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revie w
2015 International Society of Nephrology

What is nephrocalcinosis?
1

Linda Shavit , Philippe Jaeger and Robert J. Unwin

1

Adult Nephrology Unit, Shaare Zedek Medical Center, Jerusalem, Israel and UCL Centre for Nephrology, Royal Free Campus and
Hospital, University College London, London, UK

The available publications on nephrocalcinosis

are
wide-ranging and have documented multiple
causes and associations of macroscopic or
radiological nephrocalcinosis, most often located
in the renal medulla, with various metabolic and
genetic disorders; in fact, so many and various
are these that it is difficult to define a common
underlying mechanism. We have reviewed
nephrocalcinosis in relation to its definition,
genetic associations, animal models, and
putative mechanisms. We have concluded, and
hypothesized, that nephrocalcinosis is primarily
a renal interstitial process, resembling
metastatic calcification, and that it may have
some features in common with, and pathogenic
links to, vascular calcification.
Kidney International (2015) 88, 3543; doi:10.1038/ki.2015.76;
published online 25 March 2015
KEYWORDS: bone; calcium; hypercalciuria; hyperoxaluria; mineral metabolism;
urology

Correspondence: Robert J. Unwin, UCL Centre for Nephrology, Royal

Free Hospital and Campus, University College London, Rowland Hill Street,
London NW3 2PF, UK. E-mail: robert.unwin@ucl.ac.uk
Received 29 November 2014; revised 18 January 2015;
accepted 22
January 2015; published online 25 March 2015

Strictly, the term nephrocalcinosis refers

to the generalized deposition of calcium
oxalate (CaOx) or calcium phosphate (CaPi)
in the kidney. However, in most cases,
deposition seems to be interstitial, and
this
is
what
nephrocalcinosis
is
now
generally taken to mean. Although
some
authorities may limit the definition of
nephrocalcinosis to the deposition of mainly
interstitial CaPi crystals, this is not
universally acknowledged, and thus for the
purposes of this review we have retained the
broader definition of nephrocalcinosis to
include both CaPi and CaOx deposition. When
high-resolution Fourier transform infrared
microspectroscopy and electron diffraction
have
been
used
to
investigate
the
composition of Randalls plaque (areas in the
renal papillae containing interstitial apatite
deposits that can serve as a nidus for
urothelial surface CaOx deposition), CaPi
crystals
are
detected
as
their
major
component.1 However, CaOx calculi overlie
and adhere to Randalls plaque in up to 50%
of stone formers.2 Therefore, concurrent
deposition of both hydroxyapatite and CaOx
crystals may occur in the same patient with
nephrocalcinosis.
CaPi
is
present
as
crystalline apatite, but it is not known
whether amorphous deposits of CaPi also
occur.
A variety of inherited and acquired
diseases have been associated with
nephrocalcinosis and recognized as potential
Kidney International (2015) 88, 3543

causes. Nephrocalcinosis can be classified in

three ways that represent increasing degrees
of severity of renal involvement:1 molecular
or chemical, often observed in patients with
overt hypercalcemia and which is usually
reversible when hypercalce- mia is corrected;2
microscopic, which in most cases is a precursor
to macroscopic nephrocalcinosis and is diagnosed
by identifica- tion of mineral deposits on
light microscopy of renal tissue;3 and
macroscopic, in which calcification is visible
on either a plain abdominal X-ray or
ultrasound scan and/or confirmed by a
computed tomography scan, is the well-known
clinical and diagnostic feature of
nephrocalcinosis. Although the clinical
presentation and outcomes of these forms of
nephrocalcinosis can be different, in clinical
practice there is often some overlap.
Nephrocalcinosis usually involves the renal
medulla (in 97% of
patients) or, less
often,
the
cortex.
Cortical
nephrocalcinosis has been described in
patients with renal cortical necrosis
(typically, and originally described,
following postpartum
hemorrhage),
chronic
glomerulonephritis
or pyelonephritis,
primary and secondary oxalosis (which are
more often causes of medullary
nephrocalcinosis), autosomal recessive
polycystic disease, chronic renal allograft
rejection,
and benign nodular cortical nephrocalcinosis.3
35

