient for VUR. Examples include follow-up
studies in children with previously diagnosed
VUR, screening siblings of children with VUR,
and potentially for the initial evaluation of
girls with urinary tract infections. Renal US
lacks adequate sensitivity and specificity for diag-
nosing VUR [139–142], although high-grade
VUR is more likely if hydronephrosis is
present [143].

Fig. 19 Duplex system

with ectopic ureterocele.
Renal US shows mild
hydronephrosis
(arrowheads) of the upper
and lower poles. A band of
renal tissue extends across
the mid-pole, indicating a
duplex system
23 Diagnostic Imaging of the Child with Suspected Renal Disease
Fig. 20 Duplex system with ectopic ureterocele. MRU
shows hydronephrosis of the right upper pole
Hereditary Disorders
Cystic Kidney Diseases
In autosomal recessive polycystic kidney disease
(ARPKD), intravenous pyelogram (IVP) shows
enlarged kidneys with a delayed nephrogram
[144]. Radial streaks due to contrast in the dilated
collecting ducts are usually present in infancy, but
may not be visible in older children. Because of
concerns regarding intravenous contrast, a renal
US is now the usual initial diagnostic test
(Fig. 25). US shows enlarged kidneys with
increased echogenicity and poor corticomedullary
differentiation; a hypoechoic rim is often visible
[145]. Older children have increased medullary
echogenicity, which may resemble
nephrocalcinosis [146]. Multiple small cysts may
be visible by US [146, 147], but this is quite
variable. Macrocysts are sometimes visible in
older children [147].
In autosomal dominant polycystic kidney dis-
ease (ADPKD), US, CT, and MRI are useful for
685
Fig. 21 Neurogenic bladder in a 16-year-old with a spinal
cord injury. Voiding cystourethrogram shows pear-shaped
bladder with trabeculation
detecting macroscopic cysts and associated com-
plications (Figs. 26 and 27). The number and size
of cysts in children increases with age, and those
children with more cysts have increased kidney
size [148]. ADPKD rarely causes renal failure in
childhood [32, 149]. In adults, kidney size mea-
sured by MRI predicts decline in renal function
[150]. Increased renal echogenicity may be seen
in some children [147]. Children occasionally
have marked disease asymmetry, which can create
diagnostic confusion [151]. Both ARPKD and
ADPKD can have associated liver abnormalities
[152]. Extrarenal complications of ADPKD
include liver cyst infection/hemorrhage, intracra-
nial aneurysms, and valvular heart disease [153].
US in nephronophthisis shows hyperechoic
kidneys with loss of corticomedullary differentia-
tion; they are of normal or slightly decreased size.
Medullary cysts are a hallmark of the disease, but
686
they are not always detected by US. CT is a more
sensitive method for identifying medullary cysts
and thin-section CT is recommended [154].
The diagnosis of medullary sponge kidney is
based on characteristic IVP changes: stagnation of
contrast in one or more renal papillae due to
dilation of the collecting ducts. The resultant
image has been described as a “pyramidal
blush.” In medullary cystic disease, the kidneys
are usually normal or small, and US or CT may
show a few medullary cysts [155].
Fig. 22 Voiding cystourethrogram shows dilatation of the
posterior urethra (asteric) in a boy with posterior urethral
valves
Fig. 23 Renal US shows
dilatation of the right renal
pelvis, calices, and ureter in
a boy with posterior urethral
valves
J. Loewen and L.A. Greenbaum
Tuberous Sclerosis
Angiomyolipomas are the most common renal
lesion in tuberous sclerosis (TS) and are readily
seen by CT or ultrasound (Figs. 28 and 29). Chil-
dren with disruption of the contiguous PKD1 and
TSC2 genes usually have severe cystic disease and
are at high risk for hypertension and early kidney
failure [156]. Renal malignancies, including renal
cell carcinoma and malignant angiomyolipomas,
are a serious concern in TS, and both are seen
in children, sometimes in early childhood
[157, 158]. All children with TS should have a
renal ultrasound at diagnosis and follow-up renal
ultrasounds every 1–3 years, with frequency dic-
tated by the specific clinical situation
[159]. Patients with extensive or rapidly changing
lesions require more frequent follow-up. Those
with more severe kidney disease may require CT
or MRI to screen for malignant changes. Differ-
entiating angiomyolipomas from malignancy
requires careful comparison of sequential images.
Infections of the Urinary Tract
Clinical and laboratory criteria do not reliably
differentiate pyelonephritis from cystitis
[160–162]. DMSA is the gold standard for diag-
nosing acute pyelonephritis, although MRI and
CT provide similar sensitivity [163]. US may
detect loss of corticomedullary differentiation
and renal enlargement, but lacks adequate
23 Diagnostic Imaging of the Child with Suspected Renal Disease 687

Fig. 24 Voiding
cystourethrogram shows
bilateral grade
4 vesicoureteral reflux

Fig. 25 Autosomal
recessive polycystic kidney
disease in an infant. (a)
Longitudinal view shows
diffuse enlargement of the
right kidney, which is in
increased in echogenicity.
(b) Transverse image
through the mid-pole of
both kidneys

sensitivity and specificity when compared to
DMSA [164–167].
The decreased uptake using DMSA with
pyelonephritis may be focal, multifocal, or dif-
fuse. Acute pyelonephritis must be differentiated
from renal scarring, which is associated with
sharper borders and loss of cortical volume.
DMSA may allow diagnosis of pyelonephritis in
children who have received antibiotics prior to
urine culture or who have a negative culture
688 J. Loewen and L.A. Greenbaum

Fig. 26 Autosomal
dominant polycystic kidney
disease. Renal US,
longitudinal image, and
bilateral renal cysts of
varying sizes in a 15-year-
old

Fig. 27 CT in a 10-year-
old with autosomal
dominant polycystic kidney
disease and bilateral cysts of
varying sizes. Left kidney
complicated by cyst
hemorrhage and perinephric
hematoma (arrows)

Fig. 28 Angiomyolipomas
in tuberous sclerosis. Renal
US, longitudinal image,
demonstrates multiple small
echogenic lesions
throughout the kidney
23 Diagnostic Imaging of the Child with Suspected Renal Disease 689

Fig. 29 Angiomyolipomas
in tuberous sclerosis. Renal
CT, axial image, with
multiple fat-containing
lesions in the kidneys

Fig. 30 Renal scarring in a

10-year-old with
vesicoureteral reflux.
DMSA renal scan shows a
small right kidney with
diffuse renal scarring.
Uptake in the right kidney is
20 %

despite clinical and laboratory evidence 6 months after an acute infection to

suggesting pyelonephritis [168].
DMSA is the current standard modality for
diagnosing renal scarring (Fig. 30; [169]), with
increased sensitivity when compared to US or IVP
[170]. Scars appear as photopenic areas with vol-
ume loss. DMSA should be deferred at least
differentiate a transient lesion due to infection
from a scar [101, 170, 171]. MRI has the potential
to differentiate acute pyelonephritis from renal
scarring, perhaps avoiding the need for radiation
exposure and obviating the need for two diagnos-
tic tests [172].
690
Fig. 31 Acute
glomerulonephritis in a
3-year-old. Renal US shows
the large echogenic right
kidney
All boys and young girls with a first UTI are
conventionally evaluated with a renal US and
VCUG [173]. The necessity of this approach has
been questioned, especially given the lack of evi-
dence that prophylactic antibiotics are effective in
preventing UTIs or renal damage [174]. Moreover,
because of the widespread use of prenatal US in
many countries, the diagnostic yield of US exam-
ination in children with UTI is low [175]. In girls,
the radiation burden of the initial imaging can be
reduced by using RNC instead of VCUG.
An alternative strategy is to perform DMSA scin-
tigraphy as an initial test in children with UTI and
only perform VCUG or RNC in children with an
abnormal DMSA [176].
Renal Failure
US is the initial imaging modality when a patient
presents with unexplained renal failure. Imaging
must include assessment of renal size,
echogenicity, morphology, cystic disease, and
hydronephrosis [32, 177]. US generally allows
differentiation of chronic renal failure (small,
echogenic kidneys with a thin cortex) from acute
renal failure. The kidneys may be large if chronic
renal failure is secondary to polycystic kidney
disease. US readily detects hydronephrosis,
which suggests that renal failure is due to an
obstructive etiology. Hydronephrosis may also
be secondary to VUR, papillary necrosis, or high
urine output; such hydronephrosis is rarely severe.
J. Loewen and L.A. Greenbaum
Renal parenchymal disease is often associated
with increased echogenicity (Fig. 31;
[178–180]), although a normal US examination
does not exclude this possibility. The kidneys
generally have a normal appearance in prerenal
azotemia. The kidneys may be normal or have
increased echogenicity in acute tubular necrosis.
Echogenic kidneys, which may be moderately
enlarged, are often seen in glomerular disease
[179, 181]. The kidneys are enlarged and very
echogenic in HIV nephropathy [182]. The US
findings in renal parenchymal disease are rarely
specific [180, 181]. US is commonly used for
pre-biopsy localization [32].
Renal Transplant
US is the primary imaging modality for evaluation
of the renal transplant [183, 184]. US can identify
posttransplant complications, including abnormal
fluid collections (e.g., lymphoceles, urinomas)
(Fig. 32), obstruction, and mass lesions
[32, 185]. Doppler US can detect vascular disease
after kidney transplant (renal artery thrombosis,
renal artery stenosis, renal vein thrombosis, arte-
riovenous fistula) [27, 184, 186, 187]. There are a
number of potential US abnormalities seen in
patients with acute graft rejection (increased
renal size and echogenicity, decreased or absent
central sinus echoes) [188], but US does not have
adequate sensitivity or specificity; kidney biopsy
remains necessary for the diagnosis of rejection.
23 Diagnostic Imaging of the Child with Suspected Renal Disease
Fig. 32 Lymphocele in a
15-year-old with renal
transplant. Renal US shows
a complex, loculated fluid
mass adjacent (L ) to the
transplanted kidney (T )
Acute rejection, vascular thrombosis, and acute
tubular necrosis (ATN) are the most common
complications in the immediate postoperative
period. Long-term complications include arterial
stenosis, ureteral stenosis, chronic rejection, and
posttransplant lymphoproliferative disorder
[32, 189].
Radionuclide imaging (99mTc-DTPA or 99mTc-
MAG3) is useful when US does not provide an
explanation for graft dysfunction. Absence of
flow to the kidney occurs with arterial or venous
obstruction or with hyperacute rejection. In con-
trast, a graft with ATN has normal renal perfusion,
but delayed or no excretion. Radionuclide imag-
ing is useful for demonstrating urinary obstruction
or that a perinephric fluid collection is due to a
urine leak [190–192].
Urolithiasis and Nephrocalcinosis
Urolithiasis
US is a sensitive test for detecting calculi in the
kidney and the proximal and distal ureter. US does
not easily detect stones in the middle portion of
the ureter, although hydronephrosis due to
obstruction may suggest the diagnosis. Acoustic
shadowing is a useful finding, but may not be
present with smaller calculi. Twinkle artifact,
691
likely secondary to the US beam interacting with
stone, can also assist in calculus detection
[32]. US is less sensitive than CT in detecting
calculi in symptomatic children (Fig. 33; [193]).
CT is also superior to US for identification of
bladder stones, especially if the bladder is
augmented.
Nephrocalcinosis
Nephrocalcinosis is typically detected by US
(Fig. 34), although it may be seen via CT. Plain
x-ray only detects nephrocalcinosis in severe
cases [194]. Causes of cortical nephrocalcinosis
include primary hyperoxaluria [195] and cortical
necrosis, while there are many entities that may
cause medullary nephrocalcinosis [194, 196].
Trauma to the Urinary Tract
CT with contrast is the primary modality for eval-
uating a patient with suspected trauma to the kid-
ney (Fig. 35; [197, 198]). CT angiography permits
detailed evaluation of the renal artery and vein.
US is an alternative imaging modality that may
identify large lacerations, hematomas, and
urinomas; US with Doppler readily identifies
significant injuries of the renal artery or vein.
692 J. Loewen and L.A. Greenbaum

Fig. 33 Renal calculus in the mid-pole of the left kidney. (a) Coronal image. (b) Axial image

Fig. 34 Medullary
nephrocalcinosis in a
12-year-old with renal
tubular acidosis. Renal US,
longitudinal image, shows
echogenic renal pyramids

US suggests bladder laceration when there is a contrast allows for identification of tumor calcifi-
large amount of fluid in the cul-de-sac. When cations; contrast allows for better differentiation
indicated, VCUG can potentially identify the site of tumor from normal tissue. US is sensitive for
of bladder leak and detect urethral injury. the detection of bladder tumors in children [199].

Renal and Urinary Tract Tumors Imaging of Specific Renal Tumors

Role of Imaging Wilms Tumor

US and CT are complementary in the evaluation
US is especially suited for initial evaluation, while and follow-up of Wilms tumor (Figs. 36 and 37).
CT or MRI is necessary for defining tumor extent US with Doppler may provide superior imaging of
and the presence of metastases. CT without tumor in the inferior vena cava in some patients
23 Diagnostic Imaging of the Child with Suspected Renal Disease
Fig. 35 Motor vehicle collision resulting in extensive left renal laceration with a large perinephric hematoma. (a) Axial
CT. (b) Coronal CT
[200]. Radiologic staging of Wilms tumor
includes chest x-ray, US of the abdomen and
pelvis, and CT of the chest, abdomen, and pelvis.
Chest x-ray appears to be an appropriate substitute
for chest CT to identify pulmonary metastases
[201]. MRI is also an effective modality for eval-
uation of Wilms tumor [202]. US is a sensitive
approach for screening patients for tumor recur-
rence, although CT or MRI is necessary to identify
nephrogenic rests [203].
US is the preferred modality for Wilms tumor
screening, which is necessary in children with
syndromes that significantly increase the risk of
Wilms tumor (e.g., hemihypertrophy, Denys-
Drash syndrome, Beckwith-Wiedemann syn-
drome) [204]. Screening frequency and duration
varies depending on the syndrome.
Lymphoma
Children with lymphoma may have renal involve-
ment. By US, renal lymphoma has decreased
693
echogenicity [205]. CT may demonstrate “con-
trast inversion” of the lesions in renal lymphoma:
increased density compared to normal paren-
chyma without contrast and decreased density
with contrast [205].
Vascular Disease and Hypertension
Renal Artery Stenosis
Doppler US lacks adequate sensitivity for diag-
nosing renal artery stenosis in children [206–208],
although it may be a useful screening test in adults
[209]. US may demonstrate a small affected
kidney if the stenosis is severe and long-standing;
the contralateral kidney may have increased
echogenicity [210]. Renal scintigraphy before
and after captopril has good sensitivity in identi-
fying renovascular disease in children [106]. CT
and MRI angiography provide excellent visuali-
zation of the extraparenchymal renal artery
(Fig. 38), the most common site of disease, but
694
Fig. 36 Bilateral Wilms tumor in a 5-year-old with a palpable abdominal mass. (a) Axial CT. (b) Coronal CT
do not visualize distal branch vessel lesions
[65]. When clinical suspicion is high, conven-
tional angiography remains necessary to evaluate
children with suspected hypertension due to renal
artery disease.
Renal Vein Thrombosis
US is utilized to diagnose renal vein thrombosis.
The appearance on US varies depending on the
timing of imaging. Within the first few days of clot
formation, imaging may demonstrate pathogno-
monic echogenic streaks due to clot in interlobular
and interlobar veins [211, 212]. Renal enlarge-
ment occurs during the first week and is associated
with an echogenic cortex and less echogenic med-
ullary pyramids. Beyond the first week, there is a
loss of corticomedullary differentiation, and
echogenic streaks are no longer visible [211]. An
additional later finding is calcification of thrombi
within renal veins, including large or small
veins [213]. Long-term imaging outcomes vary
J. Loewen and L.A. Greenbaum
from a normal-appearing kidney to a small,
echogenic kidney with little or no function. In
perinatal renal vein thrombosis, renal length is
negatively correlated with outcome [214].
US may directly demonstrate clots in the renal
vein and inferior vena cava. Doppler US may
show absent renal venous flow and pulsatility.
The arterial RI is frequently elevated. Clot retrac-
tion and formation of collaterals after a few days
may create diagnostic confusion due to a return of
some venous flow and a decrease in arterial RI. In
neonates, adrenal hemorrhage, which is readily
seen by US, frequently accompanies renal vein
thrombosis and assists in confirming the diagnosis
[215, 216]. This association is more common on
the left due to the common drainage of the renal
vein and adrenal vein.
Pheochromocytoma
Pheochromocytoma may be strongly suspected
based on clinical and laboratory evaluation, but
23 Diagnostic Imaging of the Child with Suspected Renal Disease 695

Fig. 37 Wilms tumor in a 2-year-old. (a) Coronal CT Longitudinal US image with tumor filling the dilated IVC
image shows a large right renal mass, pulmonary nodules, extending to the diaphragm (arrows) and entering the right
and tumor extension into the IVC to the diaphragm. (b) atrium (a)

Fig. 38 Renal artery

stenosis in a 13-year-old
with neurofibromatosis and
hypertension. Axial
maximal intensity CT
image shows narrowing of
both renal arteries near their
origins from the aorta

imaging is necessary to confirm the diagnosis and
allow for staging and resection, which is usually
curative. US may identify a pheochromocytoma
in or near the adrenal gland, but US lacks the
sensitivity of CT or MRI, especially for small
lesions or tumors in other locations (Fig. 39).
Hypertensive crisis may occur if ionic contrast is
utilized, but nonionic contrast appears safe at the
doses used for CT [217]. Chest CT or MRI is
indicated if an abdominal lesion is not detected
696 J. Loewen and L.A. Greenbaum

Fig. 39 Pheochromocytoma in a 14-year-old. (a) Axial CT image shows a right pelvic mass (P). (b) Sagittal T2-weighted
MR image shows a right pelvic mass extending into the adjacent neural foramen (arrow)

MIBG scintigraphy, a complementary imaging

modality, may be appropriate as an initial test, to
confirm the diagnosis in equivocal cases or to
search for disease when the CT or MRI is negative
(Fig. 40; [218, 219]). Positron emission tomogra-
phy (PET) scan is a potential alternative if all
other studies are negative and clinical suspicion
remains high [220].

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or for staging of malignant disease. Pheochromo-
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 Pediatric Renal Pathology
24
Agnes B. Fogo

Contents Light Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 706
Renal Biopsy Indications . . . . . . . . . . . . . . . . . . . . . . . . . . . 706
Hematuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 706
Proteinuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 706
Nephrotic Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 706
Acute Nephritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 707
Acute Kidney Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 707
Rapidly Progressive Glomerulonephritis . . . . . . . . . . . . 707
Chronic Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 707
Systemic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 707
Follow-Up of Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 708
Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 708
Obtaining Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 708
General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 708
Contraindications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 709
Biopsy Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 709
Aspiration Biopsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712
Complications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712
Assessment of the Renal Biopsy . . . . . . . . . . . . . . . . . . . 712
Adequacy of Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712
Allotment of Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713
Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 714
Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 715
Basic Renal Lesions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 715
Normal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 715
Overall Pattern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 717
Specific Glomerular Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 718
Glomerular Basement Membrane . . . . . . . . . . . . . . . . . . . 720
Tubules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 720
Interstitium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 721
Vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 721
Clinical Pathological Correlations . . . . . . . . . . . . . . . . 722
Diagnostic Findings in Selected Renal Diseases . . . . 722
Anti-glomerular Basement Membrane Antibody
Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 733
Prognostic Implications of Biopsy Findings . . . . . . 737
Renal Transplant Biopsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 738
New Methods for the Future . . . . . . . . . . . . . . . . . . . . . . . 741
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 742

A.B. Fogo (*)

Department of Pathology, Microbiology and Immunology,
Vanderbilt University Medical Center, Nashville, TN, USA
e-mail: agnes.fogo@vanderbilt.edu

# Springer-Verlag Berlin Heidelberg 2016 705

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_22
706
Introduction
This chapter reviews the usual circumstances in
which biopsies are obtained, methods of obtaining
the biopsy material and analyzing the tissue, and
considerations in allocation of biopsy tissue.
Lesions in the kidney are described and specific
terminology defined, and the distinct characteristic
morphologic findings in various diseases are briefly
described and illustrated. Last, experimental tech-
niques that may provide important pathogenic,
prognostic, or diagnostic information are discussed.
Renal Biopsy Indications
The indications for renal biopsy vary according to
the ethnic and age characteristics of the popula-
tion studied and the geographic location because
these factors influence the incidence of various
renal diseases. The indications discussed below
present the most common settings in children for
which renal biopsy is undertaken.
Hematuria
Isolated hematuria (i.e., without proteinuria and
with normal function of the kidney) may be due to
hypercalciuria or familial or urologic disease
[1–3]. Once these disorders are ruled out, a glo-
merular origin of persistent isolated hematuria
should be considered. Red blood cell casts or
dysmorphic red blood cells indicate likely glomer-
ular origin of hematuria. Renal biopsy may define
the underlying abnormality in these patients. The
most common findings are mesangial proliferative
disease or IgA nephropathy. Less common disor-
ders include hereditary nephritis (Alport syn-
drome) and thin basement membrane lesion. The
latter may be familial (benign familial hematuria)
or sporadic. One-quarter to nearly one-half of the
patients with isolated hematuria have normal
biopsies [1–4]. Renal biopsy may, therefore,
define the pathology and provide assurance of a
benign prognosis in some patients or diagnose a
possible hereditary disease, which may initiate
screening of other family members. Last, the
A.B. Fogo
information can be of importance in avoiding
further repeated invasive evaluation in the patient.
Proteinuria
Isolated sub-nephrotic proteinuria may be pos-
tural or due to tubulointerstitial disease. These
possibilities should be evaluated completely
before renal biopsy is considered. Any glomerular
disease may cause mild to moderate proteinuria as
the only manifestation, and biopsy may yield the
diagnosis even at an early stage.
Nephrotic Syndrome
Numerous children with nephrotic syndrome
(NS) were studied when renal biopsy first became
available. The biopsies showed so-called minimal
change disease (MCD) in the vast majority of
cases. The efficacy of corticosteroids in this setting
has obviated the need for renal biopsies in most of
these cases. Therefore, young children (i.e., age
1–7 years) with NS will typically undergo a thera-
peutic trial of corticosteroids without a biopsy.
However, in infants with NS, in older children, or
in those with evidence of nephritis (hypertension,
hematuria, low C3, or decreased renal function) or
failing corticosteroid therapy, renal biopsy is often
performed. In these patients, disease other than
MCD (e.g., focal segmental glomerulosclerosis
[FSGS], membranoproliferative glomerulonephri-
tis [MPGN], IgA nephropathy, membranous
glomerulopathy, C3 glomerulopathy, or more
rarely, in infants less than 1 year, Finnish-type
congenital nephrotic syndrome or diffuse
mesangial sclerosis) is often present [4–9]. Children
with steroid-resistant NS and FSGS on biopsy may
have a podocyte gene mutation, such as podocin, as
cause of their disease. Genetic testing is important
to identify these children, as 10–30 % of children
with sporadic steroid-resistant NS and FSGS have
such mutations, and most do not respond to con-
tinued immunosuppression and will not have recur-
rence of the genetic disease in a kidney transplant
[10–12]. These genetically induced lesions do not
show specific renal biopsy morphologic findings.
24 Pediatric Renal Pathology
Acute Nephritis
The child with acute glomerulonephritis may need a
biopsy when the course is not typical of acute
postinfectious disease or if urinary abnormalities per-
sist. Although the primary disease process may be
evident in systemic conditions, such as Henoch-
Schönlein purpura or systemic lupus erythematosus
(SLE), renal biopsy often is indicated to assess sever-
ity of injury, to guide therapy and prognosis. Differ-
entiation of specific types of proliferative lesions
such as MPGN, the spectrum of C3 glomerulopathies
(C3GP), including dense deposit disease (DDD) and
C3-dominant glomerulonephritis is made by renal
biopsy. This distinction has important implications
for treatment. C3GP is due to complement
dysregulation, due to genetic deficiency of key com-
plement regulatory molecules and/or antibodies to
C3 convertase (C3 nephritic factor), directing a dif-
ferent approach to treatment [13]. Further, morpho-
logic recurrence of DDD in the transplant is
invariable and causes worse graft outcome [14–16].
Acute Kidney Injury
The cause of acute kidney injury may be clinically
obvious, or there may be multiple potential cul-
prits. When prerenal and obstructive causes are
not apparent, renal parenchymal disease should
be considered. When acute kidney injury is associ-
ated with nephritis, NS, or evidence of vasculitis or
systemic diseases, biopsy is usually performed.
Other common causes include acute tubular necro-
sis or injury (often caused by drug or ischemic
injury), vascular disease, and interstitial nephritis.
These conditions can often be diagnosed without
renal biopsy. However, when the cause remains
uncertain after complete evaluation, renal biopsy
may be necessary for diagnosis [17].
Rapidly Progressive
Glomerulonephritis
Renal biopsy is considered an urgent procedure in
the patient with rapidly progressive glomerulone-
phritis (RPGN). Various systemic vasculitides that
707
may be distinguished only by specific serologic
studies (see below) or renal biopsy must be treated
urgently to avoid severe chronic kidney damage.
Although serum anti-glomerular basement mem-
brane (GBM) antibody or anti-neutrophil cytoplas-
mic antibody (ANCA) titers may provide useful
information, the ANCA test in particular is not
diagnostic of a specific condition; rather, it is a
screening test for necrotizing vasculitides
[18, 19]. ANCA positivity may be detected by
indirect immunofluorescence to detect binding of
serum antibodies to test neutrophils in a perinuclear
(p-ANCA) or cytoplasmic (c-ANCA) pattern.
ANCA are now routinely tested more sensitively
and specifically by ELISA to detect antibodies to
myeloperoxidase (MPO) or proteinase-3 (PR3).
ANCA positivity can also be present in severe
crescentic immune complex disease and was
detected, in 19 % of such patients in a large series
[20, 21]. ANCA, particularly p-ANCA (MPO), are
also present in about 20 % of anti-GBM antibody
disease [21]. Thus, kidney biopsy will allow spe-
cific diagnosis not only of the cause of RPGN but
also its activity and chronicity, aiding in treatment
strategies.
Chronic Kidney Disease
Patients with chronic kidney disease of uncertain
etiology are candidates for renal biopsy. Although
renal biopsy of the small, shrunken kidney is more
risky because of the greater incidence of bleeding
complications, the diagnosis of primary disease can
be important. This information allows assessment
of existing severity of morphologic lesions, deter-
mination of risk of recurrence in eventual renal
transplant, and suitability of deceased versus
living-related donor transplantation. If the disease
has a familial basis or recurs frequently with resul-
tant graft loss, deceased donor transplantation may
be preferable to living-related donor transplant [16].
Systemic Diseases
The severity of renal involvement in systemic
disease, such as chronic or recurrent hemolytic-
708
uremic syndrome (HUS), Henoch-Schönlein pur-
pura, diabetes mellitus, or SLE, may not be appar-
ent without renal biopsy. Patients with diabetes
and renal abnormalities that are atypical for the
expected clinical course of diabetic nephropathy
are often biopsied to determine whether condi-
tions other than, or in addition to, diabetes are
affecting the kidney. Severity of lesions and
stage of chronicity and activity impart prognostic
information and may affect therapeutic decisions
(see below). The most extensively studied disease
in this regard is SLE. Differentiation of specific
class of lupus nephritis by the International Soci-
ety of Nephrology/Renal Pathology Society
(ISN/RPS) classification (see below) may be dif-
ficult without renal biopsy [22–24]. Overall, evi-
dence indicates that renal biopsy findings may be
more sensitive than clinical assessment alone in
evaluating the severity of renal involvement in
SLE [25–27].
Follow-Up of Disease
With improved therapeutic modalities available
for intervention in chronic progressive kidney
disease, sequential or follow-up biopsies are
becoming increasingly necessary to evaluate ther-
apeutic efficacy. Additional therapy may be indi-
cated, or alternatively, cytotoxic therapy with its
side effects may be withheld, if the biopsy shows
end-stage histology. Intervention with, for exam-
ple, angiotensin-converting enzyme (ACE) inhib-
itors or angiotensin type 1 receptor blockers
(ARBs) can alter the course of chronic progres-
sive renal disease [28].
Transplantation
Kidney transplant biopsies are useful in assessing
episodes of clinically suspected rejection, investi-
gating the cause of decreased renal function or
urine output, and detecting the development of
de novo or recurrent disease. Occasionally, infec-
tion may be diagnosed by renal biopsy. Drug
toxicity may be diagnosed by morphologic find-
ings. The absence of lesions in a patient with a rise
A.B. Fogo
in creatinine supports calcineurin inhibitor toxic-
ity because this drug commonly causes a decline
in the glomerular filtration rate (GFR) by vaso-
constriction and not overt structural lesions. The
absence of findings of acute rejection in renal
biopsy thus can assist in avoiding unnecessary
immunosuppressive therapy with its potential for
increased morbidity and mortality. Even a diag-
nosis of chronic allograft nephropathy (CAN, also
called IFTA; interstitial fibrosis/tubular atrophy),
which is not amenable to immunosuppressive
therapy, has important therapeutic implications
for the patient.
Diseases that recur in the transplant with high
frequency include IgA nephropathy, MPGN,
dense deposit disease (DDD, also previously
known as MPGN type II), FSGS, atypical,
diarrhea-negative HUS (caused by complement
dysregulation defect), and membranous
glomerulopathy. The latter two also occur de
novo in the transplant, likely related to antibody-
mediated rejection. Metabolic diseases such as
oxalosis and diabetic nephropathy can also cause
recurrent disease in the renal transplant, if liver or
pancreas transplantation does not cure the primary
abnormality [16]. Alport syndrome is caused by
mutation in one of the type IV collagen genes,
resulting in abnormal GBM assembly and struc-
ture. Patients with Alport may develop anti-GBM
antibody disease in the transplant because of anti-
bodies against its normal type IV basement mem-
brane collagen [29, 30].
Obtaining Tissue
General Considerations
Percutaneous renal biopsy is the most common
method for obtaining tissue for the kidney. In large
series, major complications are rare. The tech-
nique, first done in 1951 by Iverson and Brun,
allows tissue yield in 93–95 % of biopsies, with
more than 87 % of these being adequate
[31–33]. The biopsy findings altered diagnoses
in half of the cases in one series, indicating differ-
ent therapeutic approaches in approximately
one-third of those cases [31]. Although some
24 Pediatric Renal Pathology
renal diseases show diagnostic features by light
microscopy (LM) (Table 1), special studies add to
the sensitivity of the study. For the renal biopsy to
be most useful, it must be evaluated appropriately
by an experienced renal pathologist. Biopsies
must be examined by special LM, IF, and electron
microscopy (EM) for the most accurate diagnosis
[32, 34]. If the nephrologist’s hospital does not
provide these services, arrangements must be
made to send tissue in appropriate fixatives (see
below) to a reference laboratory with these capa-
bilities. If such services cannot be provided, it is
doubtful whether the institution should be under-
taking renal biopsies.
Contraindications
Contraindications (relative or absolute) to percu-
taneous biopsy are solitary, ectopic, or horseshoe
kidney; bleeding diathesis; abnormal renal vascu-
lar supply; and uncontrolled hypertension
[31–33, 35]. In the era of ultrasound guidance
and automated biopsy instruments, solitary kid-
ney may be biopsied safely in selected patients
[36]. Relative contraindications include obesity,
uncooperative patients, hydronephrosis, ascites,
and small shrunken kidneys, all associated with
greater risk for complications. Open or modified
open biopsy is preferable if the biopsy informa-
tion is crucial in these conditions. Percutaneous
biopsy is contraindicated if the kidney has tumors,
large cysts, abscesses, or pyelonephritis because
the needle track may facilitate spread of malignant
cells or infection. Open biopsy allows selection of
specific areas for biopsy in these situations.
Biopsy Technique
The patient may be brought to the hospital on the
day of the biopsy. Laboratory evaluation must
include a complete blood cell count with normal
platelet count, partial thromboplastin, and pro-
thrombin times. On rare occasions, infusions of
platelets or fresh frozen plasma or DDAVP may
be necessary to allow renal biopsy in critical clin-
ical situations in which histopathologic diagnosis
709
is essential. Adequate control of hypertension
before the procedure is important as hypertension
is a risk for post biopsy bleeding [37]. Before
biopsy is done, ultrasound examination must con-
firm that there are two kidneys in normal position.
The biopsy optimally is timed so that an experi-
enced technician or pathologist can attend to
ensure prompt processing of the biopsy tissue.
Food and drink should have been withheld for
at least 6 h before biopsy, and the child should be
lightly sedated. The child lies in the prone position
with a sandbag or rolled sheet under the abdomen,
and the skin of the flank is “prepped” and draped
in sterile fashion. Although the left kidney is
usually preferred, either side can be chosen for
biopsy. The lower pole of the kidney is marked on
the skin with a pen after localization by any of
several imaging techniques, such as computed
tomography (CT) or ultrasound, the last being
the most commonly used method. Local anes-
thetic is infiltrated first in the skin and then in
deeper tissues, taking care not to enter the kidney.
Conventional or spring-loaded needles are used
for renal biopsies. The conventional biopsy nee-
dle is inserted to the desired position as the patient
holds his or her breath. In younger children who
cannot reliably cooperate in this manner, biopsy is
done under anesthesia. The cannula is advanced
over the obturator once the needle is in correct
position, the needle is advanced, and the entire
needle with the core of tissue is then removed.
The use of an automatic spring-loaded biopsy
system is now widespread because of the simplic-
ity and ease of the technique [38, 39]. The kidney
is localized with ultrasound guidance and the
depth of the kidney as judged by ultrasound. The
biopsy needle is advanced to the kidney capsule
under ultrasound observation and guidance. The
patient may hold his or her breath for only a few
seconds while the spring-loaded needle is acti-
vated, causing the obturator to automatically
advance into the kidney, and the entire needle is
then removed with the tissue core. The speed of
automated biopsy needles, however, minimizes
the need for the patient to hold respirations,
required with conventional needles. It is important
to note that the caliber of the needle used with any
of these techniques directly impacts the adequacy
710 A.B. Fogo

Table 1 Characteristic abnormalities of glomerular diseases

Disease and IF staining
typical clinical
presentation LM pattern Mesangial Subepithelial Subendothelial EM, other findings
Hematuria/nephritis
Alport’s syndrome Early: normal    Thin and thick,
Late: sclerosis basket-weaving
GBM
Mesangial lupus Mesangial +   Immune deposits
nephritis ISN/RPS proliferation All Igs, C3, by EM, reticular
II C1q aggregates in
endothelial cells
Focal lupus Proliferative and/or + + (few) + (scattered) Immune deposits
nephritis ISN/RPS sclerosing, <50% All Igs, C3, C1q by EM, reticular
III of glomeruli aggregates in
endothelial cells
Diffuse lupus Proliferative and/or + + + (wire loop) Immune deposits
nephritis ISN/RPS sclerosing, 50% All Igs, C3, C1q by EM, reticular
IV of glomeruli, wire aggregates in
loops endothelial cells
IgA nephropathy Mesangial +   Immune deposits
proliferation Predominant by EM
or
codominant
IgA
Henoch-Schönlein Mesangial  + +/ +/ Immune deposits
purpura endocapillary  Predominant  by EM
hypercellularity,  or
crescents codominant
IgA
Postinfectious GN Endocapillary + +  Irregular, hump-
hypercellularity, Coarsely Coarsely like subepithelial
PMNs granular granular deposits on top of
IgG, C3 IgG, C3 GBM by EM
Hemolytic-uremic Glomerular/    Increased lamina
syndrome arteriolar rara interna by EM,
thrombosis swollen
endothelial cells,
fibrin tactoids, no
deposits
MPGN Endocapillary +  + Subendothelial
hypercellularity, IgG, C3 IgG, C3 immune deposits
lobular, double by EM, cellular
contour GBMs interposition
C3 GP: Mesangial,    Discontinuous
endocapillary Dominant
proliferation C3, scanty or
no Ig
C3-dominant GN Double contours in C3GN: usual C3GN: chunky, irregular C3GN:
C3GN mesangial capillary wall subendothelial/
mesangial,
occasional
subepithelial
deposits by EM
(continued)
24 Pediatric Renal Pathology 711

Table 1 (continued)
Disease and IF staining
typical clinical
presentation LM pattern Mesangial Subepithelial Subendothelial EM, other findings
DDD Ribbonlike DDD: DDD: ribbonlike, C3 DDD:
capillary wall in globular C3 intramembranous,
DDD mesangial dense
deposits by EM
Nephrotic syndrome
Minimal change Normal    Effacement of
disease podocyte foot
processes
No deposits
Focal segmental Segmental +/   Effacement of
glomerulosclerosis glomerulosclerosis, IgM, C3 podocyte foot
glomerular processes, no
hypertrophy deposits
Diabetic Increased mesangial    Thick GBM
nephropathy matrix,  nodular, without deposits
thick GBM,
hyalinized arterioles
Membranous Thick GBM, spikes Scattered +  Subepithelial,
lupus nephritis on Jones’ stain mesangial immune
ISN/RPS V deposits
+ All Igs, C3,  Reticular
C1q aggregates in
endothelial cells
Primary Thick GBM, spikes  +  Subepithelial
membranous GP on Jones’ stain  IgG, C3  immune deposits
PLA2R
RPGN
Anti-GBM Necrosis of  Linear  No deposits by EM
antibody disease glomeruli, staining of
crescents; all lesions GBM
of similar activity/ IgG linear
chronicity (C3 chunky/
granular)
Granulomatosis Focal segmental    No deposits by EM
with polyangiitis necrosis of
glomeruli,
crescents; variable
activity
Microscopic Focal segmental    No deposits by EM
polyangiitis necrosis of
glomeruli,
crescents; variable
activity
Abbreviations: GP glomerulopathy, PLA2R phospholipase A2 receptor

of the specimen [40]. When 18-gauge needles are
used with this method, the resulting cores are very
small and there can be artifact along the edges.
The use of a 16-gauge needle thus is more likely to
provide an adequate tissue sample without
distortion and with fewer passes necessary to
obtain adequate tissue.
Usually, two cores of kidney cortex are neces-
sary for optimum evaluation or three cores if
18-gauge needles are used. If tissue cannot be
712
obtained after several passes, the biopsy should be
attempted on another day. After biopsy, a dressing
is applied, and the child is kept supine in bed for
6 h and monitored with frequent checks of vital
signs and urine for hematuria.
Increasingly, percutaneous native and trans-
plant renal biopsies are performed as same day
procedures in pediatric patients so that hospital
admission is not required. Those with post biopsy
perinephric hematomas, post biopsy gross hema-
turia, or very young children can be observed
overnight so that hemostasis is assured
[41–43]. Open biopsy can be performed under
general anesthesia. The kidney can be directly
visualized even through a small incision.
Although a larger sample may be obtained with
a wedge biopsy, it is preferable to also perform a
needle biopsy to sample deeper cortex and
medulla for assessment of diseases that preferen-
tially involve juxtamedullary glomeruli (see
below).
Aspiration Biopsy
Fine-needle aspiration (FNA) biopsy technique is
used most often to diagnose mass lesion of the
kidney. FNA can also obtain material for culture.
A modified FNA technique has been described for
collection of glomeruli from either native or trans-
plant kidneys to analyze glomerular lesions. This
FNA technique has limitations in sample size and
is not suitable for study of vascular or fibrotic
processes or diagnosis of antibody-mediated
rejection. The less-invasive nature of the proce-
dure makes it amenable to serial monitoring of
interstitial cellular infiltrates in transplanted kid-
neys, but its use has declined due to less diagnos-
tic yield versus the gold standard core biopsy
approach [44, 45].
Complications
Important complications occur in 5–10 % of
patients [31, 46–54]. Major complications, usu-
ally bleeding, leading to nephrectomy occurred in
5 patients in a review of 8,081 [54] and 1 patient in
A.B. Fogo
a series of 5,120 biopsies. In a series totaling
1,820 biopsies in children, 1 nephrectomy
resulted [46–51]. Transient microscopic hematu-
ria is universal after biopsy, although macroscopic
hematuria is seen in only 5 % and requires trans-
fusion in up to 2.3 % of patients. Perirenal hema-
toma is most often asymptomatic and can be seen
by CT in up to 85 % of biopsies. Symptomatic
hematoma is rare, occurring in less than 2 %.
Arteriovenous fistulae are usually symptomatic
with hematuria, hypertension, or cardiac failure
in only 0.5 % of biopsies, although bruits may be
detected in as many as 75 % of patients. Most
fistulae heal within a few months. Other reported
complications include inadvertent puncture of
other viscera or major renal vessels, sepsis, renal
infection, and seeding of cancer. Death is rare, less
than 0.1 % in reviews of large series. Complica-
tion rates appear to be slightly higher in less
developed countries [46, 48]. Complications for
the spring-loaded needle biopsy system appear to
be similar to conventional needles if the same
gauge is used [55]. Complications relate in part
to the number of passes made to obtain tissue.
Therefore, the potential lower rate of complication
with an 18-gauge spring-loaded needle in some
studies is offset largely by the need for more
passes for adequate samples.
Assessment of the Renal Biopsy
Adequacy of Sample
Cores of fat and connective tissue will float when
placed in saline, but a core of renal parenchyma
will sink. The biopsy sample should also be visu-
ally inspected with a dissecting microscope or
hand lens. Glomeruli are visualized as small red
dots in the biopsy core. Scarred glomeruli may be
difficult to identify since they are not perfused. In
diffuse disease, such as membranous
glomerulopathy, one glomerulus may be adequate
for diagnosis. However, other diseases, such as
crescentic glomerulonephritis, FSGS, or lupus
nephritis, may be focal. The greater the number
of glomeruli sampled, the lower the probability of
missing a focally distributed lesion [56]. If only
24 Pediatric Renal Pathology
10 % of glomeruli in the kidney are involved by
the focal process, a biopsy sample of only 10 glo-
meruli has a 35 % probability of missing the
lesion, decreasing to 12 % if the biopsy contains
20 glomeruli. When one-fourth of glomeruli are
involved in the kidney, there is only a 5 % chance
of missing the abnormal glomeruli in a biopsy of
10 glomeruli. A biopsy sample of 20–25 glomer-
uli for light microscopic assessment is sufficient to
distinguish between mild disease (less than 20 %
of glomeruli involved), moderate disease (20–50
% of glomeruli involved), or severe disease (more
than 50 % of glomeruli involved). The sample site
must also be considered in evaluating the ade-
quacy of tissue. Even a large biopsy, consisting
only of superficial glomeruli, cannot exclude the
presence of early FSGS, in which the initial
involvement is in the juxtamedullary glomeruli.
Likewise, although nephronophthisis is most
often diagnosed clinically, a juxtamedullary
biopsy would be necessary for its morphologic
diagnosis.
Allotment of Tissue
Renal tissue should be studied by light micro-
scopic techniques with special stains (hematoxy-
lin and eosin, modified silver stain [periodic acid/
methenamine, also called Jones’ stain], periodic
acid-Schiff [PAS], and/or Masson trichrome), IF,
and EM [34, 57–59]. The tissue is divided so that
glomeruli are present in each portion of the sam-
ple. Optimally, the pathologist or an experienced
histotechnologist or other trained person will
attend the biopsy and inspect and allot tissue for
each study. If this is impossible, tissue may be
placed in saline and brought directly to the labo-
ratory for prompt processing. It is important to
handle the tissue gently so that artifacts do not
occur. The fresh tissue must not be picked up with
forceps because this will crush and distort the
morphology. The core can be handled carefully
with a wooden stick or pipette. The core should
not be placed on a sponge or gauze pad because
this may cause a divot-like artifact as the unfixed
tissue molds to the holes of the underlying sur-
face. The biopsy specimen is therefore placed on a
713
clean smooth surface, such as a wax board, for
cutting.
It may be difficult to identify the cortical part of
the specimen even after inspection with a hand
lens or dissecting microscope when scarring or
severe injury is present. Therefore, we recom-
mend cutting two 1-mm pieces with a sharp
blade from each end of each core for electron
microscopic studies. The remaining core is then
divided into specimens for IF and LM. When
multiple cores are obtained, this allocation should
be applied to each core, rather than allocating one
complete core to one study. This approach will
maximize chances of adequacy of tissue with
glomeruli for each study. When the tissue sample
obtained is very small, the nephrologist and the
pathologist should consider the differential diag-
nosis and allocate tissue accordingly. For exam-
ple, in a case of suspected IgA nephropathy, tissue
for IF is most important. IF studies can be
attempted on remaining fixed tissue in the paraffin
block, but are not as reliable as when performed
on frozen nonfixed material. Electron microscopic
studies can be done on other portions of tissue
(as long as mercury-based fixatives have not been
used). When tissue for EM is inadequate, portions
of the paraffin-embedded tissue left after light
microscopic examination may be cut out from
the block and processed for EM. Although the
quality is not optimal, diagnostic findings can
still be discerned. In special circumstances, when
no tissue remains in the paraffin block and a focal
lesion in one section must be studied, one may
attempt to process the tissue section from a glass
slide for EM.
Light Microscopy
Numerous fixatives are used for light microscopic
examination, and they vary from institute to insti-
tute. Satisfactory results may be obtained with
Zenker’s, Bouin’s, formalin, Carnoy’s, or parafor-
maldehyde. Material for IF studies may be snap
frozen immediately at 20 C in solutions of
isopentane, dry ice, acetone, or Freon and embed-
ded in Tissue-Tek, OCT, or other compounds for
frozen sections. If tissue cannot immediately be
714
snap frozen, it may be placed in Michel’s tissue
media, where it may be stored for up to 1 week
before freezing. This allows tissue to be sent to
reference laboratories for appropriate processing.
Tissue for EM may be fixed in glutaraldehyde,
formaldehyde, or other appropriate non-mercury
fixatives. Tissue placed in glutaraldehyde should
be promptly processed or, if stored for future
possible processing, should be transferred to an
appropriate buffer solution within a week to avoid
artifacts. Tissue for LM is routinely processed,
embedded in paraffin, and cut into 2–3-μ thick
sections. Serial sections with multiple levels are
then prepared for examination.
If water-soluble compounds are expected, such
as urate or uric acid, the tissue should be fixed in
ethanol. Lipids are best detected in frozen sections
because they are extracted during xylene
processing for paraffin sections. Hematoxylin
and eosin stains are most useful for overall assess-
ment of the interstitium and crystals. This stain
also allows particularly good visualization of
infiltrating cells, especially eosinophils. In addi-
tion, fibrin may be easily visualized by this
stain. Periodic acid-Schiff (PAS) stains glycopro-
teins and accentuates basement membranes and
matrix material and allows definition of the brush
border of proximal tubular cells. Areas of
hyalinosis and protein precipitation, including
cryoglobulin (which usually is dominantly IgM),
are also accentuated with PAS. Silver stains, such
as Jones’ stain, stain basement membrane
material but not deposits, thus allowing distinc-
tion of these components. Masson’s trichrome
stain detects areas of collagen deposition by
staining bluish. Sirius red stains all collagens
and can also be visualized and quantified under
polarized light to assess fibrosis. Other special
stains, such as immunohistochemistry for
specific molecules, may be indicated. Congo red
stain detects amyloid. Special stains can detect
bacterial or fungal organisms and acid-fast
bacilli. Special techniques may also be used on
the light microscopic material. These include
polarization to detect crystals or foreign bodies
and morphometry to assess glomerular size (see
below) and severity of interstitial fibrosis
quantitatively.
A.B. Fogo
Immunofluorescence
Immunofluorescence (IF) studies are most com-
monly done by direct IF on frozen tissue sections
with application of fluorescein-conjugated anti-
bodies directed against IgG, IgA, IgM, comple-
ment components C3 and C1q, kappa, lambda,
and often albumin and fibrinogen. Additional
antisera may be used as clinically indicated. Phos-
pholipase A2 receptor, (PLA2R), the antigen in
most cases of primary membranous GP, is
detected by IF by digestion techniques, followed
by applying antibody to PLA2R to tissue section
cut from the paraffin block [60, 61]. Additional
immunostaining studies include those for hepati-
tis B antigen, thyroglobulin, C4, C4d, specific
viruses (e.g., SV40 for polyoma virus), myoglo-
bin, and type IV collagen alpha chains. Some of
these antigens are recognized by antisera even
after fixation and can be detected by immunohis-
tochemical techniques (e.g., immunoperoxidase)
on the formalin-fixed tissue sections and then
studied with a light microscope. This technique
requires enzyme pretreatment of tissue depending
on the antigen to be studied, which must be tai-
lored exactly depending on length and type of
fixation and section thickness. Direct observation
of the digestion process, stopping when all plasma
is removed from capillary loops, has been used to
achieve reliable results [62]. These challenges
have prevented widespread use of this technique
in the USA. In general, IF on frozen tissue for
detection of immunoglobulins and complements
is more sensitive than IHC on fixed tissue.
Frozen tissue sections stained by the com-
monly used method of fluorescein-conjugated
antibodies are viewed by IF microscopy, evaluat-
ing staining in glomeruli, vessels, tubules, and
interstitium. The pattern of glomerular staining is
assessed to define granular or linear capillary wall
staining or mesangial deposits. Arteriolar staining
may be of diagnostic significance to detect, e.g.,
deposits in lupus. Tubules may show granular
deposits in lupus nephritis, pseudolinear deposits
in light chain deposition disease, or linear staining
in the rare anti-TBM antibody disease. Nuclear
staining can be seen in lupus or lupus-like diseases
as a tissue manifestation of the patient’s positive
24 Pediatric Renal Pathology
ANA. IF is more sensitive than EM in identifying
immune deposits, although EM provides more
detailed information on the exact localization of
those deposits [58, 59]. Diagnostic lesions of
interest should be photographed because fluores-
cence fades on storage and with light exposure.
Electron Microscopy
Tissue for EM is processed with postfixation in 1 %
osmium tetroxide, which enhances contrast of the
tissue, and then dehydrated and embedded. With
new, more rapidly polymerizing embedding media,
such as Spurr, tissue may be ready for examination
within 1 day. Electron microscopic study was
found to add information in 6–11 % of renal biop-
sies in a study from 1983 [32]. In a later study, EM
was needed to make a diagnosis in 21 % of cases
and provided important confirmatory data in
approximately 20 % of cases [58]. EM showed
diagnostic pathological abnormalities in 18 % of
patients with “normal” light microscopic findings.
Larger, so-called thick scout sections (1 μ) are
stained with toluidine blue to select smaller areas
for thin sectioning for the electron microscope.
Usually, the glomerulus containing the most repre-
sentative lesion is chosen, but sclerotic glomeruli
are not useful to examine by EM. If there are
irregular or focal proliferative lesions, several glo-
meruli may be sampled, including the proliferative
ones. The 60–90 Å thin sections are stained with
uranyl acetate and lead citrate to enhance contrast
before viewing in the EM scope.
Immune complex deposits are nonmembrane
bound and denser than basement membrane or
matrix materials. In specific diseases, such as
cryoglobulinemia, amyloid, immunotactoid or
fibrillary glomerulopathies, or lupus nephritis,
specific substructure of deposits may be seen.
Specific localization of immune complexes is
done by EM examination, indicating whether
deposits are subendothelial, subepithelial,
mesangial, or in all of the above compartments.
In some diseases, such as light chain deposition
disease or lupus nephritis, deposition may also be
seen in vessels and tubules. The so-called finger-
print deposits, with substructure reminiscent of
715
fingerprinting, are present in some cases of lupus
nephritis. Reticular aggregates (also called tubular
arrays) of organelle membrane material in endo-
thelial cell cytoplasm throughout the body are
characteristically seen in large numbers in patients
with SLE or HIV infection, or those treated with
exogenous interferon, thought to reflect a
response to high levels of interferon [59, 63].
EM also delineates specific basement mem-
brane abnormalities. For instance, small
subepithelial deposits without surrounding new
basement membrane material do not result in
spikes, and therefore cannot be visualized by
Jones’ stain on LM, but can still be detected
directly by EM. The lamina densa of the GBM is
markedly thickened in diabetic nephropathy. Cir-
cumferential cellular interposition is defined by
extension of monocytes and mesangial cell cyto-
plasm into the subendothelial space, with newly
formed basement membrane interpositioning
between the advancing infiltrating cells and the
endothelium with intervening basement membrane
material, thus giving rise to the classic double
contours seen with silver stain by LM in MPGN.
Increased lucent material is present in the expanded
lamina rara interna in transplant glomerulopathy,
HUS, eclampsia/preeclampsia, and other diseases
presumed to involve endothelial injury/
coagulopathy. In these conditions, deposition of
fibrin, fibrinogen, and their degradation products
may also occur. Fibrin is recognizable by its dense,
coarse sheaflike structure by EM, with periodicity
observed in favorable sections. Morphometry of
the GBM from EM prints is used to diagnose thin
basement membranes in hereditary nephritides.
EM also allows structural assessment of changes
of specific cells (see below). Diagnostic inclusions
are seen in various storage/metabolic disease, such
as Fabry’s disease.
Basic Renal Lesions
Normal
During fetal maturation, the glomerular capillary
tufts are initially covered by large, cuboidal,
darkly staining epithelial cells with only small
716
Fig. 1 Schematic illustration of glomerulus, with glomer-
ular capillary attached to a mesangial stalk area. The glo-
merular endothelium (E) is fenestrated and lines the
glomerular basement membrane (GBM), which covers
the mesangium. The outside of the GBM is covered by
Fig. 2 Immature
glomerulus with plump,
dark epithelial cells from
biopsy of a 29-week
gestation baby (PAS, 670)
lumina visible (Fig. 1). The cells lining Bowman’s
space undergo similar change from initial tall
columnar to cuboidal to flattened epithelial cells,
except for those located at the opening of the
proximal tubule, where cells remain taller. Imma-
ture nephrons may occasionally be seen in the
superficial cortex of children up to 1 year of age.
Glomerular growth continues until adulthood,
with average normal glomerular diameter approx-
imately 95 μ in a group of patients less
A.B. Fogo
the epithelial cell and its foot processes (Ep). The
mesangial cell (M ) is embedded within the mesangial
matrix (MM), with processes connecting to the GBM (Pro-
vided courtesy of Professor Wilhelm Kriz with permission
from [56])
than 5 years old (average age 2.2 years) and
140–160 μ in adulthood [64, 65].
The normal mature glomerulus consists of a
complex branching network of capillaries origi-
nating at the afferent arteriole and draining into
the efferent arteriole. The glomerulus contains
three resident cell types: mesangial, endothelial,
and epithelial cells (Fig. 2). The visceral epithelial
cells (podocytes) cover the urinary surface of the
GBM with pseudopodlike extensions called foot
24 Pediatric Renal Pathology
processes, with intervening filtration slits. Endo-
thelial cells are opposed to the inner surface of the
GBM and are fenestrated. At the stalk of the
capillary, the endothelial cell is separated from
the mesangial cells by intervening mesangial
matrix. The term endocapillary is used to describe
hypercellularity/proliferation filling up the capil-
lary lumen, contributed to by proliferation of
mesangial, endothelial, and infiltrating inflamma-
tory cells. In contrast, extracapillary proliferation
refers to proliferation of the parietal epithelial
cells that line Bowman’s capsule.
The mesangial cell is a contractile cell that also
has phagocytic properties. It lies embedded in the
mesangial matrix in the stalk region of the capil-
lary loops, attached to anchor sites at the ends of
the loop by thin extensions of its cytoplasm. Nor-
mally, up to three mesangial cell nuclei per lobule
are present. The basement membrane consists of
three layers distinct by EM, the central broadest
lamina densa and the less electron-dense zones of
lamina rara externa and interna. Thickening
occurs with maturational growth. Most investiga-
tors have found thicker basement membranes in
boys, with normal range from 220 to 260 nm at
1 year of age, 280 to 327 nm at age 5 years, 329 to
370 nm at age 10 years, and 358 to 399 nm at age
15 years [66, 67]. In our laboratory, we found a
range of GBM thickness in children with normal
kidneys from approximately 110 nm at age 1 year
to 222  14 nm at 7 years of age.
The glomerulus is surrounded by Bowman’s
capsule, which is lined by parietal epithelial cells.
These are continuous with the proximal tubule,
identifiable by its PAS-positive brush border. The
efferent and afferent arterioles can be distinguished
morphologically in favorably oriented sections or
by tracing their origins on serial sections. Segmen-
tal, interlobular, and arcuate arteries may also be
present in the renal biopsy specimen. The cortical
biopsy also allows assessment of the tubules and
interstitium. Proximal tubules are readily identified
by their PAS-positive brush border, lacking in the
distal tubules. Collecting ducts show cuboidal,
cobblestone-like epithelium. Tubules are normally
back to back with minimal interstitial cells and the
peritubular capillaries intervening. The medulla
may also be included in the biopsy.
717
Overall Pattern
Assessment of the biopsy specimen must include
inspection of all sections from different levels
because additional glomeruli may be sampled on
deeper cuts of the biopsy core and many diseases
are characterized by focal lesions [68]. The sever-
ity and patterns of lesions are assessed, and nor-
mal and affected glomeruli are counted. Lesions
are classified as focal if only some (less than half)
glomeruli are involved, diffuse if all (or most)
glomeruli are involved, segmental if only portions
of glomeruli are involved, and global if entire
glomerular tufts are involved. Characteristic glo-
merular disease patterns include lobular prolifer-
ation in MPGN, nodular proliferation of
mesangial cells, and abundant matrix material in
characteristic Kimmelstiel-Wilson lesions in dia-
betic nephropathy, focal and segmental sclerosis,
intraglomerular/arteriolar fibrin thrombi, necrotiz-
ing lesions, and crescent formation (Table 1). The
crescent, a lesion due to proliferation of mostly
parietal epithelial cells, owes its name to its shape
in well-established lesions.
Glomeruli are assessed for alterations in size
(see below). It is important to compare with a
normal control for a given age group because
marked glomerular maturational growth occurs
in children. Glomerular hypertrophy may be an
important predictor of increased risk for ultimate
development of FSGS in children with apparent
MCD (see below). Maturational pattern of glo-
meruli (see above) should be noted. Occasional
fetal glomeruli may be found in children beyond
infancy and are not of specific diagnostic
significance.
Glomeruli are assessed for glomerulosclerosis,
that is, the presence of segmental obliteration and
scarring of glomerular capillary tufts. Sclerosis
may be in a segmental or global pattern
(Table 2). Previous studies suggested that up to
10 % of glomeruli may be normally globally
sclerosed in people younger than 40 years of age
[69]. This number may be even smaller in chil-
dren, with less than 1–3 % global sclerosis
expected normally up to age 40 or 56, respectively
[70, 71]. These occasional globally sclerotic glo-
meruli are thought to represent errors of
718
Table 2 Definitions of common morphological terms
Light microscopy
Focal Involving some glomeruli
Diffuse Involving all glomeruli
Segmental Involving part of glomerular
tuft
Global Involving total glomerular tuft
Lobular Simplified, lobular
appearance of capillary loop
architecture (MPGN)
Nodular Relatively acellular areas of
mesangial matrix (diabetic
nephropathy)
Sclerosis Obliteration of capillary loop
by increased matrix (scarring)
Crescent Proliferation of parietal
epithelial cells
Spikes Projections of glomerular
basement membrane
intervening between
subepithelial immune
deposits (membranous GP)
Endocapillary Increase in mesangial and/or
proliferation/ endothelial cells, +/
hypercellularity infiltrating inflammatory cells
Hyaline Descriptive of glassy, smooth
appearing material
Hyalinosis Hyaline-appearing insudation
of plasma proteins (in, e.g.,
glomeruli in focal segmental
glomerulosclerosis or in
arterioles in, e.g.,
hypertension or diabetes)
Mesangium Stalk region of capillary loop
with mesangial cells
surrounded by matrix
Subepithelial Between podocyte and
glomerular basement
membrane
Subendothelial Between endothelial cell and
glomerular basement
membrane
Tram-track Double contour of glomerular
basement membrane due to
deposits and/or interposition
of cells (see below)
Wire loop Thick, rigid appearance of
capillary loop due to
subendothelial deposits
Activity Extent of possible treatment
sensitive lesions, based on, e.
g., extent of crescents, cellular
infiltrate, necrosis,
proliferation
(continued)
A.B. Fogo
Table 2 (continued)
Chronicity Extent of probable irreversible
lesions, based on, e.g., extent
of tubular atrophy, interstitial
fibrosis, fibrous crescents,
glomerulosclerosis
Immunofluorescence microscopy
Granular Discontinuous flecks of
staining along capillary loop
producing granular pattern
Linear Smooth continuous staining
along capillary loop
Electron microscopy
Foot process Flattening of foot processes of
effacement podocytes so that they cover
the basement membrane
Microvillous Small extensions of podocytes
transformation with villus-like appearance
Interposition Extension of cell cytoplasm
with interposition between
endothelial cell cytoplasm and
basement membrane and
underlying new basement
membrane formation
Reticular aggregates Organized arrays of organelle
membrane particles within
endothelial cells
Immunotactoid GP Large, organized
microtubular deposits,
>30 nm diameter
Fibrillary GP Fibrils 14–20 nm diameter
without organization
GP glomerulopathy
nephrogenesis. The percentage of global sclerosis
increases even with normal aging, up to half the
patient’s age, minus 10 [71]. Globally, sclerosed
glomeruli in greater percentage indicate the possi-
bility of renal disease (focal global sclerosis) [72].
The pattern of tubulointerstitial fibrosis, whether
proportional to glomerular sclerosis or not and
whether diffuse or present in a “striped” pattern
following the medullary rays or in broad patchy
zones, has diagnostic significance (see below).
Specific Glomerular Cells
Podocytes
The podocytes (glomerular visceral epithelial
cells) may show vacuolization in various diseases
24 Pediatric Renal Pathology
with severe proteinuria. Although more extensive
vacuolization of podocytes has been seen in FSGS
compared with patients with MCD [73], these
changes are only seen after established sclerotic
lesions are identifiable by LM and do not permit
distinction of these two disease processes in the
early phase in which segmental sclerosis may be
undetected. Effacement of the foot processes of
the podocytes by EM is common to any disease
with marked proteinuria, and the podocyte may
also show microvillous transformation, with long,
attenuated pseudopods. Podocytes are limited in
their ability to proliferate. However, the early
sclerotic lesion of FSGS is characterized by prom-
inence and hypertrophy of the overlying
podocytes (“capping” lesion), often associated
with endocapillary foam cells. This cellular vari-
ant of FSGS may be more common in children
than in adults with FSGS [74]. The idiopathic
collapsing variant of FSGS and HIV-associated
nephropathy both show prominent hyperplasia
and protein droplets of the visceral epithelial
cells, with overlying segmental collapse of the
glomerular capillary tuft. These activated cells
have loss of podocyte markers and demonstrate
instead parietal epithelial cell (PEC) markers by
immunostaining [75]. In the situation of recurrent
FSGS in the renal transplant, NS and foot process
effacement may be seen within weeks after
biopsy, with sclerosis becoming apparent at a
later date [76]. Even at this early stage, there are
increased activated PECs on the tuft in recurrent
FSGS, but not in native kidney biopsies of mini-
mal change disease [77]. In Fabry’s disease, there
is accumulation of glycosphingolipid because of
deficiency of alpha-galactosidase. Podocytes
show marked vacuolization by LM with charac-
teristic whorled, laminated electron-dense myelin
bodies by EM. In Fabry’s disease, these inclusions
may also be present in endothelial cells, tubular
epithelial cells, some interstitial cells, and the
vessels in early lesions in children [59, 78].
These inclusions decrease after treatment with
enzyme replacement [79].
Mesangial Cells
Hyperplasia of mesangial cells is defined as more
than three mesangial cell nuclei present per
719
mesangial region, in areas away from the vascular
pole. Increased mesangial prominence may be due
to increased cellularity, increased matrix,
deposits, or a combination. Large mesangial
deposits appear on Jones’ stain as pinkish areas
surrounded by the light silver-staining areas of
mesangial matrix. The so-called interposition
results when monocyte or mesangial cell cyto-
plasm extends outward between basement mem-
brane and endothelial cells and new matrix
accumulates between the mesangial and endothe-
lial cell bodies.
Endothelial Cells
Extreme proliferation and swelling of endothelial
cells can obliterate capillary lumina in conditions
characterized by abnormalities of coagulation.
Endothelial cells usually contain characteristic
reticular aggregates in lupus nephritis and
HIV-associated nephropathy or after exogenous
interferon therapy [63]. Endocapillary cell
hypercellularity/proliferation is characteristic of,
for example, diffuse proliferative lupus nephritis
and MPGN.
Crescents
Crescents consist primarily of proliferating parie-
tal epithelial cells with some infiltrating macro-
phages and are a manifestation of severe
glomerular injury. The name reflects the often
crescent-shaped sheet of cells filling up part or
nearly all of Bowman’s space. Crescents result
from injuries that break the GBM, leading to
exudation of plasma protein and formation of
fibrin within Bowman’s space, which then
induces proliferation of the parietal epithelial
cells and infiltration of macrophages. When cres-
cents are a prominent histologic feature, the
patient most often presents clinically with a rap-
idly progressive glomerulonephritis.
Crescents may occur in a variety of diseases.
Diseases with crescents as a primary manifesta-
tion include antibody-mediated injury (anti-GBM
antibody disease), severe immune complex dis-
eases (e.g., lupus nephritis) and pauci-immune
diseases. The latter are often, but not invariably,
associated with positive ANCA tests and may be
associated with systemic disease or be renal
720
limited. The p-ANCA (anti-myeloperoxidase)
pattern is most often associated with microscopic
polyangiitis, whereas the c-ANCA (anti-protein-
ase-3) pattern is typical in granulomatosis with
polyangiitis (Wegener’s granulomatosis). Of
note, positive ANCA tests are not sensitive in
distinguishing these categories [18–20]. Renal
biopsy is therefore critical for accurate diagnosis.
Diagnosis and appropriate treatment must occur
rapidly in this clinical situation to optimize
chances of recovery of renal function. The early
lesion of cellular crescents is responsive to cyto-
toxic therapy. Biopsy indications of irreversible
renal damage include breaks of Bowman’s cap-
sule and fibrous transformation of the cellular
crescents, periglomerular fibrosis, and scarred
glomeruli and tubulointerstitium.
Glomerular Basement Membrane
GBM abnormalities are best evaluated by
EM. The basement membrane is abnormally
thick in diabetic nephropathy [59]. Diffuse abnor-
mally thin GBMs, less than 250 nm in adults, are
seen in familial hematuria [66, 67]. GBM thinning
cannot be accurately from EM processed from
paraffin tissue, as this back-up technique causes
artifactual and variable thinning of GBMs [80].
In children, the diagnosis of thin basement mem-
branes is more difficult than in adults because
GBM increases in thickness with normal matura-
tion. Glomerular basement membrane thickness
should be compared with normal for age and sex
[see above; [66, 81, 82]]. In Alport syndrome, the
basement membrane in established lesions is char-
acterized by irregular areas of very thin and very
thick, with splitting and splintering of the base-
ment membrane [30, 83]. The GBM in nail-patella
syndrome is irregular, thickened, and split, with
electron-lucent areas containing banded collagen
type I fibers [59].
Immune deposits may localize on either side
of the GBM. Subepithelial immune deposits
are characteristically seen in membranous
glomerulopathy. Subendothelial immune deposits
are seen, for example, in proliferative lupus
nephritis or MPGN.
A.B. Fogo
The basement membrane may show double
contours on silver stain by LM in diseases other
than MPGN, C3GN, or dense deposit disease. In
transplant glomerulopathy, the double contour
appearance results from varying degrees of cellu-
lar interposition and widening, with increased
lucent material in the lamina rara interna. This is
also a characteristic finding in preeclampsia,
transplant glomerulopathy, and chronic HUS or
other chronic thrombotic microangiopathies [59].
Tubules
Morphologically evident acute tubular injury or
necrosis correlates poorly with the clinical extent
of acute kidney injury (AKI). The changes vary
from nondiagnostic vacuolization to frank necro-
sis with sloughing of tubular epithelial cells (toxic
type) and flattened epithelium characteristic of
regeneration (ischemic type). In cortical necrosis,
zones of cortex, including glomeruli, are necrotic.
In chronic kidney disease, tubules are atrophied
with dilation and flattened epithelium, presum-
ably secondary to lesions affecting the glomeru-
lus, although primary tubular and interstitial
injury mechanisms may also be involved in
these changes. Tubular atrophy is also present in
primary tubulointerstitial diseases. Tubuloin-
terstitial fibrosis is an important manifestation of
calcineurin inhibitor toxicity. The fibrosis occurs
along the medullary rays due to the more severe
ischemia occurring in these areas, resulting in a
striped, rather than diffuse, pattern of fibrosis,
with intervening preserved tubules.
Nonspecific casts of Tamm-Horsfall
(uromodulin) protein are seen in chronic kidney
disease. Other casts may have a diagnostic appear-
ance, such as the giant cells surrounding tubular
casts in light chain cast nephropathy (the so-called
myeloma kidney). Casts of myoglobin with char-
acteristic reddish-brown appearance are seen in
rhabdomyolysis, often with associated acute tubu-
lar necrosis [84]. Crystals, for example, oxalate,
dihydroxyadenine (DHA), or cysteine, may be
identified by examination under polarized light
[85, 86]. Tubules contain characteristic inclusions
in Fabry’s disease [87].
24 Pediatric Renal Pathology
Polymorphonuclear neutrophils (PMNs)
within collecting ducts and proximal tubules are
diagnostic of acute pyelonephritis. In chronic
pyelonephritis, there is tubular atrophy and inter-
stitial fibrosis, characteristically in a patchy,
regional distribution (geographic or “jigsaw” pat-
tern). The combination of segmental glomerular
sclerosis with ischemic changes of corrugation
and thickening of the GBM and periglomerular
fibrosis and patchy, regional interstitial fibrosis
and tubular atrophy is also characteristic of reflux
nephropathy [88].
Cysts may be demonstrated by biopsy,
although the diagnosis of specific cystic diseases
is usually made by combination of clinical and
ultrasound findings. The segment of the nephron
giving rise to the cysts can be identified by histo-
chemical stains [89]. Areas of low cuboidal
epithelial-lined structures surrounded by a cuff
of immature mesenchyme are present in the dys-
plastic kidney, often with cartilage, fat, or abnor-
mal blood vessels in the interstitium. The deep
medullary cystic dilation with thickened,
lamellated TBM characteristic of
nephronophthisis can be identified with a deep
biopsy. Dilation of proximal tubules with
microcyst formation in conjunction with exten-
sive foot process effacement by EM is character-
istic of congenital nephrotic syndrome of Finnish
type. FSGS in a collapsing pattern with visceral
epithelial cell hyperplasia, numerous reticular
aggregates by EM, and tubular cystic dilation
and interstitial fibrosis out of proportion to the
severity of glomerular lesions are highly sugges-
tive of HIV-associated nephropathy [63].
Viral infections, including polyoma virus and
CMV, result in characteristic nuclear inclusions in
tubular epithelia. Specific diagnosis is made by
immunostaining for viral proteins. Characteristic
viral particles may also be detected by EM [90].
Interstitium
Interstitial edema is a nonspecific change, present,
for example, in early acute transplant rejection,
renal vein thrombosis, or inflammatory processes.
Identification of eosinophils in an infiltrate is
721
suggestive of drug-induced interstitial nephritis,
although eosinophils are also present in some
cases of idiopathic interstitial nephritis [91].
Eosinophils can also be part of acute cellular
rejection in the transplant. Nonnecrotizing granu-
lomas, with or without eosinophils, most often
reflect drug-induced hypersensitivity reaction. A
pleomorphic infiltrate of lymphocytes, plasma
cells, and PMNs suggests possible polyoma
virus nephropathy in the transplant, confirmed
by finding viral nuclear inclusions and positive
immunostaining in tubules (see above)
[90]. Fibrosis results in increased spacing of
tubules because of the accumulation of
PAS-positive collagenous material. The collagen
also stains specifically blue with Masson’s
trichrome stain. Fibrosis in a striped pattern sug-
gests calcineurin inhibitor toxicity [92, 93]. Occa-
sionally, the interstitium is infiltrated by
malignancy. Hematopoietic neoplasms are espe-
cially prone to involve the kidney. In cystinosis,
characteristic rectangular or trapezoid crystals are
present mostly in monocyte/macrophages and can
be recognized by EM or when polarized on frozen
sections [85].
Vessels
Arterioles and larger segmental and interlobular
arteries are evaluated for changes in the intima and
media; the presence of deposits, fibrin, hyaline,
amyloid, or other material; or the presence of
vasculitis. Arterioles show inclusions early in
children with Fabry’s [78]. Larger vessels typi-
cally are not sampled by a biopsy, and diseases
such as classic polyarteritis nodosa that affect
these large vessels are therefore best evaluated
by other methods (e.g., arteriography). Intimal
fibrosis and medial thickening with hyperplasia
and hypertrophy of media are characteristic of
hypertension-attributable injury. Concentric
medial necrosis with nodular protein deposition
suggests acute calcineurin inhibitor nephrotoxi-
city [93]. Fibrin thrombi, when present in glomer-
uli and/or arterioles, are the essential lesion of the
thrombotic microangiopathies caused by, e.g.,
HUS [59]. Fibrin localizes predominantly within
722
glomerular lumina in disseminated intravascular
coagulation and hyperacute rejection.
Clinical Pathological Correlations
After evaluation of the structural changes of the
renal biopsy in conjunction with the clinical his-
tory, a diagnosis may be obvious. In some cases,
the biopsy specimen may show overlap features,
or there may be elements that do not correlate
clearly with the clinical setting. Close collabora-
tion by nephrologists and pathologists is essential
in arriving at the diagnosis. The pathologist must
be familiar with clinical manifestations of renal
disease, and the nephrologist should be familiar
with the terminology used by the pathologist to
describe the biopsy findings (Table 2).
When lesions are evaluated, the balance of all
elements must be considered. When a typical dis-
ease pattern is not present, one must consider
whether more than one process is taking place.
For instance, drug-induced interstitial nephritis
may be superimposed on other glomerular dis-
ease. This is especially true in the transplant set-
ting, where multiple disease processes may occur
at one time. In some instances, the biopsy findings
do not correlate with the patient’s renal function.
When such apparent discrepancies are found, one
possibility is that the biopsy specimen is not rep-
resentative of all the nephrons of the kidney. The
number of glomeruli necessary to estimate the
severity of diseases that show focal distribution
has been discussed above. However, the extent of
glomerulosclerosis may in and of itself not corre-
late with renal function. Tubular atrophy and
interstitial fibrosis may be more closely correlated
with extent of renal damage and renal function
[94–96]. One must also consider the elements
other than structure that influence the patient’s
renal function (i.e., blood pressure, filtration prop-
erties of the GBM, and the glomerular filtering
surface area). Patients with enlarged glomeruli,
either caused by compensatory hypertrophy or
by a primary pathological process, may show
less deterioration of renal function than expected
based on the extent of glomerular scarring.
Similarly, treatment of the patient with
A.B. Fogo
antihypertensive agents that affect glomerular fil-
tration rate (e.g., ACE inhibitors, which preferen-
tially dilate efferent arterioles) may actually
increase serum creatinine levels in the short run.
Compensation by remaining nephrons may mask
ongoing severe disease processes such that creat-
inine levels may remain near normal until late in
the course of disease when therapy is less likely to
have an impact on chronic progressive injury.
Diagnostic Findings in Selected Renal
Diseases
Minimal Change Disease/Focal
Segmental Glomerulosclerosis
MCD is diagnosed only after the exclusion of
abnormal findings at the light microscopic level,
with diffuse foot process effacement as the only
abnormality by EM. The disease is characteristi-
cally sensitive to glucocorticoid therapy. How-
ever, repeated renal biopsies in patients with
apparent MCD initially have shown overt FSGS,
which has a high incidence of progression to end-
stage renal disease (ESRD) [64, 97, 98]. As
discussed above, a small sample may not include
the segmentally sclerotic glomerulus, diagnostic
of FSGS. In FSGS, there is often also hyalinosis,
an insudation of plasma proteins and lipids with a
glassy, smooth hyaline appearance on LM. There
are no immune deposits, and foot process efface-
ment is present in all glomeruli by EM (Fig. 3).
The presence of IgM by IF without deposits by
EM in a biopsy that otherwise appears to be MCD
(so-called IgM nephropathy) does not have prog-
nostic value [99]. Some variants of FSGS may
have prognostic value [100]. A working classifi-
cation of morphological patterns of FSGS has
been shown to have prognostic implications
(Table 3) [101]. The usual type is diagnosed
when no special features are present. The collaps-
ing type of FSGS, characterized by collapse of the
glomerular tuft, either segmental or global with
associated podocyte hypertrophy/hyperplasia,
shows a rapid progression to end-stage disease
[102]. The cellular lesion, with endocapillary pro-
liferation with frequent foam cells and often with
podocyte hyperplasia, may represent an early
24 Pediatric Renal Pathology 723

Fig. 3 Minimal change

disease. The foot processes
are effaced (11,000)

Table 3 Working classification of FSGS FSGS studied steroid-resistant children and adults
Possible with FSGS, who were then randomized to cyclo-
prognostic sporine or mycophenolate mofetil and dexameth-
Type Key histologic feature implication asone, in addition to usual ACEI/ARB
FSGS, nos Segmental sclerosis Typical course [107]. Patients with tip lesion had better out-
Collapsing Collapse of tuft, Poor prognosis comes, even in this study where steroid respon-
FSGS GVEC hyperplasia
sive patients, expected to include larger numbers
Cellular Endocapillary ?Early stage
FSGS proliferation, often lesion of tip FSGS, were excluded. Conversely, collaps-
GVEC hyperplasia ing FSGS had worse prognosis, and FSGS nos
Tip lesion Sclerosis of tuft at Better was intermediate [107].
proximal tubule pole prognosis C1q nephropathy is characterized by either no
Perihilar Sclerosis and ?May reflect a sclerosis or segmental glomerulosclerosis or even
variant hyalinosis at vascular secondary type
pole of FSGS mesangial or endocapillary proliferation and
nos not otherwise specified, GVEC glomerular visceral
occasional crescents by LM, with mesangial C1q
epithelial cell deposits and lesser immunoglobulin components
[108]. EM shows mesangial and paramesangial
dense deposits but a lack of reticular aggregates.
Patients typically are adolescents and have
stage of FSGS and appears to occur more often in steroid-resistant NS and do not have clinical evi-
children with FSGS than adults [74, 103]. The tip dence of SLE. A recent study found this lesion in
lesion, that is, adhesion, sclerosis, or 1.9 % of all native kidney biopsies and in 9.2 % of
endocapillary foam cell lesion localized to the pediatric biopsies. Tubular atrophy and interstitial
proximal tubular pole, appears to have better fibrosis were the best correlates of renal insuffi-
prognosis [104–106]. The perihilar variant, with ciency at biopsy and at follow-up. Most patients
sclerosis and hyalinosis localized to the vascular with minimal change-like lesions at biopsy
pole, likely more often represents a secondary achieved remission versus only a third of patients
sclerosing process [101]. Several studies have with segmental sclerosis lesions. Progression to
validated the better prognosis of tip FSGS, the ESRD has occurred in some patients with sclero-
worse prognosis of collapsing FSGS, and the sis at biopsy, but long-term outcome of those
intermediate outcomes of FSGS, not otherwise without sclerosis at presentation has not yet been
specified [101–106]. The NIH Clinical Trial of established [109].
724
Fig. 4 Apparent minimal change disease (MCD) with
subsequent overt focal segmental glomerulosclerosis
(FSGS). The first biopsy from this 5-year-old girl, middle
panel, was indistinguishable from MCD, except for
marked glomerular hypertrophy versus age-matched
Because therapy and prognosis are different for
MCD versus FSGS, early distinction of these two
entities is of primary interest. We studied pediatric
patients with steroid-resistant nephrotic syndrome
and MCD on renal biopsies and compared to
patients with apparent MCD on biopsy who sub-
sequently progressed to overt FSGS [64]. Mor-
phometric analysis of initial biopsies showed that
glomerular size at the onset of disease, before
sclerosis was apparent, was remarkably larger in
patients who subsequently progressed to FSGS
(Fig. 4) [64, 110, 111]. There was a higher risk
for development of FSGS in patients <5 years old
A.B. Fogo
typical MCD with subsequent benign clinical course (top
panel). The patient’s later biopsy, bottom panel, 50 months
later, showed segmental sclerosis, diagnostic of FSGS
(Jones’ stain, 160)
with glomerular area greater than 1.5 times that of
normal age-matched controls. On the other hand,
glomerular size equal to or less than normal con-
trols in this group of patients indicated a good
prognosis. Calculated glomerular diameter for
increased risk of FSGS in these patients less than
5 years old was more than 118 μ versus 95 μ
glomerular diameter in age-matched controls.
Depending on processing and fixation, these values
may vary (our values are based on paraffin-
embedded tissue fixation in formalin or Zenker’s
fixative). Normal ranges should be established in
each laboratory assessing glomerular size.
24 Pediatric Renal Pathology
From these studies, abnormal glomerular
enlargement suggests a high probability of devel-
opment of FSGS in pediatric patients with appar-
ent MCD. Causes of abnormal glomerular
enlargement other than idiopathic FSGS, such as
diabetes mellitus, cyanotic cardiovascular dis-
ease, and massive obesity, must be excluded
before such inferences can be made. Interestingly,
the incidence of FSGS may be increased in these
settings. The association of abnormal glomerular
growth with development of glomerulosclerosis
may reflect a pathogenic linkage, in that processes
leading to excess matrix and sclerosis may be
manifested as glomerular growth. This view is
supported by the coexistence of these two pro-
cesses in many other diseases, including sickle
cell diseases, HIV infection, and reflux
nephropathy [112].
Alterations of dystroglycans, specific proteins
that are expressed along the GBM, may be of use
in differentiating MCD in FSGS. α- and
β-dystroglycans were decreased in MCD, but pre-
served in the nonsclerotic areas of FSGS in a small
study [113]. However, we have also observed loss
of dystroglycans in the proliferating visceral epi-
thelial cells in collapsing FSGS, thus indicating
caution in using this marker for definitive diag-
nostic distinction of MCD versus FSGS. Expres-
sion of CD44, a marker of activated PECs, on the
glomerular tuft was present in the early recurrence
of FSGS in the transplant with only foot process
effacement, preceding overt sclerosis, but not in
native kidney MCD [77]. This marker may thus
also be of use in diagnosing native kidney FSGS
in its early stages. Recently, molecular studies also
indicate distinct gene expression profiles in MCD
versus FSGS, with much higher ratio of podocin
to synaptopodin mRNA in the former, and
increased podocyte loss in severe proteinuric con-
ditions [114–116].
Specific gene mutations of podocyte-specific
genes have been identified in patients with famil-
ial FSGS, and also in pediatric patients with
nonfamilial FSGS. The slit diaphragms are crucial
for regulation of permselectivity, and mutations of
several slit diaphragm genes cause proteinuria and
FSGS. Autosomal recessive FSGS with early
childhood onset and rapid progression to end
725
stage is caused by mutations in NPHS2, which
encodes podocin, a key component of the slit
diaphragm [117]. Mutation in ACTN4, which
encodes alpha-actinin-4, causes adult onset auto-
somal dominant FSGS [118]. The prognosis is
poor, with progression to renal disease in 50 %
of patients by age 30. Recurrence of NS in the
transplant in patients with genetic FSGS has been
very rare, presumably related to immune events,
as the transplant does not carry the mutated gene
[118]. TRPC6, a receptor, is mutated in some
kindreds with FSGS, with variable penetrance
and adult onset [119]. Phospholipase C epsilon
mutation (PLCE1) is present in some cases of
DMS or rare cases of FSGS, with rare steroid
response [120–122]. Other rare mutations have
been found in both familial and nonfamilial
FSGS [123, 124]. These familial forms do not
have specific morphological features of the seg-
mental sclerosis. Children with steroid-resistant
NS without family history of FSGS also have
high incidence of abnormalities in crucial
podocyte-related genes. Mutations in NPHS2
may occur in up to 25 % of pediatric patients
with sporadic steroid-resistant NS [11, 12].
Genetic screening for a panel of these podocyte-
related structural genes may prove useful in
identifying those whose NS will prove steroid
resistant and who have less risk of NS after
transplant [12].
Congenital nephrotic syndrome (CNS) of
Finnish type is caused by mutation in nephrin.
Renal biopsy shows mesangial hypercellularity
or no glomerular lesions and dilated proximal
tubules by light microscopy [125]. Nephrin local-
izes to the slit diaphragm of the podocyte and is
tightly associated with CD2-associated protein
(CD2AP). Nephrin is thought to function as a
zona occludens-type junction protein. CD2AP
plays a crucial role in receptor patterning and
cytoskeletal polarity, and its absence resulted in
sclerosis and foot process effacement in mice,
supporting a role for CD2AP in the function of
the slit diaphragm. Rare case reports of CD2AP
mutation in human FSGS exist [126]. Laminin-β2
(LAMB2) is mutated in Pierson syndrome, often
with microcoria and CNS abnormalities, with
mesangial sclerosis [127, 128].
726
Fig. 5 Arteriolar fibrin
thrombi with congestion in
capillary loops in
hemolytic-uremic
syndrome (Jones’ stain,
270)
FSGS associated with mitochondrial cytopathy,
due to a mutation of mitochondrial DNA in
tRNAleu(UUR), may show multinucleated
podocytes and have abnormal mitochondria by
electron microscopic examination. Patients also
have unusual hyaline lesions in the arterioles.
Some patients with FSGS without full-blown fea-
tures of mitochondrial cytopathy (i.e., myopathy,
stroke, encephalopathy, occasionally diabetes
mellitus, hearing problems, cardiomyopathy)
have also been reported to have this mitochondrial
mutation [129]. For further discussion of MCD
and FSGS, see ▶ Chap. 27, “Idiopathic Nephrotic
Syndrome in Children: Genetic Aspects.”
Hemolytic-Uremic Syndrome
HUS is the most common disease in children that
is manifested by injury to the microvasculature
and is typically due to E. coli 0:157 verotoxin
and associated with diarrhea. The characteristic
lesion of these conditions is thrombotic
microangiopathy (TMA). By LM, thrombi in glo-
meruli and arterioles are present (Fig. 5). The
renal biopsy findings, rather than clinical param-
eters, predict long-term prognosis [130]. Patients
with cortical necrosis have a particularly ominous
prognosis. The extent of glomerular versus arteri-
olar involvement is also of prognostic
A.B. Fogo
significance. Generally, both the long-term prog-
nosis and the clinical presentation are more severe
if larger vessels are involved. Arterial involve-
ment was not seen in biopsies performed during
the first 2 weeks of hospitalization [131]. The
glomerular endothelium is markedly swollen,
nearly occluding capillary lumina. Fibrin thrombi
are visualized easily. With chronicity, these areas
may progress to segmental ischemic collapse with
sclerosis, especially when arterioles are involved.
The arterioles can become completely occluded
by thrombi, with necrosis of vessel walls. IF
shows occasional nonspecific entrapment of C3
and IgM in injured areas with fibrin and fibrino-
gen. EM shows extreme swelling of the glomeru-
lar endothelium with increased lucent material in
the lamina rara interna of the basement membrane
with entrapped platelets, fibrin, and red cell frag-
ments, without immune deposits (Fig. 6). De novo
thrombotic microangiopathy in the renal trans-
plant is indistinguishable morphologically from
HUS in the native kidney. Cyclosporine and
FK506 have both been implicated in its pathogen-
esis [132, 133]. Mutations of complement regula-
tory genes are implicated in familial and some
diarrhea-negative sporadic cases. In adults, addi-
tional causes of include thrombotic thrombocyto-
penic purpura, related to ADAMTS13 deficiency,
24 Pediatric Renal Pathology
Fig. 6 Hemolytic-uremic
syndrome. The lamina rara
interna (endothelial side of
the glomerular basement
membrane) is widened with
increased lucent material.
No immune deposits are
present (3,300)
postpartum renal failure, and various drugs. The
morphologic characteristics of the TMA lesion are
largely the same regardless of etiology. For further
discussion of HUS, see ▶ Chap. 47, “Renal
Involvement in Children with HUS.”
Henoch-Schönlein Purpura/IgA
Nephropathy
Henoch-Schönlein purpura is often viewed as the
systemic variant of IgA nephropathy (Berger’s dis-
ease) [4, 134]. The glomerular manifestations are
similar, and vary in both, likely depending on
extent and location of deposits. In some cases,
there is only focal or even diffuse mild to moderate
mesangial proliferation (Fig. 7) [135]. In more
severe cases, there is focal or even diffuse
endocapillary hypercellularity/proliferation. In
severe cases, there may also be necrosis of glomer-
ular tufts with crescents in Bowman’s space. IF by
definition shows predominance or codominance of
mesangial IgA, with capillary loop deposits in
more severe cases. IgG, IgM, and C3 deposits
may also be detected. The immunoglobulin
deposits are present diffusely, even in glomeruli
that appear normal by LM. By EM, electron-
dense mesangial deposits are present, with occa-
sional “spillover” of deposits to subendothelial
regions in the regions adjacent to the mesangium,
particularly in cases with endocapillary hypercel-
lularity/proliferative lesions (Fig. 8). Deposits are
decreased when clinical remission occurs [136]. In
Henoch-Schönlein purpura, deposits are often pre-
sent in subendothelial as well as mesangial areas,
727
associated with more severe glomerular lesions,
including crescents, and worse outcome [135].
Classification schemas analogous to those for
lupus nephritis have been proposed, but have not
had widespread use [137, 138]. Therefore, the
International Study Group of IgA Nephropathy
composed of nephrologists and pathologists
reviewed a large number of archival IgA nephrop-
athy cases to determine which biopsy lesions cor-
related with prognosis [139, 140]. Additional
studies have validated these findings, including in
children [141–145]. Mesangial hypercellularity,
i.e., more than half the glomeruli with more than
three nuclei in a mesangial area, endocapillary
hypercellularity/proliferation, segmental sclerosis,
and significant (>25 %) interstitial fibrosis were
each associated with worse long-term outcome.
Cases with crescents usually had received more
intense immunosuppression and had responded to
this therapy. More recent studies suggest that cel-
lular crescents, if left untreated, also may have
worse prognosis [141]. For further discussion of
Henoch-Schönlein purpura/IgA nephropathy, see
▶ Chap. 32, “Immunoglobulin A Nephropathies
in Children (Includes HSP).”
Membranoproliferative
Glomerulonephritis and C3
Glomerulopathies
The MPGN pattern of injury consists of
endocapillary/mesangial proliferation and/or
hypercellularity with double contours of the
GBM. This pattern may be due to chronic
728
Fig. 7 (a) Mesangial and
endocapillary
hypercellularity and small
cellular crescent in Henoch-
Schönlein purpura (Jones’
stain, 430). (b)
Immunofluorescence
demonstrated IgA
mesangial deposits
endothelial injury and thus have no immune com-
plex deposits, such as in chronic TMA (see
above). The same light microscopic pattern is
seen when immune complexes or monoclonal
proteins or cryoglobulins are deposited in the
subendothelial and mesangial areas, causing a
similar LM response. Deposit of C3 only, or dom-
inant C3, indicates the underlying etiology
involves complement dysregulation [13]. This lat-
ter group of disease is called C3 glomerulopathy
and includes dense deposit disease (DDD), where
the deposits are characteristically very dense by
EM, or C3-dominant glomerulonephritis, where
deposits have density similar to usual immune
complexes by EM. Thus, IF and EM are crucial
A.B. Fogo
to distinguish the cause of MPGN pattern lesions.
“MPGN” as a diagnosis should only be used when
the lesion is caused by immune complexes.
MPGN is characterized by a tram-track appear-
ance of the GBM on silver stain, because of dupli-
cation around intramembranous/subendothelial
deposits and interposition of mesangial cells
and/or macrophages (Fig. 9). The glomeruli are
enlarged and hypercellular with a lobular appear-
ance by LM (Fig. 10). There is marked mesangial
and endocapillary hypercellularity, and occasional
PMNs and mononuclear cells may be present. By
IF, IgG and C3 are present in a coarse granular
pattern along basement membranes, usually with
lesser IgM. Subendothelial and mesangial
24 Pediatric Renal Pathology 729

Fig. 8 Immune complex

deposits surrounding
mesangial cell in Henoch-
Schönlein purpura
(3,400)

Fig. 9 Lobular appearance

of glomeruli in MPGN
(200) (Jones’ stain, 400)

immune deposits are seen by EM [4, 59]. MPGN
may be idiopathic or secondary to any of numer-
ous chronic infections. Hepatitis C positivity,
often with associated cryoglobulins, was present
in about one-fourth of adult cases of MPGN
pattern lesions in adults in Japan and the USA
[146, 147]. This association has not been demon-
strated in children with apparent idiopathic MPGN
[148]. MPGN recurs in 20–30 % of grafts and may
lead to graft loss [16]. Secondary MPGN, more
common in adults, more often demonstrates a focal
segmental pattern of proliferation, contrasting
the more diffuse involvement seen in idiopathic
MPGN, more common in children.
C3 Glomerulopathy: Dense Deposit
Disease and C3 Glomerulonephritis
In dense deposit disease (DDD), the glomeruli
may appear similar by LM to those of immune
complex-type MPGN, and this disease has
730 A.B. Fogo

Fig. 10 (a) Double

contours of the glomerular
basement membrane
(“tram-tracking”) in
MPGN, caused by
subendothelial/
intramembranous deposits
and cellular interposition
(Jones’ silver stain, 400).
(b) Chunky irregular
capillary wall and
mesangial deposits in
MPGN, caused by
subendothelial/
intramembranous and
mesangial deposits and
cellular interposition
(anti-IgG, 200)

therefore also previously been called type II
MPGN. However, the pathogenesis is entirely
different. These patients often show circulating
IgG autoantibodies, also known as C3 nephritic
factor, or have defects in key complement regula-
tory molecules or both [13, 15, 149, 150]. Therapy
directed at abnormal complement regulation has
therefore been attempted.
The basement membranes are deeply eosino-
philic, often with a ribbon garland or sausage-
shaped contour. By IF, discontinuous smooth lin-
ear deposits of C3 along the GBM and round
globular mesangial deposits are found, typically
without immunoglobulin staining. The disease is
named dense deposit disease because of the char-
acteristic appearance by EM with strongly
electron-dense deposits within the basement
membrane, comprised of complement degrada-
tion products. A large international study of
69 cases of DDD by the Renal Pathology Society
showed mesangial proliferation was most com-
mon, followed in order by membranoproliferative
appearance with endocapillary proliferation and
crescentic or acute proliferative patterns [151,
152]. Renal survival may be worse in DDD than
in immune complex-type MPGN (median sur-
vival 8.7 vs. 15.3 years) [153]. The distinction
between these two diseases is also important
since dense deposit disease invariably recurs in
renal transplantation, with worse graft outcome
[15, 154]. C3-dominant glomerulonephritis
(C3GN) also had dominant or only C3 by IF
24 Pediatric Renal Pathology 731

Fig. 11 Diffuse lupus

nephritis (ISN/RPS Class
IV) with massive dense
mesangial and
subendothelial deposits and
fewer deposits in
subepithelial areas
(5,600)

Fig. 12 Membranous
glomerulopathy with
subepithelial deposits and
intervening lamina densa
(seen as spikes by silver
stain on LM) (15,580)

(2 steps more intense than Ig), but usual density
of deposits by EM, and often with underlying
complement regulatory molecules defects rather
than C3 nephritic factor [13]. There is high recur-
rence in the transplant, about 75 % by 10 years,
with worse outcome [14, 155]. Some patients
presenting after infection with apparent
postinfectious GN clinically and morphologically
have underlying mutations in complement regu-
latory genes [156], with classic C3GN on follow-
up biopsy, and have worse outcome than typical
postinfectious GN.
Lupus Nephritis
Lupus nephritis is not a single disease, but rather
there is a spectrum of severity of involvement of
the kidney by the immune complex characteristic
of SLE. Most patients with SLE will have mor-
phologic manifestation of renal immune deposi-
tion. However, patients who undergo renal biopsy
most often have clinical renal manifestations and
will have more pronounced changes [27]. Lupus
nephritis is characterized by deposits in all ana-
tomic compartments of the glomerulus (i.e.,
mesangial, subepithelial, and subendothelial
regions) [23–25, 157] (Figs. 11 and 12). All
immunoglobulin classes with dominant IgG, C3,
and smaller amounts of C4/C1q are usually
found in lupus nephritis deposits (Fig. 13), and
immune complex deposits are seen by
EM. Reticular aggregates (see above) are typi-
cally seen in endothelial cells in any patient with
SLE, regardless of presence of lupus nephritis
[4, 59, 157] (Fig. 14).
732 A.B. Fogo

Fig. 13 Immunofluor-
escence of chunky capillary
and small mesangial IgG
deposits in diffuse lupus
nephritis (ISN/RPS Class
IV). The larger segments of
capillary loop staining
correspond to
subendothelial deposits,
with a smooth outer edge
where deposits are molded
underneath the GBM
(4,000)

Fig. 14 Endothelial cell

containing tubular-shaped
reticular aggregates in lupus
nephritis (20,000)

The WHO classifications, either the original or
modified, were previously most commonly used
[22, 23]. However, the International Society of
Nephrology (ISN) and the Renal Pathology Soci-
ety (RPS) put forth a revised lupus nephritis clas-
sification to clarify some areas of difficulty in the
previous versions [24, 158, 159]. Greater
interobserver reproducibility occurs with the
ISN/RPS classification [158]. In the ISN/RPS
classification, Class I has minimal mesangial
deposits with normal LM. Class II is characterized
by mesangial expansion visible by LM and
mesangial deposits, with only scattered peripheral
loop deposits. In class III, focal lupus nephritis,
deposits are present in mesangial areas, and there
are lesions of either active endocapillary prolifer-
ation, necrosis, cellular crescents, or chronic
lesions with sclerosis, fibrocellular, or fibrous
crescents. These focal lesions, by definition,
involve less than 50 % of glomeruli. The
subendothelial deposits can result in thick, rigid-
appearing capillary basement membranes by LM,
the so-called wire-loop lesions. “Hyaline”
thrombi (aggregates of immune complexes) may
fill capillary lumina. When the above lesions
affect at least 50 % of glomeruli, lupus nephritis
is classified as class IV diffuse lupus nephritis.
The proliferative lesions of both class III and IV
are typically associated with subendothelial
deposits in addition to mesangial deposits, with
only rare or scattered subepithelial deposits. IF
demonstrates widespread distribution of immune
complexes. EM confirms the massive and exten-
sive immune complex deposition (Fig. 11). For
class III and IV, the extent of active versus
chronic lesions (A vs. C) is specified. The seg-
mental necrotizing lesions in class IV lupus
nephritis may have worse prognosis and are
24 Pediatric Renal Pathology
Fig. 15 Anti-glomerular
basement membrane
antibody disease with linear
staining of GBMs by
immunofluorescence for
IgG (125)
often associated with necrosis, and thus the pres-
ence of these segmental lesions versus global
(S vs. G) endocapillary proliferation is noted
[24, 159, 160].
Class V membranous lupus nephritis is char-
acterized by predominance of subepithelial
deposits in a pattern similar to that of idiopathic
membranous glomerulopathy, with added
mesangial deposits (Fig. 12). Primary membra-
nous glomerulopathy, most often due to anti-
bodies to phospholipase A2 receptor (PLA2R),
is rare in children [60]. Renal biopsy staining
can determine the presence of PLA2R in the
deposits, and this staining is negative in cases of
membranous lupus nephritis [60, 61]. In some
patients with membranous GP associated with
hepatitis B, PLA2R antibodies may also be pre-
sent, suggesting perhaps epitope spreading
[161]. Serum ELISA assays for detection of
PLA2R autoantibodies are now also available.
Subendothelial deposits are minor components
in class V lupus membranous nephritis. When
there are superimposed focal or diffuse prolifera-
tive or sclerosing lesions in addition to membra-
nous changes, both processes are diagnosed, e.g.,
combined focal or diffuse lupus nephritis and
membranous lupus nephritis, ISN/RPS Class III
+ V or Class IV + V, respectively. Widespread
chronic sclerosing lesions in a nonspecific pattern
in a case of lupus nephritis are defined as Class
VI. Tubular basement membrane deposits can
occur in any class of lupus nephritis and may
733
account in part for the tubulointerstitial injury.
Vascular lesions include immune deposits, or
thrombotic microangiopathy, often related to
antiphospholipid antibodies. Vasculitis occurs
rarely. For further discussion of lupus nephritis,
see ▶ Chap. 46, “Renal Involvement in Children
with Systemic Lupus Erythematosus.”
Anti-glomerular Basement Membrane
Antibody Disease
Light microscopic examination shows crescentic
glomerulonephritis with focal necrotizing lesions.
Patients may not always show detectable serum
levels of anti-GBM antibodies, especially after the
acute phase of illness. Serology is positive in 95 %
of patients in the first 6 months after onset. However,
in all patients, even those with negative serology,
linear IgG staining by IF of glomerular basement
membranes is present (Fig. 15). Standard EM does
not visualize the immune deposits, perhaps because
of the diffuse distribution of the antigen (α3 type IV
collagen NC-domain). Patients with more than 50 %
crescents have a worse prognosis [162].
Granulomatosis with Polyangiitis
By LM, the appearance of granulomatosis with
polyangiitis is the same as for other nonimmune
complex crescentic necrotizing glomerulonephrit-
ides, such as microscopic polyangiitis or anti-
GBM antibody disease (Fig. 16). The lesions are
734
Fig. 16 (a) Segmental necrosis and crescent in
granulomatosis with polyangiitis, with irregular focal dis-
tribution of lesions. Immunofluorescence was negative
(Jones’ stain, 100). (b) Segmental GBM breaks, necrosis,
focal and segmental. Granulomas are rare in the
kidney, and arteritis is rarely found in the small
sample inherent to the renal needle biopsy. IF stud-
ies allow differentiation of the lesion from
anti-GBM antibody disease. It shows fibrin and
fibrinogen in areas of necrosis, and nonspecific
trapping of immunoglobulin, especially IgM. By
EM, immune deposits are absent or very rare
(hence the name “pauci”-immune). Distinction
from microscopic polyangiitis cannot usually be
made by renal biopsy findings. Clinical manifesta-
tions must be used to distinguish between these
two disorders. For further discussion of
granulomatosis with polyangiitis, see ▶ Chap. 45,
“Renal Involvement in Children with Vasculitis.”
A classification of these lesions shows prognostic
value based on the extent of crescents versus scle-
rosis, with those with more than 50 % of glomeruli
affected with sclerosis having worst prognosis and
best outcome in those with focal, i.e., <50 % of
glomeruli affected, lesions [163].
Postinfectious Glomerulonephritis
Patients with typical postinfectious glomerulone-
phritis due to streptococcal infection do not usu-
ally undergo renal biopsy. Infectious agents other
than streptococci can also cause postinfectious
glomerulonephritis [164]. When the diagnosis
remains in question, when abnormalities persist,
A.B. Fogo
and cellular crescent in granulomatosis with polyangiitis,
without glomerular hypercellularity in noninvolved seg-
ment of the glomerulus. Immunofluorescence was negative
(Jones’ stain, 430)
or when the initial disease is severe, renal biopsy
may be done. Glomeruli are enlarged and
hypercellular with prominent endocapillary pro-
liferation and infiltration by neutrophils and
mononuclear cells (Fig. 17) [165]. In severe dis-
ease, crescents are present. Occasionally, large
subepithelial deposits (“humps”) can be visual-
ized by LM. These differ from those typical of
membranous glomerulopathy in being more
unevenly distributed along the capillary basement
membrane and larger in size. The deposits lie on
top of the basement membrane, rather than being
embedded within it (as in membranous
glomerulopathy), and therefore spikes are not usu-
ally present. By IF, there are coarsely granular,
discontinuous areas of IgG and prominent C3
along the capillary wall and in the mesangium.
Postinfectious glomerulonephritis due to
staphylococcal infection may have dominant IgA
rather than IgG and can be distinguished from
IgA nephropathy by EM appearance of
deposits [166]. Electron-dense subepithelial
deposits are large, variegated, hump or dome
shaped, and irregularly spaced (Fig. 18).
Occasional mesangial deposits are present in
many biopsies. For further discussion of
postinfectious glomerulonephritis, see ▶ Chap.
31, “Acute Postinfectious Glomerulonephritis in
Children.”
24 Pediatric Renal Pathology 735

Fig. 17 Postinfectious
glomerulonephritis with
endocapillary
hypercellularity/
proliferation and PMN
infiltration (PAS, 430)

Fig. 18 Electron
micrograph of subepithelial
large, irregularly spaced,
hump-shaped subepithelial
deposits in postinfectious
glomerulonephritis. The
deposits are variegated and
lie on top of the GBM
(small arrows). There is
endocapillary proliferation
with PMN (long arrow)
infiltration (7,000)

Diabetic Nephropathy efferent arteriolar hyalinization was present in

Diabetic nephropathy affects 30–40 % of patients
with diabetes mellitus, either type 1 or type 2, with
overt clinical nephropathy manifest 15–20 years
after onset of diabetes. Therefore, diabetic
nephropathy has been considered a disease of
adults. However, adolescents may have diabetic
renal lesions even after short duration of disease
[167]. In addition, obesity and type 2 diabetes
mellitus are increasing in children. The structural
changes in these diabetic children include GBM
thickening and mesangial expansion and were
associated with proteinuria, hypertension, and
decline in GFR. Overt diabetic nephropathy with
nodular glomerulosclerosis and afferent and
several of these young patients. A classification
of diabetic nephropathy lesions has been proposed
to allow stratification of patients with similar
lesions for further study, ranging from just GBM
thickening to advanced nodular lesions in most
glomeruli [168]. For further discussion of diabetic
nephropathy, see ▶ Chap. 49, “Diabetic Nephrop-
athy in Children.”
Alport Syndrome/Thin Basement
Membrane Lesion
Early in life in males with classic Alport syn-
drome, and in female carriers, the renal biopsy
may show no significant light microscopic
736
abnormalities. At later stages, glomerulosclerosis,
interstitial fibrosis, and prominent interstitial foam
cells are typical. These foam cells are not specific
for this disease and are found in numerous
proteinuric states. Glomeruli can show segmental
sclerosis. Immunofluorescence may show
nonspecific trapping of IgM. By electron micros-
copy, the diagnostic lesion consists of irregular
thinned and thickened areas of the glomerular
basement membranes with splitting and irregular
multilaminated appearance of the lamina densa,
so-called basket weaving. In between these lami-
nae, a granular, mottled material is present. Sim-
ilar GBM changes by EM, but with normal
collagen chain staining (see below), occur in
Frasier syndrome. This entity is due to WT-1
mutation and manifests as NS with FSGS by
LM, no deposits, pseudohermaphroditism, and
increased risk of gonadoblastoma [169]. At early
stages of Alport, i.e., in children or carrier women,
the glomerular basement membrane shows only
thinning. Some males with classic Alport syn-
drome only have glomerular basement membrane
thinning even at advanced clinical stages [39,
170]. Rarely, Alport patients may have prolifera-
tive lesions with IF and EM deposits within
lamellated GBM areas, suggesting trapping of
immunoglobulins rather than a superimposed
immune complex disease [171]. However, we
have also observed superimposed glomerulone-
phritides in Alport patients.
Immunostaining for type IV collagen chains
can aid in the interpretation of thin basement
membranes [170]. Heterotrimers of α3, α4, and
α5 type IV collagen are a key normal component
of the GBM. Mutation of α5 type IV collagen in
X-linked Alport syndrome, or of α3 or α4
subchains in autosomal forms, prevents incorpo-
ration of the other chains into this heterotrimer
of the GBM. Thus, in kidney biopsies, about
70–80 % of males with X-linked Alport syndrome
lack staining of GBM, distal tubular basement
membrane, and Bowman’s capsule for α3, α4,
and α5 (IV) chains [170]. In autosomal recessive
Alport syndrome (ARAS), due to mutations of
either α3 or α4, the GBMs also usually show no
expression of α3, α4, or α5 type IV collagen. In
contrast to X-linked cases, ARAS patients have
A.B. Fogo
normal expression of α5 type IV collagen in
Bowman’s capsule, distal tubular basement mem-
brane, and skin, where the α5 collagen chain is
part of other heterotrimers. Thus, skin biopsy is
not useful for diagnosis of ARAS, since the
mutated alpha chains are not normally present in
the skin. Female heterozygotes for X-linked Alport
syndrome show mosaic staining of GBM and distal
TBM for α3, α4, and α5 type IV collagen chains
and skin mosaic staining for α5 type IV collagen
due to the lyonization effect. Patients with autoso-
mal dominant Alport syndrome have not been
studied immunohistochemically. Of note, occa-
sional cases with Alport syndrome clinically and
by renal biopsy showed apparent normal α5 type
IV pattern of skin IF staining, and about 20 % of
male X-linked Alport patients show faint or even
normal staining of the GBM for α3 and α5, likely
because the antigenic site recognized by the anti-
body has not been altered by the mutation [170].
Thinning of the GBM is also the characteristic
finding in benign familial hematuria [81, 82]. Most
familial benign hematuria is caused by a carrier state
of ARAS, with mutation of either α3 or α4, causing
thin basement membranes and hematuria [172]. The
diagnosis of thin basement membranes is based on
morphometric measurements from electron micro-
scopic prints and was present in 1.9 % of a large
native kidney biopsy series [173, 174]. LM and
standard IF are normal. The GBM thickness nor-
mally increases with age. Normal thickness in
adults in one series was 373  42 nm in men versus
326  45 nm in women. GBM thickness <250 nm
has been used as a cutoff in many series [175]. In
another series, average was 330  50 nm in males
and 305  45 nm in women [173]. In children, the
diagnosis of thin basement membranes must
be made with caution, establishing normal
age-matched controls within each laboratory. In
our laboratory, we found a range of GBM thickness
in normal children, from approximately 110 nm at
age 1 year to 222  14 nm in seven year olds. As
mentioned above, thin GBM (without lamellation)
is also the early, or may be the only, manifestation in
some kindreds with Alport syndrome and in female
carriers of X-linked Alport. Thus, the presence of
thin GBM cannot per se be taken to categorically
indicate a benign prognosis.
24 Pediatric Renal Pathology
Prognostic Implications of Biopsy
Findings
When the biopsy sample is adequate, extensive,
severe, and irreversible lesions signify a dismal
prognosis for the patient. Globally sclerotic glo-
meruli are not amenable to treatment, although
evidence from human diabetic nephropathy and
animal studies indicates that the earlier stages of
sclerosis may be affected by some therapeutic
interventions and may even be reversible
[176–179]. Similarly, active lesions with ongoing
cellular crescents, necrosis, and inflammatory
infiltrate are potentially dramatically modulated
by therapy, allowing subsequent healing. There
may be minimal irreversible damage to glomeru-
lar structures when intervention occurs early.
Although the renal biopsy may yield a diagno-
sis, there is less information of prognostic indica-
tors in diseases that have a variable course.
Extensive analysis aimed at determining histo-
logic features associated with poor prognosis has
been done in some diseases discussed below.
Classification schemes, especially for lupus
nephritis and membranous glomerulopathy,
imply progression from one stage of disease to
the next. Although sequential biopsies have
illustrated progression from focal to diffuse
proliferative glomerulonephritis in lupus nephri-
tis, patients may change from any class to any
other, with or without treatment, and recurrence
in the transplant may be a different ISN/RPS class
than that observed in the native kidney [22, 23, 25,
27, 159]. In lupus nephritis, patients with less
severe proliferative disease appear to have better
prognoses than those with extensive involvement
of glomeruli, especially with necrosis.
Although the presence of cellular crescents is
associated with activity of disease clinically, the
renal biopsy offers additional prognostic informa-
tion beyond that gleaned from the clinical presen-
tation [27, 180]. Focal and diffuse proliferative
lesions (ISN/RPS III and IV) may present very
similarly clinically, but only the latter appears to
require intense, long-term immunosuppression.
Whether the segmental, more often necrotizing
lupus nephritis lesions have worse outcome than
more diffuse global proliferative lesions is
737
controversial [27, 159]. Lesions of activity in
lupus nephritis include endocapillary prolifera-
tion, necrosis, cellular crescents, and interstitial
inflammatory cells. Lesions that indicate chronic-
ity include tubular atrophy, interstitial fibrosis,
glomerular sclerosis, and fibrous crescents.
Although assessment of activity and chronicity
indices is useful for population groups, these
appear to have less absolute information to guide
assessment in individual patients. Nonetheless, in
large series, assessment of indices of activity and
severity in patients with lupus nephritis or other
diseases has shown some correlation with prog-
nosis and response to therapy. Diffuse prolifera-
tive lesions, extensive crescents, segmental
necrosis, and tubulointerstitial fibrosis are associ-
ated with progression to ESRD [27, 180,
181]. The best prognostic indicator in one study
was the proportion remaining of intact glomeruli
in follow-up biopsies [182].
IgA nephropathy was previously thought to
have a benign prognosis. In a large series of adult
patients with IgA nephropathy, poor prognosis was
indicated by segmental glomerulosclerosis, adhe-
sions or crescents, and tubulointerstitial fibrosis
[183]. Progression occurs in 11–15 % of pediatric
patients [4, 112, 135, 184]. Scoring of activity and
chronicity of lesions has been correlated with clin-
ical course. Activity was assessed by degrees of
crescent formation, mesangial proliferation, and
interstitial infiltrate. Chronicity was scored by
degrees of fibrous crescents, segmental and global
sclerosis, tubular atrophy, and interstitial fibrosis
[185]. The Oxford IgA nephropathy study identi-
fied four lesions associated with worse long-term
outcome, namely, mesangial hypercellularity (M),
i.e., more than half the glomeruli with more than
three nuclei in a mesangial area, endocapillary
hypercellularity/proliferation (E), segmental scle-
rosis (S), and significant (25 %) tubulointerstitial
fibrosis (T), also validated in children with IgA
nephropathy [139–145]. Extension of deposits to
peripheral capillary wall areas has also been
reported as a poor prognostic indicator, but did
not add independent risk beyond the four MEST
criteria of the Oxford IgA nephropathy scoring
[141]. Crescents may also have prognostic
implications [141].
738 A.B. Fogo