proteinuria,
nephrolithiasis,
In this review, we try to summarize the
extensive literature on multiple causes of
macroscopic medullary nephrocalci- nosis, and
their implication in promoting clinically
significant renal injury. We review the
recent studies that have investigated some
novel pathogenic mechanisms and the genetic
background to progressive renal calcification.
A review of molecular nephrocalcinosis due to
overt hypercal- cemia and its consequences is
beyond the scope of this review.
ETIOLOGY OF NEPHROCALCINOSIS

Several novel genetic disorders have been

described
in
association
with
metabolic
abnormalities
that
predispose
to
the
development
and
progression
of
nephrocalcinosis.
Epithelial
cell
and
paracellular
disturbances
in
calcium
transport resulting in hypercalciuria seem
to be the most important, along with an
increase
in
phosphate
or
oxalate,
and
decrease in urinary citrate excretion. In
addition, in some cases specific anatomical
abnormalities predispose to the development of
nephrocalcinosis, for example, in medullary
sponge kidney (MSK).
Recent advances in genetics have helped
identify a number of transporters, channels,
and
receptors
that
are
involved in
regulating renal tubular reabsorption of
calcium and phosphate (Table 1). Several
genes involved in rare mono- genic disorders
have been associated with hypercalciuric
nephrolithiasis with nephrocalcinosis (that
is, CLCN5, CASR, CLDN16, CLDN19, ADCY10,
SLC34A1, SLC9A3R1, GLUT2,
HSPG2, and FN1), whereas variants of
uromodulin and fetuin seem to be protective.4
12 For example, mutations with loss of function
of the calcium-sensing receptor (CaSR) have
been reported in the hypercalcemic disorders
of
familial
benign
hypocalciuric
hypercalcemia, neonatal severe primary hyperparathyroidism,
and
familial
isolated
hyperparathyroidism, whereas gain-of-function
CaSR mutations result in autosomal dominant
hypocalcemia with hypercalciuria and Bartter
syndrome
type
V.4,1316
However,
modest
hypercalcemia per se, when not accompanied by
hypercalciuria, seems to be insufficient to
trigger nephrocalcinosis, for example, in
patients with familial benign hypocalciuric
hypercalcemia;
only
those
patients
with
significant hypercalciuria appear to be at
risk of developing renal calcium deposition.
Several additional mutations have been
identified in Bartter syndrome, which usually
presents with hypokalemic alkalosis, renal
salt wasting, hyperreninemic hyperaldosteronism,
hypercalciuria,
and
hypocitraturia
(presumed to be secondary to hypokalemia and
potassium depletion): muta- tions in genes
encoding the loop diuretic-sensitive NaK2Cl
(NKCC2)
cotransporter,
the
renal
outer
medullary
potassium
(ROMK)
channel,
the
voltage-gated chloride channel, CLC- Kb, and
the
CLC-Kb
-regulatory
subunit,
barttin.12,17,18
Nephrocalcinosis
has
been
described as a clinical feature in Bartter
syndrome types I, II, and V (mutations in
NKCC2, ROMK, and CaSR) (Table 1).
Another hereditary disorder associated with
nephrocalci- nosis, which is X-linked and
characterized
by
low-molecularweight

hypercalciuria,
is

and

re v i
ew

Dents disease (now known as Dent-1) that is

caused by a mutation in the gene for the
chloride/proton antiporter 5, CLC5, that
regulates
proximal
tubular
cell
endocytosis.5 In
addition,
the
X-linked
recessive
disease
known
as
the
oculocerebrorenal syndrome of Lowe, or just
Lowe syndrome, which is due to a mutation
in the OCRL gene, a phosphatidylinositol
4,5-bisphosphate-5- phosphatase in- volved
in actin polymerization, and that presents
typically
with
congenital
glaucoma,
cataracts, mental retardation, as well as
multiple reabsorption defects in the proximal
tubule,
is
also
associated
with
nephrocalcinosis and renal stones; similar
to Dent-1, it can lead to renal failure.
There is also a clinical variant of Lowe
syndrome known as Dent-2 that presents
predominantly with a renal phenotype and is
similar to Dent-1.5
Familial
hypomagnesemia
with
hypercalciuria and nephrocalcinosis is an
autosomal recessive renal tubular disorder
that
is
frequently
associated
with
progressive kidney failure and recurrent
urinary tract infections: it was originally
linked to mutations of the claudin-16 gene
(also known as paracellin-1), a member of
the claudin family of membrane proteins that
form the intercellular tight junction barrier
in a variety of epithelia, including the
thick ascending limb of the loop of Henle,
the site of the defect in this disorder;
although other claudin mutations (claudin-19)
have also been described.8,9
Several disorders have been associated
with homozygous and compound heterozygous
inactivating
mutations
of the solute
carrier family 34, member 3 SLC34A3, the
36
3543

L Shavit et al.:
Nephrocalcinosis

gene
encoding the sodium (Na+)-dependent
phosphate
cotransporter
2c
(NPT2c).
Hereditary
hypophosphatemic
rickets
with
hypercalciuria is an autosomal recessive renal
phosphatewasting
disorder
leading
to
hypophosphatemic rickets, bowing of the legs,
short stature, as well as appropriately
elevated 1,25(OH)2 vitamin D levels, often with
hypercalciur- ia, renal calcification, and
kidney
stones.1921
Recent
studies
have
revealed that individuals with mutations
affecting both
SLC34A3 alleles have a
significantly increased risk of kidney stone
formation or medullary nephrocalcinosis (46%
compared with 6% observed in healthy family
members carrying only the wild-type SLC34A3
allele).22 Renal calcification was also more
frequent in heterozygous carriers compared
with the general population, and it was more
likely to occur in homozygous and compound
heterozygous and heterozygous individuals
with decreased serum phosphate, decreased
tubular reabsorption of phosphate, and
increased serum 1,25(OH)2 vitamin D
levels;22 however, there was no
correlation between genotype and urinary
calcium excretion. Various genetic causes have
been identified in patients with fibroblast
growth factor-23dependent hypophosphatemic
disorders that usually present with
significant hypophos- phatemia, a decreased
tubular reabsorptive threshold for
phosphate, growth
retardation,
rickets
or
osteomalacia, inappropriately normal or
suppressed 1,25(OH)2 vitamin D levels,
normal serum levels of calcium, normalto-high parathyroid hormone levels, and
normal urinary calcium
Kidney International (2015) 88,

L
S
h
a
vi
Gene/protein/inheritance mode
Clinical manifestations
t
ATP6N1B/7q33-q34, autosomal recessive -subunit
et
+
Defect
of
the
proton
secretion
affecting
the
-intercalated
cells
ATP6N1B of the H -pump
al
of the collecting duct. Hypokalemic hyperchloremic acidosis
.:
with nephrocalcinosis and nephro- lithiasis.
SLC4A1/17q21-q22, autosomal dominant anion exchanger (AE1) Defect in bicarbonate transport at the basolateral membrane of the N
e
p
intercalated cells of the collecting duct. Hypokalemic
hyperchloremic acidosis, nephrocalcinosis, and nephrolithiasis.
ATP6B1/2cen-q13, autosomal recessive subunit
Defect of the proton secretion in the -intercalated cells of the
+
collecting duct. Hypokalemic hyperchloremic acidosis with
ATP6B1 of the H nephrocalcinosis and nephrolithiasis, and neural deafness.
pump
NKCC2/15q15-q21.1, autosomal recessive NKCC2
Decreased sodium, potassium, and chloride reabsorption in the
sodiumpotas- siumchloride transporter
ascending limb of Henle loop leading to hypokalemia, alkalosis,
hypercalciuria, secondary aldosteronism, and in some cases
nephrocalcinosis.