Fig. 19 Acute rejection,

classified as type I by
Cooperative Clinical Trials
in Transplantation criteria.
There is interstitial
lymphocytic infiltrate with
tubulitis, activated
lymphocytes, tubular cell
injury, and interstitial
edema (Jones’ stain, 220)

Fig. 20 Acute vascular

rejection, classified as type
II by Cooperative Clinical
Trials in Transplantation or
also by Banff criteria. There
is subendothelial infiltration
by lymphocytes in this
artery, the so-called
endothelialitis (Jones’ stain,
220)

Focal glomerulosclerosis superimposed on
membranous glomerulopathy has been associated
with more severe tubulointerstitial nephritis and a
worse outcome. This lesion was present in 20 % of
children with hepatitis B-associated membranous
glomerulopathy [186].
Renal Transplant Biopsy
The primary use of biopsy in the renal transplan-
tation is to uncover the reason for altered renal
function. Causes of renal dysfunction in the trans-
plant can be broadly divided into those related to
rejection, drug toxicity, recurrent or de novo
disease, and the procedure itself, such as acute
tubular injury/necrosis.
Rejection
Acute rejection is diagnosed by the presence of
either interstitial inflammation with lymphocytes
and plasma cells infiltrating tubules (tubulitis, the
hallmark of acute interstitial type rejection;
Fig. 19) or, when more severe, by extension of
this process to vessels, with subendothelial arte-
rial or arteriolar infiltration by lymphocytes
(endothelialitis, the hallmark of acute vascular
rejection; Fig. 20). The interstitial changes of
acute rejection are not pathognomonic. In con-
trast, the finding of endothelialitis is highly
24 Pediatric Renal Pathology 739

specific for acute vascular rejection. Appropriate

stains, such as PAS, must be used to allow visu-
alization of the tubular basement membrane and
identification of tubulitis. An adequate specimen
for evaluation of possible rejection should contain
at least two cores, with at least seven glomeruli
and two arteries [187].
Several schemes have been used to diagnose
and classify rejection: the Banff scoring system,
based on detailed scoring of various components
of injury, and the Cooperative Clinical Trials in
Transplantation (CCTT) criteria [187–189]. In
both classification schemes, acute rejection is
based on the presence of tubulitis or
endothelialitis, i.e., lymphocytes in the tubule
under the tubular basement membrane or under-
neath the endothelium of arteries. Other inflam-
matory cells (e.g., eosinophils, neutrophils, and
plasma cells), although much fewer in number
than T lymphocytes, may also contribute to the
infiltrate in acute rejection. Type I rejection in
both schemas is diagnosed when interstitial lym-
phocytic infiltrate and tubulitis are present
(>25 % of parenchyma infiltrated in Banff,
>5 % in CCTT) (Fig. 20). Infiltrate and tubulitis Fig. 21 Antibody-mediated rejection is characterized by
C4d positivity in peritubular capillaries (200)
less than specified for type I is called “borderline”
by Banff criteria [188, 189]. Type II acute vascular
rejection is diagnosed in both schemas when there follow-up. In contrast, treatment of such subclin-
is mild or moderate endothelialitis (arteritis). ical rejection in the early time period after trans-
Severe acute vascular rejection, type III, is diag- plantation resulted in better preserved renal
nosed when there is transmural vascular inflam- function at 24 months [191].
mation and/or fibrinoid necrosis. These types are There are no specific IF or electron micro-
differentiated not only based on histologic pattern scopic immune complexes associated with acute
but also on differences in underlying mechanisms rejection. Molecular studies indicate the possibil-
and response to therapies: type I and II are likely ity of earlier, more sensitive, and specific diagno-
T-cell-dependent processes and are separated sis of acute rejection using these techniques (see
based on the likely greater clinical severity of below) [192]. In particular, the presence of C4d, a
any rejection when endothelialitis is present, complement breakdown product which binds
whereas antibody-mediated mechanisms contrib- covalently to tissue, in peritubular capillaries is
ute to type III changes. Isolated type II highly associated with anti-donor antibodies
endothelialitis lesions are often associated with (humoral rejection) [193] (Fig. 21). Diagnosis of
antibody-mediated rejection [190]. humoral, antibody-mediated rejection has impor-
Identification of acute rejection at earlier stages tant therapeutic and prognostic implications. C4d
and thus initiation of treatment at milder levels of staining can be done on frozen or fixed paraffin-
injury appear to be clinically important. Thus, processed tissue, although the latter is less
mild tubulitis that is borderline by Banff criteria, sensitive [194].
even in normally functioning grafts, was found The changes of chronic rejection include inti-
to be predictive of higher serum creatinine at mal fibrosis of arteries, interstitial fibrosis, and
740
transplant glomerulopathy [188, 195]. A previous
or baseline biopsy is necessary to prove that inti-
mal fibrosis is de novo and potentially represents
chronic rejection, rather than a preexisting,
nonspecific change in the graft. Interstitial fibrosis
is also a nonspecific finding and may result from
various injuries. Transplant glomerulopathy is a
more specific lesion indicative of chronic rejec-
tion and is particularly linked to previous
antibody-mediated rejection. By LM, the glomer-
uli show basement membrane double contours
and corrugation and even segmental sclerosis
with hyalinosis. The latter lesion likely resulted
in reports of de novo “idiopathic FSGS” in
the transplant. However, in transplant
glomerulopathy, there is widening of the lamina
rara interna of the GBM with cellular interposition
and new basement membrane formation by
EM. Reduplication of basal lamina of peritubular
capillaries is suggested to be more specific for
transplant glomerulopathy but may also occur in
some other glomerular diseases and HUS [196].
Calcineurin Inhibitor Toxicity
Calcineurin inhibitor toxicity may manifest in
various ways. Tacrolimus (FK506) has much the
same spectrum of toxicity as cyclosporine
[197]. The most common morphologic lesion in
patients with a clinical diagnosis of cyclosporine
toxicity, as verified by clinical follow-up, is that of
a normal kidney biopsy morphologically. In these
patients, renal dysfunction is due to reversible,
calcineurin inhibitor-induced vasoconstriction
and hypofiltration. Morphologic changes sugges-
tive, but not pathognomonic, of calcineurin inhib-
itor toxicity include arteriolopathy with injury to
the endothelium and vascular smooth muscle
cells. In its classic form, this injury results in
nodular IgM IF positivity along the apical side
of the arteriole, with necrosis and smooth muscle
cell injury demonstrated by EM [197]. By LM,
concentric hyalinosis is present, whereas typically
eccentric, more segmental hyalinosis is associated
with hypertension. Isometric tubular
vacuolization in a patchy distribution, although
not specific, is also indicative of cyclosporine
toxicity; calcineurin inhibitor toxicity results in a
A.B. Fogo
striped distribution of interstitial fibrosis caused
by injury along the medullary rays [195]. This
pattern often cannot be gleaned in small needle
biopsies. FSGS with ischemic, corrugated GBMs
in remaining glomeruli may also result from
calcineurin inhibitor toxicity and can be associ-
ated with significant proteinuria [197].
Calcineurin inhibitors have also been associ-
ated with thrombotic microangiopathy lesions
(see above) [197]. Of note, thrombotic
microangiopathy can occur in patients who are
recipients of transplants of kidneys or other
organs and following radiation, with or without
calcineurin inhibitor treatment. In some patients,
collapsing-type glomerulosclerosis may be asso-
ciated with cyclosporine toxicity, likely
representing a response to severe vascular injury
and ischemia and usually with less complete foot
process effacement than in idiopathic collapsing
glomerulopathy in the native kidney, and with
improvement on cessation of CNI therapy [198].
Recurrent and De Novo Disease
Recurrent and de novo diseases are important
causes of renal allograft injury, affecting approx-
imately 10 % of renal allografts [199]. Of all graft
loss, 2–4 % is due to recurrence of disease. IF
microscopy should be performed in all transplant
biopsies to rule out this possibility. When IF or
LM findings in conjunction with the clinical set-
ting indicate an undetermined lesion, electron
microscopic study should also be performed [68].
In children, the most common recurrent dis-
eases include IgA nephropathy and Henoch-
Schönlein purpura, MPGN, dense deposit disease,
and FSGS [200]. Although SLE has been reported
to recur only rarely, our experience indicates a
recurrence rate of approximately 30 %
[201]. However, morphologic recurrence of dis-
ease does not necessarily lead to graft loss
[200]. Dense deposit disease recurs morphologi-
cally in nearly all patients, with worse graft out-
come [14]. In contrast, HUS has an overall
recurrence rate of 15–25 %, reflecting largely
invariable recurrence in those with atypical HUS
due to inherited complement dysregulation, with
high rates of graft loss. Although IgA
24 Pediatric Renal Pathology
nephropathy recurs morphologically in approxi-
mately 50 % of patients, only 10 % of grafts
with recurrent disease are lost. MPGN recurs in
20–30 %, with 10–40 % graft loss. FSGS recurs
in 20–30 % of cases, resulting in graft loss in
30–50 % of these. Of note, in recurrent FSGS,
the only morphologic change found in the first
weeks after recurrence of proteinuria is foot pro-
cess effacement, with early segmental sclerosis
detectable at 6–8 weeks after recurrent NS.
De novo disease may also affect the transplant.
Membranous glomerulopathy is the most com-
mon de novo glomerulonephritis in the transplant
and is linked to antibody-mediated rejection
[202]. Glomerulonephritis related to infections,
such as hepatitis C-related MPGN, also can
occur in the transplant. Early changes of diabetic
nephropathy develop much more rapidly in the
transplant than in the native kidney and
may occur within a few years, whether diabetes
preexisted or is corticosteroid induced [203, 204].
Thrombotic microangiopathy may be related to
drug toxicity (see above) or be idiopathic in the
transplant.
Posttransplant lymphoproliferative disease
(PTLD) is due to the unrestrained proliferation
of B lymphocytes, most often because of transfor-
mation by Epstein-Barr virus, and is an aggressive
process, which, if untreated, disseminates and
may cause death [205]. PTLD may respond to
decreased immunosuppression. An expansile
lymphoid infiltrate with atypical, transformed
lymphocytes and serpiginous necrosis are features
suggestive of PTLD [205]. Immunohistochemical
studies can be used to detect Epstein-Barr virus to
further support this diagnosis. Typing studies of
the lymphocytic infiltrate are not often helpful
because most PTLD is polytypic, rather than
clonal. Of note, acute rejection and PTLD may
be present concurrently.
Polyoma virus nephropathy (PVN), most often
due to the BK virus, has increased in the last years
in the transplant, perhaps related to increased
immunosuppression [90]. PVN occurs in both
adult and pediatric transplant recipients
[206]. The biopsy shows a pleomorphic infiltrate
with lymphocytes, plasma cells, PMNs, and
741
occasional eosinophils, with enlarged tubular
cells with smudgy nuclei. Early lesions (stage A)
with minimal inflammation, fibrosis, or injury
have better prognosis than stage B with marked
viral changes and inflammation and moderate
fibrosis, or stage C, with extensive fibrosis
[90]. Polyoma virus infection is confirmed by
immunostaining for SV40 large T antigen present
in replicating polyoma virus (includes BK, JC and
SV40). In some polyoma virus nephropathy
cases, there may be associated granular tubular
basement membrane deposits staining with IgG
and C3 and visualized by EM [207]. Polyoma
virus nephropathy can respond to decreased or
changed immunosuppression and antiviral
therapy.
New Methods for the Future
With the recent surge of application of molecular
biology techniques to the study of renal disease,
candidate factors involved in pathogenesis and
progression of disease are being studied in animal
models. Studies in human beings have also com-
menced. With further development of such stud-
ies, we may identify specific abnormal processes
and thus target therapy more specifically.
Research techniques that have been advanta-
geously applied to elucidate pathogenesis of
disease include immunostaining; identifying spe-
cific antigen in deposits of membranous
glomerulopathy, e.g., PLA2R in primary membra-
nous GP or causes of secondary membranous GP
(thyroglobulin with Hashimoto’s disease and hep-
atitis B and C antigen), and light chains or
paraproteins in plasma cell dyscrasia-associated
diseases; identification of specific type IV colla-
gen abnormality in Alport syndrome, C4d as a
marker of humoral rejection, and novel insights
into C3 glomerulopathies; and elucidation of
pathogenesis of specific E. coli-associated toxins
in some forms of HUS.
Current studies are aimed at understanding
disease etiologies and mechanisms at a molecular
and proteomic level, studying renal biopsies by
laser capture microdissection (LCM), with mass
742
spectrometry, real-time reverse transcription poly-
merase chain reaction (RT-PCR), and in situ
hybridization techniques [114–116, 208, 209].
Recent efforts have expanded use of these tech-
niques to study mechanisms of injury and progres-
sion. Competitive and real-time RT-PCR have
been used successfully on small cores and even
single isolated glomeruli from human biopsies
[114, 116]. Modulation of growth factors, colla-
gens, cytokines, chemokines, and their receptors
has been investigated molecularly in renal biop-
sies. Cytokines and chemokines and their recep-
tors were upregulated in diseases with
macrophage influx and mesangial cell prolifera-
tion, supporting an important role in initiating and
perpetuating injury. Such approaches can offer
exciting new mechanistic insights into renal
diseases.
In parallel, genetic studies are targeted both at
diagnosis and identifying patients at risk for pro-
gression in diseases with a variable course, such
as diabetes and IgA nephropathy. Genetic screen-
ing for key podocyte-related mutations is often
done in children with FSGS. Noninvasive urinary
proteomic and microvesicle studies are also
explored to identify particular signature patterns
indicative or predictive of specific lesions or risks
[210–212].
Diagnostic use of RT-PCR and in situ
hybridization have focused on detection of
viruses, including hepatitis B and C, cytomegalo-
virus, polyoma virus, and Epstein-Barr virus.
Endothelial injury transcripts in kidney
biopsies may allow diagnosis and earlier
detection of specific types of injury in the
transplant [213].
Together, these approaches promise to map
risks and mechanisms of disease initiation and
progression and point to targets to achieve resolu-
tion of injury. Sequential biopsies with evaluation
of changes in structure and patterns of abnormal
factors and modulation by therapy may be neces-
sary to fully understand pathogenesis. Instead of
diagnoses of morphologic patterns recognized by
current techniques in the renal biopsy specimen,
molecular techniques may allow more precise
diagnosis of the specific diseases and identifica-
tion of injury mechanisms.
A.B. Fogo
Acknowledgments The author wishes to thank Drs. Tina
Kon, Kathy Jabs, and Tray Hunley for their suggestions.
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24 Pediatric Renal Pathology
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 Part V
Glomerular Disease
 Congenital Nephrotic Syndrome
Hannu Jalanko and Christer Holmberg
Contents
Glomerular Filtration Barrier . . . . . . . . . . . . . . . . . . . . . 755
Glomerular Capillary Wall and Podocytes . . . . . . . . . . 755
Slit Diaphragm Components . . . . . . . . . . . . . . . . . . . . . . . . 755
Primary Nephrotic Syndrome . . . . . . . . . . . . . . . . . . . . . 756
CNS of the Finnish Type (CNF, NPHS1) . . . . . . . . . . . 756
Podocin Gene Mutations (NPHS2) . . . . . . . . . . . . . . . . 760
Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760
Clinical Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760
WT1 Gene Mutations (Denys–Drash,
Isolated CNS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 761
Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 761
Clinical Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 761
Laminin β2 Gene Mutations
(Pierson Syndrome) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 762
PLC«1 Gene Mutations (NPHS3) . . . . . . . . . . . . . . . . . 763
Other Forms of Primary CNS . . . . . . . . . . . . . . . . . . . . . . . 763
H. Jalanko (*) • C. Holmberg
Hospital for Children and Adolescents, University of
Helsinki, Helsinki, Finland
e-mail: hannu.jalanko@hus.fi; christer.holmberg@hus.fi
# Springer-Verlag Berlin Heidelberg 2016
E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_78
25
Secondary Nephrotic Syndromes . . . . . . . . . . . . . . . . . . 764
Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 764
Immune Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765
Diagnosis of CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765
Clinical Findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765
Kidney Biopsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765
Gene Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 766
Management of CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 766
Immunosuppressive Medication . . . . . . . . . . . . . . . . . . . . . 766
Antiproteinuric Medication . . . . . . . . . . . . . . . . . . . . . . . . . . 767
Albumin Substitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 767
Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 767
Additional Medication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768
Unilateral Nephrectomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768
Dialysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 769
Kidney Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 769
Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 769
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 770
753
754
Renal diseases associated with nephrotic syn-
drome (NS) in the first year of life are uncommon
and make up a heterogeneous group of disorders
[73]. Congenital nephrotic syndrome (CNS) is
defined as proteinuria manifesting in the first
3 months of life. NS appearing later during the
first year (4–12 months) is defined infantile, and
NS manifesting thereafter is called childhood
NS. While this classification is used to help the
Table 1 Classification of congenital nephrotic syndrome
(CNS)
Primary CNS
Nephrin (NPHS1) gene mutations (NPHS1, CNF,
isolated NS)
Podocin (NPHS2) gene mutations (NPHS2, isolated
NS)
Phospholipase C epsilon 1 (PLCE1) gene mutations
(NPHS3, isolated NS)
Wilms tumor suppressor 1 (WT1) gene mutations
(Denys–Drash, isolated NS)
Laminin β2 (LAMB2) gene mutations (Pierson
syndrome, isolated NS)
Laminin β3 (LAMB3) gene mutations (Herlitz
junctional epidermolysis bullosa)
Lim homeobox transcription factor 1b (LMXB1)
mutations (nail–patella syndrome)
OCRL gene defect (Lowe)
ARHGDIA gene mutations (RhoGDIA defect)
Mutations in genes encoding mitochondrial coenzyme
Q10 synthesis (COQ2, COQ6, ADCK4)
Galloway–Mowat (gene defect not yet known)
Secondary CNS
Congenital syphilis
Toxoplasmosis, malaria
Cytomegalovirus, rubella, hepatitis B, HIV
Maternal systemic lupus erythematosus (SLE)
Neonatal antibodies against neutral endopeptidase
Maternal steroid–chlorpheniramine treatment
Table 2 Prevalence of genetic findings of CNS patients in four international studies
Study No NPHS1 (%)
Hinkess (6) 46 39
Heeringa (7) 42 40
Machuca (8) 117 54
Santini (9) 15 80
H. Jalanko and C. Holmberg
clinical diagnosis, it is arbitrary in the sense that
NS caused by a specific gene mutation can
manifest soon after birth or later in life. However,
since the management of CNS is often
different from the more common forms of child-
hood NS, the terminology still seems warranted
(Table 1).
Primary CNS is typically caused by mutations
in genes encoding for components of the glomer-
ular filtration barrier [100, 147, 203, 216]. The
classical form is the Finnish type of CNS (CNF,
NPHS1), which is caused by mutations in nephrin
gene (NPHS1) leading to massive proteinuria,
hypoproteinemia, and edema in the newborn
period. Other important genes causing CNS are
podocin gene (NPHS2), Wilms’ tumor factor
1 gene (WT1), laminin β2-gene (LAMB2), and
PLCe1 gene (PLCe1, NPHS3). These disorders
have a more widespread age of onset and
also more variable clinical manifestations.
Mutations in these five genes cover the
majority (>80 %) of the genetic defects observed
in CNS patients (Table 2) [83, 85, 133, 183]. Even
though CNS is a rare disorder, new genetic forms
are repeatedly discovered. These include NS
caused by mutations in ARHGDIA gene [57, 70]
and in genes encoding for enzymes taking part
in mitochondrial coenzyme Q10 (ubiquinone)
biosynthesis [45, 186]. In the genetic forms of
CNS, proteinuria can be an isolated disorder or
part of more generalized syndrome. It is important
to notice that CNS can also be caused by autoim-
mune disorders and neonatal infections, espe-
cially in Africa and Asia. Thus, the diagnosis of
a patient with early manifestations of NS must be
based on several criteria including clinical presen-
tation, family history, laboratory findings, genetic
testing, and renal histology.
NPHS2 (%) WT1 (%) LAMB2 PLCE1
39 2.2 4.4 % n.a.
17 8 n.a. 8%
15 1.7 1.7 % 1.7 %
7 13 n.a. 0%
25 Congenital Nephrotic Syndrome
Fig. 1 A cross-sectional image of one glomerular capil-
lary (left) and glomerular capillary wall (right), which
comprises three layers: fenestrated endothelium, glomeru-
lar basement membrane (GBM), and epithelial cell layer
with podocyte foot processes and interposing slit dia-
phragms (SDs). The four most important molecules in the
Glomerular Filtration Barrier
Glomerular Capillary Wall
and Podocytes
The main feature of primary CNS is the extensive
leakage of plasma proteins into urine through the
kidney filtration barrier (Fig. 1) [42, 102]. This
barrier is located in the glomerular capillary wall
and comprises three layers: fenestrated endothe-
lium, glomerular basement membrane (GBM),
and epithelial cell (podocyte) layer with distal
foot processes and interposed slit diaphragms
(SDs) (Fig. 1). The filter is a highly sophisticated
size- and charge-selective molecular sieve, and
normally only water and small plasma solutes
pass into Bowman’s space [40, 111]. As
100–200 l of protein free filtrate (primary urine)
is formed daily, one can calculate that 3–6 kg of
albumin has to be prevented from crossing the
glomerular filtration barrier each day.
The flow of glomerular filtrate follows the
extracellular route, passing across the GBM and
SD, which bridge adjacent foot processes just
above the GBM. The GBM is a protein network
formed by type IV collagen, laminin, nidogen,
and negatively charged proteoglycans. The role
of GBM in glomerular permselectivity has been
debated, but it is now known that a primary defect
755
pathogenesis of CNS are also depicted. WT1 is a transcrip-
tion factor important for podocyte functions. PLCε1 is a
cytoplasmic enzyme involved in cell signaling. Nephrin is
a major component of SD and podocin is an adapter and
scaffold protein for the SD components
in a GBM component, laminin, results in NS [35,
103]. Also, mutations a gene encoding integrin α3
(ITGA3) may lead to nephrotic syndrome indicat-
ing that the binding of the GBM to podocyte
(cytoskeleton) is important for the glomerular fil-
tration barrier [149].
Podocytes and SD are, however, even more
important in preventing proteinuria than the GBM
[40, 124, 203]. Podocytes are specialized cells that
consist of the cell body and primary, secondary, and
tertiary processes. The cytoskeleton of the cell body
and major processes consist of microtubules and
intermediate filaments, whereas actin is the major
cytoskeletal component of foot processes. Actin
network and the interacting proteins, such as
α-actinin-4, inverted formin-2, myosin-9, myosin-
1E, and ARHGDIA, maintain the architecture of the
foot processes and respond to signals from outside,
modifying the cellular functions accordingly.
Genetic defects in these five interacting proteins
cause NS manifesting in older children and adults
[11, 24, 27, 108, 115, 142, 181, 192, 212]. Some-
what surprisingly, there have so far not been reports
on CNS cases caused by these five genes.
Slit Diaphragm Components
The precise molecular structure of SD is still
unresolved. The first SD component identified
756
Fig. 2 The nephrin molecule is a transmembrane cell
adhesion protein with small intracellular and transmem-
brane domains and a larger extracellular domain. The
extracellular domain contains eight Ig-like modules and
one fibronectin type module adjacent. The extracellular
was nephrin, which is a 1,241-residue cell adhe-
sion protein of the immunoglobulin family
[92, 112, 174, 206]. The extracellular part of
nephrin contains eight immunoglobulin-like mod-
ules and one type III fibronectin domain (Fig. 2).
The intracellular domain contains nine tyrosine
residues, which may become phosphorylated by
protein kinases. This phosphorylation modulates
the interaction of nephrin with other proteins, such
as adaptor protein Nck and phosphoinositide
3-kinase (PI3K) [94, 106, 217]. These proteins
link nephrin to actin cytoskeleton, suggesting an
important role for nephrin in the regulation of the
actin cytoskeleton and podocyte morphology.
Nephrin takes part also in other cell signaling
processes involved in the podocyte cell survival
[19, 125].
In addition to nephrin, several other podocyte
proteins have been identified in the SD, such as
Neph1, Neph 2, Neph3, and FAT 1 and FAT 2 [46,
97, 173, 194]. According to the present under-
standing, nephrins associate with the Neph pro-
teins extracellularly and form the backbone of SD
[10, 58, 191]. Neph proteins are structurally
related to nephrin and they are also involved in
the cell signaling [191]. Mice with genetic defects
in Neph 1 develop heavy proteinuria, but no
humans with mutations in Neph1 have so far
been identified [46]. Another interesting SD com-
ponent is transient receptor potential cation chan-
nel 6 (TRPC6), which is a cation channel that
interacts with nephrin [207]. Mutations in this
gene lead to autosomal dominant FSGS
manifesting in adulthood but not in infants.
Nephrin and Neph proteins interact with the
adapter proteins podocin, CD2AP, and ZO-1 that
H. Jalanko and C. Holmberg
part contains three cysteine residues taking part in molec-
ular interactions. The intracellular part contains several
tyrosine residues phosphorylated by protein kinases,
which is important for cellular signaling
are located in the cytosolic part of the podocyte
foot process [23, 190, 208]. Podocin is encoded
by the NPHS2 gene and it is a small hairpin-like
protein belonging to a raft-associated stomatin
family. It is a scaffolding protein and serves in
the structural organization of the SD as well as in
the signal transduction from the SD into
podocytes [94, 95]. Genetic mutations in podocin
result in CNS as well as NS manifesting later
in life.
Primary Nephrotic Syndrome
CNS of the Finnish Type (CNF, NPHS1)
CNF originally denoted to a severe form congen-
ital nephrotic syndrome typically seen in the Finn-
ish newborns [75, 154]. After the gene (NPHS1)
responsible for this disorder was isolated, both
CNF and NPHS1 have been used as abbreviations
for the same disorder. The disease is highly
enriched in Finland, the incidence being 1:8,200
live births [96]. However, patients are reported all
over the world among various ethnic groups
[7, 16, 117, 187]. A very high incidence (1/500
live births) of CNF has been reported among the
Old Order Mennonites in Lancaster County,
Pennsylvania [22]. In recent reports of genetic
findings in CNS patients coming from all over
the world, mutations in the NPHS1 gene
have accounted for 40–80 % of the cases
(Table 2) indicating that NPHS1 mutations
are the most important cause of genetic CNS
[83, 85, 133, 183].
25 Congenital Nephrotic Syndrome
Genetics
CNF is inherited as an autosomal-recessive trait.
The NPHS1 gene is located to chromosome
19q13.1 and exon sequencing analysis has
revealed two important mutations in over 90 %
of the Finnish CNF patients (Fin-major and
Fin-minor) [112]. Both mutations result in a stop
codon and a truncated nephrin protein not
expressed in the SD. The Fin-major and
Fin-minor mutations are rare outside Finland,
but enrichment of other mutations has been
reported also in non-Finns. In Mennonites,
1481delC mutation is common and leads to a
truncated protein of 547 residues [22]. On the
other hand, a homozygous nonsense mutation
R1160X in exon 27 has been found in all Maltese
patients [117]. Six of the 16 cases with this muta-
tion had an atypically mild disease. The same
mutation has been reported in six French CNF
patients and two of them had a mild disease.
Recently enrichment of c.2131C > A missense
mutation was observed in Maori patients from
New Zealand [209]. Over two hundred NPHS1
mutations have been identified and most
non-Finns have individual mutations including
deletions, insertions, nonsense, missense, and
splicing mutations spanning over the whole
gene. While most NPHS1 mutations lead to
CNS, it has become clear that some “mild” muta-
tions can lead to NS manifesting in older children
or even in adults expanding the phenotype caused
by this gene defect [156, 163, 184].
Pathogenesis
Mutations in the NPHS1 gene result in defective
nephrin molecule and disturbed structure and
function of the glomerular filtration barrier. The
Fin-major and Fin-minor mutations cause a com-
plete absence of nephrin in SD, and these kidneys
lack the filamentous image of SD in electron
microscopy (Figs. 3 and 4) [159]. Similarly,
nephrin knockout mice lack the slit diaphragm
filaments in electron microscopy and die of severe
proteinuria soon after birth [165]. The findings
suggest that the absence of nephrin leads to leak-
age of plasma proteins into urine through the
“empty” podocyte pores. Liu et al. [130] have
shown that many missense mutations cause
757
misfolding of nephrin protein and defective intra-
cellular nephrin transport with absence of nephrin
in SD as well. This most probably explains why
even “mild” mutations can cause heavy protein-
uria and a severe form of CNS.
The expression of nephrin was first reported to
be restricted to glomerular podocytes and this was
later confirmed [112, 144]. The data obtained
especially from mice, however, suggest that
nephrin may also be expressed in some areas of
the central nervous system, pancreatic beta cells,
and testis [165]. No constant extrarenal manifes-
tations, however, are observed in CNF patients
speaking against a significant role of nephrin in
other organs than the kidney [119].
Renal Pathology
The CNF kidneys develop quite normally in the
fetal period [172, 175]. At the 16–22 gestational
weeks, the glomeruli show little changes in light
microscopy, but occasional dilated tubuli may be
seen. In electron microscopy the mature glomeruli
show effacement of podocyte foot processes, and
the SD image may be completely missing in cases
with severe NPHS1 mutations [159]. It is remark-
able that also carriers of NPHS1 mutations can
have these “proteinuric” changes making the
pathological diagnosis difficult [160].
After birth, the CNF kidneys are large with the
mean kidney weight of almost twice that of
age-matched control children [172]. The glomer-
uli show a slight to moderate increase of
mesangial matrix and mesangial hypercellularity
(Fig. 5). Degenerative changes such as shrinking
of the glomerular tuft, fibrotic thickening of the
Bowman’s capsule, and glomerular sclerosis
become evident with time [122]. Of note, the
same kidney biopsy may show variable
glomerular changes as depicted in Fig. 5. Electron
microscopy shows effacement of foot processes,
irregularities in the GBM, and some swelling of
the endothelial cells (peliosis) [110]. Dilated
proximal tubules are few at first, but radial dila-
tions of the tubules become obvious with time.
Also, interstitial fibrosis and lymphocyte
collections especially around sclerotic glomeruli
increase substantially within the first 1–2
years [120].
758 H. Jalanko and C. Holmberg

Fig. 3 Electron
microscopy of the
glomerular capillary wall.
(a) Normal kidney showing
the podocyte foot processes,
the glomerular basement
membrane, and the
fenestrate endothelium. (b)
NPHS1 kidney with
irregular podocyte foot
processes (effacement)
typical for all proteinuric
kidney diseases. (c)
Immunoelectron microcopy
of a podocyte pore in a
normal kidney. The
filamentous image of the slit
diaphragm (S) is seen
(arrow). Antibodies against
the intracellular part of
nephrin show the
localization of nephrin in
the S (gold particles). (d):
Immunoelectron
microscopy of the podocyte
slit pore in a NPHS1 kidney.
No SD or nephrin is seen

Fig. 4 Scanning electron microscopy of a capillary wall in structure is distorted. The cell body (arrow) “hang” in a
a normal and a NPHS1 kidney. (a). The podocyte cell body primary process and the ramification of secondary pro-
(arrow) and the primary, secondary, and tertiary processes cesses is absent
are clearly seen. (b). In the NPHS1 kidney, the podocyte
25 Congenital Nephrotic Syndrome
Fig. 5 Typical histological findings in CNF kidneys demonstrating the variability of glomerular lesions. The findings
range from normal to severely sclerotic glomeruli indicating that the etiological diagnosis requires genetic testing
Clinical Features
The basic problem in CNF is severe renal loss of
plasma protein, which may begin already in utero.
The early signs and symptoms are secondary to
this protein deficiency [75, 96, 159]. In a survey of
the Finnish patients, over 80 % of the children
were found to be born prematurely (<38th week),
with a mean birth weight of 2,600 g
(1,500–3,500). However, only two of the 46 new-
borns were small for gestational age (65). Most
neonates do not have major pulmonary problems,
although amniotic fluid is often meconium
stained. The placenta is larger than normal and
almost invariably weighs more than 25 % of the
baby’s birth weight. The mean ratio of placental to
infant weight (ISP) is 0.38 in babies with CNF
compared with 0.18 in normal babies. The reason
for this is not known.
In typical CNF, edema and abdominal disten-
sion become evident soon after birth. NS was
diagnosed within the first week in 82 % of the
Finnish patients and within 2 months in the rest of
the cases. In a survey of European and Turkish
patients, CNF manifested within the first 90 days
in all 18 cases [85]. Protein losses lead to severe
hypoalbuminemia (<10 g/L) before protein sub-
stitution is started [88]. Without albumin substi-
tution and nutritional support, the classical
759
features of CNF, such as generalized edema,
abdominal distension, ascites, and widened cra-
nial sutures and fontanelles, may develop during
the first weeks or months of life.
Infants with CNF do not have extrarenal
malformations. Minor functional disorders in the
central nervous system and heart, however, are
quite common during the nephrotic stage. Most
children have muscular hypotonia, and mild atro-
phic changes in the brain were observed by com-
puter tomography (CT) or magnetic resonance
imaging (MRI) in a third of the Finnish infants
[159]. In 8 % of the Finnish patients, dystonic
cerebral palsy has been diagnosed; the etiology
of this is not known. Minor cardiac findings such
as hypertrophy and mild pulmonary stenosis have
been reported in one-fourth of the Finnish patients
[159]. In a report form Malta, pulmonary valve
stenosis was found in three cases and a subaortic
stenosis in one patient [67].
Laboratory Findings
The magnitude of proteinuria depends on the
“severity” of the NPHS1 mutations. Typically,
the first urine analysis already shows proteinuria,
microscopic hematuria, and some leukocyturia. If
the serum albumin concentration is not low (<15
g/L), the urinary protein concentration usually
760
exceeds 20 g/L. In addition to albumin, many
other proteins are lost into urine: immunoglobulin
G (IgG), transferrin, apoproteins, lipoprotein
lipase (LPL), antithrombin III (ATIII), ceruloplas-
min, vitamin D binding protein, and thyroid bind-
ing globulin [3]. The serum levels of these and
their ligands (e.g., thyroxin) are low in serum,
leading to secondary metabolic disturbances.
The low thyroxin concentration leads to an
increase in thyroid-stimulating hormone
(TSH) [141].
Low serum albumin and postheparin plasma
LPL activities and high free fatty acid concentra-
tions lead to hypertriglyceridemia. Total and
low-density lipoprotein (LDL) cholesterol levels
are high but high-density lipoprotein (HDL)
levels are low, and the LDL and HDL particles
are enriched with triglycerides [4]. These lipid
abnormalities and arteriolar changes seen already
during the first year of life may lead to an
increased risk of arteriosclerosis. Deficiency in
polyunsaturated fatty acids may be prominent in
infants with CNS, which may be regarded as a
possible risk factor for the developing brains [1].
Podocin Gene Mutations (NPHS2)
Genetics
Mutations in the NPHS2 gene, encoding for a
podocyte protein podocin, are a common cause
of a steroid-resistant NS (SRNS) in children and
adults, accounting for up to 28 % of sporadic and
over 40 % of familial cases of SRNS manifesting
at various ages [30, 31, 52, 109, 157, 178]. The
podocin gene mutations, however, are also an
important cause of CNS accounting for 17–39 %
of the cases (Table 2).
NPHS2 mutations were found in 18 % of over
400 patients from a worldwide cohort of SRNS
cases with the disease onset ranging from birth to
21 years (88). Severe truncating mutations (non-
sense or frameshift) resulted in early onset of NS
(from birth to 9.1 years of age; mean 1.75 years).
This was also true for homozygous R138Q muta-
tion (from birth to 5.4 years; mean 1.77 years),
which is common in European patients. The fact
H. Jalanko and C. Holmberg
that identical mutations may result in onset of
SRNS over spectrum of several years suggests
that additional modifying factors are involved.
Since podocin is a podocyte adapter protein
required for the proper targeting of nephrin into
SD, also nephrin expression may be distorted in
CNS caused by NPHS2 mutations [66, 94, 152,
188]. Coexistence of NPHS1 and NPHS2 muta-
tions has been reported in CNS patients, but the
clinical significance of this is not clear [117, 188].
CNS caused by NPHS2 mutations is an autosomal
recessive disease requiring disease-causing muta-
tions on both alleles. Interestingly, it was recently
found that p.Arg229Gln mutation leads to disease
phenotype only when it is associated with some
30 -mutations [201].
Clinical Features
No systematic analysis of the clinical findings in
CNS patients with NPHS2 mutations has been
published. It is, however, obvious that the severity
of proteinuria and the clinical findings are more
variable than in CNF patients. While nephrotic
range proteinuria is typically detected in the first
few days of life in CNF patients, CNS often man-
ifests at the age of a few weeks in infants with
NPHS2 mutations [18]. In the study of Machuca
et al., 74 % of all patients presenting NS during
the first week of life had NPHS1 mutations,
whereas only 11 % carried NPHS2 mutations.
Conversely, the latter gene mutations accounted
for 37 % of patients in whom NS began after
1 month of life, whereas NPHS1 mutations were
less frequent (16 %) at this stage [133]. CNS with
NPHS2 mutations develops end stage renal dis-
ease (ESRD) on average 6.6 years after diagnosis
[85, 86]. Patients with p.R138Q mutations
progressed to ESRD at the median age of
79 months, whereas patients carrying the p.
V260E mutations progressed more rapidly
[133]. As podocin is only expressed in kidney
glomerulus, non-renal manifestations are unlikely
as is the case with CNF. The NPHS2 founder
mutation R138X was associated with cardiac
anomalies in six consanguineous Arabs
[51]. However, in another cohort of 12 patients,
25 Congenital Nephrotic Syndrome
10 had a normal heart condition and two presented
mild anomalies with mitral insufficiency and left
ventricular hypertrophy [32].
In general, most patients with NPHS2 muta-
tions show histology of focal segmental
glomerulosclerosis (FSGS) in kidney biopsy.
However, CNS patients with NPHS2 mutations
often show minimal histological changes or
CNF-type histology, and FSGS is seen in less
than half of the patients [133]. Thus, it is clear
that CNS caused by NPHS1 or NPHS2 mutations
cannot be differentiated by the renal histology or
clinical findings.
WT1 Gene Mutations (Denys–Drash,
Isolated CNS)
Genetics
Wilms’ tumor suppressor gene (WT1) is located
on chromosome 11p13 [29]. It encodes for a
nuclear WT1 protein, which is a transcription
factor of the zinc finger family presumed to regu-
late the expression of numerous target genes
through DNA binding [145]. WT1 plays a crucial
role in the embryonic development of the kidney
and genitalia. In mature kidney, WT1 is abun-
dantly expressed in podocytes and it is believed
to control the expression the SD proteins, such as
nephrin [198]. The WT1 gene contains 10 exons,
the first six of which encode a proline-/glutamine-
rich transcriptional regulatory region. Exons 7–10
encode the four zinc fingers of the DNA-binding
domain. Up to 24 different isoforms of WT1 may
result from the combination of alternative trans-
lations sites, alternative RNA splicing, and RNA
editing. The biological role of all these isoforms is
not known.
Clinical Features
A variety of WT1 mutations, which either affect
development or induce tumor formation, have
been identified. Developmental defects include
the Denys–Drash syndrome (DDS), Frasier syn-
drome (FS), and WAGR syndrome (Wilms’
761
tumor, aniridia, genitourinary anomalies, and
mental retardation) [148]. Missense mutations
mostly cause DDS, donor splice site mutations
in intron 9 are associated with FS, and constitu-
tional deletion of one whole copy of WTI is asso-
ciated with WAGR. FS is characterized by the
association of male pseudohermaphroditism and
glomerulopathy [9, 114]. There is complete male
to female gender reversal in 46, XY patients. FS is
associated with gonadoblastomas but not with
Wilms’ tumor. Proteinuria is detected in child-
hood, usually between 2 and 6 years of age, and
kidney biopsy reveals FSGS.
DDS is caused by heterozygous mutations in
WT1 [59, 162]. More than 60 germline mutations
(both familial and de novo) have been described in
DDS patients. Most are missense mutations
within exons 8 and 9, coding for zinc finger
domains 2 and 3, which leads to alteration in the
DNA-binding capacity of WT1 [128]. Also dele-
tions, insertions, and nonsense mutations have
been described in DDS patients leading to a trun-
cated protein [189]. The mutant protein probably
suppresses the normal allele, which explains the
more severe phenotype seen in DDS compared
with children with complete deletion of one
WT1 allele. The podocyte function is affected,
and, at the molecular level, upregulation of
PAX2 and downregulation of nephrin have been
reported [146]. Also, the expression of growth
factors that regulate glomerular capillary develop-
ment is affected in DDS [69].
DDS is a combination of NS showing a histo-
pathologic picture of diffuse mesangial sclerosis
(DMS), male pseudohermaphroditism, and
Wilms’ tumor [44, 47, 49]. DDS can be divided
into three clinical categories: (1) genotypic males
with all three abnormalities, (2) genotypic males
with nephropathy and ambiguous external and/or
internal genitalia only, and (3) genotypic females
with nephropathy and Wilms’ tumor only
[189]. While male pseudohermaphroditism is an
important diagnostic sign, the diagnosis in
females may be difficult and delayed. The renal
tumor and nephropathy may be clinically simul-
taneous, but in many cases, nephropathy either
precedes the tumor or remains the only abnormal-
ity of the disease [8, 38].
762
The nephropathy begins with proteinuria and
NS. It is usually discovered at the age of a few
months, sometimes at birth. Overall, WT1 muta-
tions account for a few percent of CNS cases
(Table 2). Renal failure is often noted concomitantly
with NS and progression to ESRD before the age of
4 years is the rule. The characteristic glomerular
lesion is DMS and in rare cases FSGS [211]. No
correlation between the genotype and the severity of
renal disease has been observed. Because the renal
disease in DDS is resistant to drug treatment, kidney
transplantation remains the only therapeutic alterna-
tive, and favorable results without recurrence of
DMS have been reported. In order to avoid the
development of Wilms’ tumor, bilateral nephrec-
tomy at the onset of terminal renal failure is
recommended. Removal of native kidneys is
recommended to be performed to all patients with
nephropathy caused by WT1 mutations [93].
Mutations in WT1 can also cause an isolated
kidney disease with NS appearing at a various
age. In many cases, the kidney biopsy reveals
DMS histology. Three historical studies reported
WT1 mutations in eight of 21 patients with iso-
lated DMS [74, 98, 99, 104, 177]. These muta-
tions were localized on exons 7, 8, and 9 and on
intron 9 in the WT1 gene. The connection of CNS
and DMS histology was described already in the
1970s [73, 126]. As the clinical picture appeared
to be distinct, DMS was thought to represent a
specific entity. In the published reports, protein-
uria was often moderate and NS was detected
soon after birth or later in infancy, often in the
second and third years. It is clear now that isolated
DMS is not a single entity but caused by several
gene defects. Besides WT1, mutations in the
PLCE1 and LAMB2 gene often result in DMS
type histology, as discussed later. It is also proba-
ble that new genetic variants causing DMS will be
found in the next few years.
Laminin b2 Gene Mutations (Pierson
Syndrome)
Pierson syndrome is a form of primary CNS
caused by loss-of-function mutations in the
LAMB2 gene [137, 205, 214, 215]. This gene
H. Jalanko and C. Holmberg
encodes for the laminin β2-chain that is specifi-
cally expressed in the GBM, ocular structures, and
neuromuscular synapses. Laminins are extracel-
lular matrix proteins with important roles in cell
adhesion, differentiation, and proliferation. The
deficiency of laminin β2-chain results in the
GBM alterations and impairment of the function
of the glomerular filtration barrier. In an animal
model, lack of laminin β2-chain resulted in
ectopic deposition of laminin and mislocalization
of anionic sites in the GBM [103]. These changes
and proteinuria preceded the lesions in podocytes
indicating that the GBM lesions were responsible
for protein leakage.
Pierson syndrome comprises CNS with the
histological presentation of DMS, associated
with complex ocular maldevelopment. Renal dis-
ease may already start prenatally resulting in ter-
minal renal failure during the first few weeks or
months of life [214, 215]. The most characteristic
ocular feature in Pierson syndrome is microcoria,
a fixed narrowing of the pupils due to a defect of
the dilator muscle. Ocular manifestations also
include abnormalities of the lens, cornea, and
retina, often leading to blindness. Patients who
survive the newborn period due to renal replace-
ment therapy develop neurological deficits, con-
sistent with the known expression of laminin β2 in
the nervous system. These include severe muscu-
lar hypotonia and psychomotor retardation [210].
Pierson syndrome is typically caused by
biallelic truncating mutations in the LAMB2
gene. Several reports have, however, revealed
LAMB2 mutations causing milder variants of
Pierson syndrome. Hasselbacher et al. described
two children with CNS and early development of
ESRD, but no other primary abnormalities
[78]. Kagan et al. described a patient with CNS,
high-grade myopia, and minor structural eye
anomalies, but no microcoria [107]. Choi
et al. reported a girl with congenital microcoria
and infantile NS, with normal renal function still
at the age of 6 years [36]. Matejas et al. reported a
large cohort of 51 patients from 39 unrelated fam-
ilies showing that in most patients (90 %) NS
started during the first 3 months of age. DMS
was the major histological finding in biopsies
with a few samples showing FSGS. ESRD
25 Congenital Nephrotic Syndrome
developed within a few weeks or months in most
cases. Microcoria or other ocular abnormalities
were detected in almost all, and severe visual
impairment was noticed in every other patient.
Microcephaly or neurodevelopmental deficits
were present in 10 and 50 % of the patients,
respectively [136].
Since missense mutations in the LAMB2 pre-
sent with a spectrum of symptoms reaching from
isolated early-onset NS to patients with CNS and
mild ocular and neurological symptoms, genetic
analysis of LAMB2 may be warranted in CNS
patients with no mutations in the NPHS1 and
NPHS2 genes. LAMB2 mutations, however, are
much less frequent as indicated in Table 2.
PLCe1 Gene Mutations (NPHS3)
Mutations in the phospholipase C epsilon
1 (PLCE1) gene cause an autosomal recessive
form of early-onset NS [87]. PLCe1 is a phospho-
lipase enzyme and a signaling protein for many G
protein-coupled receptors, including angiotensin
II. PLCe1 promotes the downstream activation of
protein kinase C enhancing calcium signaling
events. PLCe1 is abundant in kidney glomerulus
and localizes to the cytoplasm of the podocyte cell
body and major and secondary processes. It inter-
acts with IQ motif-containing GTPase-activating
protein 1 (IQGAP1), which also interacts with
nephrin [105]. This suggests that PLCe1 may be
involved in the SD signaling and the function of
the glomerular filter [34]. PLCe1 is highly
expressed in the developing glomerulus and the
absence of PLCe1 may halt kidney development
leading to DMS-type glomerular lesions. Of note,
this is associated with a major reduction in the
expression of nephrin and podocin [84].
Mutations in PLCE1 were originally reported
in 14 patients coming from seven families
[87]. Most patients were Turkish and the age at
the onset of the disease ranged from 2 months to
9 years. The patients did not have extrarenal man-
ifestations resembling the situation in patients
with NPHS1 and NPHS2 mutations. Twelve
patients had homozygous truncating mutation,
and all developed gross proteinuria and showed
763
DMS histology in the kidney biopsy. Nine of
these 12 patients progressed to ESRD by the age
of 5 years. Interestingly, two patients responded to
cyclosporine A or prednisone and have remained
in remission for many years. Two siblings had
nontruncating missense mutations and both had
FSGS histology in kidney biopsy.
Based on the original reports, Gbadegesin
et al. analyzed the relative frequency of PLCE1
mutations in a worldwide cohort of children with
“idiopathic” DMS and found truncating PLCE1
mutations in 10 of the 35 families (29 %) [61]. The
age of patients at the onset of the disease varied
between 1 and 36 months and most patients
progressed to ESRD quite fast, with an age
range of 8–60 months. Importantly, three of the
35 families had mutations in the WT1 gene and
none had LAMB2 mutations. Thus, PLCE1 muta-
tions were the most common cause of idiopathic
DMS in this cohort. PLCE1 mutations are, how-
ever, a rare cause of NS in infants (Table 2) or
older children with steroid-resistant NS. Besides
DMS, the kidney biopsy may also show FSGS
histology [25, 60]. It has also been shown that
even homozygous mutation in PLCE1 gene may
not be sufficient to cause NS, suggesting that other
factors modify the effect of PLCE1 abnormalities
[63]. Accordingly, simultaneous mutation in
PLCE1 gene and mitochondrial RC complex defi-
ciency has been reported in an infant with
NS [13].
Other Forms of Primary CNS
Nail–patella syndrome is caused by mutations in
the gene coding for Lim homeobox transcription
factor 1β (LMXB1) [21, 80]. This protein is
expressed in podocytes and regulates the synthe-
sis of alpha-chains of collagen type IV, podocin,
and CD2AP. Renal symptoms are found in a third
of these patients and a single case with CNS has
been described. NS has also been described in
single pediatric and adult patients with genetic
defect in acting-binding proteins (α-actinin-4,
inverted formin-2, myosin-9, myosin 1E, and
ARHGDIA) [11, 24, 27, 57, 70, 108, 113, 115,
142, 181, 192] and integrin α3-gene [149] as well
764
as those with Lowe syndrome [150], Herlitz junc-
tional epidermolysis bullosa [79], sialic acid stor-
age disease [196], and congenital disorders of
glycosylation [195].
During the past few years, mitochondrial dis-
orders have been associated with NS and some of
the patients have been small infants [45, 65, 76,
82, 135, 180, 186]. The metabolic problem in
these patients has been decreased levels of mito-
chondrial coenzyme Q10 (ubiquinone), which is
an essential component of the mitochondrial
electron-transport chain. Its biosynthesis requires
at least 15 genes. Mutations in eight of them
(PDSS1, PDSS2, COQ2, COQ4, COQ6,
ADCK3, ADCK4, and COQ9) cause primary
CoQ10 deficiency, a heterogeneous group of dis-
orders with variable age of onset (from birth to the
seventh decade) and associated clinical pheno-
types, ranging from a fatal multisystem disease
to isolated steroid-resistant NS or isolated central
nervous system disease. The interesting question
here is whether ubiquinone supplementation in
these patients would be helpful, which seems to
be the case. In the report by Heeringa et al., ubi-
quinone treatment (15–30 mg/kg/day) was started
in a 2-month-old infant with COQ6 mutations and
NS. During the follow-up proteinuria decreased
quite dramatically and remained low until the age
of 15 months [82].
In 1968, Galloway and Mowat [53] described
two siblings showing congenital microcephaly,
NS, and hiatus hernia. Several subsequent reports
showing various brain malformations and con-
genital or early-onset NS have been called
Galloway–Mowat syndrome. The classic
Galloway–Mowat syndrome is an autosomal
recessive disorder, but the disease-causing gene
has not been isolated [118, 197].
Since podocytes and neuronal cells share com-
mon morphology and biology with many proteins
expressed in both types of cells, the association of
CNS and brain problems is not unexpected. In
cases with CNS and brain malformation, the his-
tological finding has often been DMS, but other
histological findings have also been reported. A
wide variety of clinical anomalies have also been
reported in patients with CNS, including dysmor-
phic facial features as well as ocular, limb,
H. Jalanko and C. Holmberg
cardiac, and diaphragmatic anomalies. Most of
these case reports were published in the 1990s
when no genetic analyses of podocyte proteins
were available [54, 143, 153, 182].
Secondary Nephrotic Syndromes
NS has been described secondary to some con-
genital and infantile disorders. Syphilis, toxoplas-
mosis, some viral infections, and the infantile
form of SLE are currently the most important
secondary causes of congenital and infantile NS.
Infections
Congenital syphilis has long been known to cause
CNS. It may cause a nephritic or nephrotic syn-
drome in the newborn [151, pp. 157–159, 204,
213]. Proteinuria and hematuria are present, but
severe NS is less common. NS may manifest in
the newborn but is more often seen between 1 and
4 months of age. Membranous nephropathy is a
common finding in kidney biopsy. Antimicrobial
therapy, usually penicillin, is curative, provided
that irreversible renal lesions have not
developed [12].
An association of neonatal cytomegalovirus
(CMV) infection and CNS has also been reported
[14, 20, 170]. In these cases, DMS type histology
is often found and the disease clearly responds to
ganciclovir therapy. In these infections, manifes-
tations other than NS lead to the correct diagnosis.
Toxoplasmosis has been associated with CNS in a
few cases [193]. Congenital rubella has been
reported to cause CNS with membranous glomer-
ulonephritis [50]. The acquired immunodefi-
ciency syndrome caused by the human
immunodeficiency virus is associated with
nephropathy, including NS. This disease usually
affects children older than 1 year, but some HIV
infants with nephropathy have been reported. In a
recent report highly active antiretroviral therapy
in combination with angiotensin-converting
enzyme antagonists was shown to be effective in
decreasing proteinuria and preserving renal func-
tion [171]. Hepatitis B virus may cause
25 Congenital Nephrotic Syndrome
membranous glomerulonephritis, and infants with
nephrosis associated with hepatitis B infection
have been described [64].
Immune Disorders
Although SLE is rarely diagnosed before 5 years
of age, an infantile form of SLE has been reported.
NS was the major clinical finding in five infants
aged 6 weeks to 6 months with SLE [48]. These
patients had elevated antinuclear antibody titers,
hypocomplementemia, and diffuse proliferative
glomerulonephritis. Response to the immunosup-
pressive therapy was poor in many cases.
Nephrotic syndrome due to membranous
nephropathy has been observed antenatally in
infants from mothers who have mutations in the
metallomembrane endopeptidase gene, which
encodes the neutral endopeptidase (NEP)
expressed on podocytes. During pregnancy, the
fetal NEP protein induces a maternal alloimmune
response. Maternal antibody to NEP fetal protein
results in subepithelial immune deposits in the
fetus and the neonate. The mothers’ IgG response
to the fetal NEP determines the severity of the
neonatal disease [43, 155]. The nephrotic syn-
drome is present at birth but proteinuria decreases
with time along with a progressive decrease of the
maternal antibodies and a disappearance of the
immune deposits. However, persistent proteinuria
and chronic renal insufficiency may be observed
due to nephron loss and renal scarring.
Diagnosis of CNS
Clinical Findings
In severe forms of CNS, generalized edema, uri-
nary protein >20 g/L, and serum albumin level
<10 g/L can be detected in the newborn period.
The amount of proteinuria, however, varies in
different entities and the clinical signs may not
be evident during the first weeks of life. Also, the
true magnitude of proteinuria may be detectable
only after partial correction of hypoproteinemia
by albumin infusions. Small amounts of red blood
765
cells and leucocytes are often present in urine.
Serum creatinine and urea levels are variable.
Renal function remains quite normal for the first
months in CNF, but in other forms kidney failure
may develop faster. Blood pressure levels can be
low due to hypoproteinemia and low oncotic pres-
sure, or elevated if renal failure is already present.
In newborns, the placental weight >25 % of
birth weight is present in CNF but may be seen in
other forms of CNS [159]. The kidneys may be of
normal size or larger than normal in ultrasound
scanning, and the renal cortex is often
hyperechogenic [6]. Search for possible
non-renal malformations is important, especially
since they may give clue to the etiologic diagno-
sis. These include genital abnormalities (WT1),
eye defects (LAMB2), and neurological disorders
(Mowat–Galloway, LAMB2, mitochon-
driopathies). CNS caused by nephrin, podocin,
or PLCe1 mutations is not associated with
extrarenal malformations. Cardiac evaluation,
however, often reveals ventricular hypertrophy
but structural defects are rare. If infectious etiol-
ogy is regarded possible, search for specific anti-
bodies (e.g., syphilis, toxoplasma) or DNA/RNA
(e.g., CMV, hepatitis B, HIV) in blood samples is
indicated.
Kidney Biopsy
The different genetic entities of CNS are associ-
ated with typical histological findings, as indi-
cated above. However, since a specific gene
defect may cause several types of glomerular
lesions, such as mesangial expansion, FSGS,
MCNS, and DMS, and the findings in different
genetic entities show overlapping, renal biopsy
does not reveal the etiology. Also, the non-
glomerular findings, such as tubular dilatations
and interstitial fibrosis and inflammation, can be
seen in all forms of proteinuric diseases. Thus, the
indications for renal biopsy are not quite clear. The
knowledge of severity of glomerular sclerosis and
interstitial fibrosis may help in the assessment of
the treatment strategies. On the other hand, the
lesions can be focal and the biopsy findings may
be misleading. If immunohistochemistry for
766
nephrin and podocin is available, analysis of their
expression in biopsy sample is useful. Total lack of
either protein speaks for a severe disorder not
responding to antiproteinuric therapy.
Gene Testing
Genetic analysis is the method of choice for the
precise diagnosis of CNS. The knowledge of eti-
ology helps in assessing the management and
prognosis, in follow-up for possible associated
symptoms, and in genetic counseling of the fam-
ily. Analysis of the NPHS1 and NPHS2 mutations
is warranted in all CNS patients without a family
history or clear-cut extrarenal findings. If no
mutations are detected in NPHS1 or NPHS2
genes, analyses of WT1, LAMB, or PLCe1 are
indicated according to the clinical and histological
findings. Gene tests are readily available in sev-
eral laboratories. Updated information on these
laboratories is provided in www.genetests.org.
Besides the conventional sequencing of one gene
at a time, next-generation sequencing analysis is
now available for NS genes, which allows simul-
taneous survey of all known genes in 2–4 weeks
[132, 140].
Prenatal diagnosis in families with a known
risk for CNS should be based on genetic testing
whenever possible. The results can be obtained
fast if the mutations are known in advance. In case
of no family history or if the mutations in the
affected child were not identified, prenatal genetic
testing is more challenging but possible. Espe-
cially CNF can still be suspected prenatally
based on elevated alpha-fetoprotein (AFP) levels
in maternal serum and amniotic fluid [5]. If the
AFP concentration in amniotic fluid is very high
and the ultrasound examination does not reveal
fetal anencephaly or other malformations, CNF is
a probable diagnosis. However, heterozygous
fetal carriers of NPHS1 gene mutations may
have temporarily elevated AFP levels in amniotic
fluid and maternal serum, and repeated measure-
ment of amniotic fluid AFP before the 20th week
of pregnancy is recommended in cases with high
AFP levels [26, 160]. The problem, however, is
that the AFP levels may remain elevated after the
H. Jalanko and C. Holmberg
20th week also in carriers of the NPHS1 mutation
and the decision on a possible pregnancy termi-
nation is difficult (175). Thus, it is highly
recommended that the genetic testing of the fetus
or parents for NPHS1 mutations should be
performed before the 20th week whenever clearly
elevated AFP levels are discovered in a pregnancy
with normal ultrasound findings.
Management of CNS
In most patients with primary CNS, the therapeu-
tic decisions usually aim at renal transplantation,
and the goals during the first months are to control
proteinuria and edema, provide good nutrition,
and prevent thrombosis and infections, allowing
the child to reach a weight and body size consis-
tent with successful kidney transplantation
[77, 88, 116, 185]. With an optimal therapy,
growth and development are satisfactory and the
patients can spend most of the time at home. In
most cases, terminal uremia is to be expected
within the first years of life.
Immunosuppressive Medication
CNS is a rare problem and no larger study on the
use of immunosuppressive drugs, prednisone or
cyclosporine A (CsA), has been published. Based
on the clinical experience and the published data
on older children with genetic forms of NS, it is
very unlikely that either drug would bring primary
CNS into remission [28, 62, 176]. Accordingly,
only one of the 45 European and Turkish CNS
patients achieved a continuous remission through
the steroid treatment. The genetic defect in this
child was not known [85]. There are, however,
reports on solitary patients with “mild” mutations
in NPHS1, NPHS2, WT1, and PLCE1 who have
shown improvement after immunosuppressive
therapy, but the mechanism behind this is not
known [56, 83, 87, 134]. Both prednisone and
CsA probably have direct effect of podocytes,
which might explain the positive effect. However,
CNS children most often have “severe” mutations
not responding to therapy and should be spared
25 Congenital Nephrotic Syndrome
from long-lasting, unwarranted immunosuppres-
sive treatment. In secondary CNS, specific ther-
apy against the infectious agent, of course, is
warranted and often effective.
Antiproteinuric Medication
A reduction in the protein excretion using an ACE
inhibitor and indomethacin has been reported in
infants with primary CNS [68, 81, 164]. Blockade
of renin-angiotensin axis not only reduces glo-
merular perfusion pressure, but also exerts protec-
tive measures on podocytes [37]. In a few patients
with CNS, adding of angiotensin II receptor
(AT II) to ACE inhibitor medication has further
reduced proteinuria [129]. Thus, the first drug in
the antiproteinuric medication is captopril 1–5
mg/kg/day, and, if no response is obtained in a
few weeks, losartan (0.4–1.3 mg/kg/day) can be
added to the medication. Follow-up of kidney
function is important. Whether indomethacin
(1–5 mg/kg/day) is added in refractory cases is a
matter of opinion. The antiproteinuric treatment is
worth trying in many cases of CNS. In our expe-
rience, CNF patients with the severe (truncating)
Fin-major and Fin-minor mutations do not
respond to captopril or indomethacin, but if either
of the mutations leads to a single amino acid
change, a reduction in the protein excretion may
be observed [159].
Albumin Substitution
The magnitude of protein losses into urine is
crucial for the therapeutic decisions (Table 3).
If proteinuria is modest, only occasional or no
albumin infusions are needed. On the other
hand, heavy proteinuria (10–100 g/L) inevitably
leads to life-threatening edema, protein malnutri-
tion, reduced growth, and secondary complica-
tions. In these cases protein substitution by
parenteral albumin infusions is mandatory. Our
practice in treating CNF patients is to infuse
20 % albumin solution together with a bolus of
intravenous furosemide (0.5 mg/kg), using deep-
vein catheters that are inserted at 2–3 weeks of
767
Table 3 Management of infants with primary CNS
Protein substitution parentally
20 % albumin infusions as required
Dosing 3–4 g/kg/day when severe NS
Divided in 1–3 infusions (day or night)
Furosemide (0.5 mg/kg) together with albumin
Deep vein catheter needed
Nutrition
Hypercaloric diet (130 kcal/kg/day)
Protein supplementation (4 g/kg/day)
Lipid supplementation
Complete multivitamin preparation
Calcium and magnesium (potassium) supplementation
Nasogastric tube or gastrostomy often required for
proper feeding
Medication
Anticoagulation (warfarin, AT III infusions)
Thyroxin substitution
Parenteral antibiotics when bacterial infection
suspected
Antiproteinuric drugs (ACE inhibitor, AT II R blocker,
indomethacin)
Dialysis
Terminal renal failure
After nephrectomy in cases with early transplantation
Transplantation
After dialysis/preemptive
Extraperitoneal grafting when >10–15 kg
age. The substitution is first divided into three 2-h
infusions (starting dose of 1–5 ml/kg/infusion),
and after a few weeks, albumin is given as one
6–8-h infusion during the night (up to 15–20
ml/kg/night, which is 3–4 g/ kg of albumin). In
patients with heavy urinary losses, this substitu-
tion increases plasma albumin levels only tempo-
rarily and no target level can be used in
determining the albumin dosing. Lack of substan-
tial edema and stable weight are major goals. In
our hands, additional furosemide boluses are
given during the daytime when the maximal albu-
min dosing (4 g/kg/day) has been reached.
Nutrition
Infants with severe CNS have traditionally been
treated with a high-energy (130 kcal/kg per day)
768
and a high-protein (3–4 g/kg per day) diet
[37, 88]. Breast milk and cow’s milk-based infant
formulae are first used and the excess protein is
given as a whey- and casein-based protein prod-
uct. Glucose polymers and fat emulsion are given
to increase energy intake. The children also
receive vitamin D3 (10–40 μg/day), and alpha-
calcidiol (0.1–0.3 μg/day) is added when an
increase of parathyroid hormone level is noticed.
A complete multivitamin preparation is given
according to the recommended dietary allowances
for healthy children of the same age. Supplemen-
tary magnesium (50 mg/day) and calcium
(500–1,000 mg/day) are also given to keep the
serum levels within the normal range. The daily
water allowance is 100–130 mL/kg. Most patients
need a nasogastric tube or gastrostomy to guaran-
tee their energy intake. Modifications to this treat-
ment are, of course, necessary with deteriorating
renal function.
Additional Medication
Patients with NS often have low levels of serum
thyroid-binding globulin and thyroxin [33, 41, 55].
McLean et al. [141] reported an increase in TSH in
four of five patients with CNS and a positive
response to thyroxin substitution. We have a similar
experience. Although serum thyroxin concentra-
tion is always low, TSH may be normal in the
beginning but increases in most patients during
the first months. Thus, we have adopted a policy
of routinely giving thyroxin from birth, adjusting
the dosage according to TSH.
Urinary excretion of plasminogen and ATIII
leads to compensatory protein synthesis, resulting
in increased levels of macroglobulins, fibrinogen,
thromboplastin, and factors II, V, VII, X, and XII,
contributing to hypercoagulopathy [158]. Throm-
boses and severe coagulation problems have been
reported in children with CNS [127], and the use
of low-dose aspirin and dipyridamole therapy has
been recommended. All Finnish CNF patients are
treated with sodium warfarin from 3 to 4 weeks of
age, which has clearly decreased the incidence of
severe thrombotic events. Before surgical or vas-
cular procedures, warfarin is stopped 5 days
H. Jalanko and C. Holmberg
before the procedure and ATIII (50 IU/kg) is
given preoperatively to temporarily correct the
ATIII deficiency. Patients with CNS may also
loose serum transferrin, transcobalamin, and
erythropoietin into urine, which is associated
with development of anemia [202]. Management
of this anemia with erythropoietin is often unsuc-
cessful and repeated blood transfusions are
needed. On the other hand, some CNS patients
show thrombophilia and low-dose aspirin has
been used in individual cases.
Bacterial infections may be a major problem in
infants with NS. Because of urinary losses of
gamma globulins and complement factors B and
D [138], nephrotic children are especially prone to
infections caused by capsular bacteria such as
pneumococci, and prophylactic use of penicillin
has been recommended. Ljungberg
et al. retrospectively analyzed the incidence and
type of infection in 21 infants with CNF during
the first year of life [131]. The infants suffered
from 63 verified and 62 suspected episodes of
sepsis. The use of central venous lines had no effect
on the incidence, and the prophylactic use of anti-
biotics or immunoglobulins did not reduce the
incidence. Thus, we do not recommend prophylac-
tic medication or immunoglobulin infusions, but a
high degree of suspicion for septic infections is
warranted. The symptoms are often vague and
masked by signs of focal infections occurring at
the same time. Due to urinary losses, plasma
c-reactive protein (CRP) levels remain low. Anti-
biotic therapy should be started promptly on suspi-
cion and should cover the major hospital strains of
bacteria. Response to treatment is usually excel-
lent. Because of the urinary loss of IgG, vaccina-
tions are preferably given after nephrectomies.
Unilateral Nephrectomy
Some centers have adopted a routine of
performing unilateral nephrectomy to reduce pro-
tein losses [39, 139]. This decreases the need and
frequency of the albumin infusions and may help
in the everyday management so that the com-
mencement of dialysis and renal transplantation
can be postponed to an older age. In infants with
25 Congenital Nephrotic Syndrome
massive protein losses (up to 100 g/L), such as
those with truncating NPHS1 mutations, removal
of one kidney does not reduce protein losses dra-
matically. Based on this we proceed directly to
bilateral nephrectomy and peritoneal dialysis at an
early age in CNF patients to avoid the many
problems encountered during the nephrotic
stage. Prolonged NS is also accompanied by path-
ological lipid status, which leads to vascular
changes already during infancy.
Dialysis
Although quite normal growth can be achieved with
optimal therapy during the nephrotic stage, the
patients with severe CNS are still malnourished and
hypoproteinemic and have a risk for septic infections
and thrombotic events, which may lead to permanent
neurological complications [2]. To optimize treat-
ment we perform bilateral nephrectomy and com-
mence peritoneal dialysis or hemodialysis when the
infant weighs about 7 kg. The PD catheter is insti-
tuted 2 weeks prior to nephrectomy. CNF children
often have inguinal hernias, which are corrected
when the PD catheter is inserted. The diet and med-
ication used for a proteinuric child are switched to
that of a uremic infant. The aim is to improve the
child’s nutritional state before kidney transplantation,
which is performed when the child weighs about
10 kg. The general condition improves, muscle
mass increases, and catch-up growth can be
documented while receiving PD [90, 91, 123]. It
must be emphasized that different centers have vari-
able practices in the management of CNS infants. In
contrast to our practice, early (intraabdominal) trans-
plantation can be performed in a proteinuric infant
without dialysis, and the results are satisfactory. On
the other hand, in CNS patients with moderate pro-
teinuria, the development of ESRD is often waited
before RTx is performed. So, many patients are more
than 2 years of age at the time of RTx.
Kidney Transplantation
Renal transplantation has become an established
mode of therapy for most children with CNS.
769
The fact that CNS children are often transplanted
at 1–3 years of age using adult-size kidneys may
sometimes be surgically demanding and increase
the risk for thrombotic complications, as com-
pared to older recipients. Peri- and postopera-
tively abundant hydration of the recipient (2,500
ml/m2) is necessary to maintain optimum aortic
and renal artery blood flow and avoid low-flow
states that could damage the graft [179].
The use of immunosuppressive medication
should be balanced in order to prevent rejection
episodes, which may be clinically subtle and, on
the other hand, avoid the many side effects asso-
ciated with these drugs. Recurrence of NS in the
graft is very exceptional but has occurred in some
CNF children who have developed anti-nephrin
antibodies after transplantation. Treatment of the
recurrence with cyclophosphamide and plasma-
pheresis and more recently with anti-CD20 anti-
bodies normally leads to remission [89, 121, 161].
Recurrent NS has also been reported in a
few patients with NPHS2 mutations, but in
these cases no anti-podocin antibodies were
detected [15].
Overall, the results of kidney transplantation in
CNS are good and similar to those obtained in other
etiologies [101]. Patient survival at 5 years is over
90 % and graft survival over 80 % in registry data-
bases and in single centers [166]. Also, the quality of
life is good in most patients [71, 72]. A great major-
ity (72 %) of those who have reached the school
attend a normal class. The early CNF patients who
suffered from thrombotic complications have neu-
rologic handicaps [17, 168, 169]. Growth and puber-
tal development of CNS patients, who were
transplanted as infants, is quite normal [200], but
fertility in males who have reached the adulthood is
reduced [199]. Chronic allograft nephropathy is a
major problem also in these patients and the second
transplantation is frequent when the patients become
young adults [101, 167].
Concluding Remarks
During the past few years, our knowledge on the
genetic and molecular basis of CNS has greatly
increased. Podocyte proteins play an important
770
role in the glomerular sieving and mutations in the
nephrin, podocin, WT1, PLCε1, and laminin B2
account for most cases of CNS. It can be expected
that more genetic defects will be found in CNS
patients in the near future. Also, management of
these infants has improved, so that the outcome of
CNS patients is close to other pediatric kidney
patients requiring renal transplantation.
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 Inherited Glomerular Diseases
26
Michelle N. Rheault and Clifford E. Kashtan

Contents Hereditary Metabolic Disorders

The Glomerular Basement Membrane . . . . . . . . . . . 778
Glomerular Basement Membrane Disorders . . . . . 779
Type IV Collagen Disorders . . . . . . . . . . . . . . . . . . . . . . . 779
Alport Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 779
Thin Basement Membrane Nephropathy . . . . . . . . . . . . 786
Hereditary Angiopathy with Nephropathy,
Aneurysms, and Cramps (HANAC Syndrome) . . . . . 788
Laminin Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788
Pierson Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788
Type III Collagen Nephropathies . . . . . . . . . . . . . . . . . 789
Nail-Patella Syndrome (Hereditary Osteo-
onychodysplasia) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 789
Collagen Type III Glomerulopathy
(Collagenofibrotic Glomerulopathy) . . . . . . . . . . . . . . . . 791
Hereditary Nephritis with Thrombocytopenia
and Giant Platelets: Epstein and Fechtner
Syndromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 791
Hereditary Metabolic Disorders
with Primary Glomerular Involvement . . . . . . . . . . . 791
Fabry Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 791
Other Glomerular Lipidoses . . . . . . . . . . . . . . . . . . . . . . . . . 792
with Secondary Glomerular Involvement . . . . . . . . 792
Familial Amyloidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 792
Alpha-1 Antitrypsin Deficiency . . . . . . . . . . . . . . . . . . . . . 792
Alagille Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 793
Hereditary Lecithin-Cholesterol Acyltransferase
(LCAT) Deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 793
Lipoprotein Glomerulopathy . . . . . . . . . . . . . . . . . . . . . . . . 793
Familial Juvenile Megaloblastic Anemia . . . . . . . . . . . . 794
Other Hereditary Diseases with Glomerular
Involvement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794
Charcot-Marie-Tooth (CMT) Disease . . . . . . . . . . . . . . . 794
Cockayne Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794
Hereditary Acro-osteolysis with Nephropathy . . . . . . 794
Other Syndromes with Renal Involvement . . . . . . . . . . 794
Hereditary Glomerulopathies Without
Extrarenal Symptoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795
Fibronectin Glomerulopathy . . . . . . . . . . . . . . . . . . . . . . . . . 795
Mitochondrial Cytopathies . . . . . . . . . . . . . . . . . . . . . . . . . . 795
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795

M.N. Rheault (*) • C.E. Kashtan

Department of Pediatrics, Division of Pediatric
Nephrology, University of Minnesota Masonic Children’s
Hospital, Minneapolis, MN, USA
e-mail: rheau002@umn.edu; kasht001@umn.edu

# Springer-Verlag Berlin Heidelberg 2016 777

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_79
778
The Glomerular Basement Membrane
The structural and functional characteristics of
the normal glomerular capillary wall are discussed
in detail elsewhere in this text. For the
purposes of this chapter, this discussion will
focus on the major protein components of
glomerular basement membrane (GBM): type IV
collagen and laminin. The GBM contains
specialized isoforms of these proteins which inter-
act and form networks bridged by entactin/
nidogen and heparan sulfate proteoglycans
(agrin) [1–4].
Type IV collagen. There are six isoforms of
type IV collagen, designated α1(IV)–α6(IV). All
type IV collagen isoforms share several basic
structural features: a collagenous domain of
M.N. Rheault and C.E. Kashtan
While the interstitial collagens, such as type I
collagen, lose their NC1 domains after chain
association and form fibrillar networks, type IV
collagen trimers form open, nonfibrillar networks
through NC1-NC1 interactions between two tri-
mers and amino-terminal interactions between
four trimers.
α1α1α2(IV) trimers are present in all basement
membranes, whereas α3α4α5(IV) and α5α5α6
(IV) trimers are more restricted in their distribu-
tions (Fig. 1). In normal, mature human kidneys,
α3α4α5(IV) trimers are found in GBM, Bowman’s
capsules, and the basement membranes of distal
tubules. α5α5α6(IV) trimers are present in
Bowman’s capsules and the basement membranes
of distal tubules and collecting ducts of
normal kidneys but are not found in GBM [6, 7].
1,400 amino acids containing the repetitive trip- α5α5α6(IV) trimers are also found in normal
let sequence glycine-X-Y (Gly-X-Y, with X and Y epidermal basement membranes; however,
representing other amino acids); a α3α4α5(IV) trimers are not. α3α4α5(IV) trimers
noncollagenous carboxy-terminal (NC1) domain are also found in several ocular and cochlear base-
of 230 amino acids that includes 12 completely ment membranes, as discussed in the section on
conserved cysteine residues; and a “Alport Syndrome” below.
noncollagenous amino-terminal sequence of Each type IV collagen α chain is encoded by
15–20 residues (7S). The collagenous triplet one of six distinct genes, COL4A1–COL4A6,
sequence in each chain contains 20 interruptions.
The secreted form of type IV collagen is a
heterotrimer composed of three α chains, resulting
from self-association of NC1 domains and folding
of the collagenous domains into triple helical struc-
tures. Amino acid sequences within the NC1
domains determine the specificity of chain associ-
ation, resulting in three major trimeric species:
α1α1α2(IV), α3α4α5(IV), and α5α5α6(IV) [5].
Fig. 1 Immunofluorescence microscopy. Normal distribution of the α(IV) chains in renal (a) and epidermal basement
membranes (b)
which are arranged in pairs on three chromosomes
(Fig. 2). COL4A1 and COL4A2 encode the α1
(IV) and α2(IV) chains, respectively, and are
located on chromosome 1. The α3(IV) and α4
(IV) chains are respectively encoded by the
COL4A3 and COL4A4 genes on chromosome
2, while the α5(IV) and α6(IV) genes are encoded
by the COL4A5 and COL4A6 genes on the X
chromosome. The paired genes are arranged in a
26 Inherited Glomerular Diseases 779

3′ 5′ 5′ 3′
13q34
130 bp COL4A1
COL4A2 5′
3′ 5′ 3′
2q35
COL4A4 5′ COL4A3
3′ 5′ 3′

Xq22
450 bp
COL4A6 COL4A5

Fig. 2 Schematic representation of the distribution of type IV collagen gene on chromosomes 13, 2, and X

50 –50 orientation, separated by sequences of vary- Table 1 Type IV collagen disorders

ing length that contain regulatory elements [8, 9]. Gene
Laminin. Laminin also forms networks based Disorder Inheritance Locus product
on heterotrimeric association of α, β, and γ Alport X-linked COL4A5 α5(IV)
isoforms. Each subunit consists of an α, β, and γ syndrome Autosomal COL4A3 or α3(IV) or
recessive COL4A4 α4(IV)
chain. There are 12 known laminin chains (α1–5,
Autosomal COL4A3 or α3(IV) or
β1–4, and γ1–3), each encoded by a distinct gene, dominant COL4A4 α4(IV)
that form at least 15 heterotrimeric isomers. Thin basement Autosomal COL4A3 or α3(IV) or
Laminin-521 has the composition α5β2γ1 and is membrane dominant COL4A4 α4(IV)
the laminin form found in mature GBM [4]. nephropathy
HANAC Autosomal COL4A1 α1(IV)
syndrome dominant

Glomerular Basement Membrane

Disorders

To date, mutations affecting two of the major pro-

teins of GBM, type IV collagen and laminin, have
been identified as causes of inherited glomerular
disease. Mutations in the α3(IV), α4(IV), or α5
(IV) isoforms of type IV collagen cause Alport
syndrome and some cases of thin basement mem-
brane nephropathy (TBMN), whereas mutations
in the α1(IV) isoform cause HANAC syndrome,
comprising hereditary angiopathy, nephropathy,
aneurysms, and muscle cramps (Table 1). Muta-
tions in laminin β2 have been implicated in
Pierson syndrome.
Type IV Collagen Disorders
Alport Syndrome
Familial hematuria was first described as a clinical
entity in the early 1900s [10]. Over the next
20 years, studies of this condition in successive
generations of a single family described
development of proteinuria and renal insuffi-
ciency in affected individuals, particularly males
[11]. In 1927 Alport reported the association of
nephritis with deafness in this family [12]. Numer-
ous descriptions of families with hereditary
nephritis were published in the 1950s and 1960s,
leading ultimately to the observations that
established Alport syndrome as a heritable disor-
der of type IV collagen. These seminal events
included the identification of unique ultrastruc-
tural alterations in Alport glomerular basement
membranes by electron microscopy
(EM) [13–15], the observation that Alport GBM
exhibited abnormal reactivity with anti-GBM sera
directed against antigens associated with type IV
collagen [16–18], the mapping of the major
Alport locus to the X chromosome [19], the dis-
covery of a type IV collagen gene (COL4A5) on
the X chromosome [20], and finally the descrip-
tion of COL4A5 mutations in families with
X-linked Alport syndrome [21].
780
Genetics
Mutations in any of the COL4A3, COL4A4, or
COL4A5 genes may adversely affect the forma-
tion and composition of affected basement mem-
branes. If any of the α3(IV), α4(IV), or α5
(IV) chains are absent due to severe mutations,
then the other type IV collagen isoforms are
degraded and no α3α4α5(IV) heterotrimers are
deposited in basement membranes [22]. Milder
mutations, generally missense mutations affecting
the glycine residues involved in triple helix for-
mation, may lead to the formation of abnormally
folded trimers that are either degraded or lead to
M.N. Rheault and C.E. Kashtan
reasons that are as yet uncertain, some people
with heterozygous mutations in COL4A3 or
COL4A4 have a progressive course leading to
chronic renal failure or end-stage renal disease
and are thus considered to have ADAS.
Over 700 different mutations in the COL4A5
gene have been identified in patients and families
with XLAS [28]. Mutations can be found along
the entire 51 exons of the gene without identified
hot spots. About 10–15 % of COL4A5 mutations
occur as spontaneous events; therefore, a family
history of renal disease is not required for a diag-
nosis of XLAS. Reported mutations include large
formation of an abnormal type IV collagen rearrangements (20 %), small deletions and
network. insertions (20 %), missense mutations that alter
There are three genetic forms of Alport syn- a glycine residue in the collagenous domain of the
drome: X-linked (XLAS), autosomal recessive α5(IV) chains (30 %), other missense mutations
(ARAS), and autosomal dominant (ADAS). (8 %), nonsense mutations (5 %), and splice-
XLAS is caused by mutations in the COL4A5
gene and is the predominant form of the disease,
accounting for ~80 % of affected patients.
Affected males are hemizygotes carrying a single
mutant COL4A5 allele. Affected females carry a
normal COL4A5 allele as well as a mutant allele
and are therefore heterozygotes. Affected females
are mosaic for expression of the normal and
mutant alleles due to X-inactivation [23]. Approx-
imately 15 % of patients with Alport syndrome
have the recessive form of the disease (ARAS)
due to mutations in both alleles of either COL4A3
or COL4A4. These patients are either homozy-
gotes, who have the identical mutation in both
alleles of the affected gene (and who may have
consanguineous parents) or compound heterozy-
gotes, who have inherited different mutations in
the affected gene from their parents [24]. ADAS
caused by heterozygous mutations in COL4A3 or
COL4A4 was classically thought to be rare, affect-
ing only about 5 % of affected patients [25]. With
the advent of next-generation sequencing, how-
ever, recent studies are suggesting a higher per-
centage of patients with AS who demonstrate
autosomal dominant inheritance than was previ-
ously recognized [26, 27]. Most individuals with
heterozygous COL4A3 or COL4A4 mutations are
asymptomatic or exhibit isolated, nonprogressive
microscopic hematuria associated with thin glo-
merular basement membranes (TBMN). For
site mutations (15 %) [28–31]. The COL4A5
genotype has a powerful effect on the phenotype
of XLAS in affected males [30, 31]. Large dele-
tions, nonsense mutations and small mutations
that alter the translational reading frame are asso-
ciated with a 90 % probability of progression to
ESRD by age 30 [30, 31]. This risk is 70 % in
patients with a splice-site mutation and 50 % in
those with a missense mutation [30, 31]. The posi-
tion of a glycine substitution may also affect the
XLAS phenotype, with 50 glycine missense muta-
tions causing a more severe phenotype than 30
glycine mutations [31]. The number of side-
chain carbon atoms in the substituting amino
acid also influence the phenotype associated
with a glycine substitution [32].
A similar variety of mutation types has been
described in patients and families with ARAS
[24, 29, 33–35]. There are also genotype-
phenotype correlations in patients with ARAS
where nonsense mutations or mutations resulting
in stop codons are associated with early onset renal
failure [24].
Clinical Features
Gender and genotype are the major determinants
of the severity of renal, cochlear, and ocular dis-
ease in Alport syndrome. Males with XLAS and
patients of either gender with ARAS inevitably
progress to end-stage renal disease (ESRD) and
26 Inherited Glomerular Diseases
the majority develop sensorineural deafness
[24, 30]. The pacing of these events is influenced
by the nature of the underlying disease mutation.
While the majority of women with heterozygous
COL4A5 mutations have mild disease manifesta-
tions, ESRD and severe deafness develop in a
significant minority [36]. Patients with ADAS
exhibit relatively slow progression of renal and
cochlear dysfunction, and ocular findings are
much less common than in XLAS and ARAS
[26, 37, 38].
Renal. Persistent microscopic hematuria is the
cardinal clinical feature of Alport syndrome,
occurring in 100 % of males with XLAS, 95 %
of females with XLAS, and in all patients with
ARAS [30, 36]. Hematuria is likely present from
infancy in XLAS males and in patients with
ARAS. Episodic gross hematuria is not unusual,
especially during childhood [39]. Some children
with Alport syndrome have virtually constant
gross hematuria.
Overt proteinuria typically appears during later
childhood or adolescence in XLAS males and in
ARAS patients. Affected children first demon-
strate microalbuminuria, which progresses to
overt proteinuria and even nephrotic syndrome
with time [39–41]. About 75 % of XLAS females
ultimately develop proteinuria of some degree
[36]. Most children with Alport syndrome have
normal blood pressures, but hypertension is com-
mon in adolescent males with XLAS and teen-
aged ARAS patients.
In untreated XLAS males, the probability of
ESRD is 50 % by age 25, 80 % by age 40, and
100 % by age 60 [30]. COL4A5 genotype has a
powerful effect on rate of progression to ESRD in
XLAS males. Large deletions and nonsense muta-
tions confer a 90 % probability of ESRD before
age 30, compared to a 70 % risk with splice-site
mutations and a 50 % risk with missense muta-
tions [30]. The epidemiology of ESRD in ARAS
is probably similar to XLAS males [24]. It takes
781
asymptomatic hematuria while others develop
ESRD [42]. According to one study of a large
cohort of XLAS females, the risk of ESRD is
12 % by age 45, 30 % by age 60, and 40 % by
age 80 [36]. The explanation for the wide vari-
ability in outcomes for women with XLAS is
unclear, but likely multifactorial. Unlike in males
with XLAS, genotype does not correlate with
phenotype in females with XLAS, perhaps as a
result of the overwhelming influence of random
X-chromosome inactivation on disease course in
XLAS females [23, 36]. Risk factors for ESRD in
women with XLAS include proteinuria and sen-
sorineural deafness [36, 43]. Further studies are
required to develop accurate methods to predict
the risk of progressive kidney disease in women
with XLAS.
Cochlear. Hearing is normal at birth and dur-
ing early childhood. Symmetrical deficits in sen-
sitivity for high-frequency sounds often become
detectable by audiometry in late childhood. Over
time, the hearing deficit progresses into the fre-
quency range of conversational speech. Because
the deficit typically does not exceed 60–70 dB and
speech discrimination is preserved, hearing aids
are effective in most affected individuals. In
XLAS males, the probability of hearing loss is
50 % by age 15, 75 % by age 25, and 90 % by
age 40 [30]. COL4A5 genotype influences the
probability of hearing loss, with 90 % of those
with deletions, nonsense mutations, and splice-
site mutations exhibiting deafness before age
30 compared to 60 % of those with missense
mutations [30]. In XLAS females, the probability
of hearing loss is 10 % by age 40 and 20 % by age
60 [36]. The majority of patients with ARAS
develop deafness, although precise data on timing
is not available [24].
Ocular. Anomalies of the lens, retina, and cor-
nea are common in Alport syndrome, especially
among males with XLAS and patients with
ARAS, often becoming apparent during adoles-
50 years for 50 % of ADAS patients to develop
ESRD, twice as long as XLAS males [26, 37].
Women who are heterozygous for COL4A5
mutations (“carriers” of XLAS) demonstrate
widely variable disease outcomes with some
women demonstrating only lifelong
cence and young adulthood [38]. About 15 % of
XLAS males exhibit anterior lenticonus, in which
the central portion of the lens protrudes into the
anterior chamber [30]. While this lesion is often
asymptomatic, it may be associated with reduced
visual acuity, and cataracts and even rupture of the
782
Fig. 3 Electron
micrograph. Lead citrate
and uranyl acetate stain
(4,800). Alport syndrome.
Thin and regular GBM
(arrow) with attenuated
lamina densa. Epithelial
foot processes are
extensively fused
lens may occur. Alteration of retinal pigmentation,
consisting of whitish-yellowish perimacular
flecks, occurs in about 15 % of XLAS males
[30, 44], often in association with lenticonus.
Corneal abnormalities include recurrent corneal
erosions [45, 46] and posterior polymorphous
dystrophy [47]. Patients with severe COL4A5
mutations are more likely to develop perimacular
dot-and-fleck retinopathy [48].
Other. The association of XLAS with smooth
muscle tumors (leiomyomas) of the esophagus,
tracheobronchial tree, and, in women, the external
genitalia has been described in several dozen fam-
ilies [49–51]. Symptoms such as dysphagia, post-
prandial vomiting, epigastric or retrosternal pain,
recurrent bronchitis, dyspnea, cough, and stridor
often appear in late childhood. The Alport syn-
drome-diffuse leiomyomatosis complex arises
from a contiguous gene deletion on the X chro-
mosome involving exon 1 of COL4A5, the com-
mon promoter region that regulates gene
expression of COL4A5 and COL4A6, and the
first 2 exons of the adjacent COL4A6 gene [49].
Mental retardation, midface hypoplasia, and
elliptocytosis have been described in a small num-
ber of XLAS males who carry deletions that
extend downstream of the 30 end of the COL4A5
gene [52, 53]. Finally, abnormalities in arterial
vessels have been described in males with Alport
syndrome including aortic root dilatation and
aneurysms of the thoracic and abdominal
aorta [54].
M.N. Rheault and C.E. Kashtan
Pathology
The clinical features of Alport syndrome originate
in changes in basement membrane structure and
function initiated by absence or abnormalities of
α3α4α5(IV) and α5α5α6(IV) networks.
Renal. Light microscopic abnormalities are
unusual in children with Alport syndrome who
are less than 5 years of age. Mesangial hypercel-
lularity and matrix expansion, and eventually
focal segmental glomerulosclerosis, are common
in older children and adolescents, especially boys.
Tubular atrophy and interstitial fibrosis develop
progressively after age 10 in boys with Alport
syndrome [55].
Electron microscopy may reveal pathogno-
monic changes, depending on the patient’s age
and gender. The earliest abnormality is diffuse
attenuation of the GBM; consequently, differenti-
ation of Alport syndrome from TBMN by routine
renal biopsy processing may be difficult in chil-
dren (Fig. 3). To further complicate matters, in
some families with Alport syndrome due to
COL4A5 mutations, GBM attenuation is the only
ultrastructural abnormality. However, the great
majority of boys with XLAS and both boys and
girls with ARAS develop the classic Alport GBM
lesion, consisting of diffuse thickening accompa-
nied by “basket-weave” transformation of the
lamina densa, intramembranous vesicles and den-
sities, scalloping of the epithelial surface of the
GBM, and foot process effacement (Fig. 4). The
percentage of GBM displaying this lesion
26 Inherited Glomerular Diseases 783

Fig. 4 Electron
micrograph. Lead citrate
and uranyl acetate stain
(11,250). Alport
syndrome. Thickened GBM
showing splitting of the
lamina densa and presence
of granulations

Fig. 5 Immunofluorescence microscopy. Distribution of with X-lined Alport syndrome. (c) Absence of epidermal
the α(IV) chains in renal and epidermal basement mem- basement membrane labeling (arrow). (f) Discontinuous
branes of male (a–c) and female (d–f) patients affected epidermal basement membrane labeling (arrows)

increases progressively with age in boys with (IV), and α5(IV) chains is completely absent in
XLAS [56]. Females with XLAS display a range
of GBM alteration, from focal GBM attenuation
to diffuse thickening and basket-weaving, and
there is no consistent correlation of GBM findings
and age [56].
Routine immunofluorescence is normal or
shows nonspecific immunoprotein deposition.
Because disease-causing mutations result in
abnormal expression of α3α4α5(IV) and α5α5α6
(IV) networks in basement membranes in most
Alport patients, specific immunostaining for type
IV collagen α chains is useful for both diagnosis
and differentiation of XLAS and ARAS [57]
(Figs. 5 and 6). Expression of the α3(IV), α4
80 % of XLAS males, and 60–70 % of females
with XLAS exhibit mosaic expression of these
chains [57] (Fig. 5). In most patient with ARAS,
GBM is nonreactive with antibodies to the α3(IV),
α4(IV), and α5(IV) chains, and Bowman’s cap-
sules and tubular basement membranes are also
negative for the α3(IV) and α4(IV) chains [58]
(Fig. 6). However, immunostaining for α5
(IV) chains in Bowman’s capsules and tubular
basement membranes is present, because in these
basement membranes, expression of α5α5α6
(IV) networks is preserved. It is important to
note that altered immunostaining for the α3
(IV)–α6(IV) chains in patients with XLAS and
784
Fig. 6 Immunofluorescence microscopy. Distribution of the α(IV) chains in renal and epidermal basement membranes of
patients affected with autosomal recessive Alport syndrome
ARAS is not age-dependent. Therefore, this
method can provide diagnostic information even
in patients who are too young to display charac-
teristic abnormalities in GBM ultrastructure.
Normal epidermal basement membranes (EBM)
express the α5α5α6(IV) network but not the
α3α4α5(IV) network. EBMs show negative
staining for α5(IV) chains in about 80 % of
XLAS males, and mosaic expression of α5(IV) is
observed in 60–70 % of XLAS females, allowing
diagnosis of XLAS by skin biopsy [59, 60] (Fig. 5).
Skin biopsy is not useful for diagnosis of ARAS,
since expression of the α5α5α6(IV) network in
EBM is normal [58] (Fig. 6).
Cochlear. The hearing loss of Alport syndrome
arises from cochlear dysfunction [61]. Normal
cochleae in mice, dogs, and humans express α3
(IV), α4(IV), and α5(IV) chains in the spiral lim-
bus, in the spiral ligament, and in the basement
membrane interposed between the organ of Corti
and the basilar membrane [62–65]. However,
expression of these chains is absent in cochleae
of ARAS mice [62], XLAS dogs [63], and men
with XLAS [65]. Careful examination of well-
preserved cochleae from men with XLAS and
deafness revealed a zone of separation between
the organ of Corti and the underlying basilar
membrane, and cellular infiltration of the tunnel
of Corti and the spaces of Nuel [66]. These
changes are not observed in similarly well-
preserved cochleae obtained from normal individ-
uals or patients with other causes of deafness. The
structural changes observed in Alport cochleae
may be associated with defective attuning of bas-
ilar membrane motion and hair cell stimulation,
resulting in reduced acuity of hearing. Similar
changes have not been observed in cochleae
from mice or dogs with Alport syndrome,
M.N. Rheault and C.E. Kashtan
although deafness in these models is minimal or
absent.
Ocular. The α3(IV), α4(IV), and α5(IV) chains
are normal components of several basement mem-
branes in the eye, including the corneal basement
membrane, Descemet’s membrane, lens capsule,
the internal limiting membrane of the retina, and
the retinal pigment epithelium basement mem-
brane [64, 67–69]. The ocular manifestations of
Alport syndrome likely arise from absence or
abnormality of α3α4α5(IV) networks in these
basement membranes. Absence of the α3α4α5
(IV) network leads to increased distensibility in
the lens capsule when tested in experimental
models of AS [70]. The lens capsules of Alport
patients with anterior lenticonus exhibit marked
attenuation and focal areas of dehiscence,
suggesting that the lens capsule lacks the mechan-
ical strength to maintain normal lens shape [68,
71, 72].
Diagnostic Considerations
Accurate diagnosis of Alport syndrome and dif-
ferentiation of this condition from other causes of
familial and sporadic glomerular hematuria are
based upon careful clinical evaluation, reliable
pedigree data, and thoughtful consideration of
the relative merits of skin biopsy, kidney biopsy,
and genetic testing.
In a child with isolated hematuria, a positive
family history of hematuria in the absence of a
history of ESRD suggests a diagnosis of TBMN.
Two rare causes of familial hematuria associated
with macrothrombocytopenia, Epstein and
Fechtner syndromes, can be excluded if the plate-
let count is normal. Familial IgA nephropathy [73]
and membranoproliferative glomerulonephritis
[74] are uncommon causes of familial hematuria.
26 Inherited Glomerular Diseases
In the absence of a family history of hematuria,
the differential diagnosis of glomerular hematuria
includes Alport syndrome, TBMN, IgA nephrop-
athy, membranoproliferative glomerulonephritis,
membranous nephropathy, lupus nephritis,
postinfectious glomerulonephritis, and Henoch-
Schönlein nephritis. Associated clinical findings
(e.g., rash, arthritis) or laboratory findings (e.g.,
hypocomplementemia) will suggest diagnoses
other than Alport syndrome in many of these
patients.
While results of hearing evaluation are likely to
be normal in young children with Alport syn-
drome, audiometry may be very useful in children
over 6–8 years of age, especially boys. Ophthal-
mologic assessment may also provide valuable
information, although ocular lesions are more
prevalent in Alport patients with advanced dis-
ease, and less likely to be present in the population
of young patients in whom differential diagnosis
of hematuria may be more difficult.
Tissue studies can complement clinical and
pedigree information that is insufficient to clearly
differentiate Alport syndrome from other diagno-
ses. Skin biopsy with immunostaining for the α5
(IV) chain, as described above, may be diagnostic,
especially when clinical and pedigree data
strongly suggest a diagnosis of XLAS. Normal
expression of the α5(IV) chain in epidermal base-
ment membrane can be explained in several ways:
(1) the patient has XLAS, but his or her COL4A5
mutation does not abolish α5(IV) expression,
(2) the patient has a form of Alport syndrome
(ARAS or ADAS) in which expression of α5
(IV) in epidermal basement membranes is not
affected, or (3) the patient does not have Alport
syndrome. Whereas skin biopsy is useful only if it
provides definitive confirmation of a diagnosis of
XLAS, renal biopsy carries the advantage of
enabling the diagnosis of XLAS, ARAS, and
non-Alport kidney disease, particularly if type
IV collagen immunostaining is applied.
Mutation detection rates of 80–90 % are attain-
able in males with XLAS using direct sequencing
of COL4A5 [75]. Comparable data for detection
of COL4A3 and COL4A4 mutations in patients
with ARAS are lacking. Next-generation
sequencing allows for rapid simultaneous analysis
785
for mutations in COL4A3, COL4A4, and COL4A5
[27]. Commercially available genetic testing for
mutations in COL4A3, COL4A4, and COL4A5 is
available in many countries.
Treatment
The goal of treatment in patients with Alport
syndrome is to slow the progression of kidney
disease and delay the need for renal replacement
therapy. There have been no controlled therapeu-
tic trials in human Alport syndrome. Conse-
quently, treatment recommendations must be
derived from animal studies, anecdotal reports,
and retrospective registry reviews. In murine
ARAS, several interventions have proven effica-
cious, including angiotensin antagonism [76–78],
TGFb-1 inhibition [79], chemokine receptor
1 suppression [80], administration of bone mor-
phogenic protein-7 [81], blockade of matrix
metalloproteinases [82], and bone marrow trans-
plantation [83, 84].
Cyclosporine treatment resulted in prolonga-
tion of survival in male XLAS dogs [85]. Cyclo-
sporine treatment diminished proteinuria and
appeared to stabilize renal function in a small,
uncontrolled study of Alport males [86]. However,
apparent acceleration of renal fibrosis was
suggested by the results of other studies of cyclo-
sporine treatment in Alport patients, and this treat-
ment is no longer recommended [87, 88].
Uncontrolled studies in human Alport syn-
drome subjects have shown that angiotensin
blockade can transiently reduce proteinuria [89,
90]. Subgroup analysis of 30 children with Alport
syndrome in a larger multicenter, randomized,
double-blind study comparing losartan with pla-
cebo or amlodipine in proteinuric children dem-
onstrated a significant reduction in proteinuria in
the losartan treated group [91]. An extension of
this study showed comparable efficacy of either
enalapril or losartan in reducing proteinuria in
children with Alport syndrome [92]. A report
from the European Alport Registry, which
included 283 patients followed over 20 years,
compared renal outcomes in Alport syndrome
patients treated with ACE inhibition at various
disease time points: microalbuminuria, protein-
uria, or in chronic kidney disease (CKD) stages
786
III–IV [93]. This retrospective review demon-
strated a delay in renal replacement therapy by
3 years in the treated CKD group and by 18 years
in the treated proteinuric group [93]. Side effects
of ACE inhibition were rare and included
hyperkalemia, cough, and hypotension. Based
on these findings, a prospective, double-blind,
randomized, placebo controlled trial is under
way to compare outcomes in children with Alport
M.N. Rheault and C.E. Kashtan
without hematuria is affected. Given that by age
60 there is an estimated risk of ESRD of 30 % in
women with XLAS [36], female members of
XLAS families who have hematuria should be
discouraged from kidney donation. One study
described development of proteinuria and a
decline in renal function in heterozygous females
with XLAS after kidney donation [96], highlight-
ing the potential risks in this population.
syndrome treated with ramipril versus placebo at
an early time point (microalbuminuria or isolated
hematuria) [94].
Clinical practice guidelines have been devel-
oped to guide treatment of patients with Alport
syndrome, including heterozygous females
[40]. Treatment with ACE inhibitors or angioten-
sin receptor blockers should be considered for
affected individuals with microalbuminuria and
either a family history of ESRD at a young age
(<30 years) or a known severe COL4A5 mutation
and for all affected individuals with overt protein-
uria. Treatment of hypertension and other mani-
festations of chronic kidney disease are similar to
children with other etiologies of chronic kidney
disease.
Renal Replacement Therapy
Patients with Alport syndrome typically have
excellent outcomes following renal transplanta-
tion [95]. Two issues require the special attention
of transplant physicians involved in the care of
Alport patients. First, evaluation of potential
related donors must identify affected individuals
who may be at risk for development of significant
renal insufficiency. Second, posttransplant moni-
toring must allow early diagnosis of
posttransplant anti-GBM nephritis, a complica-
tion of transplantation that is unique to Alport
syndrome.
Familiarity with the genetics of Alport syn-
drome and the signs and symptoms of the disease
is required for informed donor evaluation. Since
100 % of males with XLAS have hematuria [30],
the absence of hematuria excludes Alport syn-
drome in male relatives of XLAS patients.
About 95 % of females with XLAS have hematu-
ria [36], so there is only a 5 % chance that a female
Anti-GBM nephritis occurs in 3 % of
transplanted Alport males [97]. The onset of
anti-GBM nephritis typically occurs during the
first year after transplantation and usually results
in irreversible graft failure within weeks to
months of diagnosis. There is a high rate of recur-
rence in subsequent allografts. In XLAS males,
the primary target of anti-GBM antibodies is the
α5(IV) chain [98, 99]. Females with XLAS who
require transplantation are at little or no risk of
developing anti-GBM nephritis. However, both
males and females with ARAS can develop anti-
GBM nephritis after transplantation. The α3
(IV) chain is the primary target of anti-GBM
antibodies in ARAS patients [98, 100].
Goodpasture autoantibodies and anti-GBM anti-
bodies from transplanted Alport patients target
distinct epitopes on the carboxy-terminal
noncollagenous (NC1) domain of the α3
(IV) chain [101].
Renal allograft biopsy with routine immuno-
fluorescence should be performed early in the
evaluation of Alport patients who develop hema-
turia or increased creatinine after transplantation
or if circulating anti-GBM antibodies are
detected. Anti-GBM nephritis after renal trans-
plantation should be treated with cytotoxic ther-
apy and plasmapheresis, although such therapy
has been unsuccessful in the majority of reported
patients.
Thin Basement Membrane
Nephropathy
Historically, families displaying autosomal domi-
nant transmission of isolated, nonprogressive
hematuria were classified as having “benign
26 Inherited Glomerular Diseases
familial hematuria” [102–104]. Affected patients
typically exhibited no renal parenchymal abnor-
malities apart from thinning of glomerular base-
ment membranes (GBM) observed by electron
microscopy [105, 106]. The more inclusive term
“thin basement membrane nephropathy” (TBMN)
has gradually displaced benign familial hematuria
as the preferred designation for hematuria associ-
ated with thin GBM, because it encompasses spo-
radic cases of hematuria associated with thin
GBM; familial or sporadic cases of thin GBM in
which hematuria is accompanied by proteinuria,
hypertension, and/or renal insufficiency; and
benign familial hematuria. The prevalence of
TBMN is estimated at 1–2 % of the
population [107].
Thinning of GBM is a pathological description
rather than a distinct entity. Depending on the
timing of renal biopsy, thin basement membranes
may be observed in patients with hemizygous or
heterozygous mutations in COL4A5 (XLAS),
biallelic mutations in COL4A3 or COL4A4
(ARAS), heterozygous mutations in COL4A3 or
COL4A4 (the carrier state of ARAS), and muta-
tions at nontype IV collagen loci [94]. The natural
history of hematuria associated with thin GBM is
determined by the underlying mutation, perhaps
in combination with remote modifier genes.
Hemizygous mutations in COL4A5 and biallelic
mutations in COL4A3 and COL4A4 result in pro-
gressive GBM thickening, proteinuria, and renal
failure. Heterozygous mutations in COL4A3 or
COL4A4 are usually associated with
persistent GBM attenuation, isolated hematuria,
and benign outcomes. Heterozygous mutations in
COL4A5 in women with XLAS are associated
with a wide range of prognostic outcomes, as
described in the section on “Alport Syndrome.”
Perhaps this spectrum of outcomes reflects
differences in the cellular responses provoked by
complete absence of α3α4α5(IV) networks from
GBM (hemizygous XLAS and ARAS), mixed
α3α4α5(IV)-positive and α3α4α5(IV)-negative
GBM (heterozygous XLAS), and homogeneous
reduction in GBM content of α3α4α5
(IV) networks (heterozygous COL4A3 or
COL4A4 mutations).
787
Clinical Features
TBMN is the most common cause of persistent
microscopic hematuria in children and adults
[108]. Persistent microscopic hematuria, associ-
ated with normal blood pressure, renal function,
and urine protein excretion, is the characteristic
feature of TBMN in childhood [3], although epi-
sodic gross hematuria may also be observed.
TBMN is not specifically associated with hearing
loss, ocular defects, or other extrarenal
abnormalities.
Proteinuria has been reported in up to 30 % of
adults with TBMN [106, 109]. About 5–7 % of
adult patients with TBMN have chronic kidney
disease with elevated serum creatinine levels
[109–112].
Pathology
Diffuse attenuation of the lamina densa and GBM,
with preservation of normal podocyte anatomy, is
the characteristic histological abnormality in
TBMN. Glomerular obsolescence or sclerosis
may be observed in adult patients with TBMN,
including some with heterozygous COL4A3 or
COL4A4 mutations [111, 113].
GBM width is dependent on age and gender.
The lamina densa and GBM increase rapidly in
width between birth and 2 years of age, followed
by a more gradual increase during childhood and
adolescence [114]. Adult men exhibit greater
GBM widths than adult women [115].
Because different investigators have used differ-
ent techniques to measure GBM width, a standard
definition of “thin” GBM does not exist. In children
the threshold is 200–250 nm [2, 116, 117],
while the adult threshold ranges from 250 to
330 nm [106, 118].
Routine immunofluorescence studies of renal
biopsy material from patients with TBMN are
typically unremarkable. Immunostaining using
specific antibodies against α3(IV), α4(IV), and
α5(IV) chains yields normal results in patients
with TBMN [105, 119, 120].
Diagnostic Considerations
IgA nephropathy, TBMN, and Alport syndrome
together account for the majority of children with
788
glomerular hematuria seen in pediatric nephrol-
ogy clinics [1–3, 116]. Thorough clinical evalua-
tion, including detailed pedigree analysis, can
assist in determining which children need tissue
studies and which can be followed prospectively
without biopsy. Obtaining urinalyses on first-
degree family members may provide valuable
information, since adults with familial hematuria
may be unaware that they are affected [121].
When a child has isolated microscopic hema-
turia, a family history of dominantly transmitted
hematuria, and a negative family history for renal
failure, a clinical diagnosis of TBMN is reason-
able, and renal biopsy is unnecessary. These chil-
dren should be followed prospectively every 1–2
years. If proteinuria or hypertension develops,
renal biopsy should be considered.
In children who have persistent microscopic
hematuria but do not have affected relatives,
renal biopsy is often informative. In those children
whose renal biopsy findings are limited to GBM
attenuation, the clinician’s challenge is to differ-
entiate TBMN and Alport syndrome. Audiometry
and ophthalmologic examination may be helpful
in older children, but these studies will usually be
normal in young children with Alport syndrome.
Abnormal results of immunostaining for the α3
(IV), α4(IV), and α5(IV) chains suggest a diagno-
sis of Alport syndrome. While normal
immunostaining results cannot entirely exclude
Alport syndrome, they can help support a
suspected diagnosis of TBMN.
The role of molecular analysis of the COL4A3,
COL4A4, and COL4A5 genes in patients with
suspected TBMN remains to be determined.
Since the finding of a heterozygous mutation in
COL4A3 or COL4A4 cannot guarantee a benign
prognosis [26, 27, 113, 122], the need for follow-
up examination of such patients would not be
precluded.
Treatment
Since TBMN usually has a benign outcome, treat-
ment is rarely indicated. Patients with TBMN who
have proteinuria are theoretically candidates for
angiotensin blockade.
M.N. Rheault and C.E. Kashtan
Hereditary Angiopathy
with Nephropathy, Aneurysms,
and Cramps (HANAC Syndrome)
Missense mutations in the COL4A1 gene cause an
autosomal dominant hereditary angiopathy asso-
ciated with nephropathy, aneurysms, and muscle
cramps (HANAC syndrome) [123]. The muta-
tions affect three highly conserved glycine resi-
dues in the collagenous domain of the α1
(IV) chain. Retinal arteriolar tortuosity and retinal
hemorrhages are common in affected individuals,
as are intracranial aneurysms [124], leukoence-
phalopathy, and elevated creatine kinase levels.
Some affected individuals have muscle cramps.
Renal findings in affected individuals include
microscopic and gross hematuria, mild renal
insufficiency, and renal cysts. Mutations in the
COL4A1 and COL4A2 genes have not been
found in patients with Alport syndrome or
TBMN [125].
Renal biopsy in affected individuals with
hematuria showed no abnormalities of GBM
structure or type IV collagen expression. How-
ever, basement membranes of Bowman’s cap-
sules, tubules, and interstitial capillaries
exhibited irregular thickening, splitting into mul-
tiple layers, and focal interruptions.
Laminin Disorders
Pierson Syndrome
The association of congenital nephrotic syndrome
with eye abnormalities in siblings was first
described by Pierson in 1963 [126]. Affected
infants died within the first year of life with
ESRD. Subsequent reports of additional patients
described variable abnormalities on fetal ultra-
sound, including kidney enlargement and
hyperechogenicity, oligohydramnios, placental
enlargement, and/or pulmonary hypoplasia
[127, 128]. Infants surviving to term exhibited
congenital nephrotic syndrome, with renal biopsy
findings of diffuse mesangial sclerosis and diffuse
26 Inherited Glomerular Diseases
GBM abnormalities, accompanied by ocular
globe enlargement (buphthalmos) with reduction
in the size and reactivity of pupils (microcoria).
Additional ocular findings in some patients
included cataract, posterior rupture of the lens
capsule, and retinal abnormalities. Some affected
children have also been found to have muscular
hypotonia, central nervous system hemangiomas,
and genital abnormalities [129, 130].
The gene for Pierson syndrome was mapped to
chromosome 3p14-p22 and identified as LAMB2,
the gene encoding the laminin β2 chain, by Zenker
and colleagues in 2004 [127]. Subsequent reports
indicate that LAMB2 mutations may be associated
with isolated congenital nephrotic syndrome and
with mild variants of Pierson syndrome [131,
132]. The absence of the laminin β2 chain in
transgenic mice results in massive proteinuria
along with retinal and neuromuscular abnormali-
ties [133]. In these mice, proteinuria precedes the
appearance of podocyte abnormalities, suggesting
that the GBM contributes to the barrier function of
the glomerular capillary wall. Genotype influ-
ences the phenotype in individuals with LAMB2
mutations. Truncating or splice-site mutations are
more likely to be associated with complete
Pierson syndrome, whereas missense mutations
or small deletions are more likely to be associated
with the absence of neurologic abnormalities and
higher age of onset of renal disease [132].
Type III Collagen Nephropathies
In the kidney, type III collagen is normally found
in the interstitium and blood vessel walls. Two
rare disorders in which pathological accumulation
of type III collagen is observed in glomeruli are
discussed in this section.
Nail-Patella Syndrome (Hereditary
Osteo-onychodysplasia)
Nail-patella syndrome (NPS) is a rare autosomal
dominant disorder characterized by dystrophic
789
nails, hypoplasia or absence of the patellae, dys-
plasia of the elbows and iliac horns, and renal
disease [134, 135]. Affected individuals may
also exhibit glaucoma and sensorineural deafness
[136, 137]. The disorder affects about 1 in 50,000
individuals. The severity of renal involvement,
which occurs in 30–40 % of patients, determines
prognosis.
Genetics
Targeted disruption of the LIM homeodomain
transcription factor gene Lmx1b in mice resulted
in hypoplastic nails and absent patellae as well as
renal disease, suggesting a possible locus for
human NPS [138]. LMX1B, the human homo-
logue of Lmx1b, and the NPS locus were found
to map to chromosome 9q34, and heterozygous
mutations in LMX1B were identified in NPS
patients [139, 140]. A variety of LMX1B gene
defects have been identified in NPS patients,
including missense, splicing, insertion/deletion,
and nonsense mutations [139–143]. The results
of in vitro experiments in which the transcrip-
tional effects of mixing wild-type and mutant
LMX1B protein are measured, along with the
variety of observed mutation types, suggest that
the NPS phenotype results from haploinsuf-
ficiency rather than a dominant-negative mecha-
nism [141, 144]. Mutations in the homeodomain
of LMX1B protein and female gender may confer
an increased risk of renal involvement [137].
LMX1B protein is specifically expressed in
glomerular podocytes, beginning at the S-shaped
body stage of glomerular development
[138]. Lmx1b-null mice exhibit marked reduction
in GBM expression of the α3 and α4 chains of
type IV collagen and the slit diaphragm proteins
podocin and CD2AP [145–147]. However,
changes in expression of these putative targets of
LMX1B were not observed in kidneys of patients
with NPS nephropathy [148]. Recently, LMX1B
was found to be essential for the maintenance of
differentiated podocytes in adult kidneys through
effects on actin cytoskeletal organization
[149]. Mutations in LMX1B have also been
described in patients with autosomal dominant
790
Fig. 7 Electron
microscopy.
Phosphotungstic acid stain
(10,500). Nail-patella
syndrome. Irregular
distribution of fibrillar
collagen within the GBM.
Inset shows the typical
periodicity of interstitial
collagen (48,000)
focal segmental glomerulosclerosis without
extrarenal involvement [150].
Clinical Features
Renal. Less than half of NPS patients have clinical
renal disease [151]. Symptoms including mild
proteinuria with or without microscopic hematu-
ria typically appear in adolescence or young adult-
hood. Occasional patients develop nephrotic
syndrome and hypertension. The nephropathy is
typically mild, with about a 5–10 % risk of pro-
gression to ESRD. Marked differences in the
severity of the nephropathy can be observed in
related patients, suggesting the influence of
superimposed factors.
Nails. Nail abnormalities occur in over 90 % of
patients and are usually apparent from birth
[152]. Fingernails, especially on the thumb and
index finger, are more severely affected than toe-
nails. The nails may be absent or dystrophic with
discoloration, koilonychias, longitudinal ridges,
or triangular lunulae.
Skeletal defects. Over 90 % of NPS patients
exhibit aplasia or hypoplasia of the patellae
[152]. Associated symptoms may include knee
pain, effusions, dislocations, and osteoarthritis.
Dysplasia of the elbows occurs in over 90 % of
patients [152]. Anomalies such as hypoplasia of
the radial head with dislocation, posterior pro-
cesses at the distal ends of the humeri, hypoplasia
of the olecranon, and elongation of the neck of the
M.N. Rheault and C.E. Kashtan
radius may be associated with mild to severe
limitation in extension, pronation, and supination
of the forearm. About 80 % of patients display
osseous processes projecting posteriorly from the
iliac wings, known as iliac horns, which are patho-
gnomonic for NPS [152].
Pathology
The nephropathy of NPS has no specific features
by light microscopy or routine immunofluores-
cence. Focal segmental glomerulosclerosis and
deposits of IgM and C3 may be observed,
depending on the severity of renal involvement.
Characteristic lesions are observed by
electron microscopy [153, 154]. With standard
staining techniques, the GBM and
mesangium exhibit multiple lucencies, imparting
a “moth-eaten” appearance. Staining with
phosphotungstic acid reveals clusters of cross-
banded collagen fibrils within these lucent
areas (Fig. 7). These collagen fibrils have been
observed in NPS patients with no clinically evi-
dent renal disease, and there is no correlation
between the extent of GBM changes and patient
age, severity of proteinuria, or degree of renal
functional impairment [141, 142]. Staining of
NPS kidneys with antibodies against type III col-
lagen produced irregular, discontinuous labeling
of GBM in normal-appearing glomeruli and
focally intense staining of sclerotic
glomeruli [148].
26 Inherited Glomerular Diseases
Treatment
There is no specific therapy for NPS renal disease.
Kidney transplantation has been carried out suc-
cessfully, and recurrence of renal disease appar-
ently did not recur. Selection of living donors
must be performed with care, since NPS is an
autosomal dominant disorder.
Collagen Type III Glomerulopathy
(Collagenofibrotic Glomerulopathy)
Renal disease associated with glomerular deposits
of type III collagen has been described in patients
who have no extrarenal abnormalities [155, 156].
In comparison to NPS patients, subjects with col-
lagen type III glomerulopathy have relatively
severe renal findings, more extensive histological
changes, and greater glomerular type III collagen
accumulation.
Clinical Features
Patients with collagen type III glomerulopathy
can present in childhood or in adulthood. Early
presentation is associated with a more severe dis-
ease course. Proteinuria is common to the juvenile
and adult forms of the disease. In affected indi-
viduals, proteinuria progresses to nephrotic syn-
drome and is accompanied by hypertension and
the development of renal failure in up to 50 % of
affected patients [156, 157]. Microangiopathic
hemolytic anemia has been described in some
patients. Patients presenting in adulthood exhibit
more gradual increase in proteinuria and loss of
renal function [158].
Pathology
Light microscopy shows enlarged glomeruli with
mesangial expansion and glomerular capillary
wall thickening due to subendothelial accumula-
tion of poorly staining material. Routine immuno-
fluorescence studies are unremarkable or show
nonspecific immunoprotein deposits. Electron
microscopy shows deposits of electron-lucent
material in mesangial matrix and GBM. Staining
with phosphotungstic acid demonstrates fibrillar
collagen in these deposits which is identified as
type III collagen by specific immunostaining.
791
Hereditary Nephritis
with Thrombocytopenia and Giant
Platelets: Epstein and Fechtner
Syndromes
Epstein and Fechtner syndromes are allelic, auto-
somal dominant disorders that have some features
in common with Alport syndrome, such as hema-
turia, progressive nephropathy and sensorineural
deafness [159, 160]. However, these conditions
are distinguished clinically from Alport syndrome
by the invariable presence of thrombocytopenia
and giant platelets. In addition, granulocytes of
patients with Fechtner syndrome display
cytoplasmic inclusions known as Dohle-like bod-
ies, and these patients may develop cataracts. In
some patients, ultrastructural changes in GBM
resemble those of Alport syndrome
[161, 162]. However, glomerular expression of
type IV collagen α3, α4, and α5 chains is normal
in these patients [162].
Despite the similarities with Alport syndrome,
Epstein and Fechtner syndromes are genetically
distinct disorders. Following the mapping of the
Epstein and Fechtner loci to chromosome 2q11-
13 [163, 164], heterozygous mutations in MYH9,
the gene that encodes nonmuscle myosin heavy
chain IIA (NMMHC-IIA), were identified in
patients with these disorders as well as in patients
with two other conditions featuring giant platelets,
Sebastian syndrome and May-Hegglin anomaly,
and in nonsyndromic hereditary deafness
(DFNA17) [165–168]. NMMHC-IIA is expressed
in podocytes [169], but the mechanism by which
mutations in this protein might adversely affect
podocyte function is unknown.
Hereditary Metabolic Disorders
with Primary Glomerular Involvement
Fabry Disease
Anderson-Fabry disease is a rare X-linked
disorder of glycosphingolipid metabolism resulting
from deficiency of the lysosomal hydrolase
a-galactosidase A.
792
Renal tissue from all hemizygous patients
demonstrates characteristic glycolipid
accumulation within every glomerular, vascular,
and interstitial cell and within distal tubular
cells, regardless of age at biopsy. Two
intermingled cell populations, normal cells and
cells exhibiting glycolipid accumulation, are
observed in heterozygous females. Degenerative
renal changes develop with age. They initially
affect vessels and consist of round
fibrinoid deposits resulting from smooth muscle
cell necrosis. These changes are followed by
nonspecific vascular, glomerular, and tubuloin-
terstitial lesions [170]. Enzyme replacement
appears to result in decreased glycolipid storage
in renal cells and stabilization of renal function
[171–173].
Other Glomerular Lipidoses
Nephrosialidosis is a rare autosomal recessive
condition caused by neuraminidase deficiency.
Clinical and radiologic features include
dysmorphic facies, visceromegaly, mental retar-
dation, skeletal anomalies, marrow foam cells,
and cherry-red spot on fundoscopy. Renal
involvement consists of proteinuria and
progression to ESRD early in life [174]. Renal
biopsy findings include podocyte and proximal
tubular cell vacuolization [175]. By electron
microscopy, many of these vesicles appear
empty, but others contain flocculent or
membrane-like electron-dense material. Wheat
germ agglutinin binds to cytoplasmic material in
podocytes and tubular cells, indicating the pres-
ence of compounds with terminal sialic
(neuraminic) acid moieties [176].
Silent accumulation of glycolipids or muco-
polysaccharides has been described in
patients with Gaucher disease, Niemann-Pick
disease, I-cell disease, and GM1
gangliosidosis [177]. Clinical renal disease during
childhood is unusual in patients with these
disorders.
M.N. Rheault and C.E. Kashtan
Hereditary Metabolic Disorders
with Secondary Glomerular
Involvement
Familial Amyloidosis
Hereditary amyloidosis encompasses a group of
autosomal dominant disorders characterized by
extracellular accumulation of protein fibrils
arranged in an antiparallel β-pleated sheet config-
uration [178]. These disorders are classified
according to the protein composing amyloid
fibrils and/or the type of mutation in the
corresponding gene. Transthyretin variants have
been found in most affected families, but variants
of cystatin C, gelsolin, apolipoprotein A1, fibrin-
ogen, and lysozyme have been described in other
kindreds. Symptomatic renal involvement is
unusual during childhood.
Familial Mediterranean fever is an autosomal
recessive disorder described primarily, but not
exclusively, in several ethnic groups originating
in the Mediterranean region. The disease is char-
acterized by recurrent episodes of fever, abdomi-
nal pain, joint pain, pleuritis, and pericarditis
[179]. Renal amyloidosis of the AA type may
result in proteinuria and eventual renal failure.
Renal amyloidosis of the AA type may be associ-
ated with the autosomal dominant disorders
Muckle-Wells syndrome and tumor necrosis fac-
tor receptor 1-associated periodic syndrome
(TRAPS) [180].
Alpha-1 Antitrypsin Deficiency
Chronic liver disease due to deficiency of alpha-1-
antitrypsin (α1AT) may be associated with glo-
merulonephritis in children [181–183]. Renal
biopsy reveals diffuse or focal segmental
membranoproliferative glomerulonephritis, type
I in most cases, associated with subendothelial
deposits composed of immunoglobulin and com-
plement. Alpha-1-antitrypsin deficiency has also
been associated with IgA nephropathy and
26 Inherited Glomerular Diseases
Fig. 8 Electron
microscopy. Lead citrate
and uranyl acetate stain
(8,600). Alagille
syndrome. Massive
accumulation of lipid
vacuoles within mesangial
cells and matrix. Irregular
distribution of lipid
vacuoles within the GBM
systemic ANCA positive vasculitis [184]. Renal
manifestations include proteinuria, hypertension,
and renal failure. Regression of nephrotic syn-
drome and glomerular lesions after liver trans-
plantation has been observed [185].
Alagille Syndrome
Alagille syndrome is an autosomal dominant dis-
order with variable penetrance that causes chole-
stasis in childhood, associated with characteristic
facies, cardiac malformations, vertebral abnor-
malities, posterior embryotoxon, hypogonadism,
growth retardation, and high-pitched voice. The
disease results from mutations in JAG1, which
encodes a ligand for the Notch1 receptor, or muta-
tions in NOTCH2 [186, 187]. JAG1 and
NOTCH2 are required for glomerular develop-
ment. Renal involvement occurs in ~40 % of
patients with JAG1 mutations and can include
renal dysplasia, renal tubular acidosis,
vesicoureteral reflux, cystic disease, and obstruc-
tion [188]. Accumulation of lipid vacuoles in
mesangial matrix, mesangial cells, and GBM has
been observed in some patients [189] (Fig. 8). The
extent of mesangiolipidosis correlates with the
severity of cholestasis. Although the glomerular
lesions are present early in life, renal symptoms in
childhood are unusual. Progression to ESRD may
occur in affected adults.
793
Hereditary Lecithin-Cholesterol
Acyltransferase (LCAT) Deficiency
LCAT deficiency is a rare autosomal recessive
disorder characterized by the inability to esterify
plasma cholesterol, leading to deposition of
unesterified cholesterol in tissues, including the
kidney [190]. Clinical manifestations include cor-
neal opacities, anemia, and proteinuria, sometimes
in the nephrotic range [191, 192]. Progression to
ESRD usually occurs in the fourth or fifth decade.
Glomerular lesions include accumulation of foam
cells of endothelial and mesangial origin and mas-
sive accumulation of lipids within the mesangial
matrix and subendothelial GBM [190, 191].
Lipoprotein Glomerulopathy
Lipoprotein glomerulopathy is characterized by
intraglomerular lipoprotein thrombosis and high
plasma concentrations of apolipoprotein E
[193]. The disease is usually detected in adults,
but onset of symptoms during childhood has been
described [194]. Renal symptoms range from pro-
teinuria to nephrotic syndrome, and progression
to ESRD occurs in some patients [195]. Recur-
rence of glomerular lesions after renal transplan-
tation has been observed [196]. Mutations in
apolipoprotein E have been found in patients
with this disorder [197].
794
Glomerular lipidosis has also been reported in
patients with familial hypercholesterolemia
resulting from defects in the LDL receptor, in
type III hyperlipoproteinemia, and in patients
with cholesterolic polycoria. Renal symptoms
usually appear in adulthood in patients with
these disorders.
Familial Juvenile Megaloblastic Anemia
Familial juvenile megaloblastic anemia (Imerslund-
Grasbeck syndrome) is a rare autosomal recessive
disorder caused by selective vitamin B12 malabsorp-
tion. Anemia is detected in infancy or early child-
hood and is associated with mild, nonprogressive
low-molecular weight proteinuria [198]. Most cases
arise from mutation in the gene for either cubilin
(chromosome 10) or amnionless (chromosome 14),
which are components of the intestinal receptor for
the vitamin B12-intrinsic factor complex as well as
the receptor that mediates proximal tubular
reabsorption of filtered protein [199].
Other Hereditary Diseases
with Glomerular Involvement
Charcot-Marie-Tooth (CMT) Disease
CMT disease is a genetically heterogeneous,
familial peripheral neuropathy resulting in pro-
gressive symmetric atrophy and weakness of dis-
tal muscles and sensory deficits. Autosomal
dominant inheritance is most commonly caused
by mutations in peripheral myelin protein
22 (PPMP22) and myelin protein zero (MPZ);
however, over 40 genes have been associated with
this disorder [200]. Proteinuria and progression to
ESRD associated with focal glomerulosclerosis
(FSGS) occur in some patients [201, 202]. Since
some of these patients are also deaf, CMT disease
could potentially be misdiagnosed as Alport syn-
drome. No specific ultrastructural changes in GBM
have been described in patients with CMT disease.
Recently, mutations in INF2, a cause of autosomal
dominant FSGS, were found in patients with CMT
and FSGS [203].
M.N. Rheault and C.E. Kashtan
Cockayne Syndrome
Cockayne syndrome is an autosomal recessive
disorder characterized by growth retardation, neu-
rologic abnormalities, premature aging, senile
facies, sensorineural deafness, cataracts, retinop-
athy, sun sensitivity, and dental caries [182]. The
disorder arises from mutations in genes involved
in DNA nucleotide excision repair (ERCC6 and
ERCC8) [204]. Renal symptoms including hyper-
tension, proteinuria, and renal insufficiency occur
in about 10 % of patients, associated with diffuse,
homogeneous GBM thickening [205].
Hereditary Acro-osteolysis
with Nephropathy
Hereditary acro-osteolysis is a rare disorder char-
acterized by arthritic episodes and progressive
resorption of carpal and tarsal bones. Familial
(dominant or recessive) and sporadic cases have
been reported. Hypertension, proteinuria, and pro-
gressive renal failure occur in some patients
[206]. Renal biopsy findings include arteriolar
thickening and sclerosis and focal
glomerulosclerosis.
Other Syndromes with Renal
Involvement
Renal abnormalities, typically cystic renal dyspla-
sia and/or tubulointerstitial lesions, are found in
most patients with Bardet-Biedl syndrome (BBS),
a genetically heterogeneous, autosomal recessive
disorder whose cardinal features included obesity,
polydactyly, mental retardation, retinal dystrophy,
and hypogonadism [207]. Glomerular symptoms
such as proteinuria and questionable glomerular
changes have been described in a few patients
[208]. Mutations in a number of genes involved
in the functioning of primary cilia have been
linked to BBS [209].
Alstrom syndrome is an autosomal recessive
disorder characterized by cone-rod dystrophy,
obesity, progressive sensorineural deafness,
dilated cardiomyopathy, insulin resistance
26 Inherited Glomerular Diseases
syndrome, and developmental delay due to muta-
tions in the ALMS1 gene [210]. Renal tubular
dysfunction and tubulointerstitial lesions have
been observed in some patients [211]. Renal dis-
ease due to vascular lesions and secondary
glomerulosclerosis occurs in some patients with
familial dysautonomia [212].
Hereditary Glomerulopathies Without
Extrarenal Symptoms
Fibronectin Glomerulopathy
Fibronectin glomerulopathy is associated with
massive accumulation of fibronectin derived
mainly from the soluble plasma isoform, transmit-
ted as an autosomal dominant trait [213–215]. A
substantial fraction of cases arises from mutations
in the gene FN1 [216], which is located on chro-
mosome 2 and encodes fibronectin, but another
locus appears to exist at chromosome 1q32
[217]. Clinically, the disease is characterized by
proteinuria of variable magnitude, typically first
observed in early adulthood. Subsequently,
patients develop hypertension and renal insuffi-
ciency, with gradual progression to ESRD. Recur-
rence after kidney transplantation has been
described [218].
Mitochondrial Cytopathies
Mitochondrial cytopathies are a heterogeneous
group of disorders resulting from genetic defects
in enzymatic complexes of the respiratory chain,
leading to impaired oxidative phosphorylation
and energy production. The most common renal
defect is the de Toni-Debre-Fanconi syndrome,
associated with nonspecific abnormalities in
tubules and interstitium on renal biopsy, but glo-
merular lesions such as FSGS, collapsing
glomerulopathy, and crescentic glomerulonephri-
tis may also occur [219]. Proteinuria and FSGS
have been found in patients with maternally
inherited diabetes and/or deafness and in patients
with complete or incomplete MELAS syndrome
795
(mitochondrial encephalopathy with lactic acido-
sis and stroke-like episodes) [220].
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 Idiopathic Nephrotic Syndrome
in Children: Genetic Aspects 27
Olivia Boyer, Kálmán Tory, Eduardo Machuca, and
Corinne Antignac

Contents Autosomal Dominant Forms of Isolated

SRNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 814
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 806 Mutations in ACTN4 Encoding α-Actinin-4 . . . . . . . . 814
Autosomal Recessive Forms of Isolated Mutations in TRPC6 Encoding the Transient
SRNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 810 Receptor Potential Cation Channel 6 . . . . . . . . . . . . . . . . 815
Mutations in NPHS2 Encoding Podocin . . . . . . . . . . . . 810 Mutations in INF2 Encoding the Inverted
Mutations in NPHS1 Encoding Nephrin . . . . . . . . . . . . 812 Formin 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 815
Mutations in PLCE1 Encoding Phospholipase C Less Commonly Mutated Genes . . . . . . . . . . . . . . . . . . . . 816
Epsilon 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 812 NGS Findings and Redefinition of Known
Less Commonly Mutated Genes . . . . . . . . . . . . . . . . . . . . 813 Gene-Associated Phenotypes . . . . . . . . . . . . . . . . . . . . . . . . 816
NGS Findings and Redefinition of Known Syndromic Forms of SRNS . . . . . . . . . . . . . . . . . . . . . . . . 818
Gene-Associated Phenotypes . . . . . . . . . . . . . . . . . . . . . . . . 814 SRNS with Genitourinary Features:
Denys–Drash and Frasier Syndromes . . . . . . . . . . . . . . . 818
SRNS with Ocular Features: Pierson
Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 818
O. Boyer
SRNS with Osseous Dysplasia: Nail-Patella
Service de Néphrologie Pédiatrique, Hôpital
and Schimke Syndromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 819
Necker-Enfants Malades, Université Paris-Descartes,
SRNS with Central Nervous System
Paris, France
Involvement: Metabolic Diseases and
e-mail: olivia.boyer@nck.aphp.fr
Galloway–Mowat Syndrome . . . . . . . . . . . . . . . . . . . . . . . . 820
K. Tory SRNS with Peripheral Neurological
Laboratory of Hereditary Kidney Diseases, Inserm UMR Involvement: Charcot–Marie–Tooth Disease
1163, Paris, France and FSGS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 823
SRNS with Cutaneous Features: Epidermolysis
Department of Pediatrics, Semmelweis University,
Bullosa and FSGS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 824
Budapest, Hungary
e-mail: kalman.tory@inserm.fr; tory.kalman@med. Familial Forms of Steroid-Sensitive
semmelweis-univ.hu Nephrotic Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 825
E. Machuca Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 825
Department of Nephrology, Medical School, Pontificia
Universidad Católica de Chile, Santiago, Chile References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 826
C. Antignac (*)
Laboratory of Hereditary Kidney Diseases, Inserm UMR
1163, Paris, France
Paris Descartes, Sorbonne Paris Cité University, Imagine
Institute, Paris, France
Department of Genetics, MARHEA reference center,
Necker – Enfants Malades Hospital, Paris, France
e-mail: corinne.antignac@inserm.fr

# Springer-Verlag Berlin Heidelberg 2016 805

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_23
806
Introduction
Over the last decades, screening of large cohorts
of pediatric patients presenting with steroid-
resistant nephrotic syndrome (SRNS) for gene
mutations has revealed the importance of genetic
disorders in the pathogenesis of proteinuric
glomerulopathies. Genetic forms of nephrotic
syndrome have been considered as infrequent dis-
orders; however, at least 66 % of the cases
presenting with SRNS during the first year of
life [1] and up to 30 % of SRNS cases with an
onset below 25 years of age have an underlying
monogenic defect [2]. From a clinical perspective,
theoretically all patients with hereditary SRNS
will be resistant to immunosuppressive agents
and will not experience relapse after transplanta-
tion [3–5]. Whereas partial remission may be
achieved under cyclosporin A, shown to stabilize
the podocyte actin cytoskeleton in vitro through
the synaptopodin pathway [6], such treatment
cannot induce complete remission in SRNS
related to podocyte gene mutations [4, 7–10].
Gene discovery efforts aimed at unraveling the
causes of Mendelian forms of nephrotic syndrome
have resulted in the identification of mutations in
novel genes that encode proteins crucial for the
establishment and maintenance of the glomerular
filtration barrier. These discoveries have helped
decipher the pathophysiologic mechanisms of
the glomerular filtration process by revealing
that most of the defective proteins are essential
for glomerular podocyte function. Podocytes are
highly differentiated epithelial cells with an
octopus-like shape maintained by a finely regu-
lated cytoskeleton. Their multiple cell extensions
named “foot processes” are interdigitated and
connected by slit diaphragms, final glomerular
filtration barriers. The list of genes mutated in
syndromic and non-syndromic forms of SRNS is
constantly evolving and consists to date of more
than 30 genes inherited in an autosomal recessive,
autosomal dominant, or mitochondrial fashion
(Table 1) [11–13]. Among these genes, NPHS1
and NPHS2 encoding nephrin and podocin,
respectively, are by far the two main genes impli-
cated in SRNS [2, 9]. Mutations in NPHS1 are
O. Boyer et al.
responsible for most of the cases with congenital
nephrotic syndrome (CNS) and might be found in
infantile forms of SRNS [14, 15]. NPHS2 muta-
tions are the most frequent causes of early-onset
autosomal recessive SRNS [16] and account for
37.5 % of the cases with NS presenting in the first
year of life among European populations [1].
Autosomal dominant (AD) forms of SRNS are
infrequent and generally observed in adults, except
for WT1-related SRNS. Variable degrees of pro-
teinuria are detected, and progression to end-stage
kidney disease (ESKD) is usually slow [17, 18].
Since most patients do not exhibit full-blown
nephrotic syndrome, these forms are usually
referred to as AD focal segmental glomerulo-
sclerosis (FSGS).
The advent of next-generation sequencing
(NGS) techniques in the past few years has tre-
mendously facilitated the identification of new
gene mutations in genetic diseases, most of them
being rare mutations involving few families.
Moreover, it has revealed mutations in unex-
pected genes and has widened the phenotypes
associated with podocyte gene mutations.
Recently, mutations in genes involved in
tubulointerstitial and developmental nephropa-
thies have been associated with isolated SRNS
such as the homozygous p.P209L TTC21B muta-
tion known to also lead to nephronophthisis [19]
or PAX2 mutations known as a cause of congenital
anomalies of the kidney and urinary tract
(CAKUT) and papillorenal syndrome [20]. More
importantly, NGS approaches have wholly modi-
fied the diagnosis screening algorithms, and
podocyte gene panels are increasingly used as
the first step of genetic analyses in selected
patients such as children or adults with familial
FSGS [2, 21, 22].
This chapter will review the available epidemi-
ologic data, genotype–phenotype correlations,
and the mechanisms by which mutations in
genes implicated in hereditary forms of isolated
SRNS lead to proteinuric glomerular disease.
A genetic overview on recently discovered genes
responsible for rarer cases of hereditary
syndromic SRNS and advances in the study of
familial SSNS are presented as well.
 27

Table 1 Genes implicated in hereditary SRNS and FSGS and associated phenotypes
Typical age
Number of families at dg of Recurrent extrarenal
Gene Desc. Trans. Encoded protein (pts) publishedc PU/NS Age at ESKD Typical histology involvement
Autosomal recessive isolated SRNS
NPHS1 [14] 1998 AR Nephrin >270(>300) 0–10 yrs 7 ms–15 yrs MGC, FSGS –
NPHS2 [16] 2000 AR Podocin >600(>850) 0–40 yrs 2 yrs–50 yrs MGC, FSGS –
PLCE1 [76] 2006 AR Phospholipase C epsilon 50(71) 0–8 yrs 5 ms–12 yrs DMS, FSGS –
1
CD2AP [101] 2007 AR CD-2 associated protein 1(1) 10 mo 3 yrs FSGS –
PTPRO [92] 2011 AR Receptor-type tyrosine- 2(5) 5–14 yrs 18 yrs (n = 1) MGC, FSGS –
protein phosphatase O
MYO1E [85] 2011 AR Myosin 1E 10(14) 2 ms–9 yrs 6 yrs to 15 yrs FSGS –
ADCK4 [238] 2013 AR aarF domain containing 8(15) <1–21 yrs 7–23 yrs FSGS Goiter
kinase 4
TTC21B [19] 2014 AR Intraflagellar transport 7(13) 9–30 yrs 1–35 yrs FSGS –
protein IFT139
CRB2 [93] 2015 AR Crumbs homolog 5(4) 9 mo to 6 yrs NA FSGS Cerebral ventriculomegaly
2 protein with congenital NS
Autosomal dominant isolated SRNS
Idiopathic Nephrotic Syndrome in Children: Genetic Aspects

ACTN4 [105] 2000 AD α-Actinin-4 12(>75) 3–54 yrs 6–59 yrs FSGS –
TRPC6 [43] 2005 AD Transient receptor >28(>62) 2–75 yrs 0–20 yrs FSGS –
potential 6 after onset
INF2d[122] 2010 AD Inverted formin 2 >60(>221) 5–72 yrs 13–70 yrs FSGS –d
LMX1Bd[129] 2013 AD LIM homeodomain 8(>28) 5–70 yrs 28–70 yrs FSGS –d
transcription factor 1B
ARHGAP24 2011 AD Rho-GTPase-activating 1(3) NA 12–29 yrs FSGS –
[126] protein 24
ANLN [127] 2014 AD Anillin 2(12) 9–69 yrs 35–75 yrs FSGS –
COL4A3/ 2014 AD Collagen IV alpha-3 and 16(57) 2–70 yrs 12–82 yrs FSGS Hearing impairment
COL4A4 alpha-4 chains
[131]
WT1[145] 1992 AD Wilms’ tumor protein 7(31) 3–59 yrs 10–69 yrs FSGS Ambiguous genitalia
PAX2d[20] 2014 AD Paired box protein 2 7(24) 7–68 yrs 30–58 yrs FSGS –d
(continued)
807
 808

Table 1 (continued)
Typical age
Number of families at dg of Recurrent extrarenal
Gene Desc. Trans. Encoded protein (pts) publishedc PU/NS Age at ESKD Typical histology involvement
Syndromic forms
WT1 [154] 1991 AD Wilms’ tumor protein Denys–Drash >170 0–10 yrs 0–15 yrs DMS Wilms’ tumor in both genders,
Sd genital anomalies in 46,XY
patients
Frasier Sd >60 7 mo to 5 yrs to 34 yrs FSGS Pseudohermaphroditism,
34 yrs gonadoblastoma in 46,XY
patients
LAMB2 [168] 2004 AR Laminin β2 subunit >60 (>70) 0–6 yrs 0–21 yrs DMS Microcoria, abnormal lens, VI,
hypotonia, motor delay
LMX1B [128] 1998 AD LIM homeodomain >35 (>54) 9–76 yrs 22–52 yrs FSGS, Hypoplastic nails/patellae,
transcription factor 1B NPS-nephropathy iliac horns
SMARCAL1 2002 AR SWI/SNF-related, (>90) 2–12 yrs 3.8–22 yrs FSGS Spondyloepiphyseal
[204] matrix-associated, actin- dysplasia, T-cell deficiency,
dependent regulator of cerebral ischemia, broad nasal
chromatin, subfamily tip, hyperpigmented macules
a-like 1
MTTL1 [208] 1990 Maternal tRNALeu(UUR) >30 (>45) 5–47 yrs 13–51 yrs FSGS MELAS: myopathy,
encephalopathy, lactic
acidosis, and stroke-like
episode, diabetes, and
deafness
COQ2 [227] 2006 AR Para-hydroxybenzoate- 10(13) 0–30 mo 0 to 5 yrsa FSGS Encephalomyopathy,
polyprenyltransferase hypotonia, seizures, lactate
acidosis
PDSS2 [233] 2006 AR Decaprenyl-diphosphate 3(3) 0–23 mo ND No data Encephalomyopathy,
synthase subunit 2 hypotonia, seizures, lactate
acidosis
COQ6 [237] 2011 AR Coenzyme Q10 8(16) 2 mo to 6.4 5 ms to 9.3 yrs FSGS SND, seizure
monooxygenase 6 yrs
O. Boyer et al.
 27

SCARB2 2008 AR Lysosome membrane 18(24) 9 to >59 yrs 9 to >59 yrs FSGS Progressive myoclonic
[241, 242] protein 2 (LIMP-2) epilepsy, cerebral and
cerebellar atrophy, peripheral
neuropathy, hearing
impairment
WDR73 [261] 2014 AR WD40-repeat-containing 2(3) 5 to >13 yrs 5 yrs FSGS Secondary microcephaly,
protein intellectual deficiency, cortical
and cerebellar atrophy, facial
dysmorphy, optic atrophy
ARHGDIA 2013 AR Rho-GDP dissociation 3(6) 0–2.4 yrs 6 wks to 3 yrs DMS Intellectual disability
[262, 263] inhibitor α
INF2 [124] 2011 AD Inverted formin 2 27(44) 10–46 yrs 12–47 yrs FSGS Charcot–Marie–Tooth
neuropathy, deafness
ITGB4 [278] 1998 AR Integrin β4 subunit 2(2) 2 mo to NA FSGS Junctional epidermolysis
10 yrs bullosa, nail dystrophy,
recurrent hemorrhagic cystitis,
desquamation of laryngeal
mucosa
LAMB3 [279] 2005 AR Laminin β3 subunit 1(1) 4 mo NA (b5 mo) DMS Epidermolysis bullosa, nail
dystrophy
ITGA3 2012 AR Integrin α3 subunit 6(6) 0–4 yrs 2 wks to 4 yrs FSGS Interstitial lung disease,
[280, 281] epidermolysis bullosa
Idiopathic Nephrotic Syndrome in Children: Genetic Aspects

CD151 [283] 2004 AR Tetraspanin CD151 2y NA NA Thickening of the Pretibial skin lesions, SND,
GBM (n = 1) lacrimal duct stenosis, nail
dystrophy
a
Besides CoQ supplementation
b
Died (at the age of)
c
Only patients with renal phenotype are considered
d
Only patients published as isolated FSGS are considered
AD autosomal dominant, AR autosomal recessive, desc. description, DMS diffuse mesangial sclerosis, ESKD end-stage kidney disease, FSGS focal segmental glomerulosclerosis, MGC
minimal glomerular changes, mo months, NA not available, NPS nail-patella syndrome, NS nephrotic syndrome, pts patients, PU proteinuria, SND sensorineural deafness, Sd syndrome, trans.
transmission, VI severe visual impairment, wks weeks, yrs years
809
810
Autosomal Recessive Forms
of Isolated SRNS
Mutations in NPHS2 Encoding Podocin
Gene Identification and Protein
Characterization
Using a positional cloning approach, Boute
et al. identified mutations in NPHS2 (MIM
#604766) encoding podocin in pedigrees of
patients from Europe and Northern Africa who
presented with childhood-onset autosomal reces-
sive non-syndromic SRNS [16]. Subsequent stud-
ies further defined the phenotype associated with
mutations in the NPHS2 gene, revealing that
patients usually develop NS from birth to 6 years
of age, do not respond to immunosuppressive
agents, and reach ESKD before the end of the
first decade of life [1, 3, 4, 23]. Histological find-
ings range from minimal glomerular changes
(MCD), in patients biopsied early, to FSGS at
later stages [16]. NPHS2 mutations are approxi-
mately responsible for 30–40 % of familial and for
10–30 % of sporadic cases of SRNS [3, 4, 24–27].
Interestingly, NPHS2 mutations were also
found to be a frequent cause of congenital NS
(CNS) manifesting in utero or within the first
3 months of life (15–39 %), infantile NS with
onset between 4 months and 1 year of age
(29–35.2 %) [1, 23, 27, 28], and also rarely
adult-onset SRNS [29–36], thereby further broad-
ening the spectrum of phenotypes attributable to
podocin mutations [27].
The NPHS2 gene spans a 25-kb region, con-
sists of eight exons, and encodes the 42-kDa pro-
tein podocin with 383 amino acid residues.
Podocin is almost exclusively expressed in the
kidney at the slit diaphragm of podocytes, from
the capillary-loop stage [16, 37, 38]. It is a lipid
raft-associated protein belonging to the stomatin
family. Podocin is predicted to be an integral
membrane protein inserted in the membrane
through a short hydrophobic region resulting in a
hairpin-like topology with intracellular NH2- and
COOH-terminal ends [16]. It also contains a
prohibitin homology (PHB) domain that, in addi-
tion to palmitoylation sites in the hydrophobic
O. Boyer et al.
region, is required for podocin to bind cholesterol
and regulate the activity of associated proteins
[39]. Podocin accumulates in oligomeric form in
lipid-raft microdomains of the slit-diaphragm
plasma membrane where it recruits nephrin
thereby augmenting the nephrin-induced activa-
tion of the AP-1 transcription factor and also
interacts with CD2AP and TRPC6 [40–43]. The
resulting protein complex represents an important
link between the slit diaphragm and the podocyte
cytoskeleton and is crucial for structural organi-
zation and regulation of filtration function of the
slit diaphragm, mechanosensory signaling,
podocyte survival, cell polarity, and cytoskeletal
organization [37, 39, 42–45].
Allelic Variants and Genotype/Phenotype
Correlations
Mutations in the NPHS2 gene include a full spec-
trum of missense changes, protein-truncating non-
sense and frameshift mutations, and splice-site
variants and involve all eight coding exons [4, 16,
24]. To date, more than 110 pathogenic mutations
and 40 variants of unknown significance have been
reported [27]. Mutations are frequently found in
pediatric patients with SRNS originating from Cen-
tral Europe, North America, Turkey, the Middle
East, North Africa, and South America. Contrari-
wise, NPHS2 mutations are rarely detected in cases
from Japan, China, Korea, and sub-Saharan Africa.
Several founder mutations have been identified
including p.R138Q in Europe [16, 24], p.R138X
in the Israeli-Arab population [46], p.V260E in the
Comoros Islands, and p.A284V in South America
[27]. The p.R138Q mutation in exon 3 is the most
prevalent mutation in European series [47]. It was
found, respectively, in 32 % and 44 % of all
affected NPHS2 alleles in two large European
series [3, 26]. The resulting protein is retained in
the endoplasmic reticulum (ER) and loses its ability
to recruit nephrin in lipid rafts [40]. Interestingly,
missense mutations leading to retention of the
mutant protein in the ER (such as the p.R138Q)
result in an earlier onset of disease than mutations
that do not disrupt the mutant protein traffic to the
plasma membrane (20.8  4 vs. 128.7  9
months) [48]. A potential therapeutic strategy that
27 Idiopathic Nephrotic Syndrome in Children: Genetic Aspects
might delay the onset and reduce the severity of the
disease in patients with gene mutations causing
ER-retention of the mutant plasma membrane pro-
tein (e.g., slit-diaphragm proteins) relies on chem-
ical chaperones. Several of these molecules have
been used in in vitro systems, allowing for targeting
of the mutant protein to the plasma membrane [49].
The mean age at onset of NS in patients carry-
ing two pathogenic mutations ranges between 2.6
and 3.4 years of age [3, 26]. Weber et al. also
found that patients with frameshift or nonsense
mutations in the homozygous or compound het-
erozygous states have an earlier onset of nephrotic
syndrome than those carrying missense mutations
[3]. In addition, individuals homozygous for the p.
R138Q mutation present with early-onset disease
[3, 26]. At least two mutations, p.V180M and
p.R238S, are associated with a milder phenotype,
including later age at onset of NS and age at
ESKD [3]. The p.R138X mutation has been asso-
ciated with a high incidence of cardiac abnormal-
ities in children [50], although this finding has not
been confirmed in patients carrying other
mutations [51].
The p.R229Q variant is the most frequently
reported non-synonymous NPHS2 variant in Cau-
casians [52], in whom the observed frequency of
heterozygotes ranges from 0.03 to 0.13 [3, 4, 34,
52–54]. In African-Americans and sub-Saharan
populations, the p.R229Q allele is infrequent
[52]. In vitro studies demonstrated decreased
binding of the p.R229Q mutant protein to nephrin,
suggesting that this variant may be pathogenic
[34]. Patients carrying the p.R229Q non-neutral
polymorphism associated with a pathogenic
mutation have a significantly later onset of disease
[3, 26] and represent almost all adult-onset cases
with NPHS2 mutations [30, 34, 36]. Indeed,
patients with such mutations develop ESKD at a
mean age of 26.1  18.9 years [36]. Our group
demonstrated that the p.R229Q variant is only
pathogenic when trans-associated to specific
C-terminal mutations, which exert a dominant
negative effect on podocinR229Q through an
altered dimerization and mislocalization [55].
On the contrary, patients bearing the p.R229Q
variant at the homozygous state are
811
asymptomatic. This has direct implications in
genetic counseling, as individuals carrying p.
R229Q are only at risk of having an affected
child if their spouse carries a dominant negative
NPHS2 mutation, when the rate of transmission
may be dramatically different from those expected
in autosomal recessive disorders [55].
Finally, tri-allelic inheritance of NPHS1 and
NPHS2 mutations has been occasionally reported
[25, 56, 57], but additional studies are needed to
better understand the complex genetics of renal
disease progression in the setting of nephrotic
syndrome.
Clinical Relevance
The identification of NPHS2 mutations in children
presenting with nephrotic syndrome may have
important clinical implications. Patients with
mutations in the NPHS2 gene do not respond to
steroid or immunosuppressive therapy, although a
partial reduction of proteinuria has been reported
with cyclosporine A in a small number of cases
[1, 4]. Avoidance or withdrawal of immunosup-
pressive therapies in these patients would spare
them from the potential risks and side effects
associated with these drugs. Moreover, patients
with two pathogenic NPHS2 mutations have a
significantly lower risk of relapse after transplan-
tation than cases in which mutations were not
identified (8 % vs. 30 %) [3, 4, 58, 59]. Patients
bearing mutations in the heterozygous state have a
risk comparable to those without mutations
[60]. In contrast with the mechanism of relapse
in cases with NPHS1 mutations, there is no evi-
dence to support a role for anti-podocin antibodies
[3, 61, 62]. The recent discovery of the nonpatho-
genic association of the p.R229Q variant with
C-terminal NPHS2 mutations has given a possible
clue to this unclear phenomenon [55]. Indeed, a
subset of patients with podocin mutation and
posttransplant recurrence may actually have an
immune form of SRNS and fortuitously bear the
p.R229Q variant and a C-terminal NPHS2 muta-
tion in trans. Other possible mechanisms of recur-
rence of proteinuria in these patients may involve
de novo glomerulopathy or drug toxicity.
812
Animal Models
In the two mouse models of constitutive Nphs2
inactivation [63] or knock-in of the murine equiv-
alent of the human mutation p.R138Q [64], mice
develop congenital nephrotic syndrome with mas-
sive foot process effacement, as well as unex-
pected lesions of mesangiolysis and mesangial
sclerosis, and succumb to ESKD within 5 weeks
of life. These effects are modulated not only by
genetic background but also, in Nphs2 null mice,
by the maternal environment [65]. Conversely, the
conditional model of podocin inactivation specif-
ically in podocytes in the mature kidney [66]
results in progressive NS and renal insufficiency
with FSGS lesions rather than diffuse mesangial
sclerosis (DMS) lesions, hence recapitulating the
phenotype of patients bearing NPHS2 mutations.
The later model is therefore an invaluable tool to
test novel therapeutic strategies in SRNS. The
crucial role of podocin in glomerular physiology
was further confirmed by the absence of slit dia-
phragm and an altered plasma filtration in
zebrafish using podocin morpholino [67]. After-
wards, it has also been shown that the Drosophila
ortholog of podocin Mec2 is required for the func-
tion of the nephrocyte, a podocyte-like cell with a
filtration slit diaphragm in flies [68].
Mutations in NPHS1 Encoding Nephrin
NPHS1 (MIM #602716) has been identified as the
major gene involved in congenital nephrotic syn-
drome of the Finnish type (CNF) [14]. The clini-
cal aspects of CNF and the identification of the
NPHS1 gene and characterization of nephrin are
extensively described in the corresponding chap-
ter. Briefly, nephrin is a transmembrane protein
with eight immunoglobulin-like modules, a fibro-
nectin domain, a single transmembrane domain,
and a cytosolic C-terminal end [69]. It localizes to
the slit diaphragm and is critical for its architec-
ture. The spectrum of NPHS1 mutations includes
protein-truncating nonsense and frameshift inser-
tion/deletion mutations, splice-site changes, and
missense variants. Most of these mutants are
retained in the ER and lead to a severe CNF
phenotype. Interestingly, Liu et al. have
O. Boyer et al.
demonstrated that in vitro treatment with a chem-
ical chaperone may allow for trafficking of
ER-retained mutants to the plasma membrane
[70, 71].
However, later findings have broadened the
spectrum of renal disease related to nephrin muta-
tions since patients with childhood-onset and even
adult-onset SRNS may rarely carry NPHS1 muta-
tions [15, 72–74]. Indeed, the p.R1160X mutation
may result in a milder phenotype in about 50 % of
cases with histological findings consistent with
CNF and either mild proteinuria or complete
remission up to 19 years of age [56]. Thereafter,
NPHS1 mutations were identified in 10/160
patients (142 families) presenting with SRNS at
a mean age of 3 years (range: 6 months to 8 years)
and MCD or FSGS on biopsy [15]. Affected cases
were compound heterozygous for at least one
“mild” missense mutation, which exhibited nor-
mal trafficking to the plasma membrane and
maintained the abilities to form nephrin
homodimers and to heterodimerize with NEPH1.
These findings may explain the lesser severity of
disease observed in these cases.
Mutations in PLCE1 Encoding
Phospholipase C Epsilon 1
A positional cloning approach coupled to gene
expression profiling in rat glomeruli identified
the PLCE1 gene (MIM #608414), encoding phos-
pholipase C epsilon 1, as a good candidate in
7 families with SRNS. PLCε1 catalyzes the
hydrolysis of phosphoinositides to generate two
second messengers – inositol 1,4,5-triphosphate
(IP3) and diacylglycerol (DAG) [75] – that initiate
intracellular pathways of cell growth and differ-
entiation. PLCε1 is highly expressed in podocytes
and interacts with the cell junction-associated pro-
tein IQGAP1 [76], a nephrin-binding partner
involved in cell morphology and adhesion
[77]. Mutational analysis subsequently revealed
truncating and missense mutations in several of its
34 exons [76]. In the initial cohort reported by
Hinkes et al., all patients with homozygous trun-
cating PLCE1 mutations had DMS as opposed to
two patients with missense mutations who
27 Idiopathic Nephrotic Syndrome in Children: Genetic Aspects
exhibited FSGS with a later onset of NS [76]. Sub-
sequently these investigators showed that PLCE1
truncating mutations accounted for 28.6 % of
cases of isolated DMS in a cohort of 40 patients
from 35 families mostly of Turkish origin [78]. In
contrast, our group observed either truncating or
missense mutations in both FSGS and DMS
patients and no clear phenotype–genotype corre-
lation in a series of 168 patients [79]. A more
recent study reported an earlier age of onset in
patients with PLCE1 splice-site mutations com-
pared with C-terminal truncating mutations or
missense mutations [2]. Interestingly in the origi-
nal description, two patients bearing truncating
mutations achieved complete remission when
treated early and remained free of proteinuria
after several years of follow-up [76, 80]. Later,
four families with transiently affected or even
asymptomatic family members – although homozy-
gous for truncating mutations – have been
published, suggesting an incomplete penetrance of
PLCE1-associated nephrotic syndrome [79, 81],
an uncommon phenomenon in autosomal recessive
diseases [82, 83]. A second-locus modifier, yet to
be identified, could explain this phenomenon,
although variations in 19 candidate genes including
16 phospholipases were ruled out [79]. The reported
incomplete penetrance is consistent with the lack
of renal phenotype observed in Plce1/ mice [76].
Less Commonly Mutated Genes
MYO1E
By homozygosity mapping and high-throughput
sequencing, MYO1E mutations (MIM #601479)
have been identified in two Italian families with
isolated SRNS [84, 85]. Among the ten families
with MYO1E mutations published thus far, most
affected children had developed ESKD between
6 and 13 years of age, although a normal renal
function could be observed in a 14-year-old child
[84–86]. Patients typically develop FSGS [84–86]
with disorganization of the glomerular basement
membrane (GBM) and tubular alterations. Con-
sistently, three of the five patients with available
data had microscopic hematuria, an uncommon
813
feature in NS [84]. MYO1E encodes myosin 1E,
a member of the ubiquitously expressed class I
myosins, implicated in podocyte actin cytoskele-
ton organization and motility. Its knocking down
in both zebrafish and mouse models causes
nephrotic syndrome [87–89], and myo1e/
mice recapitulate the GBM lesions observed in
humans [84, 88].
PTPRO
The protein tyrosine phosphatase receptor type O
(PTPRO, also called GLEPP1) was first identified
as a podocyte-specific transmembrane protein
localized to the apical side of foot processes in
rabbit glomeruli [90]. Ptpro/ mice do not
develop albuminuria but have reduced glomerular
filtration rate and hypertension after nephrectomy
[91] and exhibit altered, amoeboid-shaped
podocytes with broad and short major processes.
PTPRO (MIM #600579) mutations were identified
in two Turkish consanguineous families
by homozygosity mapping [92]. Three of the five
affected siblings had developed SRNS between the
ages of 5 and 14 years with FSGS or MCD on
histology and severe foot process effacement, and
one progressed to ESKD at the age of 18 years.
CRB2
Homozygous or compound heterozygous CRB2
mutations (MIM #609720) have been identified
by exome sequencing in four families with
childhood-onset isolated SRNS with FSGS diag-
nosed between 9 months and 6 years of age
[93]. CRB2 encodes the putative transmembrane
Crumbs homolog 2 protein. The knockout of
zebrafish Crb2b results in foot process efface-
ment, and podocyte arborization and polarity
defects [93]. In accordance with the embryonic
lethality of Crb2 knockout mice, all affected chil-
dren carried a missense mutation on at least one
allele [94]. Of note, CRB2 mutations were also
detected in five fetuses and one infant with cere-
bral ventriculomegaly and echogenic kidneys
with histopathological findings of congenital
nephrosis [95] (▶ Chap. 25, “Congenital
Nephrotic Syndrome”).
814
CD2AP
CD2-associated protein was originally cloned as an
interaction partner of CD2, a T lymphocyte signal-
ing protein expressed on the cell surface, implicated
in T-cell adhesion to antigen-presenting cells
[96]. In the kidney, CD2AP is expressed in proxi-
mal tubules, collecting ducts and podocytes where
it interacts with nephrin [97], podocin [37], and
F-actin [98] suggesting its role in anchoring the
SD to the actin cytoskeleton. Cd2ap/ mice dem-
onstrate not only an impaired T-cell function in vitro
but also congenital nephrotic syndrome leading to
ESKD by 6–7 weeks of age [97], the latter being
prevented by podocyte-specific expression of
CD2AP in Cd2ap/ mice [99]. Intriguingly,
Cd2ap+/ mice are susceptible to develop protein-
uria secondary to nephrotoxic antibodies, showing
that CD2AP haploinsufficiency may contribute to
multifactorial glomerular injury [100]. Though sev-
eral studies screened for CD2AP mutations (MIM
#604241) in SRNS, only a single patient has thus
far been identified with two mutated alleles
[101]. He carried a homozygous last-exon nonsense
mutation (p.R612*) that truncated the protein for
the last 28 amino acids and markedly diminished its
interaction with F-actin [101]. Patient lymphocytes
displayed no CD2AP expression, consistent with a
complete loss of function. He presented with failure
to thrive secondary to nephrotic-range proteinuria
and microscopic hematuria at the age of 10 months,
with FSGS on histology, progressed to ESKD by
the age of 3 years, and underwent transplantation
two years later with no recurrence. Both parents
were heterozygous for the mutation and had normal
glomerular filtration rate and no proteinuria, ruling
out a haploinsufficient phenotype in this family.
NGS Findings and Redefinition
of Known Gene-Associated Phenotypes
TTC21B
Mutations of the ciliary gene TTC21B (MIM
#612014), encoding the retrograde intraflagellar
transport-A protein IFT139, were initially identified
as a cause of nephronophthisis [102]. Unexpectedly,
its most common mutation (p.P209L), a founder
mutation in Portugal and North Africa, was recently
O. Boyer et al.
identified by exome sequencing combined to link-
age analyses in seven families with FSGS. TTC21B
is therefore the first ciliary gene involved in a glo-
merular disorder. Patients presented late-onset pro-
teinuria at ages 9–23 years, high blood pressure and
ESKD at ages 15–32 years. Moreover, characteris-
tic morphological alterations of nephronophthisis
such as tubular basement membrane thickening
were also present in these patients, indicating the
coexistence of primary glomerular and tubuloin-
terstitial alterations. Besides, various hepatic fea-
tures have been detected in one family and several
cases found mutated since the original publication
(unpublished data). In accordance to data in rat
tissues, a primary cilium was found to be present
in human podocytes in fetal kidney biopsies and
undifferentiated cultured podocytes, but not in adult
kidney sections and differentiated cells [19]. It was
further shown that IFT139 was expressed at the
cilium base in fetal and undifferentiated podocytes,
whereas in mature and differentiated podocytes,
IFT139 localized along the expanding and complex
microtubule network. In undifferentiated cells,
IFT139 knockdown induced ciliary defects that
were partially rescued by overexpression of
IFT139P209L, suggesting a hypomorphic effect of
this allele. However, this hypomorphic effect was
not sufficient to rescue the cytoskeletal disorgani-
zation yielded by IFT139 knockdown in differenti-
ated cells. The modest consequences of the
hypomorphic variant p.P209L on ciliogenesis with
more deleterious effects on cytoskeletal regulation
are in accordance with the late phenotype observed
in mutated patients [19]. Other ciliary proteins
expressed in mature podocytes and sharing addi-
tional roles in microtubule maintenance may be
involved in SRNS.
Autosomal Dominant Forms
of Isolated SRNS
Mutations in ACTN4 Encoding
a-Actinin-4
A genome-wide scan performed in a 100-member
kindred allowed Mathis et al. to map the first locus
of AD FSGS on chromosome 19q13 [103, 104].
27 Idiopathic Nephrotic Syndrome in Children: Genetic Aspects
Linkage analysis including additional families
helped to reduce the size of the region and led to
the identification of three nonconservative mis-
sense mutations in the ACTN4 gene (MIM
#604638) [105]. ACTN4 encodes the ubiquitous
actin-binding protein α-actinin-4, highly
expressed in podocytes [105]. α-actinin-4 inter-
connects actin filaments in podocyte foot pro-
cesses [106]. Most reported mutations are
missense mutations involving residues within or
next to the actin-binding site of α-actinin-4 [105,
107]. Some of them have been shown to increase
the affinity of α-actinin-4 to F-actin and/or induce
α-actinin-4 mislocalization and the formation of
α-actinin-4 and F-actin aggregates around the
nucleus with subsequent impairment of podocyte
spreading and motility [105, 107–110]. Knock-in
and knockout mouse models and in vitro data
suggest both gain-of-function and loss-of-func-
tion mechanisms [108]. Affected cases typically
present with proteinuria starting in the teenage
years or later, with FSGS lesions on kidney his-
tology, and slowly progress to ESKD in the fifth
decade of life, although earlier cases have been
seldom reported [105, 111, 112]. Disease is
incompletely penetrant. Further mutation screen-
ing of the ACTN4 gene in cases with familial and
sporadic forms of FSGS allowed the identification
of several additional mutations. Overall, ACTN4
mutations seem to account for approximately 3–4
% of AD FSGS [107, 111, 113].
Mutations in TRPC6 Encoding
the Transient Receptor Potential Cation
Channel 6
Winn et al. identified a second locus for AD FSGS
in a large family from New Zealand [114].
Affected cases presented with nephrotic-range
proteinuria in their third or fourth decade and
developed progressive renal insufficiency within
10 years after onset. Using fine-mapping and
candidate-gene screening, the same group of
investigators subsequently detected a missense
mutation in the TRPC6 gene (MIM #603652),
encoding the transient receptor potential cation
815
channel, subfamily C, member 6 [115]. Subse-
quently, TRPC6 mutations were evaluated to be
responsible for approximately 2–5 % of AD FSGS
[43, 113, 116, 117]. Notably, TRPC6 mutations
have been described in six children, one of whom
was a 2-year-old boy with collapsing FSGS and
rapid progression to ESKD due to a de novo
mutation [116–119].
TRP channels are involved in mechanosensation,
ion homeostasis, cell growth, and PLC-dependent
calcium entry into cells [120, 121]. TRPC6 is
expressed in podocytes, specifically at the slit dia-
phragm where it interacts with podocin and nephrin
[43]. Some of the mutations were shown to enhance
TRPC6-mediated calcium signals suggesting a
gain-of-function mechanism, whereas others do
not [115]. Diverse mechanisms may result in
dysregulation of the ion channel or may affect the
interaction with other slit-diaphragm proteins [43].
Mutations in INF2 Encoding
the Inverted Formin 2
M. Pollak’s group identified heterozygous muta-
tions in the INF2 gene (MIM #610982) encoding
the inverted formin 2 in 12 % of AD FSGS families
[122]. Subsequent studies confirmed that INF2
mutations are the first cause of AD FSGS [113,
123]. Age at proteinuria onset ranges from 5 to
72 years and at ESKD onset from 13 to 70 years.
Incomplete penetrance was also reported by most
authors. Conversely, mutations in this gene seem to
be responsible for <1 % of sporadic cases of FSGS
due to de novo mutations [113, 123]. Our group
later demonstrated that INF2 mutations are the
main cause of Charcot–Marie–Tooth disease
with FSGS (see below) [124]. Formins are ubiqui-
tous proteins governing various cellular phenome-
nons such as actin cytoskeleton remodeling, cell
polarity and morphogenesis, and cytokinesis [125].
INF2 belongs to the diaphanous-related formin
family, which prototypic member is mDia1. INF2
contains the formin homology domains FH1 and
FH2 involved in actin elongation and nucleation,
respectively, as well as the diaphanous
autoregulatory domain (DAD) and diaphanous
816
inhibitory domain (DID) which play a role in
INF2 auto-inhibition. The vast majority of INF2
mutations are localized in the DID region. These
results further confirmed the crucial role of an intact
actin cytoskeleton in podocyte dynamics and
function.
Less Commonly Mutated Genes
ARHGAP24
Using a candidate-gene approach, ARHGAP24
encoding the actin-regulating protein Rho-GAP
24 (MIM #610586), highly expressed in
podocytes, was sequenced in 310 patients with
FSGS [126]. A loss-of-function mutation was
identified in one family with AD FSGS. The
mother had died of ESKD at 29 years of age,
and two offsprings had reached ESKD at 12 and
20 years of age, respectively. This mutation
involved the GAP catalytic domain, very con-
served between species. This is another example
of actin-regulating genes in which mutations lead
to hereditary FSGS.
ANLN
More recently, mutations in the ANLN gene (MIM
#616027) encoding anillin were identified by
exome sequencing combined to linkage analyses
in two families with AD FSGS [127]. Patients’
phenotype was similar to FSGS related to other
AD genes. The age at proteinuria onset varied
from 9 to 69 years, with FSGS on biopsies and
ESKD occurring between 35 and 75 years. Anillin
is an actin-binding protein required for cytokine-
sis. It also interacts with the slit-diaphragm pro-
tein CD2AP and the formin mDia2. In the kidney,
anillin is mainly expressed in tubular cells but also
in podocytes of control subjects [127]. Con-
versely, anillin is overexpressed on kidney biop-
sies of patients with idiopathic FSGS. In vitro,
anillin mutants have an impaired binding to
CD2AP resulting in abnormal podocyte motility.
These results add to the expanding group of
podocyte cytoskeleton genes involved in FSGS
pathophysiology.
O. Boyer et al.
NGS Findings and Redefinition
of Known Gene-Associated Phenotypes
LMX1B Mutations and FSGS Without
Nail-Patella
LMX1B mutations lead to nail-patella syndrome
(NPS) (MIM #161200) associating dysplasia of
the patellae, nails and elbows, iliac horns, glau-
coma, and in some cases FSGS with specific
lesions characterized by the presence of type III
collagen fibrils in the GBM by electron micros-
copy (cf. below) [128]. By linkage analysis and
exome sequencing, a novel LMX1B mutation was
unexpectedly identified in a pedigree of five
patients with AD FSGS but no glomerular base-
ment membrane anomaly suggestive of nail-
patella-like renal disease by electron microscopy
and no extrarenal features [129]. Mutations in two
other families were subsequently identified by
Sanger sequencing among a cohort of 73 addi-
tional unrelated families with FSGS. The patients
presented significant albuminuria between 5 and
70 years of age, and some of them reached ESKD
between 26 and 70 years of age. Another novel
sequence variant (p.R249Q) involving the same
domain of the protein has been recently described
in an AD pedigree with similar phenotype, i.e.,
late-onset isolated FSGS [130]. LMX1B encodes a
homeodomain-containing transcription factor that
is essential during development. The two muta-
tions involve the same residue of LMX1B and are
expected to diminish the interaction between the
LMX1B homeodomain and DNA molecule by in
silico analyses. These data demonstrate that muta-
tions in genes involved in syndromic forms of
SRNS may also be responsible for isolated
FSGS and highlight the need to include these
genes in next-generation sequencing diagnosis
approaches in FSGS.
COL4A3/COL4A4 Mutations
and Isolated FSGS
Next-generation sequencing brought up several
additional surprising findings. Searching for
mutations in a pedigree of three sisters referred
for familial SRNS diagnosed between 8 and
13 years of age and FSGS on biopsy,
27 Idiopathic Nephrotic Syndrome in Children: Genetic Aspects
R. Gbadegesin’s group identified two compound
heterozygous variants of COL4A3 segregating with
the disease [131]. The three children also had
microscopic hematuria and two had hearing
impairment. COL4A3 and COL4A4 encode the α3
and four chains of collagen IV, respectively.
Mutations in these genes are responsible for Alport
syndrome of autosomal dominant (MIM #104200)
or recessive inheritance (MIM #203780) or benign
familial hematuria (MIM #141200). Unlike the
classical X-linked and the AR forms of childhood
Alport syndrome, AD forms of the disease (~10 %
cases) have a late onset and a high phenotypic
variability with inconstant gross hematuria and
hearing loss [132]. Consequently, the authors
searched for COL4A3 and COL4A4 mutations in
a cohort of 69 unrelated families with FSGS and
found a rare heterozygous variant in six of them,
translating into a mutation rate of 10 % of familial
FSGS [131]. None of the patients had characteristic
GBM lesions suggestive of Alport syndrome by
optical or electron microscopy. Most heterozygous
patients were detected with proteinuria during
adulthood (2–65 years of age). Nevertheless, all
patients with available data had associated hema-
turia. Subsequent reports confirmed that COL4A3
and COL4A4 mutations are rather frequent causes
of AD FSGS (~10–12 %) [133, 134]. Only one
patient had available electronic microscopy exam-
ination results and exhibited only segmental thin-
ning of the GBM [133]. These data emphasize the
need to perform COL4A3 and COL4A4 gene
screening in familial FSGS, especially in case of
microscopic hematuria or deafness, or in the
absence of other SRNS gene mutation.
WT1 Mutations and AD FSGS
WT1 encodes a transcription factor of the zinc
finger family that plays a crucial role in kidney
and genital tract development [135, 136]. Its
expression persists in podocytes in the adult
kidney, and its integrity is required for proper glo-
merular filtration barrier function [137]. Heterozy-
gous mutations of WT1 (MIM #607102), mostly de
novo mutations, are associated to a wide spectrum
of syndromes of autosomal dominant inheritance in
children [138]. These include Denys–Drash and
817
Frasier syndromes (cf. below) related to exon
8 and 9 mutations and specific splice-site mutations
in exon 9, respectively [139, 140]. Female patients
mostly present with isolated nephropathy (FSGS or
DMS) or nephropathy in association with develop-
mental tumors and have normal puberty although
streak dysgenetic ovaries have been seldom
described in 46,XX children [139].
Very surprisingly, through classical Sanger
sequencing and more recently NGS, WT1 muta-
tions were identified in pedigrees with isolated
AD FSGS, including males without genital abnor-
malities or developmental tumors who transmitted
the mutation [141–145]. All patients carried a
missense mutation in exon 8 or 9 (R458Q,
S461F, or R471K) affecting the second and third
zinc fingers [141–143]. These results suggest
including WT1 testing in the genetic work-up not
only of females with sporadic FSGS but also in
males with familial AD FSGS.
PAX2 Mutations and AD FSGS
NGS also allowed the identification of PAX2 muta-
tions (MIM #167409) segregating with the disease
in seven families with AD FSGS [20]. The
24 affected patients had various degrees of protein-
uria diagnosed between 7 and 68 years (mostly
second to fourth decades) and FSGS on biopsy
(10/24 patients). Nine had reached ESKD. PAX2
is a transcription factor expressed from the fourth
week of gestation in the kidney as well as otic and
optic vesicles and the hindbrain. Heterozygous
PAX2 mutations were thus far known as a cause
of CAKUT and papillorenal syndrome character-
ized by renal hypodysplasia, optic nerve coloboma,
and rarely sensorineural hearing loss. In the afore-
mentioned series from Barua et al. [20], kidney
ultrasound in patients with PAX2-related FSGS
either was normal or could reveal echoic or small
kidneys, dilated renal pelvis, or calyceal diverticu-
lum. Most patients did not exhibit any extrarenal
symptom, but papillorenal syndrome was diag-
nosed retrospectively in one family. PAX2 muta-
tions could result in FSGS secondary to nephron
loss but also in specific podocyte injury through
dysregulation of target genes such as the transcrip-
tion factor WT1 [20].
818
Syndromic Forms of SRNS
SRNS with Genitourinary Features:
Denys–Drash and Frasier Syndromes
The WT1 gene was first found to be inactivated in
Wilms’ tumor [146]. It encodes a zinc finger tran-
scription factor and translational regulator that
functions both as a tumor suppressor and as a
critical regulator of kidney and gonadal develop-
ment [135, 136, 147]. The key role of WT1 in
kidney development has been highlighted by the
development of animal and in vitro models show-
ing complete failure of kidney development in the
absence of WT1 expression [147–149]. Mutations
in the WT1 gene are associated with varied
syndromic forms of glomerular disease and geni-
tourinary abnormalities, as well as isolated cases
of SRNS.
Denys–Drash syndrome (DDS, MIM 194080)
is a rare urogenital disorder comprising nephrop-
athy due to DMS, associated with male
pseudohermaphroditism and predisposition to
Wilms’ tumors [150, 151]. Nephrotic syndrome
presents in the first months of life, may be pre-
ceded by isolated proteinuria, and is always resis-
tant to steroid therapy. Progression to ESKD
occurs most often before 4 years of age, and no
recurrence is observed after renal transplantation
[152]. Wilms’ tumor may be the first presentation
of the disease or may be discovered later during
the course of nephropathy by systematic ultra-
sound screening. Patients with DDS bear hetero-
zygous missense or truncating mutations in exons
8 and 9, mostly de novo, affecting the DNA- and
RNA-binding affinity of the second and third zinc
finger domains [153–156]. Isolated cases of DMS
have also been attributed to WT1 mutations,
mostly in 46,XX patients [139, 157].
Frasier syndrome (FS, MIM 136680) is char-
acterized by male pseudohermaphroditism with
usually normal female external genitalia but
streak gonads and increased susceptibility to
gonadoblastomas in 46,XY patients, whereas 46,
XX girls have both normal external and internal
genitalia. FS is associated with childhood-onset
proteinuria, usually between 2 and 6 years of age,
O. Boyer et al.
with FSGS on biopsy and slow progression to
ESKD toward adolescence or early adulthood
[158]. As in DDS, inheritance is autosomal dom-
inant, although most cases are sporadic due to de
novo mutations. WT1 encodes numerous
isoforms, which are products of alternative trans-
lation start sites, alternative splicing, and RNA
editing [159]. Of particular interest are WT1
(+KTS) and WT1(KTS) variants, which differ
by the presence of the three amino acids KTS
between zinc fingers 3 and 4. The presence of
this insert influences the molecular and biochem-
ical properties of the resulting protein. While
WT1(KTS) binds DNA efficiently and acts as
a transcriptional activator, WT1(+KTS) seems to
have higher affinity to RNA and splicing factors
[160]. Heterozygous mutations in the donor splice
site of intron 9 of WT1 are causative of Frasier
syndrome and lead to reduction of the +KTS
isoform of the protein [161]. This results in an
alteration of the normal ratio of +KTS/-KTS
isoforms in the cell [140].
De novo deletion of the 11p13 locus leads to
WAGR syndrome (MIM 194072), characterized
by Wilms’ tumors, aniridia, genitourinary abnor-
malities, and intellectual disability [162]. Aniridia
is due to the deletion of the PAX6 gene, which
resides in the same locus than WT1.
Noteworthily, 46,XX females carrying either
missense or splice-site WT1 mutation commonly
do not exhibit genital anomalies, have normal
puberty, and therefore present with isolated spo-
radic SRNS [8, 155, 163–165]. Genetic counsel-
ing is essential in such patients, since they have a
theoretical 50 % risk to transmit the mutation
[163, 164].
SRNS with Ocular Features: Pierson
Syndrome
Pierson syndrome (OMIM #609049) is character-
ized by congenital nephrotic syndrome with DMS
and an irregular GBM on kidney biopsy, ocular
anomalies mostly microcoria, and developmental
delay [166, 167]. Using both homozygosity map-
ping [168] and a candidate-gene approach at the
27 Idiopathic Nephrotic Syndrome in Children: Genetic Aspects
locus owing to the CNS phenotype of Lamb2 null
mice [169], LAMB2 truncating mutations were
identified in families with Pierson syndrome.
Interestingly, the current literature also includes
reports of milder LAMB2 variants resulting in
apparently isolated congenital nephrotic syn-
drome with minor or no ocular defects and normal
psychomotor development [170–172], as well as
childhood onset of Pierson syndrome in a
non-consanguineous family of seven affected
individuals, with nephrotic syndrome and ESKD
presenting between 5 and 10 years of age and
progressive blindness around 2 years of age
[173, 174]. Laminins are heterotrimeric extracel-
lular matrix proteins that provide the basic scaf-
fold for assembly of the other components of the
GBM, including type IV collagen, nidogen/
entactin, and sulfated proteoglycans [175].
Lamb2 null mice recapitulate the clinical features
observed in patients with Pierson syndrome [169].
Interestingly, proteinuria precedes podocyte foot
process effacement in these mice suggesting that
the slit-diaphragm integrity is not sufficient to
maintain proper glomerular filtration functions
[176]. Indeed, expression and localization of
podocin and synaptopodin appeared normal at
onset of proteinuria in transgenic mice expressing
the p.R246Q lamb2 mutation, whereas they were
perturbed upon development of heavy proteinuria
[177]. More importantly, overexpression of lamb1
in lamb2-null mice could prevent the develop-
ment of nephrotic syndrome and extend life span
[178], hence raising the prospect of future
therapies.
SRNS with Osseous Dysplasia: Nail-
Patella and Schimke Syndromes
Nail-Patella Syndrome: Mutations
in LMX1B
Nail-patella syndrome (NPS; MIM 161200) is an
autosomal dominant disorder with complete pen-
etrance and variable phenotypic expression, char-
acterized by pleiotropic developmental defects of
dorsal limb structures. The most characteristic
findings include absent, hypoplastic, or dystrophic
819
nails that may be observed at birth, iliac horns, and
hypoplastic patellae [179]. Nephropathy may
occur in 25–50 % of the cases [180–184]. This
manifests at any age – mostly after the second
decade – as microalbuminuria progressing to pro-
teinuria that may be intermittent, usually associ-
ated with hematuria. Overt nephrotic syndrome is
not a common feature, and progression to ESKD
occurs in 5–14 % of the cases, usually many years
after proteinuria onset [184, 185]. Light micros-
copy of renal tissue usually reveals nonspecific
changes [186], whereas GBM exhibits ultrastruc-
tural abnormalities that are the most specific his-
tological hallmarks of NPS [180, 187–189].
Typically, there is irregular thickening and split-
ting of the GBM, with electron-lucent areas, and
the presence of clusters of fibrillar type III colla-
gen within the GBM and the mesangial matrix.
Finally, primary open angle glaucoma and senso-
rineural hearing impairment have been recognized
as less frequent features of the disease [184, 190].
LMX1B is a transcription factor belonging to
the LIM homeodomain family of proteins that
plays an essential role in the normal development
of dorsal limb structures, GBM, anterior segment
of the eye, and some types of neurons. Approxi-
mately 85 % of families with NPS present muta-
tions in LMX1B. The majority of mutations results
in protein truncation. Missense mutations gener-
ally involve substitutions in the homeodomain
region critical for DNA binding [182–184, 191,
192]. Recently, entire-gene deletions were
reported by Bongers et al., confirming that
haploinsufficiency of the LMX1B transcription
factor underlies this disease [193]. The precise
role of LMX1B in the kidney is only partially
elucidated. The targeted disruption of the Lmx1b
gene (LIM homeobox transcription factor 1 β) in
mice recapitulates the NPS phenotype [194] but
only in homozygous mutant mice, whereas het-
erozygous littermates do not exhibit any evident
abnormalities. In Lmx1b-deficient mice, the abun-
dance of α3 and α4 chains of collagen IV are
markedly diminished [195], as were the levels of
podocin [196, 197], CD2AP [196], synaptopodin,
and VEGF [197]. No such anomaly has been
observed in patients bearing heterozygous
820
mutations in the LMX1B gene [198]. The latter
findings may be explained by the fact that these
patients carry one functional allele and one
mutated allele, whereas mice have two mutant
alleles.
Schimke Immuno-osseous Dysplasia:
Mutations in SMARCAL1
Schimke immuno-osseous dysplasia (SIOD,
OMIM 242900) is a rare incompletely penetrant
autosomal recessive disorder characterized by
spondyloepiphyseal dysplasia, progressive renal
dysfunction due to FSGS, and T-cell immunode-
ficiency [199]. Additional inconstant features
include cerebral ischemia, migraine-like head-
aches, deficiency of other blood cell lineages,
hyperpigmented macules, corneal opacities,
microdontia, intellectual delay, recurrent infec-
tions, premature atherosclerosis, hypothyroidism,
cerebellar atrophy, and testicular hypoplasia with
atrophy and azoospermia [200–203]. Kidney dis-
ease manifests typically with proteinuria evolving
to overt SRNS, which is diagnosed between the
first year of life and 14 years of age [200, 201].
Patients who survive infectious complications
progress to ESKD between 5 and 15 years of
age. Numerous cases have been transplanted with-
out evidence of relapse in the allograft [201];
however, the evolution of cerebrovascular and
infectious complications does not seem to
improve after transplantation.
A genome-wide scan, performed in four fami-
lies, detected significant linkage at chromosome
2q35, and mutations were identified in the
SMARCAL1 gene [204]. This gene encodes a mem-
ber of an SNF2 subfamily of proteins that mediate
DNA-nucleosome restructuring during gene
regulation and DNA replication, recombination,
methylation, and gene repair (SWI/SNF-related
matrix-associated actin-dependent regulator of
chromatin, subfamily-a-like-1 gene). The majority
of mutations identified are nonsense and frameshift
mutations, likely leading to loss of function [204].
Clewing et al. showed that SMARCAL1 biallelic
mutations accounted for the phenotype in 38 of
72 independent cases with SIOD [205], revealing
the genetic heterogeneity of this syndrome.
Patients with two missense mutations tended to
O. Boyer et al.
have a milder course of disease, surviving beyond
15 years of age. The functional targets of
SMARCAL1 remain unidentified.
SRNS with Central Nervous System
Involvement: Metabolic Diseases and
Galloway–Mowat Syndrome
Mitochondriopathies with SRNS: MELAS
and COQ10 Deficiencies
Mitochondriopathies are a diverse group of disor-
ders due to structural, biochemical, or genetic
derangements of mitochondria [206]. Renal dys-
function is a rare event and may result from muta-
tions encoded by the mitochondrial or nuclear
genomes. The mitochondrial genome codes for
13 essential subunits of the mitochondrial respi-
ratory chain, as well as the 22 transfer RNA
(tRNA) and two ribosomal RNA (rRNA)
genes [207].
The c.3243A > G point mutation in the
tRNALeu(UUR) mitochondrial gene is associated
with MELAS syndrome (myopathy, encephalop-
athy, lactic acidosis, and stroke-like episodes)
[208]. Some patients carrying tRNALeu(UUR) gene
mutations may present with diabetes and deafness
[209, 210], cardiomyopathy [211], progressive
external ophthalmoplegia, and FSGS [212–217],
with or without nephrotic syndrome
[215–221]. Although most affected cases are diag-
nosed in adulthood and have glomerular disease
associated with other manifestations of mitochon-
drial disease, some patients present with isolated
nephropathy or may have an earlier onset during
adolescence [216, 219, 221, 222]. Likewise, a
deletion in the mitochondrial DNA has been asso-
ciated with FSGS in a cohort of Japanese patients
and in a Turkish patient [220, 223].
Nuclear genes, in which mutations lead to
syndromic or isolated SRNS, encode proteins of
the coenzyme Q10 pathway. Coenzyme Q10
(COQ10, ubiquinone) is a lipid-soluble component
of all cell membranes, vital for the transport of
electrons from complexes I and II to complex III
of the mitochondrial respiratory chain, a potent
antioxidant, and a cofactor of mitochondrial dehy-
drogenases and pyrimidine synthesis proteins [224].
27 Idiopathic Nephrotic Syndrome in Children: Genetic Aspects
At least ten enzymes are required for COQ10
synthesis, which, when mutated, lead to heteroge-
neous multisystemic disorders associating myopa-
thy, seizures, ataxia, deafness, optic atrophy, and
cardiomyopathy among others. SRNS may develop
in patients with COQ10 deficiency with or without
extrarenal features, as a result of COQ2, COQ6,
PDSS2, or ADCK4 gene mutations. Altogether,
mutations in these genes are identified in about
1 % of SRNS cases [2]. The characteristic histolog-
ical finding is FSGS, often of the collapsing
type, with dysmorphic podocyte mitochondria by
electron microscopy [224]. Podocytes are not con-
sidered to have a high energetic requirement, and
the role of COQ10 in podocyte function remains to
be clarified [225, 226]. However, the beneficial
effects of early COQ10 supplementation strongly
support the primary role of COQ10 in podocyte
function.
The first COQ10 gene found to be mutated in
SRNS was COQ2 encoding para-hydroxybenzoate-
polyprenyl transferase, identified by candidate-gene
approach [227]. Most children present with severe
renal and neurological involvement, whereas only
two unrelated children had isolated SRNS, and two
infants died at the age of 5 and 6 months with no
renal involvement [227–231]. Eight of the nine
reported children developed ESKD or died within
the first 18 months of life. The ninth child, who had
an affected brother, was diagnosed early at the age
of 1 year and could benefit from COQ10 supple-
mentation since NS onset that allowed preservation
of renal and neurologic function until last examina-
tion at the age of 5 years [232].
The first child described with PDSS2 muta-
tions was diagnosed with neonatal Leigh syn-
drome associated to severe NS at the age of
7 months and succumbed to refractory status
epilepticus at 8 months of age [233]. He was
found to have COQ10 deficiency in muscle and
fibroblasts and compound heterozygous PDSS2
mutations by candidate-gene approach. Two addi-
tional patients presented SRNS at birth and
2 years of age, associated to cerebral palsy in the
oldest [2]. PDSS2 encodes the second subunit of
decaprenyl diphosphate synthase, the first enzyme
of the COQ10 biosynthetic pathway and one of
the rate-limiting enzymes [233]. The kd/kd mice, a
821
mouse model in which a spontaneous Pdss2 muta-
tion occurred, develop an isolated renal disease
with dysmorphic mitochondria and later collaps-
ing glomerular lesions [225, 234–236]. Impor-
tantly, the renal phenotype of kd/kd mice can be
at least partially rescued by CoQ10 supplementa-
tion [225, 226].
COQ6, encoding a monooxygenase, was iden-
tified by homozygosity mapping [237] in five
families from Turkey or Lebanon. Four of them
are consanguineous and carry one of two homo-
zygous founder missense mutations (p.G255R
and p.A353D). The fifth unrelated patient is com-
pound heterozygous for two truncating mutations.
The affected children manifested with proteinuria
at the median age of 1.2 years (range: 0.2–6.4) and
progressed to ESKD at a median age of 1.7 years
(range: 0.4–9.3) [237]. Renal biopsy revealed
FSGS in seven cases and DMS in one. Sensori-
neural deafness was reported in all nine individ-
uals examined. Additionally, seizures were
reported in two of the 11 patients. Two children
were supplemented with CoQ10 with a beneficial
effect in both of them [237].
The most recently identified gene implicated in
COQ10 biosynthesis is ADCK4, encoding aarF
domain containing kinase 4. Using exome
sequencing and homozygosity mapping, Ashraf
et al. identified ADCK4 mutations in 14 patients
from seven families [238]. The subsequent
targeted sequencing of ADCK4 in 400 sporadic
cases resulted in the identification of a single case
with a homozygous mutation. Affected patients
developed SRNS between the ages of 3 and
21 years, typically with FSGS on kidney histology
(collapsing variant in three cases), and progressed
to ESKD at a median age of 15 years (range:
7–23). A single patient with infantile nephrotic
syndrome and developmental delay received
COQ10 supplementation with subsequent reduc-
tion of proteinuria. Intriguingly, apart from goiter
reported in two unrelated adolescents, no other
recurrent extrarenal involvement is associated to
NS [238]. A second cohort of 26 patients from
12 families confirmed a late-onset and mostly
renal-limited phenotype of ADCK4 mutations
with a median age at onset of proteinuria of
14 years, ESKD during the second decade, and
822
mild neurological features or retinitis pigmentosa
in only 3/26 patients [304]. Knockdown of
ADCK4 orthologs in zebrafish and drosophila
recapitulates the nephrosis phenotype [238]. The
encoded aarF domain containing kinase 4 is ubiq-
uitous, expressed at high levels in podocytes
where it localizes to foot processes and mitochon-
dria [238], but its function is unknown. However,
several lines of evidence strongly support the role
of ADKC4 in CoQ10 synthesis: (1) its high
sequence similarity with ABC1/COQ8 of Saccha-
romyces cerevisiae [239], (2) low COQ10 levels
in patient fibroblasts and/or lymphocytes,
(3) ADCK4 interaction with CoQ6 and CoQ7 in
cultured podocytes, and (4) the rescue of altered
cell migration in ADCK4 knockdown cultured
podocytes by COQ10 supplementation
[238]. Given the relatively high prevalence of
ADCK4 mutations in late-onset SRNS/FSGS, the
lack of extrarenal involvement characteristic of
mitochondriopathies, and the potential benefit
from early COQ10 supplementation, ADCK4
sequencing should be considered in all adolescent
patients. Moreover, mutated patients should be
repeatedly screened for subclinical extrarenal
symptoms.
Action Myoclonus–Renal Failure
Syndrome: Mutations in SCARB2
Action myoclonus–renal failure syndrome
(AMRF, MIM 254900) is an autosomal recessive
disease characterized by progressive myoclonic
epilepsy associated with renal failure. It typically
presents at 15–25 years of age with neurological
symptoms including tremor, action myoclonus,
seizures, and later ataxia, whereas cognitive func-
tion is preserved. Proteinuria is usually diagnosed
concomitantly with the onset of neurological
symptoms at a median age of 19 years [240]. Pro-
gression to ESKD occurs generally within 5 years
after onset of proteinuria [240, 241]. The renal
pathology is characterized by FSGS, sometimes
of the collapsing type [240]. By linkage analysis
and gene expression profiling to prioritize gene
sequencing within the region, Berkovic
et al. identified homozygous truncating mutations
O. Boyer et al.
in the SCARB2 gene (encoding LIMP-2). These
mutations led to a downregulation of SCARB2
mRNA and undetectable protein levels in western
blots of cell lysates from lymphoblastoid B cell
lines from the two affected subjects [242]. LIMP-
2 (lysosomal integral membrane protein) is the
receptor for β-glucocerebrosidase, a lysosomal
enzyme deficient in Gaucher disease [243].
LIMP-2 is considered to play a role in the biogen-
esis and maintenance of endosomal and lysosomal
compartments [244]. The Limp2 knockout mouse
model displays some features of AMRF such as
proteinuria, cochlear deafness, demyelinating
peripheral neuropathy, but also uretericpelvic
obstruction [242, 245, 246]. The role of LIMP-2
in the podocytes and the pathophysiologic events
leading to NS and FSGS in patients with SCARB2
mutations remain to be elucidated.
Galloway–Mowat Syndrome: Mutations
in WDR73
Galloway–Mowat syndrome (GMS, OMIM
#251300) is a rare autosomal recessive disorder
characterized by SRNS, microcephaly, and severe
neurological impairment [247]. Disease frequency
is unknown; however, more than 60 cases
have been reported since the original description
in 1968 [247, 248]. Inconstant morphological
defects include hiatus hernia, micrognathia,
arachnodactyly, and floppy ears. The distinctive
neurological hallmark is microcephaly, which
might be congenital (primary microcephaly) or
may develop after birth (secondary microcephaly).
Structural brain abnormalities may include cerebral
atrophy and gyration defects and are associated
with severe psychomotor impairment, hypotonia,
and seizures in half of all cases. Sensorineural
blindness and deafness have also been described.
Renal manifestations of GMS range from isolated
proteinuria to overt SRNS, which rapidly pro-
gresses to ESKD after a few months. Proteinuria
is usually discovered within the first year of life,
although congenital onset is not unusual as are
cases in which the onset of NS is close to the
third year of life [247, 249–256]. Kidney histology
may reveal either MC, FSGS that might be of the
27 Idiopathic Nephrotic Syndrome in Children: Genetic Aspects
collapsing type, or DMS, the latter more frequent in
early-onset forms [251, 257–260]. The prognosis
of GMS is poor, and most affected children reach
ESKD between 36 and 72 months and die before
the age of 6 years, although there are rarer cases
with preserved renal function after this age
[256, 260].
WDR73 has been very recently reported as the
first gene mutated in Galloway–Mowat syndrome
(GMS) by homozygosity mapping and whole
exome sequencing in an index family and thereaf-
ter in a second family by testing a cohort of
30 unrelated individuals [261]. This demonstrates
the large genetic heterogeneity of GMS. Two of the
three affected children developed NS at the age of
5 and 8 years with FSGS, whereas the third had
normal urinary sediment at the age of 7. Facial
dysmorphy, optic atrophy, secondary microcephaly
with severe intellectual deficiency, and cortical and
cerebellar atrophy with thin corpus callosum were
present in all three children. An abnormal morphol-
ogy of cell nuclei was found both in the patients’
fibroblasts and in WDR73-depleted cultured
podocytes, which could be rescued in the latter
either by the reexpression of the wild-type protein
or microtubule depolymerization. WDR73 is there-
fore suggested to play a role in the organization of
the microtubule network [261].
ARHGDIA Mutations and Early-Onset
SRNS with Intellectual Disability
Mutations of ARHGDIA, encoding Rho-GDP dis-
sociation inhibitor α (Rho-GDIα), have been iden-
tified by whole exome sequencing in six children
from three consanguineous families [262, 263]
with infantile NS, DMS on histology, and ESKD
before 3 years of age. Intellectual disability was
reported in three children, one with sensorineural
hearing loss and another with seizures and cortical
blindness. Rho family GTPases are key regulators
of the actin cytoskeleton and thus cell adhesion,
migration, and division [126, 264–267]. Rho-
GDIα inhibits the three canonical Rho-GTPases
RHOA, RAC1, and CDC42, by sequestering
them in their inactive GDP-bound states in the
cytosol [262, 263, 267]. Their pivotal role in
823
podocyte physiology has been demonstrated in
animal models. The podocyte-specific knockout
of Cdc42 results in podocyte injury [267], and
inducible transgenic mice expressing either consti-
tutively active or dominant negative RhoA develop
albuminuria [266, 268]. All three identified
ARHGDIA mutations have been shown to abrogate
the interaction of Rho-GDIα with its Rho-GTPase
partners, resulting in hyperactivation of RAC1 and
CDC42 and altered podocyte motility [262,
263]. Mouse and zebrafish models recapitulate
the NS phenotype [262, 263], which can be par-
tially rescued by RAC1 inhibitors, hence opening a
window of opportunity for therapy of some forms
of hereditary SRNS [264, 269].
SRNS with Peripheral Neurological
Involvement: Charcot–Marie–Tooth
Disease and FSGS
INF2 and Charcot–Marie–Tooth Disease
with FSGS
Since the 1960s, an increased prevalence of
nephropathies, particularly FSGS, has been
documented in patients with Charcot–Marie–Tooth
disease [270]. Charcot–Marie–Tooth disease refers
to a heterogeneous group of inherited chronic
peripheral motor and sensory neuropathies, with
an estimated prevalence of 1 in 2,500 live births.
Affected individuals typically present progressive
distal muscle weakness and atrophy, reduced ten-
don reflexes, foot and hand deformities, and rarely
mild to moderate sensorineural hearing loss. INF2
which mutations account for 12–17 % of AD FSGS
cases encodes the formin INF2 known to interact
with the myelin and lymphocyte proteins MAL and
MAL2 and Cdc42 all implicated in essential steps
of myelination and myelin maintenance. Through a
candidate-gene approach, INF2 was sequenced in
16 families with FSGS and Charcot–Marie–Tooth
disease, and mutations were identified in 75 % of
them, but in none of a series of 50 patients with
Charcot–Marie–Tooth disease and no renal
involvement [124]. INF2 is expressed in Schwann
cells, where it colocalizes and interacts with MAL,
824
and INF2 mutations enhance INF2 binding to
Cdc42, thereby perturbing myelination. Since the
original publication, 32 patients from 21 families
have been described in the literature [124,
271–274]. Affected patients developed proteinuria
between 7 and 30 years of age with FSGS and
Charcot–Marie–Tooth disease of the intermediate
type (i.e., with both demyelinating and axonal
lesions and intermediate median-nerve conduction
velocities) during the second decade, and ESKD
occurred in some patients between 12 and 47 years
of age. Because of the phenotypic variability, phy-
sicians should be alert for proteinuria in all patients
with Charcot–Marie–Tooth disease, and similarly,
a careful clinical neurological evaluation should be
considered for patients with FSGS.
SRNS with Cutaneous Features:
Epidermolysis Bullosa and FSGS
SRNS with Epidermolysis Bullosa:
Mutations in ITGB4, ITGA3, CD151, or
LAMB3
Junctional epidermolysis bullosa is an autosomal
recessive skin disorder in which blisters occur at
the lamina lucida in the skin basement membrane.
The disease is genetically heterogeneous with col-
lagen, integrin, and laminin gene mutations
underlying most cases. Nephrotic syndrome and
renal failure may occur in some patients with
epidermolysis bullosa [275]. The most common
histological finding is secondary amyloidosis
[276, 277]. The association of FSGS and
epidermolysis bullosa was first reported in a
male infant with pyloric atresia, junctional
epidermolysis bullosa, nephrotic-range protein-
uria and FSGS diagnosed 6 weeks after birth,
desquamation of laryngeal mucosa necessitating
tracheostomy, and recurrent hemorrhagic cystitis
leading to the obstruction of the vesicoureteral
junction within the first year of life [278]. Muta-
tional analysis of the β4-integrin gene ITGB4
revealed a homozygous missense mutation affect-
ing the second fibronectin type III domain.
A second child, compound heterozygous for a
splice-site and a missense mutation, presented
O. Boyer et al.
with a milder phenotype and developed FSGS at
the age of 10 years [2]. The mechanisms by which
mutations in the ITGB4 gene induce glomerular
disease remain unknown, and a fortuitous associ-
ation cannot be ruled out. Similarly, although
LAMB3 mutations are a frequent cause of junc-
tional epidermolysis bullosa, renal involvement
has thus far been reported only in a single child
who developed mixed proteinuria but mostly
nephrotic syndrome with DMS on histology and
died at the age of 5 months [279]. Immunohisto-
pathologic findings in the patient’s kidney biopsy
revealed both altered expression of laminin α
chains in the GBM and lack of laminin-5 in the
tubular basement membrane that may account for
glomerular and tubular involvement, respectively.
Despite possible incidental renal features in the
abovementioned case reports, other integrin and
laminin genes expressed in both skin basement
membrane and GBM might be good candidates
to explain the dual dermo-renal phenotype.
Indeed, mutations of ITGA3 (MIM #614748)
encoding the integrin α3 subunit have been iden-
tified using homozygosity mapping and
candidate-gene approach by two different groups
in patients with epidermolysis bullosa and NS
[280, 281]. Most of the six unrelated children
reported thus far developed CNS, severe intersti-
tial lung disease, and epidermolysis bullosa lead-
ing to ESKD, respiratory failure, and death during
the first 2 years of life [2, 280, 281]. In a single
patient, SRNS was diagnosed later during child-
hood at 4 years of age [2]. The integrin α3 subunit
heterodimerizes with the β1 subunit to form
integrin α3β1 expressed at high levels in
podocytes, where it associates with the tetraspanin
CD151 [281–286] and binds laminins [287].
Consistently, mutations in CD151 (MIM
#609057) encoding the tetraspanin CD151 have
also been reported in children with SRNS and
epidermolysis bullosa from two unrelated families
[283]. The three affected children developed
pretibial epidermolysis bullosa, neurosensory
deafness, bilateral lachrymal duct stenosis, nail
dystrophy, and thickening and splitting of the
tubular basement membrane. CD151 strongly
interacts with integrin α3β1 and assures its distri-
bution along the GBM [283–286]. Cd151/
27 Idiopathic Nephrotic Syndrome in Children: Genetic Aspects
mice recapitulate the human phenotype in specific
strains [288, 289]. In contrast to LAMB3 and
ITGB4 genes, no patient with ITGA3 or CD151
gene mutations and epidermolysis bullosa without
an apparent renal phenotype has been reported
to date.
Familial Forms of Steroid-Sensitive
Nephrotic Syndrome
The incidence of steroid-sensitive NS (SSNS) in
the pediatric population ranges between 2 and
7/100,000. Although most cases are sporadic,
several reports have confirmed the existence of
hereditary forms of this disease [290–298]. The
exact incidence of familial forms of SSNS is
unknown, but according to a single survey, it
may represent up to 3 % of the cases
[291]. Based on the low proportion of the familial
over the sporadic cases, it is unlikely that SSNS
and the immune forms of SRNS were monogenic
in majority. Indeed, HLA-DQA1 and PLCG2 mis-
sense variants have been very recently identified
as risk loci in SSNS by genome-wide association
study [299]. A homozygous nonsense mutation of
EMP2, encoding epithelial membrane protein 2,
has been detected by homozygosity mapping and
exome sequencing in a consanguineous Turkish
family with two affected children [300]. Both
children had frequently relapsing SSNS at the
age of 2 years with sustained remission under
cyclophosphamide. Subsequent screening of
1,600 individuals with NS revealed EMP2 muta-
tions in two Turkish siblings who presented with
frequently relapsing SSNS, in a third Turkish
infant with SSNS, but also in an African-
American patient, who developed steroid- and
cyclosporine-resistant NS at the age of 3 years
[300]. EMP2 is a tetraspan protein that contains
four transmembrane domains and is highly
enriched in kidney glomeruli where it localizes
to both podocytes and non-podocyte glomerular
cells. The knockdown of emp2 in zebrafish results
in pericardial effusion, a surrogate of
NS. Nevertheless, as the coexistence of SSNS
and SRNS within the same family is exceptional,
their common monogenic origin is a much
825
unexpected finding. Moreover, one would expect
the underlying defect in hereditary SSNS to lie in
a gene involved in the immune system, instead of
a transmembrane podocyte protein. Therefore, the
role of EMP2 in the pathogenesis of NS needs
further clarification.
Perspectives
Hereditary forms of NS are far more common than
previously thought ten years ago. The highest
rates of mutation detection are in patients
presenting with proteinuria in the first year of
life and subsequently decrease among older
patients [2]. Most of the cases with hereditary
forms of NS have a disease onset within early
childhood, are resistant to immunosuppressive
therapy, and do not relapse after kidney transplan-
tation. As a large proportion of patients with NS
do not have an identified underlying genetic
defect, and given the complexity of the patho-
physiology of the kidney, numerous further
genes are expected to be mutated in NS. The
most recently identified SRNS genes are
extremely rarely mutated and concern a very low
number of families. It is plausible that more com-
plex patterns of inheritance, as described in
patients bearing bi- or tri-allelic variants or as
observed with the p.R229Q NPHS2 variant [55],
will be increasingly recognized. Indeed, disease
predisposing mutations may lead to variable phe-
notypic expression and penetrance depending
upon unidentified environmental and genetic fac-
tors [79]. Moreover, common variants in genes
expressed in podocytes may account for an
increased risk of FSGS and ESKD observed in
selected ethnic groups, as has been described with
the APOL1 gene, in which several haplotypes con-
fer a major risk effect for FSGS in individuals of
African ancestry [301]. The accessibility to next-
generation sequencing techniques has deeply facil-
itated the screening of mutations in a broader
approach of podocyte-specific genes [2, 302,
303]. However, these diagnosis approaches face
the difficulty of determining the causal mutation(s)
among a great number of potential pathogenic var-
iants. To date, several fascinating disorders, such as
826
familial forms of SSNS, connecting podocyte phys-
iology and the immune system, remain incom-
pletely solved. The constant advances in the
understanding of hereditary kidney diseases and
renal physiology also open the way to therapeutic
innovations. A promising therapy, still explored at a
basic level, involves protein chaperones. These
drugs redirect the trafficking of missense mutant
proteins to the plasma membrane when abnormally
retained in subcellular organelles. Additional
encouraging results have been obtained with drugs
that stabilize the podocyte actin cytoskeleton. It is
now clear that genetic diagnosis of cases with
SRNS or familial NS is necessary to allow for
accurate genetic counseling, to avoid ineffective
therapies and even start early suitable treatment
such as ubiquinone in COQ10 deficiency, and,
hopefully in the near future, to offer specific
mutation-based therapies.
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 Idiopathic Nephrotic Syndrome in
Children: Clinical Aspects 28
Patrick Niaudet and Olivia Boyer

Contents Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 853

Symptomatic Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 853
Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 840 Specific Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 854
Associated Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 841 Initial Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 855
Clinical and Biological Features . . . . . . . . . . . . . . . . . . . 841 Treatment of Relapses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 856
Urinalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 842 Steroid Side Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 857
Blood Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 842 Alternative Treatments (Fig. 9) . . . . . . . . . . . . . . . . . . . . . . 857

Complications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 843 Long-Term Outcome of Children

Hypovolemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 843 with Steroid-Sensitive Nephrosis . . . . . . . . . . . . . . . . . . 863
Acute Renal Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 843 Treatment of Steroid-Resistant INS . . . . . . . . . . . . . . . . . 863
Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 844 Recurrence of Proteinuria After Renal
Thrombosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 844 Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 867
Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 845 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 868
Chronic Renal Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 845
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 869
Renal Biopsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 846
Minimal Change Nephropathy . . . . . . . . . . . . . . . . . . . . . . 846
Diffuse Mesangial Proliferation . . . . . . . . . . . . . . . . . . . . . 846
Focal and Segmental Glomerular Sclerosis . . . . . . . . . 848
Immunofluorescence Patterns . . . . . . . . . . . . . . . . . . . . . . . 850
Pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 851
The Anionic Glomerular Basement Membrane . . . . . 851
Immunological Anomalies and Circulating
Permeability Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 851

P. Niaudet (*) • O. Boyer

Service de Néphrologie Pédiatrique, Hôpital
Necker-Enfants Malades, Université Paris-Descartes,
Paris, France
e-mail: pniaudet@gmail.com; olivia.boyer@nck.aphp.fr