Tab le 1| C l i n i c a l m a n i f e s t a t i o n s and g en e ti c b a si s of in h e r i t e d d i s o rd e rs a s s o c i a t e d with me d u l l a ry n e p h ro c a l c i n o s i s (NC)

Disorder
dRTA Autosomal recessive

dRTA Autosomal dominant

dRTA With neural deafness at
birth or late onset
Bartters syndrome type 1

Bartters syndrome type 2

ascending limb

KCNJ1/11q24, autosomal recessive ROMK1 potassium channel

Autosomal dominant
hypoparathyroid- ism Bartters
syndrome type 5

CASR/3q13.3-q21, autosomal dominant calciumsensing receptor (activating mutations)

Familial hypomagnesemia with

hypercal- ciuria and
nephrocalcinosis (FHHNC)
Familial hypomagnesemia with
hypercal- ciuria and
nephrocalcinosis with ocular
impairment
Autosomal dominant
hypocalcemia with hypercalciuria
(ADHH)

CLDN16/3q27, autosomal dominant claudin 16

progressive

Decreased sodium, potassium, and chloride reabsorption in the

of Henle loop leading to hypokalemia, alkalosis,

hypercalciuria, secondary aldosteronism, and in some
cases nephrocalcinosis.
Inhibition of calcium reabsorption in the ascending limb of Henle
loop leading to hypercalciuria, hypocalcemia,
hyperphosphatemia, and hypophosphaturia, and in some
cases hypokalemia. Nephrocalcinosis in some cases.
Urinary losses of magnesium and calcium with nephrocalcinosis, and
kidney failure in homozygotes; heterozygotes may produce kidney

stones only.
CLDN19/1p34.2, autosomal dominant claudin 19
progressive

Renal wasting of magnesium and calcium, nephrocalcinosis, and

kidney failure in homozygotes; macular colobomata, myopia, and
nystagmus.

CASR activating mutation, autosomal dominant

Hereditary hypophosphatemic
SLC34A3/ 2c (NPT2c), autosomal recessive sodiumrickets with hypercalciuria
dependent phosphate cotransporter
(HHRH)
Dents disease (also known as Dent-1) CLCN5/Xp11.22, X-linked recessive chloride
channel 5 on the
endosome membrane
Lowes syndrome (also known as Dent-2)
OCRL1/Xq26.1, X-linked recessive
phosphatidylinositol 4,5bisphosphate 5-phosphatase
X-linked hypophosphatemia (XLH)
PHEX, X-linked phosphate-regulating endopeptidase
administra-

Hypocalcemia, hypercalciuria, normal PTH, recurrent nephrolithiasis, and

nephrocalcinosis, particularly during treatment with vitamin D
and calcium supplementation. Children may present with
seizures and neuromuscular irritability during periods of
stress; low serum magnesium in some cases.
Renal phosphate wasting, hypophosphatemic rickets, leg
bowing, short stature, elevated 1,25(OH)2D levels,
hypercalciuria, nephrocalcinosis, and kidney stones.
Multiple reabsorption defects in the proximal tubule associated
with nephro- lithaisis, nephrocalcinosis, and in many cases endstage renal disease (ESRD). Multiple reabsorption defects in the
proximal tubule (cf. Dents) nephrolithaisis, nephrocalcinosis,
and many cases end-stage renal failure. Hydrophthalmia,
cataract, mental retardation (less common in Dent-2 variant).
Treatment for XLH, ADHR, and ARHR consists of the long-term oral
tion of phosphate and calcitriol, and can be complicated by
nephrocalcinosis (in up to 80% of patients with XLH)

Ki
d
n
e
y
In
te
rn
at
io
n
al
(2

3
7

Autosomal dominant
hypophosphatemic rickets
(ADHR)
Autosomal recessive
hypophosphatemic rickets
(ARHR)
MacGibbonLubinsky
syndrome (ERS, enamelrenal syndrome)
Primary hyperoxaluria (PH) type 1
of

FGF23, autosomal dominant

attributable to intermittent episodes of hypercalcemia and

hypercalciuria.

Dentine matrix protein 1 (DMP1), ENPP1, or FAM20C

FAM20A/17q24, autosomal recessive

Characteristic dental defects (amelogenesis imperfecta,

gingival hyperplasia, impaired tooth eruption) and
nephrocalcinosis.

AGXT/ 2q36-37, alanine glyoxylate aminotransferase (AGT)

Nephrocalcinosis nephrolithiasis, renal impairment, and ESRD in 50%

patients by early adulthood. Nonrenal manifestations of systemic
oxalosis
include cardiac conduction defects, bone pain, increased risk of
fractures, and diminished visual acuity.

r
e
vi
e

7q11.23 28 genes including the elastin gene, ELN, and LIMK1

WilliamsBeuren Syndrome (WBS)

HOGA1, mitochondrial 4-hydroxy-2-oxoglutarate aldolase enzyme

Primary hyperoxaluria type 3

derived neurotrophic factor

(GDNF)
and receptor
tyrosine kinase (RET) genes Sporadic (nongenetic forms)
Medullary
sponge
kidney (MSK)

38

Abbreviations: dRTA, distal renal tubular acidosis; FGF23, fibroblast growth factor-23; PTH, parathyroid hormone; ROMK, renal outer medullary potassium.

Recurrent
Elfin
Patients
Patients
face,
calcium
with
supravalvular
with
PH
nephrolithiasis
PH
type
type
3aortic
generally
2 generally
stenosis,
and nephroc
presen
have
hy
GRHPR/9p11, decreased or absent activity
of glyoxylate reductase/mode
hydroxypyruvate reductase (GRHPR)
Gene/protein/inheritance
Clinical
manifestations
recurrent
nephrolithiasis
after
the age
of 6 nephrocalciyears,
and
don
bone disease. Chronic pain, recurrent episodes
abnormalities.
of have
urinary
Episodic
tract hypercalcemia
obstruction,
andand
infection;
and
hypercalciuria;
rarely
however,
progress
normal
to ESRD.
kidney

Table 1

ry hyperoxaluria type 2 Disorder

Continued

L Shavit et al.:
Nephrocalcinosis

revi
ew
excretion. These disorders include X-linked
hypophosphate- mia (XLH; mutant phosphateregulating endopeptidase on the X chromosome
(PHEX)), autosomal dominant hypophos- phatemic
rickets
(ADHR;
mutant
fibroblast
growth
factor23),
and
autosomal
recessive
hypophosphatemic
rickets
(ARHR;
mutant
dentine matrix protein 1 (DMP1), ENPP1, or
FAM20C). Treatment for XLH, ADHR, and ARHR is
with long-term oral
phosphate
supplements
and calcitriol that is often complicated by
nephrocalcinosis (up to 80% of patients with
XLH); this is believed to be the result of
intermittent episodes of hypercalcemia and
hypercalciuria from overtreatment with active
vitamin D or poor compliance with oral
phosphate supplementation.2325
Another recently reported genetic association
is with mutations in the insulin receptor that
are known
to
cause
severe
insulin
resistance, growth retardation, reduced fat
and muscle, and soft tissue overgrowth, and are
also
associated
with
hypercalciuria
and
nephrocalcinosis, but which have not been
reported in a kidney-specific knockout (KO)
mouse model.26 Thus,
perhaps
apart
from
hypercalciuria, seeking a common underlying or
unifying mechanism from genetic disorders in
which nephrocalcinosis is a feature has raised
more questions than provided answers. Indeed,
recently described mutations in FAM20A (see
FAM20C above) have been reported in enamelrenal syndrome (ERS), a rare autosomal recessive
disorder presenting with nephrocalcinosis and
characteristic dental defects
(amelogenesis
imperfecta,
gingival
hyperplasia,
impaired
tooth eruption), in which affected patients
are
hypocalciuric
rather
than
hypercalciuric.27,28
Although there is still no defined mechanism
for the nephrocalcinosis seen in ERS, we
propose
that
dysregulation
of
calcium
homeostasis either locally within the renal
interstitium, or perhaps also systemically,
may have a key role. As the protein product
of FAM20A
is locally secreted
in low
abundance in saliva and blood, and is also
expressed in the kidney, we suggest that de
novo interstitial calcification, rather than
intratubular crystallization, is biologically
plausible and may be primarily owing to the
normally high calcium transport and flux
across the renal tubular epithelium
that
must
be
prevented
from
depositing in
the
renal
interstitium;
therefore,
nephrocalcinosis may resemble in some way the
process
of
abnormal
metastatic
tissue
calcification. FAM20A is a secreted kinase
related to FAM20C that can phosphorylate
caseins and prevent CaPi precipitation in
milk; FAM20C mutations cause Raine syndrome,
in which there is ectopic calcification.29 Both
FAM20C and FAM20A can phosphorylate the
SIBLING
proteins
MEPE
(or
its
ASARM
products), DMP1 and osteopontin involved in
biomineralization
and
found
in
the
kidney.30,31 Given that the SIBLING proteins can
promote or inhibit mineralization, depending
on their form and phos- phorylation status,
such
an interaction could underlie the
nephrocalcinosis and enamel defect seen in
enamel-renal syndrome.
Distal renal tubular acidosis, a condition
in which tubular secretion of hydrogen ions
in the distal nephron is impaired,
Kidney International (2015) 88, 3543