# Springer-Verlag Berlin Heidelberg 2016 839

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_24
840
The most common cause of nephrotic syndrome
in children is idiopathic nephrotic syndrome
(INS), also called nephrosis [1]. INS is defined
by the combination of a nephrotic syndrome (mas-
sive proteinuria, hypoalbuminemia, hyperlipid-
emia, and edema) and nonspecific histological
abnormalities of the glomeruli including minimal
changes, focal and segmental glomerular sclerosis
(FSGS), and diffuse mesangial proliferation. On
electron microscopy, glomeruli show an efface-
ment of epithelial cell (podocyte) foot processes
and no significant deposits of immunoglobulins or
complement by immunofluorescence. In a major-
ity of children, only minimal changes are seen on
light microscopy. These children are referred to as
having “minimal change disease.”
Many authors consider minimal change dis-
ease (MCD) and FSGS as separate diseases
because of differences in response to corticoste-
roids and subsequent clinical course. Indeed,
these pathologic features carry prognostic signif-
icance. Patients with FSGS have more frequently
hematuria, are often resistant to corticosteroid
treatment, and progress more often to renal fail-
ure. Recent data have comforted the belief that
MCD and FSGS are different entities. MCD is a
functional podocyte disease, whereas FSGS
appears to be a structural podocytopathy [2]. The
notion of “podocyte dysregulation” [2, 3], the
different expression cyclin-dependent kinase
inhibitors in MCD and in FSGS, the role of
these cell cycle disturbances leading to podocyte
proliferation and maturation [4], and the identifi-
cation of parvovirus B19 in glomeruli of patients
with FSGS [5, 6] are in favor of distinct entities.
The high circulating levels of the soluble uroki-
nase receptor (suPAR) found in FSGS but not in
MCD [7, 8] support the hypothesis that MCD and
FSGS as podocytopathies originating from differ-
ent glomerular permeability factors.
In the early stages, FSGS and MCD are indis-
tinguishable [9]. The best illustration of this is the
aspect of a transplant biopsy carried out shortly
after relapse of proteinuria following transplanta-
tion in a patient whose primary renal disease was
FSGS. Despite heavy proteinuria, the glomeruli
initially show minimal changes. A significant
number of patients with FSGS respond to
P. Niaudet and O. Boyer
corticosteroids, whereas some steroid-resistant
patients have no sclerotic changes on biopsy spec-
imens containing adequate tissue [10]. Therefore,
some authors believe that, although histological
variants of the INS carry prognostic significance,
they cannot at present be considered as separate
entities.
The term MCD has become synonymous with
steroid-responsive nephrotic syndrome although
renal biopsy is usually not performed in patients
who respond to steroid therapy. Indeed, in many
centers, renal biopsy is recommended only for
those patients who fail to respond to steroids.
Consequently, renal biopsy findings in recent
published series are not representative of the true
incidence of various histopathological categories
seen in INS. It is therefore more appropriate to
classify the patients according to their response to
steroid therapy. Response to steroid therapy
carries a greater prognostic weight than the histo-
logical features seen on initial renal biopsy. Thus,
two types of INS can be defined: steroid-
responsive nephrotic syndrome, in which protein-
uria rapidly resolves after starting steroid therapy,
and steroid-resistant nephrotic syndrome, in
which steroids do not induce remission.
Epidemiology
The incidence of INS varies with age, race, and
geography. INS is observed in all parts of the
world. The annual incidence of steroid-responsive
nephrotic syndrome in children in the USA and in
Europe has been estimated to 1–3 per 100,000
children below age of 16 [11–13], with a cumula-
tive prevalence of 16 per 100,000 children. Similar
figures were reported in New Zealand [16]. Geo-
graphical and/or ethnic differences are well known.
In the United Kingdom, for example, the incidence
of INS is sixfold greater in Asian than in European
children [14]; this is also true for Indians [15] and
for Japanese and Southwest Asians. INS is less
frequent in Africa [16–18]. Such differences under-
line the role of genetic as well as environmental
factors in the pathogenesis of the disease.
A male preponderance in children, with a male
to female ratio of up to 3.8:1 [11, 19], is frequently
28 Idiopathic Nephrotic Syndrome in Children: Clinical Aspects 841

reported. Both sexes are similarly affected in Table 1 Conditions associated with idiopathic nephrotic
adolescents. syndrome
The familial occurrence of INS is well known Allergy
in patients with steroid-resistant idiopathic Pollen
nephrotic syndrome (see ▶ Chap. 27, “Idiopathic Fungi
Nephrotic Syndrome in Children: Genetic Cow’s milk
Aspects”). House dust
Bee stings
Cat fur
Poison ivy
Associated Disorders
Drugs
Nonsteroidal anti-inflammatory drugs
INS is, by definition, a primary disease. Neverthe-
Ampicillin
less, in a number of cases, an upper respiratory Gold
tract infection, an allergic reaction, or another Lithium
factor may immediately precede the development Mercury
or relapse of the disease. Trimethadione
Many agents or conditions have been reported Malignancies
to be associated with INS such as infectious dis- Hodgkin disease
eases, drugs, allergy, vaccinations, and malignan- Non-Hodgkin lymphoma
cies (Table 1). The question remains whether Colon carcinoma
these factors are real causes, a simple coincidence, Bronchogenic carcinoma
or precipitating agents. Others
Allergy is associated with up to 30 % of cases Viral infection
[20–22]. Among a list of anecdotal cases, the Kimura’s disease
allergens reported include fungi, poison ivy, tim- Diabetes mellitus
othy grass pollen, house dust, medusa stings, bee Myasthenia gravis
Immunization
stings, and cat fur. A food allergen may be respon-
sible for relapses of steroid-sensitive nephrotic
syndrome, such as cow’s milk and egg. Laurent
et al. evaluated the effect of an oligoantigenic diet A number of drugs may induce a nephrotic
given for 10–15 days to 13 patients. This diet syndrome with the histopathological appearance
coincided with improvement of proteinuria in of minimal change disease. The list comprises
nine, including complete remission in five [23]. drugs such as nonsteroidal anti-inflammatory
The association between minimal change dis- agents, salazopyrin and mesalamine, D-penicilla-
ease and malignancies mainly concerns lympho- mine, lithium, rifampicin, heavy metals (gold,
matous disorders: Hodgkin’s disease and mercury), and trimethadione.
non-Hodgkin’s lymphomas [24, 25]. The
nephrotic syndrome may be the presenting feature
of the disease. It usually disappears after success- Clinical and Biological Features
ful treatment of the malignancy. Eosinophilic
lymphoid granuloma in orientals (Kimura’s dis- The disease may occur during the first year of
ease) has also been reported in association with life, but it usually starts between the ages of 2 and
INS [26, 27]. 7 years. The nephrotic syndrome is rarely dis-
Several cases of minimal change disease have covered during a routine urine analysis. The
been reported in association with the onset of onset is often preceded by an upper respiratory
insulin-dependent diabetes mellitus. The disease tract infection. The disease is characterized by a
is usually responsive to corticosteroids and fol- sudden onset, edema being the major presenting
lows a relapsing course. symptom. It becomes clinically detectable when
842
fluid retention exceeds 3–5 % of body weight.
Periorbital edema, frequently misdiagnosed as
allergy, is often the initial symptom. Edema is
gravity dependent, localized to the lower extrem-
ities in the upright position and to the dorsal part
of the body in reclining position. This edema is
white, soft, and pitting, keeping the marks of
clothes or finger pressure. Anasarca may develop
with ascites and pleural and pericardial effusions.
Although there may also be abdominal disten-
sion, dyspnea is rare. Periorbital edema may limit
eye opening, and edema of the scrotum and
penis, or labia, may be seen. A rapid formation
of ascites is often associated with abdominal pain
and malaise: these symptoms may also be related
to concomitant hypovolemia. Abdominal pain is
occasionally due to a complication such as peri-
tonitis, thrombosis, or, rarely, pancreatitis. Car-
diovascular shock is not unusual, secondary to
the sudden fall of plasma albumin, with abdom-
inal pain and symptoms of peripheral circulatory
failure with cold extremities and hypotension.
Emergency symptomatic treatment is needed.
Blood pressure is usually normal but sometimes
elevated.
Macroscopic hematuria is observed in a few
cases. The disease may also be revealed by a
complication. Peritonitis due to Streptococcus
pneumoniae is a classical mode of onset
[28]. Deep vein or arterial thromboses and pulmo-
nary embolism may also occur during the first
attack or during a relapse.
Urinalysis
Proteinuria is detected by dipstick testing 3 or 4+.
Quantitative evaluation gives figures ranging
from less than 1 g to more than 10 g/day. The
nephrotic range proteinuria is defined as >50
mg/kg/day or 40 mg/h/m2. In young children it
may be difficult to perform 24-h urine collection,
and urinary protein/creatinine ratio or U albumin/
U creatinine ratio in untimed urine specimens is
useful. For these two indices, the nephrotic range
is 200–400 mg/mmol.
In most cases, proteinuria is highly selective,
consisting of albumin and lower molecular weight
P. Niaudet and O. Boyer
proteins. The selectivity of proteinuria may be
appreciated by polyacrylamide gel electrophore-
sis or by the evaluation of the Cameron index,
which is the ratio of IgG (MW 150 kDa) to trans-
ferrin (80 kDa) clearances. A favorable index
would be below 0.10 or better below 0.05; a
poor index is above 0.15 or 0.20. Such poor
index is more often associated with steroid-
resistant nephrotic syndrome and FSGS.
However, there is a considerable overlap in
results, and the test has limited value. The amount
of protein excreted in the urine does not reflect
the quantity of protein crossing the glomerular
basement membrane since a significant amount
is reabsorbed in the proximal tubule. Some chil-
dren with severe steroid-resistant nephrotic syn-
drome and tubulointerstitial lesions have both
glomerular and tubular proteinuria with an
increased excretion of β-2 microglobulin,
retinol-binding protein, and lysozyme due to an
impaired protein reabsorption in the proximal
tubule.
The urine sediment of patients with INS often
contains fat bodies. Hyaline casts are also usually
found in patients with massive proteinuria, but
granular casts are not present unless there is asso-
ciated acute renal failure and acute tubular necro-
sis. Macroscopic hematuria is rare, occurring in
3 % of patients. Microscopic hematuria is present
in 20 % of cases and has no histopathologic or
prognostic significance.
Urinary sodium excretion is low (<5 mmol/
24 h), associated with sodium retention and
edema. Kaliuresis is usually higher than natriure-
sis, but it may be reduced in oliguric patients.
Blood Chemistry
Serum proteins are markedly reduced below 50 g/l
in 80 % of patients and below 40 g/l in 40 %.
Albumin concentration usually falls below 20 g/l
and may be less than 10 g/l. Electrophoresis shows
not only low albumin levels but also increased α-2
globulins and, to a lesser extent, β-globulins, while
γ-globulins are decreased. IgG is markedly
decreased, IgA slightly reduced, IgM is increased,
while IgE is normal or increased. Among other
28 Idiopathic Nephrotic Syndrome in Children: Clinical Aspects
proteins, fibrinogen and β-lipoproteins are
increased, and antithrombin III is decreased.
Hyperlipidemia is a consequence of (I) an
increased hepatic synthesis of cholesterol, triglyc-
erides, and lipoproteins; (II) a decreased catabo-
lism of lipoproteins due to a decreased activity of
lipoprotein lipase which normally transforms
VLDL to LDL via IDL; and (III) a decreased
LDL receptor activity and an increased urinary
loss of HDL [29, 30]. Total cholesterol and LDL
cholesterol are elevated, while HDL cholesterol
remains unchanged or low, particularly HDL2,
leading to an increased LDL/HDL cholesterol
ratio [31]. Patients with severe hypoalbuminemia
have increased triglycerides and VLDL.
Apoproteins, apo B, apo CII, and apo CIII, are
also elevated. The levels of lipoprotein Lp(a) are
elevated in nephrotic patients, and this further
contributes to an increased risk of cardiovascular
and thrombotic complications.
Serum electrolytes are usually within the nor-
mal range. A low sodium level may be related to
dilution from inappropriate renal retention of
water due to hypovolemia and inappropriate
antidiuretic hormone secretion. The mild reduc-
tion of plasma sodium concentration is often an
artifact related to hyperlipidemia. Serum potas-
sium may be high in oliguric patients. Serum
calcium is consistently low as a result of
hypoproteinemia. Ionized calcium is usually nor-
mal but may be decreased due to urinary loss of
25-hydroxyvitamin D3 [32] and normal but inap-
propriate levels of calcitriol [33]. Blood urea
nitrogen and creatinine concentrations are usually
within the normal range or slightly increased in
relation to a modest reduction in the glomerular
filtration rate (GFR).
A few patients with FSGS and a poor subse-
quent outcome present with a Fanconi syndrome:
glycosuria, aminoaciduria, urinary bicarbonate
loss, and hypokalemia [34]. A defect in urinary
acidification has also been reported [35].
Hematology
Hemoglobin levels and hematocrit are increased
in patients with plasma volume contraction. Ane-
mia with microcytosis may be observed, probably
related to urinary loss of siderophilin. The urinary
843
loss of erythropoietin may also contribute to ane-
mia [36]. Thrombocytosis is common and may
reach 5.108 or 109/l.
Complications
Hypovolemia
A few children are severely hypovolemic, and this
complication is observed typically at onset or
early during a relapse (Table 2). Sepsis, diarrhea,
or diuretics may precipitate hypovolemia. These
children often complain from abdominal pain and
have low blood pressure and cold extremities.
Hemoconcentration with a raised hematocrit
accompanies hypovolemia.
Acute Renal Failure
Renal function is usually within normal limits at
presentation. A reduction of the GFR, secondary
to hypovolemia, infection, or thrombosis, is fre-
quent [37, 38]. A reduced GFR may be found in
patients with normal effective plasma flow.
Bohman et al. showed a close relationship
between the degree of foot process fusion and
both GFR and filtration fraction, suggesting that
fusion of foot processes could lead to a reduction
of glomerular filtering area and/or of permeability
to water and small solutes [39]. This reduction is
transitory, with a rapid return to normal after
remission. Van de Walle et al. found that changes
in glomerular permeability may also have a major
role in acute renal failure [37].
Table 2 Nephrotic hypovolemia
Clinical features Precipitating factors
Abdominal pain Severe relapse
Hypotension Infection
Sluggish circulation Diuretics
Relative polycythemia Paracentesis
Acute tubular necrosis Diarrhea
Thrombosis
844
Table 3 Infections in nephrotic syndrome
Clinical syndrome Risk factors
Pneumococcal Low IgG
peritonitis
Haemophilus infection Low factor B
Gram negative sepsis Edematous tissue
Staphylococcus Impaired lymphocyte
cellulitis function
Corticosteroids
Immunosuppressive drugs
Marked oliguria may occur in children
[40]. Oliguric renal failure may be the presenting
symptom. Renal failure may be secondary to bilat-
eral renal vein thrombosis, which is recognized by
sonography or to interstitial nephritis, which has
been reported, especially with furosemide. Skin
rash and eosinophilia are suggestive of this
diagnosis.
Acute renal failure is usually reversible, espe-
cially with intravenous infusion of albumin
[41]. In some cases, where glomerular structure
is normal on renal histology, renal failure may last
for as long as a year and sometimes be
irreversible [42].
Infections
Bacterial infections are frequent in nephrotic chil-
dren (Table 3). Sepsis may occur at the onset of
the disease. The most common infection is peri-
tonitis, often with Streptococcus pneumoniae
[43]. Other organisms may be responsible:
E. coli, streptococcus B, Haemophilus influenzae,
and other gram-negative organisms. Apart from
peritonitis, children may develop meningitis,
pneumonitis, and cellulitis. Several factors may
explain the propensity of nephrotic children to
develop bacterial infections: low IgG levels due
to an impaired synthesis, urinary loss of factor B
and impaired T lymphocyte function. Factor B is a
cofactor of C3b of the alternative pathway of
complement, which has an important role in
opsonization of bacteria such as Streptococcus
pneumoniae.
P. Niaudet and O. Boyer
Table 4 Thrombosis in nephrotic syndrome
Clinical syndrome Risk factor
Pulmonary emboli Hyperviscosity
Pulmonary artery Low antithrombin III
thrombosis
Cerebral venous High fibrinogen
thrombosis
Renal vein Platelet hyperaggregability
thrombosis
Peripheral venous Underlying genetic
thrombosis thrombophilic tendency
Artery thrombosis Hypovolemia
Central venous catheter
Immobilization
Infection
Viral infections may be observed in patients
receiving corticosteroids or immunosuppressive
agents. Chickenpox is often observed in these
young children and may be life-threatening if
acyclovir is not started rapidly. Interestingly, mea-
sles infection may induce long-lasting remissions.
Thrombosis
Nephrotic patients are at risk of developing
thromboembolic complications, which may be
life threatening [44]. The majority of thromboem-
bolic complications occur within the first 3 months
after the onset of nephrotic syndrome. Children
aged >12 years are at higher risk of this compli-
cation than younger children. Several factors con-
tribute the increased risk of thrombosis, including
hypercoagulability state, hypovolemia, immobili-
zation, infection, the presence of central venous
catheter, and a possible underlying genetic
thrombophilic tendency [45] (Table 4). A number
of hemostatic abnormalities have been described
in nephrotic patients: increase in platelet count
and platelet aggregability, increase in
procoagulant proteins with high molecular weight
such as fibrinogen and factors V and VIII,
decreased level of the anticoagulant protein anti-
thrombin III is due to its urinary loss, and
decreased fibrinolytic activity due to the increased
concentrations of both α2-macroglobulin and
28 Idiopathic Nephrotic Syndrome in Children: Clinical Aspects
lipoprotein(a) [46]. The incidence of thromboem-
bolic complications in nephrotic children is close
to 3 % [45, 47]. Venous thrombosis account for
most of the cases of thromboembolic complica-
tions, while arterial thrombosis is much less fre-
quent. In a recent study including 370 children,
venous thromboembolism accounted for 97 % of
the cases [48]. Two reports suggest that the per-
centage of subclinical thromboembolic complica-
tions may be far more common [49, 50]. In the
first study, lung scintigraphic examination in
26 children showed evidence of pulmonary embo-
lism in 7 (28 %) and residual changes in another
10 (38.5 %). In the second study, 28 % of children
with nephrotic syndrome without respiratory
symptoms had subclinical pulmonary embolism.
Pulmonary embolism should be suspected in
cases of pulmonary or cardiovascular symptoms
and may be confirmed by computerized tomogra-
phy pulmonary angiography (CTPA) [51]. Renal
vein thrombosis should be suspected in cases with
sudden macroscopic hematuria or acute renal fail-
ure. Doppler ultrasonography shows an increase
in kidney size and the absence of blood flow in the
renal vein. Thrombosis may affect the arteries
such as pulmonary arteries or other deep veins.
Growth
Growth may be severely affected in children with
persistent nephrotic syndrome. Depletion of hor-
mones due to urinary losses is a possible cause of
stunting. Hypothyroidism related to urinary loss
of iodinated proteins has been observed and may
be corrected [52]. A low plasma IgF1 and IgF2
level associated with a urinary loss of the carrier
proteins has also been reported [53].
Chronic Renal Failure
The main difference between steroid responders
and steroid nonresponders is the tendency of the
latter to develop end-stage renal failure, which is
seen in less than 3 % of responders. This compli-
cation occurs in 50 % or more of the steroid-
resistant patients after a follow-up of 10 years.
845
The only “benefit” of the decrease of GFR is the
improvement of the nephrotic syndrome due to a
decrease of proteinuria.
We have retrospectively analyzed in the Enfants
Malades series the outcome of 181 children with
steroid-resistant INS who have been followed for at
least 5 years. Eighty-five percent were primary non-
responders and 15 % late nonresponders. Initial
renal biopsy had shown minimal changes in
62 cases and FSGS in 119 cases. Renal survival
rates were 65 % at 5 years, 50 % at 10 years, and
34 % at 15 years. Interestingly, the rate of progres-
sion to end-stage renal failure was similar in patients
with minimal changes or FSGS on initial biopsy.
The data reported in other series are difficult to
compare as most of them deal with patients with
FSGS. The Southwest Pediatric Nephrology Study
group reported 75 children with FSGS followed for
periods of 7–217 months [54]. Twenty-one percent
had progressed to end-stage renal failure, 23 % had
decreased glomerular filtration rate, 37 % had a
persistent nephrotic syndrome, and 11 % were in
remission. Paik et al. retrospectively analyzed
92 children with steroid-resistant FSGS and found
renal survival rates at 5, 10, and 15 years of 84 %,
64 %, and 53 %, respectively [55]. Poor prognostic
factors of chronic renal failure were asymptomatic
proteinuria at presentation, initial renal failure, and
higher proportion of glomeruli with segmental
sclerosis. Gipson et al. reported that the response
to treatment was a good predictor of long-term
renal survival in children with FSGS [56]. Indeed,
more than 50 % of patients who had not achieve
complete or partial remission of proteinuria had
progressed to ESRF after 3 years, whereas com-
plete remission was associated with a 90 %
decreased risk of ESRF.
Progression to end-stage renal failure has been
reported to be more rapid in patients of African or
Hispanic descent when compared with Cauca-
sians. Ingulli and Tejani found that among 57 Afri-
can American and Hispanic children, 50 % of
them had reached end-stage renal failure within
3 years, and 95 % had reached this stage after
6 years [57]. In addition, among the children
with INS, the proportion of those with steroid-
resistant FSGS tends to be more important in
African American and Hispanic children.
846
Renal Biopsy
Renal biopsy is usually not indicated in a child aged
1–10 years with typical symptoms and a complete
remission with corticosteroids. However, it is indi-
cated in children less than 1 year of age, when the
child has macroscopic hematuria or hypertension or
low C3 levels or persistent renal failure. The main
indication is the failure to respond to corticosteroid
therapy. A renal biopsy may be indicated in patients
who relapse before considering alternative therapy,
namely, anticalcineurin agents.
Light microscopy shows three morphological
patterns: minimal changes, diffuse mesangial pro-
liferation, and FSGS. Minimal changes are found
in the majority of children, whereas FSGS is
found in 5–7 % of them and diffuse mesangial
proliferation in 3–5 % [11, 54].
Minimal Change Nephropathy
On light microscopy, glomeruli may be normal
with normal capillary walls and normal cellularity
(Fig. 1). Swelling and vacuolation of epithelial
cells and a slight increase in mesangial matrix
are often observed. A mild mesangial hypercel-
lularity may be noted as well as scattered foci of
tubular lesions and interstitial fibrosis [58].
Ultrastructural changes are always present,
involving podocytes and mesangial stalks.
Fig. 1 Minimal change
disease. Glomeruli appear
normal by light microscopy
with no tubulointerstitial
lesions
P. Niaudet and O. Boyer
Podocyte foot process fusion is generalized and
constant (Figs. 2 and 3); its extent is closely
related to the degree of proteinuria [59]. Other
epithelial changes consist of microvillus forma-
tion and the presence of numerous protein
reabsorption droplets. The glomerular basement
membranes are normal with no parietal deposits.
The endothelial cells are often swollen
[60]. Mesangial alterations include mesangial cell
hyperactivity, increased mesangial matrix, and
occasionally finely granular, osmiophilic deposits
located along the internal side of the basement
membrane. These ultrastructural alterations are
nonspecific and are probably related to massive
proteinuria. Vascular changes are absent in children.
Diffuse Mesangial Proliferation
Some patients with steroid-resistant INS show a
marked increase in mesangial matrix associated
with hypercellularity (Fig. 4) [58, 61, 62]. How-
ever, peripheral capillary walls are normal, and
immunofluorescence microscopy is negative.
Electron microscopy shows foot process fusion
similar to the changes observed in minimal
change disease. The presence of mesangial
hypercellularity has been found to have prognos-
tic significance with a higher rate of progression to
renal failure [61], but these findings were not
confirmed by other authors.
28 Idiopathic Nephrotic Syndrome in Children: Clinical Aspects 847

Fig. 2 Electron microscopy. On the left, normal aspect with the podocyte foot processes attached to the glomerular
basement membrane. On the right, minimal change disease with effacement of foot processes

Fig. 3 Scanning electron microscopy showing the normal aspect of podocytes with their foot processes on the left and
their effacement in minimal change disease on the right

Fig. 4 Diffuse mesangial

proliferation with an
increased number of
mesangial cells and
mesangial matrix
848 P. Niaudet and O. Boyer

Fig. 5 FSGS. In early

stage, there is a segmental
collapse of glomerular
capillaries surrounded by
hypertrophic podocytes
containing intracytoplasmic
vacuoles

Fig. 6 FSGS. Segmental

lesion of the tuft
characterized by the
deposition of hyaline
material at the inner side of
the glomerular basement
membrane with a ring of
podocytes separated from
the glomerular basement
membrane by a clear “halo”

Focal and Segmental Glomerular
Sclerosis
The glomerular lesions affect a variable propor-
tion of glomeruli [58, 63]. The focal changes are
limited to a part of the tuft, the other capillary
loops showing no modification. The lesions
always predominate in the deeper cortex at the
corticomedullary junction [64]. The segmental
lesion affects a few capillary loops, which stick
together either at the hilum or at the periphery of
the tuft or at both (Fig. 5) [65, 66]. The clinical
course has been found to be more benign when the
location of these sclerotic lesions is peripheral (the
tip lesion), although such findings have not been
confirmed by other authors [64, 67–69]. Hyaline
material is often present within the sclerotic
lesions. A clear “halo” zone is observed at the
periphery of the sclerotic segments (Fig. 6). The
segmental lesion has a different aspect depending
on whether it affects a group of capillary loops free
in Bowman’s space or is adherent to Bowman’s
capsule. The “free” sclerotic segments are always
surrounded by a “crown” of flat or hypertrophied
podocytes. The podocytes form a continuous layer
overlying the damaged areas of the tuft and in close
apposition to the clear “halo.” When the sclerotic
lesion is adherent to Bowman’s capsule, there is a
direct synechia between the collapsed capillary
loops and Bowman’s basement membrane. The
rest of the tuft and the nonsclerotic glomeruli
show either “minimal changes” or “diffuse
28 Idiopathic Nephrotic Syndrome in Children: Clinical Aspects
mesangial proliferation” both with foot process
fusion. Glomerular hypertrophy is common in
FSGS, and when such hypertrophy is found in
minimal change disease, it is somewhat predictive
of further development to FSGS [70, 71].
Tubular atrophy and interstitial fibrosis are often
present and apparently proportional to the glomer-
ular damage [63, 72]. Focal glomerular lesions
should therefore be suspected when focal tubular
and interstitial changes are found associated with
minimal glomerular changes. Erkan et al. found
apoptosis in proximal and distal tubular cells of
children with idiopathic FSGS [73]. There was a
correlation between the degree of proteinuria and
the number of apoptotic cells. An elevated tubule
cell apoptosis rate at the time of initial biopsy was
found to be an independent predictor of progres-
sion to end-stage renal disease.
On electron microscopy, the lesion is character-
ized by the presence of paramesangial and
subendothelial, finely granular, osmiophilic
deposits [72, 74, 75] with either disappearance or
swelling of endothelial cells and an increase in
mesangial matrix material. Fatty vacuoles may be
seen, either in the middle of the abnormal deposit or
in the cytoplasm of endothelial and mesangial cells.
The peripheral synechia, located between
podocytes and basement membrane, is formed by
the apposition of acellular material in which thin
and irregular layers of newly formed basement
membranes are visible. Modifications of the
podocytes consist of focal cytoplasmic degenera-
tion, breakdown of cell membranes, and detach-
ment of epithelial cells from basement
membranes, with filling of the resulting space by
cell debris and new membranes [76].
FSGS is characterized by important changes in
the podocytes, with major cell cycle derangement
[2, 3]. The normal mature podocyte does not divide
and does not express proliferative markers such as
PCNA and Ki-67. The podocyte expresses several
cell surface proteins such as WT-1, C3b receptor,
glomerular epithelial protein-1 (GLEPP-1),
podocalyxin, synaptopodin, and vimentin. The
first stages of FSGS are characterized by the loss
of the cell surface proteins (dedifferentiation) and
the expression of macrophage markers and
cytokeratin (transdifferentiation). Proliferations
849
markers (PCNA and Ki-67) are expressed, indicat-
ing a mitotic activity. This “podocyte
dysregulation” is accompanied by podocyte detach-
ment from the glomerular basement membrane.
A pathologic classification of FSGS, desig-
nated the Columbia classification, has been pro-
posed with five histological variants [77]:
1. FSGS not otherwise specified (NOS) or classic
FSGS, the most frequent variant.
2. The perihilar variant with lesions located at
the vascular pole of the glomerular tuft.
3. The cellular variant with podocyte hyperplasia
and endocapillary hypercellularity, foam cells,
and leukocyte infiltration.
4. The tip variant, where the lesion involves the
tubular pole, is the most benign form of FSGS
with presenting features and outcome more
close to MCD [78].
5. Collapsing variant or collapsing
glomerulopathy characterized by segmental
or global wrinkling and collapse of the glomer-
ular capillary walls with prominent hypertro-
phy and hyperplasia of the overlying
podocytes (Fig. 7) [77].
All variants share podocyte alterations. This
classification has clinical implications in terms of
response to therapy and risk of progression to renal
failure. For example, glomerular tip lesions have
been associated with better outcomes and collaps-
ing variant with worse outcomes [67, 69]. The
glomerular tip lesion was found to be a predictor
of a favorable response to therapy in a pediatric
series [79]. Patients with collapsing
glomerulopathy have a severe nephrotic syndrome
and rapidly progress to renal failure. The rate of
response to steroids is poor. The incidence of
collapsing glomerulopathy seems to have
increased in the recent years. The main cause of
secondary collapsing glomerulopathy is
HIV-associated nephropathy [80]. Many authors
consider that “collapsing glomerulopathy” is a
distinct form of FSGS which may be “idiopathic,”
also observed in patients with recurrent nephrotic
syndrome after renal transplantation or associated
with HIV, parvovirus B19 infection, or CMV infec-
tion [81–83]. Idiopathic collapsing glomerulopathy
850
Fig. 7 Collapsing
glomerulopathy
Fig. 8 IgM deposits
predominates in blacks and has a poor
prognosis [82].
FSGS is an irreversible scarring process in the
glomeruli, as shown by the analysis of repeat biop-
sies [74, 75, 84]. Studies in experimental animals
[85] as well as in nephrotic patients have shown
that proteinuria precedes the development of focal
sclerotic lesions. The same sequence was reported
in patients with recurrence of the disease after
transplantation. Within weeks following recurrence
of proteinuria, podocytes observed by electron
microscopy appear swollen and vacuolated. The
podocytes exhibit strong mitotic activity, with
multinucleation and expression of the PCNA and
Ki-67 proliferation markers.
FSGS is not a specific histopathological lesion:
similar alterations may be seen in persistent idio-
pathic proteinuria and heroin-associated nephropathy
and, independently, in association with HIV
P. Niaudet and O. Boyer
infection, Alport’s syndrome, hypertension, pyelone-
phritis, and obesity. It has also been reported in renal
hypoplasia with oligomeganephronia, after partial
nephrectomy and in other conditions with a reduction
in nephron number, including reflux nephropathy or
obstructive uropathy.
Immunofluorescence Patterns
IgM-associated nephropathy. Immunofluores-
cence microscopy is usually negative [86]. How-
ever, mesangial deposits of IgM, IgG, C3, and
more rarely IgA have been reported. The most
commonly found is IgM (Fig. 8) and Cohen
et al. found that patients with IgM deposits in the
mesangium had a poor response to corticosteroids
[87]. Other studies did not confirm these findings.
Habib et al. reported on immunofluorescence studies
28 Idiopathic Nephrotic Syndrome in Children: Clinical Aspects
in a series of 222 children with INS [88]. Although
IgM was the immunoglobulin most frequently pre-
sent in the glomeruli (54 of 222), there was no
correlation between IgM deposits, the initial response
to steroid therapy or the final outcome. IgM deposits
have also been described in association with diffuse
mesangial proliferation and with FSGS. IgM is a
large molecule that can be nonspecifically trapped
in an injured glomerulus, and mesangial IgM
deposits may represent an epiphenomenon.
IgA deposits and minimal change disease.
A number of patients with INS display mesangial
deposits of IgA. They are classified by some as
IgA nephropathy. Others consider that mesangial
IgA in patients with nephrotic syndrome and min-
imal changes, that is, without cellular proliferation
is coincidental [88–90]. This applies to INS asso-
ciated with mesangial IgA deposits and explains a
rapid response to steroids, which would not be the
case in true IgA nephropathy.
C1q glomerulopathy. C1q glomerulopathy
refers to a disorder in which mesangial prolifera-
tion is associated with mesangial deposits on elec-
tron microscopy and prominent C1q deposits on
immunofluorescence microscopy [91–93] and no
clinical and laboratory evidence of systemic lupus
erythematosus.
C1q nephropathy has been thought to be a
subgroup of primary FSGS. However, many
reports describe different symptoms, histopathol-
ogies, therapeutic responses, and prognoses,
suggesting that C1q glomerulopathy may be a
combination of several disease groups rather
than a single disease entity [94]. C1q
glomerulopathy may be associated with MCD,
FSGS, or proliferative glomerulonephritis.
Pathophysiology
The Anionic Glomerular Basement
Membrane
The permeability of the glomerular basement
membrane (GBM) to proteins is determined not
only by the size but also by the charge of the
protein. It is believed that the anionic charge of
the glomerular basement membrane is responsible
851
for the charge selectivity of filtration. The anionic
(negative) charges of the glomerular basement
membrane repulse the negatively charged albu-
min molecules, whose isoelectric point is 4.6.
The mechanism of proteinuria in the absence
of histological alterations on light microscopy has
suggested an electrochemical disorder of the
GBM. Indeed it was shown that the glomerular
Kf is diminished despite increased permeability to
serum albumin due to a loss of glomerular nega-
tive charges [95–98].
Immunological Anomalies
and Circulating Permeability Factors
In 1974, Shalhoub postulated that minimal change
disease might be secondary to a disorder of the
immune system [99]. He hypothesized that the
production of a lymphokine may increase the
permeability of the glomerular filtration barrier
to proteins. The evidences supporting this hypoth-
esis were the response of the disease to corticoste-
roids and to alkylating agents; the remission
occurring in association with measles, which
depresses cell-mediated immunity; the suscepti-
bility of patients to pneumococcal infections; and
the occurrence of minimal change nephrotic syn-
drome in patients with Hodgkin’s disease. Since
then, several facts have sustained this hypothesis
[99–101]: in SSNS/MCD, the triggering of
relapses by allergy or infections; the unique obser-
vation of the remission of proteinuria when a
kidney from a donor with INS in relapse was
transplanted to a recipient without INS [102]; in
SRNS/FSGS, the rapid recurrence of proteinuria
after renal transplantation in 30 % of patients
[103] that may respond to plasma exchanges or
immunoadsorption [100, 104]; and the induction
of proteinuria in rat following injection of serum
from patients with posttransplant recurrence of
nephrotic syndrome [105]. The Buffalo/Mna
strain of rats spontaneously develops proteinuria
with FSGS at 2 months of age. The nephrotic
syndrome recurs when these rats receive a kidney
from a healthy LEW.1W rat [106]. Conversely,
proteinuria and renal lesions regress when kid-
neys from a Buffalo/Mna rat are transplanted
852
into normal LEW.1W rats. A transient neonatal
nephrotic syndrome was observed in several chil-
dren born to an affected mother with SRNS
[107, 108].
It has long been postulated that the circulating
permeability factor was a T-cell secreted cytokine
[109]. Cytokine profile studies during relapses
suggest the implication of Th2-mediated cyto-
kines [110, 111] more specifically IL-13
[112, 113] or IL-4 [114]. The work of Sahali
et al. demonstrating a dysregulation of the
NFκB/lκB pathway [115] and upregulation of
the Th2-specific factor c-maf during relapses
[116] is in accordance with these results. Aside
from cytokines, other factors have been suspected
to be involved in the pathogenesis of INS includ-
ing cardiotrophin-like cytokine-1, hemopexin,
and suPAR. Savin et al., using isolated rat glomer-
uli incubated in vitro with the sera of patients with
recurrence of proteinuria after renal transplanta-
tion, described a glomerular permeability factor
[100]. Using this assay, the same group identified
the cardiotrophin-like cytokine 1(CLC-1), a pro-
tein of the IL-6 family [117]. Its concentration is
100 times higher in the sera of patients with recur-
rent proteinuria after transplantation compared to
normal subjects. The injection of CLC-1 to rats
induces proteinuria. CLC-1 decreases nephrin
expression in cultured podocytes. Hemopexin, a
heme-scavenging protein secreted by the liver dur-
ing inflammation, induces reversible proteinuria
and foot process effacement when infused into
rats [118, 119]. Hemopexin has a serine protease
activity during relapses in children
[120]. Hemopexin might exist in an altered isoform
with increased activity in patients during relapses
as compared with patients in remission.
Hemopexin causes nephrin-dependent podocyte
cytoskeleton rearrangements and disruption of the
glomerular filtration barrier permselectivity, both
of which are reversed by incubating cultured
podocytes with normal human sera [121]. How-
ever, the mechanisms of hemopexin activation dur-
ing relapses and their consequences on podocyte
structure and function are not yet fully understood.
Recent findings have suggested the role of
soluble urokinase receptor (suPAR) in the patho-
genesis of FSGS [8]. The group of Jochen Reiser
P. Niaudet and O. Boyer
detected significantly high suPAR levels in FSGS
patients, especially those with posttransplant
recurrence, but not in patients with MCD or mem-
branous nephropathy [8]. The role of suPAR-
induced αvβ3 integrin activation on proteinuria
was further studied in three animal models:
recombinant suPAR injection to suPAR knockout
mice (Plaur/), LPS injection in wild-type mice
transplanted with Plaur/ mouse kidney, and a
transgenic mouse model overexpressing suPAR
through repeated cutaneous plasmid
electroporations. The authors also showed an acti-
vation of the suPAR-αvβ3 integrin pathway in
cultured podocytes incubated with patient sera.
However, these data have not been confirmed by
other groups [122]. Cathelin et al. showed that the
administration of recombinant suPAR to C57BL/
6J wild-type mice does not result in albuminuria
nor podocyte architecture alterations although
leading to suPAR glomerular deposition
[123]. In addition, suPAR levels are elevated in
patients with reduced glomerular filtration rate
and in other non-proteinuric diseases such as can-
cer and HIV infection [124, 125] with MCD or
membranous nephropathy [8]. Conversely, some
patients with normal suPAR plasma levels
develop posttransplant recurrent FSGS [8]. Last,
suPAR levels are not increased in children with
MCD suggesting distinct pathophysiological
mechanisms underlying MCD and FSGS.
Although high pre-transplant suPAR levels seem
to be associated with an increased recurrence risk
after transplantation, the potential role of suPAR as
a valuable marker in INS is debated [122, 126, 127].
Three studies including a total of 1,151 patients,
212 of whom with primary FSGS, concluded
that levels of suPAR do not discriminate
between primary FSGS and other causes of glomer-
ular diseases [128–130]. Altogether these data
argue against suPAR being per se the FSGS
factor [131].
The type of immune cell producing the circulat-
ing factor also remains elusive. T-cells have long
been incriminated. More recent data suggest the
role of B-cells in INS [132, 133]. Indeed, the effi-
ciency of rituximab in preventing relapses in
patients with steroid dependency strongly advo-
cates the role of B-cells in INS [134–136]. B-cells
28 Idiopathic Nephrotic Syndrome in Children: Clinical Aspects
could be involved by secreting the permeability
factor (i.e., that could be an immunoglobulin) or
through a T-cell to B-cell communication
dysregulation [137]. However, Fornoni
et al. showed that rituximab also has a direct effect
on podocyte cytoskeleton stabilization [138].
It was also reported that CD34(+) hematopoietic
progenitors from patients injected to NOD/SCID
immunodeficient mice could induce proteinuria
and foot process effacement, whereas peripheral
CD34() mononucleated cells could not [139].
Accumulating data sustain the role of one or
several permeability factors. However, their
effects on podocyte structure and function and
the mechanisms driving foot process effacement
and proteinuria remain unclear. Aside from
suPAR-αvβ3 integrin pathway, B7.1 (CD80) and
c-mip signaling have been incriminated. B7.1
(CD80), a T-cell co-stimulator present at the sur-
face of antigen-presenting cells, is increased in
urine of children with MCD [140]. LPS injection
in mice induces B7.1 (CD80) expression on
podocytes [141] and thereby podocyte signaling
cascades that could lead to foot process efface-
ment and proteinuria [142]. Differential cloning
of patient lymphocytes in remission or relapse
suggested the role of c-mip, expressed in
podocytes during relapses, T-lymphocytes, and
Hodgkin lymphoma cells from patients develop-
ing nephrotic syndrome [116, 143]. C-mip is also
activated upon LPS or Adriamycin administration
to mice in experimental models of nephrotic syn-
drome [144]. Transgenic mice overexpressing
c-mip in podocytes develop massive proteinuria
and foot process effacement [144]. C-mip silenc-
ing by RNA interference reduces LPS-induced
proteinuria [144]. These results are a first step
toward targeted therapies in INS.
In summary, it is currently unclear whether
INS is caused by one or multiple factors and
whether this/these putative factors are identical
in MCD and FSGS. Indeed, urinary CD80 is
elevated in MCD patients in relapse compared
with FSGS patients. In contrast, serum suPAR
is significantly elevated in FSGS patients
suggesting that these two conditions might have
distinct underlying mechanisms [145]. Further
studies are needed to confirm and better refine
853
the roles of the putative circulating factors and
their effects on downstream podocyte signaling
pathways.
Treatment
Symptomatic Treatment
Diet
Dietary therapy should include a protein intake of
around 130–140 % of the recommended daily
allowance according to statural age. Salt restriction
is advised for the prevention and the treatment of
edema. A very low salt diet is only necessary in
cases of massive edema. Fluid restriction is
recommended for moderate to severe hyponatremia
(plasma sodium concentration less than 125 meq/l).
A reduction of saturated fat is recommended. Car-
bohydrates should be given preferentially as starch
or dextrin-maltose, avoiding sucrose, which
increases lipid abnormalities.
Hypovolemia
Hypovolemia occurs as a consequence of rapid
loss of protein and is sometimes aggravated by the
use of diuretics. This complication needs emer-
gency treatment by rapid infusion of plasma
(20 ml/kg) or albumin 20 % (1 g/kg) administrated
with control of heart rate, respiratory rate, and
blood pressure.
Diuretics
Diuretics should only be used in cases of severe
edema, after hypovolemia has been corrected.
Furosemide is administered at a dose of 1–2
mg/kg. If not effective, spironolactone (5–10
mg/kg) or amiloride (0.2–0.5 mg/kg) may be pre-
scribed if the plasma creatinine concentration is
normal [146]. Patients with severe edema may be
treated with furosemide or, if necessary, furose-
mide plus albumin to increase the rate of diuretic
delivery to the kidney. This approach is immedi-
ately effective, but not long lasting. Moreover,
respiratory distress with congestive heart failure
has been observed in some patients [147].
Refractory edema with serious effusions may
require drainage of ascites and/or pleural
854
effusions. Immersion of the body up to the neck in
a bath may be helpful in these cases [148].
Thromboemboli
Nephrotic patients are at risk for thromboembolic
complications. Prevention of this complication
includes regular ambulation, avoidance of
hemoconcentration due to hypovolemia, avoid-
ance of central venous catheter if possible, and
early treatment of sepsis or volume depletion.
There are no controlled trials on the efficacy and
safety of prophylactic anticoagulation to prevent
thromboembolic complications in children with
nephrotic syndrome. However, several authors
propose prophylactic measures in children with
risk factors. For example, prophylactic warfarin
therapy may be given to patients with a plasma
albumin concentration below 20 g/l, a fibrinogen
level over 6 g/l, or an antithrombin III level below
70 % of normal. Patients at risk may also be
treated with low-dose aspirin and dipyridamole,
although there is no evidence that it is
beneficial [45].
Heparin is given initially if thrombi do occur,
alone or with thrombolytic agents. Anticoagulation
is most often initiated with low molecular weight
heparin, such as enoxaparin at a starting dose of
1 mg/kgBW every 12 h, with the antifactor Xa
assay as a therapeutic drug monitoring. Its pharma-
cokinetics is more predictable than unfractionated
heparin, and it is given subcutaneously, avoiding
venous catheter. In situations where short half-life
and reversible anticoagulation is necessary,
unfractionated heparin is utilized. The heparin
dose necessary to obtain a therapeutic effect is
often greater than normal due to decreased anti-
thrombin III level.
Antihypertensive Drugs
Any arterial hypertension has to be carefully con-
trolled, using preferably a β-blocker or a calcium
channel blocker during acute episodes. In cases of
permanent hypertension, an angiotensin-
converting enzyme inhibitor is preferred.
Infections and Immunizations
Prophylaxis of S. pneumoniæ with oral penicillin
is often applied in patients during the initial
P. Niaudet and O. Boyer
treatment with corticosteroids. Pneumococcal
vaccine may be performed. It is effective even in
children receiving high doses of steroids, and it is
not associated with an increased risk of relapse
[149, 150]. In cases of peritonitis, antibiotics
against both S. pneumoniæ and gram-negative
organisms are started after peritoneal liquid sam-
pling. Varicella is a serious disease in patients
receiving immunosuppressive treatment or daily
corticosteroids. Varicella immunity status should
be checked in these patients. In cases of exposure,
early preventive treatment by acyclovir must be
instituted. Varicella vaccination is safe and effec-
tive if the child is in remission even if he/she is on
low-dose alternate-day steroids [151, 152].
Hyperlipidemia
Persistent hyperlipidemia is a risk factor for ath-
erosclerosis and may play a role in the progression
of chronic renal failure. Experience with
hypolipidemic drugs in nephrotic patients is still
limited, but it seems that statins are effective and
able to decrease hypercholesterolemia [153, 154].
Although long-term side effects of these drugs are
not known, it is reasonable to consider a lipid-
lowering regimen in children with a persistent
nephrotic syndrome [155].
Miscellaneous
Calcium metabolism may be altered by the uri-
nary loss of 25-hydroxycholecalciferol and its
carrier protein. Preventive treatment with vitamin
D supplements is therefore useful, but does not
completely prevent bone loss [156]. Thyroxine
substitution may be indicated, but only in patients
with documented hypothyroidism due to urinary
loss of iodinated proteins.
Specific Treatment
Steroid therapy is applied in all cases of INS
whatever the histopathology, even in patients
with FSGS. The majority of patients are steroid
responsive (Table 5). Urine protein profile (prote-
ome) may in the future help to predict the response
to steroid therapy [157]. Steroid responders may
relapse, but the majority still responds to steroids
28 Idiopathic Nephrotic Syndrome in Children: Clinical Aspects
Table 5 Definitions
Nephrotic syndrome: proteinuria >40 mg/h/m2 or >50
mg/kg/day or protein/creatinine ratio >0.2 g/mmol
(>2 g/g) and hypoalbuminemia <25 g/l with or
without edema
Remission: proteinuria <4 mg/h/m2 or 0-trace on
Albustix for 3 consecutive days
Steroid responsive: complete remission achieved with
steroid therapy
Steroid resistant: failure to achieve remission following
4-week prednisone 60 mg/m2  followed by three
methylprednisolone pulses
Relapse: proteinuria >>40 mg/h/m2 or >50 mg/kg/day
or Albustix +++ for 3 consecutive days after having been
in remission
Frequent relapses: 2 or more relapses within 6 months of
initial response or 4 or more relapses within a period of
1 year
Steroid dependence: 2 consecutive relapses during
corticosteroid therapy or within 14 days after cessation of
therapy
Early non responder: steroid resistance during the first
episode
Late non responder: steroid resistance in a patient who
had previously responded to corticosteroid therapy
over the subsequent course. Only 1–3 % of
patients initially steroid-sensitive subsequently
become steroid-resistant and are defined as “late
nonresponders” [158].
Initial Treatment
Although steroid therapy is often started immedi-
ately following the diagnosis of nephrotic syn-
drome, it should be stressed that spontaneous
remission occurs in 5 % of cases within 1 or
2 weeks. Therefore, initiation of steroid therapy
may be delayed for a few days [159]. Some of
these early spontaneous remissions are definitive.
Infection must be treated before starting steroids,
not only to prevent the risk of overwhelming
sepsis during treatment but also because occult
infection may be responsible for steroid
resistance [160].
Steroid therapy is started when the diagnosis of
INS is most likely in a child older than 1 year and
younger than 11 years of age, without hyperten-
sion, gross hematuria, or extrarenal symptoms and
855
normal complement levels. In some cases, the
treatment is started after a renal biopsy has been
performed. Prednisone remains the reference
drug. Prednisolone has the advantage of being
soluble in water, making treatment easier in
young children, but it may fail to induce remission
in some patients who respond quickly to the same
dosage of prednisone. Indeed, the intestinal
absorption of prednisolone is less than that of
prednisone, and this may explain a lesser efficacy
in some children.
The ISKDC regimen consists of prednisone,
60 mg/m2/day with a maximum of 80 mg/day, in
divided doses for 4 weeks followed by 40 mg/m2/
day with a maximum of 60 mg/day in divided
doses, on three consecutive days per week for
4 weeks [161]. The Arbeitsgemeinschaft f€ur
pädiatrische Nephrologie showed that an
alternate-day regimen (40 mg/m2 every other
day for 4 weeks) resulted in a significantly lower
number of patients with relapses and fewer
relapses per patient [162]. It also showed that on
alternate days, prednisone could be given in a
single dose rather than in divided doses.
A response occurs in most cases within 10–15
days (median 11 days). According to the Interna-
tional Study of Kidney Disease in Children,
approximately 90 % of responders enter in remis-
sion within 4 weeks after starting steroids,
whereas less than 10 % go into remission after
2–4 more weeks of a daily regimen. A few more
patients go into remission after 8–12 weeks of
daily steroids [11, 13], but prolongation of daily
steroid treatment beyond 4 or 5 weeks increases
the risk of side effects. An alternative for patients
who are not in remission after 4 weeks is to
administer three to four pulses of methylprednis-
olone (1 g/1.73 m2). This additional regimen
seems to be associated with fewer side effects
than prolongation of daily high-dose steroids and
probably produces remission more rapidly in the
few patients who would have entered into remis-
sion during the second month of daily
therapy [163].
A working committee recently developed the
following KDIGO guidelines [164]. Initial pred-
nisone therapy consists of 60 mg/m2 or 2 mg/kg
administered as a single daily dose for 4–6 weeks
856
(maximum dose of 60 mg/day), followed by
alternate-day prednisone of 40 mg/m2 or 1.5
mg/kg (maximum dose of 40 mg/day) continued
for 2–5 months with tapering of the dose.
The duration of initial steroid therapy influ-
ences the risk of relapse. The APN compared a
standard regimen of 4-week daily prednisone and
4 weeks of alternate-day prednisone with a longer
initial course of 6 weeks of daily prednisone at a
dose of 60 mg/m2/day followed by 6 weeks
alternate-day prednisone at a dose of 40 mg/m2/
day [165]. The subsequent relapse rate within
12 months following discontinuation of therapy
was lower with the prolonged course of therapy
compared to the standard course (36 % vs. 61 %).
Following an 8-week steroid regimen, 50–70 %
of children experience relapses. Several controlled
studies have compared the 8-week regimen with
longer duration of steroid regimen (3–7 months)
including 4–8 weeks of daily prednisone followed
by alternate-day prednisone [165–169]. With a
follow-up of 2 years, a significant reduction of
25–30 % in the relapse rate was observed with a
prednisone regimen of 3 months or more
[170]. Conversely, Teeninga et al. reported the
results of a randomized, double-blind, placebo-
controlled trial involving 150 children presenting
with nephrotic syndrome who received either
3 months of prednisolone followed by 3 months
of placebo or 6 months of prednisolone with both
groups receiving equal cumulative doses of pred-
nisolone [141]. This study concluded that
extending initial prednisolone treatment from 3 to
6 months without increasing cumulative dose did
not benefit clinical outcome. Therefore, prolonged
treatment regimens that reduce relapses most likely
result from increased cumulative dose rather than
treatment duration. More recently, Yoshikawa
et al. reported the results of an open-label, random-
ized controlled trial comparing a 2-month prednis-
olone therapy with a 6-month therapy in
246 children with steroid-sensitive nephrotic syn-
drome [171]. Time to frequent relapse and time to
first relapse were also similar in both groups
allowing the authors to conclude that 2 months of
initial prednisolone therapy for steroid-sensitive
nephrotic syndrome, despite less prednisolone
exposure, is not inferior to 6 months of initial
P. Niaudet and O. Boyer
therapy in terms of time to onset of frequently
relapsing nephrotic syndrome. Another random-
ized control trial from India showed that extending
the initial prednisolone treatment from 3 to
6 months does not influence the course of the
disease [172].
There are no data showing that treating for
more than 7 months is beneficial. However, a
study showed that an alternate-day regimen over
a year did not reduce the rate of relapse compared
to a 5-month alternate-day regimen [173].
Although the studies were not designed to analyze
the side effects of glucocorticoids, the authors did
not report increased toxicity with longer duration
of treatment.
A slow tapering phase to avoid adrenal sup-
pression may maintain long-term remission as a
study showed that moderate to severe adrenal
suppression was associated with an increased
risk of relapse [174]. Some authors have
suggested a possible prevention by low-dose
maintenance hydrocortisone [174, 175]. Another
study also concluded that adrenocortical suppres-
sion increases the risk of relapse in children on
long-term alternate-day steroid therapy [176].
Increasing initial immunosuppression by
adding cyclosporine to steroid therapy does not
change the 2-year relapse rate [177].
Treatment of Relapses
About 30 % of children experience only one
attack and are definitively cured after a single
course of steroids. Persistent remission for
18–24 months after stopping treatment is likely
to reflect definitive cure, and the risk of later
relapses is low. Ten to twenty percent of patients
relapse several months after stopping treatment
and are most often cured after three or four epi-
sodes, which respond to a standard course of
steroid therapy. The remaining 50–60 % experi-
ence relapses as soon as steroid therapy is stopped
or when dosage is decreased. In some cases, exac-
erbation of proteinuria is only transient, and spon-
taneous remissions are observed [193]. The risk of
relapse is greater in children aged less than 5 years
at onset. A recent study found an association
28 Idiopathic Nephrotic Syndrome in Children: Clinical Aspects
between the GR-9b haplotype of the glucocorti-
coid receptor gene and the outcome of children
with steroid-responsive nephrotic syndrome.
Children with this haplotype showed increased
incidence of first relapse, frequent relapses, and
steroid dependence as compared with noncarriers
[178]. Steroid-dependent patients often raise
difficult therapeutic problems.
Steroid-dependent patients may be treated with
repeated courses of prednisone, 60 mg/m2/day,
continued 3 days after the urine has become pro-
tein free, followed by alternate-day prednisone,
40 mg/m2, for 4 weeks as proposed by the Inter-
national Study of Kidney Disease in Children
[161]. Another option consists of treating relapses
with daily prednisone, 40–60 mg/m2, until pro-
teinuria has disappeared for 4–5 days. Thereafter,
prednisone is switched to alternate days, and the
dosage is tapered to 15–20 mg/m2 every other day,
according to the steroid threshold, that is, the
dosage at which the relapse has occurred. Treat-
ment is then continued for 12–18 months. The first
approach allows better definition in terms of num-
ber of relapses but is associated with more
relapses. The latter regimen is associated with
less steroid side effects as the cumulative dosage
is lower. Prolonged courses of alternate-day ste-
roid therapy are often well tolerated by young
children, and growth velocity is not affected.
However, prednisone dosage must be as low as
possible in order to reduce the side effects. In
adolescents, steroid therapy is often accompanied
by decreased growth velocity.
A controlled trial has shown that deflazacort
reduces the risk of relapse in comparison with
equivalent doses of prednisone, without addi-
tional side effects [179]. Unfortunately,
deflazacort is not available in many countries.
The role of upper respiratory tract infections in
exacerbating nephrotic syndrome has been
highlighted in all series: 71 % of relapses were
preceded by such an event in a prospective study,
although only 45 % of respiratory infections were
followed by an exacerbation of proteinuria
[180]. The risk of relapse is decreased during
upper respiratory tract infections when steroid
therapy is given daily for 5–7 days rather than
on alternate days [181–183].
857
Steroid Side Effects
Side effects of prolonged steroid therapy are well
known and are observed in children with a steroid-
dependent course. Growth retardation is observed
with prolonged daily steroid therapy, and alternate-
day therapy may preserve growth [184]. However,
when the dose needed to maintain remission is too
high, growth may be impaired [185, 186]. Osteopo-
rosis has been reported in adults who had suffered
from nephrotic syndrome during childhood [187].
However, a study in children and adolescent with
steroid-dependent nephrotic syndrome failed to
find a deleterious effect of alternate-day steroid
therapy on bone mineral content [188]. Biyikli
et al. reported that steroid treatment causes a
dose-dependent decrease in bone formation, as
shown by the changes in osteocalcin and alkaline
phosphatase levels and low 25-hydroxyvitamin D
levels [189]. A randomized controlled trial com-
pared vitamin D and calcium supplements with no
prophylaxis in children receiving high-dose steroid
therapy during a relapse. The authors found a
decrease in bone mineral content in both groups but
less pronounced in the treated group (4.6  2.1 %
vs. 13.0  4.0 %, respectively; P < 0.001)
[156]. Another controlled study showed that
bisphosphonates are effective in preventing steroid-
induced osteoporosis in children receiving long-term
steroid therapy [190]. The other side effects include
weight gain, cataracts, behavior disturbances, and
hypertension.
Alternative Treatments (Fig. 9)
An alternative treatment is required when
children develop severe side effects of steroid
therapy, in children at risk of toxicity (diabetes
or during puberty), in children with severe
relapses accompanied by thrombotic complica-
tions or severe hypovolemia, and in those with
poor compliance.
Alternative treatments include levamisole,
which has a steroid-sparing effect, and alkylating
agents such as cyclophosphamide or
chlorambucil, mycophenolate mofetil, cyclospor-
ine, and, more recently, rituximab (Fig. 9).
 858

2
4 weeks prednisone, 60 mg/m /day (1)
Complete remission

No relapse Relapse > 3 months Relapse while on alternate day steroid therapy
(30%) after steroid withdrawal or less than 3 months after steroid withdrawal (60%)
(10%)

Daily steroid therapy and switch

Sart again as (1) to alternate day 3-4 days after remission

Prolonged alternate day steroid therapy

and/or levamisole

Persistent remission More relapses and steroid side effects

without steroid side-effects

Course of alkylating agents or MMF

Continue the treatment for 12 to 18 months

Relapse + steroid side effects

No relapse Relapse
Anti-calcineurin ± low dose alternate day steroid therapy

alternate day steroid therapy

for 12-18 months
Anti-calcineurin toxicity, steroid toxicity and relapses

Rituximab

Fig. 9 Proposed algorithm for the treatment of steroid-dependent idiopathic nephrotic syndrome
P. Niaudet and O. Boyer
28 Idiopathic Nephrotic Syndrome in Children: Clinical Aspects
Levamisole
The beneficial effect of levamisole was first
reported by Tanphaichitr et al. [191]. Levamisole
was subsequently reported to reduce the risk of
relapse in steroid-dependent patients [192–196].
A significant steroid-sparing effect at a dose of 2.5
mg/kg every other day was demonstrated in a
prospective controlled trial of the British Associ-
ation for Paediatric Nephrology [197, 211].
Another controlled study confirmed the efficacy
of levamisole for preventing relapses [198, 212].
Levamisole given for 6 months was compared
with cyclophosphamide given for 8–12 weeks
in a retrospective study involving 51 children
with steroid-dependent nephrotic syndrome
[199, 213]. The relapse rate and the cumulative
dose of prednisone were reduced to the same
extent with both drugs. However, the beneficial
effect of levamisole is not sustained after stopping
treatment.
Levamisole is well tolerated in most children.
Side effects occasionally include neutropenia,
agranulocytosis, vomiting, cutaneous rash, vascu-
litis, and neurological symptoms including insom-
nia, hyperactivity, and seizures [200–202].
However, levamisole is not widely available [201].
Alkylating Agents
Alkylating agents, such as cyclophosphamide and
chlorambucil, have been used for more than
50 years to achieve long-lasting remission.
The efficacy of alkylating agents is illustrated
by a meta-analysis that compared alkylating
agents with prednisone in maintaining remission.
In three trials including 102 patients, oral cyclo-
phosphamide reduced the risk of relapse at 6–12
months (RR 0.44; 95 % CI 0.26–0.73). Similarly,
chlorambucil compared with prednisone alone
reduced the risk of relapse at 12 months in 32 chil-
dren (RR 0.13; 95 % CI 0.03–0.57) [203]. Several
studies involving patients with frequently relaps-
ing or steroid-dependent nephrotic syndrome
showed that cyclophosphamide resulted in
sustained remission in 57–93 % of patients at
1 year, 31–66 % at 5 years, and 25 % at 10 years
[204–207]. The duration of remission is higher
when cyclophosphamide is given in association
with steroids compared to cyclophosphamide
859
given alone [204, 208, 209]. The incidence of
relapse after cyclophosphamide is significantly
higher in patients with FSGS (73 %) or mesangial
proliferation, compared to children with minimal
change (22 %) [210].
The Arbeitsgemeinschaft fur pädiatrische
Nephrologie reported that a treatment for
12 weeks at a daily dose of 2 mg/kg was more
effective than an 8-week course, with 67 % as
compared to 22 % remaining in remission after
2 years [211]. However, a randomized trial
showed that prolonging the course of cyclophos-
phamide from 8 to 12 weeks did not further reduce
the proportion of children experiencing
relapses [212].
However, more recent data show disappointing
results of cyclophosphamide treatment for
steroid-dependent patients. Kemper
et al. reported that only 6 out of 20 children had
a sustained remission following a 12-week course
of cyclophosphamide [213]. In a retrospective
study, Vester et al. reported that 24 % of 106 chil-
dren who had received a course of cyclophospha-
mide were still in remission after 10 years [207].
A younger age than 3 years at onset is associated
with lower response rate [214]. A recent series
reported lower remission rates of 44 %, 27 %,
and 13 % at 1, 2, and 5 years after cyclophospha-
mide therapy [215]. In another study of 90 chil-
dren with a steroid-dependent course, sustained
remissions were observed in 31 % of patients at
5-year follow-up [216]. These variations are prob-
ably due to differences in patient populations as
steroid-dependent patients have a lower response
rate than frequently relapsing patients. The degree
of steroid dependency also affects remission rates
[217], and cyclophosphamide is less effective in
patients with steroid dependency compared to
patients with frequent relapses [218].
Cyclophosphamide has also been administered
as monthly boluses at 500 mg/m2 for 6 months.
Gulati et al. reported a remission rate of 38 % at
5 years among the 29 steroid-dependent patients
[219]. However, Donia et al. reported a remission
rate of only 5 % at 2 years in a group of 20 steroid-
dependent patients [220]. Prasad et al. compared
cyclophosphamide given orally or as boluses in
47 children [221]. The remission rate 6 months
860
after the end of treatment was higher following the
cyclophosphamide boluses (73 % vs. 38 %) but
was similar with a follow-up of 2 years.
Cyclophosphamide toxicity includes bone
marrow depression, hemorrhagic cystitis, gastro-
intestinal disturbances, alopecia, and infection
[222]. Leukopenia is frequently observed, but
weekly hematological monitoring may limit its
severity and concomitant steroids help blunt mar-
row depression. Hemorrhagic cystitis rarely
occurs. Alopecia, which is variably pronounced,
remits a few weeks after stopping treatment. Viral
infections can be overwhelming if cyclophospha-
mide is not stopped in due time.
Long-term toxicity includes malignancy, pul-
monary fibrosis, ovarian fibrosis, and sterility.
Gonadal toxicity is well established, and the risk
of sterility is greater in boys than in girls. The
cumulative threshold dose above which
oligospermia/azoospermia may be feared lies
between 150 and 250 mg/kg [223–225]. In
females, the cumulative dose associated with ste-
rility is greater, but not well defined. Pregnancies
have been reported after treatments longer than
18 months [226].
Most authors would prescribe a 12-week
course of oral cyclophosphamide at a daily dose
of 2 mg/kg.
Beneficial results have also been achieved with
chlorambucil in steroid-responsive nephrosis
[227–229]. The response to chlorambucil is
related to the clinical pattern of relapse as for
cyclophosphamide. A meta-analysis of 26 con-
trolled trials and cohort studies found that the 2-
and 5-year relapse rates following treatment with
cyclophosphamide or chlorambucil were 72 %
and 36 % in frequently relapsing nephrotic syn-
drome compared with 40 % and 24 % in steroid-
dependent nephrotic syndrome [222].
Acute toxic effects are less frequent with
chlorambucil than with cyclophosphamide. Leu-
kopenia and thrombocytopenia may occur and are
reversible within 1–3 weeks. Severe microbial
and viral infections have been reported, including
malignant hepatitis and measles encephalitis.
Long-term toxic effects include the risk of
developing cancer or leukemia, which has only
been reported in patients who had prolonged
P. Niaudet and O. Boyer
courses of treatment. Gonadal toxicity, as with
cyclophosphamide, essentially affects boys. Azo-
ospermia is total and probably irreversible at
cumulative doses above 10–20 mg/kg. No case
of azoospermia was reported in patients given less
than 8 mg/kg.
Cyclosporine
Cyclosporine has been shown in a number of
uncontrolled studies to reduce the incidence of
relapses in 75–90 % of patients with steroid-
dependent INS [230]. However, most patients
experience relapses when the dosage is tapered
or when cyclosporine is withdrawn [231]. The
patients thus behave with cyclosporine as they
did with steroids; that is, they become cyclospor-
ine dependent. The relapse rate usually returns to
the pretreatment rate. Hulton et al. found that
patients in whom cyclosporine had been
discontinued and later restarted had more
relapses, requiring steroids in addition to cyclo-
sporine in order to maintain remission [232].
The effects of cyclosporine have been evalu-
ated in two comparative trials in steroid-sensitive
patients. Cyclosporine at a dosage of 6 mg/kg/day
for 3 months, then tapered over 3 months, was
compared with chlorambucil given for 2 months.
At 12 months, 30 % of patients who had received
chlorambucil and only 5 % of those who had
received cyclosporine were still in remission
[233]. A multicenter randomized controlled trial
compared cyclosporine for 9 months then tapered
over 3 months, with oral cyclophosphamide for
2 months [234]. After 2 years, 25 % of the patients
(50 % of adults and 20 % of children) who had
received cyclosporine had not relapsed, while
63 % of those treated with cyclophosphamide
(40 % of adults and 68 % of children) were still
in remission. During the year following treatment,
the relapse rate (1.8 vs. 0.7) and the steroid dosage
required (109 vs. 23 mg/kg/year) were signifi-
cantly higher in children who had received
cyclosporine.
Tejani et al. performed a randomized con-
trolled trial comparing low-dose prednisone and
cyclosporine versus high-dose prednisone for
8 weeks as first-line treatment in 28 children
[235]. Thirteen of the 14 children receiving the
28 Idiopathic Nephrotic Syndrome in Children: Clinical Aspects
combined treatment went into remission com-
pared to only 8/14 receiving prednisone alone
( p < 0.05). The duration of remission after ending
treatment was comparable in both groups. Severe
hypercholesterolemia may inhibit cyclosporine
efficacy and require higher dosages for similar
results [236, 237].
A prospective, open multicenter trial from
Japan compared the efficacy and safety of two
cyclosporine regimen, a dose adjusted to maintain
trough level between 60 and 80 ng/ml (group A)
and a fixed dose of 2.5 mg/kg (group B) in chil-
dren who had initially received cyclosporine for
6 months with a trough level of 80–100 ng/ml
[238]. After 2 years, the rate of sustained remis-
sion was significantly higher in group A.
Considering cyclosporine dependency, this
treatment must be pursued to prevent new relapses.
Indeed, cyclosporine exposes to nephrotoxicity. In
case of decreased renal function, it is advisable to
reduce dosage or even stop the treatment. Renal
function improvement of is in favor of functional
renal insufficiency or drug nephrotoxicity. Never-
theless, lesions of chronic nephrotoxicity can
develop without any appreciable decline of the
glomerular filtration rate [239–241]. As it is often
necessary to continue treatment for a long time,
repeat renal biopsies are highly advisable to detect
these lesions. They most often consist of tubuloin-
terstitial injury, characterized by stripes of intersti-
tial fibrosis containing clusters of atrophic tubules
and by vascular lesions.
Other side effects are of less concern: hyper-
tension, hyperkalemia, hypertrichosis, gum
hypertrophy, and hypomagnesemia are common
but easily manageable. Cyclosporine treatment
has no deleterious effect on bone mineral
content [242].
Tacrolimus
In a retrospective study of ten children, it was
observed that tacrolimus was not better than cyclo-
sporine for the management of severe steroid-
dependent nephrotic syndrome [243]. In a series
of five children treated with tacrolimus, two devel-
oped insulin-dependent diabetes mellitus, which
resolved after stopping tacrolimus therapy [244].
However, one advantage of tacrolimus over
861
cyclosporine is a reduced cosmetic side effects
(hypertrichosis, gum hypertrophy).
Mycophenolate Mofetil
During the past 10 years, several reports have
shown that mycophenolate mofetil (MMF) treat-
ment may have a beneficial effect in children with
steroid-dependent INS [245–251]. These studies
have shown that MMF allowed decreasing or
stopping steroid therapy in 40–75 % of children.
However, relapses were nearly constant after ces-
sation of treatment. MMF was shown to have a
significant cyclosporine and/or steroid-sparing
effect in children with cyclosporine-dependent
INS with a beneficial effect on renal function
[248, 252].
In a small randomized trial comparing MMF to
cyclosporine, sustained complete remission was
achieved in 7 of 12 patients who received MMF
and in 11 of 12 patients treated with cyclosporine
suggesting that cyclosporine is more effective
than MMF [253]. A Bayesian study was
conducted in 23 children with steroid-dependent
idiopathic nephrotic syndrome having received
prior alkylating agent treatment and 2/3 of them
levamisole [254]. They were treated with MMF
and prednisone according to a defined schedule
[reduction of alternate-day dose to 50 % of
pre-MMF dose at 3 months, 25 % at 6 months].
Twenty-three children completed the study. Four
relapsed during the first 6 months and two at
months 8 and 11.5. In the 19 patients free of
relapse during the first 6 months, median predni-
sone maintenance dose decreased from
25 (10–44) to 9 (7.5–11.2) mg/m2 and cumulative
dose from 459 (382–689) to 264 (196–306)
mg/m2/ before and on MMF, respectively. The
authors concluded that MMF reduces relapse
rate and steroid dose in children with steroid-
dependent nephrotic syndrome and should be
proposed before cyclosporine.
These studies confirm the efficacy and safety
of MMF in patients with steroid-dependent
nephrotic syndrome and support its use for a lon-
ger duration than 12 months. Doses of 450–600
mg/m2/day in two divided doses are usually
given. Side effects including gastrointestinal dis-
turbances (abdominal pain, diarrhea) and
862
hematologic abnormalities are rare. Many authors
now recommend the use of MMF rather than
alkylating agents in children with steroid-
dependent nephrotic syndrome who suffer from
side effects of steroid therapy. However, random-
ized trials should be performed before such
recommendations can be made.
Rituximab
Several retrospective series suggest that rituximab
(RTX), a chimeric anti-CD20 monoclonal anti-
body that depletes B-cell lymphocytes, is effec-
tive in steroid-dependent or calcineurin inhibitor-
dependent patients allowing to reduce or stop
immunosuppression [255–257]. In a case series
of 22 children with severe steroid or cyclosporine-
dependent NS, the administration of RTX allowed
the discontinuation of one or more immunosup-
pressive agents [134]. The response to rituximab
appears to be better if the patient is in remission at
the time of the infusion. Seven patients were
nephrotic at the time of RTX treatment. Remission
was induced in three of the seven proteinuric
patients. RTX was effective in all patients when
administered during a proteinuria-free period in
association with other immunosuppressive
agents. Therefore, rituximab should not be admin-
istered during a relapse but after remission has
been induced by increased doses of steroids. One
or more immunosuppressive treatments could be
withdrawn in 19 patients, with no relapse of pro-
teinuria and without increasing other drug dosage.
When relapses occurred, they were associated
with an increase in CD19 cell count. Adverse
effects were observed in 45 % of cases, but most
of them were mild and transient.
Ravani et al. randomized 54 children (mean
age 11  4 years) with INS dependent on predni-
sone and calcineurin inhibitors for >12 months
[135]. RTX with lower doses of prednisone and
calcineurin inhibitors was compared to current
therapy alone. Three-month proteinuria was
70 % lower in the RTX arm as compared with
standard therapy arm (intention to treat); relapse
rates were 18.5 % (intervention) and 48.1 % (stan-
dard arm) (P = 0.029). Probabilities of being
drug-free at 3 months were 62.9 % and 3.7 %,
respectively (P < 0.001); 50 % of RTX cases were
P. Niaudet and O. Boyer
in stable remission without drugs after 9 months.
The authors concluded that rituximab and lower
doses of prednisone and calcineurin inhibitors are
non-inferior to standard therapy in maintaining
short-term remission in children with INS depen-
dent on both drugs and allow their temporary
withdrawal.
Iijima et al. performed a multicentre, double-
blind, randomized, placebo-controlled trial
involving 48 patients with complicated frequently
relapsing or steroid-dependent nephrotic syn-
drome who received rituximab (375 mg/m2) or
placebo once weekly for 4 weeks [258]. All
patients received standard steroid treatment for a
relapse at screening and stopped taking immuno-
suppressive agents by 169 days after randomiza-
tion. The median relapse-free period was
significantly longer in the rituximab group
(267 days, 95 % CI 223–374) than in the placebo
group (101 days, 70–155; hazard ratio: 0.27,
0.14–0.53; p < 0.0001). The rate of relapse by
day 85 was 42 % in the rituximab group and 83 %
in the placebo group, and all patients in the trial
had relapsed by 19 months.
Indeed, a significant proportion of patients
relapse after rituximab administration. Most
relapses occur simultaneously with the recovery
of B-cell lymphocytes. Maintenance therapy with
MMF has been shown to be effective in
preventing relapse after treatment with rituximab
[259]. Fujinaga et al. found that cyclosporine was
even more effective than MMF for maintaining
remission after a single infusion of RTX [260].
Although rituximab is well tolerated in most
patients, it may be associated with adverse effects
including infusion-related reactions (hypotension,
fever, skin rash, diarrhea, and bronchospasm).
Patients may develop serious infections second-
ary to leukopenia and/or hypogammaglo-
bulinemia. Patients with minimal change disease
have depressed serum IgG levels. This is more
pronounced during relapses, but persists during
remission [261]. It was recently reported that
rituximab prolongs this IgG depletion [262]. Sev-
eral cases of progressive multifocal leukoence-
phalopathy caused by JC polyomavirus have
been reported in patients with hematological dis-
orders or lupus. In addition, there is one published
28 Idiopathic Nephrotic Syndrome in Children: Clinical Aspects
case report of death due to lung fibrosis [263] and
another one of severe myocarditis requiring heart
transplantation [264] in two children with
nephrotic syndrome treated with rituximab. Addi-
tional severe adverse effects reported in childhood
nephrotic syndrome include Pneumocystis carinii
pneumonia [134, 265] and severe immune-
mediated ulcerative gastrointestinal disease
[266]. These life-threatening complications may
be underestimated in the literature. Moreover, the
long-term immunological consequences of
repeated rituximab infusions in children are not
known.
The KDIGO Glomerulonephritis Workgroup
suggested that rituximab be considered only in
children who have continuing relapses despite
optimal combinations of prednisone and steroid-
sparing agents or in patients who had developed
calcineurin inhibitor nephrotoxicity [267]. Well-
defined randomized control studies are mandatory
to examine which drug has the best risk/benefit
profile and to better define the place of rituximab
in the treatment of childhood steroid-sensitive
nephrotic syndrome [268], knowing that
rituximab does not cure the disease and repeated
courses may be necessary [269].
Long-Term Outcome of Children
with Steroid-Sensitive Nephrosis
Steroid-dependent patients may have a prolonged
course. However, if the patient continues to
respond to steroids, the risk of progression to
chronic renal failure is minimal.
Schärer and Minges found that 22 % of patients
had only one attack and 35 % of the relapsing
patients continued to relapse after 10 years
[270]. Trompeter et al. reported the late outcome
of 152 children steroid-responsive nephrotic syn-
drome after a follow-up of 14–19 years:
127 (83 %) were in remission, 4 had hypertension,
10 were still relapsing, and 11 had died [271]. The
duration of the disease was longer in children who
had started before the age of 6 years. Wynn
et al. found that 15 % of 132 patients had a
persistent relapsing course with a mean follow-
up of 27.5 years [272]. Lewis et al. reported on
863
26 patients over the age of 20 years, of whom
5 were still relapsing in adulthood [273].
Koskimies et al. reported on the follow-up of
children with INS observed in Finland from 1967
to 1976: 94 of 114 cases had responded to corti-
costeroids. Twenty-four percent of steroid
responders had no relapse, 22 % had infrequent
relapses, and 54 % had frequent relapses. More
than two-thirds were in remission at time of report
[274]. None of these patients developed renal
insufficiency, and none died from the disease.
Lahdenkari et al. reported a 30-year follow-up of
the patients reported previously by Koskimies
et al. [275]. Of 104 patients, 10 % had further
relapses in adulthood.
Fakhouri et al. reported on the outcome in
adulthood of 102 patients born between 1970
and 1975 [187]. Forty-two percent presented at
least one relapse in adulthood. A young age at
onset and a high number of relapses during child-
hood were associated with a high risk of relapse in
adulthood. Similarly, Ruth et al. in a study of
42 patients found that 14 (33 %) relapsed in adult-
hood [276]. The higher relapse rates in these two
reports probably reflect patient selection with
more steroid-dependent cases compared to
Koskimies’ series.
This generally good long-term prognosis is
also observed in patients with steroid-responsive
focal and segmental glomerulosclerosis: the
19 children reported by Arbus et al. remained
responders, and none had renal insufficiency
after a mean follow-up of 10 years [277].
Treatment of Steroid-Resistant INS
Resistance to steroid therapy is defined by the
absence of remission after 1 month of daily pred-
nisone therapy at a dose of 60 mg/m2/day
[161]. Some authors continue the treatment for
1 or 2 weeks, while others favor a series of three
methylprednisolone pulses (1,000 mg/1.73 m2)
every other day [163]. Indeed, the side effects of
this regimen are less than those induced by an
increase in the daily prednisone dose. Persistence
of proteinuria 1 week after this treatment defines
steroid resistance. A recent report found that
864 P. Niaudet and O. Boyer

prednisone, 60 mg/m2/day, 4 weeks

+ 3 methylprednisolone pulses

Persistent nephrotic syndrome

Renal biopsy

Cyclosporine or tacrolimus + prednisone

Complete remission No response

stop cyclosporine or tacrolimus and prednisone

No relapse Relapse
often steroid
responsive
Symptomatic therapy MMF + MP pulses?
(ACEI, statins) IV cyclophosphamide?
Rituximab?
Galactose?

Fig. 10 Proposed algorithm for the treatment of steroid-resistant idiopathic nephrotic syndrome

children with initial steroid resistance show
decreased glucocorticoid receptor expression in
peripheral blood mononuclear cells before
starting therapy compared to steroid-responsive
children. This low expression may be one of the
pathophysiological mechanisms of steroid resis-
tance in children [278].
Overall, the prognosis of steroid-resistant idi-
opathic nephrotic syndrome is poor, with a high
proportion of children progressing to end-stage
renal failure. This explains that intensive treat-
ment regimens have been tried. The results of
immunosuppressive treatments should take into
account the fact that children with genetic forms
of INS most often fail to respond to any therapy.
However, many published trials include patients
who had not been tested for mutations in the
different genes involved in steroid-resistant INS.
Moreover, most studies are nonrandomized and
include a small number of patients.
The interpretation of treatment is also compli-
cated by the fact that many studies include
patients with minimal change disease, diffuse
mesangial proliferation, and FSGS, while others
only include patients with FSGS.
The treatment of these patients remains diffi-
cult. Several therapeutic options have been pro-
posed (Fig. 10). The results of these treatments are
difficult to analyze, as a significant proportion of
the patients progress to cure gradually, with a slow
decrease of proteinuria, and in such cases,
relapses are rare.
Pulse Methylprednisolone
Mendoza et al. have proposed methylpredniso-
lone pulse therapy. It consists of methylpredniso-
lone (30 mg/kg intravenously), administered
every other day for 2 weeks, weekly for
8 weeks, every other week for 8 weeks, monthly
for 9 months, and then every other month for
6 months in association with oral prednisone
and, if necessary, cyclophosphamide or
chlorambucil [279]. At an average of over
6 years of follow-up, 21 of 32 children were in
complete remission, and the 5-year incidence of
end-stage renal disease was approximately 5 %
versus 40 % in historical controls [280]. Two pub-
lications reported similar results [281, 282].
Although these results are better than those seen
with any other therapy, other reports described
less favorable results [283, 284].
Calcineurin Inhibitors
A combination of calcineurin inhibitor with
low-dose steroid therapy for at least 6 months is
presently the best option as a first-line therapy.
28 Idiopathic Nephrotic Syndrome in Children: Clinical Aspects
Cyclosporine
The rate of complete remission is significantly
higher when cyclosporine is given in combination
with steroids [230].
The French Society of Pediatric Nephrology
reported the results of a prospective trial including
65 children treated with cyclosporine, 150–200
mg/m2, and prednisone, 30 mg/m2/day for
1 month and on alternate days for 5 months there-
after [285]. Twenty-seven patients (42 %) went
into complete remission (48 % of those with min-
imal changes and 30 % of those with FSGS) and
four (6 %) partial remission, whereas 34 (52 %)
failed to respond to the combined treatment. The
remission rate was higher in patients who had
initially responded to steroids but had developed
late steroid resistance (71 %) compared with ini-
tially steroid nonresponders (33 %). Interestingly,
eight patients who relapsed after cyclosporine
treatment responded to steroid and experienced a
steroid-dependent course. Progression to chronic
or terminal renal failure was observed only in
patients who had had a partial remission (1 patient)
or in those who did not respond to the treatment
(12 patients including 5 with minimal change
disease and 7 with FSGS).
Ingulli et al. reported that prolonged cyclospor-
ine treatment in children with steroid-resistant
FSGS reduces proteinuria and blunts the progres-
sion to end-stage renal failure [286]. The dose of
cyclosporine (4–20 mg/kg/day) was titrated to the
serum cholesterol level to achieve a remission. In
this study, only 5 of the 21 treated patients (24 %)
progressed to ESRF compared to 42 of 54 patients
from an historical group who had not received this
treatment. Gregory et al. treated 15 children with
steroid-resistant INS with an association of mod-
erate doses of cyclosporine and prednisone. They
observed a remission in 13 children after a mean
duration of treatment of 2 months [287]. Singh
et al. reported the effect of cyclosporine in 42 chil-
dren with steroid-resistant FSGS [288]. The mean
proteinuria decreased from 7.1 to 1.8 g/day, while
the serum albumin increased from 2.1 to 3.5 g/dl.
The mean serum creatinine increased from
0.85 to 1.26 mg/dl. Twenty-five patients achieved
complete remission. Ehrich et al. reported a retro-
spective study including 25 children with steroid-
865
resistant FSGS who received prolonged and inten-
sified treatment with combined cyclosporine and
steroids including methylprednisolone pulses.
This treatment resulted in sustained remission in
84 % of children with non-genetic forms of
steroid-resistant INS [289].
Three randomized trials involving 49 children
showed that complete remissions and partial
remissions were observed in 31 % and 38 % of
patients which was significantly higher than in the
control arms [290–292].
Tacrolimus
There is evidence from case series that tacrolimus
is effective in children with steroid-resistant INS
[293–295]. Gulati et al. found that tacrolimus and
prednisone are more effective than cyclophospha-
mide pulses in children with steroid-resistant
nephrotic syndrome, including those with mini-
mal change disease [296]. Tacrolimus was found
to be as effective as cyclosporine in a randomized
trial involving 41 children [297]. Both groups
received alternate-day steroids and enalapril. The
rate of remission at 6 months was 85.7 % with
tacrolimus and 80 % with cyclosporine. Interest-
ingly, the proportion of patients with relapses was
significantly higher in the cyclosporine group.
Conversely, Wang et al. found that tacrolimus
was associated with higher efficacy and lower
renal toxicity in comparison to CsA, with no dif-
ference in the rate of relapse [298].
Alkylating Agents
Although alkylating agents have little therapeutic
effect in steroid-resistant FSGS, for unknown rea-
sons, they are still widely used either alone or in
combination with corticosteroids. The Interna-
tional Study of Kidney Disease in Children
reported on 60 children with steroid-resistant
FSGS who were randomly allocated to receive
either prednisone 40 mg/m2 on alternate days for
12 months (control group) or cyclophosphamide,
2.5 mg/kgBW for 3 months plus prednisone
40 mg/m2 on alternate days for 12 months
[299]. Complete remissions were observed in
28 % of children in the control group and in
25 % of children who received cyclophospha-
mide. The authors concluded that there was no
866
beneficial effect of cyclophosphamide in these
patients. Rennert et al. treated ten children with
steroid-resistant FSGS with cyclophosphamide
pulses. Only two of the five initial nonresponders
went into remission, whereas all five late nonre-
sponders achieved complete remission [300]. In a
prospective study of 24 patients, Bajpai et al. also
found that therapy with intravenous cyclophos-
phamide had limited efficacy in patients with ini-
tial corticosteroid resistance, while sustained
remission was likely to occur in patients with
late resistance and those with absence of signifi-
cant tubulointerstitial changes on renal histology
[301]. Intravenous cyclophosphamide pulses
were found to be more effective than oral cyclo-
phosphamide in children with steroid-resistant
minimal change disease. In this trial, patients
receiving cyclophosphamide pulses had more
sustained remissions despite lower cumulative
dose [302]. Another study found that finally,
Gulati et al. found that tacrolimus and predniso-
lone were superior to IV cyclophosphamide and
prednisolone with a rate of complete remission of
52.4 % and 14.8 %, respectively [296].
Mycophenolate Mofetil
There is no convincing data for the beneficial
effect of mycophenolate mofetil in children with
steroid-resistant FSGS. Mendizabal et al. treated
five patients, and only one achieved complete
remission [250]. Mycophenolate mofetil in asso-
ciation with methylprednisolone pulses and
angiotensin-converting enzyme inhibitors was
reported to significantly reduce proteinuria
[303]. Another study involving 52 patients found
a rate of complete remission of 23 % and partial
remission of 35.5 % following therapy with MMF
[304]. A prospective trial of the NIH compared
cyclosporine with low-dose alternate-day predni-
sone to a combination of oral pulse dexametha-
sone and mycophenolate mofetil [305]. Partial or
complete remission was achieved in 22 of the
66 patients in the mycophenolate/dexamethasone
group and 33 of the 72 cyclosporine-treated
patients at 12 months. Thus, this study did not
find a difference in rates of proteinuria remission
following 12 months of cyclosporine compared to
mycophenolate/dexamethasone, although
P. Niaudet and O. Boyer
cyclosporine was more effective in reducing pro-
teinuria. The authors concluded that the small
sample size might have prevented detection of a
moderate treatment effect.
Rituximab
At present, there is no evidence that rituximab is
effective in patients with steroid-resistant
nephrotic syndrome, although retrospective case
series report that treatment with rituximab is effec-
tive in some patients [255, 306–310]. Bagga
et al. reported three complete and two partial
remissions in five patients receiving rituximab
[306]. Kamei et al. treated ten steroid and
calcineurin inhibitor-resistant patients with addi-
tional rituximab. Seven patients achieved com-
plete remission, one partial remission, and two
failed to respond. The seven patients with com-
plete remission preserved normal renal function
without proteinuria [308]. However, subsequent
reports did not confirm such good results [307,
311–313]. Gulati et al. reported that rituximab had
induced a complete remission in 27 % of 33
steroid-resistant patients and a partial remission
in 21 % of them. The rate of remission was
higher in patients with minimal changes on renal
biopsy and in patients who were late nonre-
sponders [307]. Magnasco et al. reported the
results of an open-label randomized trial including
31 children aged 2–16 years. All received
calcineurin inhibitors and prednisolone and 16 of
them received in addition two rituximab infu-
sions. Proteinuria remained unchanged in these
patients, and none entered partial or complete
remission [312].
Galactose
Savin et al. showed that galactose binds to the
focal segmental glomerulosclerosis permeability
factor and that oral administration of galactose
decreased the activity of the plasma permeability
factor [314]. Following this observation, De Smet
et al. reported the remission of proteinuria in a
patient with a nephrotic syndrome resistant to
corticosteroids, immunosuppression, and plasma-
pheresis [315]. However, Sgambat et al. did not
observe a decrease of proteinuria in seven chil-
dren with steroid-resistant nephrotic syndrome
28 Idiopathic Nephrotic Syndrome in Children: Clinical Aspects
despite a reduction of the plasma permeability
factor activity [316].
Nonsteroidal Anti-inflammatory Drugs
These drugs may decrease proteinuria. Several
authors have used indomethacin in the treatment
of INS with variable results. Donker et al. found a
reduction of proteinuria in patients with FSGS but
with a simultaneous reduction of glomerular fil-
tration rate [317]. Velosa et al. also found a clear
reduction in proteinuria in patients treated with
meclofenamate [318].
The detrimental effect of nonsteroidal anti-
inflammatory agents on renal function is well
known, and patients with renal disease seem more
vulnerable [319, 320]. Positive sodium balance,
increased edema, and risk of arterial hypertension
are recognized complications. The decrease of glo-
merular filtration rate observed with nonsteroidal
anti-inflammatory drugs is usually reversible in
salt-sodium-depleted patients. However, irrevers-
ible renal failure has been reported with a high
incidence in a prospective study in children with
steroid-resistant FSGS [321].
Angiotensin-Converting Enzyme
Inhibitors
Numerous studies in adults have demonstrated
that angiotensin-converting enzyme (ACE) inhib-
itors and angiotensin II receptor blockers (ARBs)
slow the rate of progression of proteinuric chronic
renal disease. Captopril was reported to decrease
dramatically nephrotic range proteinuria due to
renovascular hypertension and secondary FSGS
[322]. A decrease in proteinuria, and even com-
plete remission, has also been observed in patients
with chronic glomerulopathies with or without
hypertension [323]. A 50 % decrease of protein-
uria without a concomitant decrease in glomerular
filtration rate was reported in children with
steroid-resistant INS [324]. Marked benefits with
an ACE inhibitor and/or ARB therapy, plus
mycophenolate, were observed in nine children
with steroid-resistant FSGS [303]. At 6 months,
mycophenolate plus angiotensin blockade
resulted in a 72 % decrease in proteinuria from
baseline values, a benefit that was maintained for a
minimum period of 24 months.
867
Lipid-Lowering Agents
Hyperlipidemia has been shown in experimental
animals to accelerate the progression of glomeru-
lar sclerosis. Controlled trials in adult patients
have shown that lipid-lowering agents can prevent
the decline of renal function. No such studies have
been performed in children.
Recurrence of Proteinuria After Renal
Transplantation
The major problem of patients with INS who
progress to end-stage renal failure is the risk of
recurrence of proteinuria after renal transplanta-
tion. In children, recurrence of proteinuria occurs
in most cases within the first hours or the first days
after transplantation. A high proportion of patients
with immediate recurrence show delayed graft
function [325, 326]. In some patients, proteinuria
recurs several months later. Proteinuria is most
often associated with a nephrotic syndrome.
Transplant biopsy when performed early shows
minimal glomerular changes with foot process
fusion [327, 328]. Podocyte foot process efface-
ment can be observed within minutes after reper-
fusion and correlates with proteinuria [329].
Lesions of FSGS appear after several days or
weeks, which is a strong argument to consider
these lesions as secondary rather than the cause
of heavy proteinuria [327]. Therefore, it is more
appropriate to speak of recurrence of nephrotic
syndrome rather that recurrence of FSGS.
The overall risk of recurrence is estimated to be
between 20 % and 30 % [330]. The risk is higher in
children compared to adults [331]. Initial steroid
sensitivity in children with steroid-resistant
nephrotic syndrome is associated with a higher risk
of recurrence. In a large series of children without
genetic or familial steroid-resistant nephrotic syn-
drome, 26 of 28 late nonresponders (92.9 %) expe-
rienced recurrence versus 26 of 86 initial
nonresponders (30.2 %) [332]. In children, recur-
rence is more frequent when the disease has started
after the age of 6 years than before [333, 334].
The most likely explanation is that the incidence of
genetic forms of steroid-resistant INS is higher in
younger children and these patients have a very low
868
risk of recurrence. A rapid progression of the disease
to end-stage renal failure is a major factor associated
with recurrence: in most series, when the duration of
disease has been shorter than 3 years, the nephrotic
syndrome recurs in half of the patients [333, 335].
Recurrence is less frequent in African-Americans
than in whites and Hispanics [330]. The histopatho-
logical pattern observed on the first biopsy during
the course of the disease is also an important predic-
tive factor [331, 333, 336, 337]. Recurrence occurs
in 50–80 % of patients in whom initial biopsy
showed diffuse mesangial proliferation but in only
25 % of patients with minimal changes on first
biopsy. Most patients who experience a recurrence
in a first graft also show recurrence in a second graft
[326, 338, 339]. Conversely, when the graft has been
lost due to rejection without recurrence, a second
graft can be performed safely as recurrence is excep-
tional in this setting [343]. Donor type (living
or deceased donor) does not change recurrence
risk [340].
Recurrence increases the risk of delayed graft
function and early and late graft losses. Graft
failure occurs in about 50 % of patients with
recurrence [330, 341, 342]. However, some
patients may show good renal function for several
years despite persistent nephrotic syndrome.
The beneficial role of cyclosporine in recurrent
steroid-resistant INS is still debated. There is no
evidence that cyclosporine can prevent the recur-
rence of nephrotic syndrome following transplan-
tation [343–348]. Following the introduction of
cyclosporine, the incidence of recurrence did not
change, but graft survival was improved. In
patients who have recurrent disease, high doses
of oral cyclosporine or intravenous cyclosporine
may be effective [349–353]. This beneficial effect
may be related to the immunosuppressive effect of
cyclosporine and also to the direct effect of cyclo-
sporine on podocytes by blocking calcineurin-
mediated dephosphorylation of synaptopodin
and the resulting stabilization of actin [354]. In
our group, 17 children with recurrence were
treated with intravenous cyclosporine at an initial
dose of 3 mg/kg/day, which was afterward
adapted in order to maintain permanent whole
blood levels between 250 and 350 ng/ml. In
14 of the 17 cases (82 %), proteinuria completely
P. Niaudet and O. Boyer
disappeared after 20.8  8.4 days (range: 12–40
days). Persistent remission was observed in
11 patients with a follow-up of 3.7  3 years.
Actuarial graft survival was 92 % and 70 % at
1 and 5 years [352].
Most patients with recurrence are treated with
plasma exchanges or immunoabsorption. In a
review of the literature, Ponticelli found that
49 of 70 children who had received plasma
exchanges had entered complete or partial remis-
sion of proteinuria [355–361]. The best results are
observed when plasma exchanges are performed
early after recurrence. Complete and sustained
remission was observed in nine of ten adult
patients treated with high dose of oral steroids,
14 days of intravenous cyclosporine, and plasma
exchanges for 9 months [362]. A few studies of
preemptive plasma exchanges have been reported
that show inconstant efficacy.
Nozu et al. first reported the case of a child with
posttransplant lymphoproliferative disease in
whom rituximab induced a complete remission
of a recurrent nephrotic syndrome [363]. Pescovitz
et al. reported the case of a child with recurrent
FSGS resistant to plasma exchanges and cyclo-
sporine who developed at month 5 a posttrans-
plantation lymphoproliferative disease and had a
complete remission of proteinuria following six
infusions of rituximab [364]. Other reports con-
firmed the beneficial effect of rituximab given
alone or more often with plasma exchanges
[365–368], but failures have also been reported
[369, 370].
Conclusion
Most children with idiopathic nephrotic syndrome
respond to corticosteroid therapy. Although about
half of them experience relapses requiring long-
term steroid therapy and several corticosteroid-
sparing agents, these children maintain normal
renal function, and the severity of the disease is
mainly related to the complications of the treat-
ments needed to maintain remission.
There is evidence to suggest that minimal
changes and FSGS represent a spectrum of dis-
eases with FSGS at the severe end [367]. Indeed,
28 Idiopathic Nephrotic Syndrome in Children: Clinical Aspects
several disease entities are included in the term
“steroid-resistant INS.” Defects in podocyte pro-
teins have been identified in some cases, and these
patients do not respond to any therapy. In other
patients, circulating factor(s) produced by
immune cells increase the glomerular basement
membrane permeability to proteins. More than
50 % of steroid-resistant patients progress to
end-stage renal failure. Recurrence of the
nephrotic syndrome occurs in 30–40 % of them
after renal transplantation.
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 Immune-Mediated Glomerular
Injury in Children 29
Michio Nagata

Contents Growth Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 914

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 884
Glomerular Components . . . . . . . . . . . . . . . . . . . . . . . . . . . 885
The Glomerular Basement Membrane (GBM) . . . . . . 887
Mesangial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 887
Podocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 889
Endothelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 890
Parietal Epithelial Cells (PECs) . . . . . . . . . . . . . . . . . . . . . 891
Immune System and Animal Models . . . . . . . . . . . . . 893
Platelet-Derived Growth Factor (PDGF) . . . . . . . . . . . . 914
Transforming Growth Factor (TGF) . . . . . . . . . . . . . . . . . 914
Fibroblast Growth Factor (FGF) . . . . . . . . . . . . . . . . . . . . . 915
Vascular Endothelial Growth Factor (VEGF) . . . . . . . 915
Connective Tissue Growth Factor (CTGF) . . . . . . . . . . 915
Epidermal Growth Factor (EGF) . . . . . . . . . . . . . . . . . . . . 916
Platelets and the Coagulation Cascade . . . . . . . . . . . 916
Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 916
Fibrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 917

Antibody-Mediated Immunity . . . . . . . . . . . . . . . . . . . . . 894 Tissue Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 918

Circulating Immune Complexes . . . . . . . . . . . . . . . . . . . . . 894 The Enzymatic Fibrinolysis System . . . . . . . . . . . . . . . . . 918
In Situ Immune Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . 896 Eicosanoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 919
Cell-Mediated Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . 896 Reactive Oxygen Species (ROS) . . . . . . . . . . . . . . . . . . . 921
T-Cell Component of Adaptive Nitric Oxide (NO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 922
Immune Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 897
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 922
Inflammatory Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 899
Leukocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 899
Polymorphonuclear Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 900
Monocytes/Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . 901
Mast Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 904
Toll-Like Receptors (TLRs) . . . . . . . . . . . . . . . . . . . . . . . . . 904
Complement Cascade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 905
Soluble, Secreted Peptides . . . . . . . . . . . . . . . . . . . . . . . . . 909
Interleukins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 909
Interferons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 911
Tumor Necrosis Factor (TNF) . . . . . . . . . . . . . . . . . . . . . . . 911
Chemokines and Their Receptors . . . . . . . . . . . . . . . . . . . 911

M. Nagata (*)
Graduate School of Comprehensive Human Sciences,
University of Tsukuba, Tsukuba, Japan
e-mail: nagatam@md.tsukuba.ac.jp

# Springer-Verlag Berlin Heidelberg 2016 883

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_25
884 M. Nagata

Overview components and the subsequent activation of sec-

ondary inflammatory mediators. The glomerular
Glomerular injury is the central basis of renal deposition of antibodies is promoted by the
insufficiency, and immunologic mechanisms humoral immune process of Th2 activation, which
play critical roles for initiation and progression leads to antibody production via B-lymphocyte
of glomerular diseases. activation. This deposition may be composed of
Two axes of adaptive immune system involve preformed immune complexes in the circulation,
the pathogenesis of glomerular injury: antibody- which are trapped in the glomerulus, or antibodies
mediated and cell-mediated immunity. In children, (autoantibodies) that bind to intraglomerular anti-
popular immune-mediated glomerular diseases gens or interact with intrinsic glomerular cell-
include poststreptococcal acute glomerulonephri- surface antigens, as a result of changes to the cell
tis, IgA glomerulonephritis, membranoproli- surface (in situ immune complex).
ferative glomerulonephritis (type 1), membranous Antibody deposition on glomerular capillary
glomerulonephritis, ANCA-related glomerulone- walls or the mesangium activates the complement
phritis, and lupus nephritis, all of which reveal cascade, and intrinsic cell-derived chemotactic
various autoimmune features (Table 1). Although factors result in inflammatory cell influx, which is
ANCA-related glomerulonephritis is caused by a crucial event in the glomerular injury by produc-
Th1-predominant T-cell-mediated immunity in a tion/activation of proinflammatory cytokines,
pauci-immune fashion, remainders are thought to chemokines, growth factors, reactive oxygen species
be processed by antibody-mediated immunity. (ROS), eicosanoids, and nitric oxides (NO). These
The major steps of antibody-mediated glomeru- secondary mediators not only stimulate intrinsic
lar injury are immune deposition on glomerular cells to proliferate and/or produce matrix materials,
but they also induce the synthesis of similar mole-
cules by intrinsic glomerular cells. The secondary
Table 1 Autoimmune features of various renal mediators involve severe endothelial injury which
diseases [1] potentially results in dysregulation of local coagula-
Disease Autoimmune features tion system, accompanied by exudation and the
Poststreptococcal GN Anti-C1q, IgG, AECA, anti- formation of cross-linking fibrins. These end
DNA, ANCA, PDI, C3Nef products of the extrinsic coagulation cascade also
IgA nephropathy Anti-glycan, endothelial cells, damage glomeruli, particularly tuft necrosis, the
mesangial cells, IgG
common forerunner of crescentic formation.
Anti-GBM nephritis Anti-GBM, ANCA (20 %)
ANCA-positive Anti-MPO, PR3, cPR3, NET,
An alternative immune mechanism is cell-
nephritis DNA, endothelial cells, mediated immunity, with Th1 activation promot-
LAMP2 ing cytotoxic glomerular injury by influx of
Lupus nephritis Anti-dsDNA, annexin, MPO, delayed-type hypersensitivity (DTH) effectors,
PR3, nucleosome, IgG, C1q, i.e., macrophages, T cells, and fibrin. Proliferative
cardiolipin, MBL, NET
glomerulonephritis in the absence of immune
MPGN I Anti-C3 Nef, C4Nef, C1q
complexes or antibodies suggests a role for cell-
MCD/FSGS None identified
Membranous Anti-PLA2R, DNA, NEP,
mediated immune injury. This is supported by the
nephropathy aldose reductase, SOD2 experiment showing that transfer of sensitized T
Dense deposit disease C3Nef, C4Nef, anti-CHF, cells successfully induced glomerular injury in the
factor B, C1q host. Cytokines secreted by DTH effectors are
C3 nephropathy C3Nef, anti-CHF considered to injure the glomerular cells. It is
AECA anti-endothelial cell antibody, PDI protein disulfide clear that the antibody- and cell-mediated immune
isomerase, NET neutrophil extracellular trap, LAMP2 systems are not independent and often interact in
lysosomal membrane protein 2, MBL mannose binding
lectin pathway, NEP anti-neutral endopeptidase, SOD2 glomerular injury. The complex mechanism of
anti-manganese superoxide dismutase, CHF complement immune-mediated glomerular injury is integrated
H factor (Modified by Ref. [1]) by a recent review by Couser (Fig. 1) [1].
29 Immune-Mediated Glomerular Injury in Children
Etiologic events
PAMPS/DAMPS
Complement TLRs, NLRs
Auto antibodies,
C5b-9 C5a antigen-antibody
complexes
Resident Circulating
glomerular cells inflammatory cells
Proteases Chemokines
Oxidants Growth factors
Cytokines Eicosanoids
Fig. 1 Schematic overview of both the innate and adap-
tive immune mechanisms that mediate tissue injury in
GN. Etiologic events expose immunostimulatory PAMPs
or DAMPs that activate both the innate (red) and the
adaptive (antigen specific, blue) immune systems, which
also interact with each other. Activation of the innate
immune system occurs immediately and involves TLRs
or NLRs on both circulating inflammatory cells and resi-
dent glomerular cells. TLR activation results in release of
inflammatory mediators that cause glomerular injury.
Some PAMPs and DAMPs can activate complement
directly through the innate immune system. TLRs are
also required to activate the adaptive immune system
through antigen-presenting cells that promote
This chapter provides a review of our basic
understanding of and updated information regard-
ing immune-mediated glomerular injury, with
emphasis on the pathogenic mechanism(s).
Although numerous scientific advances have
come out of animal experiments, it should be
kept in mind that experimental models of
immune-mediated glomerular injury are largely
relevant to the acute phases of anti-GBM antibody
nephritis, nephrotoxic serum nephritis, or anti-
Thy1 nephritis of mesangial proliferation. Fur-
thermore, the molecular profiles of the immune
systems of mice and humans are significantly
different. This review of our basic knowledge of
885
Antigen-
presenting cell
CD4 T cells
B cells
TREGs
TH1
TH2
TH17
Glomerular tissue injury
differentiation of CD4 helper cells, B cell activation, and
antibody production. Antibodies lead to circulating com-
plex trapping or in situ formation of immune complexes
that can activate both the TLR and complement compo-
nents of the innate immune system. Complement activa-
tion generates the chemotactic factor C5a that attracts
circulating inflammatory cells (including neutrophils, mac-
rophages, basophils, and natural killer cells), which release
mediators and damage glomeruli and C5b-9 that activates
resident glomerular cells to do the same. CD4 Th1 and Th2
cells cause tissue injury primarily thorough macrophages
and basophils, respectively, whereas Th17 cells can medi-
ate glomerular damage directly. CD4 regulatory cells
(Tregs) downregulate the adaptive immune response [1]
immune-mediated glomerulonephritis may help
to explain some aspects of the mechanism(s)
underlying chronic glomerulonephritis in
humans.
Glomerular Components
Glomerulonephritis arises from the responses of
intrinsic glomerular cells to inflammatory reac-
tions. Glomerular deposition, hypercellularity
(intrinsic and inflammatory cells), and capillary
destruction are typical features of glomerular
injury (Fig. 2). The biologic characteristics of
886
Fig. 2 Typical features of immune-mediated glomerular
injury seen in human biopsy samples by LM and IF. (a)
Mesangial proliferation in IgA glomerulonephritis (PAS
stain), (b) crescentic formation with tuft necrosis and fibrin
deposition in ANCA-related glomerulonephritis (PAM
the intrinsic glomerular cells are implicated in this
process and described here. Although the cellular
components of the glomerulus are anatomically
defined by three cell types, mesangial cells, endo-
thelial cells, and podocytes, parietal cells are also
M. Nagata
stain), (c) subendothelial immune deposition in lupus
nephritis (PAM stain), (d) granular and peripheral pattern
of IgG in membranous glomerulonephritis, (e) fringe pat-
tern of C3 deposition in MPGN, (f) subendothelial IgG
deposits in lupus nephritis
involved in glomerular pathology, particularly in
cellular crescent formation and adhesion (the
renal corpuscle comprises these four cell types).
In addition, the biochemical profiles of the extra-
cellular matrices, glomerular basement
29 Immune-Mediated Glomerular Injury in Children
membrane, and mesangial matrices are unique
and may be targets for antibody deposition, lead-
ing to effects on the corresponding intrinsic cells.
Ultrastructural analyses provide a good opportu-
nity to define the involvement of extracellular
matrices in glomerular injury.
The Glomerular Basement Membrane
(GBM)
The GBM, which is a central component of the
filtration barrier based on charge and size, is com-
posed of extracellular matrix materials, including
type IV collagen, nidogen (entactin), heparan sul-
fate proteoglycans (HSPG), laminin, and perlican.
GBM is synthesized by podocytes and endothelial
cells; however, the details of the formation of the
membrane from the individually synthesized pro-
teins remain unknown. Nevertheless, this unique
permeability system traps macromolecules and
antibodies and is responsible for their glomerular
deposition.
The GBM also contains potentially harmful
antigens. Pathogenic autoantibodies directed
against the C-terminal globular noncollagenous
domain 1 (NC1) of the α3-chain of type IV colla-
gen, which has a distinct GBM molecular compo-
sition, promote severe glomerulonephritis in
patients with Goodpasture syndrome by epitope-
specific autoantibody binding and effector T-cell
reaction (Fig. 3; [3]). Similar epitopes are targeted
by alloantibodies in a subset of patients with
Alport nephritis who have received allografts.
GBM is also the target of bioactive substances
released from inflammatory cells and intrinsic
cells during inflammation. GBM changes may
directly influence the physiologic stability of fil-
tration, thereby reducing the glomerular filtration
rate and causing proteinuria. Electron microscopy
has revealed GBM alterations in association with
immune deposits, and inflammatory cell influx
can directly alter the morphology of the GBM
(Fig. 4). Proteolytic enzymes derived from neu-
trophils may directly alter the GBM, resulting in
membranolysis, lamination, gaps, and edema
(typical features of GBM injury), which may
lead to urinary abnormalities. Immune deposition
887
on the GBM, as seen in membranous glomerulo-
nephritis and lupus nephritis, can also alter the
GBM by changing its barrier of charge and size
properties.
Mesangial Cells
The mesangium is the axis of the glomerulus,
holding the capillaries in a developed dense net-
work of matrix, which includes microfibrils and
fibronectin. Mesangial cells are derived from the
metanephric mesenchyme, and there is one or two
cells within each mesangial area. The biologic
properties of mesangial cells have some similari-
ties with vascular smooth muscle cells. Both cell
types show contraction and cell proliferation in
response to vasoactive substances and cytokines
in vitro.
The lack of intact GBM between endothelial
cells and the mesangium enables bone marrow-
derived inflammatory cells and macromolecules
to access the mesangium (Fig. 5). Early studies
showed that resident cells in the mesangium
expressed the phenotype of bone marrow cells
(Ia positive) in the normal state and that their
numbers were increased in glomerulonephritis.
GFP-tagged bone marrow cell transplantation in
rat anti-Thy1 nephritis, a model of mesangial pro-
liferative glomerulonephritis, has provided evi-
dence that bone marrow-derived cells are
responsible for mesangial hypercellularity.
The mesangial fluid pathway was discovered
in an experiment involving the ligation of lymph
vessels around the renal artery. This leaky barrier
and fluid pathway allow circulatory macromole-
cules, including antibodies, to be trapped in the
mesangium as mesangial deposits. The pheno-
types and behaviors of cells in the mesangium
are also modulated by the glomerular scaffold of
self-produced extracellular matrix materials.
In glomerular injury, deposition, cell prolifera-
tion, matrix production, and cell death occur in the
mesangium. Mesangial proliferation is a typical
feature of glomerular injury. Activation of cell
cycle molecules, as revealed by immunohisto-
chemical detection of proliferating cell nuclear
antigen or Ki-67, is detected in the mesangial
888 M. Nagata