resulting in metabolic acidosis, an alkaline

urine pH, hypocitraturia and hypercalciuria
with nephrocalcinosis, and metabolic bone
disease, can be due to mutations of the
erythrocyte anion exchanger (band 3, AE1) in
its autosomal dominant form and mutations of
subunits of the H+-ATPase (proton) pump in its
autosomal recessive forms.3234 As with Bartter
syndrome, hypokalemia in distal renal tubular
acidosis may contribute to hypocitraturia and
tendency to renal calcification and stones.
Another
multisystem
genetic
disorder,
WilliamsBeuren
syndrome,
is
caused
by
hemizygous
deletion
of
1.51.8
Mb
on
chromosome 7q11.23, encompassing 28 genes,
includ- ing
the
elastin
gene.
Affected
patients have a variety of
phenotypes
that
include
narrow
facial
features, supravalvular aortic stenosis or
other
vascular
disorders,
hypertension,
impaired
cognition,
short
stature,
and
endocrine, genitour- inary, auditory, dental,
ophthalmologic,
and
dermatologic
abnormalities. It has been reported that 5
50% of patients with WilliamsBeuren syndrome
experience
one
or
more
episodes
of
hypercalcemia that are generally mild and
asymptomatic. Hypercalciuria can accompany
the
hypercalcemia,
but
isolated
hypercalciuria can also occur, especially in
adults. Nephrocalcinosis is relatively rare,
found in o510% of patients undergoing renal
ultrasonography.35
However,
although
the
optimal
imaging
test
for
detecting
nephrocalci- nosis reliably is yet to be
determined, the available evidence suggests
that a combination of computed tomography
scan with either ultrasonography or kidneys
uretersbladder X-ray yields the most accurate
results.36
In MSK, which was originally considered to
be a sporadic disorder that can occur rarely
in families with developmental and growthrelated defects such as hemihypertrophy, a
recent study has shown that 50% of MSK
stone-forming patients have relatives with
milder
forms
of
MSK.37
Mutations
or
polymorphisms in genes that are primarily
involved in nephrogenesis, such as those
encoding
a
glial
cell
linederived
neurotrophic factor and receptor tyrosine
kinase, have been