Type IV collagen chains

Protomers
1 1
2
1
2 α1.α2.α1
3
3 4
5
α3.α4.α5
4
5
6
5
α5.α6.α5 5

6 7S NC1 Trimer

Auto-antibody

GPA

T A

α3 α5 I
P
S
C P V P
E L
GPA G Y
T
region S

α3.(IV) NC1

α3.α4.α5(IV)
Protomer

Fig. 3 Structure of Goodpasture antigen epitopes in col-
lagen type IV. (a, c) GP epitope is localized in the NC1
domain of α3(IV) chain of type IV collagen. In the GBM,
the α3(IV) chain is associated with the α4(IV) and α5
(IV) chains, forming protomers and networks. The GP
epitopes are cryptic within the α3
α4
α5(IV) NC1 hexamer GBM glomerular basement membrane) (Modified figure
and inaccessible for autoantibody binding unless the NC1
hexamer dissociates. Several hydrophobic amino acids
were found critical for the epitope of the immunodominant
GPA autoantibodies (inset), which appears to be seques-
tered by an interaction with an α5(IV) chain in the same
protomer (b). Linear staining pattern of IgG in the glomer-
ulus from a patient with GP syndrome (GP Goodpasture,
from Borza and Hudson [2] with permission)

cells in glomerulonephritis, indicating mesangial those produced by inflammatory cells, as well as

cell proliferation in situ.
Mesangial activation occurs though receptor-
mediated mechanism. Mesangial cell proliferation
is stimulated by various cytokines and growth
factors, including interleukins (ILs), PDGF, and
fibroblast growth factor (FGF). In addition, Fc
fragments of IgG and IgA bind the receptor in
mesangium and stimulate the cells. For example,
PDGF binds to a tyrosine kinase receptor, which
activates a number of downstream effectors,
including the Ras/MAPK pathways, to activate
various transcription factors (c-fos, c-myc, c-jun)
and proto-oncogenes. Activated mesangial cells
synthesize cytokines and growth factors similar to
various vasoactive substances, such as angioten-
sin, endothelin, and vasopressin. As soluble cyto-
kines and growth factors act in autocrine,
paracrine, and juxtacrine manners, mesangial
inflammatory cells effectively activate resident
cells, and both cell types accelerate cell prolifera-
tion and matrix production in situ. During active
glomerulonephritis with mesangial proliferation,
mesangial cells frequently express α-smooth mus-
cle actin (α-SMA), desmin, calponin, and matrix-
binding integrin receptors. This may not only be
an example of phenotypic changes in mesangial
cells reflecting cell activation, but may also repre-
sent involvement in further glomerular injury.
29 Immune-Mediated Glomerular Injury in Children
Fig. 4 Changes of filtration barrier in glomerulonephritis
by EM. (a) Lytic change in GBM is accompanied with
endothelial swelling with loss of fenestra on the back-
ground of inflammatory cells in the capillary. Note foot
process effacement in podocytes with actin accumulation
in podocyte. (b) Subepithelial dense deposits with
Mesangial proliferation is recognized as a fore-
runner of glomerulosclerosis, and several in vitro
experiments have revealed that mesangial cells
synthesize matrix components in response to var-
ious inflammatory cytokines and growth factors.
Hyperplastic mesangial cells reduce cell numbers
by apoptosis mediated via a variety of signaling
mechanisms.
Podocytes
Podocytes have many intrinsic properties
sustained by actin cytoskeleton and some unique
molecules that participate in the glomerular path-
ophysiology. Podocytes are located outside of
glomerular capillaries and do not affect directly
the bloodstream; instead, they face the primary
ultrafiltrate, which contains various biochemical
889
thickening and irregularity in GBM and foot process
effacement. (c) Thickening of GBM due to membranolysis
containing cellular debris, deposits, and cellular infoldings.
(d) Endothelial cell swelling and podocyte detachment
from GBM
substances. Podocytes are an important compo-
nent of the filtration barrier, in that they have slit
membranes and synthesize GBM components.
Podocytes are highly specialized postmitotic
cells that are derived from the metanephric mes-
enchyme. Indeed, podocytes have a negligible
capacity to proliferate, in that they undergo
nuclear division, but cytokinesis is not evident
in vivo. Actin dynamics in podocytes has been
intensively investigated, and its role for cell integ-
rity and function emerged. Podocytes are rich in
actin filaments, and the cytoskeletons of their
intermediate filaments are linked to foot process
maintenance and slit membrane function. Several
podocyte markers, such as WT1, CALLA, CD10,
synaptopodin, podocalyxin, podoplanin, and
nestin, have been identified. The slit membrane-
related molecules such as nephrin and podocin are
also sometimes used as specific markers.
890
m
m
pa
e
p
Fig. 5 Structure of the glomerulus and monocyte influx.
(a) Schematic presentation of the glomerular profile. The
glomerulus is composed of four cell types: mesangium (m),
endothelial cell (e), podocyte ( p), and parietal cell (pa).
In addition, the podocyte is a major source of
growth factors, such as VEGF, TGF-β, and
CTGF. Podocyte also expresses angiotensin II
type 1 receptor which mediates local renin-
angiotensin system for podocyte function.
Podocyte injury is the base of proteinuria, and
podocyte loss from GBM (podocytopenia) may
lead to glomerulosclerosis.
Immunologic mechanisms including GBM
deposition, complement activation, and inflam-
matory mediators involve podocyte damage, and
alteration of podocyte molecules results in pro-
teinuria and sclerosis. Podocyte injury is visible
by morphology. Electron microscopy reveals
cytoplasmic vacuolation, pseudocyst formation,
and foot process effacement with actin accumula-
tion and detachment. In addition, phenotypic
changes, namely, expression of desmin, also
represent podocyte injury in the rodents. These
changes are induced not only by hemodynamic
effects, but also by immune-mediated properties.
Membranous glomerulonephritis is an example of
immune-mediated podocyte injury, which results
from complement activation. The antigen
responsible for experimental membranous
glomerulonephritis (Heymann nephritis) is the
M. Nagata
m
p
e
Mf
m
Note lack of GBM between endothelial cell and
mesangium. Original illustration is courtesy from
Dr. W. Kriz with permission, (b) invasion of macrophages
(Mφ) into mesangium in the biopsy of in MPGN
podocyte-membrane protein megalin. Similarly,
the recently identified neutral endopeptidase
(NEP) and M-type phospholipase A2 receptor,
which are located in podocytes, are potentially
intrinsic antigens that form immune complexes
in situ that cause membranous glomerulonephritis
in humans [4, 5]. Podocytes express B7-1, which
is a costimulatory molecule for T cells that is
upregulated in injured podocytes and causes
dysregulation of the actin cytoskeleton, leading
to proteinuria [6].
Endothelial Cells
The glomerular endothelial cell is the only intrin-
sic glomerular cell type that directly faces the
circulation. These endothelial cells are different
from those in other organs because of their
extraordinary flatness and highly fenestrated fea-
tures, allowing effective filtration. The fenestra-
tion (70–100 nm in size) is maintained by vascular
endothelial growth factor A (VEGF-A), which is
derived from the podocytes. The endothelial cell-
surface layer provides a negatively charged mesh
of polysaccharide moieties of glycoproteins,
29 Immune-Mediated Glomerular Injury in Children
Table 2 Neutrophil endothelial cells express various adhesion molecules for interaction
Endothelial molecules Leukocyte molecules
P-selectin Sialyl-Lewis X-modified
protein
E-selectin Sialyl-Lewis X-modified
protein
GlyCam-1, CD34 L-selectin
ICAM-1 (immunoglobulin CD11/CD18 integrins
family) (LFA-1, Mac-1)
VCAM-1 VLS-4 integrin
(immunoglobulin family)
CD31 CD31
glycosaminoglycans (heparan sulfate, sialic acid,
chondroitin sulfate, hyaluronan) which determine
vascular rheology and are probably involved in
permselectivity. The charge and various adhesion
molecules on the cell surface permit interactions
between endothelial cells and blood-borne fac-
tors, including leukocytes, platelets, the third
component of complement, antigens, and immune
complexes (Table 2). Endothelial cells physiolog-
ically synthesize several coagulation proteins,
growth factors, extracellular matrix components,
and various substances, including nitric oxide,
prostaglandins, and endothelin, to maintain cell
integrity and the vascular environment.
Angiotensin-converting enzyme (ACE) and
endothelin-converting enzyme participate in the
local formation of bioactive angiotensin II and
mature endothelin, respectively.
Endothelial cells are involved in immune-
mediated glomerular injury in many aspects,
including characteristic cell surface which is a
feasible target of inflammatory reaction, expres-
sion of MHC proteins, and changes of variety of
bioactive/physiologic substances.
Endothelial cell proliferation, typically
observed in endocapillary proliferative glomerulo-
nephritis and preeclampsia (also known as
endotheliosis), is considered as post-injury regen-
erative responses. Since endothelial cells actively
regenerate and cell loss is replaced either by neigh-
boring cells or bone marrow-derived cells, endo-
thelial cell injury may be usually reversible.
Endothelial cell injury is also evidenced by fibrin
deposition, as seen in thrombotic microangiopathy.
An additional feature of endothelial injury is the
891
Major function
Rolling (neutrophils, monocytes, lymphocytes)
Rolling and adhesion PMNs, monocytes, T cells)
Rolling (neutrophils, monocytes)
Adhesion, arrest, transmigration (neutrophils,
monocytes, lymphocytes)
Adhesion (eosinophils, monocytes, lymphocytes)
Transmigration (all leukocytes)
double contour of the GBM, as typically seen in
transplant glomerulopathy. The mechanism of this
humoral-mediated rejection is unclear, but MHC
class II antigens on the endothelial surface interact
with anti-donor antigens and form an in situ
immune complex that persistently activates or
injures endothelial cells, resulting in matrix pro-
duction and subendothelial widening. These fea-
tures have also been noted in MPGN, in which
persistent complement activation is the trigger for
endothelial cell injury.
Parietal Epithelial Cells (PECs)
Parietal cells, also known as Bowman’s epithe-
lium, are located in the continuous epithelial lin-
ing between the podocytes and proximal tubular
cells. PECs and podocytes share a common phe-
notype during glomerular differentiation until the
S-shaped body stage. Although PECs seem to
play a role in glomerular injury, little is known
about their biologic and physiologic properties.
PECs express receptors for PDGF and FGF, and
their ligands derived from inflammatory or intrin-
sic cells can stimulate cell proliferation and matrix
synthesis. PECs are the major constituent of the
cellular crescent, which expresses connective tis-
sue growth factor (CTGF) mRNA, suggesting a
growth factor-mediated mechanism for glomeru-
lar scar formation. An in vitro study revealed that
FGF-2 stimulated PECs proliferation and PDGF
stimulated matrix production in PECs in culture.
In addition, tissue factor or cross-linked fibrin
stimulates proliferation in PECs in vitro.
892 M. Nagata

a IL-4 IL-12 TGF-β

IL-6
IL-21

GATA3 T-bet RORγT Foxp3

STAT-6 STAT-4 STAT-3

IL-23

Th2 Th1 Th17 Treg

IL-17A, IFN-γ Tregs acquiring
producing affector function
IL-4 IFN-γ Th cell IL-17A IL-10
IL-5 TNF IL-17F TGF-β
IL-13 LT-α IL-21 LT-35
IL-22

lgE, neutralising lgG Macrophage activation Neutrophil activation Regulation of

Eosinophil activation Complement fixing lgG Direct tissue interactions Th subsets
High FcR affinity lgG

Parasitic infection Intracellular infection Extracellular infection Limiting tissue injury

Allergy Classical DTH Tissue injury Peripheral tolerance
Autoimmunity Autoimmunity Graft tolerance
Rejection Rejection

b APC
Th17

IL-23
IL-22

IL-17A, IL-17F

Neutrophil Direct tissue effects,

activation chemokine production
Macrophage
TNF, IL-1β

Fig. 6 (a) Simplified diagram of the Th1, Th2, Th17, and
Treg subsets, showing key cytokines produced by these
cells and the cytokines and transcription factors critical to
the development and expansion of these subsets. The bio-
logic functions of Th cell subsets are briefly summarized.
There is evidence that Th17 cells can acquire the capacity
to produce IFN-, and Tregs can, at sites of inflammation,
lose their Foxp3 expression and acquire effector function.
(b) Summary of potential mechanisms by which Th17
cytokines could mediate immune renal injury [9]
29 Immune-Mediated Glomerular Injury in Children
Table 3 Characteristics of T-helper subsets
Characteristics Th1 cells
Inducing stimuli IL-12, INF-γ, IL-18, IL-27, PRR signaling
Transcription factors STAT4, STAT 1, T-bet, NFκB
Cytokine produced INF-γ, IL-2, TNF, lymphotoxin-β
Chemokine receptor CXCR3, CCR5, CCR1
expression
Antibody isotypes Human: IgG1, IgG2, IgG3
Mouse: IgG2a, IgG2b, IgG3
Effector response Cell-mediated immunity, macrophage activation, antibody-
mediated cellular cytotoxicity
The experimental model of crescentic glomerulo-
nephritis has shown that PECs undergo an epithe-
lial mesenchymal transition, which is involved in
scar formation in glomerular crescents [7]. PEC
proliferation has also been demonstrated in
nonimmune-mediated glomerulonephritis, partic-
ularly in focal segmental glomerulosclerosis and
other immune-/nonimmune-mediated glomerulo-
sclerosis conditions. Nitch-1-mediated PEC
migration has shown to be a glomerular wound
healing process in response to severe injury of
filtration barrier [8]. Cell proliferation, matrix pro-
duction, and distortion of functional architecture
in PECs participate in the glomerulosclerosis by
immune or nonimmune mechanisms.
Immune System and Animal Models
Innate and adaptive immune systems are the path-
ogenic background of immune-mediated glomer-
ular injury. Although molecular and cellular
mechanism of immune system is substantially
complex, it may be convenient to follow the
mechanism of glomerular injury by two steps,
initiation and promotion. Lymphocytes mainly
drive an initiation, and neutrophils and mono-
cyte/macrophages largely participate in promo-
tion as secondary mediators.
Lymphocyte-mediated injury is processed by
antibody-mediated (B cell driven) or cell-
mediated (T cell driven) mechanisms, and they
usually interact and are synergistically involved
in the tissue damage. B-cell system promotes pro-
duction and in situ deposition of antibodies
(majority forms antigen-antibody complexes) in
893
Th2 cells
IL-4
STAT6, GATA3
IL-4, IL-5, IL-13
CCR3, CCR4, CCR8
IgG4, IgE
IgG1, IgE
Eosinophil
activation, allergy
the glomerulus. This initiation induces the sec-
ondary inflammatory mediators, including cyto-
kines and chemical mediators, derived from
activated leukocytes. T-cell-mediated glomerular
injury may include rather wide-ranged processes,
since T cell regulates B-cell system. However
when we concern about T-cell-dependent glomer-
ular injury as a narrow meaning, it is less com-
mon. One example of this mechanism is cytotoxic
action of T cells which may damage the intrinsic
glomerular cells or alter glomerular filtration bar-
rier, causing proteinuria. This complex immune
system profiled by T-cell subsets (Th1/Th2/Th17/
Treg) may determine the type of glomerular injury
(Table 3).
There are varieties of experimental models of
immune-mediated glomerulonephritis that have
been used to analyze the nephritogenic mecha-
nisms. Nephrotoxic serum nephritis, a classical
model initially established by Masugi in 1933, is
induced by injection of duck anti-rabbit kidney
antisera to rabbits resulting in crescentic glomer-
ulonephritis. Anti-GBM antibody nephritis is
induced by administration of anti-GBM IgG
showing proliferative (usually crescentic) glomer-
ulonephritis. Both are the basic and widely exam-
ined models of glomerulonephritis. There are two
phases of immune reaction in these models, het-
erologous and autologous phase. The former is
provoked by the binding of injected alloantibody
to GBM, whereas the latter is caused by a host-
produced autoantibody that binds to the GBM.
Heterologous phase alone causes mild and self-
limiting nephritis, but prolongation or accelera-
tion of autologous phase by pre-immunization
with IgG of immunized animals provokes severe
894
glomerulonephritis. Autoantibody may be impor-
tant to induce severer nephritis. Several mice
models of lupus, including (NZBXNZW) F1,
BXSB, and MRL/lpr mice, reveal spontaneous
and proliferative glomerulonephritis with immune
complex deposition, similar to the human lupus
nephritis. Heymann nephritis is a model of mem-
branous glomerulonephritis, and a responsible
antigen is identified as megalin. In this model,
complement activation plays significant roles for
proteinuria, and proliferative nephritis is not usu-
ally seen. All these models are immune complex-
mediated glomerulonephritis as revealed by
immunoglobulin and complement deposition,
regardless of whether circulating or in situ
immune complex formation. On the contrary,
widely used model of anti-Thy1 nephritis reveals
mesangial proliferative glomerulonephritis but
pauci-immune. In this model, antibody against
thymus immediately binds to receptor on
mesangial cells resulting in cell lysis
(mesangiolysis) associated with transient activa-
tion of complements; however, immune complex
and complements are lost immediately. Mesangial
proliferation in this model may reflect repair pro-
cess from mesangiolysis to reconstruct glomerular
architecture mimicking glomerulogenesis, rather
than the result of mitogenic stimuli of inflamma-
tory mediators (unlike immune-mediated glomer-
ulonephritis). Since majority of experimental
works using these models investigated an acute
phase of the diseases, it is necessary to understand
the pathogenetic difference in each model and
recognize the limitation of the results to translate
human chronic glomerulonephritis.
Antibody-Mediated Immunity
Antibody (humoral)-mediated immune glomeru-
lar injury is initiated by antigen-antibody complex
formation within the glomerulus. B-cell activation
by interleukins (IL-4, IL-5), which are synthe-
sized by antigen-specific CD4+ T cells, is a pre-
requisite for antigen-specific antibody production.
Many glomerular lesions result from the glomer-
ular binding of antibodies. Immune-mediated glo-
merular injury is seen in a wide spectrum of
M. Nagata
glomerular diseases, with various histologic pat-
terns, and the clinical outcomes may be related to
the nature of the antigen(s) and their glomerular
binding site(s).
Circulating Immune Complexes
If the immune system produces autoantibodies,
immune complexes can be formed. Circulating
immune complex-mediated glomerulonephritis is
primarily seen as a type III hypersensitivity reac-
tion (Fig. 7). As known in the serum sickness
disease model, high amount of foreign serum
injection results in the appearance of immune
complex in the circulation which accumulates in
the glomerulus and induces acute glomerulone-
phritis and vasculitis. Regardless of whether they
are endogenous or exogenous, the antigens are not
glomerular in origin. Exogenous antigens can be
bacterial (Streptococcus and Staphylococcus
spp.), viral (hepatitis B), parasitic (Plasmodium
spp.), or spirochete (Treponema spp.) in origin.
In contrast, DNA and histones are endogenous
antigens that are typically seen in lupus nephritis.
The highly permeabilized glomerular filter is
susceptible to passive trapping of preformed
immune complexes from the circulation. The
highly negative charge of the capillary wall and
endothelial glycocalyx surface also attracts circu-
lating macromolecules.
The biologic activities of circulating immune
complexes depend largely on the biochemical and
quantitative features of the circulating antigens.
The size of an immune complex is determined by
the ratio of the antigen to antibody. When the
antibody is present in excess, cross-linking of
immune complexes occurs, and the complex
becomes large. The primary site of such a large
immune deposition is either the subendothelium
or the mesangium and efficiently activates the
complement cascade by binding to Fc receptors
(the Arthus reaction), thereby further stimulating
the inflammatory processes. In contrast, when an
antigen bears few epitopes or when there is an
excess of antigen relative to antibody, the immune
complexes formed tend to be smaller in size and
soluble. These small immune complexes may be

Fig. 7 Mechanism of immune deposition and inflammatory reaction in the glomerulus.
(a) Circulating immune complex interaction with endothelium results in deposition in
GBM. (b) In case of anti-GBM antibody glomerulonephritis (Goodpasture syndrome),
fixed antigen is a component of GBM (NC1 domain of α3 (IV) chain of type IV
collagen). Thus antibody binds GBM and shows linear distribution. (c) In case of
membranous glomerulonephritis (Heymann nephritis), antigen is a podocyte compo-
nent (megalin). Antigen-antibody complex is formed in situ and complex shift to
29
Immune-Mediated Glomerular Injury in Children
accumulate at subepithelial area (d). Antigen-antibody complex in the circulation
deposits on the endothelial surface is an initiation of immune-mediated glomerular
injury. Local activation of complement system chemoattracts inflammatory cells
followed by secretion of secondary messengers which injure glomerular component
(The figure was published in Kumar et al. Robbins basic pathology, 8th edn. 2007,
p. 545 and 127, Copyright Elsevier)
895
896
deposited within the GBM or subepithelial areas,
but they often fail to induce inflammation,
because the deposition and complements are iso-
lated from circulation. When the antigen to anti-
body ratio nears equivalence, the immune
complexes formed tend to be of maximal size
and insoluble. The net charge of the complex is
an additional determinant of immune complex
deposition, based on the interactions with the neg-
atively charged GBM and endothelial surface.
The levels of circulating immune complexes
have been shown to correlate with glomerular
injury in lupus glomerulonephritis. The size and
biochemical characteristics of immune complexes
may change with disease activity or with thera-
peutic modulation. Indeed, immune complex
deposition in subendothelial or mesangial cells
occasionally shifts to the subepithelial cells in
lupus nephritis, such as Class IV to Class V trans-
formation. Acceleration of glomerular capillary
permeability in the process of glomerulonephritis
may be an alternative factor to change the site of
immune deposition. Although experimental
models suggest induction of glomerulonephritis
by glomerular immune complex deposition, it
remains unclear as to whether passive trapping
of preformed immune complexes alone is suffi-
cient to induce glomerulonephritis in humans.
In Situ Immune Complexes
In situ immune complex formation is basically the
result of the combination of unbound circulating
soluble antibody and antigen within the tissue. In
situ tissue antigens are either fixed (components
of the glomerulus) or planted (exogenous)
(Fig. 7). The best example of an in situ immune
complex disease is anti-GBM antibody-induced
glomerulonephritis (anti-GBM antibody nephritis).
In this condition, antibodies are directed against
a fixed antigen of a component of the GBM,
i.e., the noncollagenous domain of the α3 chain of
collagen type IV. These antibodies occasionally
cross-react with the lung alveolar basement mem-
brane, and the resulting simultaneous inflammation
of the kidneys and lungs represents a severe clinical
phenotype, known as Goodpasture syndrome.
M. Nagata
Another example of an in situ immune complex
disease is Heymann nephritis, which serves as a
model of membranous glomerulonephritis in
humans. Combine this model with isolated per-
fused kidney system provided first evidence for
in situ immune complex-induced glomerulonephri-
tis [10]. Autoantigenic target in this model is
516 kD protein megalin that binds receptor-
associated protein (RAP), a podocyte-membrane
glycoprotein [11]. In humans, megalin is not
expressed, but phospholipase A2 receptor has
been identified as the antigen in human membra-
nous glomerulonephritis [5].
In clinical practice, various antibodies are
detected in the blood, while circulating immune
complexes are generally undetectable. It seems
that in situ immune complex formation is the
distinct process for immune deposition in various
glomerular diseases in humans.
Cell-Mediated Immunity
Cell-mediated immunity can be summarized as
the T-cell-dependent interaction of antigen-
presenting cells and CD4+ helper T cells through
the T-cell receptor (TCR), which promotes cyto-
kine release and stimulates the proliferation and
activation of effector cells, macrophages, and
CD8+ cytotoxic T cells [12].
T-cell-mediated glomerular injury includes sev-
eral mechanisms, such as T-cell-mediated B-cell
activation, cytotoxic effects, and the direct actions
of lymphokines on the glomerular filtration barrier.
The first process is T-cell-driven antibody-mediated
immunity as described above, and the latter two
processes are narrowly defined, basically because
of the paucity of antibody deposition in situ.
Inappropriate T-cell responses may cause hyper-
sensitive immune reactions and provoke glomeru-
lonephritis. In humans, T-cell infiltration is
observed in the glomeruli in pauci-immune glo-
merular diseases, such as ANCA-related glomeru-
lonephritis, and therapeutic interventions suppress
glomerular inflammation and T-cell infiltration.
Anti-GBM antibody nephritis in Wistar Kyoto
rats revealed infiltration of CD8+ T cells in the
glomeruli, and suppression of circulating CD8+ T
29 Immune-Mediated Glomerular Injury in Children
cells resulted in decreased glomerular inflamma-
tion. However in mice model of anti-GBM anti-
body nephritis, glomerular injury is CD8+ T cell
independent. These observations provide circum-
stantial evidence to establish the basis of a T-cell-
dependent glomerular injury mechanism. Transfer
of rat CD4+ cell line specific for Col4α3NC1 suc-
cessfully induced severe nephritis without glomer-
ular IgG and C3 deposition, which may be the
direct evidence for T-cell-mediated glomerular
injury [13]. Conversely, T-cell-mediated injury
cannot be excluded, even when antibodies
(reflecting a B-cell response) are present.
T-Cell Component of Adaptive
Immune Response
The complex immunologic network-driven
immune-mediated glomerular injury involves the
primed CD4+ helper T-cell-derived subsets,
including Th1, Th2, Th17, and Treg subsets.
Th1 and Th2 cells can be distinguished based on
their cytokine (Th1; INF-γ, Th2; IL-4), chemo-
kine, and antibody isotype profiles and on which
generate different immune effector responses.
Th17 was the newly identified subset by expres-
sion of IL-17A and IL-17F. These cell types dif-
ferentiate from naïve precursors, “Th0” cells,
following specific antigen stimulation by pleiotro-
pic cytokines via their α/β T-cell receptors
(Fig. 8). Antigen-presenting dendritic cells in
humans are derived from monocytes or
plasmacytoid cells, with a bias towards the stim-
ulation of either INF-γ or IL-4, IL-5, and IL-10
production by naïve T cells, as demonstrated
in vitro (Table 2, Fig. 6). Although every pathway
may interact, the predominance of a particular
subset may explain the different patterns of glo-
merular histology and disease activity, which are
related to disease outcome [15].
Th1 development is promoted primarily by
INF-γ and IL-12, and partially by IL-18, IL-27,
and IL-23, indicating complex and overlapping
mechanisms for Th1 immunologic activities.
Th2 responses are driven by IL-4 and IL-10, and
they inhibit Th1 responses. IL-10 appears to be
more active in inhibiting Th1 differentiation than
897
in promoting Th2 responses. Differences in che-
mokine receptor expression between Th1 and Th2
cells also influence the activation patterns of these
cells by different chemokines. CXCR3, the recep-
tor for INF-γ-inducible chemokines, is expressed
at high levels on INF-γ-producing Th1 cells and at
low levels on Th2 cells. The CCR3 and CCR4
chemokine receptors are expressed by Th2 cells
that produce IL-4. Th17 cells are differentiated
from naïve CD4+ T cells by TGF-β and IL-6 and
produce IL-17 which promotes autoimmune dis-
ease, such as multiple sclerosis and rheumatoid
arthritis. Th17 cells also secrete IL-21, IL-22, and
IL-9.
The roles of Th1 and Th2 cells in glomerular
injury have been studied using different strains for
Th1 (mice: C57BL/6, rats: Lewis) and Th2 (mice:
BALB/c, rats: Brown Norway) dominance.
Induction of nephrotoxic serum nephritis in
Th1-predominant C57BL/6 mice results in severe
glomerular injury. Anti-GBM antibody nephritis
in Lewis (Th1) and Brown Norway (Th2) rats
resulted in more severe glomerulonephritis, with
monocytic and T-cell influx and Th1-profile cyto-
kine production, in Lewis rats than in Brown
Norway rats. Mice deficient in Th2 cytokines
show more pronounced crescentic glomerulone-
phritis, while the administration of Th2 cytokines
ameliorates this disease. Mercuric chloride-
induced autoimmune glomerulonephritis in
congenic strains of Brown Norway rats resulted
in a membranous-like form of glomerulonephritis.
These experiments indicate that Th1 predomi-
nance is related to crescentic glomerulonephritis
and that Th2 predominance tends to be involved
in membranous glomerulonephritis.
In human anti-GBM antibody nephritis, there
is an INF-γ-predominant, antigen-specific effector
cell response in the active stage and IL-10 pre-
dominance in remission. In humans, CD4+ cells
and macrophages are present in the cellular cres-
cents of crescentic glomerulonephritis. In ANCA-
related glomerulonephritis, a T-cell response with
Th1 predominance is involved. In the renal tissues
of ANCA-related glomerulonephritis, the level of
INF-γ mRNA is high, and the level of IL-4 mRNA
is low. Peripheral blood T cells show a high INF-γ/
IL-4 ratio in patients with ANCA-associated
898 M. Nagata

NaÏve CD4+ T cell

TGF-β

IFN-γ, IL-12 IL-4

IL-6, IL-23 Retinoic acid

STAT1+4 STAT6 STAT3 FoxP3

T-bet GATA3 RORγ t

Th1 Th2 Th17 Treg

Intracellular bacteria Helminth infections Extracellular Downregulation of
Cell-mediated Allergic disease bacteria, fungi the immune response
autoimmunity Cell-mediated in autoimmunity and
IL-4, IL-5, IL-13 autoimmunity infection
IFN-γ, TNF-α
TGF-β, IL-10, IL-35
IL-17, IL-17F, IL-21
IL-22, TNF-α

Fig. 8 Differentiation of CD4t T-cell subsets. After the
encounter of antigen and costimulatory molecules
presented by antigen-presenting cells, naive CD4t T cells
can differentiate into different subpopulations. The lineage
commitment of CD4t T cells depends on the cytokine
milieu accompanying the T-cell activation process in sec-
ondary lymphoid organs. The diverse T-helper cell
subtypes show differential activation of transcriptional
programs and are characterized by the secretion of certain
effector cytokines that enable them to provide protection
against different classes of pathogens and to mediate auto-
immunity. IFN-γ, interferon-γ; IL, interleukin; TGF-β,
transforming growth factor-β; Th1, T-helper 1 cell;
TNF-α, tumor necrosis factor-α [14]

glomerulonephritis, as compared with patients
with nonproliferative glomerulonephritis. Corti-
costeroids diminish this ratio, which suggests
that Th1 predominance is linked to ANCA-
associated glomerulonephritis in humans.
For full activation of CD4+ cells, engagement
of T-cell receptor with the antigenic peptide-MHC
molecule complex in antigen-presenting cells
needs secondary costimulatory signals for full
activation. T-cell costimulatory molecules include
CD28-B7 family/CD80 (B7-1) + CD86 (B7-2)
and CD40 (TNF receptor on B cells)/CD154
(ligand of CD40 on T cells) that determines Th1
and Th2 polarity. Protection of anti-GBM nephri-
tis in CD28 deficient mice is due to inhibition of
autologous antibody production [16]. Selective
blockade of B7-1 prevented the development of
crescentic glomerulonephritis in anti-GBM
nephritis in Wistar Kyoto (WKY) rats [17]. In
the same nephritis in mice, administration of
either CD80 or CD86 revealed no effect on
glomerulonephritis; however, administration of
anti-CD80/CD86 antibody attenuated glomerulo-
nephritis [18]. Anti-GBM antibody nephritis in
CD80 (B7-1)-deficient mice reveals attenuation
of crescentic glomerulonephritis in concert with
29 Immune-Mediated Glomerular Injury in Children
reduction of glomerular CD4+ cell accumulation.
By contrast, same nephritis in CD86 (B7-2)-defi-
cient mice shows disease exacerbation [19]. Thus
CD80 is pathogenic in crescentic glomerulone-
phritis by enhancing survival and proliferation of
CD4+ cells, whereas CD86 is protective by
enhancing Th2 and attenuating Th1 responses.
Inducible costimulatory molecule (ICOS) and
ICOS ligand (ICOSL) are a receptor-ligand pair
that belongs to the CD28/B7 family. Administra-
tion of antibody against ICOSL in mice with
anti-GBM nephritis increased glomerular accu-
mulation of T cells and macrophages without
affecting systemic immune response [20].
This suggests that ICOSL plays protective roles
during induction of anti-GBM nephritis by locally
reducing accumulation of T cells and macro-
phages. Blockade of CD154-CD40 costimulatory
signals in anti-GBM antibody nephritis in WKY
rats prevented glomerulonephritis [21]. In addi-
tion, anti-GBM nephritis in CD40 chimeric mice
(CD40 is absent in glomeruli but intact in bone
marrow) revealed suppression of glomerulone-
phritis accompanied by reduced level of MCP-1,
IL-10, mRNA, and T-cell and macrophage influx.
This indicates that CD40 expression in intrinsic
glomerular cells promotes Th1 effector cell
response in glomerulonephritis [16].
Comparing to Th1 and Th2 subsets, the role of
Th17 cells on the glomerulonephritis is under
investigation. Th17-derived IL-21, IL-22, and
IL-9 induce proinflammatory cytokines and
chemokines in resident cells resulting in recruit-
ment and activation of leukocytes and macro-
phages in situ [14]. Although presence of Th17
cells in the interstitial nephritis and induction of
cytokines and chemokines by tubular epithelial
cells in vitro has been noted earlier, its importance
in glomerulonephritis was firstly shown by atten-
uation of nephrotoxic nephritis in IL-17 knockout
mice. Transfer of Th-17-polarized ovalbumin-
specific CD4+ effector cells induced proliferative
glomerulonephritis [22]. In addition, cross-
regulation between Th17 cells and Th1 cells was
suggested by nephrotoxic nephritis in mice
lacking Th1 response [23].
Regulatory T cells (Treg) suppress effector T
cells through receptor-mediated mechanism, and
899
thus, failure of Treg to control T-cell activity can
provoke autoimmunity. Transfer of CD4+CD25
Treg inhibited murine anti-GBM antibody nephri-
tis via T-cell and macrophage suppression, with
significant reductions in the levels of INF-γ,
TNF-α, and TGF-β mRNA. This may be the
mechanism of the protective roles of Treg in
autoimmune-mediated glomerulonephritis. How-
ever, Treg transfer to lupus-prone mice
suppressed autoantibody production but not glo-
merulonephritis [24]. Treg suppresses Th1 and
Th17 cells by secreting IL-10, and Treg deletion
aggravated T-cell-mediated glomerulonephritis
through INF-γ induction and promotion of
delayed-type hypersensitivity [25, 26]. The
T-cell-mediated immune mechanisms of glomer-
ulonephritis have been analyzed in an acute
nephritis model, and the role of T cells in chronic
glomerulonephritis, particularly in humans,
remains largely unknown.
Inflammatory Cells
Leukocytes
Leukocytes are bone marrow-derived cells that
include granulocytes, lymphocytes, and mono-
cytes/macrophages. Granulocytes include neutro-
phils, eosinophils, and basophils. Neutrophils
(polymorphonuclear cells, PMNs) and macro-
phages play central roles in the acute inflamma-
tion process. Neutrophils and macrophages have
common effects in terms of tissue injury, but mac-
rophages are more actively involved in glomeru-
lar injury by their immunologic functions.
Usually, PMNs predominate in the acute phase
of glomerulonephritis, e.g., in acute poststrep-
tococcal glomerulonephritis and ANCA-related
glomerulonephritis. Macrophages appear in
acute and chronic glomerulonephritis, and both
cell types are sometimes copresented in the two
phases of glomerulonephritis, particularly acute
phase on chronic glomerulonephritis.
In the variety of pathogeneses of glomerulone-
phritis, leukocytes are involved in the loss of
immune tolerance, T cell-directed adaptive
immune responses, cellular effectors inducing
900
injury in delayed-type hypersensitivity-like reac-
tions, and macrophage/neutrophil recruitment via
the deposition of circulating or in situ immune
complexes through receptor-mediated fashion
[27]. Leukocytes express Fc-γ receptor on cell
surface, and it binds the Fc portion of IgG, and
lack of Fc-γ R protects immune complex-
mediated glomerulonephritis [27]. In addition,
ANCA have been postulated to activate neutro-
phils, leading to glomerular capillaritis.
Polymorphonuclear Cells
PMNs are implicated in glomerular injury owing
to their numerous bioactive/toxic products. PMNs
are chemoattracted by anaphylatoxin (C5a and
C3a), CXC chemokines, the immune adherence
of complement receptors, and Fc receptor-
dependent binding to antibodies. The
corresponding receptors include FcR for IgG,
CR1, CR2, and CR3 for complement fragments
and C1q for the collectins. Locally activated
PMNs express sialyl-Lewis X-modified proteins
and interact with endothelial cells, through
selectin or integrin ligands on the endothelial sur-
face. Various cytokines stimulate endothelial cells
to express these adhesion molecules (Table 2).
Occasionally, PMNs on endothelial cells migrate
to the mesangium and stimulate cells there,
through bioactive or toxic products. Among the
PMN-derived substances, lysosomal enzymes and
reactive oxygen species (ROS) are the most harm-
ful for the glomerulus. Lysosomal enzymes
include metalloproteases and other proteases,
which along with cathepsin G exert proteolytic
activities and damage endothelial cells or promote
lysis of the GBM and mesangium. Microbes,
cytokines, immune complexes, and various
inflammatory stimuli promote ROS synthesis in
PMNs through the NADPH oxidase pathway.
ROS ordinarily function to destroy phagocytosed
microbes and bring about cell death. Low-level
secretion of ROS accelerates the inflammatory
cascade by upregulating the expression of cyto-
kines, chemokines, and adhesion molecules. At
high levels, ROS damage endothelial cells, acti-
vating the coagulation cascade and increasing
M. Nagata
glomerular permeability, with consequent break-
down of the ECM, which links mesangiolysis and
GBM changes, and injuring the intrinsic cells.
Activation of antioxidant mechanisms that
involve catalase, superoxide dismutase, and glu-
tathione peroxidase ordinarily protects tissues
from oxidant stress. The role of ROS in glomeru-
lar injury is separately discussed.
A good example of neutrophil-mediated glo-
merular injury is ANCA-related glomerulonephri-
tis [28]. ANCA activates cytokine-primed
neutrophils and monocytes through Fab’2 binding
and Fc receptor engagement in vitro. These PMNs
adhere to endothelial cells and release ROS, pro-
teolytic enzymes, and inflammatory cytokines,
which promote cytotoxic tissue damage. In addi-
tion to these danger enzymes and ROS, comple-
ment- and cytokine-induced neutrophils
occasionally form extracellular fibrillar networks
called neutrophil extracellular traps (NETs). The
trap is composed of nuclear chromatin with
embedded granule proteins which are antimicro-
bial peptides and enzymes. Anti-MPO CD4 cells
recognize MPO as a planted glomerular antigen
and act with macrophages to amplify severe glo-
merular injury [29]. It has been suggested that
NETs involve tuft necrosis in ANCA-related
glomerulonephritis [30].
Intravenous injection of mouse antibodies or
splenocytes specific for mouse MPO alone
induces necrotizing crescentic glomerulonephritis
in Rag2 mice (lack of functioning B cells and T
cells). In this model, it appeared that PMNs’ infil-
tration was conspicuously associated with glo-
merular necrosis and crescent formation with
macrophages, whereas lymphocytes were not
prominent. Mice depleted of circulating PMNs
with rat monoclonal antineutrophil antibody
(NIMP-R14) were completely protected from
anti-MPO IgG-induced necrotizing crescentic
glomerulonephritis [31]. Local pretreatment of
cremaster muscle with TNF-α followed by
systemic administration of anti-MPO IgG in
wild-type mice leads to enhancement of
leukocyte-endothelial cell interaction in the
cremasteric microvessels, whereas this effect
was not seen in Fc receptor γ chain-deficient
mice. In addition, coadministration of anti-MPO
29 Immune-Mediated Glomerular Injury in Children
Fig. 9 Macrophage infiltration in IgA glomerulonephritis.
(a) Immunohistochemical detection of macrophages by
CD68 in IgA glomerulonephritis. Note many
immunolabeled cells (brown) locate predominantly in the
antibody with anti-CD18 antibody (CD18;
integrin β2) has no effect on the local leukocyte-
endothelial cell interaction [32]. This indicates
Fc-γ receptor and integrin β2 are important for
processing MPO-induced small vessel vasculitis.
Immunization of MPO in mice with bone marrow
transplantation of MPO-positive cells to
MPO-deficient recipient (chimera) developed
crescentic glomerulonephritis, whereas chimeric
mice of MPO-negative bone marrow cells into
wild-type mice did not [33], indicating that bone
marrow cells are the necessary target to mediate
MPO antibody-induced crescentic glomerulone-
phritis. The local inflammation induced by cyto-
kine (e.g., TNF-α)-primed PMNs and anti-MPO
antibody via FcR-γ effectively promotes cyto-
toxic endothelial cell damage and activates coag-
ulation cascade. This occasionally results in
fibrinoid necrosis followed by the breakdown of
GBM and subsequent crescentic formation.
Monocytes/Macrophages
Macrophages are derived from circulating blood
monocytes. In normal rat mesangial areas, tissue-
resident macrophages are occasionally present,
but they are uncommitted. When inflammation
occurs, monocyte-derived macrophages are
recruited, programmed, and activated, largely by
901
mesangial proliferation and less in the capillary (PAS
stain), (b) EM reveals monocytes/macrophages in the
mesangium and in the capillary (arrowhead)
INF-γ. Examination of anti-Thy1 nephritis reveals
that infiltration per se is insufficient to promote
macrophage programming, but this is completed
shortly after localization to the appropriate local
microenvironment.
Human renal biopsies and experimental
models of glomerulonephritis reveal glomerular
macrophage accumulations, which may be indic-
ative of disease activity or severity. IgA glomeru-
lonephritis in children represents relatively acute
inflammation, and the mesangial influx of macro-
phages leads to mesangial proliferation, as dem-
onstrated by electron microscopy and
immunohistochemistry (Fig. 9). Local activation
of macrophages also occurs in various glomerular
diseases, including nonimmune-mediated condi-
tions, such as diabetic glomerulosclerosis.
Macrophages produce a wide range of
potentially cytotoxic products, including
proinflammatory cytokines and chemokines, pro-
teolytic enzymes, ROS, eicosanoids, and growth
factors (Table 4, Fig. 10). Experiments on the sup-
pression or depletion of macrophages in acute and
chronic glomerulonephritis models have demon-
strated reduced glomerular injury [34, 35]. In cres-
centic glomerulonephritis in rats, liposome-MDP
(dichloromethylene diphosphonate) attenuated
glomerular macrophage influx accompanied by
suppression of CD8 and expression of adhesion
molecule. These findings circumferentially support
902 M. Nagata

Table 4 Macrophage activation status

Status Alteration Product
Classically activated (M1) Upregulation iNOS, IL-1β, TNF-α, IL-6, HMGB1, IL-8, MCP-1, MHC class I
Alternatively activated (M2) Upregulation Mannose receptor, MHC class II, arginase, FIZZ1/Ym1, IL-ra
Downregulation iNOS, TNF-α, IL-6
IL10 activated Upregulation Soluble TNF receptors, IL1ra
Downregulation IL-b, TNF-α, IL-12, IL-6, IL-8, iNOS, MHC class I
Type II activated Upregulation IL-10, TNF-α, IL-6, MHC class I
Downregulation IL-12
Uptake of apoptotic cells Upregulation TGF-b, PGE2
Downregulation TNF-α, IL-8, MCP1
Steroid treated Upregulation IL-10, uptake of apoptotic cells
Downregulation IL-12, TNF-α, IL1-β, IL-6

Fig. 10 Activation of Circulating Adherent Emigrating

macrophages and monocyte
production of various
cytokines. Circulation
monocytes transmigrate to
the site of inflammation,
and T-cell-derived
cytokines (INF-γ) stimulate
activation in monocytes/
macrophage and produce
many soluble factors for
tissue cell injury/tissue
fibrosis. In the glomerulus, Tissue macrophage
macrophages are able to
migrate into mesangium,
subendothelial space, or Immune
urinary space (The figure response:
was published in Kumar activated T cell
et al. Robbins Basic
Pathology, 8th edn. 2007, Activation Cytokine
p. 55, Copyright Elsevier) by microbes, (IFN-γ)
dead cells,
etc.
Activated macrophage

Tissue injury and Fibrosis

inflammation
• Reactive oxygen and
nitrogen species
• Proteases
• Cytokines, including
chemokines
• Coagulation factors
• AA metabolites
• Growth factors
(PDGF, FGF, TGFβ)
• Fibrogenic cytokines
• Angiogenesis factors
(FGF)
29 Immune-Mediated Glomerular Injury in Children
Monocyte
Circulation
Injured tissue
Macrophage
M2 polarization
Alternative activation by IL-4 or IL-13
Increased endocytic activity; increased
expression of mannose receptor
dection-1 and arginase; increased cell
growth; increased tissue repair;
increased parasite killing
Fig. 11 Macrophage heterogeneity during inflammation.
Monocytes attracted to the injured tissue are thought to be
able to acquire distinct phenotypes and physiological func-
tions. Largely there are two major phenotypes in macro-
phages, M1 and M2. In M1 polarization, macrophages are
stimulated with INF-γ and reveal high microbicidal
the role of macrophages in the glomerulonephritis.
In this context adoptive transfer of macrophages in
leukopenic rats with anti-GBM nephritis revealed
proteinuria and glomerular cell proliferation and
indicates direct evidence for macrophage to induce
glomerulonephritis [36]. Given these results, it has
long been suggested that the scale of macrophage
influx promotes glomerular injury. However, it has
recently become clear that it is the functional prop-
erties, rather than the total number, of macrophages
that determine the severity of inflammation. In
vitro studies have led to the hypothesis of func-
tional heterogeneity in macrophages. Polarized
macrophages can be subdivided into several phe-
notypes, particularly classically activated (M1) and
alternatively activated (M2) macrophages
[37]. The difference in activators determines the
heterogeneous phenotype (Fig. 11). Innate activa-
tion is promoted by Toll-like receptor (TLR)
ligands, which induce the production of
903
Innate activation by TRL ligands
Increased production of
proinflammatory cytokines,
iNOS and ROS
M1 polarization
Classical activation by IFN-γ TNF-α
and LPS
Increase production of
proinflamamtory cytokines, iNOS and
ROS: increased expression of MHC
class II molecules and CD86;
increased antigen presentation;
increased microbicidalactivity
activity and produce ROS and iNOS for proinflammatory
action. By contrast M2 polarization is promoted by IL-4,
IL-10, IL-13, or TGF-β, and it acted on the tissue repair and
parasite killing. However, this hypothesis has been drawn
by the in vitro experiments and still remains undetermined
in vivo (Modified figures from Gordon et al. [37])
proinflammatory cytokines, NO, and ROS. INF-γ
TNF-α and/or LPS primes macrophages to convert
classical activation (M1 polarization) and leads to
increased production of proinflammatory cyto-
kines, iNOS, and ROS, as well as the upregulation
of MHC class II molecule expression and
costimulatory CD86 molecules, thereby promoting
antigen presentation. In contrast, IL-4- or IL-13-
primed macrophages undergo alternative activa-
tion (M2 polarization), which is associated with
increases in endocytosis, arginase, anti-
inflammatory cytokines, and the synthesis of extra-
cellular matrix, thereby promoting angiogenesis.
M2 polarization also leads to decreased iNOS and
NO production and is involved in anti-
inflammatory effects, cell growth, and tissue repair.
Recent findings suggest that M2 macrophages are
induced by IL25 resulting protection in renal dis-
eases [38]. This phenotypic heterogeneity of mac-
rophages may be switched during the course of
904
chronic glomerulonephritis, depending on the
background pathophysiology.
Macrophage migration inhibition factor (MIF)
is a kind of proinflammatory cytokine to induce
cytokines, chemokines, and adhesion molecules
in the inflammatory environment. Antagonist for
MIF resulted in the prevention of lupus nephritis
[39]. Activator protein (AP-1) transcription factor
JUND is a major determinant of macrophage
activity and associated with susceptibility to glo-
merulonephritis [40]. During the process of glo-
merular injury, macrophages take up apoptotic
cells, most of which are infiltrated inflammatory
cells. This uptake causes the macrophages to exert
anti-inflammatory effects, through increased
expression of TGF-β and prostaglandin E2
(PGE2) and decreased production of IL-8,
TNF-α, and IL-1β. Suppression of T-cell prolifer-
ative responses also results from the anti-
inflammatory effects of macrophages that have
taken up apoptotic cells.
Mast Cells
Mast cells are bone marrow-derived cells that play
significant roles in innate and adaptive immune
responses. Mast cells secrete various inflamma-
tory mediators, including chemical vasomotor
mediators, cytokines, growth factors, and eicosa-
noids. These substances attract inflammatory cells
and are involved in mediating tissue remodeling.
Mast cell detection in tissues is typically
performed by staining with toluidine blue, Alcian
blue, and safranin, all of which enable the visual-
ization of cell granules. Immunohistochemistry to
detect enzymes specific for mast cells, such as
tryptase and chymase, can be used to subdivide
mast cells into MCT (containing tryptase) and
MCTC (containing tryptase plus chymase) cells.
This enzyme visualization method reveals many
more mast cells than classic toluidine blue
staining. Mast cells are present in human glomer-
ulonephritis, primarily in the interstitium, and are
probably involved in fibrosis. The role of mast
cells in glomerulonephritis has been examined in
anti-GBM antibody nephritis in congenitally mast
cell-deficient mice (W/Wv) showing a higher
M. Nagata
mortality rate and enhanced disease activity, as
compared with wild-type mice [41]. This condi-
tion can be rescued by mast cell reconstitution,
suggesting a protective role for mast cells in
immune complex-mediated glomerulonephritis.
Toll-Like Receptors (TLRs)
The recently identified TLR families provide
receptor-mediated pathogen recognition and acti-
vation of the immune system. Ten human and nine
murine TLR proteins related to the original Dro-
sophila Toll gene have been identified to date.
Inflammatory cytokines and chemokines are
induced through the activation of TLRs by a vari-
ety of ligands. The biologic activities that result
from TLR binding include activation of antigen-
presenting cells, dendritic cell maturation, B-cell
activation, and different cytokine production pro-
files. The responsibility of TRL in the innate and
adaptive immune response may be a likely back-
ground in the immune-mediated glomerular
injury. Small nuclear RNA and autoantigen for
lupus activate B cells and dendritic cells via
TLR-7. TLR-7 overexpression is associated with
antinuclear autoantibody production and lupus-
like disease in mice, and backcrossing TLR-7-
deficient mice with MRL lpr/lpr mice attenuated
kidney and lung disease. Administration of syn-
thetic oligodeoxynucleotides with immunoregula-
tory sequences (IRS) which specifically blocks
TLR-7 suppressed autoimmune kidney and lung
diseases. Endogenous or exogenous CpG-DNA
activates TLR-9 and administration of CpG
DNA to MRL-Fas(lpr) mice resulted in disease
progression accompanied by MCP-1 and
RANTES expression and leukocyte recruitment
[42]. In ddY mice of spontaneous IgA glomeru-
lonephritis, genome-wide scan revealed myeloid
differentiation factor 88 (MyD88) as a candidate
gene for disease progression, and TRL-9, the
receptor for MyD88, was increased in a conven-
tionally housed condition. Nasal challenge with
CpG oligonucleotide (a ligand for TRL-9) aggra-
vated renal injury with strong Th1 polarization
and increased mesangial IgA. TRL-9 polymor-
phism in the TLR-9 gene associated with the
29 Immune-Mediated Glomerular Injury in Children
disease progression of IgA in humans. TLR-9
pathway may be involved in the pathogenesis
and determination of severity of IgA glomerulo-
nephritis [43]. HSPN and IgA glomerulonephritis
in children showed upregulation of TLRs and
Treg depression in peripheral blood cells,
suggesting autoimmune proteasome switch in
these diseases [44].
TLR-3 recognizes dsDNA of viral origin and is
expressed in mesangial cells. Cytokines stimulate
TLR-3 mRNA in mesangial cells in vitro.
TLR-3mRNA is significantly upregulated in glo-
meruli associated with enhanced mRNA of
RANTES/CCL5 and MCP-1/CCL2 in hepatitis
C-associated glomerulonephritis in humans. This
suggests that immune complex containing viral
RNA activates mesangial cells via TLR-3-medi-
ated chemokine/cytokine effects [45].
Complement Cascade
The complement system, which is a cascade of
plasma proteins, involves approximately
30 plasma and membrane-bound proteins that
play a pivotal function in antimicrobial defense
and immune complex clearance. Complement
components are largely liver derived and are pre-
sent in inactive forms (numbered C1 through C9)
in the plasma, and local complement production
occurs in the inflammatory milieu involved in
tissue damage, including leukocyte recruitment,
cell necrosis, and apoptosis. Immune complex
clearance and proinflammatory functions of com-
plement system provide its complex participation
in immune-mediated glomerulonephritis.
The biologic actions of complement ultimately
generate a pore-like membrane attack complex
(C5-9, MAC), which punches holes in invading
microorganisms. Among the several important
complement fragments produced in the
MAC-generating processes are the cleavage prod-
ucts of C3 and C5 (C3a, C5a), collectively termed
anaphylatoxin, which promote vascular perme-
ability (C3a, C5a) and leukocyte chemotaxis and
activation (C5a).
The presence of complement components in
the tissue of glomerular diseases represents local
905
activation of complement system that is involved
in the pathogenesis of glomerular injury. Classi-
cally, inhibition of the complement cascade by
cobra venom factor attenuated various glomeru-
lonephritis models. Complement depletion, by
aggregated human IgG, protected against
complement/PMN-dependent glomerular injury
in anti-GBM antibody nephritis, with marked
attenuation of leukocyte influx. In addition, defec-
tive complement regulation system, as seen in
human and animal models with specific comple-
ment component deficiencies, has provided evi-
dence of a complement-mediated/complement-
dependent pathway in glomerular injury. This
includes certain forms of hemolytic uremic syn-
drome and MPGN type II [46].
The complement system consists of three dis-
tinct pathways: the classical, mannan-lectin, and
alternative pathways (Fig. 12). These pathways
sometimes are synergistically involved in the glo-
merulonephritis, such as simultaneous activation
of lectin pathway and alternative pathway in IgA
glomerulonephritis and purpura nephritis [47].
Of the four classical types of hypersensitivity
reactions, complement is thought to be involved
in types II and III. Type II hypersensitivity and
antibody-mediated hypersensitivity target intrin-
sic glomerular cells or extracellular matrix, pro-
voking tissue damage by a complement-
dependent mechanism. This mechanism typically
includes Goodpasture syndrome and Heymann
nephritis. In type III hypersensitivity, antigen-
antibody complexes within the glomerulus (glo-
merular capillary endothelium or mesangium) and
complement bind the C3b receptor or the Fc
receptor of immune-competent cells and activate
the production of chemical mediators or cytokines
that lead to tissue injury. Lupus nephritis and
poststreptococcal acute glomerulonephritis are
promoted by this mechanism.
The complement activation-involved glomeru-
lar injury is thought to be mediated by leukocyte
recruitment via C5a, which accelerates the inflam-
matory process (leukocyte dependent) or through
direct attack by complement fragments on intrinsic
glomerular components (leukocyte independent).
Leukocyte-dependent tissue injury mecha-
nisms are involved in injury to the glomerular
906
Fig. 12 Schematic depiction of the three pathways of
complement activation as they relate to GN. In the adaptive
immune system, complement-fixing antibodies (IgG1,
IgG3, IgM) in immune complexes and CRP initiate classic
pathway activation through C1q, C4, and C2 to form
C4b2a, the classic pathway C3 convertase. In the innate
immune system, PAMPs and DAMPs activate complement
through the MBL or alternative pathways. Some infectious
PAMPs and DAMPs bind MBL leading to activation of
MBL-associated serine proteases (MASPs), which activate
C3 via C4, C2, and the classic pathway C3 convertase. In
the alternative pathway, direct activation of C3 occurs
spontaneously (“C3 tickover”) and by foreign surfaces,
capillary. C5a affects the chemotaxis of neutro-
phils, monocytes/macrophages, and eosinophils
and accelerates the release of inflammatory medi-
ators. C5a also increases the adhesion of leuko-
cytes to endothelium, by activating the leukocytes
and increasing the avidity of the surface integrin
for the ligands C3b and C3bi deposited on endo-
thelial surface. C5-deficient mice show reduction
of glomerular PMNs infiltration in cryoglobulin-
induced immune complex glomerulonephritis
[48]. Complement anaphylatoxins bind several
receptors including G protein-coupled receptors
(C3aR and C5aR). C5a binds two receptors, C5aR
M. Nagata
damaged cells, and IgA. Cleavage of C3 produces C3b,
which combines with factor B and properdin to form the
alternative pathway C3 convertase that is regulated by
factors H and I to prevent excess C3 activation. Both
classic and alternative pathway C3 convertases cleave C3
leading to release of C3b, which allows C5 convertase
formation. Cleavage of C5 produces the chemotactic factor
C5a and C5b, which combines with C6, C7, C8, and
multiple C9 molecules to form the lipophilic C5b-9 mem-
brane attack complex that can activate resident glomerular
cells to become effector cells. C5b-9 formation is regulated
by cell-bound CRPs such as CD59 [1]
and C5L2, and most of the functional effects of
C5a are mediated by C5aR. Transplantation of
C5aR-deficient marrow suppressed glomerulone-
phritis in MPO-deficient mice immunized with
MPO, indicating that C5a involves leukocyte acti-
vation via C5aR [49]. This inflammatory process
involves GBM breakdown and activation of the
coagulation system, due to endothelial injury;
leads to proliferative and matrix changes in the
mesangium and parietal cells; and accelerates
podocyte damage. MAC stimulates NF-κB,
which is linked to the activation of the
proinflammatory cytokines IL-8 and MCP-1.
29 Immune-Mediated Glomerular Injury in Children
In humans, C3aR is upregulated in the glomerular
endothelial region in lupus nephritis, but not other
glomerular diseases [50].
Leukocyte-independent mechanism of glomer-
ular injury is seen in Heymann nephritis. Like
type II mechanisms, antigen (megalin) on
podocyte surface is a target of complement cyto-
toxicity and results in direct injury for filtration
barrier and complement depletion-attenuated
Heymann nephritis. In addition, sublyric levels
of MAC damage the DNA in podocytes [51].
Glomerulonephritis in complement deficiency
provides a clue to understand the roles for com-
plement in glomerular injury. C6-deficient/C6-
depleted rats with anti-Thy1 nephritis or
Heymann nephritis show attenuated disease activ-
ity. By contrast occurrence of IgA glomerulone-
phritis even in the hereditary C6 or C9 deficiency
in humans indicated that MAC per se is not essen-
tial in the IgA glomerulonephritis.
Interestingly, C1q or C4 deficiency develops
lupus-like disease in mice, and C1q, C2, and C4
deficiency in humans increased risk of developing
SLE. Although the phenomenon may contradict
to the many literatures suggesting complement
system injures the glomerulus, it may be
explained by the scenario that complement defi-
ciency may lead to autoimmune phenomena due
to defective clearance of apoptotic cells which are
a rich source of autoimmunity in SLE.
Glomerular cells are normally protected from
complement-mediated injury by cell-surface
defense mechanisms, and complement regulatory
proteins are expressed in intrinsic glomerular
cells. CR1 (CD35) preferentially binds to C3b
and C4b, which are normally expressed in
podocytes and protect against complement-
dependent cellular damage. CR2 is also expressed
in intrinsic glomerular cells, including podocytes
and mesangial and endothelial cells, and is
upregulated in membranous nephropathy. In addi-
tion, ER (endoplasmic reticulum) stress protein
may have protective role for MAC-mediated cell
injury as shown in podocytes [52].
In human glomeruli, decay accelerating factor
(DAF or CD55) and membrane cofactor protein
(MCP or CD 46) regulate C3 and C5 activation.
CD59, which is a membrane protein inhibitor of
907
the MAC of complement, inhibits at the level of
C8 activation, preventing MAC formation.
Glomerular-resident cells normally express MCP
and CD59, and these molecules are upregulated in
glomerular disease. Nephrotoxic serum nephritis
in mice deficient for either DAF1 or CD59a or
both DAF1 and CD59a genes reveals that mice
lacking DAF1 gene developed severer proteinuria
and histology compared to wild-type or CD59a-
deficient mice. Severe disease in DAF1 gene defi-
ciency is associated with glomerular C3 and C9
deposition, whereas immunoglobulin deposition
is comparative. Crry is a rodent homologue with
activities similar to DAF and MCP. Blocking
CD59 or Crry accelerates complement-dependent
glomerulonephritis, and the soluble forms of these
molecules effectively inhibit complement and
attenuate glomerular damage.
Alternative mechanism of complement-
dependent glomerular injury is defective comple-
ment regulatory proteins. Protective proteins
against the complement cascade include
factor H, factor H-like protein, factor I, and C4
binding protein, and functional defects in these
proteins are the background. Factor H is a soluble
glycoprotein that regulates complement, both in
the fluid phase and on the cellular surface. It acts
in the amplification loop of the alternative path-
way to regulate C3bBb through dissociation of
C3bBb into inactive factor Bb and C3bfH,
followed by irreversible inactivation by factor
I. Factor H has affinity for polyanions on the cell
surface and protects the cell from alternative
pathway-mediated injury.
Dense deposit disease (DDD, synonym
as MPGN type II) is a peculiar disease with
persistent complement activation without immu-
noglobulin deposition, as revealed by immunoflu-
orescence [53]. Electron microscopy shows a
characteristic continuous, electron-dense deposi-
tion along the lamina densa of the GBM. The
disease has a poor prognosis, with chronic renal
failure and frequent relapses after renal transplan-
tation, which suggests that genetic or humoral
factors cause the disease. Systemic and persistent
alternative pathway activation in DDD is gener-
ally caused by either the presence of autoanti-
bodies to C3 convertase (C3bBb), C3NeF, which
908
stabilizes it, or by inherited mutations in or the
presence of antibodies against factor H, resulting
in the inhibition of convertase inactivation [54].
C3NeF-mediated convertase stabilization may be
dependent on the factor H-mediated inactivation
of the convertase. Similar to DDD, glomerulone-
phritis with isolated C3 deposition (C3 glom-
erulonephritis) has been recently categorized
and needs differential diagnosis with DDD
and poststreptococcal acute glomerulonephritis.
C3 glomerulonephritis is a heterogeneous disease
which includes abnormalities of complement reg-
ulatory protein and others [55].
The genes for the factor H family of proteins
localize to the “regulators of complement activa-
tion” (RCA) region at 1q32. Few reports have
addressed the potential association between
human DDD and factor H mutations, although
such mutations are important in the pathogenesis
of the disease. A 13-month-old Native American
boy with MPGN type II, who had a C518R muta-
tion in the factor H short consensus repeat (SCR)
9 in trans with a C941Y mutation in factor H
SCR16, showed retention of the defective factor
H in the endoplasmic reticulum. A relationship
between MPGN type II and factor H has also
been demonstrated in animal models. Norwegian
Yorkshire piglets with the I1166R mutation in
SCR20 developed MPGN type II; this mutation
prevented extracellular release of factor H,
resulting in intracellular accumulation. Factor
H-deficient mice show significant reductions in
C3 and reproducible MPGN type II, with C3
deposition in the GBM, whereas combined factor
H and factor I deficiency or factor I deficiency
alone did not show MPGN II [56]. These findings
in humans and animals suggest that mutated factor
H is not delivered to the cell surface and/or is
unable to bind to surface-bound C3b, resulting in
incomplete convertase inactivation. Cases with
DDD with C3 mutation revealed defect of C3
structure which is critical for recognition of the
substrate C3 by the alternative pathway
convertase and for the regulatory activities of
factor H, DAF, and MCP [57]. Spontaneous
MPGN in factor H-deficient mice was attenuated
in additional deficient with C5, but not those with
C6. Severe nephritis of anti-GBM nephritis in
M. Nagata
factor H-deficient mice is blocked by administra-
tion of anti-C5 antibody [58]. Notably, the site of
pathologic lesions is limited to the glomerulus,
suggesting a unique requirement in the GBM for
factor H-mediated protection against complement
attack.
Another type of glomerular injury with persis-
tent complement activation is atypical hemolytic
uremic syndrome (aHUS), which is typically seen
as a familial condition. A factor H mutation has
been implicated in aHUS, and the HUS database
(http://www.fh-hus.org) lists more than 100 muta-
tions related to factor H: 163 factor H mutations,
57 factor I mutations, and 49 MCP mutations
linked to aHUS, as well as 6 factor H mutations
linked to MPGN type II, as of July 2014. Factor H
mutations apparently generate different glomerular
injury phenotypes in MPGN type II and aHUS.
In contrast to the factor H mutations in patients
with MPGN type II, aHUS patients with factor H
mutations are usually heterozygous (few cases
with homozygous mutation are reported) for the
mutation, and these patients have normal levels of
circulating factor H protein [59]. In addition, factor
H mutations in aHUS patients are typically located
in the C-terminal region, which is important for
binding to the cell surface. Transgenic mice that
lack the exons encoding the C-terminal region of
factor H develop aHUS, closely resembling the
human disease. These mice show preservation of
fluid-phase complement activation, associated
with defects in endothelial protection against com-
plement attack, leading to persistent endothelial
injury. This corresponds to the pathogenic
background of aHUS, unlike MPGN type II. Sim-
ilarly, heterozygous mutations in MCP have been
reported in cases of familial aHUS. These muta-
tions resulted in three amino acid changes (posi-
tions 233–235) and the insertion of a premature
stop codon, resulting in loss of the transmembrane
domain of the protein and severely diminishing
the cell-surface expression of MCP [60]. Recently
developed anti-C5 antibody (eculizumab) has
been shown to be effective for children
with not only aHUS, but also Shiga toxin-
producing Escherichia coli (STEC-HUS), who
are negative for complement regulatory protein
abnormalities [61].
29 Immune-Mediated Glomerular Injury in Children
Soluble, Secreted Peptides
Following the initiation of immune-mediated glo-
merular injury, regardless of whether it is cell
mediated, antibody mediated, or both, soluble
and secreted peptides are significantly involved
in the promotion of glomerular injury as second
messengers. Not only interactions between these
molecules occur among inflammatory cells, but
their direct actions on intrinsic cells and cellular
responses are also important in the pathogenic
background of glomerular injury. Various cyto-
kines, chemokines, and growth factors are solu-
ble, secreted peptides that promote the responses
of intrinsic cells, including cell proliferation, cell
death, and matrix production/degradation. The
cellular responses of intrinsic cells and the fea-
tures of inflammatory cells lead to the glomerular
pathology that defines the stage and grade of
glomerulonephritis.
Interleukins
Interleukins (ILs), initially identified as cytokines
secreted by leukocytes, are important signaling
molecules. To date, more than 35 subtypes have
been identified. ILs are known to mediate cellular
immune/inflammatory reactions, and they play
key roles in immune-mediated glomerular injury.
Many cells synthesize ILs that act locally,
resulting in the proliferation or maturation of T
cells, T-cell interactions with B cells, antibody
production by B cells, and changes in the Th1
and Th2 subset balance.
In the kidneys, ILs stimulate mesangial cells
and endothelial cell proliferation and stimulate the
production of oxygen radicals, collagenase, cyto-
kines, chemokines, adhesion molecules, and
extracellular matrix, and they may accelerate cell
injury/death. Among the ILs, IL-1, IL-2, IL-4,
IL-6, IL-8, IL-10, IL-11, IL-12, and IL-13 are
known to be involved in many aspects in glomer-
ular injury.
IL-1, IL-6, IL-8, IL-12, and IL-18 primarily act
to promote glomerular injury, whereas IL-4,
IL-10, IL-11, and IL-13 have anti-inflammatory
actions. These molecules act on glomerulus by
909
similar but occasionally different mechanisms,
and their roles are sometimes disease specific.
For example, IL-10, IL-12, and IL-18 are
pathogenesis-specific ILs in lupus nephritis and
are involved in glomerular injury by mediating
leukocyte infiltration.
IL-1 is produced by macrophages, neutrophils,
and dendritic cells and accelerates helper T-cell
costimulation, B-cell maturation, and macrophage
and endothelial cell activation, via the specific
receptors Cd121a/IL1R1 and CD121b/IL1R2.
IL-1β is involved in macrophage-mediated glo-
merular injury, as is TNF-α. IL-1 is transiently
expressed during acute glomerular injury and cor-
relates with the degree of glomerular inflamma-
tion; it is not expressed during chronic glomerular
damage. IL-1 accelerates leukocyte adherence to
endothelial cell surface, resulting in mesangial
proliferation, activation of procoagulant activity,
and the synthesis of prostaglandins. Indeed, IL-1
mRNA is expressed in acute glomerular injury,
and the administration of a soluble IL-1 receptor
antagonist attenuates the damage. In addition,
IL-1 stimulates mesangial VEGF synthesis via
the PI3-K/mTOR pathway and stimulates matrix
production by epithelial cells [62].
IL-6 is produced by several types of blood-
borne cells, glomerular endothelial cells, and
mesangial cells. IL-6 is a multifunctional cytokine
that is important for B-cell differentiation and
maturation, immunoglobulin synthesis, acute-
phase protein production, bone marrow progeni-
tor stimulation, mesangial proliferation, and the
activation of monocytes/macrophages. IL-6 is
detected in the serum and urine in both animals
and humans with glomerulonephritis. IL-6-trans-
genic mice demonstrate glomerulonephritis, and
blockade of IL-6 receptors using a neutralizing
antibody ameliorates lupus glomerulonephritis in
NZB/W F1 mice. The promotive role of IL-6 for
mesangial proliferation or glomerulonephritis
may largely be mediated by activation of mono-
cyte/macrophages.
IL-8 is synthesized by macrophages, lympho-
cytes, epithelial cells, and endothelial cells. IL-8
stimulates the activation and chemotaxis of
granulocytes via CXCR1/IL8RA. In addition,
activated mesangial cells in glomerular injury
910
synthesize IL-8, and neutralization of IL-8 by a
specific antibody attenuates immune complex-
type glomerulonephritis, concomitant with the
suppression of leukocytes. In lupus nephritis
patients, urinary biomarkers such as TNF-like
weak inducer of apoptosis (TWEAK),
osteopontin, and MCP1 were positively correlated
with renal involvement but not that of IL-8
[63]. In poststreptococcal acute glomerulonephri-
tis in children, increased levels of IL-8 were
detected in the urine and plasma particularly in
the acute phase, and upregulation of glomerular
IL-8 is associated with endocapillary proliferative
lesions [64]. IL-8 may relate to the acute and
active phases of glomerulonephritis.
IL-12, which is a heterodimeric molecule, com-
posed of the p35 and 40 subunits, plays a key role
in uncommitted T cells of the Th1 phenotype.
Antigen-presenting cells are the dominant source
of the IL-12, but intrinsic renal cells are also the
effector to produce IL-12. Anti-GBM antibody
nephritis induced in IL-12p40/ mice reveals
attenuation of glomerulonephritis associated with
suppression of leukocyte infiltration [65].
IL-18 is produced by macrophages and stimu-
lates INF-γ synthesis by Th1 cells and NK cells,
resulting in NK cell activation. IL-18 is
overexpressed in patients with lupus nephritis
associated with higher INF-γ and lower IL-4 pro-
duction [66]. Anti-GBM nephritis in IL18/
mice reveals reduction of local synthesis of TNF,
IL1β, IFN-γ, and MIP1. IL-18 deposition in tis-
sues is observed in ANCA-related glomerulone-
phritis, and IL-18 is involved in the priming or
recruiting of neutrophils in this disease [67]. IL-
18-primed neutrophils synthesize MPO and PR3
ANCA via p38 MAP kinase activation in vitro.
IL-4 acts by promoting Th2 responses, and it
inhibits the various proinflammatory effects of
macrophages. Macrophages primed with IL-4
become unresponsive to proinflammatory cyto-
kines, including INF-γ and TNF-α. Glomerular-
resident cells, i.e., mesangial cells and podocytes,
synthesize IL-4. IL-4 ameliorated crescentic glo-
merulonephritis in Wistar Kyoto rats, and this is
associated with macrophage suppression, and
transfection of IL-4-expressing macrophages in
rats with nephrotoxic serum nephritis prevented
M. Nagata
glomerulonephritis with reduction of ED-1-posi-
tive macrophage influx [68]. The aggravation of
glomerulonephritis in IL-4-deficient mice is atten-
uated by treatment with recombinant IL-4. These
findings indicate a renoprotective role for IL-4 in
glomerulonephritis.
IL-10, a pluripotent cytokine, is produced by
various activated immune cells, including
T-helper cells, B cells, monocytes/macrophages,
and keratinocytes. IL-10 downregulates the
costimulatory molecules that participate in T-cell
activation. IL-10 suppresses mesangial cell prolif-
eration in anti-Thy1 nephritis and also attenuates
glomerular injury in nephrotoxic serum nephritis.
C-reactive protein suppresses nephrotoxic serum
nephritis in B6 mice, and the effect is abolished in
IL-10-deficient mice [69]. Renal dendritic cells
stimulate CD4+ T cells, inducing IL-10 production.
Nephrotoxic serum nephritis in mice depleted renal
dendritic cells by diphtheria toxin revealed
advanced tubulointerstitial and glomerular lesions,
without any effects on the macrophages. Produc-
tion of IL-10 by renal dendritic cells is mediated by
promotion of the IL-10 inducer molecule, inducible
costimulatory molecule ligand (ICOSL). Long-
term expression of IL-10 by a recombinant
adeno-associated virus modulated glomerulo-
sclerosis in a subtotal nephrectomy model accom-
panied by suppressing the INF-α and IL-2 mRNA
levels. This suggests a renoprotective role for IL-10
in nonimmune-mediated chronic models of
glomerulosclerosis [70].
IL-11 is a bone marrow-derived,
multifunctional, anti-inflammatory cytokine that
is involved in acute inflammatory responses medi-
ated by acute-phase protein production. Rat
mesangial cells and macrophages express the
IL-11 receptor α-chain. IL-11 suppresses the
expression of the proinflammatory cytokines
TNF-α, IL-1β, and IL-12 in LPS-stimulated peri-
toneal macrophages. Recombinant human IL-11
attenuates crescentic glomerulonephritis in WKY
rats, showing reduced necrosis, macrophage infil-
tration, and apoptosis [71]. In human glomerulo-
nephritis, daily urinary IL-11 was correlated with
proteinuria and suggested IL-11 acts as a counter
cytokine against Th1-mediated inflammatory glo-
merular diseases [72].
29 Immune-Mediated Glomerular Injury in Children
Interferons
Interferon (INF) stimulates the immune response,
activating NK cells and macrophages and increas-
ing antigen presentation to lymphocytes, and is
now known to comprise three subtypes: α, β, and
γ. INF-γ is a key molecule for Th1 cells and is
synthesized by many cell types under various con-
ditions, although macrophages are the most impor-
tant source in inflammation. INF-γ is important in
the effector phases of nephritogenic immune
responses, including IgG2a generation, T-cell and
macrophage recruitment, and macrophage activa-
tion. Evidence suggests that INF exerts different
effects in various nephritogenic conditions.
In human lupus nephritis, laser microdissection
of glomeruli shows upregulation of glomerular
INF-γ in association with IL-10, IL-12, and IL-18,
and that was correlated with glomerular leukocyte
influx [73]. INF-γ is also upregulated in the mice
model of lupus nephritis, and blocking INF-γ atten-
uated the kidney disease. INF-γ enhanced autoim-
munity, generating damaging autoantibodies in the
lupus model. Lymphocyte depletion in the anti-
Thy1 nephritis model revealed attenuation of glo-
merular injury through the suppression of INF-γ.
Endogenous INF is pathogenic in anti-GBM anti-
body nephritis. In contrast, anti-GBM antibody
nephritis in INF-γ receptor-deficient mice showed
aggravation of the nephritis and progressive cres-
centic glomerulonephritis [74]. Both aggravating
and protective effects for INF-γ in glomerulone-
phritis are probably depending on the mechanistic
background of the diseases. INF-γ may be an effec-
tor for other cytokines or may modify the reactions
of macrophages, rather than having direct actions
on glomerular cells.
Tumor Necrosis Factor (TNF)
TNF is a monocyte-/macrophage-derived,
proinflammatory cytokine and an important media-
tor of inflammatory tissue damage. There are three
family members, TNF-α, TNF-β, and lymphotoxin-
β, and TNF-α is the best characterized TNF among
them. Two receptors for TNF (TNFR1 and TNFR2)
have been identified and are involved in mediating
911
local inflammatory injury and systemic immune-
regulatory functions in the kidney. Many experi-
mental studies and clinical observations support
roles for TNF-α in the pathogenesis of acute and
chronic renal disease. TNFR1 attenuates
LPS-enhanced nephrotoxic nephritis with
downregulation of IL-1β. Anti-GBM glomerulone-
phritis is attenuated in TNF-α-deficient mice, and
blocking TNF-α in nephrotoxic serum nephritis in
Wistar Kyoto rats attenuated glomerulonephritis
accompanied by downregulation of urinary
MCP-1 [74]. Wild-type bone marrow transplanta-
tion to TNF-deficient recipients (intact bone mar-
row but absent renal-derived TNF) resulted in
attenuation of anti-GBM nephritis, whereas the
effect was not seen in mice with TNF-deficient
leukocytes but intact intrinsic renal cells. This sug-
gests intrinsic renal cells are the major source of
TNF and participate in TNF-mediated renal injury.
Likewise TNF stimulates cytokine production in
mesangial cells in vitro. TNF-α probably activates
proinflammatory cytokines and participates in the
progression of glomerulonephritis.
Although TNF-α generally plays a stimulatory
role in glomerulonephritis, it can also act as an
immune modulator. The fact that blockade of
TNF-α in human rheumatoid arthritis or Crohn’s
disease leads to the development of autoanti-
bodies, a lupus-like syndrome, and glomerulone-
phritis in some patients suggests a B-cell
modulator of TNF-α [75].
Given these apparent dual functions of TNFs,
balance between the proinflammatory and immu-
nosuppressive effects of TNF may participate in
immune-mediated glomerulonephritis.
Chemokines and Their Receptors
Chemokines (chemotactic cytokines) are small
proteins (8–10 kDa) that are now known to con-
stitute a superfamily of leukocyte subset-specific
activating and/or chemoattractant cytokines. The
function is to guide the migration of inflammatory
cells into the site of inflammation. The four clas-
ses of chemokines (CXC, CC, C, and CX3C) are
classified based on similarities in their amino acid
sequences; specifically, they share patterns of
912
Fig. 13 Chemokines and
their receptors. Four
subfamilies of chemokines
have been characterized,
including the CC subfamily,
the CXC subfamily, the XC
subfamily, and the CX
subfamily [76]
paired cysteine repeats and two internal disulfide
bridges. Many chemokines bind multiple recep-
tors, and most receptors bind multiple chemokines
(Fig. 13). In kidney diseases, chemokines bind
variable receptors on the leukocyte surface and
promote acute kidney injury and chronic kidney
diseases (Fig. 14; [76]).
The CC and CXC chemokines have been exten-
sively investigated in kidney diseases. In humans,
urinary CC chemokines are detected in crescentic
glomerulonephritis and glomerulosclerosis.
Increased levels of urinary CXC chemokines are
associated with glomerular deposition in cases of
active glomerulonephritis. The CC chemokines
(β-chemokines), comprising to date a family of
27 members, are involved in the recruitment of
monocytes/macrophages and T cells. Monocyte
chemoattractant protein (MCP-1/CCL2), macro-
phage inflammatory protein (MIP1/CCL3), and
regulated upon activation normal T cells expressed
and secreted (RANTES/CCL5) are the major CC
chemokines involved in glomerulonephritis. There
M. Nagata
are five receptors for CC chemokines. CCR1 is
expressed on monocytes, CCR3 and CCR5 are
expressed on Th1 cells, and CCR3 and CCR4 are
expressed on Th2 cells. Th17 cells produce CCL20
and induce monocyte and Th1 cells as well as
additional Th17 cells.
MCP-1 accelerates glomerular injury. Expres-
sion of MCP-1 has been observed in proliferative
glomerulonephritis and crescentic glomerulone-
phritis. Urinary and serum MCP-1 levels are
increased in poststreptococcal acute glomerulone-
phritis (PSAGN) in children, and urinary but not
serum MCP-1 levels correlate with proteinuria in
acute phase [64]. MCP-1 and MIP1-α have been
detected in human crescentic glomerulonephritis
and correlate with the degree of macrophage
influx into the glomeruli [77]. MCP-1-deficient
MRL-Fas/lpr mice showed decreased disease
severity, with suppression of macrophage and
T-cell recruitment, resulting in decreased local
levels of cytokines and chemokines. An MCP-1
antagonist attenuated glomerulonephritis in
29 Immune-Mediated Glomerular Injury in Children
IL-1β, TNF-α, ROS, LPS,
IL-17, IL-23, IFN-γ, TGF-β...
CXCL1–3/5–8 CCL2–5/8/13–16
CX3CL1
CCR1/2/5/8
CXCR1/2 CX3CR1
Neutrophils Monocytes
AKI
Fig. 14 Interaction of chemokines and receptors on leu-
kocyte subsets during acute and chronic kidney injury.
Kidney diseases are characterized by the accumulation of
various leukocyte subsets that are controlled by
chemokines through their corresponding receptors
MRL-Fas/lpr mice, and a neutralizing antibody to
MCP-1 attenuated the disease in a mouse model of
crescentic glomerulonephritis. A Spiegelmer
(L-enantiomeric RNA oligonucleotide, mNOX-
E36) that binds to murine CCL inhibited its function
and improved the survival rate of lupus mice by
modulating nephritis [78]. In addition, P38MAPK
ameliorates crescentic GN by suppressing MCP-1,
independently of TNF-α and IL-1β [79].
RANTES binds CCR1 and CCR5 in mono-
cytes. Inhibition of RANTES leads to protective
effects in experimental glomerulonephritis, and
CCR5 deficiency aggravates T-cell-dependent
nephrotoxic serum nephritis via enhanced CCL3/
CCL5-CCR1-driven renal T-cell recruitment [80].
CCR1 antagonist prevents lupus nephritis by
inhibiting interstitial inflammation but not affect
913
CXCL4/9–11/16 CCL17/19/21/22
CCL1–5/7/8/11/12
CCL14–17/19/21–27
CXCR3/6
CCR2–10 CCR4/6–8
T cells Dendritic cells
CKD
expressed by the different subsets. Although infiltration
of neutrophils and monocytes mediates acute kidney
injury, activation of T cells, macrophages, and dendritic
cells promotes progression of chronic kidney disease [76]
on glomerular injury, suggesting the role of
CCR1 is different in the pathogenic background.
CXC chemokines exert chemoattractant effects
on PMNs by intracellular phosphatase activation,
via CXCR1 and CXCR2. IL-8 interacts with
CXCR1 and CXCR 2, advancing PMN chemo-
taxis. Immunohistochemistry revealed expression
of CXCR1 predominantly in MPGN and lupus
nephritis in humans and that CXCR-1-expressing
cells are mainly PMNs [81]. The INF-γ-inducible
protein of 10 kDa (IP-10) binds to CXCR3. Renal
microvascular endothelial injury model provided
evidence that neutralizing antibody against IP-10
attenuated renal dysfunction through the reduced
number of infiltrating tubulointerstitial T cells
without affecting monocytes/macrophage
migration [82].
914
Cytokines stimulate glomerular-resident cells
to express chemokines, including IL-8, MCP-1,
MIP-1α, and macrophage inflammatory protein
(MIL)-1α, which can lead to local inflammation
and glomerular injury. Chemokines are also
involved in nonimmunologic renal injuries,
including ischemia-reperfusion models and inter-
stitial nephritis [83].
Growth Factors
Growth factors are cytokines that function as cell-
to-cell signaling molecules, regulating cellular
processes, such as maturity, proliferation, differ-
entiation, and polarity. Growth factors bind to
specific receptors on cell membranes. Several
growth factors are involved in acute and chronic
glomerular injuries and affect the survival, prolif-
eration, migration, and matrix production in both
bone marrow-derived cells and intrinsic glomeru-
lar cells.
Platelet-Derived Growth Factor (PDGF)
PDGF acts primarily on mesenchymal cells, driv-
ing cell migration and mitogenesis, extracellular
matrix production, anti-inflammatory mediator
production, tissue permeability, and hemody-
namic regulation. Intrinsic glomerular cells that
are mesenchyme derived are plausible targets of
PDGF. PDGF is released by activated platelets,
activated macrophages, endothelial cells, and
smooth muscle cells. PDGF exists in five isoforms
(A, B, C, D, AB), which bind two receptor chains
(α, β) on the cell surface (receptor tyrosine
kinases). PDGF-B is a ligand for PDGF α-and
β -receptor, whereas PDGF-D binds β receptor.
PDGF activates intranuclear proto-oncogenes
and immediate early gene expression, which are
effectors of the receptor tyrosine kinase and
responsible for particular downstream functions.
PDGF-A and PDGF-B are secreted as homo- or
heterodimers, whereas PDGF-C and PDGF-D are
homodimers. The impressive glomerular struc-
tures seen in PDGF-B- and PDGFR-β-deficient
mice reveal a lack of mesangium and aberrantly
M. Nagata
developed glomerular tufts, indicating a key role
for PDGF in glomerular angiogenesis and cell
migration. The intrarenal distribution of PDGF
varies among species. PDGF-A is absent from
the glomerulus in humans and rodents, whereas
PDGFR is expressed in mesangial cells and endo-
thelial cells. In experimental glomerular diseases,
intense upregulation of PDGF-B and PDGFR-β is
observed. PDGFR-β is expressed in normal
human glomeruli, and this expression is
upregulated in all resident glomerular cells. Anti-
Thy1 nephritis shows expression of PDGF in
areas of mesangial proliferation, and blockade of
PDGF by neutralizing antibodies or aptamers
(PDGF receptor IgG) attenuates mesangial prolif-
eration in the acute and chronic phase. PDGF
antagonists also prevent glomerular damage in
anti-Thy1 nephritis, nephrotoxic serum nephritis,
and lupus nephritis models. PDGF-C is expressed
in podocytes and mesangial cells in areas of glo-
merular injury. PDGF-D has a similar action as
PDGF-B by β-receptor-mediated activation
[84]. Treatment with PDGF-D-neutralizing anti-
body in chronic progressive anti-Thy1 nephritis
resulted in attenuation of glomerulosclerosis
accompanied by suppression of monocytes/
macrophage influx and complement activation.
Stimulation of mesangial cells in vitro with differ-
ent PDGF ligands leads to cellular proliferation
and migration. PDGF induces extracellular matrix
production in mesangial cells, parietal cells, and
tubular cells. PDGF-AB stimulates the production
of TGF-β, CCL2, CXC3CL1, plasminogen acti-
vator inhibitor-1 (PAI-1), IL-6, endothelin, and
iNOS in mesangial cells. The role of PDGF for
kidney diseases is extensively reviewed [85].
Transforming Growth Factor (TGF)
TGF is known to be a superfamily of cytokines,
consisting of about 40 molecules, including the
TGF-β, activin, and BMP subfamilies. Several
family members involve many aspects in the kid-
ney, i.e., morphogenesis, cell differentiation,
inflammation, and remodeling. TGF-β is pro-
duced by a variety of cell types in an inactive
(latent) form, and proteolytic cleavage renders it
29 Immune-Mediated Glomerular Injury in Children
functional. TGF-β exerts its functions through its
type I and type II receptors. An intracellular sig-
naling pathway, involving Smad proteins, trans-
duces the TGF-β signal to the target genes,
including PAI-I and collagen type I (α2). TGF-β
has pleiotropic functions, and the effects differ by
cell type and dosage. It acts as a growth inhibitor
in epithelial cells, whereas it sometimes stimulates
cell proliferation and matrix synthesis in the
mesenchyme.
TGF-mediated glomerular injury includes inhi-
bition of cell growth/proliferation, apoptosis, cell
differentiation, angiogenesis, and fibrosis. TGF-β
stimulates proteoglycan, biglycan, and decorin pro-
duction in mesangial cells in vitro. The role of
TGF-β in immune-mediated glomerular injury is
not well understood. Latent TGF-β transgenic
mice have no renal disease, despite high levels of
circulating TGF-β. Overexpression of active TGF-β
results in severe renal damage. Neutralizing anti-
bodies to TGF-β attenuate anti-Thy1 nephritis.
Latent TGF-β overexpression in anti-GBM anti-
body nephritis showed suppression of inflammatory
cells and cytokines, by Smad7-mediated inhibition
of NF-κB-dependent inflammation [86]. In contrast,
blockade of TGF-β by adenovirus-mediated gene
transfer of the TGF-β IIR failed to attenuate anti-
GBM antibody nephritis. Blockade of TGF-β sig-
naling in the T cells of Smad7 transgenic mice
inhibited glomerulonephritis, only with a medium
dosage of anti-GBM antibodies. Smad7 gene ther-
apy ameliorates autoimmune crescentic glomerulo-
nephritis [87]. The pleiotropic and complex
functions of TGF-β are thought to be differentially
regulated in the phases and pathogenetic back-
ground of the renal diseases.
Fibroblast Growth Factor (FGF)
The FGFs are a family of growth factors involved
in angiogenesis, wound healing, and embryonic
development. The FGFs are heparin-binding pro-
teins that interact with cell-surface-associated
heparan sulfate proteoglycans, which are a pre-
requisite for FGF signal transduction. FGFs are
key players in the processes of proliferation and
differentiation in a wide variety of cells and
915
tissues. Among the 22 FGFs identified to date,
FGF-2 (basic-FGF) is the major factor investi-
gated in glomerular injury.
As a potent angiogenic factor, FGF-2 mediates
proliferation and matrix synthesis through binding
with receptors FGF-R1 to FGF-R4. FGF-2 is
increased in mesangial proliferative glomerulone-
phritis. Recombinant FGF-2 injection in rats leads
to podocyte mitosis, without cytokinesis, resulting
in severe nephrosis and focal segmental glomerulo-
sclerosis. Parietal epithelial cells express FGF-R2,
and FGF-2 stimulates matrix synthesis. FGF-1 and
its receptor are expressed in the glomeruli, but
precise function is still unknown.
Vascular Endothelial Growth Factor
(VEGF)
VEGF is known to regulate vasculogenesis and
angiogenesis, through effects on endothelial
mitogenesis. It is involved in the chemotaxis of
macrophages and granulocytes. Vasomotor activity
for VEGF has also been noted.
The VEGF family consists of five members
(A, B, C, D, PIGF), and VEGF-A is the most
intensively investigated. VEGF-A has four
isoforms, VEGF121, 165, 189, and 206; the for-
mer two are soluble and the latter two are cell
bound. In glomeruli, VEGF is constitutively and
most abundantly expressed by podocytes and
occasionally by activated mesangial cells. The
VEGF receptors, flt and flk-1, are expressed on
endothelial cells [88]. Inhibition of VEGF
worsens anti-Thy1 nephritis, and treatment with
VEGF attenuates thrombotic microangiopathy.
Systemic administration of VEGF 165 in anti-
GBM antibody nephritis reduced inflammation
and advanced glomerular repair [89]. Administra-
tion of sFlt-1 affected neither the infiltration of
macrophages nor the crescents.
Connective Tissue Growth Factor
(CTGF)
CTGF (CCN2) is a secreted, cysteine-rich,
matrix-associated, heparin-binding protein of
916
38 kDa. The primary function of CTGF is to
modulate and coordinate signaling responses
involving cell-surface proteoglycans. CTGF
operates the downstream of TGF-β and directly
enhances the TGF-β/Smad signaling pathway or
independently acts to promote fibrosis and wound
healing [90]. CTGF is expressed in the intrinsic
glomerular cells and tubular cells and potentiates
to promote renal scar formation. TNF-α is an
upregulator of CTGF, and CTGF rapidly activates
intracellular signaling and transcription of TGF-β
inducible early gene through neurotrophin recep-
tor TrkA in human mesangial cells. CTGF is
expressed in podocytes and PECs in normal glo-
meruli and binds perlican, a known component of
the GBM. Increased CTGF in PECs is associated
with matrix production. Upregulation of CTGF
mRNA is found during crescent formation in
humans and rats, and the expression is diminished
when the crescents become scars [91].
Epidermal Growth Factor (EGF)
EGF is a 53-kDa aminopeptide that was initially
identified in mice submandibular glands and
human urine. The renal production sites of EGF
are Henle’s loop and distal convoluted tubule.
EGF is known to be involved in cell survival and
the differentiation of tubular cells. Although the
urinary levels of EGF are a marker of renal injury
and urinary MCP-1/EGF seems to be a prognostic
factor in IgA nephropathy, the function(s) of EGF
in the glomeruli and its role in glomerular injury
are shortly reviewed [92]. EGF induces mesangial
cell contraction in vitro. Heparin-binding EGF is a
strong mitogen that stimulates mesangial prolifer-
ation in vitro and involved in the crescentic
glomerulonephritis.
Platelets and the Coagulation Cascade
As for vascular endothelial cells in general, glo-
merular endothelial cells normally possess
antiplatelet, anticoagulant, and fibrinolytic prop-
erties, to maintain the microcirculation in the glo-
merulus, and this is a very consistent background
M. Nagata
that coagulation and the fibrinolysis system are
involved in immune-mediated glomerulonephri-
tis. Local activation of the coagulation cascade of
the extrinsic pathway eventually produces fibrin
or thrombi (Fig. 15). Glomerular thrombi are
occasionally observed in the active phase of glo-
merulonephritis, reflecting severe endothelial
damage. Fibrin deposition is more frequently
noted in glomerular injury, with tuft necrosis and
thrombotic microangiopathy. The influences of
these products in glomerular injury are to acceler-
ate inflammation, rather than ischemia due to glo-
merular capillary obstruction. In human
glomerulonephritis, localization of coagulation
system-related molecules has been described,
and their functions have been tested in experimen-
tal models, using neutralizing antibodies or
knockout mice.
Platelets
Platelets are involved in glomerular injury,
through leukocyte recruitment, increased vascular
permeability, and the vasoactive effects of their
intrinsic chemical substances. Platelets contain
two types of cytoplasmic granules, α and σ. The
α granules express P-selectin on their membranes
and contain fibrinogen, fibronectin, factors V and
VIII, platelet-activating factor (PAF), platelet fac-
tor 4, PDGF, and TGF-αl. The σ granules contain
adenosine diphosphate (ADP), calcium ions, his-
tamine, and serotonin. In local injury in glomeru-
lar capillaries, platelets adhere to the extracellular
matrices at sites of endothelial injury and become
activated. Activated platelets secrete granular
products and synthesize TXA2, PDGF, and PAF,
which together stimulate clot formation, cell pro-
liferation, and matrix production. PAF is bioactive
and increases leukocyte chemotaxis, vascular per-
meability, and vascular spasms. Platelets include
phospholipid complexes that are important in the
intrinsic coagulation pathway. Injured or activated
endothelial cells trigger aggregation of platelets,
forming a clot, and exposing tissue factor, which
provokes capillary necrosis and fibrin exudates,
leading to GBM breaks and extraglomerular
lesions and cellular crescent formation.
29 Immune-Mediated Glomerular Injury in Children 917

Fig. 15 Coagulation is the Tissue injury

cascade promoting the Resident cells
transformation of soluble macrophages
fibrinogen into insoluble Inflammatory
fibrin monomers. The cells
cascade is initiated in the
injured tissue by exposure
of tissue factor (TF) at the Tissue factor
surface of the injured
endothelium. TF synthesis TFPI
is stimulated by VIIa, Ca2+
inflammatory cells, which
activated resident cells or Factor X Factor Xa
macrophages for
production. Tissue factor Thrombin
activated conversion of X to
Xa, which further converts Factor V Factor Va Ca2+
prothrombin to thrombin. Xlll
Thrombin participate Ca2+
conversion of fibrinogen to
fibrin, which finally forms Prothrombin Thrombin Xllla
cross-linked fibrin by XIIIa. (II) (lla)
TF pathway inhibitor Ca2+
(TFPI) is a potent inhibitor
Fibrin
of this cascade. Fibrinogen
(IIa)
(I)

Cross-linked fibrin

In addition, platelets activate the complement as
well as the intrinsic coagulation pathway and
release vasoactive amines which potentially mod-
ulate pathological response in glomerular-resident
cells and inflammatory cells.
Fibrin
Fibrin is an insoluble fibrous protein that is the
end product of a locally accelerated coagulation
system. Fibrin stabilizes and anchors aggregated
platelets. In glomerular diseases, fibrin is occa-
sionally deposited in the capillary or mesangium.
In IgA glomerulonephritis soluble fibrin deposi-
tion is located in the mesangial proliferation of
active stage [93]. Fibrin is detected in the capillary
necrosis in cellular crescentic formation. Cross-
linked fibrin, immunoglobulin deposition, and C3
are occasionally seen in glomerulonephritis, and
co-localization of factor V with fibrin in the
mesangium indicates local activation of the coag-
ulation cascade in glomerulonephritis.
Fibrin deposition is mediated largely by the
stimulation of macrophage procoagulant activity.
Most of the fibrin deposition associated with
immunologic glomerular injury is mediated by
activation of the extrinsic coagulation cascade,
commencing with tissue factor procoagulants
[94]. In crescentic glomerulonephritis, fibrin
deposition, renal pathology, and dysfunction are
synchronous with glomerular macrophage and
T-cell influx. In experimental models, glomerulo-
nephritis is attenuated by the administration of
anti-factor V antibody. Defibrinogenation sup-
presses proteinuria in crescentic glomerulonephri-
tis, although it has no effect on the inflammatory
influx, indicating that the coagulation system pro-
motes crescentic glomerulonephritis indepen-
dently of the inflammatory reaction [95].
918
Activation of the local coagulation system can be
initiated by immunologic effects, although sec-
ondary events, which include intrinsic glomerular
cell proliferation, microcirculation defects, and
cell necrosis, are promoted by bioactive mole-
cules of the coagulation system. In the activation
of coagulation system, the fibrinolytic cascade
also limits clot formation. Fibrinolytic agents
have shown relatively consistent efficacy in atten-
uating immune-mediated glomerular injury in
several models.
Tissue Factor
Tissue factor is a cellular lipoprotein that is present
at sites of tissue injury and activates extrinsic coag-
ulation processes. In glomerular injury, inflamma-
tory cell-derived cytokines induce tissue factor
secretion by resident cells and macrophages. Endo-
thelial tissue factor release initiates glomerular
fibrin deposition, through activation of the extrinsic
coagulation pathway, by binding circulating factor
VII and facilitating its allosteric conversion to fac-
tor VIIa. Tissue factor converts X to Xa, and factor
V is a cofactor for the Xa, which is responsible for
prothrombin activation. Factor Va then converts
prothrombin to thrombin, which further converts
fibrinogen to fibrin (Fig. 15). Tissue factor also has
fibrin-independent proinflammatory effects, medi-
ated by MHC class II protein expression and leu-
kocyte accumulation.
Specific Xa inhibitor ameliorates anti-Thy
1 nephritis by inhibiting macrophage infiltration
and fibrin accumulation, suggesting that tissue
factor promotes mesangial proliferation by
function of proinflammatory and procoagulant
mechanism of factor X [96]. Administration of
anti-factor V neutralizing antibody suppressed
profibrotic cytokines in glomeruli. Inhibition of
tissue factor activity by an antibody or tissue
factor pathway inhibitor (TFPI) significantly
ameliorates the development of crescentic
glomerulonephritis, accompanied by decreased
glomerular inflammatory cell influx. Infusion of
recombinant TFPI ameliorates glomerular fibrin
deposition and glomerular pathology in experi-
mental glomerulonephritis.
M. Nagata
The Enzymatic Fibrinolysis System
In addition to the progression of coagulation pro-
cesses in tissues, the clotting cascade simulta-
neously sets into motion a fibrinolytic cascade
that restricts final clot size, which may influence
tissue scarring. The fibrinolytic system consists of
an inactive proenzyme (plasminogen), which can
be converted to an active enzyme (plasmin) that
degrades fibrin into soluble fibrin products. This
system is under the control of tissue-repair-related
molecules, including plasmin. Plasmin is derived
from the enzymatic breakdown of its inactive
circulating precursor, plasminogen, either by a
factor XII-dependent pathway or by plasminogen
activators. Plasmin is involved in fibrinolysis.
Activation of plasmin significantly involves
matrix degradation and fibrinolysis, which influ-
ences the fate of tissue injury. Activation of plas-
min inhibits fibrosis by enhancing matrix
metalloproteinase 2 activation, thereby promoting
ECM degradation. Plasminogen activator (PA) is
a serine protease that converts plasminogen to
plasmin to regulate ECM tissue deposition. Two
plasminogen activators, tissue type (t-PA) and
urokinase-type (u-PA), are known. t-PA is synthe-
sized primarily by endothelial cells and is active
when attached to fibrin, which it dissolves. Plas-
minogen binds to the plasminogen activator
receptor (PAR), which belongs to the G protein-
coupled receptor family, and activation of PAR
leads to cytoskeletal rearrangements, proliferation
of fibroblasts, and glomerular epithelial cell pro-
liferation. Thrombin also interacts with PAR.
PA and plasmin are suppressed in some glomer-
ulonephritis conditions and may be involved in
mesangial matrix accumulation via α1-antitrypsin,
α2-macroglobulin, or plasminogen activator inhib-
itor (PAI). PAI is synthesized by endothelial cells to
modulate the coagulation/anticoagulation balance
by blocking fibrinolysis. PAI-1 is a single glyco-
protein of about 45 kDa consisting of 379 or
381 amino acids. It is a member of the serpin family
with reactive site peptide bound Arg345-Met346.
Activation of PAI-1 requires complex processes.
PAI-1 promotes fibrosis by inhibiting plasmin gen-
eration, through the suppression of plasminogen
activator. PAI-1 is also activated by interaction
29 Immune-Mediated Glomerular Injury in Children
with TGF-β, and it can modulate fibrosis and
PAI-1, downregulating TGF-β activity in a nega-
tive feedback loop. PAI-1 deficiency leads to path-
ogenic over-activation of TGF-β.
PAI-1 is increased in glomerulonephritis, and
plasmin-independent profibrotic action of PAI-1
in glomerular injury involves the recruitment of
fibroblasts and macrophages. Experimental
cryoglobulin-associated mesangial proliferative
glomerulonephritis reveals upregulation of the
protease Nexin-1, t-PA, and PAI-1 [97]. Induction
of crescentic glomerulonephritis in PAI-1-defi-
cient mice resulted in improvements in nephritis,
accompanied by depressed inflammatory infil-
trates and collagen accumulation.
Protease-activated receptor 2 (PAR-2), receptor
of factor Xa, is expressed in epithelial, mesangial,
and endothelial cells and macrophages. PAR-2 is
activated by serine proteases, such as trypsin,
tryptase, and factors VIIa and Xa and increases
PAI-1 expression and macrophage-derived cytokine
induction. PAR-2 is involved in human mesangial
cell proliferation. Experiments with PAR-2-defi-
cient mice revealed that PAR-2 promoted glomeru-
lar fibrin deposition and the progression of
crescentic glomerulonephritis, due to stimulation
of renal PAI-1 expression and MMP-9 activation
[98] Thrombin-activatable fibrinolysis inhibitor
(TAFI) is a potent inhibitor of plasmin generation,
and immune complex-mediated glomerulonephritis
is ameliorated in TAFI-deficient mice.
Although the coagulation cascade/fibrinolysis
system is substantially involved in the acute and
exudative phases of glomerular injury, by interacting
with inflammatory cells, its roles in chronic progres-
sive glomerulonephritis remain unknown. Further
studies are needed to investigate the coagulation/
fibrinolysis-independent actions of PAI-1 and tissue
factor, such as inflammatory responses, cell migra-
tion, cell proliferation, angiogenic actions, and
matrix remodeling in glomerular injury.
Eicosanoids
Eicosanoids are short-acting, hormonelike sub-
stances. They are arachidonic acid (AA) metabo-
lites and have effects in inflammation and
919
hemostasis. AA is a 20-carbon polyunsaturated
fatty acid that is present mainly in cell membrane
phospholipids. Mechanical, chemical, and physi-
ologic stimuli release AA from the cell membrane
into the cytoplasm, and specific enzymes then
synthesize the metabolites. The AA cascade
branches into two major pathways, the cyclooxy-
genase and lipoxygenase pathways, both of which
are involved in glomerular injury (Fig. 16).
The cyclooxygenase pathway products include
prostaglandins (PG) E2, PGD2, PDF2a, PGI2 (pros-
tacyclin), and thromboxane (TX) A2. The
lipoxygenase pathway begins with 5-lipoxygenase,
a well-characterized AA-metabolizing enzyme.
Three major products, 5-hydroxyeicosatetraenoic
acid (HETE), the leukotriene (LT) family of com-
pounds, and the lipoxins, are known.
The restricted distribution of enzymes may
determine the cell-specific synthesis of certain
substances and cell to cell transaction to produce
active end products. For example, the AA in neu-
trophils is converted into LTA4 followed by
LTB4, which is the end product in neutrophils,
since neutrophils lack the further converting
enzyme of LTC4 synthase, and it is then secreted
from the neutrophils. In contrast, platelets lack the
5-lipoxygenase enzyme but have 12-lipoxygenase
and LTC4 synthase. Thus, in platelets, the LTB4
derived from neutrophils is converted into lipoxin
and LTC4, both of which are final platelet prod-
ucts. This transcellular interaction of AA metab-
olites promotes inflammation in situ, resulting in
biologic responses by the cells. Inflammatory
mediators, such as complement components,
also accelerate the release of these metabolites.
Common functions among the eicosanoids are
vasomotor effects; TXA2 and LTs cause vasocon-
striction, whereas PGs, PGI2, and lipoxin partici-
pate in vasodilatation. LTB4 and lipoxins also
promote chemotaxis and leukocyte adhesion.
LTs affect vascular permeability. All these func-
tions of AA products are involved in the processes
of glomerular inflammation and renal function.
Considering wide-ranged actions of AA
metabolites for inflammation and tissue damage/
repair, their roles for glomerular injury are likely
important. AA involvement in glomerulonephritis
has been extensively investigated before 2000
920
Fig. 16 The arachidonic acid cascade and metabolites.
Generation of arachidonic acid metabolites and their roles
in inflammation. Note enzymatic activities whose inhibi-
tion through pharmacologic intervention blocks major
pathways (Denoted with COX-1 and COX-2
mainly using cell lines to demonstrate prolifera-
tion and matrix production or administration of
inhibitors in experimental models.
There are two forms of cyclooxygenase,
COX-1 (constitutive) and COX-2 (inducible).
COX-1 is constitutively expressed in the kidney,
including in the mesangial cells, arterioles, PECs,
and cortical and medullary collecting ducts.
COX-2 is an inducible protein; its mRNA is
detectable in the macula densa and the cortical
thick ascending limb cells immediately adjacent
to the macula densa, whereas it is not detected in
the glomeruli. COX-2 expression in the glomeruli
may appear in glomerulonephritis. In particular,
podocyte expression of COX-2 is prominent in
renal ablation models and anti-Thy1 nephritis.
Furthermore, podocyte-specific COX-2 expres-
sion, driven by the nephrin promoter, aggravates
proteinuria and upregulates endogenous COX-2.
M. Nagata
cyclooxygenase 1 and 2, HETE hydroxyeicosatetraenoic
acid, HPETE hydroperoxyeicosatetraenoic acid The figure
was published in Kumar et al. Robbins Basic Pathology,
8th edn. 2007, p. 48, Copyright Elsevier)
Podocyte COX-2 expression is induced by glo-
merular inflammation or various stresses and ren-
ders podocytes susceptible to further injury [99].
PGE2 is primarily produced by mesangial
cells. In acute glomerular inflammation, PGE2 is
increased to maintain effective renal circulation
by relaxing vascular smooth muscle cells.
Another important function of PGE2 is its anti-
inflammatory actions in suppressing inflamma-
tory cell recruitment, chemokine synthesis, and
NO production. PGI2 is a vasodilator and a potent
inhibitor of platelet aggregation. Glomerular dam-
age with endothelial injury may result in
decreased PGI2 in situ and may promote further
glomerular injury. TXA2 has largely opposite
effects to PGI2 and is abundantly synthesized in
nephritic glomeruli. TXA2 is produced by inflam-
matory cells and intrinsic glomerular cells and
accelerates glomerular injury, through platelet
29 Immune-Mediated Glomerular Injury in Children
aggregation and vasoconstriction. The LTs, a fam-
ily of compounds converted by 5-lipoxygenase,
include LTA4, LTB4, LTC4, LTD4, and LTE4.
LTB4 is a potent chemotactic compound that pro-
motes the recruitment of neutrophils and is ele-
vated in experimental models with glomerular
damage. LTC4, LTD4, and LTE4 participate in
vasoconstriction and increase vascular permeabil-
ity. Receptor antagonists of LTs attenuate glomer-
ular injury.
Lipoxins are the products of a two-step
lipoxygenation, i.e., 5-lipoxygenase followed by
12-lipoxygenase. Lipoxins are produced locally at
sites of inflammation by the transcellular interac-
tion of neutrophils, platelets, and intrinsic cells.
The platelets themselves cannot produce lipoxin,
so they take up neutrophil-derived LTA4 and con-
vert it into lipoxin. Lipoxins have both pro- and
anti-inflammatory actions. Lipoxin A4 inhibits
cytokine-induced mesangial cell proliferation
in vitro and has been implicated as anti-
inflammatory function in glomerulonephritis.
Aspirin and nonsteroidal anti-inflammatory drugs
(NSAIDs) inhibit all the steps upstream of COX
activity. In acute poststreptococcal glomerulone-
phritis in children, lipoxin A [5] and
15-lipoxygenase (15-LO) were expressed in leuko-
cytes and glomeruli, and 15-LO products play anti-
inflammatory roles in this disease [100]. Dietary
manipulation, leading to changes in eicosanoid
metabolites, has been reported to affect glomerulo-
nephritis in humans. Dietary fish oil that contains
omega-3 fatty acids (eicosapentaenoic acid and
docosahexaenoic acid) modulates both COX and
lipoxygenase metabolism. In addition, dietary
restriction of essential fatty acids impairs renal
production of a specific lipid-derived macrophage
chemoattractant and LTB4, resulting in decreased
production of cytokines, ROS, and NO in
macrophages.
Reactive Oxygen Species (ROS)
ROS are chemical species with a single unpaired
electron in an outer orbital. They are extremely
unstable and readily react with various chemicals.
Generation of ROS can occur via the rapid
921
activation of NADPH oxidase, which oxidizes
NADPH and reduces oxygen to the superoxide
anion (O2). ROS are produced within the lyso-
some and may be released from leukocytes by
exposure to chemotactic agents, immune com-
plexes, complement, and other factors. In addi-
tion, ROS are occasionally released by intrinsic
glomerular cells. ROS are inherently unstable and
generally decay spontaneously. The influence of
oxygen-derived free radicals in inflammatory
reactions depends on the balance between the
production and inactivation of the related
metabolites.
The biologic actions of ROS include the stim-
ulation of expression of chemokines, cytokines,
and endothelial leukocyte adhesion molecules.
In addition, ROS stimulate intracellular signaling
molecules that are involved in mitogenic path-
ways. The superoxide anion, hydrogen peroxide,
and hydroxyl radical are major species of ROS.
The superoxide anion typically is the most abun-
dant, though its potential for tissue injury is lim-
ited. Hydroxyl radicals induced by the Haber
Weiss reaction promote considerable tissue dam-
age. Activated leukocytes in the inflammatory
milieu synthesize ROS and stimulate endothelial
ROS production through xanthine oxidation,
resulting in increased vascular permeability. Acti-
vation of NADPH oxidase is involved in cell
proliferation and extracellular matrix production,
mediated by intracellular signaling that includes
the mitogen-activated protein kinase (MAPK) and
activator protein 1 (AP-1) or TGF-β. In the acute
phase of anti-Thy1 nephritis, ROS- and NADPH-
dependent oxidase activities are increased. Anti-
oxidants suppress proliferative anti-GBM glomer-
ulonephritis [101]. Direct infusion of H2O2 in rat
renal arteries causes proteinuria, and H2O2 deple-
tion by catalase reduces neutrophil-dependent
proteinuria, suggesting that ROS play a particu-
larly significant role in the promotion of
neutrophil-dependent glomerular injury. Like-
wise, ROS are involved in ANCA-associated
glomerulonephritis.
Tissue fluids and the target cells of oxygen-
derived radicals have antioxidant mechanisms to
protect against harmful species. Several enzy-
matic and nonenzymatic antioxidant systems
922
inactivate free radicals. Superoxide dismutase
(SOD) significantly accelerates the spontaneous
decay of ROS. Glutathione peroxidase also pro-
tects against ROS-mediated cell injury by catalyz-
ing free radical breakdown. Catalase, which is
present in cytoplasmic peroxisomes, directs the
degradation of hydrogen peroxide. Exogenous
antioxidants (vitamins E, A, C, β-carotene) block
the formation of free radicals or scavenge such
products. The effects of these antioxidants on
glomerulonephritis have been reported, although
presently there is insufficient evidence regarding
their effects.
Nitric Oxide (NO)
NO, which is a pleiotropic mediator of inflamma-
tion initially discovered as the endothelial cell-
derived factor relaxing vascular smooth muscle
cells, produces vasodilatation. NO is a soluble gas
that is produced by endothelial cells and macro-
phages and is a typical cytotoxic substances of
macrophages. NO production is stimulated by
acute inflammatory molecules, including TNF-α,
ILs, and INFs. As the half-life of NO is extremely
short, NO action is limited to the immediate prox-
imity of the production site. NO acts on target cells
through a paracrine mechanism and induces stable
products, such as nitrates, nitrites (NO2), and cyclic
GMP-mediated intracellular alterations.
Three NO synthase isoforms have been distin-
guished in mammalian species: the constitutive
calcium-dependent neuronal NOS (nNOS) and
endothelial NOS (eNOS) and the inducible
calcium-independent iNOS. In the glomerulus,
eNOS is constitutively expressed in endothelial
cells, and occasionally, iNOS transcripts are
detected.
In acute-phase glomerulonephritis, macro-
phages are the major source of NO. iNOS is
induced in glomerular capillary inflammation, and
glomerular NO2 is increased in anti-GBM antibody
nephritis models. NO2 expression is relatively
acute and transient. Early studies showed that
decreasing NO production by L-arginine depletion
in rat nephrotoxic serum nephritis exacerbated pro-
teinuria. Increased NO synthesis observed in acute
M. Nagata
models of glomerular injury is not only a promo-
tive factor, but also has protective roles. In fact,
iNOS inhibitor in Wistar Kyoto rats with nephro-
toxic serum nephritis showed reduced crescentic
formation, and eNOS-deficient mice with anti-
GBM nephritis reveal attenuation of nephritis indi-
cating protective roles for NOS in glomerular
injury. Anti-Thy1 nephritis shows an association
between NO synthesis and mesangiolysis, and sup-
pression of NO resulted in reduced mesangiolysis.
Another experiment using anti-GBM antibody
nephritis in iNOS-deficient mice revealed no effect
on glomerular histology, macrophage-mediated
proteinuria, or glomerular tissue damage. In
humans, iNOS expression has been observed in
proliferative lupus nephritis and IgA glomerulone-
phritis, although iNOS expression is not associated
with the presence of macrophages and its function
remains to be investigated more thoroughly. Endo-
thelial NOS reduced glomerular injury through
suppression of MCP-1 and ROS production in
lupus glomerulonephritis model [102].
Acknowledgment The author’s research and academic
efforts are supported by Grants-in-Aid for Scientific
Research (C) 19,590,932 and Scientific Research
(S) 16,109,005 from the Japan Society for the Promotion
of Science. Given a limited capacity for citation, the refer-
ence includes recent review articles, despite numerous
important original works published before 2005. The
author suggests the readers to reference Chap. 29 of fifth
and sixth edition of Pediatric Nephrology (Immune mech-
anism of glomerular injury) which referenced earlier
published works.
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 Complement-Mediated Glomerular
Injury in Children 30
Zoltán Prohászka, Marina Vivarelli, and George S. Reusz

Contents Cell Death and Recovery in Response

to Complement Injury: The Lytic Terminal
Pathophysiology of the Complement Pathway Complement Complex . . . . . . . . . . . . . . . . . . . . . 944
System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 928
Outline of the Complement System . . . . . . . . . . . . . . . . . 928 Illustrative Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 945
Complement Activation Pathways . . . . . . . . . . . . . . . . . . 928 An Overview of the Mechanisms of
Control of Complement Activation . . . . . . . . . . . . . . . . . . 936 Complement-Mediated Glomerular Injury . . . . . . . . . . 945
Downstream Effects of Complement The Clinical Presentation of Glomerular
Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 938 Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 945
Complement Activation in Kidney Diseases . . . . . . . . 939 Antibody-Mediated Allograft Rejection
(AMR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 952
Molecular Mechanisms of Complement- Tubulointerstitial Injury in Progressive
Mediated Renal Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 941 Kidney Disease: The Role of Complement . . . . . . . . . 953
Role of Structural Properties of
Kidney Components and of Local Production Therapeutic Inhibition of Complement
of Complement Factors and Regulators . . . . . . . . . . . . . 942 in Glomerular Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 953
Cellular Activation and Proliferation
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 954
in Response to Complement Activation:
The Non-lytic Terminal Pathway Complement
Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 942

Z. Prohászka (*)
3rd Department of Medicine, Faculty of Medicine,
Semmelweis University, Budapest, Hungary
e-mail: prohoz@kut.sote.hu
M. Vivarelli
Division of Nephrology and Dialysis, Children’s Hospital
Bambino Gesù-IRCCS, Rome, Italy
G.S. Reusz
1st Department of Pediatrics, Faculty of Medicine,
Semmelweis University, Budapest, Hungary

# Springer-Verlag Berlin Heidelberg 2016 927

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_26
928
Pathophysiology of the Complement
System
Outline of the Complement System
The complement system comprises about 30–40
proteins, glycoproteins, and membrane receptors
that are present in body fluids and on cell surfaces
(Tables 1, 2, and 3). Nearly all the soluble proteins
are produced by the liver, but most are also syn-
thesized in other tissues, including cells of the
myeloid lineage and renal cells. Complement is
considered part of the immune system, as its main
biological function is the lysis and removal of
pathogens by opsonization. In the past decade,
new studies have revealed a pivotal role of com-
plement in immune surveillance, the generation of
inflammation, the initiation of antibody produc-
tion, and tissue repair [1–3].
Complement recognition proteins can bind to a
variety of targets. They can trigger enzymatic
cascade reactions (complement activation path-
ways), can opsonize and lyse particles or cells,
and can release several biologically active small
proteins, termed anaphylatoxins. The activity of
this system is tightly controlled by a number of
soluble and surface-bound regulatory proteins.
The pathophysiological alterations of the comple-
ment system include overactivation and consump-
tion, secondary to the sustained presence of
activators, deficiencies secondary to missing com-
plement factors, or dysregulation, secondary to
altered regulatory proteins [3].
All complement activation pathways result in
the formation of the C3 convertase complex,
which activates the central C3 component
(Fig. 1). This in turn triggers the formation of the
C5 convertase, resulting in C5 cleavage, which
marks the beginning of the terminal pathway.
Complement components also interact with
other systems [4]. Activated proteases of the coag-
ulation system, for example, can activate C3
directly, bypassing traditional complement activa-
tion pathways. Likewise, recognition molecules
of the lectin pathway bind fibrinogen or fibrin
[5], whereas complement protease MASP-2 can
activate prothrombin [6]. Other observations
also suggest that complement plays an important
Z. Prohászka et al.
role in platelet activation and cell contact sys-
tem [7]. Moreover, complement components are
important regulators of cellular immunity mecha-
nisms through their role in pathways involved
in pattern recognition (e.g., toll-like receptors)
or can stimulate cell-surface receptors through
anaphylatoxins [8].
Overall, the complement system has general
sensing functions that trigger powerful biological
actions under tight control from regulatory pro-
teins. It represents an essential triage mechanism
that constantly protects the organism against
threats such as pathogenic microorganisms or
cell debris. When properly activated, complement
promotes cellular innate immunity and initiates
protective immunological processes. Improper or
insufficient activation can lead to loss of self-
tolerance, to the development of autoimmunity,
or to tissue damage [3].
Genetic variants and differences in copy num-
ber of genes encoding for complement proteins
and regulators together establish the complotype
of any given individual. The intensity of the
complement response and of the downstream
inflammatory reaction is largely dependent on
this complex haplotype, which can predispose
to a number of complement-related diseases,
including autoimmune, inflammatory, auto-
inflammatory, degenerative, hematologic, ische-
mic, or renal disorders [9].
The aim of this chapter is to provide an over-
view of the complement system and of the molec-
ular mechanisms of complement-mediated
glomerular damage, including diagnostic and
therapeutic tools.
Complement Activation Pathways
The general mode of activation of the complement
system is similar to that of other plasma protease
cascades (e.g., to the coagulation system). Struc-
tural changes in recognition complexes trigger the
activation of proteases that in turn activate other
zymogen proteases through limited proteolysis,
leading to a cascade of reactions that result in the
formation of heterocomplexes and in the release
of anaphylatoxins (Fig. 1, Table 1). Complement
30 Complement-Mediated Glomerular Injury in Children 929

Table 1 Components of the complement pathways

Average
serum
Activator(s) of this This component concentration Molecular
Component Attributes component activates (mg/L) mass (kDa)
C3 The central C3 convertases of the C5 (as part of the C5 1,200–1,300 190
component of all classical/lectin convertase)
three (C4b2a) or alternative
complement (C3bBbP) pathways
pathways
Factor D Serine protease Activity of FD is FB 1 93
increased if bound to
C3bB
Factor B Serine protease, FD C3 200 25
binds to C3b
Properdin Recognition Binds to target surfaces and stabilizes the AP C3 25 55
component and convertase (monomeric
positive form)
regulator of the
AP. Does not
have enzymatic
activity
C1q 18 chain (6  IgG and IgM immune C1r 100–180 460
[ABC]) complexes, C-reactive
recognition protein, viral proteins,
component of cardiolipin,
the CP. Does not lipopolysaccharide,
have enzymatic polyanions, etc.
activity
C1r Serine protease Facilitation of C1s 50 85
autoactivation by the
conformational
change of C1q
C1s Serine protease C1r2 C4 50 85
C4 Common C1r2s2/MASP- Component of the C3 300–600 210
component of 12MASP-22 convertase
the classical and
lectin pathways
C2 Serine protease C1r2s2C4b or C3 20–25 110
MASP-12MASP-
22-C4b
MBL Carbohydrate Carbohydrates MASP-1 0.002–10 96 (trimer)
recognition (mannose > N-
macromolecule acetylglucosamine >
(2  (3  6) L-fucose > N-
chains) acetylmannosamine
> glucose)
Collectin Carbohydrate Carbohydrates MASP-1 0.28 200 (dimer
11 recognition (mannose > N- of trimeric
molecule acetylglucosamine > subunits)
L-fucose)
Ficolin-1 Recognition of Microbial surfaces, MASP-1 0.06 35
Ficolin-2 carbohydrate apoptotic bodies, 5 34
Ficolin-3 structures and altered self-structures 30 33
acetylated
molecules
(continued)
930 Z. Prohászka et al.

Table 1 (continued)
Average
serum
Activator(s) of this This component concentration Molecular
Component Attributes component activates (mg/L) mass (kDa)
MASP-1 Serine protease Facilitation of MASP-2 1.5–13 93
autoactivation by
MBL
MASP-2 Serine protease MASP-1 C4 0.5 76
C5 Binds C6 C5 is cleaved by the Components of the 70 190
C6 Binds C7 classical/lectin terminal pathway lack 60 110
C7 Binds C8 pathway (C4b2a3b) enzymatic activity, 55 10
or by the alternative conformational
C8 Binds C9 55 150
pathway (C3bBb3b) change of C5 upon
C9 Polymerization, C5 convertases cleavage results in 60 70
pore formation sequential binding of
C6–C9,
polymerization of C9
and pore formation

activation is heavily influenced by multiple fac-
tors, such as complement regulators, composition
of target surfaces, and blood flow.
Complement Component 3 (C3): The
Pivotal Molecule of Complement
Activation Pathways
Complement component C3 is a pivotal molecule
that is common to the three main complement
pathways [10]. C3 is the most abundant comple-
ment protein in human plasma. It is produced
mainly by the liver but also by other cell types.
C3 plasma concentrations are approximately
1.2–1.3 g/L. C3 is produced as a pre-pro single-
chain molecule including a 22-amino-acid leader
peptide and a four-amino-acid cleavage site.
Mature C3 has two chains, alpha and beta, that
are linked by a disulfide bond. Two major mech-
anisms for C3 activation have been recognized,
namely spontaneous and proteolytic activation
mechanisms, both resulting in conformational
changes that are essential for the downstream
activation of the terminal pathway. The C3
convertase activates C3 by cleaving the alpha
chain, thereby releasing C3a anaphylatoxin and
generating C3b. Active C3 convertases form as a
result of the activation of all three complement
pathways. Alternatively, spontaneous activation
of C3 can also take place through the hydrolysis
of the internal thioester bond, yielding C3(H2O).
Once formed, C3b can bind covalently to cell-
surface carbohydrates or to other macromole-
cules, via its reactive thioester group. C3b also
has opsonizing and immunoregulatory functions.
Surface-bound C3b is rapidly broken down by
factor I into the inactivated form of C3b (iC3b,
see below) and into C3f (Fig. 2a). iC3b is further
degraded by several proteases into C3dg and C3d,
as well as into C3c (a large fragment cleaved off
from the surface). Complement regulators may
facilitate the dissociation of the enzyme complex,
thereby accelerating its decay (Fig. 2b).
The Alternative Pathway
The alternative pathway (AP) of complement is
phylogenetically the oldest of the complement
pathways. Until recently, initiation of the AP
was not attributed to the recognition of specific
molecules [11]. The AP relies primarily on the
spontaneous, slow hydrolysis of C3 in the plasma
to form C3(H2O), a mechanism also termed the
tickover mechanism. Tickover of C3 results in
conformational changes that increase its binding
to factor B, allowing cleavage of factor B by factor
D. Factor D is a self-inhibitory serine protease
that is produced by adipocytes [12]. Factor B is a
serine protease that is produced in a zymogen
form. When bound to C3b, factor B becomes
30 Complement-Mediated Glomerular Injury in Children 931

Table 2 Soluble (fluid-phase) complement control proteins

Serum Molecular
concentration mass
Component Attributes Binds to Effect (mg/L) (kDa)
C1-inhibitor Serine protease C1r2s2 and Dissociation and 200–240 105
inhibitor (Serpin) MASPs covalent binding of
serine proteases of
recognition
complexes
C4b-binding protein CCP domain C4b, heparin Inactivation of CP C3 150–300 67 (alpha
containing convertase, cofactor chain)
molecule of factor I
(7 chains)
Factor H CCP domain Glycans with Inactivation of AP C3 200–600 150
containing terminal sialic convertase, cofactor
molecule acid, of factor I
(1 chains) C-reactive
protein, C3b,
heparin
Factor I Serine protease C3b and C4b Cofactor-dependent 30–50 88
cleavage of C4b and
C3b
Vitronectin Multidomain C5, C6, C7, Competes with MAC 300–500 85
regulator of C8, C9, and for binding to
coagulation, beta-1 membranes and
fibrinolysis, and integrins, inhibition of C9
complement heparin incorporation into
cascades MAC
Clusterin Highly conserved C7, C8, and Inhibition of MAC 50–100 80
heterodimer with C9 formation
five disulfide
bonds
Carboxypeptidase N Serum C3a, C4a, C5a Inactivation of 35 310
metalloprotease anaphylatoxins and
kinins by the
cleavage of terminal
basic amino acids

susceptible to cleavage by factor target surfaces. The composition of target cell

D. Conformational changes occur after formation
of C3bB, resulting in an active enzyme form. This
results in the production of a large, proteolytically
active Bb molecule and of a smaller Ba
fragment [1].
The fluid-phase C3bBb protein complex forms
the alternative pathway C3 convertase, which in
turn will cleave more C3 in the plasma. C3b
molecules that are generated bind covalently to
cell-surface molecules. Factor B attaches to bound
C3b to form C3bB. Factor D then produces a
surface-bound alternative pathway convertase,
which in turn can cleave more C3 and initiate
the alternative pathway amplification loop on
surfaces, the presence of complement regulators,
and the characteristics of blood flow over cell
surfaces are all important determinants of magni-
tude of local alternative pathway activation.
C3bBb is an unstable enzyme with a dissociation
half-life of Bb from C3b of approximately 2–3
min. Properdin binding to the C3bBb stabilizes
the complex and prolongs its half-life to 20–30
min. Properdin is a multimeric molecule (3 
7 monomers arranged in a triangular array) with
multiple binding sites that recognizes charge clus-
ters and probably other motifs on zymosan, rabbit
erythrocytes, and late apoptotic or necrotic cells,
which are all known activators of the AP. As first
932 Z. Prohászka et al.

Table 3 Cell-surface (membrane-bound) complement control proteins and receptors

Molecular
mass
Receptors Attributes Ligand(s) Function Expression (kDa)
CR1 (CD35) Single-chain C3b, C4b Cofactor for factor I Erythrocytes, cells of 190–260
transmembrane Decay-accelerating myeloid and lymphoid
receptor with activity lineages, dendritic
CCP domains cells, follicular
dendritic cells,
podocytes
Membrane Single-chain C3b, C4b Cofactor for factor I Nucleated cells 45
cofactor protein transmembrane
(MCP, CD46) receptor with
CCP domains
Thrombomodulin Single-chain Prothrombin, Increases CFH-dependent Endothelial cells 60
transmembrane protein C, and factor I-mediated
receptor with plasminogen degradation of C3b,
C-type lectin activator facilitates the degradation
domain inhibitor-1 of anaphylatoxins
Decay- GPI-anchored C3bBb and Facilitation of dissociation Nucleated cells 70
accelerating single-chain C4b2a of CP and AP C3 and C5
factor (DAF, molecule convertases
CD55)
CD59 (protectin) GPI-anchored C8, C9 Inhibits MAC formation, Nucleated cells 18
single-chain protection of “innocent
molecule bystander” cells from
action of MAC
CR2 (CD21) Single-chain C3d, C3dg Facilitation of antigen B lymphocytes, 120
transmembrane presentation and B-cell activated T
receptor with responses lymphocytes, follicular
CCP domains dendritic cells
CR3 (CD11b/ Heterodimer iC3b Facilitation of Monocytes, 165/95
CD18) phagocytosis macrophages,
neutrophil
granulocytes, NK cells,
and T cells
CR4 (CD11c/ Heterodimer iC3b Facilitation of Monocytes, 165/95
CD18) phagocytosis macrophages,
neutrophil
granulocytes, NK cells,
and T cells
C3aR 7TM GPCR C3a Cell activation (see the Mast cells, neutrophil, 53
text) eosinophil, basophil
granulocytes,
monocytes,
macrophages,
platelets, neurons
C5aR (CD88) 7TM GPCR C5a, Cell activation (see the Mast cells, basophil 40
C5a-desArg text) granulocytes,
monocytes,
macrophages, platelets
cC1qR CD91 and Collagen part Facilitation of Leukocytes 500 and 48
calreticulin of defense phagocytosis
lectins
GPI glycosylphosphatidylinositol, 7TM GPCR 7-transmembrane domain G-protein-coupled receptor
30 Complement-Mediated Glomerular Injury in Children
Activators of classical pathway: Activators of lectin pathway:
Antibodies, acute phase proteins, Repetitive carbohydrate structures
Apoptotic and necrotic bodies Altered self structures
C1q, C1r-C1s MBL, Ficolins, MASPs
C1-INH
C4, C2
Factor I
C4BP
C3 convertases
Action of defence collagens, C3b, C3d, C3dg:
Opsonisation, facilitation of phagocytosis
Cell activation
Regulation of adaptive immunity
C5 convertases
CD59
Terminal pathway
Membrane attack complex
Fig. 1 A simplified scheme of the complement system.
The activators (boxes with arrows) of the complement
recognition complexes of the classical and lectin pathways
induce conformational changes. These result in the auto-
and hetero-activation of zymogen serine proteases (C1s,
C1r, and MASPs) and in the formation of C3 convertase
enzyme complex of the CP and LP. The surfaces initiating
alternative pathway activation are characterized by proper-
din binding. The amplification of C3 activation may follow
– mediated by factor B and factor D (alternative pathway
amplification loop), but C3 may undergo spontaneous con-
stant activation by the tickover mechanism as well. The
three complement activation pathways converge on the
suggested by Pillemer at the end of the 1950s,
properdin acts as an initiator and amplificator of
the AP [13].
If C3b molecules bind covalently to the C3
convertase, it generates the AP C5 convertase
(C3bBbC3b).
The Classical Pathway
The classical pathway (CP) of complement is
initiated by the binding of immunoglobulins to
their target antigens, resulting in conformational
933
Activators of alternative pathway:
Surfaces allowing the binding of
properdin and C3b
Properdin, Factor D
C3b, Factor B C3(H2O)
Factor Factor
H I
MCP CR1
DAF Action of anaphylatoxins C3a, C5a:
Proinflammatory action
Regulation of adaptive immunity
CPN
Vitron-
ectin
Clusterin
Action of membrane attack complex:
Induction of cell activation and proliferation
Lysis of target cells
level of C3 activation. The latter may give rise to the
formation of C5 convertase, the initiation of the terminal
complement pathway, and the polymerization of C9, con-
cluding by the formation of the membrane attack complex.
Multiple fluid-phase and plasma membrane-bound regula-
tors of complement activation (gray triangles) prevent the
uncontrolled activation of the cascade. The biological
actions of complement are mediated by surface-bound
molecules (opsonins), small peptide mediators
(anaphylatoxins), and the cellular effects of membrane
attack complex (indicated in boxes). See the molecular
details of complement components and activation in the
text and in Tables 1, 2, and 3
changes in the hinge region and in the Fc domains
[10]. These changes enable the C1 complex (com-
prising one recognition molecule C1q and two
pairs of zymogen serine proteases C1s and C1r)
to bind to antibodies. Upon binding of the C1
complex to antibodies, C1q undergoes a confor-
mational change, which activates the C1r zymo-
gen. In turn, active C1r cleaves and activates C1s.
Activated C1 complex cleaves C4 and C2 and
generates the classical pathway C3 convertase
(C4b2a). The complement component C4 is
934
Fig. 2 The principles of complement regulation. Panel
a: Control of C3 convertases by cofactor-mediated
cleavage. Factor I mediates the degradation of surface-
bound C3b by cofactor-mediated cleavage. Cofactors are
fluid-phase proteins (factor H or C4BP able to bind to neg-
atively charged structures on cell surfaces) or transmembrane
complement regulators present on mammalian cells (CR1
and MCP). The formation of a molecular complex with its
cofactors enhances the protease activity of factor I in a
substrate-induced (C3b or C4b) manner, and C3b (and
C4b) degradation (release of C3f) occurs. Panel b: Control
of C3 convertases by decay-accelerating activity. The
molecules with decay-accelerating activity are factor H,
DAF, and CR1. These mediate the destabilization of C3
convertases and facilitate the dissociation of Bb from C3b.
Z. Prohászka et al.
Panel c: Control of active serine proteases by serpins
(serine protease inhibitors, example of C1 inhibitor).
Multiple serine protease inhibitors (including C1-inhibitor,
antithrombin, alpha-2 macroglobulin) act on active serine
proteases of the complement system. The archetypal serpin
inhibitor of CP and LP is C1-INH acting in a canonical
(“suicidal”) manner on C1r, Cs, and MASPs. By forming
covalent complexes with these active proteases, as well as by
facilitating the dissociation of recognition complexes,
C1-INH plays a pivotal role in the control of both the CP
and the LP. Panel d: Control of terminal complement
pathway (example of CD59). The mammalian cell-surface
regulator CD59 of the terminal pathway inhibits the incor-
poration and polymerization of C9 into the terminal comple-
ment complex, in order to protect host surfaces from attacks
30 Complement-Mediated Glomerular Injury in Children
structurally and functionally similar to C3 in that
it has an internal thioester bond and it is capable of
covalent binding to surfaces. Activated C1 com-
plex splits C4 into a large C4b fragment that binds
to target surfaces and has an enhanced affinity to
C2 and a smaller C4a fragment that has weak
anaphylatoxin activity. Complement component
C2 is a zymogen serine protease that, once
cleaved by activated C1 complex into C2a (large
proteolytic fragment) and C2b, forms together
with C4b the C3 convertase complex of the CP
(C4b2a). As for the AP, C3b can make covalent
bonds with the C3 convertase to form the CP C5
convertase complex (C4b2a3b).
In addition, C1q can also bind directly to repeat
structures of non-immunoglobulin activators of
the CP, such as nucleic acids, chromatin, cyto-
plasm intermediate filaments, mitochondrial pro-
teins, cardiolipin, ligand-bound C-reactive
protein, protein aggregates (e.g., amyloids), bac-
terial polysaccharides, and artificial surfaces (car-
bon nanotubes and polymers) [14]. These
activators represent important potential mediators
of glomerular damage.
The Lectin Pathway
Two collectins – collagen-containing C-type
lectins (i.e., mannose-binding lectin, MBL) and
collectin 11 – and three ficolins (fibrinogen colla-
gen lectins, i.e., ficolin-1, ficolin-2, and ficolin-3)
compose the recognition molecules of the lectin
pathway [15]. These oligomeric molecules circu-
late in the plasma as complexes formed with
zymogen serine proteases termed MBL-associ-
ated serine proteases (MASPs). MASP-1 and
MASP-3 are alternatively spliced products of the
masp1/3 gene and have identical recognition
domains but different serine protease domains,
whereas MASP-2 is encoded by the masp2 gene.
Recognition molecules of the lectin pathway bind
only one protease at a time, and in contrast to C1r
and C1s, activation of MASPs only involves
autoactivating mechanisms. The autoactivation
of MASP-1 is much faster than that of MASP-2,
suggesting that MASP-1 is the primary player in
the activation of the lectin pathway [16]. Active
MASP-2 can cleave C4 and C2; the exact func-
tions of MASP-1 and MASP-3 are unclear. In
935
addition, MASPs may cleave proteins of the coag-
ulation pathway [6] and have a stimulatory activ-
ity on endothelial cells [17].
Activators of the lectin pathway interact with
MBL in a calcium-dependent manner through the
carbohydrate recognition domain of MBLs.
Repeat sugar motifs terminating with mannose,
glucosamine, or fucose can bind oligomeric
MBLs and induce conformational changes. Bac-
terial and viral structures, for example, have been
shown to activate MBLs. Surface-bound MBLs
may also act as opsonins. The lectin pathway does
not require antibodies; however, MBLs can bind
certain glycan structures of IgG and IgA [18].
Similar to the collectins, ficolins are oligomeric
recognition molecules of the lectin pathway. They
have collagen-like domains, form complexes with
zymogen MASPs, and, upon binding, facilitate
their autoactivation. In contrast to collectins,
ficolins contain fibrinogen-like carbohydrate rec-
ognition domains and exhibit specific differences
in target preference. Three ficolins have been
identified in humans until now, namely, ficolin-1,
ficolin-2, and ficolin-3 (formerly M-ficolin,
L-ficolin, and H-ficolin). Ficolin-3 is the most
abundant, with a plasma concentration of approx-
imately 30 mg/L. Few binding targets for ficolin-3
have been shown, including N-acetylated struc-
tures present on apoptotic cells [19] and other
bacterial targets. Ficolin-2 binds preferentially to
acetylated sugars, to some bacterial targets, and to
C-reactive protein. Ficolin-1, the least abundant
plasma ficolin, plays a major role in facilitating
phagocytosis [20].
Once recognition molecules of the lectin path-
way have bound to their targets and conforma-
tional changes have autoactivated MASPs, C4
and C2 are cleaved. From there on, the lectin
pathway converges with the classical pathway to
form the LP C3 convertase (C4b2a), subsequently
activating the terminal pathway.
The Terminal Pathway
The formation of the membrane attack complex
(MAC, C5b-9) begins with the cleavage of C5 by
C5 convertases. C3 convertases of all pathways
can cleave efficiently C3 but have weak activity
toward C5. However, after incorporating an
936
additional C3b molecule, C3 convertases form C5
convertases, and C5 is cleaved into C5a, the most
potent anaphylatoxin, and C5b. Since C5 lacks an
internal thioester bond, it does not bind covalently
to the target surface. However, upon binding,
conformational change occurs, allowing subse-
quent aggregation of C5b with C6, C7, C8, and
multiple copies of C9. Binding and fusion of
terminal pathway components in the lipid bilayers
is reversible and involves ionic and hydrophobic
interactions. The pore assembly takes place
through well-defined heteropolymeric complexes,
collectively termed terminal complement com-
plexes (TCC). The final pore-forming complex
(C5b-9n), which can incorporate as many as
16 C9 molecules, is located deep in the lipid
bilayer and is a most effective inducer of cell
activation and death.
Control of Complement Activation
The complement system is strictly regulated to
prevent uncontrolled activation and tissue dam-
age. Since the AP has the inherent ability of C3
tickover-based amplification, tight control is of
particular importance. The major complement
control proteins can be subdivided in fluid-phase
and surface-bound molecules (Tables 2 and 3).
Their activities include inhibition of active serine
proteases, facilitation of C4 and C3 breakdown,
acceleration of C3 and C5 convertase decay, and
prevention of MAC formation on target surfaces
(Fig. 2, panels a–d) [21]. The activity of comple-
ment control proteins is often mediated by com-
plex molecular mechanisms that also require the
presence of cofactors, of other proteases, and of
surface receptors.
Because of its central role, C3 is under partic-
ularly strict control. First, C3b is rapidly
inactivated by factor I, which after cleavage of
three peptide bonds, generates iC3b from C3b,
which can no longer interact with factor B and
with C5 (Fig. 2a). Factor I is a serine protease that
requires cofactors to function [22]. The catalytic
activity of factor I is regulated by an internal inhib-
itory domain and by the temporary presence of its
substrate (C3b and C4b). Cofactors of factor I
Z. Prohászka et al.
include factor H, C4-binding protein, and surface-
bound regulators, such as the membrane cofactor
protein (CD46 or MCP) and complement receptor
1 (CD35 or CR1). A common structural property of
these cofactors is the presence of multiple comple-
ment control protein (CCP) domains, each
consisting of 60 amino acids. By way of
their multiple binding domains, these cofactors
bring a regulatory protease, such as factor I, and
C3b in close spatial vicinity and facilitate the
proteolytic breakdown of C3b. Of notice, several
factor H-related proteins (CFHRs) belong to
factor H family [23]. These proteins lack strong
complement inhibitory function. However, they
may play an important role in fine-tuning
C3 activation.
A second mechanism for controlling C3 acti-
vation relies on the decay-accelerating activity of
complement control proteins such as cofactors
(factor H, CR1) and the decay-accelerating factor
(CD55 or DAF), which competitively dissociate
the C3 convertase enzyme complexes (Fig. 2b).
The control of the classical and of the lectin
pathway recognition molecules is mediated by the
serine protease C1 inhibitor (C1-INH), belonging
to the family of serpins. C1-INH can dissociate C1
complexes (Fig. 2c) and can inactivate MASPs.
Alpha-2-macroglobulin has very similar activities
on complement molecules. C4b, the common
component of the classical and lectin pathways,
is also regulated by factor I-mediated cleavage,
similarly to C3. The C4-binding protein (C4BP)
serves as cofactor. It circulates in complex with
protein S, a vitamin K-dependent cofactor for the
breakdown of the coagulation factors Va and
VIIIa by protein C. Hence, C4BP represents a
link between regulation of the coagulation and of
the complement cascades.
Finally, the control of the terminal pathway on
cell surfaces is largely achieved by the abundant
expression of the membrane-bound glycosylpho-
sphatidylinositol-linked protein CD59 (protectin)
and by two fluid-phase regulators, namely,
vitronectin and clusterin. These molecules control
the formation of the membrane attack complex by
binding to various intermediate complexes
preventing the recruitment of C9 molecules
(Fig. 2d). Furthermore, by inhibiting access to
30 Complement-Mediated Glomerular Injury in Children 937

a Endothelial injury mediated by immunoglobulin

induced complement activation

Swelling and
detachment of
endothelial cells

b Endothelial injury mediated by

dysregulation of complement activation on the
surface of endothelial cells

Enlarged area
Podocyte

Slit diaphragm Swelling and

detachment of
endothelial cells

Platelet
Pedicles of and fibrin
Endothelial podocytes rich thrombi
Glomerular glycocalyx
basement membrane c Deposition of complement fragments in the
Fenestrated endothelial cell GBM with or without immunoglobulins

Partial cross section

of a glomerular capillary

Thickening of GBM,
double contour
Heavy deposition of
complement fragments

d Local complement activation by deposited

autoantibodies and immune complexes

Penetration and
deposition of
antibodies to
variable depth
Damage of podocytes

Fig. 3 Schematic representation of complement- glomerular basement membrane, and the podocytes with their
mediated glomerular injury. A section of the three-layered interdigitating pedicles. The enlarged areas show the charac-
filtration barrier in the glomerulus is presented: exhibiting the teristic initial alterations of this filtration barrier in four distinct
fenestrated endothelium with the mesh-like glycocalyx, the types of complement-mediated glomerular injury. The yellow
938
binding sites on phospholipid bilayers, these reg-
ulators protect neighboring host cells from spread-
ing of MACs from one cell to another.
Downstream Effects of Complement
Activation
Once particles or a cell surface are recognized by
complement recognition molecules, several mol-
ecules of C3b attach. C3 activation results in
multiple biological effects aimed at defending
the host, including opsonization, activation, and
migration of inflammatory cells, release of inflam-
matory molecules, and lysis of the target cells [3].
These aspects are briefly illustrated below.
C3 activation promotes clearance of apoptotic
bodies and of immune complexes by opsonization.
Fig. 3 (continued) color marks the dominant site of depos-
ited complement fragments, as revealed by immunofluo-
rescence, and the green color marks immunoglobulins. Of
note, proliferative cells and mesangium are not represented
in the enlarged areas. Enlarged area A: Acute endothelial
injury (endocapillary glomerulonephritis) is caused by
immunoglobulin-induced complement activation early in
the disease process, followed by IgG deposition. Cellular
infiltration is the consequence of the liberation of
complement-derived anaphylatoxins. Later, in the disease
course, immunoglobulins and/or immune complexes may
penetrate the basement membrane and form subepithelial
deposits as in postinfectious glomerulonephritis or in the
presence of donor-specific antibodies after transplanta-
tion. Enlarged area B: Failing complement regulation
due to loss of complement regulators in the endothelial
glycocalyx (such as in case of complement-mediated atyp-
ical HUS) with resulting complement activation may also
lead to capillary damage. Anaphylatoxins and the terminal
complement complex both contribute to the activation and
damage of endothelial cells. This leads to changes in phe-
notype, increased adhesion of leukocytes and platelets, loss
of cellular integrity, and detachment from the basal mem-
brane. Endothelial swelling, activated platelets, and fibrin-
rich microvascular thrombi may occlude the lumen of
glomerular capillaries, reducing perfusion. As a rule, com-
plement C3 deposits without immunoglobulins but with
fibrin. Enlarged area C: Immune complexes with specific
properties may bind to the basement membrane and cause
complement activation, inflammation, mesangial cell
interposition between the endothelium and the basement
membrane, and production of extracellular matrix leading
to thickening of the basement membrane with the charac-
teristic double contour appearance. Complement and
Z. Prohászka et al.
C3b-coated particles in the bloodstream bind to
CR1 on erythrocyte membranes. Since CR1 is a
cofactor to factor I, C3b is gradually converted
into iC3b, which increases binding to complement
receptors 3 and 4 (CR3 and CR4) expressed by
phagocytes. Uptake of iC3b-coated particles by
phagocytic cells, mainly macrophages, occurs
predominantly in the spleen and in the liver.
Impaired activation and degradation of C3 com-
promises clearance of apoptotic bodies and
immune complexes. Moreover, C3b-coated parti-
cles can also be converted into C3d and C3dg,
which are recognized by complement receptor 2
(CR2) molecules present on the surfaces of B
cells, activated T cells, and follicular dendritic
cells. Thus, opsonization of target particles also
plays a pivotal role in antigen presentation, elicits
germinal center reactions, and contributes to the
immunoglobulins are detected by immunofluorescence
microscopy (immune-complex-mediated membranoproli-
ferative glomerulonephritis). On the other hand, fluid-
phase complement activation (such as in the presence of
C3 nephritic factor) or the lack of an effective alternative
pathway regulation (in various forms of complement-medi-
ated C3 glomerulopathies) may result in the presence of
high amounts of activated C3 (C3b) in the plasma. This
may accumulate (together with complement activation
products of the terminal pathway, i.e., SC5b-9) within the
GBM. The result is similar thickening, deposition of
electron-dense material, and the formation of the charac-
teristic double contour of the GBM. Typically, the comple-
ment deposits (mostly activated and/or degraded forms of
C3) without immunoglobulins can be detected by immuno-
fluorescence. Cell proliferation and mesangial cell interpo-
sition may accompany these processes. Enlarged area D:
Autoantibodies or immune complexes circulating in the
blood penetrate through the endothelial fenestrae and the
GBM. Subsequently, they can (1) get deposited on the GBM
in a linear pattern (e.g., in anti-GBM-mediated disease) or
(2) penetrate into and reside in the subepithelial space after
an initial phase (such as in case of advanced lupus nephritis)
or (3) recognize their targets on the podocytes (such as in the
case of anti-PLA2R autoantibodies in membranous
nephropathy). The deposited immunoglobulins, as well as
the components (C1q, C4, C3) and the activation products
(C4d, SC5b-9) of the classical and terminal pathways, can be
detected in the subepithelial space by immunofluorescence.
Complement activation is largely separated from the capil-
lary lumen; hence, recruitment of inflammatory cells is
limited but direct complement-mediated damage of the
podocytes occurs
30 Complement-Mediated Glomerular Injury in Children
development of memory B cells. Additional
receptors for C1q and other recognition molecules
of the lectin pathway promote attachment and/or
uptake by phagocytes and antigen-presenting
cells.
Complement activation facilitates and
enhances inflammatory reactions. The most
potent inflammatory mediators, anaphylatoxins
C5a and C3a, act on a wide variety of immune
and nonimmune cells. These small peptides with
short half-lives exert their effects by binding to
7-transmembrane domain G-protein-coupled
receptors. Downstream effects on cells include
metabolic changes, degranulation, activation,
and enhanced cell migration. For the most part,
C3a and C5a have similar pro-inflammatory
effects and can activate eosinophils, basophils,
and mast cells. However, C3a has no effect on
neutrophils, as opposed to C5a, which is a potent
chemoattractant and activator of these cells. Acti-
vated mast cells release preformed TNF-alpha and
increase histamine and eicosanoid production.
This results in vasodilation, enhanced vascular
permeability, and recruitment of other immune
cells at the site of complement activation. Further-
more, in conjunction with TNF-alpha,
anaphylatoxins increase expression of cell adhe-
sion molecules (e.g., E-selectin, ICAM-1,
VCAM-1) on endothelial cells [24], facilitating
adhesion and migration of leukocytes. The activ-
ities of anaphylatoxins are controlled by carboxy-
peptidase N, a serum metalloprotease that inhibits
their biological effects by cleaving basic amino
acids at the terminal ends.
Classically, cell lysis mediated by MAC is
considered the most important downstream effect
of complement activation. Deficiencies of termi-
nal pathway components, however, do not support
the view that MAC is a critical defense mecha-
nism in humans. Patients with C6, C7, C8, or C9
deficiencies, for example, demonstrate increased
susceptibility only to a restricted number of bac-
teria, mainly some Neisseria species. Pathogens
and tumor cells can in fact activate escape mech-
anisms to prevent damage from complement
attack. Sublytic amounts of terminal pathway
complexes, however, may play an important role
in nonlethal signaling events (see below).
939
It is increasingly clear that the complement
system has multiple interactions with other sys-
tems, such as the immune and coagulation sys-
tems. As new drugs that modulate the
complement activity are being developed, their
effects on these pathways need to be carefully
scrutinized.
Complement Activation in Kidney
Diseases
Kidneys are particularly susceptible to
complement-mediated attacks, due to their exten-
sive endothelial surface, high blood flow, vascular
organization, and local production of complement
components. Broadly speaking, there are two
major groups of complement-mediated kidney
diseases. The first (1) is associated with increased
activation and consumption of complement and
involves the classical (and in certain cases the
lectin) pathway. The second (2) is characterized
by complement dysregulation and involves the
alternative pathway. In the clinical practice, func-
tional, immunological, biochemical, and genetic
tests help distinguish these two disease mecha-
nisms. This also affects protocols for treatment
and monitoring. The term complement profile is
often used to describe the multitude of comple-
ment parameters measured in a patient at a given
time. This “snapshot” reflects the activity of com-
plement pathways (from the activation of a given
pathway to the polymerization of C9), along with
the levels of complement proteins and activation
markers, and the presence of autoantibodies
against complement proteins (Table 4).
Mechanisms leading to complement activation
in kidney diseases are:
1. The sustained presence of antibodies, leading
to overactivation and consumption of the clas-
sical (and in certain cases the lectin) pathway.
These antibodies may be generated in response
to persistent stimulation by antigens (such as
in infections) and by malignant cells (mainly
in B-cell neoplasia) or may be secondary to
autoimmune disease (immune complexes).
If present in excessive amount or proportion,
940 Z. Prohászka et al.

Table 4 Characteristic alterations and interpretation of complement parameters in pediatric kidney diseases
Interpretation of the
Package Tests Indication results Comments
Baseline C3, C4, and All pediatric Low levels of all three: Required for all patients
complement profile CH50 glomerulonephritides activation of CP
Decreased C3 with
normal C4: activation
of AP
Decreased C4 with
normal C3: suspected
cryoglobulins and/or
HCV infection
Functional Classical and Suspected May help to identify the Global functional
screening of alternative complement missing factor in the testing of complement
complement pathway deficiency case of complement
activity activity deficiency
Classical pathway “Baseline” Suspected (auto) Low levels of C1q, C3, May be particularly
and C1q, anti- antibody-mediated and C4 activity points helpful for the
C1q IgG, C2 glomerular to activation and differential diagnosis of
pathology consumption of CP antibody- versus
Low level of a single dysregulation-mediated
factor (C1q, C4 or C2) pathologies
and decreased CP
activity indicates factor
deficiency
Anti-C1q IgG positivity
causes acquired
deficiency of CP and is
indicative for lupus
nephritis or, in the
presence of urticaria, of
hypocomplementemic
urticarial vasculitis
syndrome
Alternative “Baseline” Thrombotic Low levels of C3, In the presence of AP
pathway and factors B, microangiopathies factor B, and AP dysregulation and TMA
I, H, anti- (TMA), C3 activity indicate or C3 glomerulopathy,
factor H IgG glomerulopathy dysregulation, genetic causes must be
activation, and investigated in
consumption of AP specialized laboratories
Anti-FH IgG positivity (see below)
causes acquired
dysregulation of AP
Proliferative C3 nephritic C3 glomerulopathy Low C3 and C3NeF These tests are available
glomerulonephritis factor, anti- positivity are frequently in specialized
autoantibodies factor B IgG, found in C3 laboratories only
anti-factor H glomerulopathy,
IgG particularly in DDD
Complement C3a, C3d, Differentiation Elevated levels indicate
activation products C5b-9 between complement ongoing complement
activation and activation
complement Low levels suggest
deficiency, therapy complement factor
monitoring deficiency
Low levels of C5b-9
imply therapeutic
efficacy of eculizumab
(continued)
30 Complement-Mediated Glomerular Injury in Children
Table 4 (continued)
Package Tests Indication
Genetic analysis of Bidirectional Thrombotic
complement sequencing of microangiopathies
components and CFH, CFI, (TMA), C3
AP regulators CD46, C3, glomerulopathy
CFB, and
THBD
Copy number
determination
of CFH-
related genes
such immunoglobulins may accumulate in
specific regions of the kidney. The local-
ization of these complement-activating sub-
stances (subendothelial vs. subepithelial)
and of the resulting inflammatory reaction
(reflecting the extent of complement activation
and cell proliferation) are decisive determi-
nants of the histological and clinical conse-
quences. Overactivation of the CP in these
forms of glomerulonephritis is accompanied
by hypocomplementemia. The characteristic
alterations of the serum complement profile
include decreased C3, C4, CH50, and C1q
levels. Typically, the complement regulators
factor H and I are not affected (Table 4).
2. The impaired function of complement regula-
tory proteins, leading to dysregulation of the
alternative pathway. As complement con-
sumption affects just the one pathway, or a
few of its components controlled by the miss-
ing regulator, alterations of the serum comple-
ment profile are less marked and, if present,
indicate overactivation and consumption of the
alternative pathway – i.e., low C3 level. Defi-
ciencies of factor H or factor I are often accom-
panied by low factor B level (Table 4). It is
important to note that in this group of renal
diseases, circulating C3 levels may or may
not be reduced, partly depending on whether
the complement alternative pathway
dysregulation is in the fluid phase or on the
cell membranes. When the alternative pathway
dysregulation involves the fluid phase,
941
Interpretation of the
results Comments
Indicated for all patients
with atypical HUS
May be indicated for
patients with C3
glomerulopathy
circulating C3 levels are usually significantly
and persistently reduced, as in C3
glomerulopathies (111). When the AP
dysregulation involves membrane-bound com-
plement, circulating C3 may or may not be
reduced, as happens in atypical hemolytic ure-
mic syndrome (aHUS). Recent data show that
in patients with aHUS, circulating C3 levels
are low in only 30–50 % of patients, excepting
those with C3 or CFB mutations who mostly
(70–100 %) present low C3 [25].
Table 4 summarizes the complement tests cur-
rently offered by specialized diagnostic laborato-
ries, complete with comments for the
interpretation of their findings.
The next two sections of this chapter outline
two aspects of complement-mediated glomerular
injury. The first section describes the molecular
mechanisms of damage to renal cells and function,
whereas the last one provides a brief clinical over-
view of complement-mediated glomerular
diseases.
Molecular Mechanisms
of Complement-Mediated Renal Injury
The glomerular filtration barrier can be damaged
after local complement activation or by the depo-
sition of systemically activated complement
products.
942
Role of Structural Properties of Kidney
Components and of Local Production
of Complement Factors and Regulators
The glomerular filtration barrier is a three-
layered structure separating the bloodstream and
the urinary space (Fig. 3) within the glomerulus, a
tight aggregate of capillaries surrounded by the
interdigitated extensions of podocytes. Two cel-
lular layers – one epithelial (belonging to the
podocyte) and one endothelial – are separated by
the glomerular basement membrane (GBM), a
specialized form of extracellular matrix. The
structure of these layers together with high glo-
merular blood flow makes this filtration barrier
susceptible to complement-mediated injury.
The glomerular capillaries are lined by special-
ized endothelial cells bearing large fenestrations
that allow the passage of fluid across the endothe-
lium. These fenestrations most probably allow the
direct flow of plasma (hence, also of complement
components and activated fragments, if present)
from the blood, across the proximal layer of the
filtration barrier, toward the GBM. Recent find-
ings suggest a major role for the glomerular endo-
thelium and its glycocalyx in the maintenance of
an intact barrier, with the latter forming a mesh-
like, negatively charged, hydrated structure
[26]. This negatively charged glycocalyx is
important in recruiting fluid-phase complement
regulators and in maintaining effective control of
complement at the filtration barrier [27]. The
GBM is unusually thick and formed by specific
isoforms of proteins including laminin, type IV
collagen, nidogen, and heparan sulfate proteogly-
cans. Importantly, the GBM does not contain
complement regulatory proteins [28], and hence,
its surface is susceptible to opsonization by com-
plement. Besides, covalently fixed complement
components may persist longer, since the cofac-
tors necessary for C3b cleavage are missing.
Different cell types of the kidney appear to be a
rich local source of complement proteins, as
shown by various in vitro gene or protein expres-
sion studies [21]. Genes of C1q and factor D are
strongly expressed in the glomeruli, whereas C2,
C4, C3, and factor B are detected in the renal
tubules [29]. The production of C2 and C3
Z. Prohászka et al.
proteins in podocytes and their positive regulation
by interleukin-1 and tumor-necrosis factor alpha
have been demonstrated [30, 31]. The crucial
importance of locally produced C3 in ischemia-
reperfusion-induced kidney damage has been
shown in a complement-deficient animal model
[32]. In addition to components of the comple-
ment pathways, complement control proteins are
also expressed and produced by the various cell
types present in the kidney. As reviewed in detail
elsewhere [33], the expression of MCP and CD59
is ubiquitous in glomerular and tubular cells,
whereas that of DAF appears to be lower. In
vitro, the glomerular endothelium has a lower
baseline expression of CD59 and DAF when com-
pared to other endothelial cell types, and this
partially explains renal susceptibility to
complement-induced damage [34]. Of note, the
anticoagulant protein thrombomodulin – an acti-
vator of the factor I-mediated degradation of C3b
– is another important constituent of the endothe-
lial glycocalyx in the glomerulus. Hence, the com-
position of the endothelial glycocalyx with
binding sites for soluble complement regulators
and sufficient expression of membrane-bound
complement control proteins (including
thrombomodulin) may play an important role in
protecting the glomerulus against complement
attacks. The composition of the glycocalyx is
subject to inflammatory changes [35]. Therefore,
it can be supposed that the inflammatory process,
complement activation, and related changes of the
glycocalyx are among the most important mecha-
nisms driving complement-mediated kidney
disease.
Cellular Activation and Proliferation
in Response to Complement Activation:
The Non-lytic Terminal Pathway
Complement Complex
Following complement activation, the terminal
complement complex is formed. The assembly
of the terminal pathway complement complex
(TCC or C5b-9) in the plasma membrane takes
place in different ways. It is important to distin-
guish lytic TCC (C5b-9n complexes with C9
30 Complement-Mediated Glomerular Injury in Children
Protective mechanisms
against complement attack:
Shedding or endocytosis of
terminal complement complex
Massive complement activation
High amount of lytic complex
Loss of ATP, ADP, AMP, increase of icCa
Loss of mitochondrial inner membrane potential
Cell death
Fig. 4 Possible consequences of complement activation
mediated by the terminal complement complex.
Depending on the amount and composition of the terminal
complement complex and on cell type, complement acti-
vation may result in cell death or proliferation. Moreover,
the autocrine and paracrine effects of biologically active
present in various concentrations), which forms
pores within the cell membrane leading to cell
death, and non-lytic TCC (C5b-678 complexes
without polymerization of C9), which triggers
multiple signal transduction pathways that may
induce cell activation, as well as upregulate
metabolism and proliferation [36, 37] (Fig. 4).
The non-lytic complex has been shown to activate
the small G protein Ras, phosphatidylinositol
3-kinase, and also the ERK1, JNK1, p38, and
MAPK pathways.
Depending on the composition of the TCC and
on the cell type studied, these activities may con-
tribute to the cellular activation and to the initia-
tion of cytoprotective mechanisms or lead to cell
death. The non-lytic terminal pathway complex
may, for example, induce tissue factor expression
in macrophages after treatment with anti-
thymocyte globulin [38]. C5b-9 in sublytic con-
centration has been implicated in the cell cycle
943
Lytic terminal complement complex
Non-lytic
C5b-9n
terminal complement complex
C5b-678
Mammalian cell surface
C9
Moderate complement activation
Low amount of lytic complex and non-lytic complex
++
Signal transduction
Activation of Ras, PI3K and NFkB
Cell proliferation Increased expression
Transcription of cell cycle genes; of cell surface
Anti-apoptosis; Protein synthesis adhesion molecules
Facilitation of inflammation
Release of biologically active mediators,
growth factors and cytokines
Autocrine and paracrine effects
mediators, cytokines, and growth factors may significantly
contribute to the consequences of complement attack.
Nucleated cells possess effective defense mechanisms,
including endocytosis and the shedding of membrane ves-
icles, to protect themselves against complement attack
activation in various types of nucleated cells and
in the proliferation of smooth muscle cells. More-
over, C5b-9-induced growth factor release (such
as the induction of platelet-derived growth factor
and basic fibroblast growth factor in endothelial
cells) may also contribute to cell cycle activation
[39]. Through its stimulatory action on nuclear
factor kB, C5b-9 is also able to induce the secre-
tion of pro-inflammatory cytokines including IL-6
in smooth muscle cells [40], as well as IL-8 and
MCP-1 in endothelial cells [41]. Further, it can
promote surface adhesion molecule expression
such as neutrophil-endothelial adhesion via the
induction of osteoprotegerin [42] and intercellular
adhesion molecules (ICAMs), as shown in a por-
cine arterial endothelial cell model [43]. These
responses may be important for cell survival dur-
ing inflammation accompanied by significant acti-
vation of the terminal complement pathway, such
as in glomerular diseases with complement
944
activation. In addition to its endothelial and epi-
thelial effects, the deposition of the C5b-9 com-
plex may play a dual role in mesangial and in
tubular cells. In particular, TCC may promote
tissue fibrosis via its stimulatory effect on
TGF-beta1 and collagen expression [44, 45] on
one hand. On the other hand, it can also induce
apoptosis of these cells [46, 47].
Parallel to the generation of C5b, equimolar
quantities of the small anaphylatoxin C5a are
also produced. Acting on its G-protein-coupled
receptor, C5a elicits a multitude of biological
actions and changes that are essential in innate
and adaptive immunity. These include an increase
of vascular permeability, the chemotaxis of
inflammatory cells (neutrophils), the induction of
the respiratory burst, the release of cytokines and
chemokines, and phagocytosis [48, 49]. C5a has
been shown to act on the expression of adhesion
molecules both in neutrophils and in endothelial
cells, thereby enhancing their binding to each
other [50]. It is important to note that these
C5a-initiated responses are rather similar to
those triggered by the terminal complement com-
plex and may significantly contribute to glomeru-
lar damage through induction of cellular
proliferation, fibrosis, and scarring in case of com-
plement activation. Therefore, significant atten-
tion has been paid to the pharmacological
targeting of the C5a receptor. In recent years, a
variety of different compounds have been tested
in clinical trials of patients with kidney
diseases [51].
Cell Death and Recovery in Response
to Complement Injury: The Lytic
Terminal Pathway Complement
Complex
A single terminal complement complex (with
multiple copies of incorporated C9 proteins)
forms the TCC lytic complex, which is able to
lyse red blood cells in vitro; however, nucleated
cells possess multiple protective mechanisms
against C5b-9n-mediated destruction. A limited
Z. Prohászka et al.
number of C5b-9 complexes are efficiently elim-
inated from the plasma membrane through the
formation of microvesicles. Therefore, a large
quantity of these complexes (i.e., intense or
uncontrolled complement activation) is
required to inflict cell death via the terminal
pathway, through a process best described as the
“multiple-hit” mechanism [52] (Fig. 4). This pro-
cess is characterized by the loss of ATP,
ADP, and AMP and of the mitochondrial
membrane potential before cell lysis by the
C5b-9 complex occurs [53]. The rapid and
considerable intracellular calcium influx,
mediated by the C5b-9 channels, contributes to
the quick loss of inner mitochondrial
membrane potential, which is followed by acute
cell death. This process has several features
resembling those of apoptosis, such as TUNEL
positivity and DNA fragmentation. Nevertheless,
the cell destruction initiated by the attack of the
terminal complement pathway is primarily
necrotic in nature [54].
In nucleated cells, several regulatory mecha-
nisms exist to overcome the effects of a limited
complement attack. The two most important
defense mechanisms are membrane shedding to
the extracellular space through the formation of
microvesicles and endocytosis (Fig. 4). The time
of removal of terminal complement complexes
shortens with the incorporation of C8 and multiple
copies of C9 into the complex: C5b-8,9n has a
half-life of 1–3 min only. By contrast, complexes
without C8 and C9 persist in the membrane of
red blood cells for several hours [55]. The elimi-
nation rate of TC complexes also increases with
the elevation of intracellular calcium level, lead-
ing to activation of the PKC pathway which in
turn induces membrane vesiculation and endocy-
tosis. In case of a complement attack on
podocytes, the C5b-9 complex-containing mem-
brane vesicles that are released into the extracel-
lular space can be detected in the urine. Therefore,
measuring urinary C5b-9 levels may be useful for
the assessment of complement activation in the
kidney, as has been shown recently in
preeclampsia [56].
30 Complement-Mediated Glomerular Injury in Children
Illustrative Examples
An Overview of the Mechanisms of
Complement-Mediated Glomerular
Injury
The complement system plays a key role in a
number of glomerular pathologies. The nature
and the extent of complement-mediated glomeru-
lar damage will depend on the trigger and on the
pathway of complement activation, as well as on
its localization to the subendothelial, mesangial,
or subepithelial space (Fig. 3).
Soluble immune complexes formed in the cir-
culation may be trapped in subendothelial and
mesangial areas of the glomerulus. Glomerular
immune deposits may be formed by free circulat-
ing antibodies binding to antigens trapped at
subendothelial or mesangial sites or underneath
the podocytes in the subepithelial space. Finally,
autoantibodies may bind to normal glomerular
components like the Goodpasture’s glomerular
basement membrane antigen, as well as to
podocyte antigens, such as the phospholipase A2
receptor and neutral endopeptidase.
In immune-complex glomerulonephritis, the
structural properties of the immune complexes or
of the trapped antigens will determine their final
location within the glomerular structure and also
the biopsy findings and the clinical consequences.
(a) Subendothelial distribution of the immune
complexes and activation of the complement cas-
cade lead to massive endothelial damage and exu-
dative infiltration by inflammatory cells.
(b) Immune-complex deposition in the
mesangium induces mesangial cell proliferation
and matrix expansion. (c) If the immune com-
plexes are deposited in the subepithelial area, the
complement-generated chemotactic factors are
isolated from the capillary space, and thus, the
resulting lesion is rather a noninflammatory reac-
tion with podocyte injury, effacement, and heavy
proteinuria [57, 58].
The clinical consequences will be influenced
also by the biologic properties of the antibody
(or antigen), its Fc receptor affinity, and
945
complement-activating capacity. Its ability to
aggregate and the quantity of the deposits are
additional influencing factors.
As a variety of etiological factors may con-
verge to similar final pathways capable of causing
glomerular damage, the clinical and histological
appearance of the latter may be similar. The rec-
ognition of the underlying mechanisms may help
to understand the similarities and differences
between the specific forms of nephropathy –
such as the one occurring in systemic lupus
erythematosus – that may mimic other character-
istic histological patterns, including membrano-
proliferative glomerulonephritis or membranous
nephropathy.
Finally, complement activation may occur also
in some nonimmune-complex-mediated renal dis-
eases that are not primary glomerular disorders.
Uncontrolled complement activation may either
lead to some forms of C3 glomerulopathies or to
renal diseases that are not primary glomerular
diseases including among others hemolytic ure-
mic syndrome, thrombotic thrombocytopenic pur-
pura, and renal vasculitis.
The Clinical Presentation of Glomerular
Injury
In the following section, we shall focus on the role
of the complement system in glomerular injury
through some typical examples. For brevity, we
have omitted ischemia-reperfusion injury, a model
of complement-mediated injury that plays a central
role in acute kidney injury (reviewed in [59]) and in
renal graft rejection (reviewed in [60]).
Poststreptococcal Glomerulonephritis
Poststreptococcal glomerulonephritis (PSGN) is
characterized by an acute glomerular lesion with
proliferation of endothelial, mesangial, and epi-
thelial cells, as well as the presence of an inflam-
matory exudate. C3 deposition occurs early in the
disease process, followed by IgG deposition. This
pattern of immune-complex deposition is seen
also in other forms of postinfectious GN [61, 62].
946
The discovery of streptococcal pyrogenic exo-
toxin B (SpeB) may explain the possible mecha-
nism of complement activation and binding in
PSGN. SpeB is a small, cationic cysteine protease
with complement-activating and plasmin-binding
properties [62, 63]. The level of the antibody to
SpeB correlates with disease activity in PSGN,
and the antibody co-localizes with IgG and C3 in
subepithelial humps, the histological hallmark of
PSGN [64]. The peculiarity of SpeB is that it may
induce inflammation in the kidney, manifested by
cytokine production, the expression of adhesion
molecules, and leukocyte proliferation followed
by immune-complex deposition. SpeB exhibits
plasmin-binding properties that might cause pro-
teolysis in the glomerular basement membrane.
This may facilitate the transit of dissociated
subendothelial immune complexes, which can
then form subepithelial humps. Some
subendothelial deposits may be present in PSGN
possibly because the antibody to SpeB also
exhibits molecular mimicry with endothelial
cells [58, 62, 65].
Other antigens such as nephritis-associated
streptococcal plasmin have also been studied
intensively as possible etiological factors of
PSGN. Its nephritogenicity may be related to its
plasmin-binding activity, which induces inflam-
matory reaction and breakdown of the glomerular
basement membrane (GBM). It co-localizes in the
glomeruli with plasmin, but, in contrast to SpeB,
not with IgG or C3 [66].
Complement Profile and Diagnostics
During the initial 2 weeks of the disease, the C3
level is significantly reduced in about 90 % of
patients. In contrast, C4 and C2 levels are
usually normal or only slightly decreased. C3
and CH50 return to normal within 4–8 weeks
after onset. It is important to stress the
need for measuring C3 level early in the course
of PSGN. After the first 2 weeks of the onset of
urinary abnormalities, it may be impossible to
diagnose PSGN with certainty without a
kidney biopsy, which may be indicated if a
decrease in renal function, nephrotic-range
proteinuria, or severe hypertension is present. If
low circulating C3 persists more than 4–8 weeks
Z. Prohászka et al.
from onset of urinary symptoms, especially if
there is no increasing trend, this could
indicate that the “PSGN” was in fact the initial
event of a more chronic process such as C3
glomerulopathy (see below). Therefore, referral
for diagnostic evaluation is justified in such
a case.
Immunoglobulin A Nephropathy (IgAN)
Immunoglobulin A nephropathy (IgAN) is the
most common form of glomerulonephritis in
both children and adults. The clinical course is
variable, with episodes of recurrent gross hema-
turia concomitant with infectious (most frequently
upper respiratory tract) episodes. Following
macrohematuria, microscopic hematuria and
mild-to-severe proteinuria may persist. In some
instances, IgA nephropathy may present
with heavy proteinuria and full-blown nephritis
[67, 68]. The histopathologic lesion is variable,
with focal or diffuse mesangial hypercellularity
and an increase in the mesangial matrix in most
cases. Endocapillary lesions are rare, just like
crescentic glomerulonephritis. In patients with
progressive disease, tubular injury often results
in fibrosis [69, 70]. Besides the IgA and C3, less
intense IgG and/or IgM deposits are seen in up to
50 % of biopsies.
IgA nephropathy is the consequence of a sys-
temic abnormality in IgA synthesis, resulting in
incompletely glycosylated IgA1 molecules that
lack the galactose residues at the antibody’s
hinge region. Aberrant IgA1 has a strong tendency
to spontaneous aggregation, which unmasks MBL
binding sites. This leads to complement activation
and binding to other molecules, such as fibronec-
tin, IgG, and collagen IV. Further, the aberrant
exposure of the misglycosylated IgA1 hinge
region may induce the production of antiglycan
IgG autoantibodies, facilitated by concurrent
infections by viruses (e.g., the Epstein-Barr
virus) or bacteria (e.g., streptococci) that express
GalNAc-containing moieties.
The circulating macromolecular form of
underglycosylated IgA evades removal from the
circulation by asialoglycoprotein and CD89
receptors, and this facilitates its mesangial
deposition [71].
30 Complement-Mediated Glomerular Injury in Children
Complement Profile and Diagnostics
Neither soluble monomeric nor dimeric human
IgA can activate complement directly, through
the classical pathway. In IgA nephropathy,
C5b-9 is generated by complement activation
induced by the interaction of aberrantly
glycosylated IgA1 aggregates with MBL. Alter-
natively, in situ formation of immune
complexes with IgG antiglycan antibodies may
occur [18, 72, 73].
Thus, two distinct immunohistological pat-
terns may be observed in IgA nephropathy. In
the majority of cases (75 %), biopsy specimens
show the deposition of IgA1, C3, and the terminal
complement complex, with the absence of other
immunoglobulins or complement fragments, indi-
cating membrane-bound activation of the alterna-
tive pathway. Another pattern, showing the
presence of IgA1 and C3 in association with
MBL and mannose-associated serine protease
1 (MASP-1), is seen in about 25 % of the cases.
The presence of MBL and MASP-1 correlates
with disease severity, increased proteinuria,
decreased GFR, and poor histological prognostic
features [69, 74, 75].
C3 and mannose-binding lectin are also syn-
thesized locally, by mesangial cells and
podocytes. Therefore, the binding of polymeric
IgA to mesangial cells may induce local comple-
ment activation independent of the systemic com-
plement activity [76]. Mesangial cells also
synthesize complement regulatory proteins,
which may explain why the generation of C5b-9
does not usually result in mesangiolysis in IgA
nephropathy.
As complement activation in IgAN is
primarily local and membrane bound, most
frequently circulating levels of C3, C4, and
CH50 are normal. However, there is some
evidence of systemic activation of
complement in a subpopulation of patients with
IgAN. Elevated levels of the split products
of activated C3 have been reported, suggesting
activation of the alternative pathway in the fluid
phase [77]. The precise relationship between
systemic markers of complement activation and
disease activity is currently incompletely
understood.
947
Systemic Lupus Erythematosus (SLE)
Kidney involvement in pediatric SLE is frequent
and may be more severe than in adult-onset dis-
ease [78, 79]. A number of different mechanisms
leading to kidney damage are active in SLE. This
explains some of the clinical and histological fea-
tures shared with other glomerulopathies, such as
membranous nephropathy or membranoproli-
ferative glomerulonephritis.
Defective T-cell regulation, the persistence of
self-antigens in apoptotic bodies, and the loss of
tolerance to such antigens trigger B-cell hyperac-
tivity and lead to an overproduction of autoanti-
bodies. Typically, IgG anti-double-stranded DNA
antibodies (anti-DNA) are found in the serum and
in glomerular deposits of patients with SLE. Some
of the monoclonal anti-DNA antibodies also
exhibit cross-reactivity with capillary wall anti-
gens, especially laminin and alpha-actinin. When
internalized by cells, these may directly alter cell
functions leading to apoptosis. Mesangial
deposits have also been associated with antibodies
to mesangial cell constituents like annexin, which
co-localizes with IgG and C3 and correlates with
disease activity [80]. Deposited anti-DNA may
also react with extracellular DNA present in the
form of nucleosomes. These consist of an anionic
segment of DNA associated with a highly cationic
histone core, resulting in a net positive charge
and a high affinity for glomerular anionic sites
[81, 82].
The complement system is involved in glomer-
ular injury at different levels in SLE.
First, deficiencies in the early components of
the classical pathway (C1q, C1r, C1s, C2, or C3)
and low copy numbers of C4 predispose to the
development of SLE. Inherited or acquired C1q
deficiency is particularly important due to func-
tional consequences including the defective clear-
ance of apoptotic bodies and reduced
phagocytosis. The cellular debris may persist lon-
ger and contribute to the loss of self-tolerance and
to the development of autoimmunity. The pres-
ence of C1q autoantibodies suggests lupus
nephritis with a sensitivity and specificity of
90 % [83, 84].
Once the complement system is activated in
SLE, the activation products contribute directly
948
to glomerular injury (Fig. 3, enlarged area, D).
Proliferative lupus nephritis is associated with
IgG, IgM, and IgA (“full house”), and C3 deposi-
tion localized primarily in the mesangium
(mesangial lupus nephritis, classes I and II) in
mild disease. The deposits are visible along the
subendothelial aspect of the capillary wall,
accompanied by increasing proliferation and
inflammation (focal or diffuse proliferative lupus
nephritis class III and IV). In membranous lupus
nephritis (class V), by contrast, the deposits are
subepithelial. These could result from the dissoci-
ation of subendothelial immune complexes mov-
ing across the glomerular basement membrane
and reforming in the subepithelial space. Alterna-
tively, lupus autoantibodies specific for podocyte
antigens may occur just as in idiopathic membra-
nous nephropathy [84–86].
Subendothelial complement activation is a
major mediator of tissue injury through both the
intracapillary generation of chemotactic factors
and the formation of C5b-9. The intense inflam-
matory reaction with leukocyte infiltration and the
destruction of glomerular structures are due to the
simplicity of the interaction between the
anaphylatoxins generated by the complement cas-
cade and the circulating leukocytes. In membra-
nous lupus nephritis, the basement membrane
isolates the capillary space from the subepithelial
site of complement activation and thus prevents
the recruitment of inflammatory cells [86].
Complement Profile and Diagnostics
In SLE, complement activation through the clas-
sical pathway leads to low C3 and C4, whereas
factor B of the alternative pathway is usually
normal. Low C3 values correlate closely with
disease severity, especially with that of
renal involvement, and with the levels of anti-
bodies to double-stranded DNA. The normaliza-
tion of low C4 and C3 levels during treatment is a
good prognostic sign: therefore, C4 and C3 are
valuable biomarkers of disease activity in
SLE [84].
Moreover, patients undergoing immunosup-
pressive therapy for SLE may be susceptible to
infection by encapsulated bacteria. This may be
Z. Prohászka et al.
partly due to hypocomplementemia-related
immunodeficiency, although the relative contribu-
tion of hypocomplementemia is unclear [87].
The measurement of complement fragment
deposition (such as C3d and C4d) on peripheral
blood cells (i.e., red blood cells, platelets, and
lymphocytes) may serve as a biomarker of lupus
disease activity [88, 89]. An acquired reduction in
the numbers of complement receptor one (CR1,
also known as CD35 and the C3b receptor) on the
erythrocytes commonly occurs in immune-
complex-mediated syndromes. This is a possible
marker of disease activity and particularly of renal
flares [90, 91].
In pediatric patients with SLE, an in-depth
analysis of the levels of complement factors and
of complement pathway activity may identify
some patients with inherited complement defi-
ciencies (Table 4).
Atypical Hemolytic Uremic Syndrome
The hemolytic uremic syndrome (HUS) belongs
to the group of thrombotic microangiopathies
(TMAs), defined by the clinical triad hemolytic
anemia, thrombocytopenia, and renal failure. The
typical, diarrhea-associated form of HUS is
caused by the exotoxins of certain bacteria, most
frequently enterohemorrhagic Escherichia coli.
By contrast, the atypical forms of HUS (aHUS)
include HUS caused by defects in the regulation
of the alternative complement pathway on vascu-
lar endothelial cells [92]. Recently, mutations of
diacylglycerol kinase epsilon have been described
in patients aged less than a year, who developed
aHUS with persistent hypertension, hematuria,
and proteinuria [93]. Drug-mediated or disease-
associated forms, collectively classified as sec-
ondary HUS, have also been reported.
In patients with aHUS, the characteristic clin-
ical and laboratory features of TMA result from
the dysregulation of the complement alternative
pathway on cell surfaces (membrane bound). Sev-
eral studies have reported that about 50–60 % of
patients with aHUS carry mutations in the genes
of complement regulators (CFH, CFI, MCP, and
thrombomodulin) or of alternative pathway com-
ponents (CFB or C3). Functional characterization
30 Complement-Mediated Glomerular Injury in Children
of the mutations revealed the dysregulation of
complement activation (decreased expression of
regulators, loss of surface binding of regulators,
decreased cofactor-mediated degradation of C3b,
low decay-accelerating activity) or increased
activity of C3 convertase (resistance to degrada-
tion, increased enzymatic activity). In addition to
mutations, anti-factor H autoantibodies in aHUS
accelerate factor H clearance through immune-
complex formation, the diminished binding of
factor H to cell surfaces, and the reduced factor
H-mediated breakdown of C3b [25, 92]. However,
such genetic or acquired abnormalities should be
regarded only as risk factors for the disease. Addi-
tional disease triggers, which activate comple-
ment, are required for aHUS to develop,
including – among others – infections or preg-
nancy. If complement activation is not properly
controlled on the surface of endothelial cells, it
may promote endothelial damage and microvas-
cular thrombosis characterized by the formation
of fibrin- and platelet-rich thrombi (Fig. 3,
enlarged area B).
The histological hallmark of acute aHUS is
capillary thrombosis with wall thickening, endo-
thelial swelling, and the deposition of a floccular
material between the glomerular basement mem-
brane and the endothelial cell lining.
Mesangiolysis commonly occurs. Platelet- and
fibrin-rich thrombi and fibrinoid necrosis of the
arterioles may occlude the lumen of capillaries
and small arterioles. Fragmentation of red blood
cells may be observed within the vessel wall.
Immunofluorescence studies reveal immunoglob-
ulin M and complement deposits primarily in the
sclerotic areas [94].
Complement Profile and Diagnostics
In aHUS, complement activation may contribute
to endothelial injury and thrombosis through mul-
tiple mechanisms. Active fragments of C5b-9 and
C5a (see above), generated during complement
activation, may directly contribute to the activa-
tion, dysfunction, and destruction of the endothe-
lium. Moreover, the same factors may promote the
activation of platelets and neutrophils, further
enhancing local thrombosis and inflammation.
949
In addition, such complement activation frag-
ments directly enhance the expression of surface
adhesion molecules as well as reduce the expres-
sion of vascular endothelial growth factors and
their receptors on the endothelial cells.
The group of non-Shiga toxin-mediated HUS
comprises diseases with different etiologies, and
complement activation in the fluid phase does not
necessarily accompany the mutations of comple-
ment genes. Therefore, plasma and serum markers
of complement activation (i.e., low levels of cir-
culating C3) are seen in only about 30–50 % of
patients with acute aHUS, excepting those with
C3 or CFB mutations who mostly (70–100 %)
present low C3 [25]. However, screening of the
complement profile with genetic analysis is indi-
cated for all patients with acute aHUS. The first
step in acute cases of thrombotic microangiopathy
is to investigate ADAMTS13 metalloproteinase
activity and EHEC microbiology [95]. For all
acute TMA patients with non-deficient
ADAMTS13 activity, especially if age is below
2 and without acute gastroenteritis, investigation
of the complete complement profile and molecu-
lar genetic analysis are indicated. The former
should include appraisal of the activity of the
classical and alternative pathways by measuring
circulating levels of C3and C4; factors H, I, and
B; and autoantibodies to factor H. The genetic
analysis should comprise screening for mutations,
the determination of the risk haplotypes of factors
H and MCP (Table 4, [96]). Low C3 levels may be
present in patients with mutations in the CFH,
CFI, MCP, C3, and CFB; however, a normal com-
plement profile does not necessarily exclude
complement-mediated aHUS. The presence of
anti-factor H autoantibody indicates the autoim-
mune form of aHUS, and the level of the autoan-
tibody is a valuable laboratory marker of
therapeutic efficacy. The complement activation
marker of the terminal pathway C5b-9 can
be elevated in acute complement-mediated
aHUS, whereas its decrease (together with the
deficient activity of CP and AP) is a sensitive
marker of the therapeutic inhibition of the termi-
nal pathway by the anti-C5 monoclonal antibody
eculizumab [97].
950
Membranoproliferative
Glomerulonephritis and C3
Glomerulopathy
The term “membranoproliferative glomerulone-
phritis” (MPGN) denotes a spectrum of glomeru-
lar pathologies. These are characterized by
mesangial and endocapillary hypercellularity,
accompanied by the thickening of the basement
membrane and the interposition of cellular ele-
ments between the glomerular basement mem-
brane and the endothelial cell layer and by the
formation of a new basement membrane [98].
MPGN is frequently idiopathic in children. In
adults, however, it is commonly associated with
chronic viral, bacterial, or parasitic infections
(most commonly HBV, HCV, and HIV), malig-
nant hematological diseases (multiple myeloma,
B-cell chronic lymphoid leukemia), and
cryoglobulinemia [98].
The original morphological classification
(MPGN type I, subendothelial; type II, GBM;
and type III, subepithelial depositions) was
based on histology and on typical electron micros-
copy features. However, recent advances in the
understanding of the pathogenesis of MPGN have
confirmed that different underlying causes may
lead to similar EM findings, and a single pathoge-
netic pathway may result in different EM charac-
teristics. Thus, the reclassification of the subtypes
of the conditions designated as MPGN is based on
the underlying pathogenesis. Histologically, the
pattern of immunofluorescence is most closely
correlated with the underlying cause of disease.
Accordingly, MPGN may present with predomi-
nant immunoglobulin immunofluorescence
resulting from chronic immune-complex deposi-
tion with activation of the classical complement
pathway (“immune-complex-mediated” form) or
with predominant, albeit not exclusive, C3 immu-
nofluorescence resulting from uncontrolled acti-
vation of the alternative pathway in the fluid phase
(“complement alternative pathway-mediated”
form) [99]. These latter forms are defined C3
glomerulopathies (Fig. 5).
Immune-complex MPGN is the result of
chronic inflammation caused by persistent viral
or bacterial infection, antigenemia, or
Z. Prohászka et al.
autoimmune diseases with the prolonged, prefer-
ential deposition of immune complexes within the
glomeruli. This leads to C1q deposition and acti-
vation of the classical pathway, resulting in the
chemoattraction of leukocytes and direct cell
injury caused by terminal complement com-
plexes. The cytokines and proteases produced by
leukocytes stimulate mesangial cell proliferation
and matrix expansion. These modify the integrity
of the filtration barrier and cause nephritic syn-
drome with occasional nephrotic features
[100–103].
In general, there are no differences in the clin-
ical presentation of the various forms of MPGN.
There is an exception, however, in that C3
glomerulopathies may be associated with retinal
drusen formation and with partial
lipodystrophy [104].
Presumably, the uncontrolled activation of the
alternative complement pathway in C3
glomerulopathies is most often caused by differ-
ent mechanisms that stabilize the fluid-phase C3
convertase. A specific autoantibody called the C3
nephritic factor (C3NeF) that binds C3 convertase
is found in many patients, as well as acquired or
inherited deficiencies of the alternative
pathway components or of the regulatory proteins
[105, 106]. A C3 mutation that renders activated
C3 resistant to factor H-mediated degradation has
also been described [107]. Recently, mutations in
the CFHR5 gene have been shown to cause an
endemic form of MPGN in Cyprus (CFHR5
nephropathy) [108, 109].
Chronic, dysregulated fluid-phase C3 activa-
tion generates a variety of complement activation
products of the alternative pathway. These accu-
mulate along the inner aspect of glomerular
basement membrane and may form the classic
dense deposits seen by electron microscopy
(Fig. 3, enlarged area C). The accumulated pro-
teins damage the structure and the integrity of the
filtration barrier. This may cause heavy protein-
uria and lead to nephrotic syndrome. Dense
deposit disease may be associated with other dis-
orders mediated by the alternative pathway, such
as retinal drusen formation and partial
lipodystrophy [106].
30 Complement-Mediated Glomerular Injury in Children 951

Clinical diagnosis of glomerulonephritis

Evaluation of baseline complement profile

Two times if C3 is low, 4-6 weeks apart

Temporary low C3 Prolonged low level of C3

Suspected C3-glomerulopathy
1. Full evaluation of complement with autoantibodies
2. Biopsy, immunofluorescence, electron microscopy
3. Molecular genetic investigations

C3, C4 and CH50 decreased

C3, AP activity and Factor B decreased
Complement and immunoglobulin
C3NeF, anti-FH or anti-FB can be positive
positive staining on immunofluorescence
Dominant complement staining on immunofluorescence
C3NeF or anti-FH negative
C4 not decreased
Factor B not decreased

Immune-complex-mediated
Poststreptococcal GN Dense deposit disease C3-glomerulonephritis
glomerulonephritis

C3-glomerulopathies
Antibody- and complement-mediated glomerular damage
Complement-mediated glomerular damage

Fig. 5 Role of laboratory evaluation of complement in
the classification of glomerulonephritis. Determination
of baseline complement profile (circulating C3, C4) is
indicated for all patients with glomerulonephritis (GN).
Repeat measurements help differentiate between transient
and prolonged decrease of C3. Patients with evidence of
streptococcal disease (or other infections) and temporary
C3 decrease most probably have poststreptococcal
(or postinfectious) GN. Patients with prolonged decrease
of C3 and persistent glomerulonephritis despite therapy
may require full evaluation of complement system includ-
ing serum complement profile with C3 nephritic factor and
anti-factor H autoantibody and genetic investigation of
complement regulators. Kidney biopsy, when performed,
will show in immune-complex-mediated GN immuno-
globulin and complement positivity by immunofluores-
cence with signs of classical pathway activation, whereas
in C3 glomerulopathies (DDD or C3-GN), it will show
predominant C3 staining on immunofluorescence, even
though immunoglobulin may be positive

Complement Profile and Diagnostics level does not exclude complement-mediated

Persisting hypocomplementemia (low C3 level) is
common in all types of MPGN.
In immune-complex-mediated MPGN, com-
plement activation occurs via the classic pathway.
Typically, this is manifested by decreased (some-
times mildly) serum C3, C1q, and C4 concentra-
tion [108, 109].
In complement-mediated MPGN, i.e., C3
glomerulopathy (including cases with C3NeF
positivity), serum C3 levels are usually low
(often extremely low) due to dysregulation of the
alternative pathway, but C4 levels are normal. It
should be noted, however, that normal serum C3
MPGN, as normal C3 concentration is not excep-
tional in C3 glomerulopathies [110, 111]. Remark-
ably, poststreptococcal glomerulonephritis is also
characterized by this pattern, which can produce a
similar clinical syndrome. However, in C3
glomerulopathies, the decrease in C3 is usually
persistent, whereas it is transient in poststrep-
tococcal glomerulonephritis, resolving most fre-
quently in about 8 weeks from initial presentation.
Membranous Nephropathy
Membranous nephropathy is characterized by
heavy proteinuria, followed by the onset of
952
full-blown nephrotic syndrome. It is caused by the
subepithelial accumulation of immune deposits,
which triggers local complement activation.
Recently, phospholipase A2 receptor antibodies
have been shown as the underlying cause in a
significant proportion of patients with idiopathic
membranous nephropathy [112].
In children, secondary membranous nephropa-
thy may occur more frequently than in adults. It is
caused by a variety of chronic infections, inflam-
mation, or antigenemia (HBV, HCV, syphilis,
malaria, sickle-cell anemia, D-penicillamine treat-
ment, etc.) [113]. Lately, cationic bovine serum
albumin that may be absorbed in an undigested or
partially digested form from the gastrointestinal
tract has been described as an environmental trig-
ger of early-childhood membranous nephropathy
[114, 115].
As the basement membrane isolates the capil-
lary lumen from the subepithelial space, the acti-
vation products of the complement cascade
cannot interact with circulating inflammatory
cells. This in part explains the lack of any prolif-
erative reaction and the relatively bland urine
sediment seen in membranous nephropathy.
Podocyte injury occurring in membranous
nephropathy is entirely complement dependent
[36]. In animal models, the inhibition of terminal
complement complex formation prevents
podocyte injury and proteinuria but does not influ-
ence antibody deposition [112, 116].
The terminal complement complex resulting
from terminal pathway activation binds the
podocyte cell membrane. However, in contrast
with erythrocytes and similar to endothelial cells,
the podocyte has the capacity to shed the terminal
complement complex from its cell membrane and
excrete it into the urine. Podocyte apoptosis may
result from direct injury following insertion of the
terminal complement complex. Alternatively, it
may be secondary to the toxic effects of locally
produced cytokines and reactive oxygen radicals
(Fig. 3, enlarged area D).
It is of interest that the majority of phospholi-
pase A2 receptor antibodies belong to the IgG4
subclass with a poor complement fixing capacity.
A possible explanation for complement activation
could be that small quantities of IgG1 and IgG3
Z. Prohászka et al.
are usually present in the deposits. Another pos-
sibility could be that the IgG4 antibodies bound to
podocytes activate the lectin pathway and induce
sublytic podocyte injury [117].
Complement Profile and Diagnostics
As heavy proteinuria in membranous nephropathy
may persist long after the immune response has
been inhibited and immune deposits do not form
any longer, there is a need for validated bio-
markers to monitor disease activity.
The glomerular deposition of C3 and C5b-9
has been shown to correlate with the immune
deposit formation in progress. However, the direct
measurement of anti-PLA2R antibody, which
reflects disease activity and the therapeutic
response, has now replaced their determination
in clinical practice [118, 119].
Antibody-Mediated Allograft Rejection
(AMR)
Antibody-mediated rejection (AMR) of the renal
graft is due largely to the presence of circulating
antibodies directed against the human leukocyte
antigens (HLA) of the donor kidney (donor-
specific antibodies, DSA).
DSA-induced damage to the graft endothelium
and microvasculature depends on the magnitude
of the injury and the repair capacity of the endo-
thelial cells (EC). EC injury ranges from cell
necrosis to adaptive changes allowing the EC to
survive but with altered morphology and function
eventually leading to the occlusion of the
microvasculature [74].
In early acute AMR, massive exposure of the
EC to DSA and the ensuing complement activa-
tion predominate with the formation of numerous
terminal complement complexes (Fig. 3, enlarged
area A). The EC can eliminate terminal comple-
ment complexes. However, if the number of ter-
minal complement complexes exceeds their
eliminating capacity, the ECs may be destroyed.
This leads to the exposure of the underlying
matrix and to the activation of the coagulation
cascade and of the platelets, resulting subse-
quently in mesangiolysis in the glomeruli and in
30 Complement-Mediated Glomerular Injury in Children
the formation of microthrombi throughout the
microvasculature. The production of complement-
derived chemotactic factors induces the recruitment
and adhesion of inflammatory cells, most com-
monly neutrophils, within the capillary lumina.
Histological features include swelling and necrosis
of the EC, denudation of the underlying matrix,
platelet aggregation, thrombotic microangiopathy,
and neutrophil infiltration [120].
Sublytic injury, as detailed above, represents a
fragile equilibrium between damage and repair.
The cycles of cell injury and repair are character-
ized by EC swelling, loss of fenestrations, and the
expression of growth and mitogenic factors. All
these result in proliferative changes manifested as
mesangial matrix expansion, duplication of the
glomerular basement membrane, and multilayering
of the basal lumina of peritubular capillaries (trans-
plant glomerulopathy and capillaropathy) [121].
Throughout the course of AMR, lytic and sublytic
EC injury coexist and provide the basis for the
morphologic and clinical heterogeneity of AMR.
Complement Profile and Diagnostics
Activation of the classical pathway plays a pre-
dominant role in AMR. The deposition of DSA
within the transplanted kidney leads to C1q bind-
ing. The presence and extent of peritubular C4d
staining is of diagnostic and prognostic impor-
tance. It should be noted, however, that
C4d-negative AMR has also been reported
recently. This is more common in patients with
late-onset, chronic AMR and in AMR due to
non-HLA DSA [122, 123]. Interestingly, the
Table 5 Therapeutic targeting of complement, selected groups of drugs, and examples
Target Name, type of the drug(s)
C5 (terminal Eculizumab (humanized anti-C5
complement monoclonal antibody)
pathway)
C3 Multiple compounds (recombinant
constructs, antibodies, minibodies,
etc.)
C5a receptor Multiple compounds (peptidic,
(CD88) non-peptidic, small molecule receptor
inhibitors)
953
intensity of C4d staining may vary significantly
if multiple biopsies are taken from the same
patient. This may reflect the concomitant activity
of the processes of injury and repair [74, 124].
Tubulointerstitial Injury in Progressive
Kidney Disease: The Role of
Complement
As reviewed in Noris M et al. [21], complement
components also appear to contribute to the
tubulointerstitial damage which is seen in all
forms of progressive kidney disease, regardless
of the initial pathogenetic mechanism. In
nonselective glomerular proteinuria, various pro-
teins of the complement system appear in the
urine, leading to intratubular deposition of C3
and of C5b-9. Their activation has been proposed
to contribute to tubulointerstitial damage via
cytotoxic, pro-inflammatory, and fibrogenic
effects [125].
Therapeutic Inhibition of Complement
in Glomerular Diseases
The improved understanding of the role of com-
plement in a number of glomerular diseases stim-
ulated the development and clinical application of
various compounds targeting C3 or C5 or acting at
different levels of complement activation
(Table 5) [128]. The availability of multiple drug
candidates ensures the potential for rapid progress
Currently licensed
indications in pediatric
kidney diseases References, comments
Atypical hemolytic uremic [97, 126, 127]
syndrome
None Preclinical and clinical
studies are in progress
[128]
None Preclinical and clinical
studies are in progress
[51]
954
in this field with clinical applicability in the next
few years.
Eculizumab, a humanized monoclonal anti-
body inhibiting the terminal complement pathway
at the level of C5, has revolutionized the treatment
of aHUS [97]. However, this agent has not proved
similarly effective in all cases of C3
glomerulopathy [129]. This heterogeneous group
of complement-mediated glomerular diseases
may benefit from complement inhibitors acting
at the level of C3 [130]. The group of compounds
(peptidic, non-peptidic, and natural inhibitors)
that target C5a receptor are promising anti-
inflammatory drugs that have the advantage of
leaving the cascade intact for host defense
[51]. Improved understanding of the role of com-
plement in the pathogenesis of glomerular dis-
eases together with the improvement of
complement diagnostics and therapy is of pivotal
importance in allowing better control of these
severe diseases in the near future.
Acknowledgments The authors are grateful to Lilan
Varga and Eniko Barnucz for their helpful suggestions.
Research in the author’s laboratories was supported by
the following grants: Hungarian Scientific Research Fund
of Hungary OTKA 100909 to GSR, and OTKA 100687,
OTKA 110909 to ZP.
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 Acute Postinfectious
Glomerulonephritis in Children 31
Bernardo Rodríguez-Iturbe, Behzad Najafian,
Alfonso Silva, and Charles E. Alpers

Contents Shunt Nephritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 974

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 960 Syphilis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 975
Acute Poststreptococcal Glomerulonephritis . . . . . 960 Other Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 975
Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 961
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 975
Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 961
Clinical Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 963
Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 967
Prognosis of APSGN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 969
IgA-Dominant Glomerulonephritis
Associated with Staphylococcal Infections . . . . . . . 969
Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 969
Clinical Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 970
Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 970
Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 970
Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 970
Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 971
Renal Disease Associated with Infective
Endocarditis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 971
Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 971
Etiology and Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 972
Clinical Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 972
Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 973
Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 974

B. Rodríguez-Iturbe (*) • A. Silva

Nephrology Service, Hospital Universitario, Maracaibo,
Estado Zulia, Venezuela
e-mail: brodrigueziturbe@gmail.com; afsilvaes@yahoo.es
B. Najafian • C.E. Alpers
Department of Pathology, University of Washington,
Seattle, WA, USA
e-mail: najafian@uw.edu; calp@uw.edu

# Springer-Verlag Berlin Heidelberg 2016 959

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_27
960
Introduction
Postinfectious glomerulonephritis (PIGN) com-
prises a large group of glomerulonephridities that
are caused by infectious agents. Acute glomerulo-
nephritis is a term that defines a pathological lesion
that may be asymptomatic or it may be manifested
clinically with the acute nephritic syndrome, or the
nephrotic syndrome, or with a rapidly progressive
renal failure. In this chapter we will deal with the
most common glomerulonephridities that result
from bacterial infections: poststreptococcal glomer-
ulonephritis, IgA-dominant glomerulonephritis
associated with staphylococcal infections, and glo-
merulonephritis associated with bacterial endocardi-
tis, infected atrial ventricular shunts, syphilis,
and deep-seated infections. Since poststreptococcal
glomerulonephritis is the best known of the acute
postinfectious glomerulonephritis and its most
frequent clinical picture is the acute nephritic syn-
drome, the terms poststreptococcal glomerulone-
phritis, acute glomerulonephritis, and acute
nephritic syndrome are often, and incorrectly, used
interchangeably. The incidence of PIGN is declining
in affluent societies where most cases now occur in
elderly individuals, many with comorbid debilitat-
ing conditions such as alcoholism, HIV infection,
drug abuse, and diabetes. Nevertheless, PIGN, and
specifically poststreptococcal glomerulonephritis, is
still frequent in children in poor communities with
limited access to medical care.
Postinfectious glomerulonephritis results from
antigen–antibody reactivity. Nephritogenic
immune complexes are formed in the circulation
or, in situ, in the glomerular basement membrane
(GBM) and, in either case, cause local activation
of the complement system and the coagulation
cascade. Classic experimental studies in the
acute (one-shot) and chronic serum sickness
models [1–3] have demonstrated that glomerular
deposition of circulating immune complexes
depend on the size of the complexes (300–500
kDa) and on antigen load and the intensity of the
antibody response (antigen/antibody ratio). The
development of glomerular inflammation and
severity, as well as the progression to chronic
renal lesions, depends, on a large measure, on
the duration of antigen exposure and the host’s
B. Rodríguez-Iturbe et al.
capacity to remove the deposited complexes. In
situ formation of immune complexes results from
the deposition of the inciting antigen in the GBM
and its penetration to the epithelial side of the
GBM where it is met by the corresponding anti-
body to form immune complexes that present
characteristic subepithelial electron-dense
deposits. In situ formation of immune complexes
occurs in conditions of antigen excess and is typ-
ical of cationic antigens that have electrostatic
charge-facilitated penetration of the polyanionic
GBM [4]. In specific types of glomerulonephritis,
the pathogenesis involves the participation of bac-
terial wall superantigens causing intense T cell
activation that recruit cytokine-mediated poly-
clonal B cell responses; this is the case in glomer-
ulonephritis associated with staphylococcal
infection and predominant IgA deposition.
Immune complexes may be predominantly
located in the mesangium or in the glomerular
basement membrane (GBM) or in both, and the
immunoglobulin and complement deposition is of
granular (not linear) appearance as revealed by
immunofluorescence microscopy.
Some histological characteristics are often asso-
ciated with some clinical features: mesangial pro-
liferative glomerulonephritis usually has a sell-
limited course characterized by microscopic or
macroscopic hematuria and non-nephrotic protein-
uria. Heavy Ig deposition in the GBM and abun-
dance of electron-dense subepithelial deposits are
frequently associated with massive proteinuria.
Acute Poststreptococcal
Glomerulonephritis
Dark urine and suppression of urine output was a
feared complication of the convalescent period of
scarlet fever as recorded in the medical literature
more than six centuries ago [5]. It was then a
common disease, and the death of Mozart in
1791 was probably due to acute postscarlatinal
glomerulonephritis [6]. The findings that acute
glomerulonephritis followed streptococcal upper
respiratory and skin infections [7, 8] permitted the
definition of the disease as acute poststreptococcal
glomerulonephritis (PSGN), and the seminal
31 Acute Postinfectious Glomerulonephritis in Children
theoretical contribution of Clemens von Pirquet
[9], attributing acute PSGN (APSGN) to altered
reactivity (allergy) caused by “harmful”
antigen–antibody complexes of nephritogenic
potential (as opposed to protective antibodies
induced by vaccination), defined the pathogenesis
of this condition and opened the era of immune
complex-mediated diseases [10].
Epidemiology
In the last decades the epidemiology of APSGN
has changed along with a decline in its incidence
worldwide and particularly in industrialized coun-
tries where the disease is now rare and associated
with debilitating conditions such as alcoholism or
diabetes [11]. The reduction in the incidence of
APSGN is attributed to a variety of factors,
including easier and earlier access to competent
medical treatment of streptococcal infections and
the widespread use of fluorination of water since
virulence factors in Streptococcus pyogenes are
reduced with fluoride exposure [12]. Not only the
incidence but the severity of APSGN in children
appears to be diminishing in developed countries
[13], while in high-risk aboriginal populations,
such as the French Polynesia aboriginal commu-
nities, massive proteinuria, congestive heart fail-
ure, and renal failure are frequent features of
APSGN [14].
The incidence of PSGN is also decreasing in
developing countries but is not uncommon.
Endocapillary glomerulonephritis, assumed to be
of poststreptococcal etiology, is the most common
glomerulonephritis in children in underprivileged
societies [15] and particularly in aboriginal com-
munities [16, 17]. Two studies [18, 19] have cal-
culated the global burden of APSGN and came to
similar conclusions, with lower estimates of
9.3–9.8 cases per 100,000 population per year in
developing countries and higher estimates that
could be three times these values. The importance
of educational programs has been evidenced by
the reduction in the incidence of APSGN
observed after the implementation of programs
designed to correct the high incidence of rheu-
matic fever in the Caribbean islands [20].
961
APSGN may occur in sporadic cases or in
epidemic outbreaks. Earlier epidemics have been
found to appear periodically in closed populations
as the Red Lake Indian Reservation in Minnesota
and in specific areas in less developed countries as
Port of Spain in Trinidad and in Maracaibo, Ven-
ezuela. The risk of nephritis in epidemics ranges
from 5 % in throat infections to as high as 25 % in
pyoderma caused by M-type 49 streptococci
[21–23]. The most recent epidemic has been
reported from Nova Serrana, Brazil, and was
caused by Streptococcus zooepidemicus and asso-
ciated with the consumption of unpasteurized
milk from cows with mastitis [24]. Similar etiol-
ogy has been found in clusters of cases reported in
poor communities in industrialized countries [25].
Pathogenesis
The recognition that acute rheumatic fever and
glomerulonephritis are noninfectious complica-
tions of streptococcal infection with different epi-
demiological and biological characteristics led to
the concept that there existed rheumatogenic and
nephritogenic streptococcal strains [26, 27].
Among these differences, the rarity of recurrence
of APSGN (in contrast to the frequency of recur-
rence in rheumatic fever) suggested that
nephritogenic antigen(s) induced a long-lasting
protective immunity and raised the possibility of
a potential vaccination.
Streptococcal Antigens. APSGN has an
immune complex pathogenesis. The identity of
nephritogenic antigens has been the subject of
considerable investigation and controversy
(reviewed in Rodriguez-Iturbe and Batsford
[10]). Group A streptococci were traditionally
considered the culprit of APSGN. M types
47, 49, 55, and 55 corresponded to streptococci
causing nephritis associated with pyodermitis,
and M types 1, 2, 4, and 12 corresponded to
streptococci causing epidemics associated with
upper respiratory infection. However, more
recently, it has been found that glomerulonephritis
may result from group C streptococci (Streptococ-
cus zooepidemicus) both in epidemic [24] and
sporadic cases [25, 28] making evident that
962
antigenic fractions capable of causing nephritis
are not exclusively derived from group A
streptococci.
Recent research has focused in two antigenic
fractions: the so-called nephritis-associated plas-
min receptor (NAPlr), identified as glyceraldehyde
3-phosphate dehydrogenase (GAPDH) [29, 30],
and the streptococcal pyrogenic exotoxin
(erythrotoxin) B (SPEB) and its zymogen precur-
sor (zSPEB) [31, 32]. NAPlr and SPEB have been
identified in early biopsies and elicit antibody
responses that may be detected in the vast majority
of convalescent sera. The frequency of the anti-
body response to SPEB/zSPEB has now been
shown to be the best antistreptococcal antibody
response for the purposes of identifying a poten-
tially nephritogenic infection [33]. Simultaneous
testing of both these fractions in renal biopsies of
APSGN patients and convalescent sera indicated
that in the population of patients tested, antibody
response to and glomerular deposits of SPEB were
frequent while those to NAPlr were unusual
[34]. The discordant results may be due to the
role of host-related factors in the development of
APSGN since NAPlr studies were done in a rela-
tively homogenous Japanese population while the
SPEB studies were done in populations of patients
in Latin America and Europe. In fact, studies in
biopsies of Japanese patients did demonstrate the
deposits of both putative nephritogens [29].
GAPDH is localized in the areas of the glomer-
uli with plasmin-like activity. Increased plasmin
binding may promote the local inflammatory reac-
tivity and facilitate the penetration of nephritogenic
antigen–antibody complexes. The finding of
increased urinary plasmin activity in APSGN is
suggestive of increased urinary GAPDH
[35]. SPEB is co-localized in the glomeruli with
complement and Ig deposits and is the only strep-
tococcal antigen that has been localized within the
electron-dense subepithelial deposits (humps) [34]
that are characteristic of APSGN; the cationic
(pk > 8.0) nature of this component favors the
penetration of the anionic barrier in the GBM.
Histones are other cationic elements that may also
be relevant in the pathogenesis of APSGN since
after streptococcal lysis they enter in circulation,
trigger an in situ formation of immune complexes,
B. Rodríguez-Iturbe et al.
and induce the production of pro-inflammatory
cytokines [36]. SPEB/zymogen induces leukocyte
infiltration in the glomerulus, possibly as a result of
the induction of chemotactic and migration inhibi-
tion factor activities [37], and increases angiotensin
II production in mesangial cells [38]. The possibil-
ity of inducing a protective immunity to poststrep-
tococcal sequelae has been recently raised in
studies that determined that the Fc portion of anti-
bodies directed to SPEB bind to the C-terminal
domain (rSPEB 345–398) and immunization with
this domain prevents group A streptococcal infec-
tion in mice [39].
Finally, the possibility that more than one
streptococcal antigen is nephritogenic was made
evident by the demonstration that the gene
encoding for SPEB was absent in the
S. zooepidemicus causing the recent APSGN epi-
demic in Brazil which would indicate that this
antigen was not involved in this epidemic [40].
Immune Reactivity. Immune complexes
formed against nephritogenic streptococcal anti-
gen(s) may be formed in the circulation and
deposited in the glomeruli or formed in situ
against antigenic fractions planted in the glomer-
uli. Both mechanisms are likely present in
APSGN. The relevance of the circulating immune
complex mechanism derives from the classic
experiments of the glomerulonephritis that
develops in experimental models of acute serum
sickness [1, 2]. The participation of an in situ
formation of immune complex is likely because
the subepithelial electron-dense deposits (humps)
that are a typical lesion of acute PSGN are difficult
to produce experimentally by the injection of
preformed immune complexes while they are
common when cationic antigens are injected [4].
Consequences of the deposition of the
antigen–antibody complexes are the local activa-
tion of the complement and coagulation cascades.
Complement Activation in PSGN. Reduction
of circulating complement levels is a constant fea-
ture in APSGN. The classical pathway of comple-
ment activation is partially blocked by Ig-binding
proteins in streptococcal surface [41, 42] and
is the alternate pathway, the one that is usually
activated, perhaps by a transient expression of
C3Nef autoantibody [43]. Yet, 15–30 % of patients
31 Acute Postinfectious Glomerulonephritis in Children
with APSGN have reduced levels of C1 and C4
components. Relevant to these findings are the
demonstration that Protein H, a surface protein of
Streptococcus pyogenes in combination with IgG,
can activate the classic complement pathway [44].
The lectin pathway of complement activation may
also be activated in PSGN [45], but individuals
genetically unable to activate this pathway may
still develop PSGN [46].
Cellular Immunity and Cytokines. Cell-
mediated mechanisms are also involved in the
development of APSGN. Glomerular infiltration
of lymphocytes and macrophages has long been
recognized as a feature of the disease
[47]. Intercellular leukocyte adhesion molecules,
such as ICAM-1, are overexpressed in glomeruli
and tubulointerstitial regions and correlated with
the intensity of the inflammatory infiltration
[48]. Cytokine production plays an important
role in the renal injury in PSGN. SPEB, one of
the putative nephritogenic antigens, induces
mesangial proliferation, and increased IL-6 pro-
duction [49] of glomerular IL-8 expression corre-
lates with neutrophil infiltration and transforming
growth factor –β with mesangial expansion in
acute PSGN [50].
Autoimmune Reactivity. A number of auto-
immune findings have been reported in APSGN;
among them, anti-IgG reactivity is the best stud-
ied. High titers of rheumatoid factor are present in
about two-thirds of the patients in the first week of
the disease; furthermore, anti-IgG glomerular
deposits are frequently found in biopsies (see
later), and anti-IgG reactivity has been
documented in the IgG eluted from the kidney in
a fatal case of the disease [10, 23]. The anti-IgG
reactivity may be the result of autoantigenic mod-
ification of Ig that occurs with its desialization
caused by streptococcal neuraminidase. This pos-
sibility was raised by experimental studies show-
ing that injection of neuraminidase-treated
autologous IgG causes the generation of anti-
IgG reactivity and received support from the dem-
onstration of plasma neuraminidase activity and
increased free sialic acid levels in patients with
APSGN [51, 52]. Since desialized leukocytes
infiltrate more easily the glomeruli, neuramini-
dase may have the additional effect of facilitating
963
renal inflammation [53]. Streptococcal neuramin-
idase was considered responsible for the simulta-
neous presentation of acute PSGN and thrombotic
microangiopathy [54]. Another potential cause of
anti-Ig reactivity is the binding of IgG to type II
receptors in the streptococcal wall. This binding
produces intense anti-IgG reactivity that may play
a role in PSGN [55].
Additional autoimmune phenomena have been
found in APSGN patients. Anti-DNA antibodies,
anti-C1q antibodies, and antineutrophil cytoplas-
mic antibodies (ANCA) are present in some
patients. Interestingly, the latter has been found
in two-thirds of the patients with azotemia and
70 % of the patients with APSGN that develop
crescentic glomerulonephritis [56].
Genetic Factors. The familial incidence of
acute PSGN is indicated by the presentation of
the disease (clinical and subclinical) in 38 % of the
siblings of index cases [57]. This incidence is
higher than the attack rate in the general popula-
tion in epidemics that range between 5 % and
28 %. APSGN has been associated with
HLA-DR4 and DR-1 [23] but a definite genetic
link has not been established. Genetic mutations
in the complement factor H (CFH) gene have been
suggested as a cause for inducing dense deposit
disease whose manifestations may be triggered by
an episode of PSGN [58] with a clinical course
atypical for APSGN characterized by glomerulo-
nephritis and chronic kidney disease persisting
after streptococcal infection [59].
Clinical Characteristics
Children from 4 to 14 years are more frequently
affected by acute PSGN. It is rare below the age of
2 and above the age of 20 and is twice more
frequent in males than in females. The usual
sites of antecedent infection are the skin and the
throat, although any location of streptococcal
infection is possible. The latent period between
infection and nephritis is longer after skin infec-
tions (3–5 weeks) than after upper respiratory
infections (7–15 days). During the latent period,
asymptomatic children may have microscopic
hematuria.
964
APSGN may have a subclinical course or may
present with the acute nephritic syndrome and
more rarely with nephrotic syndrome or, excep-
tionally, with a rapidly progressive (crescentic)
glomerulonephritis [23]. Subclinical disease is
characterized by a reduction of serum comple-
ment, microscopic hematuria, and normal or
increased blood pressure in asymptomatic
patients. In epidemic conditions this presentation
occurs 1.5 times more frequently than clinically
apparent disease [21]. Prospective studies in
household members of index cases have shown
that in non-epidemic situations the patients with-
out symptoms are four to five times more frequent
than symptomatic patients [57, 60]. The most
typical clinical picture in APSGN is the acute
nephritic syndrome (hematuria, edema, hyperten-
sion, and moderate proteinuria). Glomerular
hematuria is an almost universal finding, and
gross hematuria is present in one-third of the
patients. The absence of red cells in the urine is
usually due to delays in the examination of the
urinary sediment since red cells are rapidly
destroyed, especially in alkaline urine. As a rule,
red cell casts are present in association with dys-
morphic of red cells, frequently presenting dough-
nut shape with one or more blebs. Macroscopic
hematuria usually disappears after a few days, but
microscopic hematuria may persist for a year and
occasionally exacerbates during febrile episodes
and more rarely after strenuous exercise. Edema is
the chief complaint more frequently in children
(90 %) than in adult patients (75 %). Younger
children tend to have anasarca more often than
adolescents or adult patients, but ascites is uncom-
mon, except in the cases with nephrotic syndrome.
Hypertension is present in 60–80 % of the chil-
dren and is severe enough to require specific anti-
hypertensive treatment in about half of the cases.
Edema and hypertension typically disappear in
5–10 days. Oliguria is identified at initial presen-
tation by the patient or their family in less than
half of the patients. Other nonspecific symptoms
include general malaise, weakness, headache, dull
lumbar pain, and nausea. Massive proteinuria
with or without other features of the nephrotic
syndrome are found in about 2 % of the cases in
children, and its persistence is a risk factor for
B. Rodríguez-Iturbe et al.
progression to chronic renal disease. Azotemia
occurs in 25–30 % of the children, but the need
of dialysis is infrequent. A rapidly progressive
azotemia may occur exceptionally and when pre-
sent is usually to the development of crescentic
glomerulonephritis.
Pathophysiology of the Acute Nephritic
Syndrome. The acute nephritic syndrome results
from the reduction of the glomerular filtration rate
caused by the inflammatory reaction in the glo-
meruli. The renal blood flow is usually normal,
and therefore the filtration fraction is depressed,
frequently below 1 %. The reabsorption is appro-
priately reduced in the proximal tubule and
assumed to be maintained at the distal areas of
the nephron inaccessible to micropuncture. While
the reduction in glomerular filtration has long
been assigned a central role in the fluid retention
in acute glomerulonephritis, the participation of
additional undefined distal factor(s) is required. It
is obvious that the reduction of glomerular filtra-
tion is a feature of renal conditions that are not
necessarily associated with fluid retention and,
conversely, severe fluid retention may occur with-
out reduction in glomerular filtration rate. Poten-
tial influences that may modulate sodium and fluid
retention at more distal levels are endothelial or
mesangial factors released by glomerular injury
[61], overexpression of the epithelial sodium
channel [62], and interstitial inflammatory cells
capable of maintaining an increased intrarenal
angiotensin activity [63]. At any rate, the net result
of the hemodynamic changes in glomeruli and
tubules is a tendency to sodium and water retention
that results in expansion of the extracellular vol-
ume, edema, and hypertension. The potential addi-
tional effects of hypoalbuminemia are not present
in the acute nephritic syndrome and, accordingly,
the plasma levels of hormones that regulate the
extracellular volume (renin–angiotensin, aldoste-
rone, and atrial natriuretic peptide) show an
appropriate response for an expanded extracel-
lular volume. The suppression of plasma renin
activity and aldosterone and the stimulation of
atrial natriuretic peptide are correlated with
the severity of edema. Renal prostaglandin
and kallikrein activities are suppressed [23].
In concordance with this pathophysiology, the
31 Acute Postinfectious Glomerulonephritis in Children
improvement of volume retention with the
development of spontaneous or induced diuresis
results in a prompt reduction of the edema and
correction of the hypertension.
Serological Findings. The most constant sero-
logical finding is the reduction in serum comple-
ment levels that occurs in more than 90 % of the
cases. The activation of the complement system is
usually via the alternative complement pathway
and reduced C3 with normal C1 and C4, but, as
mentioned earlier, the classic and the lectin path-
ways of complement activation may also be
engaged in some patients. There are no clinical
characteristics associated to a specific comple-
ment activation modality, and complement levels
return to normal usually within 1 month of the
development of acute glomerulonephritis. IgG
and IgM serum levels are elevated in 80–90 %
of the patients with APSGN. Two-thirds of the
patients with APSGN in the first week of the
disease present cryoglobulins and elevated
IgG–anti-IgG rheumatoid factor titers [23].
Serum antistreptococcal antibody titers are
discussed later (see “Diagnosis”).
Pathology. At the present time, renal biopsy is
seldom done in patients with APSGN with a well-
defined clinical picture. Biopsy is indicated when
specific features raise the possibility of a different
diagnosis. For instance, if the serum complement
is normal, a biopsy may define if glomerulone-
phritis has a different etiology, such as IgA
nephropathy, and if the serum complement
remains low after 1 month, the biopsy may help
to rule out hypocomplementemic mesangio-
capillary glomerulonephritis or lupus nephritis.
In patients with proteinuria in the nephrotic
range, a biopsy may serve to confirm the diagnosis
of APSGN and to raise awareness of a worse long-
term prognosis. Finally, if the patient has a rising
serum creatinine, the diagnosis of crescentic glo-
merulonephritis needs biopsy confirmation.
The classic pathologic manifestations of
postinfectious glomerulonephritis (PIGN) are suf-
ficiently distinctive at both the histological and
ultrastructural studies that these disorders can
often be reliably diagnosed in a renal biopsy.
APSGN is the prototype of the PIGN resulting
from immune complex deposition and the
965
demonstration of immune complexes in the glo-
meruli by immunofluorescence or electron
microscopy, preferably by both, and is key to
establishing the diagnosis in a renal biopsy. By
light microscopy, all glomeruli are affected, and
generally all to a similar extent. The glomerular
capillaries are dilated and hypercellular. In many
cases there is an increase of endothelial and/or
mesangial cells, and the endothelial cells in par-
ticular appear swollen (a constellation of findings
sometimes referred to as “endocapillary prolifer-
ation” or “endocapillary hypercellularity”). Glo-
merular capillaries typically contain a prominent
influx of inflammatory cells, especially neutro-
phils and monocytes (Fig. 1a). Because of the
large numbers of neutrophils, the descriptive
term “exudative glomerulonephritis” has been
applied to these lesions. Eosinophils and lympho-
cytes may be present, but not in large numbers.
Overt necrosis is rarely identified, despite the
severe inflammation within the glomeruli. How-
ever, extracapillary proliferation with formation
of crescents and/or adhesions (synechiae) is
often present in severe cases and presumably is a
consequence of glomerular basement membrane
rupture due to the severe inflammatory injury.
Erythrocytes and sometimes neutrophils may be
present in Bowman’s space, often concurrently
with crescents. In renal biopsies obtained a few
weeks after the appearance of clinical symptoms,
the picture may be less inflammatory, and the
number of neutrophils will have decreased, the
swelling of endothelial cells will have subsided,
and the number of humps seen by electron micros-
copy decreased. Nevertheless, at later stages in the
evolution of disease, diffuse mesangial hypercel-
lularity may still be seen, and this sometimes lasts
for several months. The combination of accentu-
ation of the normal glomerular lobular architec-
ture as a result of glomerular cell proliferation, the
overall hypercellularity of the glomeruli, and the
endocapillary changes involving the glomerular
capillaries can lead to situations where PIGN is
difficult to distinguish from membranoproli-
ferative glomerulonephritis by light microscopy.
Immunofluorescence and electron microscopy
usually allow the distinction between the two
diseases to be made. Other important
966
Fig. 1 Poststreptococcal glomerulonephritis. (a) Light
microscopy: global endocapillary hypercellularity/prolif-
eration with prominent influx of inflammatory cells,
including many neutrophils in capillary lumina and swol-
len endothelial cells (Jones methenamine silver stain, 40
objective). (b) Immunofluorescence: Coarse granular pat-
tern of C3 deposition in peripheral capillary walls and
considerations in the differential diagnosis
include lupus nephritis, which usually can be dis-
tinguished on clinical grounds and by the constel-
lation of associated immunofluorescence and
electron microscopic findings. Some cases of
acute glomerulonephritis morphologically indis-
tinguishable from postinfectious glomerulone-
phritis but with an atypical, non-resolving
clinical course have proven to be cases of the
recently recognized entity C3 glomerulopathy
[59, 64].
Immunofluorescence studies in biopsies taken
during the first 2–3 weeks of the diseases most
often show diffuse, irregular, coarse granular
deposits of immunoglobulin G (IgG), IgM, and
C3 along the glomerular capillary walls (Fig. 1b).
The C3 deposits resolve later than immunoglobu-
lin, and biopsies obtained late in the disease
course may show predominantly or only C3 by
immunofluorescence. Antihuman IgG deposits
can be demonstrated in 20–30 % of the cases
and less extensive deposits of C1q may occasion-
ally be present [15, 65, 66]. Based on the distri-
bution and appearance of the immune deposits,
Sorger et al. [67] recognized by immunofluores-
cence heavy “garland-type” immune deposits in
the capillaries and immune deposits presenting a
“starry sky” and “mesangial” staining patterns.
The garland type of immune deposits has been
B. Rodríguez-Iturbe et al.
mesangial regions of a glomerulus, showing in some
areas a “starry sky” pattern. (c) Electron microscopy:
Characteristic subepithelial hump-like deposits (arrows)
over the subepithelial aspect of the glomerular basement
membranes (asterisk). Abbreviations: PMN polymorpho-
nuclear, PC podocyte, E endothelial cell (11,000)
associated with heavy proteinuria [67, 68]. The
other types of immune deposits are not clearly
related to clinical behavior or prognosis. The pat-
terns of immune deposits likely represent the
changing distribution of immune complexes as
the disease evolves over time.
The ultrastructural changes in glomerular cells
observed by electron microscopy closely corre-
spond to the changes seen by light microscopy.
Swelling of glomerular endothelial cells and
increased mesangial cellularity are common. The
glomerular basement membranes are usually of
normal contour and thickness, although locally
some thickening may occur. The most consistent
and classic change is the presence of glomerular
subepithelial dome-shaped electron-dense
deposits, widely referred to as “humps” (Fig. 1c).
These are especially numerous during the first
weeks of acute glomerulonephritis, and their num-
ber decreases thereafter. Podocyte foot processes
overlying the humps are often effaced, with con-
densation of cytoplasmic microfilaments.
Early in the evolution of disease, the immune
deposits may form in the subendothelial aspect of
glomerular capillary walls and after undergoing
some process of dissociation as the disease
evolves reform as humps at the subepithelial sur-
face of the glomerular capillary walls. This migra-
tion process is characteristic of the in situ immune
31 Acute Postinfectious Glomerulonephritis in Children
complex formation shown experimentally to
result from cationic antigens [69]. The early
subendothelial deposits are uncommonly visual-
ized in electron micrographs of renal biopsies, but
in a minority of cases, these may be prominent and
impart a predominantly membranoproliferative
pattern of injury. Such cases require careful clin-
icopathological correlation to distinguish them
from other diagnostic entities, principally
membranoproliferative glomerulonephritis of
noninfectious origin, lupus nephritis, and rarely
IgA nephropathy. These cases also overlap with
systemic or deep infection-related glomerulone-
phritis, such as nephritis due to infected ventricu-
loatrial shunts, deep tissue abscesses, and
endocarditis. In addition, some small irregular
electron-dense deposits may be seen in the lamina
densa of glomerular basement membranes and the
mesangium.
After resolution of the acute disease, residual
subepithelial electron-dense deposits may persist
for extended periods of time. These are typically
located at so-called mesangial “waists,” where the
glomerular capillary walls overlay the mesangium,
and may result in an immunofluorescence pattern
of predominately mesangial staining of immune
reactants. A few of these deposits may persist for
very long periods of time, and these are typically
discovered as incidental findings in renal biopsies
performed for other clinical indications unrelated to
PSGN [65, 70].
Diagnosis. The typical clinical presentation is
that of a child of 4–15 years who suddenly
develops dark and scanty urine, swelling of the
face and legs, and high blood pressure. The initial
diagnostic approach should attempt to define if the
acute nephritic syndrome is associated with a sys-
temic condition or if it is resulting from a primary
renal disease. It is then necessary to establish an
etiologic diagnosis of glomerulonephritis. A his-
tory of a precedent upper respiratory infection
and/or skin infection suggests a poststreptococcal
etiology that may be confirmed by a positive
culture or rising antistreptococcal antibody titers.
Positive streptococcal cultures are frequent in epi-
demic conditions but only in less than 25 % of the
sporadic cases; consequently, the evidence of pre-
vious streptococcal infection usually resides in the
967
demonstration of a rising titer of serum antistrep-
tococcal antibodies. Anti-NAPlr and antiSPEB-
zSPEB titers have a greater specificity for
nephritogenic infections [33] but are not generally
available. Antistreptolysin O titers and anti-
DNAse B titers are the most frequently elevated
in upper respiratory infections and pyodermitis,
respectively. Streptozyme test which includes
four antigens (DNAse B, streptolysin O, hyal-
uronidase, and streptokinase) is reported to be
positive in more than 80 % of the cases [23, 33].
Complement levels are a first-line diagnostic
test that permits consideration of acute glomer-
ulonephritides that usually present with low
serum complement (APSGN, lupus nephritis,
shunt nephritis, endocarditis, cryoglobulinemia,
hypocomplementemic membranoproliferative
glomerulonephritis) and those that usually present
with normal complement levels (IgA nephropa-
thy, mesangioproliferative GN, hemolytic uremic
syndrome, Henoch–Schonlein purpura, vasculitis,
anti-GBM disease). The finding of profoundly
depressed C4 levels in association with normal
or mildly reduced C3 suggests the diagnosis of
cryoglobulinemia type II and the reduction of C1,
C4, and C3 levels, indicating activation of the
classic pathway of complement, is characteristic
of lupus erythematosus. Serum complement
levels return to normal in less than 1 month, and
persistence of low complement levels should raise
suspicion of a diagnosis different from APSGN.
Physical examination findings suggestive of
nephrotic, rather than nephritic syndrome, are a
soft, paperlike consistency of the ear cartilage and
the existence of ascites, since both these signs are
associated with long-standing hypoalbuminemia.
Inasmuch as the nephrotic syndrome is rare in
APSGN, the demonstration of a massive protein-
uria favors the diagnosis of lupus nephritis,
nephritis associated with visceral abscesses, and
membranoproliferative glomerulonephritis.
Treatment
Antibiotic Treatment. The first question to be
considered is when to give antibiotic treatment to
a suspected nephritogenic streptococcal infection.
968
From a clinical viewpoint, the diagnosis of active
skin infection is usually straightforward. However,
clinical judgment may miss half of the streptococ-
cal pharyngitis and may incorrectly diagnose a sore
throat as due to streptococcal infection in 20–40 %
of the cases [71]. Several clinical scores have been
proposed to increase the accuracy of this diagnosis,
and among the most popular of them is the one
proposed by McIsaac et al. [72]. This score has a
range from 0 to 4 and incorporates age as one of the
criteria. The score gives a +1 to each one of the
following: temperature >38 C, cervical
adenopathy, no cough, tonsillar exudate, and age
between 3 and 14 years. Age >44 years is assigned
1 point. Sensitivity and specificity of the score is
85 % and 95 %, respectively. Antibiotic treatment
is recommended (without culture confirmation)
when the score is 4, and antibiotic treatment is not
indicated (and cultures unnecessary) when scores
are 0–1.
Rapid, high-sensitivity streptococcal tests are
good guides to treat if they are positive, but a
negative test requires confirmation [73]. Neverthe-
less, a recent report indicates that a decision to
treat or not to treat based on the results of these
tests is not associated with a higher incidence of
poststreptococcal complications [74]. More
recently, evaluation of a home score for the diag-
nosis of streptococcal pharyngitis in patients
15 years and older, based on patient-reported clin-
ical variables, has been calculated that avoided
230,000 doctor visits each year in the United
States [75].
The diagnosis of PSGN carries with it the indi-
cation of treatment with penicillin or, in allergic
individuals, erythromycin. If infection is present at
the time of diagnosis, it requires treatment. Early
administration of penicillin is reported to prevent or
ameliorate the severity of acute glomerulonephri-
tis, and at least one report suggests that APSGN
patients that receive antibiotic treatment have a
milder clinical course [76]. If infection is not appar-
ent at the time of diagnosis, antibiotic treatment
should be given anyway because positive cultures
are sometimes obtained in apparently healthy
patients and cross infection of household members
and siblings of index cases is very high [57].
B. Rodríguez-Iturbe et al.
1.2 million units of benzathine penicillin in adults
and adolescents and half this dose in small chil-
dren, or alternatively, oral phenoxymethyl or
phenoxymethyl penicillin G 125 mg, every 6 h
for 7–10 days, are adequate treatment. Erythromy-
cin (250 mg every 6 h and 40 mg per kg in children,
for 7–10 days) is the treatment of choice in patients
allergic to penicillin. It should be noted that the
existence of erythromycin-resistant strains may be
increasing in developing countries. The incidence
of resistance to erythromycin in isolates of Strep-
tococcus pyogenes is 9.7 % in Japan [77], 21.7 % in
Spain [78], 6.8 % in the United States [79], and
11.6 % in bacterial isolates from several European
countries [80].
Preventive antibiotic treatment is indicated in
epidemic situations and to household members of
index cases in non-epidemic conditions since
most of them present evidence of recent infection
and about one-third of them develop nephritis
[57]. In high-risk communities the strategy of
treating household contacts has resulted in a
decrease in the number of cases of PSGN [81].
Treatment of the Acute Nephritic Syn-
drome. Patients with subclinical disease may be
followed as outpatients but patients with the acute
nephritic syndrome require hospitalization. Bed
rest is difficult to enforce and is of unproven
value, yet most children keep it on their own
while they are in the acute phase. Restrictions of
fluid and sodium intake are the cornerstones of the
treatment of patients with the acute nephritic syn-
drome. Cases that present significant edema,
hypertension, and circulatory congestion benefit
from the administration of loop diuretics (40 mg
IV or orally every 12 h). This therapy facilitates
the resolution of edema and ameliorates the hyper-
tension that is driven by extracellular volume
expansion [82]. Diuretic therapy seldom if ever
is required for longer than 48 h. Other diuretics are
without effect (thiazide diuretics) or dangerous
because of the possibility of hyperkalemia (aldo-
sterone antagonists).
Patients who present with severe hypertension
may require antihypertensive treatment, and nifed-
ipine (5 mg in children, every 4–6 h) is usually
effective. Parenteral hydralazine may be required
31 Acute Postinfectious Glomerulonephritis in Children
but the possibility of tachycardia requires close
observation. Angiotensin-converting enzyme
inhibitors and type 1 receptor blockers carry the
risk of hyperkalemia. Exceptionally, nitroprusside
is required to control hypertensive encephalopathy.
Another potential complication, frequently
associated with hypertension, is the posterior
reversible leukoencephalopathy that has recently
been reported in acute PSGN [83]. This compli-
cation is manifested clinically with mental distur-
bances, visual hallucinations, headache, and
convulsions and may be confused with hyperten-
sive encephalopathy. The diagnosis requires the
use of nuclear magnetic resonance image studies.
Pulmonary edema is rare and should be treated
with oxygen therapy, rotating tourniquets and
loop diuretics. Digitalis is not indicated because
it is ineffective and may result in intoxication.
Rarely overt heart failure and pulmonary edema
may complicate the clinical course. Hemodialysis
or peritoneal dialysis may be required occasion-
ally in children for the treatment of hyperkalemia,
uremia, or severe circulatory congestion.
A rapidly progressive azotemia is usually asso-
ciated with crescentic APSGN. While of
unproven efficacy, there are anecdotal reports of
beneficial effects of pulse methylprednisolone
therapy.
Prognosis of APSGN
The short-term prognosis of APSGN is excellent
in children. Fatalities may occur as a result of
hyperkalemia or pulmonary edema, but they are
exceedingly rare. The long-term prognosis of
APSGN has been the subject of many reports
since the initial studies in the first half of the
twentieth century reported essentially a complete
recovery, but the follow-up periods were short and
the patients were only children in the majority of
the studies. Subsequent observations gave widely
different results, and abnormal urinary findings in
patients followed for extended periods of time
ranged from 3.5 % [84] to 60 % [85]. The discrep-
ancies may be due in part to the lack of discrim-
ination between adult and children populations in
969
the various studies since the long-term prognosis
of APSGN is substantially worse in adults, partic-
ularly those with persistent proteinuria in the
nephrotic range. An unfavorable course was also
observed in the patients in the Nova Serrana epi-
demic, most of whom were adults. Chronic renal
failure developed in 8 % of these patients after
5 years [86]. In contrast, children have in most
studies a much more favorable long-term progno-
sis. A combined analysis of reported data indi-
cates that 20 % of the children followed for 10–20
years after acute PSGN have an abnormal urine
analysis but azotemia was found in less than 1 %
of the patients, and in our own follow-up studies
of 110 children followed for 15–18 years after the
acute attack, non-nephrotic proteinuria was found
in 7.2 %, microhematuria in 5.4 %, hypertension
in 3.0 %, and azotemia in 0.9 % [19]. Neverthe-
less, the subsequent contribution of additional risk
factors, such as diabetes and obesity, worsens the
late prognosis of these patients. Studies in
Australian aboriginal communities where these
risk factors are prevalent indicate that patients
who had APSGN have an increased risk for albu-
minuria (adjusted odds ratio 6.1 %, 95 % CI
2.2–16.9) and hematuria (OR 3.7, CI 1.8–8.0) in
relation to controls that did not have APSGN
[87]. The history of PSGN was significantly asso-
ciated (odds ratio 3–4) with increased incidence of
reduced (<60 ml/min) glomerular filtration
rate [88].
IgA-Dominant Glomerulonephritis
Associated with Staphylococcal
Infections
Epidemiology
In 1995 Koyama et al. [89] described a severe
form of glomerulonephritis associated with meth-
icillin-resistant Staphylococcus aureus (MRSA)
infections that was characterized by dominant or
codominant IgA deposits and was caused by
staphylococcal superantigens similar to those
causing the toxic shock syndrome. The incidence
of IgA-dominant glomerulonephritis has been
970
estimated in 1.6 % of the biopsies in adult patients
[90], but in recent reports from Japan, the fre-
quency appears to be decreasing, in association
with the observed reduction in the number of
MRSA infections [91]. IgA-dominant glomerulo-
nephritis is usually observed in hospital-acquired
staphylococcal infections in diabetic patients, but
it may occur in community-acquired staphylococ-
cal infections [92] and is not exclusive of staphy-
lococcal infections or diabetic patients [93].
Clinical Characteristics
The average age of the patients is 60 years; how-
ever, it has been reported in children [94]. The
disease is three to four times more frequent in
males, and the usual clinical presentation is acute
renal insufficiency. Massive proteinuria is present
in 51 % of the patients and gross hematuria in
20 % [66, 95]. Approximately half of the cases
occur in diabetic patients, and the existence of
diabetes is a risk factor for the development of
chronic renal disease: two-thirds of the patients
that developed end-stage renal disease in this con-
dition are diabetic. The most common site of
infection is the skin, followed by surgical wounds
and intravenous lines, but in some instances the
site of the original infection is not apparent.
Serum complement levels are frequently low,
but they may be normal and IgA serum levels are
frequently increased [93]. The number of circulat-
ing Vβ2(+) T cells expressing CD45RO, a marker
of activation, is increased, and high levels of sev-
eral cytokines, including tumor necrosis factor-
alpha, interleukin-1 beta, IL-2, IL-6, IL-8, and
IL-10, may be found [94, 96].
Pathogenesis
Glomerulonephritis associated with staphylococ-
cal infections can result from immune complex-
mediated renal disease, but the pathogenesis
of the IgA-dominant glomerulonephritis is the
result of superantigen-induced immune reactivity.
Staphylococcal superantigens are exoproteins
that bind directly to MHC class II molecules in
B. Rodríguez-Iturbe et al.
antigen-presenting cells and induce a massive
proliferation of T cells bearing specific V beta
determinants. In some cases there may be a selec-
tive IgA response or, in some cases, the intense
T cell activation of B cells results in polyclonal
IgA and IgG production. Koyama et al. [97]
have identified an antigen in S. aureus that is
co-localized with glomerular IgA deposits and
developed an experimental model of the disease
immunizing Balb/c mice with this antigen [98].
Pathology
IgA-dominant postinfectious glomerulonephritis
is defined by deposition of IgA in both glomerular
capillary walls and mesangial regions as the sole
or predominant immune reactant (Fig. 2b), in
contrast to the typical pattern of IgG and/or C3
deposition that is characteristic of other PIGN
[89, 90, 95, 99]. By light microscopy (Fig. 2a)
the histological characteristics may be quite
variable. They may be similar to those of PSGN
or resemble mesangial proliferative GN or
membranoproliferative GN with or without cres-
cents. By electron microscopy, the characteristics
and distribution of electron-dense deposits in IgA-
dominant GN resemble those in PSGN ([95];
Fig. 2c). Since IgA-dominant PIGN may be
superimposed on other chronic nephropathies,
such as diabetic nephropathy, these cases may
present a hybrid morphologic appearance.
The differential diagnosis between IgA-
dominant PIGN and IgA nephropathy may be
quite challenging, and one helpful feature is
the finding of hump-like subepithelial electron-
dense deposits by electron microscopy in
IgA-dominant PIGN.
Diagnosis
IgA-dominant glomerulonephritis caused by
MRSA infection may present similarities with
conditions in which there are dominant or codom-
inant IgA deposits in the kidney such as
Henoch–Schonlein purpura and IgA nephropathy.
Staphylococcal infection frequently precedes
31 Acute Postinfectious Glomerulonephritis in Children
Fig. 2 IgA-dominant postinfectious glomerulonephri-
tis in a patient with diabetic nephropathy. (a) Light
microscopy: Mesangial regions are expanded by increased
matrix (as a feature of diabetic nephropathy) and cellular-
ity. There is also an influx of inflammatory cells into
glomerular capillaries and endothelial swelling, creating
segmental endocapillary proliferation (arrowhead) (Jones
methenamine silver stain, 40 objective). (b)
Henoch–Schonlein nephropathy, but the finding
of a purpuric rash in the legs and pains in the
abdomen and joints are absent in most patients
with IgA-dominant PIGN. On the other hand,
Henoch–Schonlein purpura seldom presents with
massive proteinuria or glomerular crescent
formation [100].
Patients with severe IgA nephropathy may be
at times confused with IgA-dominant glomerulo-
nephritis. Characteristics that may be helpful in
the differential diagnosis are the lack of associa-
tion with staphylococcal infection, the normal
serum complement levels, and the absence of
massive proteinuria and of glomerular crescent
formation in IgA nephropathy.
Treatment
Antibiotic treatment of the staphylococcal infec-
tion is the cornerstone of treatment in
IgA-dominant PIGN resulting from staphylococ-
cal infection. Corticosteroid treatment is
contraindicated while there is active infection;
therefore, it is important to make the differential
diagnosis with IgA nephropathy in which treat-
ment with corticosteroids may be indicated in
severe cases. In selected cases with persisting or
progressive deterioration of renal function, steroid
treatment and immunosuppression has been used
when infection is no longer present [101].
971
Immunofluorescence – IgA: strong granular and predom-
inantly mesangial staining. (c) Immunofluorescence –
C3: concomitant granular mesangial and peripheral capil-
lary wall staining. (d) Electron microscopy: subepithelial
hump-like deposits (red arrows) over the thickened glo-
merular basement membrane (white asterisk). Abbrevia-
tions: PMN polymorphonuclear, PC podocyte (11,000)
Renal Disease Associated
with Infective Endocarditis
Infective endocarditis is the underlying infectious
process in 10 % of the cases of PIGN [65], and
renal involvement is present in 25–50 % of the
patients with infective endocarditis but is fre-
quently asymptomatic [102, 103]. The association
of focal, embolic, and nonsuppurative glomerulo-
nephritis with bacterial endocarditis was first
described by Max Lohlein in 1910 [104]. Subse-
quent descriptions two decades ago emphasized
the immune complex pathogenesis causing dif-
fuse glomerulonephritis [102, 105], and more
recent autopsy studies have disclosed that local-
ized infarcts, glomerulonephritis (without demon-
strable immune complex deposits), and interstitial
nephritis, mostly attributed to antibiotic therapy,
are all frequent renal lesions in endocarditis-
associated renal disease [106].
Epidemiology
Approximately 15,000 new cases of infective
endocarditis (hospital and community acquired)
occur in the United States each year [107], and the
incidence appears to be steadily increasing [108].
However the incidence of community-acquired
native-valve endocarditis is essentially unchanged
972
at 1.7–6.2 cases per 100,000 person years in the
United States and Europe [109] but with impor-
tant epidemiologic changes. The proportion of
adolescents and young adults that have endocarditis
associated with rheumatic heart disease has
decreased and, in contrast, the proportion of older
patients with endocarditis associated with prosthetic
heart valves, implantable devices, and drug abuse
has increased [110]. While the incidence of MRSA
infections has increased in community-acquired
cases [111], the incidence of methicillin-susceptible
cases may be increasing in healthcare-associated
cases [112]. One of the most important epidemio-
logical changes in recent years has been the
increment in cases associated with healthcare inter-
ventions and, particularly, the cases associated with
hemodialysis and caused by staphylococcus.
Bacterial endocarditis is 20–60 times more com-
mon in hemodialysis patients than in the general
population and carries a near 50 % mortality risk
[113, 114]. In the pediatric patient, congenital heart
disease and cardiac surgery represent the most com-
mon risk factors, and in reports from a single insti-
tution, the incidence of pediatric infective
endocarditis has not changed over time but the
mortality has decreased from 38 % in historical
cohorts to 4 % in modern age [115].
Etiology and Pathogenesis
Infecting bacteria are Staphylococcus aureus, and
Staphylococcus epidermidis, Streptococcus
viridans and pyogenes, Enterococcus faecalis,
and less commonly E. coli, Proteus, and microor-
ganisms of the Bartonella and Candida species. In
pediatric populations Streptococcus viridians and
Staphylococcus aureus are the most common
infecting agents and together represent more
than 50 % of the cases [115]. Peaks of bacteremia
as well as a cumulative exposure to low-level
bacteremia are genuine risks for endocarditis
[116]. In hospital-acquired endocarditis the most
common bacteria are Staphylococcus aureus and
Staphylococcus epidermidis, while in
community-acquired endocarditis, streptococcal
infections are the most frequent [117, 118]. Mitral
valve disease and mitral prolapse are the heart
B. Rodríguez-Iturbe et al.
lesions most frequently associated with
endocarditis [119].
When an immune complex pathogenesis is
involved, bacterial antigens may be demonstrated
in the glomeruli [120] and the nephritogenic mech-
anisms are similar to those operating in APSGN.
Polyclonal (type III) cryoglobulinemia may be
found in roughly half of the patients. In addition,
superantigen-driven T cell activation with poly-
clonal gammopathy may occur in bacterial endocar-
ditis caused by methicillin-resistant Staphylococcus
aureus. In experimental studies, superantigen-
associated endocarditis is regularly associated with
acute kidney injury [121]. Microembolization is
responsible for the localized infarcts, and tubuloin-
terstitial nephritis is usually secondary to antibiotic
treatment. In the patients in hemodialysis programs,
the infection is usually located in the vascular access
and when present synthetic arteriovenous grafts or
venous dialysis catheters have been usually in place
for more than 1 year [122, 123]. In these patients
calcifications of heart valves are risk factors and
treatments directed to reduce the risk of ectopic
calcification are important preventive measures of
infectious endocarditis [124].
Clinical Characteristics
The classic clinical picture of subacute bacterial
endocarditis includes Osler’s nodules, Janeway
lesions, and splinter hemorrhages; however,
these findings are rare at the present time. Fever
may be the only manifestation, accompanied or
not with arthralgias, leukocytosis, increased sedi-
mentation rate, and purpura. The lack of classic
symptoms is likely the reason why 38 % of the
patients with endocarditis found at autopsy were
not diagnosed clinically [125].
The cardiac lesion is best demonstrated by
transesophageal echocardiography. Factors usu-
ally associated with higher mortality are a vegeta-
tion size 20 mm, age less than 1 year, the
existence of heart failure, and Staphylococcus
aureus as a causative organism [126]. Emboliza-
tion to the central nervous system with stroke may
occur in 6 % of the children [127]. Systemic
embolism maybe microscopic or large; the latter
31 Acute Postinfectious Glomerulonephritis in Children
Fig. 3 Glomerulonephritis associated with endocardi-
tis. (a) Light microscopy: Widespread glomerular
basement membrane duplication creates a membranopro-
liferative pattern of injury with influx of inflammatory cells
(less prominent than APSGN) and swollen endothelial
cells (Jones methenamine silver stain, 40 objective).
(b) Immunofluorescence: Granular mesangial and
peripheral capillary wall staining for C3 and IgG.
usually corresponds to endocarditis caused by
fungus or Haemophilus.
Microscopic hematuria and proteinuria are
indicative of a renal lesion. Urinary findings usu-
ally persist for weeks or months. Azotemia may
develop in one-thirds of the patients [128] but is
usually mild except in cases that develop crescen-
tic glomerulonephritis; nevertheless even mild
renal dysfunction is associated with a poor prog-
nosis [129]. The existence of eosinophilia and
eosinophiluria should raise the suspicion of
antibiotic-induced interstitial nephritis. Nephrotic
syndrome is unusual.
Serological findings include reduced C3 and
C4 (except in superantigen-mediated nephritis),
high titers of rheumatoid factor, cryoglobulinemia
[130], and, on occasion, positive anti-PR3 ANCA
antibodies in the serum [131, 132]. When ANCA
are present in association with lung and kidney
involvement, the clinical course resembles
granulomatosis with polyangiitis (formerly
known as Wegener’s granulomatosis) [133]. The
complement levels return to normal after the
infection has been eradicated, and this finding is
associated with good prognosis of the renal lesion.
Pathology
Three different types of renal disease have been
identified in infective endocarditis: embolic renal
973
(c) Electron microscopy: Abundant subendothelial
deposits (red arrows), associated with duplication of glo-
merular basement membrane (the original basement mem-
brane is marked by asterisk and the thin new membrane is
shown by black arrows). There are also a few mesangial
deposits (white arrow). Abbreviations: E endothelial cell,
M mesangial cell (11,000)
disease, immune complex glomerulonephritis,
and drug-induced interstitial nephritis. Early
investigations come from autopsy studies that
described embolic complications [104, 134], and
subsequent studies reported glomerulonephritis in
nearly 80 % of the cases [102]. More recent stud-
ies showed, in cases studied at autopsy, a higher
incidence of localized embolic infarct than glo-
merulonephritis, while in cases studied by renal
biopsy, acute glomerulonephritis was the most
frequent lesion and renal infarcts were absent
[106, 125]. Of interest, 25 % of the renal biopsies
in cases of infective endocarditis reported from
the United Kingdom demonstrated acute intersti-
tial nephritis [106].
Infective endocarditis-related PIGN differs
from APSGN in that involvement of glomeruli
may not be uniform (i.e., the distribution of abnor-
mal glomeruli may be focal rather than diffuse).
The histological alterations can be varied and
range from a predominately mesangial prolifera-
tive glomerulonephritis to a membranoproli-
ferative pattern of injury (Fig. 3a). When
present, such a membranoproliferative pattern of
injury may be histologically indistinguishable
from membranoproliferative glomerulonephritis
type I due to other causes such as hepatitis C
virus infection and systemic autoimmune diseases
such as lupus. The glomeruli may have a mixed
and predominately mononuclear leukocyte influx;
neutrophils may be present but their presence is
974
not typically florid as it is in APSGN. Features
that overlap with those of APSGN include glo-
merular hypercellularity and endocapillary prolif-
eration with endothelial swelling. Foci of
segmental glomerular necrosis are not uncommon
and are similar to those found in ANCA-positive
vasculitis, but only a fraction of these patients
have positive serum ANCA [106]. Crescents,
when present, usually involve a minority of glo-
meruli and have been found in 10–22 % of the
patients in various series [65, 102, 106, 135, 136].
Immunofluorescence studies typically show a
granular pattern of deposition of immune com-
plexes in peripheral glomerular capillary walls,
with variable but usually less pronounced
involvement of mesangial regions (Fig. 3b). The
immune deposits typically contain some combi-
nation of IgG, IgM, and C3, but deposits of IgA
have also been described.
Corresponding electron-dense deposits are
found by electron microscopy, usually in
subendothelial locations in the capillary walls
(Fig. 3c), particularly in cases where a membrano-
proliferative glomerulonephritis pattern is pre-
sent. However, subepithelial deposits, including
occasional humps similar to those of PSGN, may
be found. All of the histological and immunoflu-
orescence and electron microscopy changes are
potentially reversible and may resolve with suc-
cessful treatment of the underlying infection.
Treatment
The high incidence of endocarditis in hemodialy-
sis patients has caused a renewed emphasis on the
need for strict infection control policies and
prompt conversion to arteriovenous access from
catheters and appropriate antibiotic prescriptions
[137]. Antibiotic treatment needs to be given for
4–6 weeks and the cessation of fever is not an
indication that antibiotics may be stopped before
this time [138].
In patients who present with crescentic glomer-
ulonephritis and a rapidly progressive course, pulse
steroid therapy and plasmapheresis have been used
with success [139], but their overall value needs to
be confirmed in more definite studies.
B. Rodríguez-Iturbe et al.
Shunt Nephritis
Congenital or acquired hydrocephalus may
require the placement of a shunt between cerebral
ventricles and the atrium (ventriculoatrial, VA) or
the peritoneum (ventriculoperitoneal, VP) to ame-
liorate increased cerebrospinal fluid pressure.
Infection is a recognized complication of these
shunts that was initially reported to occur in
one-third of these patients [140, 141]. More recent
studies have shown that the incidence of infection
in these shunts has decreased to 6.1 % [142]. The
development of nephrotic syndrome in a patient
with infected VA shunt was reported by Black and
coworkers two decades ago [160], and glomeru-
lonephritis is now a recognized complication that
develops a few months to many years after inser-
tion in 0.7–2 % of the infected AV shunts. In
contrast with VA shunts, VP shunts are rarely
complicated with glomerulonephritis.
The infecting microorganisms are usually
Staphylococcus epidermidis and, less frequently,
Staphylococcus aureus. Propionibacterium acne,
diphtheroids, and Pseudomonas and Serratia spe-
cies are infecting organisms in rare occasions. The
etiology of the renal disease has been demonstrated
by the finding of bacterial antigens in the glomeruli
of patients with shunt nephritis [143, 144].
The clinical manifestations are recurrent epi-
sodes of fever, anemia, hepatosplenomegaly, skin
rash, and cerebral symptoms related to increased
intracranial pressure. Renal involvement is indi-
cated by proteinuria that in most cases is in the
nephrotic range hematuria and hypertension. The
combination of skin and renal manifestation may
lead to incorrect diagnosis, and the possible exis-
tence of urinary tract infection frequently accom-
panying a neurogenic bladder that may afflict
these children may make the interpretation of the
urinary findings difficult. Serological findings
include reduced C3 and C4 levels, indicating acti-
vation of the classic complement pathway,
cryoglobulinemia, anemia, elevated sedimenta-
tion rate, and high rheumatoid factor titers.
Serum anti-P3 ANCA titers are sometimes present
[140, 145].
The renal lesion demonstrated at biopsy is
indistinguishable from the range of lesions
31 Acute Postinfectious Glomerulonephritis in Children
encountered in settings of endocarditis, as
described earlier.
Treatment of shunt nephritis requires intrave-
nous antibiotic therapy and removal of the
infected shunt and, if possible, substituting it for
a VP shunt. Rarely, if ever, are antibiotics alone
capable of eliminating the infection. The long-
term prognosis of the renal function is good if
the shunt is removed within a few weeks of
detecting the infection [140]. If the removal of
the shunt is delayed, the renal damage may pro-
gress to irreversible end-stage renal failure.
Hemodialysis is the preferred modality in
patients with end-stage renal disease because peri-
tonitis associated with peritoneal dialysis carries
the risk of meningitis if a VP shunt is in place.
Given the high rate of complications in the VA
shunts, the VP shunt is the preferred initial proce-
dure, but VA shunts may be the selected method in
children with chronic renal failure in whom renal
transplantation is considered.
Syphilis
Congenital and acquired syphilis may cause glo-
merulonephritis. Congenital syphilis is usually
manifested by rash, rhinitis, and osteochondritis,
but 8–10 % of the patients may develop a full-
blown nephrotic syndrome that presents as anasarca
4–12 weeks after birth [146, 147]. Later in life,
syphilis is a reversible cause of nephrotic syndrome
[148] that presents in 1–2 % of the patients with the
disease [149] and may masquerade as membranous
nephropathy. The most common histological
appearance is that of membranous nephropathy,
and electron microscopy shows electron-dense
deposits along the subepithelial aspect of the
GBM [150]. Other types of glomerulonephritides,
including mesangioproliferative, membranoproli-
ferative, and diffuse proliferative glomerulonephri-
tis, have been reported, especially in adult patients.
Treponema antigens have been demonstrated in the
glomerular immune deposits [151]. Serological
tests for syphilis are positive and complement levels
are normal. Treatment of the syphilis is associated
with improvement of the renal disease, but recovery
usually takes several months.
975
Other Infections
Deep-seated infections such as osteomyelitis and
intra-abdominal abscesses may be associated with
glomerulonephritis manifested clinically by the
nephrotic syndrome and progressive azotemia or
present only by mild urinary abnormalities [152].
In children, pneumonia caused by Mycoplasma
pneumoniae infections [153, 154] and Streptococ-
cus pneumoniae [155, 156] has been found to cause
immune complex-induced glomerulonephritis.
Pneumococcal antigen type 14 has been demon-
strated in the glomeruli. Hematuria and
non-nephrotic proteinuria are the usual clinical man-
ifestations in cases of glomerulonephritis associated
with pneumonia. In infections with neuraminidase-
producing pneumococcus, the patient may develop
hemolytic uremic syndrome due to exposure of the
Thomsen–Friedenreich antigen [157].
The most common histological pattern in these
glomerulonephritis is that of diffuse proliferative
glomerulonephritis, often with crescent formation
[152, 158]. Membranoproliferative glomerulone-
phritis has also been found in some cases. Immune
deposits of C3 with little or no immunoglobulin
deposits are common findings [152, 159].
Glomerulonephritis improves with the treat-
ment of infection if the therapy is started early
and eradicates the infection completely [152].
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 Immunoglobulin A Nephropathies in
Children (Includes HSP) 32
Koichi Nakanishi and Norishige Yoshikawa

Contents Laboratory Investigation . . . . . . . . . . . . . . . . . . . . . . . . . 1003

Biomarkers in IgAN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1004
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 984
Differential Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1005
Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 984 Relationship Between IgAN and HSP . . . . . . . . . . . . . 1005
Etiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 985 Chronic Liver Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1005
Predisposing Genetic Factors . . . . . . . . . . . . . . . . . . . . . . . 986 Idiopathic Nephrotic Syndrome . . . . . . . . . . . . . . . . . . . . 1006

Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 987 Natural History and Prognosis . . . . . . . . . . . . . . . . . . . 1006

Nature of Mesangial Immunoglobulin A Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1008
Deposits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 988 Fish Oil/Omega 3 Fatty Acids . . . . . . . . . . . . . . . . . . . . . . 1009
Immunoglobulin A Glycosylation . . . . . . . . . . . . . . . . . 990 Coagulation Modifying Agents . . . . . . . . . . . . . . . . . . . . 1009
Immunoglobulin A Immune System . . . . . . . . . . . . . . . 991 Angiotensin-Converting Enzyme Inhibitors
Multi-hit Mechanism for Pathogenesis of IgAN . . . 993 and Angiotensin II Receptor Blockers . . . . . . . . . . . . . 1009
MicroRNAs (miRNAs) and Pathogenesis Corticosteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1009
of IgAN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 993 Immunosuppressants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1010
Progression Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . 993 Clinical Trials for Combination Therapy
Initiation and Progression Mechanism of Including Corticosteroids and
Glomerular Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 993 Immunosuppressant in Japan . . . . . . . . . . . . . . . . . . . . . . . 1010
Progression Mechanisms from Glomerular Injury Chinese Herbal Medicine (Sairei-to) . . . . . . . . . . . . . . . 1011
to Tubulointerstitial Injury . . . . . . . . . . . . . . . . . . . . . . . . . . 995 Tonsillectomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1011
Renal Fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 996 Japanese Guidelines for the Treatment
of Childhood IgAN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1011
Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 996
Immunohistologic Findings . . . . . . . . . . . . . . . . . . . . . . . . 996 Clinical Manifestations and Laboratory
Light Microscopic Findings . . . . . . . . . . . . . . . . . . . . . . . . 997 Features in HSP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1011
Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 998 Extrarenal Manifestations . . . . . . . . . . . . . . . . . . . . . . . . . . 1011
Repeat Renal Biopsy Findings . . . . . . . . . . . . . . . . . . . . . 999 Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1011
Differences Between Childhood and Adult Joints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1012
Patients with IgAN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000 Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1012
The Oxford Classification of IgAN . . . . . . . . . . . . . . . . 1000 Renal Manifestations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1012
Laboratory Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1013
Pathology in HSN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1001 Clinicopathologic Correlations . . . . . . . . . . . . . . . . . . . . . 1013
Clinical Features in IGAN . . . . . . . . . . . . . . . . . . . . . . . . 1003 Differential Diagnosis of HSP . . . . . . . . . . . . . . . . . . . . 1014
Treatment of HSP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1014
K. Nakanishi (*) • N. Yoshikawa Prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1015
Department of Pediatrics, Wakayama Medical University, Recurrence in Renal Allograft . . . . . . . . . . . . . . . . . . . . . . 1017
Wakayama City, Japan
e-mail: knakanis@wakayama-med.ac.jp; References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1017
nori@wakayama-med.ac.jp

# Springer-Verlag Berlin Heidelberg 2016 983

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_28
984 K. Nakanishi and N. Yoshikawa

Introduction Table 1 Immunopathologic similarities between IgA

nephropathy and Henoch-Schönlein nephritis
Immunoglobulin A (IgA) nephropathy (IgAN) IgA Henoch-Schönlein
was first described in 1968 by Berger and Hinglais nephropathy nephritis
[1] and is now recognized as a distinct clinico- Tissue deposition
Glomeruli IgA1, J chain, IgA1, J chain
pathologic entity with a higher frequency world-
anionic
wide than any other primary glomerulopathy [2].
Skin IgA IgA (includes
It was initially considered a benign condition, but uninvolved skin)
extended follow-up of patients indicated that Serum IgA
20–50 % of adults would ultimately progress to Total " (50–70 % of " 50 % (early in
end-stage renal failure [2, 3]. Likewise, the favor- cases) disease)
able prognosis initially attributed to children Polymer " "
with IgAN must be questioned in the light of IgA1 " "
studies [4–8]. Circulating IgA
Henoch-Schönlein purpura (HSP) is a clinical Immune " "
complexes
syndrome and multisystem disorder that affects
IgA Present Present
predominantly the skin, joints, gastrointestinal rheumatoid
tract, and kidneys, although other organs can be factors
involved [9–12]. The triad is not necessarily pre- IgA1 O- Abnormal Abnormal
sent in every case. glycosylation
In 1801, Heberden reported a 5-year-old boy Polymeric IgA1 production
with abdominal pain, vomiting, melena, joint Duodenal # ?
mucosa
pain, petechial hemorrhages on his legs, and
Marrow " ?
urine “tinged with blood.” In 1837, Schönlein Tonsil " ?
first described the condition that he called IgA clearance Impaired ?
“peliosis rheumatica,” purpura rheumatica, in IgA response to immunization
which arthralgia was associated with purpura. In Systemic Exaggerated ?
1868, Henoch described the gastrointestinal man- Mucosal Reduced ?
ifestations of purpura rheumatica, and 30 years Transplant 50 % Common
later, he referred to nephritis as a complication. recurrence
Osler attributed the symptoms to anaphylaxis
[13], and the term anaphylactoid purpura, intro-
duced by Frank in 1915, remains popular in the and/or proteinuria) [17]. Recently, the second
United States [14], whereas most European and International Chapel Hill Consensus Conference
Japanese writers favor the eponymous term. Other (CHCC2012) was convened to improve the
terms used include purpura rhumatoide [15] and CHCC1994 nomenclature [18, 19]. HSP was
Schönlein-Henoch syndrome [16], the latter replaced with IgA vasculitis in CHCC2012.
acknowledging historical precedence. However, Although IgA vasculitis is a more descriptive
HSP remains popular as well as being traditional term, it does not still come into wide use.
and is used in this chapter. There are many similarities between IgAN and
The European League against Rheumatism/ Henoch-Schönlein nephritis (HSN) (Table 1).
Pediatric Rheumatology European Society
(EULAR/PReS) proposed as classification criteria
for HSP the presence of palpable purpura (man- Epidemiology
datory criterion) and at least one of the following
features: diffuse abdominal pain, any biopsy IgAN has been diagnosed all over the world, but
showing predominant IgA deposits, arthritis or its prevalence varies widely from one country to
arthralgia, and renal involvement (any hematuria another. In the Pacific Rim (e.g., Japan,
32 Immunoglobulin A Nephropathies in Children (Includes HSP)
Singapore, Australia, and New Zealand), IgAN
accounts for as many as one half of cases of
primary glomerulonephritis. In Europe, it
accounts for between 20 % and 30 % of all pri-
mary glomerulonephritis, whereas in North
America, it is responsible for only 2–10 %. The
explanation for this apparent variability in inci-
dence is uncertain, but it may be due to a racial
difference or to differences in biopsy selection
practices [20].
Genetic factors and environmental influences
could contribute to geographic differences in
prevalence. A lower prevalence among blacks
than whites has been reported in the United States.
However, in American children, similar
incidences of IgAN in Caucasian and African-
American children from Shelby County, Tennes-
see, have been reported [21]. In Australia [22],
where the population is heterogeneous and
includes many immigrants from Third World
countries, all racial groups seem to be equally
affected.
The high incidence of IgAN in certain coun-
tries may reflect the practice of routine urinalysis.
In Japan, all children between the ages of 6 and
18 years are screened annually, and those found to
have urinary abnormalities are referred for further
investigation. Thus, IgAN is the most common
primary glomerulonephritis in children seen in
Kobe University and Wakayama Medical Univer-
sity Hospitals, detected in approximately 30 % of
biopsy specimens.
HSP occurs mainly among children, but adults
can be affected [23–25]. It is rare among children
younger than 2 years and has a peak incidence
around 4–5 years of age, followed by a gradual
decline toward adolescence. The larger childhood
series indicate a slight male preponderance
[26, 15, 27, 28]. The annual incidences estimated
by Nielsen [29] in Denmark and by Stewart
et al. [30] in Ireland were 1.4 and 13.5 cases per
100,000 children, respectively. Forty years ago,
HSP was less than half as common as poststrep-
tococcal nephritis in the United Kingdom [31],
but whereas the latter is now uncommon in devel-
oped countries, the incidence of HSP seems to
have remained stable. There is significant sea-
sonal variation in incidence, the peak occurring
985
during winter [15, 32, 23]. Although HSP has a
wide geographic distribution, there is appreciable
variation. Clinical reports based on records of
large numbers of patients have originated mostly
from Europe, United Kingdom [26, 33–35],
France [15], and Finland [36], and Asia, Japan
[32, 27] and Singapore [24]. The condition
appears to be less common in North America
and Africa. These differences may be both envi-
ronmental and racial, because HSP is rare among
blacks in the southern United States [37, 38] and
among persons from the Indian subcontinent
[39]. A familial instance of HSP is rare [26, 40],
suggesting that genetic factors are not of etiologic
significance. There appears to be no association
between HLA antigens [41] or immunoglobulin
heavy-chain switch-region genes [42] and HSP.
The proportion of patients with renal involve-
ment has been reported to be 20–100 % [26, 32,
23, 43–47]. These variations may depend on the
method of detection of nephritis [48]. In many of
the early studies, serial routine urinalysis was not
used, and transient microscopic hematuria proba-
bly was missed. Two studies in which routine
urinalysis was performed for all patients admitted
to the hospital for HSP showed renal involvement
in 41 % [45] and 61 % [32] of cases.
Etiology
The pathogenesis of IgAN is not fully uncovered.
IgAN is generally considered to be an immune
complex-mediated or aggregated (polymerized)
IgA-mediated glomerulonephritis. Because IgA
is the main immunoglobulin directed against anti-
gens (viral and bacterial) in the exocrine system,
and because of the frequent association between
upper respiratory tract or gastrointestinal infection
and the onset of macroscopic hematuria, it has
been suggested that certain viral or bacterial infec-
tions may lead to IgAN. Considerable effort has
been directed toward the search for antigens and
for the antibody specificity of the mesangial IgA,
but with limited success. Many antigens, includ-
ing herpes simplex virus, cytomegalovirus,
Epstein-Barr virus nuclear antigen, adenovirus,
and milk antigen, have been identified. Although
986
much of this work has been performed in adults,
there is no evidence to suggest that the findings
cannot be extrapolated to children.
Evidence that IgAN is not a disease restricted
to the kidney but is a systemic disorder is provided
by the findings of IgA deposits in unaffected skin
[49], recurrent mesangial IgA deposits in 50 % of
transplant recipients whose primary disease has
been IgAN [50], and the disappearance of IgA
from cadaver kidneys found to have unexpected
IgA deposits when these are transplanted into
recipients with other primary renal disease [49].
The observation of numerous antigenic sub-
stances in the glomeruli indicates that the anti-
genic materials in IgAN may be heterogeneous.
The presence of Haemophilus parainfluenzae
antigens in a diffuse and global distribution in
the glomerular mesangium and the presence
of IgA antibody against Haemophilus
parainfluenzae in sera of Japanese patients with
IgAN have been demonstrated [51, 52]. It has
been reported that Staphylococcus aureus cell
envelope antigen was localized in the glomeruli
in 68.1 % (79/116) of renal biopsy specimens
from patients with IgAN and the antigen was
colocalized with IgA antibody in the glomeruli.
Staphylococcus aureus cell envelope antigen may
be a new candidate for the induction of IgAN
[53]. These findings remain unconfirmed in chil-
dren with IgAN yet. Schmitt et al. have reported
that antibody levels to the IgA-binding regions
were significantly higher in IgAN patients than
controls, particularly in patients with recent strep-
tococcal infection, suggesting that some children
with IgAN had a previous infection with a strep-
tococcal strain expressing an IgA-binding M
protein [54].
In order to present an antigen to T cells, the
antigen must first be degraded by proteasomes.
Coppo et al. have reported that a significant switch
in the expression of trypsin- and chymotrypsin-
like proteasome subunits to corresponding
immuno-proteasome subunits was found in
patients with IgAN as compared to healthy
controls [55].
The cause of HSP also remains unknown. The
illness is preceded by upper respiratory infection
in 30–50 % of cases [26, 15], but evidence of
K. Nakanishi and N. Yoshikawa
streptococcal infection has not been substantiated
[26, 15, 56, 32]. Our findings suggest that
Haemophilus parainfluenzae may be involved
for a subset of patients [52]. Other infections
implicated for a few children include varicella
[26], measles [16], rubella [26], adenovirus infec-
tion [57], hepatitis A [58] and B [59], Yersinia
infection [60], Shigella infection [61], Myco-
plasma pneumoniae infection [62], and staphylo-
coccal septicemia [63], but without strong
evidence of a causal relation. HSP has been
reported to follow smallpox and influenza vacci-
nation [64, 65], insect bites [66], exposure to cold
[67], and trauma [68]. Among adult patients, HSP
has been reported in association with human
immunodeficiency virus infection [69], Behcet
disease [70], uveitis [71], squamous cell carci-
noma [72], and rheumatoid arthritis [73].
Interestingly, it has been reported 10 of 33 chil-
dren (30 %) with HSN showed segmental or
global mesangial staining with nephritis-
associated plasmin receptor (NAPlr, a group A
Streptococcal [GAS] antigen) antibody, whereas
only 2 of 48 children (4 %) with IgAN were
positive [74]. These findings suggest that the
deposition of NAPlr in the mesangium, induced
by Group A Streptococcal infection, may have a
role in the pathogenesis of HSN. Schmitt
et al. [75] have reported that deposits of the
IgA-binding region of three different streptococ-
cal M proteins were detected in 7/13 HSP
kidneys as well as 10/16 IgAN kidneys. The
IgA-binding region would access the circulation,
encounter circulatory IgA, and form a complex
with IgA-Fc that could deposit in tissues and
contribute to the pathogenesis of HSP as well as
IgAN [75].
Predisposing Genetic Factors
Predisposing genetic factors have been suggested
as important in the development of IgAN [76].
Moreover, it has been suggested that
genetic factors may not only determine suscepti-
bility to glomerulonephritis but also influence the
pathological severity and natural course of IgAN
[77, 78].
32 Immunoglobulin A Nephropathies in Children (Includes HSP)
Evidence for genetic factors in IgAN is pro-
vided by family studies [77–81]. Rambausek
et al. reported that 9.6 % of patients with
mesangial IgAN in Germany had one or more
siblings with glomerulonephritis [77]. Julian
et al. described kindred from eastern Kentucky
in which six patients with IgAN descended from
one ancestor and eight other patients belonged to
potentially related pedigrees [79]. Moreover, they
indicated that at least 48 (60 %) of 80 IgAN
patients who were born in same region were
related to at least one other patient [80]. Scolarim
et al. reported that 26 (14 %) of 185 patients with
IgAN investigated in Italy were related to at least
one other patient with the disease [81]. These
studies suggest that familial predisposition is a
very common finding and genetic factors are
involved in the pathogenesis of IgAN. Gharavi
et al. [82] demonstrated linkage of IgAN to
6q22-23 under a dominant model of transmission
with incomplete penetrance. Further linkage-
based studies have identified significant or sug-
gestive loci on chromosomes 4q26-31, 17q12-22
[83] and 2q36 [84], but no causal gene has yet
been identified. Genome-wide association studies
have been performed in IgAN [85–87]. Five dis-
tinct susceptibility loci including three indepen-
dent loci in the major histocompatibility complex
(MHC) as well as a common deletion of the com-
plement factor H gene cluster (CFHR1 and
CFHR3) at chromosome 1q32 and a locus at chro-
mosome 22q12 were identified [86]. Another
study identified associations at 17p13 and 8p23
that implicated the genes encoding tumor necrosis
factor (TNFSF13) and α-defensin (DEFA) as sus-
ceptibility genes. In addition, the study has found
multiple associations in MHC region and con-
firmed a previously reported association at
22q12 [87].
Schena et al. [88] reported that familial IgAN
had a poorer prognosis than sporadic IgAN,
suggesting that genetic factors are implicated in
both disease susceptibility and disease progres-
sion [76]. Polymorphisms of Ig heavy-chain
switch-region gene [89], Iα1 germ-line transcript
regulatory region gene [90], genes of the renin-
angiotensin system (RAS) [91–95], transforming
growth factor-beta1 [96], platelet activating factor
987
acetylhydrolase gene [97], constant region of the
alpha chain for the T-cell receptor [98], miR-146a
[99], core 1 beta1,3-galactosyltransferase
(C1GalT1) [100, 101], and so on [102] have
been reported. Associations among IgAN and
genetic polymorphisms of other molecules, in
which relation to disease susceptibility and dis-
ease progression are suggested, have been
reported in sequence [103]. Although some asso-
ciations have emerged, they have been inconsis-
tent [104]. Some of the discrepancies may be
caused by different sample sizes and different
geographical regions of the patients included in
the studies [105].
In Japan, single-nucleotide polymorphisms
have been screened on a genome-wide scale to
clarify various complex diseases, including IgAN.
As a part of the benefit of this project, case-control
association studies were designed, using single-
nucleotide polymorphisms found in the selectin
gene [106], the class II region of MHC [107], and
the polymeric immunoglobulin receptor gene
[108], and haplotypes of these genes that might
serve to identify regions containing loci responsi-
ble for IgAN phenotypes were estimated.
Several publications have focused on genetic
factors predisposing to HSP, many of which are
polymorphisms in genes related to cytokines and
cell adhesion molecules that are involved in the
pathways for inflammatory responses and endo-
thelial cell activation [109]. The associations of
HLA-DRB1 01, 07, and 11 with HSP susceptibil-
ity are reported [110]. Genetic regulation of endo-
thelial function, such as polymorphisms in genes
coding RAS components [111], endothelial nitric
oxide synthases [112], intercellular adhesion mol-
ecule 1 (ICAM1) [113], and vascular endothelial
growth factor (VEGF) [114], could also confer
effect on HSP. In addition, MEFV, whose muta-
tions cause familial Mediterranean fever, could be
a candidate gene for HSP [115–118].
Pathogenesis
Although the pathogenesis of IgAN remains
uncertain, there is substantial evidence that it is
an immune complex disease [2, 119]. Granular
988
electron-dense deposits are observed in the glo-
merular mesangial areas by electron microscopy
and contain IgA and C3 by immunofluorescence
microscopy. Circulating IgA immune complexes
have been detected by several different assays
often associated with IgG immune complex.
Many immunological abnormalities that may
lead to the formation of IgA immune complex
have been reported in patients with IgAN.
Recurrence of IgAN frequently occurs in allo-
grafts [120], and a rapid disappearance of glomer-
ular IgA deposits is observed when kidneys with
mesangial IgA deposits are transplanted to
patients without IgAN. These clinical observa-
tions have provided strong support for the notion
that IgAN is a systemic disease. Although much
of this work was performed in adults, there is no
evidence to suggest that the findings cannot be
extrapolated to children. Moreover, glomerular
IgA deposits associated with histologic lesions
similar to those of human IgAN can be induced
in laboratory animals by passive administration of
preformed IgA immune complex or by active
immunization [121–125].
Although the pathogenesis of HSP also
remains uncertain, there is substantial evidence
that it is also an immune complex-mediated dis-
ease. The mechanisms of formation of glomerular
immune deposits likely are similar to those in
IgAN, generally regarded as a kidney-limited var-
iant of HSP. Granular electron-dense deposits in
the glomerular mesangial areas are observed with
an electron microscope and contain IgA and C3
by immunofluorescence microscopy. Circulating
IgA immune complexes have been detected, often
associated with IgG immune complexes
[126–128, 49]. Many immunologic abnormalities
that lead to formation of IgA immune complexes
have been found in patients with HSP. Mesangial
IgA deposits recur frequently in allografted kid-
neys of patients with HSP [129–131]. This evi-
dence indicates that the mesangial IgA must be of
host origin. Moreover, glomerular IgA deposits
associated with histologic lesions similar to
those of human HSN can be induced among lab-
oratory animals by means of passive administra-
tion of preformed polymeric IgA-concanavalin A
complexes [132].
K. Nakanishi and N. Yoshikawa
Nature of Mesangial Immunoglobulin A
Deposits
IgA is the second most common immunoglobulin
and contributes to immunity at the level of the
external secretory system. IgA exists in mono-
meric and polymeric forms. Monomeric IgA rep-
resents approximately 90 % of the serum IgA and
is produced mainly by the circulating lympho-
cytes and plasma cells in the spleen and bone
marrow. Polymeric IgA is produced mostly by
lymphocytes and plasma cells in the gastrointes-
tinal and respiratory tracts, where it is synthesized
as monomers and then secreted as dimers linked
by the J-chain, which is also produced within the
plasma cells. During the passage of dimeric IgA
molecules through the mucosal epithelium toward
the external lumen, the secretory component is
attached through specific noncovalent interac-
tions; this component appears to protect the
dimeric IgA from the proteolytic enzymes present
in the external secretions. IgA has two subclasses,
IgA1 and IgA2. Approximately 90 % of serum
IgA is composed of IgA1 mostly produced in the
bone marrow, whereas IgA2 is mostly derived
from the local mucosa of the gastrointestinal and
respiratory tracts. Both IgA1 and IgA2 are pro-
duced in the mucosae.
The most prominent finding in the glomeruli of
renal biopsy specimens from patients with IgAN
and HSN is mesangial IgA deposition. The major-
ity of investigators have indicated that IgA1 is the
predominant subclass present in the glomeruli
[133]. The J-chain has also been identified in the
mesangium of patients with IgAN [134]. The
secretory component is not present in the
mesangial deposits, but it binds to the mesangial
areas in vitro [135, 136]. These observations sug-
gest that the mesangial IgA deposits are poly-
meric, a hypothesis further supported by the
immunochemical characterization of IgA eluted
from renal biopsy sections [137].
Most investigators have found that IgA1 is the
predominant subclass in the glomeruli of patients
with HSN [133, 138–140]. J-chain, independent
of IgM, has been found in the mesangium of
patients with HSN [133, 141]. Secretory compo-
nent is not present in the mesangial deposits
32 Immunoglobulin A Nephropathies in Children (Includes HSP)
[139]. Although the capability of the mesangial
deposits to bind the secretory component has not
been examined in HSN, the mesangial IgA
deposits in HSN are also thought to be polymeric.
The onset of IgAN may be associated with
infections in the upper respiratory tract. It has
therefore been proposed that IgAN results from
hyperactivity of the mucosal immune system.
However, assessment of polymeric IgA1 produc-
tion by in situ hybridization for J-chain messenger
RNA in IgA plasma cells shows downregulation
in the mucosa [142] and upregulation in the bone
marrow [143]. Impaired mucosal IgA responses
allowing enhanced antigen challenge to the mar-
row shown by de Fijter et al. [144] could be the
primary abnormality in IgAN, although this
remains unproven [10].
A number of studies have suggested that the
alternative complement pathway has a pathoge-
netic role in IgAN and HSN. This hypothesis is
consistent with the typical immunohistologic
demonstration of C3 and properdin in a pattern
and distribution similar to that of IgA in the glo-
meruli and the absence of C1q and C4. The detec-
tion of the membrane attack complex of
complement further supports the role of comple-
ment activation in this disease [145, 15, 24, 146].
Certain types of IgA aggregates or IgA from
patients with myeloma have been shown to acti-
vate complement in vitro [147]. IgA has been
reported to activate the complement system via
the mannan-binding lectin pathway [148]. How-
ever, there is no direct evidence that IgA deposits
in the glomeruli mediate complement activation.
Activation of C3 is observed in the majority of
adult and pediatric patients with IgAN, but the
mediator as well as the pathophysiologic signifi-
cance of this complement activation remains to be
determined. C3 is deposited in the kidney but is
also produced by mesangial cells in IgAN [149].
Detection of the membrane attack complex of
complement further supports the role of comple-
ment activation in HSN [150]. The alternative
complement pathway is activated in patients
with HSN [151, 152], and increased serum termi-
nal complement complex has been found during
the active phase of disease [153]. However, there
is no direct evidence that the activation is
989
mediated by IgA deposits in the glomeruli, and
the mediator and the pathophysiologic signifi-
cance of this complement activation remain to be
determined.
With regard to antibody specificity, IgA eluted
from cryostat sections of IgAN biopsies has been
reported to react with mesangial areas of its own
and other IgAN patients’ biopsies, but not with
normal kidney [154]. Such eluates have also been
shown to contain antibodies that react with tonsil-
lar cells and cultured fibroblasts obtained from
patients with IgAN [155].
Because IgA is the main immunoglobulin
directed against viral and bacterial antigens in
the exocrine system, and because of the frequent
association between upper respiratory tract infec-
tion and the development of HSP, it has been
suggested that certain viral or bacterial infections
may cause HSP and that IgA may be the antibody
to viral or bacterial antigens. Little effort has been
directed at the search for antigens and for the
antibody specificity of the mesangial IgA in
HSP. Diffuse global deposition of
H. parainfluenzae was found in one third of
patients with IgAN and HSN [52]. Patients with
IgAN and HSN had significantly higher levels of
plasma IgA1 antibody against H. parainfluenzae
than did patients with other renal diseases
[52]. Emancipator [156] described the develop-
ment of intestinal lesions, purpura, glomerular
lesions, and hematuria in a bacterial polysaccha-
ride model induced in mice and suggested that
HSP may be an expression of an antigen-
dependent process. With regard to antibody spec-
ificity, IgA eluted from cryostat sections of HSN
obtained at biopsy has been reported to react with
mesangial areas of these biopsy specimens and
with biopsy specimens from other patients with
HSN [157, 158].
Despite intensive investigation, the mechanism
underlying glomerular IgA deposition in IgAN
has not been elucidated. Proposed possible mech-
anisms of glomerular IgA deposition are as fol-
lows. Decreased IgA response to mucosal
antigens may promote increased production of
polymeric IgA1 by the bone marrow, leading to
increased serum levels of IgA1. Defective
galactosylation of IgA1 (later described in detail)
990
may decrease hepatic clearance of IgA1 and pro-
mote binding of IgA1 complexes to glomerular
mesangial cells. Aberrant IgA1 deposits in the
kidney trigger the production of a variety of cyto-
kines and growth factors by renal cells and by
circulating inflammatory cells, leading to the char-
acteristic histopathological features of mesangial
cell proliferation and extracellular matrix deposi-
tion [159]. Recently it has been proposed a model
whereby two types of IgA receptors participate in
sequential steps to promote the development of
IgAN, with soluble IgA-Fc receptor I (FcαRI/
CD89) being initially involved in the formation
and/or amplification of the size of circulating IgA
immune complexes and, subsequently, mesangial
transferrin receptor (CD71), in mediating
mesangial deposition of nephritogenic IgA
immune complexes [160–164].
An animal study has demonstrated that
tetraspanin (CD37) controls the formation of
IgA-containing immune complexes and glomeru-
lar IgA deposition, which induces influx of
inflammatory myeloid cells. Therefore, CD37
may protect against the development of
IgAN [165].
In summary, the immunochemical nature of the
mesangial deposits in IgAN and HSN is consistent
with antigen-polymeric IgA complexes predomi-
nantly of IgA1 subclass, and perhaps,
multispecific for ubiquitous mucosally derived
antigens.
Immunoglobulin A Glycosylation
IgA glycosylation has received recent attention as
a putative nonimmune feature of IgA, which may
explain its abnormal behavior and glomerular
deposition in IgAN [166] and HSN [167, 168].
IgA is a heavily glycosylated protein. IgA1 carries
sugars at the hinge region, which is most unusual
in a circulating protein. IgA2 has no hinge region.
Changes in these hinge region sugars may have
functional effects on the interaction of IgA1 with
specific antigen and with effector mechanisms
such as complement and Fc receptors. Circulating
IgA1s have a decreased amount of terminal galac-
tose in the hinge region in IgAN and HSN. As yet
K. Nakanishi and N. Yoshikawa
there is no proof that this abnormality directly
promotes mesangial IgA1 deposition. IgA1 is
unique among all Igs in its possession of a hinge
region rich in proline, serine, and threonine and
characterized by O-glycosylation sites (Fig. 1).
These O-glycosylation sites consist of N-acetylga-
lactosamine O-linked to the serine or threonine
residues of hinge region. In patients with IgAN,
mesangial IgA1 and a small fraction of circulating
IgA1 contain incompletely galactosylated
O-linked glycans in the hinge region
[169–173]. This glycosylation abnormality is
absent in other glycoproteins with O-linked gly-
cans, such as complement C1 inhibitor and IgD,
indicating a defect specific for IgA1 [171,
174]. Abnormal galactosylated IgA1 increases
affinity for glomerular fibronectin, laminin, and
collagen IV [175] and may lead to accumulation
of IgA in the mesangium [172]. Preliminary data
indicate that deficient galactosylation of hinge
region glycans may be detected even in family
members of patients with IgAN [169]. Altered
amino acid sequence of the IgA1 hinge region is
a possible mechanism to consider for abnormal
galactosylation of IgA1. However, the hinge
region is a highly conserved region of IgA1 mol-
ecule. There is no evidence for any nucleotide
sequence alteration or transcriptional abnormality
of the hinge region in IgAN [176]. It has also been
postulated that altered galactosylated IgA1 in
IgAN may be due to a deficiency of structural
modification of β 1,3-galactosyltransferase, the
enzyme responsible for the terminal
galactosylation of GalNAc on O-linked glycans
[177]. This structural or functional deficiency may
be genetically determined. The recent sequence of
β1,3-galactosyltransferase may help us to under-
stand the genetic basis of these abnormalities
[178, 179]. Circulating IgA1 has reduced terminal
galactose on O-linked hinge region sugars in
IgAN [171], apparently because of a B-cell defect
in β1,3-galactosyltransferase, the enzyme respon-
sible for placing terminal galactose on O-linked
sugars [177]. The IgA1 O-glycan chains are
truncated in IgAN [180]. Circulating immune
complexes in IgAN consist of IgA1 with
galactose-deficient hinge region, and the defi-
ciency of galactose may result in the generation
32 Immunoglobulin A Nephropathies in Children (Includes HSP) 991

CH1 β1,3 - Gal - α2,3 - Sialic acid

I
Pro
I
Ser←O-glycan → Ser/Thr -O- GalNAc
I
Thr ←O-glycan
I
Pro α2,6 - Sialic acid
I

Light Heavy Pro Normal

I
chain chain Thr ←O-glycan
I
Pro
I
CH1 Ser←O-glycan
I Ser/Thr-O-GalNAc
Hinge Pro
I
region Ser←O-glycan
I α2,6 -Sialic acid
Thr ←O-glycan
CH2 I
Gal-deficient O-linked glycan
Pro
I
Pro
I
CH3 Thr ←O-glycan
I
Pro
I
Ser←O-glycan
I
Pro
I
Ser←O-glycan
I
CH2

Fig. 1 IgA1 molecules with hinge region O-glycosylation
sites. IgA1 molecules have two heavy chains with three
constant region domains CH1 to CH3 and a hinge region
between CH1 and CH2. Each serine (Ser) and threonine
(Thr) residue is a potential site for an O-glycan side chain.
Although there are nine potential glycosylation sites, IgA1
contains up to 6 O-glycans per hinge region. O-glycosyl-
ation sites consist of N-acetylgalactosamine (GalNAc)
O-linked to Ser or Thr residues of hinge region. The largest
O-linked saccharide of IgA1 is a tetrasaccharide with
GalNAc, galactose (Gal), and two sialic acid residues.
Aberrant IgA1 molecules with Gal-deficient O-linked gly-
cans can contain terminal GalNAc or sialylated GalNAc at
one or more sites. Pro proline

of antigenic determinants that are recognized by
naturally occurring IgG and IgA1 antibodies
[170]. Sano et al. demonstrated that enzymatically
deglycosylated human IgA1 molecules accumu-
late and induced inflammatory cell reaction [181];
Amore et al. showed glycosylation of circulating
IgA-modulated mesangial proliferation in
IgAN [182].
It has been showed that EBV-immortalized
IgA1-producing cells from peripheral blood cells
in IgAN patients secreted mostly polymeric IgA1
with galactose-deficient O-linked glycans [183].
Although it had not been previously known
whether the aberrant glycosylation is the result
of an acquired or inherited defect or whether the
presence of aberrant IgA1 glycoforms alone can
produce IgAN, to date studies have demonstrated
that abnormal IgA1 glycosylation is an inherited
rather than acquired trait and that abnormal IgA1
glycosylation clusters in most but not all families
with IgAN suggest the possibility of IgAN
patients with different pathogenic mechanisms of
disease [184].
Immunoglobulin A Immune System
Serum IgA levels are increased in 50–70% of
patients with IgAN, with elevations in both mono-
meric and polymeric IgA. There is an increase in
polymeric IgA1-producing plasma cells in the
bone marrow [143] and in the tonsils [185] of
patients with IgAN. The proportion of IgA-λ in
serum IgA is also increased. Serum IgA is more
anionic, owing to the increased anionicity of
λ-light chain compared with κ-light chain [185].
The binding of IgA to mesangial cells is charge
dependent, and anionic charge may play an
992
important role for in IgA1 deposition in the
mesangium [186]. In addition to the increased
levels of serum IgA, various types of autoanti-
bodies of the IgA class have been recognized.
These IgA autoantibodies include rheumatoid fac-
tor [187], antinuclear antibodies [188], and
anticollagen antibody [189]. However the IgA
may be polyspecific, indicating a polyclonal
increase rather than true antigen-specific autoan-
tibodies [190]. IgA immune complexes are also
frequently detected [191]. Cultured peripheral
blood lymphocytes from patients with the disease
produce more IgA than those of normal individ-
uals, either spontaneously or after polyclonal
stimulation in vitro. We also demonstrated an
increased spontaneous and pokeweed mitogen-
stimulated IgA production by peripheral blood
lymphocytes of children with IgAN [192]. This
increased IgA production remained stable during
the follow-up period in patients with persistent
urinary abnormalities, but decreases toward nor-
mal in patients with clinical remission.
IgA production is T cell dependent, and the
increased production in IgAN may indicate
altered T-cell function. An increased circulating
OKT4 to OKT8 cell ratio, due to increased OKT4
helper T lymphocytes and decreased OKT8 sup-
pressor T lymphocytes, has been reported in
patients [193]. Increased IgA-specific helper
T-cell activity and decreased IgA-specific sup-
pressor T-cell activity have also been reported
[194, 195].
Defective clearance of immune complexes
from the circulation may also be important
[196], but this seems more likely to be a conse-
quence rather than the cause of the increased
immune complex load.
Serum IgA levels are increased in one half
of patients with HSP during acute illness
[15, 197, 198]. Serum IgA levels return to normal
with recovery. In addition, various types of auto-
antibodies of the IgA class have been found in
HSN. These autoantibodies include IgA rheuma-
toid factor [199–201], IgA antineutrophil cyto-
plasmic antibody [202, 203], IgA anti-IgG
antibody [204], and IgA anticardiolipin antibody
K. Nakanishi and N. Yoshikawa
[205]. The presence of IgA rheumatoid factors has
been related to disease activity [28]. IgA immune
complexes are also frequently detected [126–128,
49]. Coppo et al. [206] found a high prevalence of
immune complexes during acute disease.
Levinsky and Barratt [126] found that immune
complexes also contained IgG, and only the
IgG-containing immune complexes were
nephritogenic in HSP. Codeposition of IgG in
the mesangium and the presence of circulating
IgG-containing immune complexes suggested
that IgG immune complexes may have an impor-
tant role in renal damage [207]. Not all patients
with HSP have circulating immune complexes.
There are at least two explanations for the absence
of circulating immune complexes. These patients
may have immune complexes only intermittently,
or formation of mesangial IgA deposits in patients
without circulating immune complexes may be
caused by in situ formation of the immune com-
plex directly in the mesangium. O’Donoghue
et al. [208] found IgG autoantibody binding
specificality to glomerular antigens present on
the mesangial cell only in IgAN and HSP. Circu-
lating IgA-fibronectin aggregates, macromole-
cules aggregated on a nonimmune basis, have
been found in patients with HSP [209, 210]. The
presence of fibronectin in the circulating aggre-
gates may have an important role in preferential
deposition of nephritogenic IgA-containing
immune complexes in the mesangium [209].
A large number of circulating IgA-producing
lymphocytes have been found during the acute
phase of HSP [211–213], but not when the disease
is clinically inactive [213, 214]. Production of IgA
is highly T cell dependent, and the increased pro-
duction in HSP may indicate altered T-cell func-
tion. However, Casanueva et al. [213] found the
existence of persistent abnormalities in the cellu-
lar immune mechanisms that regulate immuno-
globulin synthesis (impaired T-cell suppressor
activity) in IgAN, but not in HSP. Defective clear-
ance of immune complexes from the circulation
may be important, but this seems more likely to be
a consequence rather than the cause of the
increased immune complex load.
32 Immunoglobulin A Nephropathies in Children (Includes HSP)
Multi-hit Mechanism for Pathogenesis
of IgAN
A multi-hit mechanism for the pathogenesis of
IgAN has been proposed [215]. A high circulating
load of galactose-deficient IgA1 (Hit 1) alone does
not induce the renal injury. Rather, several sequen-
tial processes or hits are necessary for the clinical
expression of IgAN. Synthesis and binding of anti-
bodies directed against galactose-deficient IgA1
are required for formation of immune complexes
that accumulate in the glomerular mesangium (hits
2 and 3). The detail is as follows [215]. Hit 1:
Production of galactose-deficient IgA1 by a sub-
population of IgA1-secreting cells. IgA1 produc-
tion may be affected by the IgAN-associated locus
on chromosome 22q12.2 [86]. Hit 2: Formation of
anti-glycan antibodies with specific characteristics
of the variable region of the heavy chain that rec-
ognize galactose-deficient IgA1. Hit 3: Formation
of immune complexes from autoantigen
(galactose-deficient IgA1) and O-glycan-specific
antibodies. Hits 2 and 3 may be regulated by the
three MHC loci on chromosome 6p21 associated
with risk of IgAN [86]. Hit 4: Deposition of path-
ogenic immune complexes in the mesangium, acti-
vation of mesangial cells, and induction of
glomerular injury. Hits 3 and 4 may be affected
by genotype at the complement factor H locus on
chromosome 1q32 that regulates the alternative
complement cascade [86]. The first pathway
assumes formation of immune complexes in the
circulation and their subsequent mesangial deposi-
tion [216, 170, 217, 218]. An alternative theory
proposes that some of the aberrantly glycosylated
IgA1 molecules are in the mesangium as lanthanic
deposits and are later bound by newly generated
anti-glycan antibodies to form immune complexes
in situ that activate mesangial cells [219].
MicroRNAs (miRNAs) and Pathogenesis
of IgAN
Accumulating evidence indicates that miRNAs
have a role in many human diseases, including
993
IgAN [220]. MiRNAs are important mediators of
tissue fibrosis under various pathological condi-
tions and are of potential therapeutic relevance
[221]. Although to date, limited data are available
on the role of miRNAs in the pathogenesis of
IgAN, miRNAs may have important roles in the
pathogenesis and progression of IgAN [220,
222–227, 221, 228–230]. Some alterations seem
to be disease specific, whereas others are appar-
ently damage related [220]. As miRNAs in uri-
nary sediment are relatively stable and easily
quantified, they have the potential to be used as
biomarkers for the diagnosis and monitoring of
disease [220].
A number of miRNAs are involved in the
development of fibrosis and progression of
chronic kidney disease (CKD) in general [220].
For example, miR21 [231], miR192 [232, 233],
miR29a [234, 235], miR377 [236], and miR377
[237] were reported. Although these miRNAs
may have a role in IgAN, none of them,
however, has been specifically studied in the
disease [220].
Progression Mechanism
Renal fibrosis is the final common manifestation
of CKDs [238]. There is little to suggest that the
mechanisms of mesangial proliferative glomeru-
lonephritis, progression, and scarring are distinct
in IgAN compared with other types of chronic
glomerulonephritis. A simplified scheme of pro-
gression mechanism in IgAN is shown in Fig. 2.
Initiation and Progression Mechanism
of Glomerular Injury
As described above, deposition of polymeric IgA,
but not of monomeric IgA, can induce the produc-
tion and local release of a variety of cytokines,
growth factors, complements, and angiotensin II
by renal resident cells and by circulating inflam-
matory cells leading to inflammatory injury, char-
acteristic histopathological features of mesangial
994 K. Nakanishi and N. Yoshikawa

Glomerular injury
Events TGF-b /Smad
PDGF
Glomerular IgA1 deposition ↑Cytokines/growth factors Angiotensin II
↑Complements Interleukins
Reactive oxygen species
Mesangial activation Inflammatory cell infiltration MicroRNAs

Mesangial cell proliferation
Podocyte depletion
Proteinuria
Tubulo-interstitial injury
Glomerulotubular cross-talk
Fibroblast activation
Epithelial to mesenchymal transition

Imbalance of apoptosis

Mesangial-matrix expansion Imbalance of AT1R and AT2R

Extracellular-matrix deposition

Glomerulosclerosis Renal fibrosis Tubulo-interstitial fibrosis

End-stage renal disease

Fig. 2 A simplified scheme of progression mechanism in IgAN

cell proliferation, and extracellular matrix deposi-
tion [239–242]. IgA alone also appears to be
sufficient to provoke injury in susceptible individ-
uals [243]. Studies in vitro and in animal models
of mesangial proliferative glomerulonephritis
have shown the key role of cytokines and growth
factors, particularly platelet-derived growth factor
(PDGF) and TGF-β, in the induction and progres-
sion of mesangial injury, and there is evidence that
these are also involved in IgAN [244–246]. It has
been reported that the MAPK/ERK signaling
pathway was activated in the mesangium of
patients presenting with heavy proteinuria [247].
IgA1-dependent ERK activation required RAS,
and ERK activation alters mesangial cell
podocyte cross talk, leading to renal dysfunction
in IgAN [247]. Although the role of infiltrating
activated monocyte/macrophage into glomeruli is
also thought to be important in glomerular
injury [248], it has been shown that resident glo-
merular cells (mesangial and endothelial cells) are
predominantly the major source of upregulated
growth factor production in IgAN [244].
Studies in children with IgAN suggest that
mesangial proliferation may in part be the result
of local production of cytokines, interleukin-1,
interleukin-6, tumor necrosis factor (TNF),
PDGF, TGF-β, and vascular permeability
factor/endothelial growth factor (VPF/VEGF)
[249–251]. C3 deposits in mesangium and activa-
tion of complement proteins in mesangium [252]
and excessive oxidant stress [253] may mediate
glomerular injury in IgAN.
Glomerular injury and proteinuria in IgAN are
related to the degree of podocyte depletion in
humans [254]. Recent study has supported for
the concept that podocyte depletion could be a
major mechanism driving glomerulosclerosis in
human glomerular diseases [255]. Dysregulation
of apoptosis may have the important role in
podocyte depletion. It has been shown that
downregulation of Bcl-2 by podocytes is associ-
ated with progressive glomerular injury and clin-
ical indices of poor renal prognosis in human
IgAN [256]. Recently, it has been reported that
IgA1 from IgAN patients may induce apoptosis of
32 Immunoglobulin A Nephropathies in Children (Includes HSP)
podocytes through direct and indirect pathways
and that IgA1 may accelerate progression of IgAN
by inducing apoptosis of podocytes [257].
It has been showed that enhanced gene expres-
sion for RAS is detected in glomerular mesangial
cells in IgAN [258]. Recent studies have demon-
strated an altered angiotensin II subtype 1 receptor
expression in human mesangial cells in response
to raised intrarenal angiotensin II in IgAN. In vitro
studies have also support that an imbalance of
angiotensin II subtypes 1 and 2 receptor activity
in human mesangial cells following exposure to
polymeric IgA plays a significant pathogenetic
role in the inflammatory injury in IgAN [259].
Progression Mechanisms from
Glomerular Injury to Tubulointerstitial
Injury
It remains in part unclear how mesangial IgA
deposition leads to tubulointerstitial injury in
IgAN. Several mechanisms of tubulointerstitial
injury may operate independently or synergisti-
cally [242]. Recently, a glomerulotubular cross-
talk has been proposed in addition to monocytic/
macrophage infiltration, proteinuria, complement
activation, and direct inflammatory effect of IgA
[260]. The importance of infiltrating inflamma-
tory cells in the tubulointerstitium in mediating
tubular injury and renal fibrosis in IgAN has been
demonstrated [261, 242]. Especially, recent atten-
tion has been focused on the role of proximal
tubular epithelial cells in organization inflamma-
tory cell infiltration and renal fibrosis via produc-
tion of inflammatory mediators upon
activation [242].
Proteinuria is the major stimulus of proximal
tubular epithelial cell activation and subsequent
chemotaxis of infiltrating immunocompetent cells
in most glomerular diseases [262]. Endothelin-1
synthesis was enhanced in culture proximal tubu-
lar cells exposed to high concentrations of albu-
min, IgG, or transferrin. Similarly, monocyte
chemoattractant protein (MCP-1) gene was
upregulated by albumin and transferrin. Very sim-
ilar findings were reported for albumin-induced
upregulation of RANTES (regulated upon
995
activation, normal T cell expressed and secreted),
an immunoregulatory cytokine with chemotactic
properties for monocytes and memory T cells in
culture proximal tubular cells [263]. Albumin is
also a strong stimulus for tubular interleukin-
8 expression, which occurs with nuclear factor-
kappaB-dependent pathways [262]. Consistent
with these in vitro studies, vasoactive and
proinflammatory molecules were enhanced in
renal tissue from rat models with proteinuric
renal disease, particularly at proximal tubular
level [264, 265].
Activation of complement proteins in the prox-
imal tubule has major proinflammatory potential
in interstitial damage with proteinuric conditions.
Intracellular C3 staining was detected in proximal
tubules of proteinuric rats with remnant kidneys
early after 5/6 nephrectomy and preceded the
appearance of inflammation. C3 colocalized with
IgG to the same tubular cells [266]. Induction of
tubular C3 during protein overload has also been
reported [267].
Excessive protein reabsorption by proximal
tubular cells promotes fibrogenesis by release
of chemoattractants, which leads to local recruit-
ment of mononuclear cells. Interstitial accumula-
tion of inflammatory cells by release of TGF-β,
PDGF, and other cytokines leads to interstitial
cell transformation into myofibroblasts. In addi-
tion, proximal tubular epithelial cells interact
with surrounding interstitial fibroblasts to pro-
mote fibrogenesis by paracrine release of
profibrogenic molecules such as TGF-β, PDGF,
and endothelin-1 [268].
The other possible contributory factor is the
direct toxic effect following tubular binding of
IgA. IgAN patients have increased urinary IgA
concentration that correlates with serum creati-
nine concentration, as well as the urinary protein
excretion [269].
A glomerulotubular cross-talk, a mechanism in
which mesangial IgA deposition may lead to
tubulointerstitial injury in IgAN, has been pro-
posed. It has been documented that inflammatory
cytokines, including angiotensin II, are released
from mesangial cells following binding to IgA
from patients with IgAN. These mediators may
alter the glomerular barrier pore size that allows
996
the passage of these inflammatory mediators to
the tubular lumen. These mediators then activate
proximal tubular epithelial cells, which may
amplify the inflammatory cascade by local pro-
duction of chemotactic mediators, which attract
more inflammatory cells. This glomerulotubular
cross-talk will generate a positive feedback loop
of activation in the renal tubules that leads to
the overproduction of extracellular matrix
components, resulting in fibrosis. This hypothesis
was tested by conducting an experiment in
which proximal tubular epithelial cells were cul-
tured with medium prepared from mesangial cells
incubated with IgA from IgAN patients [260].
Contrary to the absent stimulatory effect on
proximal tubular epithelial cells upon direct incu-
bation with IgA, increased proliferation and
enhanced expression of inflammatory mediators
(including interleukin-6, TNF-α, soluble
ICAM-1, and angiotensin II) in proximal tubular
epithelial cells cultured with medium prepared
from mesangial cells incubated with IgA from
IgAN patients were observed. Tubular and
interstitial ICAM-1-positive cells may
participate in adhesive interactions with
interstitial leukocytes [270, 271]. Upregulation
of renal interleukin-6 expression correlates well
to the degree of tubulointerstitial damage in
IgAN [272].
Renal Fibrosis
Glomerulosclerosis, tubulointerstitial fibrosis,
inflammatory infiltration, and loss of renal paren-
chyma characterized by tubular atrophy, capillary
loss, and podocyte depletion are constituent path-
ologic findings of renal fibrosis [238]. The cellular
events leading to these histologic findings include
mesangial and fibroblast activation, tubular EMT,
monocyte/macrophage/T-cell infiltration, and
apoptosis [238]. At present, renal fibrogenesis
process is thought to be similar to wound healing
response to injury [273, 274, 238]. The reason
why the difference between a healthy wound
healing and fibrotic response occurs remains
unknown.
K. Nakanishi and N. Yoshikawa
Pathology
Immunohistologic Findings
The diagnostic immunopathological pattern of
IgAN is the presence of IgA in the glomerular
mesangium as the sole or predominant Ig. IgA
deposits often extend just beyond the mesangio-
capillary junctions into the adjacent capillary
walls (Fig. 3). There are also deposits of IgG
and/or IgM with the same staining pattern as IgA
but with lesser intensity and frequency. In our
series, mesangial IgA deposits were associated
with IgG in 32 % of patients, IgM in 8 %, and
both IgG and IgM in 11 % [275]. C3 deposits were
observed in a similar distribution pattern in 64 %
of cases. The early components of the classical
complement pathway, C4 or C1q, are absent.
Fibrin- or fibrinogen-related antigens are found
in a diffuse mesangial distribution in 25–70 % of
patients and are believed to be one of the injurious
agents in the glomeruli [276]. Although, in most
patients, IgA is present only in the mesangial
regions, in approximately 10 % of patients it is
also observed in the peripheral capillary walls.
Such peripheral capillary wall deposits, whether
documented by immunofluorescence or electron
microscopy, have been associated with more
Fig. 3 Immunofluorescence micrograph showing
mesangial IgA deposits in a patients with IgAN. Original
magnification,  400
32 Immunoglobulin A Nephropathies in Children (Includes HSP)
Fig. 4 Light micrograph showing mesangial proliferation
in patients with IgAN. Four types of mesangial change are
identified: (a) Mesangial hypercellularity is more promi-
nent than the increase in matrix. (b) The degrees of
mesangial hypercellularity and matrix increase are similar.
severe clinical manifestations and a poor renal
outcome [277–280].
Light Microscopic Findings
Various glomerular changes are observed. The
most characteristic abnormality is mesangial
enlargement, caused by various combinations of
hypercellularity and increase in matrix (Fig. 4).
Occasionally, small eosinophilic and
PAS-positive fibrinoid mesangial deposits are
also seen. Biopsies can be graded according to
the amount of mesangial cell proliferation on
the basis of the World Health Organization
criteria [281]:
997
(c) The increase in matrix is more prominent than the
mesangial cellularity. (d) Progression of IgAN is associ-
ated with the development of global glomerulosclerosis
accompanied by reactive cell invasion. Original magnifi-
cation,  400
1. Minimal glomerular lesions. The majority of
glomeruli appear optically normal, although a
few may show a slight increase of mesangial
matrix, with or without accompanying
hypercellularity. The number of mesangial
cells per peripheral mesangial area does not
exceed three. There are also small foci of tubu-
lar atrophy and interstitial lymphocyte infiltra-
tion in some patients.
2. Focal mesangial proliferation. Up to 80 % of
glomeruli show moderate or severe mesangial
cell proliferation (i.e., more than three cells per
peripheral mesangial area). The degree of
mesangial cell proliferation varies consider-
ably among glomeruli as well as segmentally
within individual glomeruli. The proliferation
998
is usually associated with increased matrix.
Small cellular or fibrocellular crescents are fre-
quently found but rarely affect more than 20 %
of the glomeruli. Capsular adhesions are fre-
quently seen overlying lobules showing
mesangial proliferation. Segmental capillary
collapse is often observed in association with
crescents. A small number of glomeruli show-
ing global sclerosis are often present. Tubular
atrophy, interstitial fibrosis, and interstitial
lymphocyte infiltration are frequently present
but are not extensive.
3. Diffuse mesangial proliferation. More than
80 % of glomeruli show moderate or severe
mesangial cell proliferation, which varies in
intensity in different regions of the mesangium
in a given glomerulus as well as from one
glomerulus to another. Mesangial cell prolifer-
ation is always accompanied by increased
mesangial matrix. Cellular and fibrocellular
crescents are often found, usually affecting
less than 50 % of the glomeruli, although in
approximately 10 % of patients, more than
50 % are involved. Capsular adhesions are
frequently seen in the absence of crescents.
A small number of globally sclerosed glomer-
uli are often present. Tubular atrophy, intersti-
tial fibrosis, and interstitial lymphocyte
infiltration are frequently present and are
extensive in 10 % of patients.
Four types of mesangial change are identified
in children with IgAN [5] (Fig. 4): (a) mesangial
hypercellularity is more prominent than the
increase in matrix, (b) the degrees of mesangial
hypercellularity and matrix increase are similar,
(c) the increase in matrix is more prominent than
the mesangial cellularity, and (d) progression of
IgAN is associated with the development of
global glomerulosclerosis accompanied by reac-
tive cell invasion.
The first type of lesion is seen in biopsies in
which the interval between onset of disease and
biopsy is short. Serial pathologic observations
reveal that prominent mesangial hypercellularity
is almost exclusively seen in initial biopsies and
disappears in follow-up biopsies. These observa-
tions suggest that predominant mesangial
K. Nakanishi and N. Yoshikawa
hypercellularity is characteristic of the early lesion
of childhood IgAN and may disappear within a
matter of months. An increase in mesangial cells,
although sometimes present, is seldom striking in
adult patients [282]. In contrast, biopsies with a
predominant matrix increase show a long interval
between onset of disease and biopsy and a high
percentage of glomerular sclerosis. Serial patho-
logic observations reveal that this type of change
is usually seen in follow-up biopsies. An increase
in the amount of mesangial matrix with duration
of the disease has also been noted in adult patients
[283]. These findings suggest that progression of
IgAN leads to gradual resolution of mesangial
hypercellularity and an increase of matrix associ-
ated with the development of sclerosis [284].
The severity of tubulointerstitial changes usu-
ally reflects the severity of glomerular damage.
Vascular lesions, such as arterial or arteriolar scle-
rosis, are reported to be common in adults [285]
but are very unusual in children with IgAN
[279]. This difference may be related to the age
at biopsy and the duration of disease before
biopsy.
Endocapillary hypercellularity is defined as an
increased number of cells within glomerular cap-
illaries, causing narrowing of the lumina. It may
be present, in either focal segmental or diffuse
distribution, and is typically associated with
extension of deposits to peripheral loops. The
hypercellularity might reflect proliferation,
inflammatory cell infiltration, or endothelial cell
swelling. Endocapillary lesions are more common
in children and associated with active disease and
high levels of proteinuria [286]. In IgAN, as in
other proliferative glomerulonephritides, many of
the glomerular cells are leukocytes [287]. The
presence of glomerular macrophages correlates
with endocapillary hypercellularity and
sclerosis [288].
Electron Microscopy
Electron microscopic abnormalities are mainly
observed in the mesangium, which is variably
enlarged by a combination of increased cytoplasm
and matrix. Electron-dense deposits in the
32 Immunoglobulin A Nephropathies in Children (Includes HSP)
Fig. 5 Electron
micrograph showing
numerous electron-dense
deposits in the mesangium
in a patient with IgAN.
Original magnification,
 5,000
mesangium are the most constant and prominent
feature and are seen in almost all patients (Fig. 5).
They are granular masses situated immediately
beneath the lamina densa in the perimesangial
region and expanded mesangium. The size and
extent of mesangial deposits vary from patient to
patient; in some patients, they are large and pro-
duce localized protrusions. Peripheral glomerular
capillary wall deposits are also found in the
subendothelial and subepithelial regions.
Subendothelial deposits occur most frequently in
the capillary wall adjacent to the mesangium,
although they are also observed in the peripheral
part of the loop. Subepithelial deposits are
reported to be unusual in adult patients but are
frequently found in children with IgAN. They are
generally small and flat and localized to a few
capillary loops; the humps typical of acute
poststreptococcal glomerulonephritis are never
observed. Lysis of the glomerular basement
membrane is also seen quite frequently in
children [289]. In affected areas of the glomerular
capillary walls, the lamina densa is thin and irreg-
ular, and the epithelial aspect of the glomerular
basement membrane shows irregular segments of
low electron density with an expanded, washed-
out appearance. The epithelial foot processes are
generally well preserved, but diffuse foot process
effacement may be seen in patients with the
nephrotic syndrome.
999
Repeat Renal Biopsy Findings
There have been only a few reports on the results
of repeat renal biopsies [290, 291]. We previously
reported our results in children with IgAN [6]. At
the time of the second biopsy, 23 patients had
showed clinical remission, defined as complete
disappearance of proteinuria and hematuria with
normal renal function, whereas 38 had persistent
urinary abnormalities with normal renal function.
There were no differences between the two groups
with regard to the initial clinical findings and the
pathologic findings in the initial biopsy. The sec-
ond biopsy of patients who were in clinical remis-
sion showed improvement of the glomerular
changes on light microscopy, a disappearance of
or a decrease in mesangial IgA deposits, and a
decreased amount of electron-dense deposits.
Conversely, light microscopy showed a progres-
sion of histologic lesions and the persistence of
both mesangial IgA deposits and electron-dense
deposits in patients with persistent urinary abnor-
malities. Clinical remission and histologic regres-
sion have been reported in adults with
IgAN [292].
Shima et al. [293] have retrospectively
screened and analyzed 124 consecutive children
(age 18 years at first biopsy) with newly diag-
nosed severe IgAN showing diffuse mesangial
proliferation, who received combination therapy
1000
(prednisolone + azathioprine or mizoribine + war-
farin + dipyridamole, 90 patients) or prednisolone
alone (34 patients) for 2 years and underwent
repeat biopsies. After 2 years of treatment, 27 of
the patients (21.8 %) showed disappearance of
glomerular IgA.
Differences Between Childhood
and Adult Patients with IgAN
Significant differences in the early glomerular
lesions of IgAN between children and adults
have been demonstrated [294, 295]. Glomerular
hypercellularity in mesangial area is prominent in
children and significantly greater than in adults. In
contrast, glomerular matrix expansion, crescent
formation, and interstitial damage are more severe
in adults compared to children. Glomerular
hypercellularity correlated with proteinuria in
children but not in adults, whereas glomerular
matrix correlated with proteinuria and renal func-
tion in adults but not in children [294].
The Oxford Classification of IgAN
During past decades, there have been many histo-
logic classifications for IgAN, and their utility in
predicting renal outcome has been tested in stud-
ies. These classifications are mostly presented
using semiquantitative or single-grade systems
[296, 297]. The former is generally well correlated
with renal outcome, but it limits its clinical utility
due to a time-consuming process [298]. The two
single-grade scoring systems widely used in clin-
ical practice are the Lee classification [299] and
the Haas classification [300]. However, both clas-
sifications have been criticized because of the lack
of reproducibility [297]. Although pathologic
classifications provide valuable information for
determining prognosis, there is much controversy
as to whether histopathological features are
superior to clinical factors in risk prediction of
IgAN [298]. In 2009, the Oxford classification
of IgAN in expectation of a high reproducibility
and high predictive power of renal outcome
was published for international consensus
K. Nakanishi and N. Yoshikawa
[296, 301]. The Oxford classification involves ana-
lyses of data from patients with a wide variety of
age [296, 301, 302]. This classification has
obtained a considerable level of worldwide accep-
tance. However, the value of this classification
system remains to be definitively established [303].
The Oxford classification identified four path-
ological features (mesangial hypercellularity, M;
endocapillary hypercellularity, E; segmental
glomerulosclerosis, S; and tubular atrophy/inter-
stitial fibrosis, T, resulting in a MEST score) that
predicted renal outcome independently of clinical
indicators at the time of renal biopsy and during
follow-up [296, 301]: M0 or M1, indicating
mesangial hypercellularity in 50 % versus
>50 % of glomeruli; E0 or E1, indicating
endocapillary hypercellularity in 0 versus 1 glo-
meruli; S0 or S1, indicating segmental sclerosis
in 0 versus 1 glomeruli; and T0, T1, or T2,
indicating tubular atrophy/interstitial fibrosis in
25 %, 26–50 %, or >50 % of renal cortex,
respectively. These findings were generally valid
both in adults and children [302]. However, the
limited number of patients (265 cases) and their
heterogeneous origin (from 11 countries in four
continents) indicated a need for validation in other
cohorts.
Validation studies have demonstrated their
clinical benefits and limitations and highlighted
questions and difficulties of interpretation of the
biopsy sample [304–315, 303]. Recently, the
VALIGA study including 1,147 patients from
13 European countries provides a validation of
the Oxford classification in a large European
cohort of IgAN patients across the whole spec-
trum of the disease. However, the independent
predictive value of pathology MEST score is
reduced by glucocorticoid/immunosuppressive
therapy [316].
To date, none of the validation studies of the
Oxford classification including children as well as
adults completely confirmed the results from the
original Oxford cohort regarding the predictive
value of M, S, and T scores [303], and only one
study confirmed the association between E score
and response to immunosuppressive therapy
[304]. Differences between studies are likely to
be accounted for, at least in part, by differences in
32 Immunoglobulin A Nephropathies in Children (Includes HSP)
inclusion or exclusion of patients with very mild
or very severe disease (both excluded from the
original Oxford cohort), proportions of patients
treated with angiotensin-converting enzyme
inhibitors and/or angiotensin receptor blockers
and duration of this therapy, use of immunosup-
pressive therapy, length of follow-up, and other
factors. Some of these differences appear to be
potentially instructive. Notably, crescents, not a
significant predictor of outcomes in the original
Oxford cohort that excluded patients with rapidly
progressive glomerulonephritis, may in fact have
predictive value in patients with severe disease
(eGFR <30 ml/min) and in children. This ques-
tion deserves further study.
Recently, Shima et al. [317] have reported that
a shorter time from IgAN onset to renal biopsy
was associated with higher glomerular ratios
of M, E, and crescents and lower ratios of S and
G in 250 children with proteinuria 0.5 g/day/
1.73 m2 at biopsy, matching the inclusion criteria
for IgAN of the original Oxford cohort. These
findings indicate that acute lesions, such as M
and E, reflect disease activity, with shorter times
from onset to biopsy generally indicating more
severe disease activity. Crescents also seem to
reflect disease activity, because crescents were
acute lesions in their cohort. In contrast, chronic
lesions such as S and G tend to be absent early in
the course of disease, even in patients with severe
acute lesions. Taken together, these findings sug-
gest that each variable of the Oxford classification
of IgAN may be influenced by the time from
disease onset to renal biopsy and that differences
in the significance of S and crescents between the
original Oxford classification and their data may
be due, at least in part, to differences in the timing
of renal biopsy [317].
Finally, the most important clinical question is
how we can make the Oxford classification more
clinically relevant. One of key issues should be
considered is that there is sufficient evidence to
warrant combining M, E, S, and T (and possibly
other) scores into a grading system (I, II, III, and
so on) designed to indicate increasing levels of
disease severity and perhaps simplify this classi-
fication system for clinicians [303]. Tanaka
et al. [318] have developed and validated a new
1001
prediction rule using clinical measures and the
Oxford classification for developing ESRD in
patients with IgAN and verified its validity in an
independent cohort. This prediction rule provides
a useful guide to estimate the individual risk for
ESRD in patients with IgAN and may be effective
at identifying those at high risk for the future
development of ESRD.
Pathology in HSN
The renal histopathology of HSN is indistinguish-
able from that of primary IgAN. Although as
IgAN various glomerular changes are observed
at light microscopic examination of renal biopsy
specimens, there are two basic types of glomerular
lesions, mesangial proliferation and epithelial
crescent formation. The two lesions typically
coexist (Fig. 6).
Severe mesangial proliferation may also be
associated with mesangial interposition in which
cell and matrix migrate into the capillary walls
between the basement membrane and endothelial
cytoplasm, giving the capillary walls a double
contour appearance on silver staining. Mesangial
interposition is only rarely diffuse in HSN, in
which it may superficially mimic the appearance
of mesangiocapillary (membranoproliferative)
glomerulonephritis [15].
Because the mesangial proliferation usually
resolves without causing permanent damage, the
morphologic severity of HSN is best assessed on
the basis of the extent of glomerular involvement
with crescents and segmental lesions, including
necrosis and sclerosis. The classification used
most is that evolved by the pathologists of the
International Study of Kidney Disease in Children
(Table 2) [27, 33, 34]. Each of the severity grades
is based on the percentage of glomeruli with cres-
cents and segmental lesions. The clinicopatho-
logic correlations of this classification have been
effectively tested in follow-up studies (Table 3)
[26, 27, 33–35].
The characteristic immunopathologic pattern
of HSN is the same as that of IgAN with the
presence of IgA in the glomerular mesangium
(Fig. 3). Although in most patients IgA is present
1002 K. Nakanishi and N. Yoshikawa

Fig. 6 Glomeruli from a

patient with HSN.
Circumferential crescents
are evident (Periodic acid-
methenamine-silver stain,
original magnification,
 200)

Table 2 Morphologic classification of Henoch-Schönlein Table 3 Correlations between ISKDC grade and clinical
nephritis evolved by the International Study of Kidney presentation/outcome in Henoch-Schönlein nephritis
Disease in Children
ISKDC Approximate risk
Minimal glomerular grade Clinical presentation of renal failure (%)
I abnormalities I Hematuria only 0
II Pure mesangial (a) focal or II Hematuria and <5
proliferation (b) diffuse proteinuria
III Minor glomerular (a) focal or III Hematuria and <10
abnormalities or (b) diffuse mesangial proteinuria
mesangial proliferation, proliferation Acute nephritic
with crescents or syndrome
segmental lesions
Nephrotic syndrome
(sclerosis, adhesions,
thrombosis, necrosis) in IV Hematuria and 25
fewer than 50 % of proteinuria
glomeruli Acute nephritic
IV As III but with crescents (a) focal or syndrome
or segmental lesions in (b) diffuse mesangial Nephrotic syndrome
50–75 % of glomeruli proliferation Rapidly progressive
V As III but with crescents/ (a) focal or nephritic syndrome
segmental lesions in more (b) diffuse mesangial V Hematuria and >50
than 75 % of glomeruli proliferation proteinuria
VI Membranoproliferative- (a) focal or Acute nephritic
like lesion (b) diffuse mesangial syndrome
proliferation Nephrotic syndrome
Rapidly progressive
nephritic syndrome
VI

only in the mesangial regions, in approximately

10 % of patients, it is also present in the peripheral
capillary walls. Such peripheral capillary wall
deposits, whether documented with immunofluo-
rescence or electron microscopic examination,
have been associated with more severe clinical
manifestations and a poor renal outcome [34].
Electron microscopic abnormalities of HSN
are also the same as that of IgAN (Fig. 5).
Niaudet et al. [319] found good correlation
between histologic changes with time and both
clinical status and outcome.
32 Immunoglobulin A Nephropathies in Children (Includes HSP)
Clinical Features in IGAN
IgAN occurs at all ages but is most common
during the second and third decades of life; it
affects boys more often than girls, with the
reported male to female ratio varying from less
than 2:1 to 6:1 [2]. In a study of Japanese children
[275], the mean age at presentation was 9.3 years
in boys and l0.3 years in girls, and the male to
female ratio was 3:2. The clinical presentation of
IgAN varies. Some patients have asymptomatic
microscopic hematuria with or without protein-
uria. Other patients have recurrent episodes of
macroscopic hematuria. Some patients present
with acute nephritic syndrome and, more rarely,
with acute renal failure.
Sixty-two percent of our 258 Japanese children
were found to have microscopic hematuria with or
without asymptomatic proteinuria [275]. Twenty-
six percent presented with macroscopic hematuria
and 12 % with an acute nephritic syndrome or
nephrotic syndrome. Several studies from Europe
and the United States reported that more than
80 % of the patients have episodes of macroscopic
hematuria, and recurrent macroscopic hematuria
is traditionally regarded as the hallmark of child-
hood IgAN [320–322] (Southwest Pediatric
Nephrology Study Group [323]). However, it
was the initial feature in only 26 % of our series,
presumably because of the school screening pro-
gram that detected a high prevalence of asymp-
tomatic urinary abnormalities rather than regional
variation in the expression of IgAN. During the
observation period, 60 % of patients had one or
more episodes of macroscopic hematuria,
whereas the other 40 % remained asymptomatic.
Macroscopic hematuria often occurs in associ-
ation with upper respiratory tract infections; less
frequently, it occurs in association with other
infections involving the mucosal system (e.g.,
diarrhea and sinusitis). Episodes of macroscopic
hematuria are sometimes associated with loin
pain. The interval between the precipitating infec-
tion and the appearance of hematuria ranges from
1 to 2 days compared with 1 or 2 weeks in acute
postinfectious glomerulonephritis. Many patients
have recurrent episodes of macroscopic hematu-
ria, each often associated with the same type of
1003
infection. The number of recurrences and the
intervals between different episodes are variable.
The incidence of macroscopic hematuria is lower
in adults (Southwest Pediatric Nephrology Study
Group [323, 324]). Emancipator et al. [3] summa-
rized the previous reports of IgAN, in which most
the patients were adults, and reported that 43 %
had macroscopic hematuria. However, in Japan,
only 18–32 % adult patients have been reported to
have macroscopic hematuria [324, 325]. The rea-
son for the age-related differences in the incidence
of macroscopic hematuria has yet to be elucidated.
In asymptomatic patients, microscopic hema-
turia is almost always present and persistent. Pro-
teinuria is common. The blood pressure and renal
function at onset are normal.
Patients with a nephritic or nephrotic onset have
the most severe glomerular damage. Hypertension
is infrequent and usually mild to moderate. Malig-
nant hypertension is not a presenting feature in
childhood. Nephrotic edema is reported in approx-
imately 10 % of patients. Acute renal failure is
occasionally associated with episodes of macro-
scopic hematuria and is usually reversible. How-
ever, a number of investigators have documented a
subset of patients with IgAN that is characterized
by extensive crescents and a rapidly progressive
course [325–327]. A review of published cases of
crescentic IgAN revealed that 41 % of patients with
this rapidly progressive form of disease were
16 years of age or younger [327].
Laboratory Investigation
Serum IgA levels are increased in 30–50 % of
adult patients but in only 8–16 % in children
with IgAN [275]. For this reason, it is seldom of
diagnostic significance. Serum complement com-
ponent concentrations are usually normal, but the
C3 level should be measured routinely if the
patient has been referred for investigation after
the first attack of hematuria to eliminate a diagno-
sis of postinfectious glomerulonephritis or
membranoproliferative glomerulonephritis. Like-
wise, the antistreptococcal antibody titers should
be determined after initial hematuria. These inves-
tigations are of little value when hematuria is
1004
known to have been present for more than
3 months. The serum creatinine should be mea-
sured routinely to estimate renal function; if nec-
essary, the glomerular filtration rate should be
determined. If present, proteinuria should be
quantified, as proteinuria is associated with histo-
logic lesions and a risk of progression. The plasma
proteins should be measured routinely in the pres-
ence of heavy proteinuria.
Biomarkers in IgAN
IgAN has been the focus of several studies aimed
at identifying specific biomarkers. Serum
galactose-deficient IgA1 level and glycan-specific
autoantibody levels are prime candidates to
become diagnostic biomarkers for IgAN because
of their central role in the earliest stages of disease
pathogenesis [328]. However, although the serum
level of galactose-deficient IgA1 is frequently ele-
vated in patients with IgAN [329], the sensitivity
and specificity of this laboratory finding are insuf-
ficient for the test to replace kidney biopsy as the
diagnostic standard [330]. The serum level of
glycan-specific IgG antibodies is correlated with
the level of urinary protein excretion [216] and the
risk of progression to end-stage renal disease or
death [331]. This biomarker may prove useful for
monitoring disease progression or the response to
therapy [330]. Yanagawa et al. have reported that
serum levels of galactose-deficient IgA1-specific
antibodies are elevated in most IgAN patients, and
their assessment, together with serum levels of
galactose-deficient-IgA1, improves the specificity
of the assays. These observations suggest that a
panel of serum biomarkers may be helpful in
differentiating IgAN from other glomerular
diseases [332].
It has reported that increased plasma levels of
activated complement C3 [333], advanced oxida-
tive protein products [334], and fibroblast growth
factor 23 [335]; an increased serum level of uric
acid [336, 337] and decreased serum levels of
CD89-IgA complexes [338] are associated with
severe histologic changes, severe proteinuria, or a
poor clinical outcome. However, these findings
may not be unique to IgAN [330].
K. Nakanishi and N. Yoshikawa
Assays for serum levels of complement C3
may be readily available in clinical practice, either
alone or in tandem with total serum IgA or serum
galactose-deficient IgA1 levels, as prognostic bio-
markers for patients with IgAN [328]. Several
researchers have reported that serum C3 levels in
patients with IgAN are significantly lower
than those in patients with non-IgAN
[339–341]. Serum IgA levels in patients with
IgAN are significantly higher than those in
patients with non-IgAN, and the serum IgA/C3
ratio seems to be a more reliable marker than
serum IgA to distinguish IgAN from non-IgAN
[339–345]. Recent studies have shown that ele-
vated serum IgA/C3 ratio is not only a robust
diagnostic marker for IgAN but also a predictor
of prognostic grading in patients with IgAN in
Japanese patients [339–345]. Komatsu
et al. have reported that IgAN patients with high
serum IgA/C3 (>4.5) have a significantly poorer
renal outcome in Japanese patients [341]. How-
ever, to date, these findings have not been con-
firmed in children.
Urinary cytokine excretion may reflect histo-
logic changes in IgAN, and their measurement
can give information about disease outcome
[346]. Increased urinary excretion of epidermal
growth factor (EGF) [347], MCP-1 [346], IL-6
[346], podocytes [348], low-molecular-mass pro-
teins [349], and mannose-binding lectin [350]
were reported in IgAN. Conversely, Stangou
et al. have reported that decreased urinary EGF
levels may represent histology in IgAN and that
EGF excretion can be a predictive marker [346].
Urinary proteomic data are also candidates for
diagnostic biomarkers because this approach can
successfully differentiate patients with IgAN from
healthy controls and from patients with other
forms of renal disease [328, 330]. Analyses of
urinary samples by capillary electrophoresis with
mass spectrometry have differentiated patients
with IgAN from healthy controls and patients
with minimal change disease or IgA immune
complex nephritis due to chronic hepatitis C
infection, even in association with nonpathologic
proteinuria [351–353]. Furthermore, the urinary
proteomic profile predicts the response to treat-
ment with an angiotensin-converting enzyme
32 Immunoglobulin A Nephropathies in Children (Includes HSP) 1005

inhibitor [354]. However, additional studies are Relationship Between IgAN and HSP
needed to determine the potential and cost-
effectiveness of urinary proteomic analysis in The relation between IgAN and HSP is complex.
establishing the diagnosis of IgAN and making There seems to be a close relationship between
decisions about treatment [330]. IgAN and HSP [355]. The morphologic and
immunopathologic features are similar in the two
conditions [355, 48]. The two disorders have been
Differential Diagnosis reported to coexist in different members of the
same family, including a pair of monozygotic
The diagnosis of IgAN is based on the presence of twins who developed the disorders simulta-
IgA as the sole or predominant Ig in the glomer- neously after a well-documented adenovirus
ular mesangium. Because diffuse mesangial IgA infection [11, 38, 356, 57]. Moreover, the evolu-
deposits are observed in a variety of other disor- tion of IgAN into HSN in the same patient is
ders (Table 4), the diagnosis of IgAN can be made described in both adults and children
only by exclusion. The IgA deposits are often [357–359]. It has been suggested that the two
incidental findings, and the pathogenesis and clin- conditions are variants of the same process and
ical significance are unclear. that IgAN is HSN without the rash [48, 57, 359].
Although there are similarities in their pathologic
and immunologic features, the two conditions are
clinically different, and the pathogenesis is not
Table 4 Diseases associated with diffuse mesangial IgA clear. Our study suggests that HSN is an acute
deposits disease, with glomerular lesions nonprogressive
Primary after the onset [27]. Therefore, in most patients,
IgA nephropathy the prognosis is associated with the severity of
Secondary glomerular change at the onset. In contrast,
Multisystem disease IgAN is a chronic, slowly progressive glomerular
Henoch-Schönlein purpura lesion, which may eventually lead to chronic renal
Systemic lupus erythematosus
failure, whatever the presentation. A few patients
Cystic fibrosis
with HSN have recurrent episodes of macroscopic
Celiac disease
hematuria and a progressive renal disease on
Crohn’s disease
repeat renal biopsies. Finally, HSN occurs mostly
Dermatitis herpetiformis
Ankylosing spondylitis
in young children and is rare in adulthood,
Neoplasms whereas IgAN affects mainly older children and
Carcinomas of the lung and colon young adults. It is therefore reasonable that IgAN
Monoclonal IgA gammopathy and HSN are treated as different clinicopathologic
Mycosis fungoides entities until pathogeneses of the two conditions
Non-Hodgkin’s lymphoma are better understood.
Infectious diseases
Mycoplasma infections
Leprosy Chronic Liver Disease
Toxoplasmosis
Others Glomerular IgA deposits may be observed in
Chronic liver disease patients with various types of chronic liver dis-
Thrombocytopenia eases [360]. Mesangial proliferation and IgA
Pulmonary hemosiderosis deposits are the most common findings. The
Mixed cryoglobulinemia
light, electron, and immunofluorescence micro-
Polycythemia
scopic features in patients with liver disease are
Scleritis
similar to those in patients with primary IgAN.
1006
100%
50%
0%
onset 5y 10y
Follow-up period
Fig. 7 Long-term prognosis of the 169 Japanese children with IgAN followed more than 10 years
Most patients with chronic liver disease have clin-
ically asymptomatic renal disease. Microscopic
hematuria and mild proteinuria may be observed,
but macroscopic hematuria or a nephrotic syn-
drome is rare. Renal functional impairment is
also rare. Glomerular lesions in children with
chronic liver disease are very unusual. The path-
ogenetic mechanisms that contribute to mesangial
IgA deposition in chronic liver diseases remain
unknown. Significant elevations of the serum
monomeric and polymeric IgA levels have been
reported in patients with chronic liver disease
[361, 362]. Impaired hepatic clearance and
increased synthesis of polymeric IgA, abnormali-
ties of IgA metabolism, and portosystemic
shunting of antigens and immune complexes
have been suggested as possible causes of
mesangial IgA deposition in chronic liver
disease [362].
Idiopathic Nephrotic Syndrome
A few patients with steroid-sensitive nephrotic
syndrome show mesangial deposits of IgA on
renal biopsy. They are classified by some as
IgAN, whereas others consider that mesangial
IgA in patients with minimal changes (i.e., with-
out cellular proliferation) is coincidental. This
probably applies to idiopathic nephrotic
K. Nakanishi and N. Yoshikawa
9%
8%
21% Renal failure
Nephrotic syndrome
5%
Proteinura ≥ 1 g/day
Proteinura < 1 g/day
Hematuria only
57%
Normal urine
15y
syndrome associated with mesangial IgA deposits
occurring in Asians and explains a favorable
response to steroids, which is not the case in true
IgAN [363].
Natural History and Prognosis
In adult series, the incidence of renal insufficiency
varies from less than 10 % to as high as 45 % in
patients followed up for more than 1 year. In long-
term follow-up of adult patients, 30–35 % have
been found to develop progressive renal insuffi-
ciency 20 years after the initial discovery of dis-
ease [2, 364–366]. It can be estimated that 1–2 %
of adult patients will enter end-stage renal failure
each year from time of diagnosis [367]. The long-
term prognosis of the 169 Japanese children with
IgAN followed more than 10 years indicates that
9 % of the patients had developed chronic renal
failure by 15 years (Fig. 7).
Because of the variable rate of progression to
chronic renal failure, there have been attempts to
identify features present at the time of diagnosis
that would predict the outcome. The following
clinical findings are regarded as poor prognostic
indicators in adult patients [2]: persistent hyper-
tension, persistent heavy proteinuria, and reduced
glomerular filtration rate at presentation
[364–366, 368–373]. In children, several studies
32 Immunoglobulin A Nephropathies in Children (Includes HSP)
have shown that the degree of proteinuria corre-
lates with the severity of morphologic glomerular
lesion [322, 374, 279], and heavy proteinuria at
the time of biopsy predicts a poor outcome [280,
375]. In contrast, slight proteinuria or its absence
at the time of biopsy predicts a favorable outcome.
Acute renal failure at onset is usually transient and
associated with macroscopic hematuria and
reversible tubular lesions. Male gender has also
been considered an unfavorable prognostic fea-
ture by some investigators [365], but we [280]
and others [278] could not confirm it in large
cohort of adult and pediatric patients. Schena
et al. reported an increased risk of end-stage
renal disease in familial IgAN [88].
Several pathologic features are associated with
a poor outcome: diffuse mesangial proliferation; a
high proportion of glomeruli showing sclerosis,
crescents, or capsular adhesions; the presence of
moderate or severe tubulointerstitial changes; the
presence of subepithelial electron-dense deposit;
and lysis of the glomerular basement membrane
by electron microscopy [280]. Patients with dif-
fuse mesangial proliferation have been reported to
have a significantly worse prognosis than those
with focal proliferation or minimal lesions by light
microscopy in adults [364, 369, 300]. In many
adult studies [278, 364, 372, 285], glomerular
sclerosis and crescents have also been associated
with poor renal outcome. Levy and associates
found that mesangial proliferative glomerulone-
phritis with crescents was associated with poor
prognosis in children [322]. As the severity of
the tubulointerstitial changes is usually related to
the severity of the glomerular changes, tubuloin-
terstitial changes in IgAN are believed to be sec-
ondary to the glomerular injury. Vascular lesions,
such as arterial or arteriolar sclerosis, have been
reported to play an important role in the progres-
sion of IgAN in adults. However, vascular
changes are very unusual in children [5] (South-
west Pediatric Nephrology Study Group
[323, 326]). This difference may be related to the
age at biopsy and the duration of disease before
biopsy.
A strong predictive relationship of low glomer-
ular density (nonsclerotic glomerular number per
renal cortical area) with progression was observed
1007
in a study by Tsuboi et al., suggesting that glo-
merular density may serve as an early histopatho-
logical marker of long-term renal prognosis in
IgAN [376]. Activation of the lectin pathway of
complement is associated with more severe renal
disease. Glomerular deposition of C4d is a marker
of activation of the lectin pathway of complement.
Espinosa et al. have reported that negative
mesangial C4d staining in glomeruli in patients
with IgAN helps to identify patients with a good
long-term prognosis [377, 378].
Recurrence of mesangial IgA deposits is often
observed in transplant recipients whose original
disease was IgAN [120, 50]. Recurrence of IgA
deposits is most often clinically asymptomatic,
and graft survival is considered good [120].
At the beginning of the 1990s, the use of
angiotensin-converting enzyme inhibitors for
focal mesangial proliferation and combined ther-
apies including corticosteroids for diffuse
mesangial proliferation increased dramatically in
Japan. Our recent retrospective cohort study of
500 children with IgAN has clarified an improved
renal survival in Japanese children with IgAN
[379]. Among all patients, the actuarial renal sur-
vival was 96.4 % at 10 years, 84.5 % at 15 years,
and 73.9 % at 20 years. Diagnosed in 1976–1989,
the renal survival was 94.0 % at 10 years, 80.1 %
at 15 years, and 70.1 % at 20 years. Diagnosed in
1990–2004, the renal survival was 98.8 % at
10 years and 98.8 % at 15 years ( p = 0.008).
With diffuse mesangial proliferation, both the
10- and 13-year renal survivals were 97.8 % in
1990–2004, compared with 78.5 % and 68.6 %,
respectively, in 1976–1989 ( p = 0.0003). In the
same study, prognostic factors for end-stage renal
disease-free survival were analyzed [379].
Mesangial proliferation degree and initial renal
biopsy year were significant in both the univariate
and the multivariate analysis. For children with
IgAN, the most influential prognostic variable
was mesangial proliferation degree. Proteinuria
at diagnosis was significant in the univariate, but
not in the multivariate analysis. Multivariate anal-
ysis showed that initial renal biopsy year was a
significant factor for renal survival independently
of mesangial proliferation degree, proteinuria at
diagnosis, and estimate creatinine clearance at
1008
diagnosis (hazard ratio = 0.08, 95 % CI
0.004–0.43).
Some patients with IgAN achieve spontaneous
remission even when not receiving medication.
The retrospective study of 96 children with
minor glomerular abnormalities or focal
mesangial proliferation who did not receive med-
ication from the 555 patients with childhood
IgAN by Shima et al. [380] showed that
57 (59.4 %) achieved spontaneous remission.
The cumulative spontaneous remission rates
among these patients were 57.5 and 77.4 % at
5 and 10 years, respectively, from onset. The
mean time from onset to remission was 5.9 
0.4 years. Of the 57 patients with spontaneous
remissions, ten (17.5 %) also developed a recur-
rence of urinary abnormalities. The cumulative
recurrence-free rates were 79.9 and 67.9 % at
5 and 10 years, respectively, after remission. The
spontaneous remission rate in childhood IgAN
with minor glomerular abnormalities or focal
mesangial proliferation was higher than expected
[380]. These results suggest that physicians
should consider the potential for spontaneous
remission and refrain from aggressive treatment
in IgAN patients with minor glomerular abnor-
malities or focal mesangial proliferation.
Treatment
IgAN is a leading cause of chronic renal disease
and end-stage renal disease in adult patients, and
recent long-term studies assessing the prognosis
in children have challenged earlier views that the
condition represents a benign disorder. Thus,
IgAN presents a therapeutic challenge in both
adults and children. The most appropriate treat-
ment for patients with IgAN is still a matter of
controversy [381]. At present, there is no curative
therapy for IgAN [382]. Because of the variable
rate of progression to renal failure and because of
the probable multifactorial pathogenesis of the
disease, the effectiveness of any treatment can
only be properly evaluated by means of a random-
ized controlled trial [381]. When considering
treatment protocols, an issue of great importance
K. Nakanishi and N. Yoshikawa
is the selection of appropriate patients in whom
the treatment is to be evaluated. Patients with
heavy proteinuria at biopsy and the most severe
glomerular lesions on renal biopsy appear to be at
greatest risk of progressive renal deterioration
and, therefore, the most appropriate candidates
for specific therapeutic interventions. Patients
with long-standing disease and extensive, irre-
versible glomerular damage are unsuitable for
such treatments.
In a randomized controlled trial, another
important issue is to select adequate endpoints.
Although in a clinical trial of progressive IgAN
the ultimate endpoint is development of chronic
renal insufficiency, most pediatric patients do not
develop it during the study period. Thus, studies
of pediatric patients with IgAN may differ mark-
edly from studies of adults with regard to the
apparent risk of progressive disease [381]. There-
fore, due to the long time period from clinical
onset of disease until progression to end-stage
renal disease, surrogate markers of outcome
must be used to evaluate efficacy of therapy for
IgAN in clinical trials. It is a noteworthy fact that
validation of these surrogate markers may be
lacking, resulting in the potential for inappropriate
conclusion with regard to therapeutic efficacy.
Therefore, the careful investigation in detailed
long-term outcome of previous randomized con-
trolled trials is important.
With regard to treatment of IgAN, it is impor-
tant to consider the differences in the nature of
IgAN between children and adults. An “evidence-
based” therapy is important in both children and
adults. However, available evidence for treatment
is partially, clearly, different between children
and adults. Generally and roughly speaking, the
evidence for treatments of IgAN in adults sup-
ports relatively passive treatments, whereas that
in children supports relatively active treatments.
The reason of this difference is unknown.
Differences in the diagnosis timing and
histological differences may be associated.
Although we should refrain from radical treat-
ments that are not based on evidence, however,
appropriate active treatments that are based on
evidence are important in treatments for children
with IgAN.
32 Immunoglobulin A Nephropathies in Children (Includes HSP)
Fish Oil/Omega 3 Fatty Acids
Controlled double-blind trial in adult patients with
IgAN [383–385] showed that treatment with fish
oil for 2 years retarded the rate at which renal
function was lost, but a meta-analysis showed
that there was only a 75 % probability that fish
oil was beneficial [386]. A randomized, placebo-
controlled, double-blind trial by the Southwest
Pediatric Nephrology Study Group in the United
States and Canada evaluated the role of omega
3 fatty acids in children and young adults with
IgAN, and the treatment group did not showed
benefit over the placebo group with respect to time
to failure, defined as estimated GFR <60 % of
baseline [387]. Furthermore, from analysis of
these clinical trials, it was reported that efficacy
of omega-3 fatty acids in children and adults with
IgAN is dosage and size dependent [388].
Coagulation Modifying Agents
Warfarin, urokinase, and antiplatelet agents have
all been assessed for the treatment of IgAN.
At present there is no sufficient evidence to sup-
port the use of coagulation modifying agents
[389, 390]. However, coagulation modifying
agents may have a role in combination therapy.
Angiotensin-Converting Enzyme
Inhibitors and Angiotensin II Receptor
Blockers
As already demonstrated in nondiabetic chronic
nephropathies [391] (GISEN Group [392, 393]),
studies including randomized controlled trials
have indicated that angiotensin-converting
enzyme inhibitors reduce urinary protein excre-
tion [394, 395] and preserve renal function [396]
in adult patients with IgAN. However, there is no
randomized controlled study only in children with
IgAN demonstrating that angiotensin-converting
enzyme inhibitors preserve renal function [397,
398]. A randomized controlled trial has proved a
significant benefit of angiotensin-converting
enzyme inhibitor on the progression of IgAN in
1009
children and young people when it was assessed
as a composite end point of 30 % reduction in
GFR or an increase of proteinuria over the
nephrotic range. It has also showed the benefit of
angiotensin-converting enzyme inhibitor on
remission of proteinuria below what is generally
considered a harmful level [399]. In adult setting a
randomized controlled trial has showed that com-
bination therapy with angiotensin-converting
enzyme inhibitor and angiotensin II receptor
blocker has an additive dose-dependent
antiproteinuric effect compared to monotherapies
in patients with IgAN [400]. The COOPERATE
trial, a large randomized study of 336 patients
with nondiabetic renal disease, in which 50 % of
subjects had IgAN, has demonstrated that combi-
nation treatment safely retards disease progres-
sion compared with monotherapy [401]. A
randomized controlled trial in 109 adults with
IgAN showed that valsartan significantly slowed
renal deterioration compared with placebo
[402]. Although there is no randomized controlled
trial for angiotensin II receptor blockers in chil-
dren with IgAN, some studies have demonstrated
their antiproteinuric effect in combination thera-
pies with angiotensin-converting enzyme inhibi-
tor and angiotensin II receptor blocker [403, 404].
Corticosteroids
Corticosteroids have been widely used to treat
moderate to severe IgAN, particularly in pediatric
patients. To date, information concerning not only
the effectiveness but also safety of corticosteroid
therapy over a long time course has been largely
defective. It has been difficult to assess the results
of treatment trials with these agents in terms of
preservation of renal function, due in part to wide
variations in the length of therapy and the dosing
regimens employed and also to the use of cortico-
steroids in combination with other drugs [381].
At present, however, some evidence has been
obtained for the role of corticosteroids in the treat-
ment of IgAN [405–407]. In adults with IgAN, an
Italian prospective randomized controlled trial
demonstrated that a 6-month course of steroid
treatment protected against deterioration of renal
1010
function with no notable adverse effects during
follow-up [405]. Recently, the long-term follow-
up data of the trial showed that corticosteroids
significantly reduced proteinuria and protected
against renal function deterioration [406].
An English single-center randomized controlled
trial has demonstrated that the value of combined
immunosuppressive treatment with prednisolone
and cytotoxic agents in reducing the occurrence of
renal failure in IgAN [407]. A homogeneous
cohort of 38 patients who had mean blood pres-
sure within 10 % of current targets and estimated
GFR 50 % normal but losing 10 % estimated
GFR/year were randomly assigned; no patient
had crescentic disease. Renal survival improved
12-fold at 5 year, with remission of nephritis by
urinalysis.
With regard to children, our previous studies
[389, 408] are the only randomized controlled
trials so far to have demonstrated that treatment
including corticosteroid for 2 years early in the
course of disease reduces immunologic renal
injury and prevent any further increase of glomer-
ular sclerosis. Up to now, however, it has been
unclear whether corticosteroid alone is sufficient
for treatment of IgAN in children, and there has
been no reliable evidence for its effectiveness in
this group of patients [381]. High-dose intrave-
nous methylprednisolone were shown to delay
development of renal failure in a randomized con-
trolled trial in adult patients [405]. However, no
convincing evidence has been published to date to
support the use of high-dose intravenous methyl-
prednisolone for the treatment of children with
IgAN.
Immunosuppressants
The use of immunosuppressants other than corti-
costeroids for treatment of IgAN has not been
sufficiently evaluated, even in adult patients.
Although the results of prospective trials of cyclo-
phosphamide [407] and mycophenolate mofetil
[409–411] for IgAN have been published, their
efficacy is controversial. It has been difficult to
assess the results of treatment trials with these
agents in terms of preservation of renal function,
K. Nakanishi and N. Yoshikawa
due in part to wide variations in the length of
therapy and the dosing regimens employed and
also to the use of corticosteroids in combination
with other drugs [381]. There is currently insuffi-
cient evidence to support the use of immunosup-
pressants alone for treatment of children with
IgAN. Recently a meta-analysis of immunosup-
pressive treatments for IgAN suggested a benefit
of corticosteroids and immunosuppressants [412].
Clinical Trials for Combination Therapy
Including Corticosteroids and
Immunosuppressant in Japan
Prospective trials by The Japanese Pediatric IgAN
Treatment Study Group have provided some use-
ful data regarding the treatment of children with
IgAN [389, 408]. In the trials, treatment was
started early in the course of disease because the
duration of the disease before treatment was short
and the extent of glomerulosclerosis was low as a
result of the Japanese school screening program.
In the trials, the majority of patients presented
with asymptomatic proteinuria and microscopic
hematuria detected by this school screening
program.
A randomized controlled trial by The Japanese
Pediatric IgAN Treatment Study Group demon-
strated that treatment of children with severe
IgAN showing diffuse mesangial proliferation
with prednisolone, azathioprine, heparin-warfa-
rin, and dipyridamole for 2 years early in the
course of disease prevents immunologic renal
injury and progression of the disease [389]. The
follow-up study of this randomized controlled
trial revealed that 2-year combination therapy
not only ameliorated the activity of the acute
phase of nephritis but also improved the long-
term outcome of severe childhood IgAN [413].
In a randomized controlled trial carried out
sequentially by The Japanese Pediatric IgAN
Treatment Study Group, the effects of predniso-
lone, azathioprine, warfarin, and dipyridamole
(combination) with those of prednisolone alone
were compared in 80 children with newly diag-
nosed IgAN showing diffuse mesangial prolifera-
tion [408]. Patients were randomly assigned to
32 Immunoglobulin A Nephropathies in Children (Includes HSP)
receive either the combination or prednisolone
alone for 2 years. It was concluded that the com-
bination treatment may be better for severe IgAN
than prednisolone alone [408].
Based on the two previous randomized con-
trolled trials and the follow-up study described
above, the immunosuppressant is considered to
be important for the treatment in children with
IgAN. However, azathioprine regimen had often
to be stopped due to toxicity. Therefore, a differ-
ent but effective immunosuppressant may be
worth trying. Mizoribine, like azathioprine, is an
antimetabolite that exerts its immunosuppressant
effect by inhibiting lymphocyte proliferation. In a
recent prospective pilot study by The Japanese
Pediatric IgAN Treatment Study Group,
mizoribine was administered instead of azathio-
prine as part of the combination therapy for treat-
ment of 23 children with severe IgAN. The
efficacy and safety of the mizoribine combination
seem to be acceptable for the treatment of children
with severe IgAN [414].
Chinese Herbal Medicine (Sairei-to)
To determine the effect of the Chinese herbal
medicine, Sairei-to (TJ-114), in children with
newly diagnosed IgAN showing focal/minimal
mesangial proliferation, a randomized controlled
trial was undertaken by The Japanese Pediatric
IgAN Treatment Study Group [415]. One hundred
and one patients were randomly assigned to
receive Sairei-to for 2 years or no drug for
2 years. The trial has demonstrated that 2-year
Sairei-to treatment early in the course of disease
is effective in children with IgAN showing focal/
minimal mesangial proliferation.
Tonsillectomy
Several retrospective studies have analyzed the
role of tonsillectomy in the treatment of IgAN
mainly in adult [416, 417]. Treatment was not,
however, homogeneous between the study
groups; patients who underwent tonsillectomy
were also treated with steroid pulses,
1011
cyclophosphamide, antiplatelets, and warfarin.
At present studies provide conflicting data. The
results of a randomized controlled trial for adults
in Japan, which compares tonsillectomy com-
bined with steroid pulses versus steroid pulses
monotherapy, indicated that tonsillectomy has no
beneficial effect over steroid pulses alone to atten-
uate hematuria and to increase the incidence of
clinical remission. Although the antiproteinuric
effect was significantly greater in combined ther-
apy, the difference was marginal, and its impact on
the renal functional outcome remains to be clari-
fied [418]. Therefore, it cannot be recommended
for widespread use for treatment of IgAN patients,
especially for children with IgAN [419].
Japanese Guidelines for the Treatment
of Childhood IgAN
Recently, the Japanese Society for Pediatric
Nephrology has developed “Guidelines for the
treatment of childhood IgAN.” The disease sever-
ity has been divided into two categories, i.e., mild
[397] and severe [389, 408, 414, 413] IgAN. A
summary of the guidelines is shown in Table 5.
Clinical Manifestations and Laboratory
Features in HSP
Extrarenal Manifestations
The extrarenal manifestations of HSP are charac-
teristic and can occur in any order and at any time
over several days or weeks.
Skin
The skin lesion typically begins with erythema-
tous macules, some of which develop into slightly
raised (palpable), urticarial papules, which soon
become purpuric (Fig. 8). The eruption is of sym-
metric distribution and predominantly affects the
buttocks and the extensor surfaces of the lower
legs and forearms but spares the trunk. Purpura
occasionally affects the earlobes, nose, and
1012
Table 5 Summary of guidelines for the treatment of child-
hood IgA nephropathy (ver. 1.0, by the Japanese Society
for Pediatric Nephrology)
Mild cases
Definition: patients who meet both clinical findings AND
histological findings as follows
Clinical findings: slight proteinuria (early morning
urinary protein to creatinine ratio <1.0)
Histological findings: <80 % of glomeruli showing
moderate or severe mesangial cell proliferation, crescent
formation, adhesion, or sclerosis and <30 % of glomeruli
showing crescent formation
Treatments: Either lisinopril or Sairei-to should be given
for more than 2 years
Severe cases
Definition: patients who meet either clinical findings OR
histological findings
Clinical manifestations: heavy proteinuria (early
morning urinary protein to creatinine ratio 1.0)
Histological findings: 80 % of glomeruli showing
moderate or severe mesangial cell proliferation, crescent
formation, adhesion, or sclerosis or 30 % of glomeruli
showing crescent formation
Treatments: the combination therapy of prednisolone,
azathioprine or mizoribine, warfarin, and dipyridamole
should be given for 2 years
Fig. 8 Palpable purpura in a patient with HSP
external genitalia but in typical, mild cases is
confined to the ankles. Among older children,
purpura can be the sole cutaneous manifestation.
Among preschool children, urticaria can occur
without purpura, although localized edema of the
dorsal surfaces of the hands and feet, face, and
scalp can occur [16, 420]. As the primary lesions
fade, new crops of purpura commonly recur as
long as 3 months after onset and occasionally
much longer [26, 15, 23] and sometimes are
K. Nakanishi and N. Yoshikawa
associated with the recurrence of abdominal and
joint symptoms.
Joints
Approximately 70 % of children [26, 15, 420]
have joint involvement, and joint symptoms may
be the initial manifestation. Joint involvement
mainly affects the knees, ankles, elbows, and
wrists. This consists of arthralgia and periarticular
edema without overlying erythema, tenderness, or
joint effusions. It is transient and leaves no resid-
ual damage.
Gastrointestinal Tract
Gastrointestinal manifestations occur among
50–70 % of affected children [420], but the inci-
dence increases to more than 90 % among those
who have renal involvement [26, 15]. The most
common symptom is abdominal colic, which
often is severe and accompanied by vomiting,
although melena occurs in one half of cases
[420]. A mass occasionally is palpable in the
upper abdomen, suggesting intussusception.
When the abdominal symptoms precede the
other manifestations, laparotomy sometimes is
considered necessary [26]. Intussusception due
to severe purpura of the intestine is common
among older children [48]. Protein-losing enter-
opathy has been reported [421, 422] and may
account for the occasional observation of
hypoalbuminemic edema in the absence of
heavy proteinuria. Vasculitic lesions have been
found in many other organs and may explain
rare manifestations such as hemorrhage in the
calf, the testis, the lung, acute hemorrhagic pan-
creatitis or parotitis, neurologic manifestations,
and myocardial or muscle involvement [464].
Renal Manifestations
Renal manifestations occur at any time. The first
urinary abnormality usually is noticed after other
symptoms, but hematuria sometimes is the initial
32 Immunoglobulin A Nephropathies in Children (Includes HSP)
feature. Among 80 % of children with a urinary
abnormality, the first abnormality is detected
within 4 weeks of onset of the illness. In most of
the remainder, the urinary abnormality develops
within the next 8 weeks [15]. Recurrences of renal
manifestations are common and appear to be par-
ticularly so among patients with severe renal dam-
age. The relapses can occur at the time of an upper
respiratory tract infection. There are no correla-
tions between the severity of extrarenal manifes-
tations and the severity of renal symptoms.
Several studies have intended to identify pre-
dictive factors for developing of nephritis in child-
hood HSP [423–427]. These studies revealed that
an older age at onset, persistent purpura, severe
bowel angina, and relapse were identified as fac-
tors associated with the occurrence of nephritis.
A recent prospective study [428] clarified that the
risk factors for developing nephritis were age over
8 years at onset, abdominal pain, and recurrence
of HSP disease. This study also showed that
patients with two or three risk factors developed
nephritis in 63 % and 87 % of cases,
respectively [428].
Minimal renal involvement may cause no more
than transient microscopic hematuria, detectable
only with routine urinalysis during the acute ill-
ness. The illness also presents with initial macro-
scopic hematuria that usually lasts a few days but
can last several weeks followed by microscopic
hematuria that can persist for months or years.
Macroscopic hematuria may be accompanied by
transient heavy proteinuria. Persistence of heavy
proteinuria of more than 1 g/day per square meter
of body surface area with microscopic hematuria
usually is usually associated with significant his-
tologic lesions. In these cases, blood pressure and
renal function are normal. Impairment of renal
function is rare and occurs with acute nephritic
syndrome associated with combination
hypervolemia, oliguria, and hypertension [26].
Massive proteinuria can cause nephrotic syn-
drome (serum level of albumin less than 25 g/l)
with pitting edema of the face and ankles and
sometimes ascites. The most severe clinical pre-
sentation is mixed nephritic-nephrotic syndrome
in which hematuria, hypertension, and impair-
ment renal function are combined with proteinuria
1013
and hypoalbuminemic edema [26]. Such clinical
presentation is most often associated with severe
crescentic glomerulonephritis.
Laboratory Features
There are no specific laboratory findings of HSP.
Laboratory investigation needed is mainly
directed at assessment of the extent of renal
involvement. If purpura is absent but has been
suggested by the patient’s recent history, the
feces are tested for occult blood, the finding of
which supports a diagnosis of HSP. If there is any
doubt about the cause of the purpura, a full blood
cell count and coagulation screen is performed.
The level of clotting factor XIII, a fibrinstabilizing
factor, is markedly low among children with HSP.
Although the serum level IgA is high in a large
number of cases of HSP, measuring IgA for
individual patients does not contribute to
management.
In every case of HSP, the urine is routinely
tested for blood and protein at onset and at least
weekly until systemic signs have resolved. If uri-
nary abnormalities are found, proteinuria is quan-
tified, and serum levels of total protein and
albumin are measured. Serum creatinine level is
measured to estimate renal function.
Clinicopathologic Correlations
Renal biopsy rarely is needed for diagnosis, but it
is used to assess the severity of glomerulonephri-
tis. Biopsy is required only in the care of patients
with severe initial manifestations (nephritic,
nephrotic, or decreased renal function) or with
heavy proteinuria >1 g/day/m2 that persists lon-
ger than 1 month. The biopsy indication for with
slight proteinuria <1 g/day/m2 that persists longer
than 3 month may be considered. The more severe
the clinical presentation, the larger is the percent-
age of glomeruli affected by crescents and seg-
mental lesions. The findings [26, 27, 33, 35],
summarized in Table 6, show that (a) patients
with initial macroscopic or persistent microscopic
hematuria who do not have persistent heavy
1014 K. Nakanishi and N. Yoshikawa

Table 6 Clinicopathologic correlations in Henoch- Among older children and adults, the combi-
Schönlein nephritis nation of joint symptoms and purpura makes it
Approximate necessary to consider systemic lupus
Biopsy risk of renal erythematosus and microscopic polyangiitis as
Clinical presentation grade failure (%)
alternative diagnoses [23]. The light microscopic
Macroscopic or I–II, <5
microscopic hematuria, rarely findings of glomeruli in patients with systemic
proteinuria minimal or III lupus erythematosus and those with HSN are sim-
absent ilar and may be indistinguishable. In systemic
Hematuria and I–IV 15 lupus erythematosus, however, glomerular IgA
persistent heavy deposits, when present, are less prominent than
proteinuria
IgG deposits, and C1q deposits almost always are
Acute nephritic II–IV 15
syndrome present. Antinuclear antibodies usually are absent
Nephrotic syndrome II–IV, 40 from patients with HSN. The differential diagno-
rarely I sis between HSN and the microscopic polyangiitis
or V is difficult. Mesangial IgA deposits are absent in
Nephritic-nephrotic II–V, >50 polyarteritis nodosa. Therefore the diagnosis of
syndrome mostly
V the microscopic polyangiitis is considered when
From White and Yoshikawa [463]
any patient has clinical signs of HSP if mesangial
IgA deposits are not found [48].

proteinuria have glomerular lesions of grade III or

less; (b) those with nephritis or nephrosis at onset
of disease or persistent heavy proteinuria have a
10–20 % likelihood of having grade IV to V
lesions; (c) a mixed nephritic-nephrotic presenta-
tion carries a 60 % risk of development of grade
IV to V lesions; and (d) most grade I to III lesions
resolve, whereas an increasing proportion of
grade IV to V lesions do not.
Differential Diagnosis of HSP
The distribution of the rash and the accompanying
gastrointestinal and articular lesions usually is so
characteristic that the diagnosis is not in doubt.
Difficulty arises in rare instances when the skin
eruption consists entirely of urticarial lesions
without purpura. Although it is not feasible to
diagnose HSP in the absence of cutaneous mani-
festations, there is anecdotal evidence that it
occurs without skin lesions, albeit rarely [464].
Hypoalbuminemic edema sometimes is found in
the absence of heavy proteinuria and can be
caused by proteinlosing enteropathy [421]. Quan-
titative assessment of proteinuria therefore is
essential.
Treatment of HSP
Although corticosteroids enhance the rate of res-
olution of the arthritis and abdominal pain, their
effectiveness in the prevention of nephritis is
debated. Saulsburry in an uncontrolled study of
severe HSP cases concluded the corticosteroid
therapy does not prevent nephritis [429]. Con-
versely, Mollica et al. found that early prednisone
treatment reduced the incidence of microscopic
hematuria, but this symptom is not associated
with a high risk of progression [430]. Ronkainen
et al. performed a randomized control study and
concluded that prednisone does not prevent the
development of renal symptoms but is effective in
treating them [431]. Dudley et al. in a large ran-
domized, double-blind, placebo-controlled trial
found no evidence that early treatment with pred-
nisolone reduces the prevalence of proteinuria
12 months after disease onset [432]. Therefore,
the existing evidence does not support prednisone
in the prevention of nephritis.
The clinical spectrum of HSN ranges from the
relatively common transient isolated microscopic
hematuria to nephrotic syndrome, rapidly pro-
gressive glomerulonephritis, and renal failure.
32 Immunoglobulin A Nephropathies in Children (Includes HSP)
In mild cases, no treatment is needed if the
urine is adequately evaluated, and ambulatory
care may suffice. The prolonged bed rest custom-
ary in the past occasionally led to complications
such as femoral venous thrombosis and psycho-
social problems [26]. Although severe abdominal
pain and vomiting are self-limiting, resolution can
be hastened by means of administration of gluco-
corticoids [433]. Utani et al. [434] found that
factor XIII concentrate replacement resolved
severe abdominal symptoms in the care of adults
with HSP associated with a decreased level of
factor XIII activity.
There is no therapy of proven value for HSN,
although some regimens have been proposed and
tested, with controversial results. These include
(a) glucocorticoids and immunosuppressive drugs
and (b) plasma exchange to remove circulating
IgA immune complexes. Retrospective studies
suggested that oral glucocorticoids were of no
benefit [435, 26, 15, 33]. Similarly, oral glucocor-
ticoids with azathioprine, chlorambucil, or cyclo-
phosphamide do not seem to be beneficial [15, 26,
435, 436]. A beneficial effect of methylpredniso-
lone pulse therapy followed by oral prednisone
was reported in a prospective case series of severe
HSN in comparison with historical controls at the
same institution [437]. The beneficial effect of
methylprednisolone pulses in combination with
immunosuppressive agents such as cyclophos-
phamide or mizoribine has also been reported
[438, 439]. Cyclosporine A therapy has given
promising results in case series, although some
patients become cyclosporine dependent
[440–443]. Plasma exchange combined with glu-
cocorticoids and immunosuppressive drugs may
be of value in the care of patients with rapidly
progressive crescentic disease [444–447].
The use of angiotensin-converting enzyme
inhibitor or angiotensin receptor blockers may be
useful in patients with moderate HSN. Recently,
Ninchoji et al. [448] reported the results of retro-
spective study for the efficacy of treatment for
HSN. Renal biopsy was performed in patients
with nephrotic syndrome or persistent proteinuria
for more than 3 months, and patients were classi-
fied by treatment. Patients (n = 31) with moder-
ately severe HSN (histological grade I–III and
1015
serum albumin >2.5 g/dl) were treated with
angiotensin-converting enzyme inhibitors and/or
angiotensin receptor blockers. Patients (n = 19)
with HSN exceeding grade III or Alb 2.5 g/dl
received combination therapy comprising pred-
nisolone, immunosuppressants, warfarin, and
dipyridamole. All patients showed resolution of
proteinuria without renal dysfunction during the
observation period (3.8  0.4 years). Their find-
ings support those of some earlier reports that
treatment strategies for HSN should depend on
the histologic and clinical severity. Furthermore,
aggressive therapies, particularly combination
therapies, are unnecessary for moderate to
severe HSN.
A recent meta-analyses [449, 450] has evaluated
the benefits and harms of different agents (used
singularly or in combination) compared with pla-
cebo or no treatment or another agent for the pre-
vention or treatment of kidney disease in patients
with HSP. It has revealed that data from randomized
controlled trials for any intervention used in improve
kidney outcomes in children with HSP are very
sparse except for short-term prednisone and that
there was no evidence of benefit of prednisone in
preventing serious long-term kidney disease in HSP.
The Kidney Disease Improving Global Out-
come (KDIGO) initiative published guidelines for
the treatment of HSN [465]. Due to the lack of
evidence-based data for HSN treatment and simi-
larities between HSN and primary IgAN, the
KDIGO guidelines proposed the same treatment
for both diseases when the clinical conditions are
similar. Although the KDIGO guidelines have been
introduced, clinicians following the KDIGO guide-
lines on the treatment of HSN may face the risk of
delaying the initiation of effective treatment and
increasing the risk of CKD over the long term.
Consequently, it should be expected that pediatric
nephrologists would be reluctant to follow those
guidelines for fear of undertreatment [451, 452].
Prognosis
The long-term morbidity and mortality of HSP are
almost exclusively attributable to renal disease.
Several centers have reported the correlation of
1016
Table 7 Correlations between biopsy grade and outcome in Henoch-Schönlein nephritis
ISKDC biopsy Total number of
grade patients Normal urine
I 22 14
II 22 15
III 53 38
IV 12 5 3
V 12 0 2
Total 121 72
All values are numbers of patients
both clinical presentation and renal morphology
with outcome [15, 26, 32, 33, 35, 289,
453–457]. We found 14 (11 %) of 122 children
with renal involvement [27] to have chronic renal
failure or to have died as a result. A further seven
patients (6 %) had active disease. The overall
prognosis of HSP is better appreciated with results
from two large series in which the conditions of
patients with or without nephritis were followed;
the estimated incidences of end-stage renal failure
were 2 % [36] and 5 % [32].
The results of long-term follow-up studies
show that the clinical presentation has no prog-
nostic value for individual patients. It is clear,
however, that children who come to medical
attention with slight proteinuria or hematuria
have an excellent prognosis and that patients
with persistent heavy proteinuria, acute nephritic
syndrome, or nephrotic syndrome are at risk of
chronic renal failure. Results of a 23-year follow-
up study by Goldstein et al. [35] indicated that
approximately 15 % of patients with nephritis at
onset of HSP or persistent heavy proteinuria, 40 %
of those with nephrosis, and fewer than 50 % of
those with a mixed nephritic-nephrotic onset have
ongoing urinary abnormalities and that many of
these patients ultimately have renal failure
(Table 6). As noteworthy facts, it has been
reported that some children with mild renal symp-
toms have poor long-term outcome [458–460].
The proportion of glomeruli with crescents or
segmental lesions (sclerosis, adhesions, thrombo-
sis, or necrosis) seems to be the most important
prognostic indicator. The larger the number of
glomeruli affected by crescents or segmental
lesions, the poorer is the prognosis. The relation
K. Nakanishi and N. Yoshikawa
Slight proteinuria, Heavy Renal
hematuria, or both proteinuria failure
8 0 0
6 1 0
10 4 1
1 3
1 9
29 7 13
between histologic grade and outcome among our
121 children with HSN who underwent biopsy
and whose conditions were followed for at least
2 years is summarized in Table 7. The table shows
a progressively worsening outcome with increas-
ing histologic severity, as judged by the percent-
age of patients in each grade with either active
disease or chronic renal failure: 0 % for grade I,
4 % for grade II, 9 % for grade III, 33 % for grade
IV, and 83 % for grade V. Of the 72 patients who
completely recovered, 67 had fewer than 50 % of
their glomeruli affected by crescents or segmental
lesions. In contrast, among the 13 patients with
chronic renal failure, 12 had more than 50 % of
glomeruli affected by crescents or segmental
lesions.
Goldstein et al. [35] observed that of 78 chil-
dren followed for 23 years, 17 (22 %) had deteri-
orated clinically. Included in this group were
seven children who had apparently completely
recovered at 10 years. Furthermore, 36 % of
44 pregnancies were complicated by hypertension
and/or persistent proteinuria. The late develop-
ment of hypertension and proteinuria after a
period of normality and the proteinuria of preg-
nancy, together with the progressive decline in
renal function that can follow initial improve-
ment, are consistent with a sequence of glomeru-
lar hyperfiltration followed by secondary
glomerulosclerosis [461] in which glomerular
destruction due to the original disease has been
extensive. Because most patients found by Gold-
stein et al. [35] to have residual abnormalities had
no symptoms, it follows that children who have
HSN with a severe clinical onset or biopsy grade
need almost indefinite follow-up care.
32 Immunoglobulin A Nephropathies in Children (Includes HSP)
Recurrence in Renal Allograft
In HSN, clinical recurrence in renal allografts is
rare, but recurrence of the mesangial IgA deposits
is frequent [129–131, 462]. Recurrent disease
appears to be strongly associated with living-
related donor transplantation. The significance is
unclear, but the association of disease recurrence
with living-related donor transplantation may
imply a genetic predisposition. In a report by
Hasegawa et al. [131], there were 9 histologic
recurrences among 12 living-related donor grafts,
5 of them associated with clinical manifestations.
In contrast, clinical recurrence was not observed
in 5 cadaveric grafts, and 4 of these had no IgA
deposits at biopsy. Habib et al. [48] found asymp-
tomatic IgA deposits in 11 of the 13 cadaveric
grafts, but only 1 of 13 patients had a clinical
recurrence.
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 Membranoproliferative and
C3-Mediated GN in Children 33
Christoph Licht, Magdalena Riedl, Matthew C. Pickering,
and Michael Braun

Contents Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1045

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1036
Histopathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1037
Complement System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1037
Lessons Learned from Animal Models . . . . . . . . . . 1040
Differential Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1045
Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1046
Supportive Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1046
Immunosuppressive Therapy . . . . . . . . . . . . . . . . . . . . . . . 1046
Complement-Targeting Therapy . . . . . . . . . . . . . . . . . . . 1047

Lessons Learned from Patients . . . . . . . . . . . . . . . . . . 1041 Clinical Outcomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1047

Autoimmune Forms of C3G . . . . . . . . . . . . . . . . . . . . . . . . 1041 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1048
Genetic Forms of C3G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1042
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1049
Clinical Manifestation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1043

C. Licht (*)
Division of Nephrology, The Hospital for Sick Children,
University of Toronto, Toronto, ON, Canada
Research Institute, Cell Biology Program, The Hospital for
Sick Children, Toronto, ON, Canada
Department of Paediatrics, University of Toronto, Toronto,
ON, Canada
e-mail: christoph.licht@sickkids.ca
M. Riedl
Research Institute, Cell Biology Program, The Hospital for
Sick Children, Toronto, ON, Canada
Department of Paediatrics, Innsbruck Medical University,
Innsbruck, Tyrol, Austria
M.C. Pickering
Centre for Complement and Inflammation Research,
Imperial College, London, UK
M. Braun
Renal Section, Department of Pediatrics, Texas Children’s
Hospital, Balyor College of Medicine, Houston, TX, USA

# Springer-Verlag Berlin Heidelberg 2016 1035

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_29
1036 C. Licht et al.

and capillary wall remodeling with formation of

List of Abbreviations
double contours. This injury pattern is a result of
aHUS Atypical hemolytic uremic
deposition of immunoglobulins (Ig)/immune
syndrome
complexes (IC) and/or complement proteins in
AMD Age-related macular degeneration
the mesangium and/or along the capillary wall of
AP Alternative pathway
the glomerulus [1, 2]. The increasing understand-
(of complement)
ing of the underlying pathophysiology has
aPL Acquired partial lipodystrophy
recently resulted in a reclassification of MPGN
C Complement
into (i) a primary complement-mediated C3
C3G C3 glomerulopathy
glomerulopathy (C3G) with predominant C3
C3GN C3 glomerulonephritis
deposition and (ii) a secondary Ig-/IC-mediated
C3NeF C3 nephritic factor
glomerulonephritis with mainly IgG staining on
CFB Complement factor B
immunohistochemistry [2, 3]. C3 glomerulopathy
CFH Complement factor H
enables the inclusion of cases with C3 deposition
CFHR1-5 Complement factor H-related
without a membranoproliferative pattern on light
protein 1–5
microscopy [3] and encompasses all glomerular
CFI Complement factor I
lesions in which there is predominant C3 staining
CFP Properdin
as defined as an immunohistochemistry C3c
CP Classical pathway
intensity 2 orders of magnitude more than any
(of complement)
other immune reactant (e.g., IgG/IgA, C1q) using
DDD Dense deposit disease
a scale ranging from 0 to 3 [3]. MPGN, in distinc-
DEAP-HUS Deficiency of CFHR plasma
tion, may result from Ig/IC deposition in the kid-
proteins and CFH autoantibody
ney and may be either idiopathic or secondary to
positive HUS
infections, autoimmune diseases, malignancies,
ESRD End-stage renal disease
or monoclonal gammopathy (Table 1) [4–6].
FFP Fresh frozen plasma
C3G is considered a primary complement disease,
GN Glomerulonephritis
where complement deposition is a result of defec-
IC Immune complexes
tive control of the complement AP [7, 8]. Of note,
Ig Immunoglobulins
complement activation – primarily of the classical
IVCP Intravenous cyclophosphamide
pathway (CP) – also occurs in Ig-/IC-mediated
LP Lectin pathway
glomerulonephritis [9]. The diagnosis of C3
(of complement)
glomerulopathy is based on the result of light
MCP Membrane cofactor protein
and electron microscopy including immunohisto-
MPGN Membranoproliferative
chemistry [3]. Treatment approaches include
glomerulonephritis
immunosuppressant therapy and treatment of the
NeF Nephritic factor
underlying condition in secondary MPGN and
PE Plasma exchange
targeting the complement system for primary
PI Plasma infusion
C3G. Some patients may even recover spontane-
PIGN Postinfectious glomerulonephritis
ously without specific treatment. Evidence for
SCR Short consensus repeat
treatment-specific benefits is still lacking, and
TMA Thrombotic microangiopathy
formal treatment trials will be required to establish
treatment guidelines and define long-term treat-
ment benefits.
Introduction While focusing on C3G, references used
in this chapter are mainly based on the
Membranoproliferative glomerulonephritis (MPGN) traditional nomenclature but are interpreted as
describes a histopathological pattern character- best as possible in light of the new consensus
ized by mesangial and endocapillary proliferation guidelines.
33 Membranoproliferative and C3-Mediated GN in Children
Table 1 Causes of secondary MPGN
Infectious diseases: bacterial/viral/protozoal
Hepatitis B, C, EBV, HIV
Endocarditis/visceral abcesses
Infected ventriculoatrial shunts/empyema
Malaria, schistosomiasis, mycoplasma
Tuberculosis, leprosy
Epstein-Barr virus infection
Brucellosis
Systemic immune diseases
Cryoglobulinemia
Systemic lupus erythematosus
Sjögren’s syndrome
Rheumatoid arthritis
Hereditary deficiencies of complement components
X-linked agammaglobulinemia
Neoplasms/dysproteinemias
Plasma cell dyscrasia
Fibrillary and immunotactoid glomerulonephritis
Light-chain deposition disease
Heavy-chain deposition disease
Light- and heavy-chain deposition disease
Leukemias and lymphomas (with cryoglobulinemia)
Waldenstrom macroglobulinemia
Carcinomas, Wilms’ tumor, malignant melanoma
Chronic liver disease
Chronic active hepatitis (B, C)
Cirrhosis
Alpha-1-antitrypsin deficiency
Miscellaneous
Thrombotic microangiopathy
Sickle-cell disease
Partial lipodystrophy
Transplant glomerulopathy
Niemann-Pick disease (Type C)
Histopathology
C3G presently includes two entities: C3 glomer-
ulonephritis (C3GN e.g., CFHR5 nephropathy)
and dense deposit disease (DDD), former classi-
fied as MPGN Type II (Fig. 1) [3]. On light
microscopy, C3GN and DDD have an
overlapping spectrum of features including
membranoproliferative glomerulonephritis, dif-
fuse proliferative glomerulonephritis, mesangial
1037
proliferative glomerulonephritis, or even a necro-
tizing and crescentic glomerulonephritis [3, 10].
Electron microscopy (EM) is needed to differen-
tiate C3GN and DDD. In C3GN, discrete C3
deposits are located in the mesangium and along
the capillary walls, whereas in DDD, C3 deposits
are more intense in the mesangium and within the
glomerular basement membrane (GBM) forming
a unique ribbon-shaped band [3, 11].
Mesangial C3 with at least codominant IgG
deposition compared to C3G with mesangial,
subendothelial, and subepithelial humps by EM
are characteristic for postinfectious GN (PIGN), a
major differential diagnosis of C3G [3]. Some
patients showed only the combination of C3 depo-
sition without IgG and humps. As clinical signs
such as low C3, hematuria/proteinuria, or deterio-
ration of renal function were persistent and defects
in the AP were detected, these patients received the
diagnosis of “atypical” PIGN, very likely being
patients with C3G [12]. As infections – including
streptococcal – are a common trigger for C3G and
humps also occur in biopsies of patients with C3G,
the differential diagnosis is difficult and mostly
relies on clinical observation.
Complement System
The complement system (Fig. 2) is an integral part
of the innate immune system for antimicrobial
defense and immune complex clearing
[13–15]. However, defective regulation or
overactivation caused by mutations and autoanti-
bodies has been linked with a variety of renal
diseases, including thrombotic microangiopathy
(TMA) especially atypical hemolytic uremic syn-
drome (aHUS), systemic lupus erythematosus
(SLE), antibody-mediated rejection (AMR),
ANCA-associated vasculitis (AAV), and membra-
nous nephropathy (MN) [16, 17]. The classical
pathway (CP) is activated by immunoglobulins
or immune complexes and thus especially
involved in autoimmune diseases and the Ig-/IC-
mediated form of MPGN. The lectin pathway
(LP) is activated by repetitive carbohydrate
1038
Fig. 1 Histological features of C3G. Panel highlighting the
microscopic features of MPGN Type I (a–c), C3G (d–f) and
DDD (g–i) by light microscopy (top row), immunofluores-
cence (middle row) and electron microscopy (bottom row).
(a–c) Typical MPGN results in a cellular glomerulus with
expanded mesangial regions resulting from increased matrix
and cellularity, as well as immune complex deposition. Cap-
illary loops are thickened with narrowed lumina from
enlarged endothelial cells, mesangial interposition, and
immune complex deposition. There is strong staining for
both IgG and C3 by immunofluorescence (only IgG is
shown). By electron microscopy, capillary loops show prom-
inent subendothelial deposits and mesangial interposition.
(d–f) C3G can have a wide spectrum of appearances ranging
from mesangial proliferative, membranoproliferate, to a nec-
rotizing crescentic glomerulonephritis (illustrated here).
C3 deposition predominates by immunofluorescence. By
electron microscopy, immune complex deposition may be
C. Licht et al.
scanty (as illustrated here) but can include large subepithelial
and/or subendothelial deposits, with or without mesangial
interposition (also seen here). (g–i) DDD imparts a uniform
ribbonlike appearance to the glomerular basement mem-
brane, highlighted here by PAS staining. Mesangial regions
show increased matrix and cellularity, but mesangial interpo-
sition along capillary loops is an inconsistent feature. By
immunofluorescence, C3 deposition can be detected,
although usually less than seen in C3G. Electron microscopy
is diagnostic with the presence of an intramembranous
electron-dense alteration to the basement membranes along
capillary loops and sometimes in mesangial regions. Early in
the disease, this change involved loops partially but becomes
confluent as the disease progresses. (a, d, f) PAS staining,
original magnification 400, (b) IgG, original magnification
400, (e) and (h) and B3: C3, original magnification 400,
(c, f, i) original magnification 10,000)
33 Membranoproliferative and C3-Mediated GN in Children 1039

Fig. 2 (a) Activation and a C3(H2O)

regulation of the complement
alternative pathway (AP): CFH
Physiological balance DAF
between spontaneous C3(H2O)Bb
activation and regulation of AP initation C3 convertase
the AP. Mutations in CFH
and C3 or autoantibodies to C3bBb
CFH, C3bBb (i.e., C3NeF) C3 C3b AP amplification
or CFB cause excessive C3 convertase
fluid-phase C3 activation CFH
resulting in the glomerular CFI
accumulation of C3 MCP
split products giving rise C3bBbC3b
iC3b C5 convertase
to the specific
immunofluorescence
patterns found in DDD and
C3GN. (b) Complement C3d, C3e, C3f, C3g C5b-9
dysregulation in C3G:
Mutations and
autoantibodies (red) b C3(H2O)
involved in C3G
CFH
DAF
C3(H2O)Bb C3NeF
AP initation C3 convertase CFB
C3b
C3bBb
C3 C3b AP amplification
CFH C3 convertase
(mut. + abs.)
CFI
MCP C3bBbC3b
iC3b
C5 convertase

Deposition of Injury / cell death

C3 split products (C5b-9)
(C3d, C3e, C3f, C3g)

structures found, e.g., on the surface of bacteria
[13–15]. Different from the CP and LP, the AP is
constitutively active and requires tight (negative)
regulation to maintain the balance between phys-
iological activation of complement when and
where needed and to prevent harmful
overactivation. Initiation of the CP and LP results
in the activation of C3 to C3b, which – in the
AP – occurs spontaneously via a process called
“tick over.” Together with complement factor B
(CFB) – activated by complement factor D
(CFD) – and properdin (CFP), C3b forms the AP
C3 convertase (C3bBb), which drastically
enhances C3 activation (“amplification”). C3b is
deposited on target surfaces (“opsonization”) and
allows for the formation of the C5 convertase
(C3bBbC3b), activation of C5, and induction of
the terminal complement cascade (TCC), ulti-
mately resulting in the formation of the membrane
attack complex (MAC) C5b-9 (Fig. 2a) [13–15].
A number of soluble and membrane-anchored
regulators limit complement activation via inacti-
vation of C3b or acceleration of the physiological
decay of the C3 convertase, C3bBb. The most
important complement regulator, fluid-phase
complement factor H (CFH), provides comple-
ment regulation via three different mechanisms:
it limits C3b binding to the endothelial surface and
1040
formation of the C3 convertase by competition,
C3b cleavage (cofactor activity), and acceleration
of the natural decay of the C3 convertase (decay-
accelerating activity) (Fig. 2a).
While the C-terminal region of CFH is respon-
sible for surface recognition and binding to C3b, the
N-terminal region of CFH provides cofactor and
decay-accelerating function [18]. It is not surprising
that aHUS – a disease characterized by uncontrolled
complement activation on endothelial cells – is
associated with mutations in the C-terminal region
of CFH, while in C3G – a disease associated with
enhanced C3 conversion – mutations are rather
detected in the N-terminal region [19].
Besides CFH, there are five proteins
sharing sequence and structural homology with
CFH – the complement factor H-related proteins
(CFHR) 1–5. CFHR 1 contributes to complement
regulation on the level of the C5 convertase [20].
CFHR1, CFHR2, and CFHR5 share a dimeriza-
tion motif, which allows them to form dimers,
which can antagonize CFH at physiological con-
centrations [21]. By contrast, recent studies have
shown that CFHR1 [22], CFHR5 [21], and
CFHR4 [23] unlike CFH do not have the ability
to negatively regulate C3 at physiological concen-
trations. In fact, by competing for surface ligands
with CFH, they can prevent the ability of CFH to
negatively regulate C3 activation. This process
has been termed CFH deregulation [21].
In addition, cellular surfaces are protected via
complement receptor 1 (CR1) and membrane
cofactor protein (MCP; CD46), both acting as
cofactors to CFI; via decay-accelerating factor
(DAF; CD55); and via CD59 which prevents
assembly of the C5b-9 complex (Fig. 2a) [13–15].
C3 inactivation results in the formation of
iC3b, C3dg, and C3d, split products which can
be used to detect complement activation but are
also of biological relevance, as they interact with
the adaptive immune system (Fig. 2a) [24].
Lessons Learned from Animal Models
A lesion consistent with MPGN type 2 developed
spontaneously in pigs with complete genetic defi-
ciency of CFH [25]. Notably, administration of
C. Licht et al.
CFH (through either plasma or purified protein)
delayed disease progression [26, 27]. Similarly,
C3G developed spontaneously in mice with com-
plete CFH deficiency (cfh/), generated
through gene targeting [25]. In both the
CFH-deficient pig and mouse models, C3 accu-
mulates along the GBM in the setting of systemic
hypocomplementemia. In both animal models,
immune electron microscopy identified comple-
ment components (i.e., C3, C5, C5b-9) within
the electron-dense deposits [25, 26]. An essential
role for the AP was demonstrated by the observa-
tion that cfh/ mice did not develop renal dis-
ease if they were also genetically deficient in the
AP activation protein, factor B [25]. The develop-
ment of C3 along the GBM in complete CFH
deficiency is dependent of CFI since cfh/
mice lacking CFI (cfh/; cfi/) developed
mesangial and not GBM-associated C3
deposition [28].
Importantly, the phenotype associated with
complete CFH deficiency in humans included
not only C3 glomerulopathy but also atypical
hemolytic uremic syndrome (aHUS) indicating
that defects in the complement AP can give rise
to at least two distinct renal pathologies. The
majority of aHUS-associated CFH mutations do
not result in complete deficiency of CFH but
selectively impair the surface recognition
domains of the protein. These mutations were
modeled experimentally by creating a
CFH-deficient mouse strain that expressed
transgenically a mutant CFH protein that func-
tionally mimicked the human aHUS-associated
CFH mutations. The mutant protein lacked the
terminal 5 surface recognition domains (denoted
CFHΔ16-20). When this protein was expressed in
CFH-deficient mice (cfh/. CFHΔ16-20), sys-
temic hypocomplementemia was ameliorated
since the mutant protein had intact complement
regulatory domains. However, the animals spon-
taneously developed aHUS and not C3G, and the
aHUS phenotype was dependent on C5 activation
[29, 30]. The conclusions of these studies were
that for aHUS to develop, defective complement
regulation along the renal endothelium was
required but so too was an intact (or partially
intact) complement system that enabled sufficient
33 Membranoproliferative and C3-Mediated GN in Children
C3 and C5 to be available to mediate the
complement-induced endothelial damage. In the
cfh/. CFHΔ16-20 animals both were present.
In contrast in the cfh/ animals, while there
was clearly no CFH-mediated regulation of
complement along endothelium since the mice
lacked CFH, the deficiency also resulted in
secondary depletion of C3 and C5. The auto-
depletion of these components, and consequent
lack of an intact complement system, meant
that these mice were protected from the develop-
ment of spontaneous aHUS. Conversely, the
observation that cfh/. CFHΔ16-20 animals
did not develop C3G suggested that the
systemic hypocomplementemia due to
complete fluid-phase dysregulation of the com-
plement AP was driving the C3G seen in the
cfh/ mice. There are examples of human
C3G where there is and is not systemic
hypocomplementemia, so fluid-phase comple-
ment AP dysregulation is relevant for some but
not all C3G.
Lessons Learned from Patients
There has been a long recognized role for com-
plement in patients with MPGN, as low C3 is a
hallmark feature of the clinical presentation.
Mutations or autoantibodies result in the loss of
control of AP C3 convertase via one or more of
the following scenarios (Fig. 2b):
– Antibodies stabilizing the AP C3
convertase thus prolonging the natural decay
of the AP C3 convertase and rendering it
overactive
– Mutations or antibodies to CFH that result in
the absence or loss of function of CFH
resulting in the loss of control of the AP C3
convertase and its enhanced activity
– Mutations in C3 or CFB that result in an
exceedingly stable C3 convertase with
prolonged decay and enhanced function
In all scenarios, the functional consequence is
an enhanced activation rate of C3. Of note
1041
enhanced levels of TCC activation were also
found in C3G patients [19].
The pathogenetic role of complement was
further supported by recent work by Sethi
et al., who was able to confirm the occurrence
of proteins of the alternative and terminal path-
way in the glomerulus of patients with C3G
using a combination of laser microdissection
and mass spectrometry [7, 8]. Of note, since
CFB was not detected, the authors concluded
that AP activation occurred in the fluid phase
but not locally.
Autoimmune Forms of C3G
In 1965, the first association of low C3 and
MPGN was reported, followed by the hypothesis,
that a factor in serum of patients with GN leads to
increased cleavage of C3 [31]. Subsequently, an
antibody binding and stabilizing the AP C3
convertase (C3bBb), thus enhancing C3 activa-
tion and C3b generation, was detected and
named C3 nephritic factor (C3NeF) [32]. C3NeF
was detected in 86 % of DDD and 46 % of C3GN
patients, respectively, and is associated with
decreased C3 levels [19]. C3NeF levels can be
fluctuant during the clinical course of a patient
and do not necessarily reflect disease activity or
treatment status [33]. Of note, in 1 case, C3NeF
was reported to disappear after renal
transplantation [34].
As C3NeF are heterogeneous, reliable detec-
tion is challenging. Current assays involve mea-
surement of IgG binding to the C3 convertase or
the detection of the ability to stabilize the AP C3
convertase [35]. The latter was reported as
the (currently) most sensitive test [36]. Efforts
creating an international standard for the detection
of C3NeF are currently under way (Michael
Kirschfink – personal communication). C3NeF
was also reported in other renal diseases, such
as SLE [37–39], PIGN [12], patients with
meningococcal meningitis [40], and healthy
individuals [35]. The finding of C3NeF does
not exclude the coexistence of complement
mutations, and despite C3NeF positivity,
1042 C. Licht et al.

Table 2 Diagnostic workup for C3G
Global complement CH50, APH50
function
Specific complement C3, C4, C3d
activation
Terminal pathway SC5b-9
activation
Complement protein CFH, CFI, CFB
levels
Autoantibody screening C3 Nephritic Factor
CFH/CFB/C3b
Autoantibodies
Mutation screening CFH, CFI, MCP/CD46, C3
CFHR1-5 (MLPA)
Adapted from Pickering et al., Kidney Int 2013 (Ref. [3])
MLPA multiplex ligation-dependent probe amplification
Italic: rare conditions
The authors acknowledge that not all tests may be available
to all clinicians and that local/national clinical practice
recommendations for the diagnostic workup of C3G
may apply
comprehensive complement diagnostics (Table 2)
should be performed [41].
Other autoantibodies including anti-CFB and
anti-C3b antibodies have been reported, all of
which result in the loss of AP C3 convertase
control [42]. Strobel et al. reported in a patient
with DDD an antibody binding to factors B and
Bb, which decreased terminal pathway activation
by inhibiting the C5 convertase [43]. Factor H
antibodies, frequently associated with aHUS
[44], are reported but rare in MPGN. A CFH
mini-autoantibody (i.e., antibody fragment-like λ
light-chain dimer) was reported to block C3b
binding of CFH thus abolishing CFH regulatory
capacity [45, 46].
Genetic Forms of C3G
In one series of 134 patients in which the CFH,
CFI, and CD46 genes were screened, mutations
were detected in 8/48 (16.6 %), 5/29 (17.2 %), and
11/56 (19.6 %) patients with MPGN 1, C3G, and
DDD, respectively [19]. Note that this
indicated that in the majority of patients with
MPGN 1, C3G, and DDD, it is not
possible to detect a mutation in CFH, CFI, or
CD46. To date, complement mutations have
been reported in the following complement
genes: CFH, CFHR5, CFI, MCP/CD46, C3, and
CFB [19, 21, 22, 47–59]. Additional mutations or
internal duplications in complement factor
H-related proteins (CFHRs), or the formation of
hybrid genes, were associated with familial C3G
[60, 61]. Risk haplotypes were identified in CFH,
C3, and MCP/CD46, with the CFH Y402H hap-
lotype more frequently reported in DDD and the
MCP -652A4G polymorphism in C3G and
MPGN1 [19, 47]. The presence of two or more
complement haplotypes significantly increased
the risk of disease manifestation [19, 47].
Table 3 gives an overview of mutations, rare
variants, and polymorphisms associated with
C3G. Interestingly, several mutations have
already been described in patients with aHUS
[19]. Compared to aHUS, mutations in CFH asso-
ciated with C3G are preferentially located in the
N-terminal region of CFH or affect Cys
residues throughout the CFH protein. In either
case, these mutations result in the loss of function
of CFH complement regulatory activity similar to
a CFH null genotype [19, 55, 62]. Deletions of
CFHR3-CFHR1, unlike in (DEAP-) aHUS
patients, have not been associated with CFH
antibodies in patients with C3GN [42]. Most CFI
mutations associated with MPGN were
already reported in patients with aHUS, and the
functional consequences in fluid phase are not
clear [19].
In 2009, Gale et al. described in 26 of 84 people
of Cypriotic ancestry with unexplained renal dis-
ease, a mutation in CFHR5 comprising a duplica-
tion in the dimerization domain of CFHR5
[51]. Several more mutations and hybrid variants
in CFHR genes have been detected and are asso-
ciated with C3G (Table 3). The role of CFHRs in
C3G was unclear until Goicoechea de Jorge
et al. reported that CFHR1, CFHR2, and CFHR5
form homo- and heterodimers among themselves
[21]. In the dimer form, these proteins are able to
compete with CFH for C3b binding and protect
C3b from inactivation and the AP C3 convertase
from its decay. This process, termed deregulation,
33 Membranoproliferative and C3-Mediated GN in Children 1043

Table 3 Overview of mutations, rare variants, common normal variants (CNV), and polymorphism associated with C3G
Gene/ Pheno-
protein Mutation/variant Function type References
CFH Mutations • Intact surface binding DDD/ Levy et al. [57], Vogt et al. [54], Ault
• Homo-/compound • Reduced C3b binding C3G et al. [48], Dragon-Durey et al. [50],
heterozygous • Loss of CFH cofactor and Licht et al. [55], Habbig et al. [52]
• SCRs 1–4 decay-accelerating
(regulatory activity
domain)
• Cys residues
(tertiary structure)
CFH Polymorphisms • Impaired C3b/heparin DDD Hageman et al. [53], Abrera-Abeleda
• Y402H (SCR 7) binding et al. [47], Abrera-Abeleda et al. [58]
• Impaired CFH cofactor
activity
CFI Type I and II • Decreased activity on cell DDD/ Servais et al. [19]
surface C3GN
MCP/ Rare SNP • Defective control on cell C3GN Servais et al. [19]
CD46 surface
CFHR1 Internal duplication • Alters FHR DDD Tortajada et al. [22]
oligomerization
• Greater degree of CFHR-
mediated deregulation
CFHR3-1 CNV • Greater degree of CFHR- C3G Malik et al. [61], Goicoechea de Jorge
• CFHR3-1 hybrid mediated deregulation et al. [21]
gene
CFHR5 CNV • Greater degree of CFHR- C3G Gale et al. [51], Goicoechea de Jorge
• Duplication in mediated deregulation et al. [21]
CFHR5 exons 2/3
CFHR5 Polymorphisms • Not tested DDD Abrera-Abeleda et al. [47], Abrera-
Abeleda et al. [58]
CFHR2-5 CFHR2-CFHR5 • Stabilizes C3 convertase, DDD Chen et al. [49]
hybrid gene reduced CFH-mediated
decay
C3 Mutations • C3mut – resistant to DDD Martinez-Barricarte et al. [56]
• Heterozygous cleavage by C3bBb
deletion • 3mut convertase – resistant
to CFH inactivation,
normal MCP/CD46
regulation
C3 Polymorphisms • Not tested DDD Smith et al. [59], Abrera-Abeleda
et al. [47]

was reported to be increased in patients with a
CFHR1-3 hybrid genes or CFHR5 mutations
[21, 51, 61].
Clinical Manifestation
MGPN, C3GN, and DDD are rare diseases with
an individual annual incidence estimated at 1–2
per million per total population (both pediatric
and adult) [63, 64]. Once thought to be primarily
pediatric diseases, it is clear that they also com-
monly present in the adult population [19].
The recent French series noted that only 39 % of
patients were less than 16 years at the time of
diagnosis, with the mean age at diagnosis for
MPGN, DDD, and C3GN reported to be 20.7
years  16.8, 18.9 years  17.7, and 30.3 years
 19.3, respectively [19]. Estimates range from
43 % to 58 % of patients with DDD and 25–54 %
1044 C. Licht et al.

of patients with C3GN being under the age of
16 at diagnosis. Within the pediatric population,
DDD tends to present at a younger age with 70 %
of patients diagnosed prior to age 15 [65]. With
respect to gender, there may be a slight bias with
males predominating in MPGN, DDD, and C3GN
[19, 63]. As the large case series that discriminate
MPGN, DDD, and C3GN are predominately from
Northern European populations, it is not clear at
present if there are ethnic or racial differences in
disease frequency.
The clinical presentations of MPGN, DDD,
and C3GN are nonspecific and require a high
index of suspicion. There is significant overlap
with regard to clinical features at presentation.
Depending on the case series, nephrotic syndrome
has been reported as the presenting feature in
40–65 % of patients with MPGN, 30–40 % with
DDD, and 25–40 % with C3GN. Regardless of
disease type, microscopic hematuria has been
reported in 40–75 % of patients, gross hematuria
in 10–20 %, and non-nephrotic proteinuria in
30–40 %. Presentations with acute glomerulone-
phritis are common in MPGN, DDD, and C3GN,
with hypertension noted in 30–60 % of patients
and renal insufficiency in 20–50 % of patients [19,
63, 66, 67]. Although rare, rapidly progressive
glomerulonephritis has been reported in MPGN,
DDD, and C3GN (Table 4).
As the classification system for MPGN has
recently evolved, our understanding of the differ-
ences in the clinical presentations of MPGN
Table 4 Clinical manifestation and outcome of DDD
and C3G
DDD C3G
Pediatric onset (<16 years) 43–58 % 25–54 %
Mean age at onset (years) 19  18 30  19
Clinical presentation
Nephrotic syndrome 38–43 % 27–44 %
Microhematuria 76 % 65 %
Arterial hypertension 21–60 % 40 %
Impaired renal function (>1.5 29 % 50 %
mg/dL creatinine)
Low C3 (<75 mg/dL) 59–79 % 40–48 %
Long-term outcome
Duration to ESRD (years) 10  11 11  10
Adapted from Refs. [19, 63]
versus C3GN is limited by the small number of
case series that have been reported. Doubtless,
there will be refinements regarding variations in
presentation; however, it is unlikely that there will
be unique features that clearly distinguish MPGN,
DDD, and C3GN clinically. The exception to this
appears to CFHR5-associated C3GN which pre-
sents in childhood with persistent microscopic
hematuria, synpharyngitic gross hematuria, and a
strong family history of ESRD. At present, this
form of G3GN has been reported primarily in the
Cypriot population but also in two patients with
no cypriotic heritage [51, 68–70].
Historically hypocomplementemia has been
regarding as a distinguishing feature of MPGN I
(low C3 and low C4) and other types of MPGN
(low C3 and normal C4) [71]. In the current clas-
sification system, MPGN is regarded as a form of
immune complex disease, and depressed C4
levels are commonly held as distinguishing fea-
ture; C3GN is thought primarily to be due to
dysregulation of the complement alternative path-
way, and therefore C4 levels are presumed to be
normal. Recent retrospective case series compar-
ing complement levels in patients with MGPN,
DDD, and C3GN generally confirm the utility of
C3 and C4 levels in distinguishing these disease
types [19, 63]. There were, however, important
caveats noted in these studies. In the French series
(115 patients), at diagnosis, only 46 % of patient
with MPGN had C3 levels less than 66 mg/dl and
only 2.4 % had C4 levels less than 9.3 mg/dl; low
C3 levels were noted in 59 % of DDD patients and
39.6 % of C3GN patients, with 1 DDD patient
noted to have a low C4 level and none of the
C3GN patients [19]. The series from England
(80 patients) reported low C3 levels, <65 mg/dl,
in 79 % of DDD and 48 % of C3GN patients, with
suppressed C4 levels, <16 mg/dl, in 15 % of
DDD and 36 % of C3GN patients [63]. It appears
based on these and other reports that while assess-
ment of C3 and C4 levels is suggestive of a diag-
nosis of MPGN, DDD, or C3GN, suppressed C3
or C4 levels are neither sufficient nor essential in
the presentation of MPGN, DDD, or C3GN
[19, 63]. It is important to recognize that of the
combined patients reported in these case series
with biopsy proven MPGN, DDD, or C3GN,
33 Membranoproliferative and C3-Mediated GN in Children
43 % had normal C3 levels at the time of diagnosis
[19, 63]. Furthermore, the patients with CFHR5
associated C3GN are also reported to have normal
serum C3 concentrations [72].
Extrarenal manifestations are uncommon
in C3G. These include acquired partial
lipodystrophy (aPL) and ocular C3 deposits, sim-
ilar to soft drusen seen in age-related macular
degeneration (AMD) [53]. Interestingly, polymor-
phisms in complement genes are also associated
with the risk of developing AMD [73]. These
drusen consist of C3 split products and CFH
[74, 75]. The long-term risk for visual problems is
about 10 % [76]. aPL usually precedes the diagno-
sis of MPGN and may present with low C3 levels
and C3NeF. It is presumably caused by comple-
ment dysregulation on adipocytes [77, 78].
Diagnosis
Physicians must maintain a high index of suspi-
cion when considering a diagnosis of MPGN,
DDD, or C3GN. The clinical presentations of
MPGN, DDD, and C3GN are variable, while
most commonly seen in the setting of acute
nephritis or nephrotic syndrome, more subtle pre-
sentations are also seen, and there is considerable
clinical overlap with the presentations of many
other forms of renal disease. Analysis of the com-
plement system can be useful particularly C3 and
C4 levels. However, as noted above, normal com-
plement levels do not exclude the diagnosis of
MPGN, DDD, or C3GN [19]. The measurement
of nephritic factor is also supportive of a diagnosis
of C3GN or DDD; however, these assays are
neither standardized, widely available, nor have
diagnostic specificity. Genetic testing of comple-
ment components may provide insight into under-
lying pathogenic mechanisms; however,
mutations in the complement system that are spe-
cific from a diagnostic standpoint have yet to be
identified. The exception to this is the CFHR5
mutation seen in a specific subset of patients
with C3GN [51]. Ultimately, a diagnosis of
MPGN, DDD, or C3GN is made by renal biopsy
in conjunction with complement diagnostics
(Table 2).
1045
Differential Diagnosis
In the setting of hypocomplementemia, low C3
levels, the differential diagnosis is narrow, and
depressed C4 levels can refine the differential
further. In the pediatric population, low C3 and
C4 levels are seen primarily in patients with either
MPGN or SLE and in some patients with PIGN.
A throughout history and physical examination
with attention to extrarenal disease manifestations
such as arthralgias, rashes, and neurologic, hepatic,
lung, or cardiac involvement combined with labo-
ratory evidence of immune dysregulation including
antibody-mediated hemolysis, leukopenia,
coagulopathy, myositis, or hepatitis strongly sup-
port a diagnosis of SLE as opposed to MPGN.
Although exceedingly rare in the pediatric popula-
tion, essential cryoglobulinemia may also present
with multi-organ involvement and systemic find-
ings. Of note, secondary forms of MPGN may
present with low C3 and C4 levels [79]. The dis-
eases presenting with low C3 and normal C4 levels
have traditionally been limited to PIGN, the nephri-
tis of chronic bacteremia, shunt nephritis, and
DDD. The presence of persistent fevers or V-A
shunts should alert the physician to the possibility
of the nephritis of chronic bacteremia or shunt
nephritis. The finding of isolated low C3 levels
with clinical features of acute GN presents a par-
ticular challenge to the clinician. Historically, the
history of a recent episode of pharyngitis, elevated
ASO or anti-DNase titers, and low C3 levels has
defined the clinical features of postinfectious
GN. DDD or C3GN is considered only if the clin-
ical course is atypical or if the C3 level does not
return to normal within 8–10 weeks of presentation
[12]. The report by Medjeral-Thomas et al. noted
elevated ASO titers in 43 % of patients with DDD
and 57 % of patients with C3GN; further compli-
cating matters was the finding that of the
DDD/C3GN patients with elevated ASO titers,
21 % had a documented antecedent upper respira-
tory tract infection [63]. Normalization of C3 levels
has been used to differentiate PIGN from other
forms of nephritis; however, current data suggests
that a significant percentage of patients with C3GN
and DDD have normal C3 levels at the time of
diagnosis [19]; it is not clear what percentage of
1046
the C3GN patients with normal C3 levels at the
time of diagnosis had previously been
hypocomplementemic [63]. As the vast majority
of patients with presumed PIGN are never
biopsied, the clinician should have a low threshold
for biopsy if the patient has a clinical course that is
atypical for PIGN or there is a family history of
unexplained ESRD. [12] Because MPGN, DDD,
and C3GN may also present with normal comple-
ment levels, they should also be considered in any
differential diagnosis for glomerular disease even if
the patient’s C3 and C4 levels are normal. Until it is
clear what the frequency of CHFR5 mutations are
in the population outside of Cyprus, C3GN should
be considered in the differential diagnosis of
patients with a family history of ESRD, persistent
microscopic hematuria, and synpharyngitic gross
hematuria (Table 1).
Treatment
No treatment standard for patients with MPGN or
C3G exists, and current treatment recommenda-
tions are mainly based on small-size single-center
studies and expert opinions.
Supportive Therapy
Angiotensin-converting-enzyme inhibitors (ACEi)
or angiotensin II receptor antagonists (ARB) are
used in many patients with MPGN due to their anti-
proteinuric and antihypertensive effect. The use of
ACEi or ARB was associated with better renal
survival in MPGN patients [19]. The treatment of
arterial hypertension and CKD/ESRD follows
established standards.
Immunosuppressive Therapy
In children, there are multiple studies reporting on
the use of steroids in MPGN. A beneficial effect of
steroids for proteinuria and long-term renal func-
tion was detected by using long-term low-dose
prednisone. However, only a subgroup of patients
formerly classified as MPGN I will respond, and it
C. Licht et al.
may only be a partial response [80, 81]. Predni-
sone is considered the first-line agent in patients
with Ig-/IC-mediated GN who present with
nephrotic-range proteinuria with or without renal
failure. A reasonable approach would be
60 mg/m2 [2] (maximal 60 mg/d)  4 weeks
(induction) followed by 40 mg/m2 [2] on
alternate-day for 6–12 (24) months (mainte-
nance). No beneficial effect was shown in patients
with DDD.
Mycophenolate mofetil (MMF) was adminis-
tered alone or in combination with prednisone in
idiopathic MPGN and generated encouraging
results. In 13 patients with steroid-resistant pri-
mary MPGN, the addition of MMF resulted in
sustained improvement (1-year follow-up) of pro-
teinuria and renal function [82]. In 51 patients
with primary GN, including 15 patients with
MPGN, a partial or complete remission was
reported to be 70 % after 1 year [83]. In a small
case series of nine children with MPGN I, treat-
ment with MMF and prednisone for a mean time
of 40 months resulted in complete or partial
response in 5. However, all patients with low C3
levels were treatment failure [84]. No reports on
the effectiveness of MMF in patients with DDD
are available.
Calcineurin inhibitors (e.g., cyclosporine A
and tacrolimus) were used in prednisone-resistant
MPGN patients. One study demonstrated its effi-
cacy in a small trial including 18 patients with
refractory MPGN and additional low-dose pred-
nisone therapy, by inducing long-term (2 years)
reduction of proteinuria and stable renal function
in 94 %. Only one patient showed a recurrence
after treatment discontinuation [85]. In 11 adult
patients with steroid resistant, in five also
cyclophosphamide-resistant MPGN, tacrolimus
plus prednisone in six resulted in partial/complete
remission in nine [86, 87]. In two children with
MPGN and suboptimal response to long-term
prednisone, a rapid and complete remission was
achieved with tacrolimus [88]. In two patients
with DDD, low-dose prednisone and cyclosporine
A was able to induce remission [89, 90]. On the
other hand, Appel et al. were not able to see a
benefit of calcineurin inhibitors in the treatment of
DDD [76].
33 Membranoproliferative and C3-Mediated GN in Children
The detection of C3NeF has prompted the use
of B-cell depleting agents like rituximab, a chi-
meric monoclonal CD20 antibody. Several case
reports in patients with MPGN I, especially with
IC-mediated disease, indicate partial or complete
remission after administration of rituximab
(in half of the cases in addition to steroids) in
11 of 13 patients [91–95]. Despite a decrease in
C3NeF levels, two patients with DDD did not
show any change in proteinuria or renal function
[96, 97]. Of note, both patients were rescued by
eculizumab therapy (below).
Complement-Targeting Therapy
With the emerging role of complement in C3G
pathogenesis, complement-targeting treatment
strategies are increasingly considered for patients
with C3G. Several case reports exist on the use of
plasma exchange (PE) in patients with MPGN I
and DDD for disease manifestation and recur-
rence in native and graft kidneys. A partial or
complete remission was observed in 17 out of
21 patients (81 %) reported in the literature
[93, 98–109]. In one patient with Ig-mediated
GN (MPGN I), despite a transient improvement
of renal function and proteinuria, her clinical con-
dition worsened including seizures, respiratory
distress, and sustained anuria. Due to a CFHR1
deletion (without CFH antibodies), low C3 and
elevated SC5b-9, indicating alternative and termi-
nal complement pathway activation, and
TMA-related symptoms, the patient was switched
from PE to eculizumab. This resulted in a dra-
matic improvement of her clinical condition, pro-
teinuria and renal function [100]. One case report
also shows the successful treatment of MPGN
recurrence during pregnancy and enabled the
delivery of a healthy child [110].
Three case reports, including two siblings with
DDD on the background of a CFH mutation in
SCR4 and one patient with MPGN I and
MCP/CD46 mutation, were treated for >3 years
with chronic infusion of fresh frozen plasma
(FFP). FFP as maintenance therapy, individual-
ized according to the patient needs (e.g., intercur-
rent infection), was able to prevent disease
1047
progression and stabilized kidney function
[52, 55, 100]. However, plasma infusion might
not be enough as induction therapy [111].
In 2012, several reports of the successful use of
eculizumab in patients with MPGN were
published [97, 100, 112, 113]. Eculizumab is a
monoclonal antibody binding C5 and therefore
preventing the assembly of the terminal comple-
ment complex (TCC, C5b-9) [114]. This complex
is integrated in cell membranes and can lead to
cell lysis. Its role in the pathogenesis in C3G is not
known. However, elevated soluble C5b-9 levels
were detected in patients with C3G, and in mice
deficient in C5, anti-murine C5 antibody amelio-
rated the disease [115].
At present, 13 patients with different forms of
C3G treated with eculizumab have been reported
in the literature. Six are part of a small clinical
trial, while the others are single case reports
[96, 97, 100, 112, 113, 116–118]. A significant
response was reported in 10, a partial response in
one and no response in two of these patients, as
reviewed by Vivarelli et al. [119]. These patients
include cases of C3G and DDD, in native and
transplanted kidney, and both cases with C3NeF
and genetic mutations. Although numbers are
small, a better outcome seems to be associated
with elevated SC5b-9 levels and shorter disease
duration [119]. Of note, treatment with
eculizumab in C3G is not always successful
[112] (personal unpublished observation), and
criteria clearly identifying individuals with the
potential to respond to eculizumab treatment are
currently lacking.
Clinical Outcomes
Long-term renal outcome data with the new clas-
sification system is limited for C3GN and DDD.
Sethi, in a small case series of 12 of primarily
adult C3GN patients, reported stable short-term
renal function with a mean follow-up of 26.4
months [67]. Medjeral-Thomas, comparing out-
comes of DDD and C3GN in a cohort of
80 patients, reported progression to ESRD in
47 % of DDD patients and 23 % of C3GN patients
with a median follow-up of 28 months; there was
1048
no difference in cumulative renal survival by
Kaplan-Meier analysis [63]. The French cohort
of 134 patients reported a 10-year renal survival
rate of 63.5 % with no differences in renal survival
between groups (MPGN, DDD, C3GN)
[19]. These data are in agreement with previous
reports indicating that progression to ESRD
occurs in 40–50 % of patients with MPGN regard-
less of type within 10 years of diagnosis. Analysis
by both the French and English groups suggested
that both eGFR and age at presentation were
strongly negatively correlated with long-term
renal survival [19, 63]. Medjeral-Thomas also
reported that the presence of glomerular crescents
or DDD by biopsy was a strong independent
predictor of disease progression [63] (Table 4).
While disease recurrence of MPGN and DDD
in renal transplants has been well described, with
recurrence rates ranging from 10 % to 100 %
depending on the case series, the impact of disease
recurrence on allograft survival has been contro-
versial, as a number of small case series report
accelerated rates of graft loss from disease recur-
rence, while larger studies suggest a more limited
impact. Analysis of the UNOS database
(811 patients with MPGN I and 179 patients
with DDD) reported a modest reduction in
10-year graft survival for patients with MPGN I,
56.2 % versus 60 %, compared to the dataset as
whole with allograft failures from disease recur-
rence in 14.5 % patients [120]. The 10-year allo-
graft survival for DDD was not significantly
different from the general population, 57.5 %,
despite almost 30 % of graft failures reported to
be from disease recurrence; however, the authors
noted significantly worse allograft survival in
pediatric DDD patients compared to patients
transplanted as adults, with age at transplantation
being positively associated with graft survival
[120]. Data supporting a modest impact of
MPGN I disease recurrence has also been reported
from the ANZDATA registry [121]. A more recent
report from the ESPN/ERA-EDTA registry indi-
cated that there was significant increase risk of
allograft loss, 32.4 %, at 5 years posttransplant
in pediatric patients with MPGN [122]. Data
suggesting that pediatric patients with DDD are
at greater risk of graft loss from disease recurrence
C. Licht et al.
has also been reported in the NAPRTCS database
[123]. The etiology of this apparent increased risk
in the pediatric population has not been identified.
As it is not possible to reclassify patients in the
existing databases, it is not clear how the new
classification system will impact the relative risk
of disease recurrence and allograft survival. There
are, however, a number of limited case series that
confirm increased risk of disease recurrence for
C3GN and the potential negative impact on allo-
graft survival. Zand et al. reported outcomes on
21 patients with C3GN; 14 (66.7 %) had disease
recurrence with a median time to graft failure of
77 months (6.4 years) [124]. Using the new clas-
sification system, the French group reported recur-
rence rates of 43 %, 55 %, and 60 % for MPGN,
DDD, and C3GN, respectively [19]. The impact on
allograft survival was not reported and the total
number of transplants was small, 35 [19]. The
English group reported data on 13 transplants,
6 with DDD and 7 with C3GN. All of the DDD
patients had recurrence of disease and 50 % failed
due to disease recurrence. Of the 7 C3GN patients,
57 % had disease recurrence; three of the four were
noted to have recurrent disease contributing to
allograft failure [63]. Overall, the allograft survival
at 5 years was 69 % (95 % CI: 65–99 %). CFHR5-
associated C3GN has also been reported to recur in
renal transplants [125].
Summary
Lessons learned from animals and humans have
helped to significantly advance our understanding
of the pathogenesis of the disease spectrum
previously labeled as MPGN. While so far classi-
fied based on morphology, today the identification
of a central role of the complement AP has
allowed for a pathomechanism-based new classifi-
cation system introducing the term C3
glomerulopathy (C3G), which reflects the predom-
inant finding of C3 in kidney biopsy specimens –
C3 glomerulonephritis (C3GN) or dense deposit
disease (DDD) – or in combination with immuno-
globulins – secondary MPGN. Complement
dysregulation resulting in C3G seems – different
from aHUS with uncontrolled complement
33 Membranoproliferative and C3-Mediated GN in Children
activation on the vascular endothelium – to be
characterized by the overshooting activation of
C3 and generation of C3 split products. Identifica-
tion of a central role of complement dysregulation
in C3G pathogenesis has for the first time identified
a specific treatment strategy for C3G, a break-
through in the management of this disease spec-
trum historically characterized by its progressive
nature and poor outcomes.
Acknowledgments The authors wish to thank Dr. Paul
Thorner, The Hospital for Sick Children and Department of
Laboratory Medicine and Pathobiology, University of
Toronto, Toronto, ON, Canada for preparing Fig. 1 and
discussing pathohistological aspects of C3G.
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 Membranous Nephropathy in Children
34
Rudolph P. Valentini

Contents Other Secondary Causes of MN . . . . . . . . . . . . . . . . . . . . 1063

Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1056
Incidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1056
Histopathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1056
Idiopathic Membranous Nephropathy . . . . . . . . . . . . . 1056
Secondary Membranous Nephropathy . . . . . . . . . . . . . 1059
Clinical Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1060
Pathogenesis of Idiopathic MN . . . . . . . . . . . . . . . . . . . 1060
Historical Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . 1060
M-Type Phospholipase A2 Receptor
Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1061
Neutral Endopeptidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1061
Other Causes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1062
Pathogenesis of Secondary MN . . . . . . . . . . . . . . . . . . 1062
Hepatitis B Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1062
Systemic Lupus Erythematosus (SLE) . . . . . . . . . . . . . 1062
Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1064
Treatment of Idiopathic Membranous
Nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1064
Corticosteroid Monotherapy . . . . . . . . . . . . . . . . . . . . . . . 1065
Alkylating Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1066
Alkylating Agents + Corticosteroids . . . . . . . . . . . . . . . 1066
Calcineurin Inhibitors + Corticosteroids . . . . . . . . . . . 1067
Mycophenolate Mofetil (MMF) . . . . . . . . . . . . . . . . . . . . 1068
Rituximab (RTX) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1070
ACTH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1070
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1071
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1071

R.P. Valentini (*)

Pediatric Nephrology, Children’s Hospital of Michigan,
Detroit, MI, USA
Wayne State University School of Medicine, Detroit, MI,
USA
e-mail: rvalenti@dmc.org

# Springer-Verlag Berlin Heidelberg 2016 1055

E.D. Avner et al. (eds.), Pediatric Nephrology,
DOI 10.1007/978-3-662-43596-0_30
1056
Background
Membranous nephropathy (MN), also known as
membranous glomerulonephritis, is a pathologic
condition associated with an immune complex
deposition within the glomerulus resulting in glo-
merular dysfunction. Clinical manifestations of
MN include proteinuria, hematuria, overt
nephrotic syndrome, and in some cases renal dys-
function. In adults, MN is the most common pri-
mary glomerulopathy associated with the
nephrotic syndrome, accounting for approxi-
mately 25–35 % of adults diagnosed with this
condition [1–3]. In children, MN may present as
the nephrotic syndrome or as asymptomatic pro-
teinuria and is considered to a much less common
condition than that seen in adults [4, 5]. MN may
be idiopathic or secondary to infections, autoim-
mune diseases, or medications. Recent break-
through discoveries regarding the pathogenesis
of idiopathic MN have reenergized the field and
should aid clinicians going forward with regard to
developing novel means to monitor and treat
patients with membranous nephropathy [6–8].
Incidence
A comprehensive review of 40 studies estimated
the incidence of primary glomerulonephritis
worldwide; it was determined that MN occurs in
1.2/100,000/year in adults, and approximately
0.05–0.1/100,00/year in children and adolescents
[9–11]. With regard to idiopathic MN in the set-
ting of nephrotic syndrome, despite being respon-
sible for 25–35 % of adults with new onset
disease, MN accounts for <5 % of children with
nephrotic syndrome. A multicenter, prospective
study conducted by International Study of Kidney
Disease in Children analyzed 521 children
presenting with the nephrotic syndrome from
1967 to 1974; in that era, pretreatment biopsies
revealed MN in only 1.5 % of children [12]. Other
more recent reports still show the incidence of
MN to be <5 % of children with nephrotic syn-
drome [9, 13]. However, when one focuses on a
more select demographic, the adolescent with
R.P. Valentini
nephrotic syndrome, MN appears to increase
markedly. Moxey-Mims reported the incidence
of MN in the setting of nephrotic syndrome
increases from 1 % in children 1–12 years of age
to 22 % in children 13–19 years of age [14]. Sim-
ilarly, an 18.5 % incidence of MN was reported
from the Southwest Pediatric Nephrology Study
Group in 65 adolescents having undergone a renal
biopsy in the setting of nephrotic syndrome [15].
Further data in support of a rising incidence of
MN in adolescents comes from a recent study
from Pakistan involving 538 patients with
nephrotic syndrome including 173 adolescents
(13–18 years of age); MN was found in 3 %
of children 12 and under, but 18.5 % of adoles-
cents [16]. The United States Renal Data Systems
(USRDS) 2013 report indicates that 36 pediatric
patients (0.6 %) had MN as an underlying cause of
their end-stage renal disease over the 5-year
period (2007–2011). These patients had a median
age of 16 years and were nearly equally split
between Caucasians and African Americans at
nearly 40 % each without any gender bias [17].
Histopathology
Membranous nephropathy is a pathologic entity
with common histologic features, but does not
represent one disease state [7, 18]. The histologic
feature characteristic of MN is a normocellular
glomerulus with prominent capillary loops sec-
ondary to subepithelial immune complex
deposits. To best delineate the subtle histologic
features that aid in the distinction between idio-
pathic and secondary forms of MN, they will be
described separately.
Idiopathic Membranous Nephropathy
The pathologic features of membranous nephrop-
athy were initially described in 1968 by
Ehrenreich and Churg [19]. This staging system
consisted of four stages seen as the evolution of
immune complex formation in relation to the glo-
merular basement membrane (GBM):
subepithelial deposits and normal GBM usually
34 Membranous Nephropathy in Children 1057

Fig. 1 Idiopathic
membranous
nephropathy: hematoxylin
and eosin,  200; arrow
points out thickened
capillary loop, without
cellular proliferation

Fig. 2 Idiopathic
membranous nephropathy:
Periodic Acid Schiff stain,
 200; glomerulus with
widely patent capillary
loops, without cellular
proliferation

only seen on EM (Stage 1), global subepithelial
deposits with projections of newly formed matrix
material (Stage 2), incorporation of the deposits
into the GBM seen as an envelopment of the
subepithelial deposits by the projections of the
GBM (Stage 3), and a remodeling phase with
thickening of the GBM and electron-lucent or
absent deposits (Stage 4) [7, 19, 20]. Adult and
pediatric studies have failed to provide convinc-
ing evidence that the Ehrenreich-Churg classifica-
tion system for MN correlates with clinical
presentation or is helpful in predicting renal
outcomes [7, 21, 22]. Work by Olbing et al.
enlightened the pediatric community on idio-
pathic MN as it was described in a cohort of
9 children [18]. Exquisite details of the histopa-
thology allowed one to see the characteristic light
and electron microscopic features [18, 23]. Light
microscopy is characterized by widely patent cap-
illary loops with normal mesangial matrix and
normal mesangial cellularity, and almost “exag-
gerated appearance of normality” [20] (Figs. 1
and 2). The capillary wall thickening can be seen
by various light stains: hematoxylin and eosin,
methenamine silver-PAS (Jones), and trichrome
stain. However, the methenamine silver-PAS
stain demonstrates the characteristic spikes from
projections of the glomerular basement membrane
(GBM) between the nonargyrophilic subepithelial
deposits.
1058 R.P. Valentini

Fig. 3 Idiopathic
membranous nephropathy:
electron microscopy, 
5492; arrows point out
subepithelial electron-dense
deposits

Fig. 4 Idiopathic
membranous nephropathy:
electron microscopy, 
12,960; arrows point out
electron-dense deposits
situated beneath podocyte
foot process

Electron microscopy has helped best
characterize this disease. Subepithelial or
epimembranous electron-dense deposits resting
upon the GBM with projections of cytoplasm
between them are characteristic features (Figs. 3
and 4). These deposits are typically diffusely dis-
tributed, but can be segmental in some cases.
Intramembranous deposits are also seen in
idiopathic MN. Mesangial deposits, on the other
hand, are uncommon in idiopathic MN in
adults, and when seen, secondary MN should
be entertained [20]. However, a pediatric
study performed by the Southwest Pediatric
Nephrology Study Group in the United States
revealed that 31 % of patients classified as idio-
pathic MN had mesangial deposits; while this was
far lower than the 100 % seen in those with MN
associated with systemic lupus erythematosus
(SLE), the presence or absence of mesangial
deposits appear to lack specificity in the
pediatric patient with MN [24]. Subendothelial
deposits are even less commonly seen in idio-
pathic MN; in the same study, subendothelial
deposits occurred in only 13 % of idiopathic MN
versus 78 % of lupus associated MN in this pedi-
atric cohort [24].
34 Membranous Nephropathy in Children 1059

Fig. 5 Membranous
nephropathy secondary to
lupus nephritis:
hematoxylin and eosin,
 400. Note: thickened
capillary loops and focal
minimal increase in
mesangial cellularity at
11–12 o’clock position

Fig. 6 Membranous
nephropathy secondary to
lupus nephritis: electron
microscopy,  12,500; long
thin arrows point out
subepithelial electron-dense
deposits; short thick arrow
(on right) shows
tubuloreticular inclusion
within glomerular
endothelial cell

Immunofluorescence is characterized by a Secondary Membranous Nephropathy

granular, glomerular capillary wall distribution
of immune deposits, comprised of immunoglobu-
lin G (IgG) and to a lesser extent complement
component 3 (C3). Further characterization of
IgG reveals a predominance of subclass
IgG4 and IgG1 within the deposits, at 81 %
and 75 %, respectively, in idiopathic MN
[25]. Subclass IgG3, which is not seen in idio-
pathic MN, is present in 50 % of patients with
lupus associated MN [26]. A large pediatric
pathology study revealed granular, capillary wall
staining for IgG in 100 % and C3 staining in
77 % [27].
Light microscopic features more commonly seen
with secondary MN include glomerular lobula-
tion, mesangial hypercellularity, segmental
involvement (scars or inflammation), and necrosis
[20] (Fig. 5). By electron microscopic, mesangial
deposits and subendothelial deposits are much
more commonly seen than in idiopathic
MN. Some findings speak to the underlying
cause of secondary MN such as SLE where
tubuloreticular inclusions (TRI) may be seen
within the glomerular endothelial cells [4]
(Figs. 6 and 7). The formation of these subcellular
1060
Fig. 7 Membranous nephropathy secondary to lupus
nephritis: electron microscopy,  40,000; short thick
arrow shows tubuloreticular inclusion within glomerular
endothelial cell
organelles within the endoplasmic reticulum is
induced by alpha interferon (IFN-α) [28]. While
a “full house” immunostaining pattern of featur-
ing glomerular deposition of IgG, IgM, IgA, C3,
and C1q can be seen in proliferative forms of SLE,
this is uncommon in membranous lupus nephritis.
The subclass IgG3 predominance, noted above,
aids in the distinction from idiopathic MN, where
IgG3 is not typically seen [26].
Clinical Features
Most children with MN present with some degree
of proteinuria. This can be asymptomatic protein-
uria picked up incidentally on a urinalysis speci-
men. This moderate proteinuria is accompanied by
microscopic hematuria in the majority (70–90 %)
of patients [13, 29]. Nephrotic syndrome has been
the clinical presentation in 25–100 % of pediatric
studies [13, 18, 21, 22, 29–34]. A study from South
Korea noted gross hematuria in up to 40 % of
R.P. Valentini
children [33]. Hypertension occurs in <10 % of
children with MN, and thromboembolic events in
<5 % of cases [4, 7, 35].
Pathogenesis of Idiopathic MN
An animal model was developed in the 1950s by
Heymann et al. that replicates the autoimmune
disease which results in nephrotic syndrome
[36]. A rat autoantibody model which is triggered
by the injection of a rat proximal tubular antigenic
preparation. This results in a subepithelial,
immune complex deposition, the activation of
complement, and sublethal injury to the
podocytes. This topic is reviewed in greater detail
elsewhere [7, 37].
Historical Animal Models
The Heymann nephritis animal model, while
developed many decades ago, has been instru-
mental in gaining an understanding to the evolu-
tion and pathogenesis of human, idiopathic
membranous nephropathy. This mammalian
model of immune complex nephritis consists of
immunization of rats with an antigenic fraction of
rat proximal tubular brush border (Fx1A). One
key pathophysiologic aspect of this immune com-
plex disease was determining whether circulating
immune complexes were deposited into the
subepithelial space. It had been hypothesized
that unique physicochemical properties of these
immune complexes allowed them to move across
the endothelial and glomerular basement mem-
brane to allow access beneath the podocytes.
Alternatively, circulating antibodies could react
to in situ antigens in the subepithelial region
resulting in immune complex formation in that
location. Subsequent studies would later validate
this latter theory that antigen-antibody immune
complexes were formed locally and could occur
in the absence of circulating antigen thereby prov-
ing the in situ (“fixed”) antigen theory in this rat
model [38, 39].
Later studies would reveal that megalin, a large
glycoprotein involved in protein reabsorption in
34 Membranous Nephropathy in Children
the proximal tubule, is the target antigen in the rat
(Heymann nephritis) model. This same protein is
also contained on the rat podocytes foot process –
thereby explaining the immune complex location
in the subepithelial space. Subsequent to immune
complex deposition, complement fixation occurs,
leading to C5-C9 insertion into the podocyte cell
membrane resulting in sublethal injury and pro-
teinuria. While this model led to a more advanced
understanding of human disease, it was not
completely applicable. While humans have a
megalin analogue expressed in the brush border
of the proximal tubule, unlike the rat, humans do
not express this megalin like protein on their
podocytes. Moreover, this megalin like protein
was absent from immune deposits in patients
with membranous nephropathy [40, 41].
The search for the antigen in human membra-
nous nephropathy continued.
M-Type Phospholipase A2 Receptor
Antibodies
In 2009, a major breakthrough in the understand-
ing of the pathogenesis of idiopathic membranous
nephropathy occurred with the identification of
phospholipase A2 receptor (PLA2 R) antibodies.
These PLA2 R antibodies are directed at this
transmembrane glycoprotein expressed on the
podocyte and have been shown to be present in
70 % of adults with idiopathic MN but not sec-
ondary MN [6]. These anti-PLA(2)R autoanti-
bodies were mainly IgG4, the predominant IgG
subclass found in the glomerular deposits in idio-
pathic MN but not in the secondary forms of
MN. It should be noted that the antibodies eluted
from biopsy samples from patients with idiopathic
MN, but not from secondary forms, recognized
PLA2 R(6). A Chinese study of patients with
idiopathic MN, membranous lupus nephritis, and
MN associated with hepatitis B virus (HBV)
infection found that anti-PLA2 R antibodies were
present in 82 % of adults with idiopathic MN, and
only 5 % and 6 % of those with MN secondary to
lupus and HBV, respectively [42].
PLA2 R antigen may be identified in the
immune deposits by immunofluorescence or
1061
immunochemistry, even in patients with no or
low levels of circulating anti-PLA2 R antibodies
[43], which demonstrate that staining for PLA2 R
is more sensitive than circulating antibodies
[44, 45]. However, PLA2 R have been detected
in the immune deposits of a few patients with
membranous nephropathy-associated NSAIDS
(naproxen and piroxicam), hepatitis B, and sar-
coidosis, but was not detected in MN secondary to
SLE [46, 47].
PLA2R staining has been reported in 45 % of
children with membranous nephropathy, the
youngest of which was 10 years old. None of the
negative patients went on to show signs of sec-
ondary MN at >3 years of follow-up, which sug-
gest that PLA2R staining sensitivity is much
lower in the pediatric than the adult primary mem-
branous glomerulopathy population [27].
Anti-PLA2 R antibody titers have been shown
to be an excellent marker of disease activity being
elevated in a relapse state and absent in a state of
remission [41]. The antibody titer following
rituximab predicts the clinical response [48].
Neutral Endopeptidase
Neutral endopeptidase (NEP), which is expressed
on human podocytes, has been shown to be the
target antigen in some rare cases of antenatal
MN. This is the case when a mother, genetically
deficient for neutral endopeptidase, has been
alloimmunized against NEP present on the fetal
podocyte during a prior pregnancy. The transpla-
cental passage of the anti-NEP antibodies is
responsible for the subepithelial immune deposits
in the fetus and the neonate. The severity of the
nephrotic syndrome correlates to the anti-NEP
antibody titer in the mother. The nephrotic syn-
drome is present at birth but proteinuria decreases
with time with a progressive decrease of the
maternal antibodies and a disappearance of the
immune deposits [49]. However, residual protein-
uria and chronic renal insufficiency may occur in
relation to nephron loss and renal scarring, per-
haps in relation to renal arterial lesions, which are
observed in addition to the subepithelial immune
deposits. Interestingly, the IgG fraction from the
1062
mother serum produces proteinuria and immune
deposits when injected into the rabbit
Other Causes
Another antigen has been identified in young
pediatric patients with MN – cationic bovine
serum albumin (cBSA). In young infants with
MN, immune complexes were identified with
subepithelial deposits, which colocalized both
cBSA protein and cBSA IgG antibodies. In this
study, the BSA antibodies were specific to bovine
serum albumin, in that they did not react to human
serum albumin. It was hypothesized that cBSA
was a modified, food-derived antigen from dietary
cow milk protein which is absorbed through the
relatively immature alimentary tract, and the cat-
ionic nature of BSA allows it to cross the nega-
tively charged glomerular basement membrane
and allows it to be “planted” into the subepithelial
region for in situ formation of immune
complexes [50].
Pathogenesis of Secondary MN
The 2012 clinical practice guidelines from the
Kidney Diseases Improving Global Outcomes
(KDIGO) estimated that secondary MN is more
common in children, accounting for 75 % of chil-
dren with MN versus 25 % of adults [51]. It is
generally accepted that systemic lupus
erythematosus (SLE) and chronic infection with
hepatitis B virus (HBV) are the most common
secondary causes of MN in both children and
adults [7]. Both disease entities will be discussed
along with the less common causes of secondary
MN as it relates to children.
Hepatitis B Virus
R.P. Valentini
Taiwan [52]. Both the causes of nephrotic syn-
drome and the presence of hepatitis B carrier state
were assessed in this study. Remarkably, the fre-
quency of HBV-associated membranous nephrop-
athy (HBVMN) decreased steadily over the
25-year study period: 11.6 % (1974–1984), 4.5
% (1984–1994), 2.1 % (1994–2004), and 0 %
(2004–2009). At the same time, this Taiwanese
study showed a marked decline in the prevalence
of HBV surface Ag (+) carrier state, which was
consistent with earlier studies [52, 53]. Others
have shown the consequences of HBV infection
with hepatitis B e Antigen (HBeAg) detected in
88 % of the glomerular deposits in children diag-
nosed with HBV MN [54].
Liao’s study also revealed some interesting
epidemiologic information as it relates to HBV
MN in Taiwan – as they studied 471 children
with nephrotic syndrome from 1974 to 2009. Of
the 35 HBV seropositive patients, 25 had biopsy-
proven HBV MN [no evidence of SLE or other
secondary causes of MN, characteristic histologic
features of MN on light, immunofluorescence,
and electron microscopy and positive serum for
hepatitis B surface antigen (HBsAg)]. These
patients were predominantly male (84 %), and
96 % were infected via horizontal transmission
in that their mothers were HBsAg seronegative.
Also, it was shown that horizontal and not vertical
transmission of HBV is important in the develop-
ment of HBV MN, raising the question of whether
or not the response to HBV and perhaps HBeAg is
different in the neonatal and infant kidney as
opposed to the older child [52].
Children with HBV MN usually present with
nephritic syndrome, hematuria, and normal renal
function. Blood tests show the presence of
HBsAg, HBeAg, and anti-HBc antibodies. Com-
plement C3 level is often low. The presence of
anti-HBe antibodies correlates with the disappear-
ance of HBeAg and clinical remission of