found in a cohort of 55 apparently sporadic

MSK patients, supporting the hypothesis of a
possible disruption of the initial events
involved in nephrogenesis at the ureteric
bud
metanephric
mesenchyme
interface.
Although not all MSK patients have glial cell
linederived neurotrophic factor variants, and
some do not have a family history of the
disease,
both
genetic
heterogeneity
and
nongenetic forms of MSK are likely to exist.38
Finally, several genetic forms of primary
hyperoxaluria
have been described and associated with a
different
incidence
and
severity
of
nephrocalcinosis and renal impairment (Table
1). A recent study of over 100 patients with
confirmed primary hyperoxaluria looked at the
prevalence of nephro- calcinosis and the
relationship between stone burden and stone
recurrence, and nephrocalcinosis and renal
function.
The
findings
of
this
study
suggested
that
the
prevalence
of
nephrocalcinosis was 34% and its presence
was
associated
with
more
severe
renal
impairment.39
The contribution of monogenic disorders to
the
overall prevalence of kidney stone
disease and nephrocalcinosis has been studied
recently in a cohort of 272 genetically
unresolved individuals (106 children and 166
adults)
from
268
families
with
nephrolithiasis
(n = 256)
or
isolated
nephrocalcinosis (n = 16).40 A total of 50
likely causative mutations in 14 of 30
analyzed genes was detected, leading to a
molecular diagnosis in 14.9% of all cases. The
percentage of monogenic cases was notably high
in both the adult (11.4%) and pediatric
cohorts (20.8%). The cystinuria gene SLC7A9
(n = 19) was most frequently mutated in this
series.
Many other diseases have been described in
association
with
nephrocalcinosis.
The
largest clinical series is that of Wrong3 in
the United Kingdom that consists of 375
patients
with
macroscopic
(radiological)
nephrocalcinosis collected over more than 40
years
of
clinical
practice
in
London,
Manchester, Newcastle, and Dundee. Figure 1
shows
that
autonomous
hyperparathyroidism
(all
patients
had
primary
hyperparathyroidism), distal renal tubular
acidosis, and MSK
Oxalosis
Dent's disease
Renal papillary necrosis
Others

MacGibbonLubinsky
syndrome (ERS)
Idiopathic hypercalciuria
dRTA
Hypomagnesemiahypercalciuria

MSK

Sarcoidosis
Hypervitaminosis D
Milk-alkali syndrome

Autonomous
hyperparathyroidism

Undiscovered cases
Acetazolamide
Progressive
osteoporosis
Cortical NC

Figure 1 | Frequencies of the main clinical causes of nephrocalcinosis (NC) in a series of 375 patients presenting with
radiological NC. This chart is based on the largest clinical series of Wrong3 and consists of 375 patients with
macroscopic NC collected over more than 40 years of clinical practice in London, Manchester, Newcastle, and
Dundee. It shows that autonomous hyperparathyroidism, distal renal tubular acidosis (dRTA), and medullary sponge

kidney (MSK) are the most frequent clinical diagnoses in patients with radiological NC; 7% of patients had no
clear diagnosis. Others refers to WilliamsBeuren syndrome (WBS), Bartters syndrome, idiopathic renal Fanconi
syndrome, hypothyroidism, glucocorticoid-suppressible hyperaldosteronism, and severe acute tubular necrosis
(ATN).
Kidney International (2015) 88, 3543

are the most frequently associated clinical

diagnoses in this series.
Nephrocalcinosis has also been described in
kidneys
after
transplantation.
Rebound
hyperparathyroidism
and
associated
hypercalcemia are common after successful
renal transplantation and have been reported
to adversely affect graft function either
acutely, by inducing vasoconstriction, or
chronically, by leading to calcification in
the tubulointer- stitium of the transplanted
kidney.41
In
a
recent
prospective
observational cohort study of 303 incident
renal transplant recipients, with 21 on
cinacalcet
treatment
at
the
time
of
transplantation, CaPi deposits in the renal
transplant were observed in 33.3% and 25.7% of
posttransplant protocol biopsies at months 3
and 12, respectively.42 Interestingly, both
pretransplant
parathyroid
hormone
and
fibroblast growth factor-23 were found to be
independent predictors of nephrocalcinosis
that might be related to their phosphaturic
effect. However, pretransplant treatment with
cinacalcet did not seem to affect either the
need for posttransplant para- thyroidectomy
or the incidence of posttransplant nephrocalcinosis
in
this
study.42
Although
a
disturbance
of
mineral
metabolism
after
transplantation is likely to explain the
propensity to crystal formation, exposure to
potentially nephrotoxic immunosuppressive drugs
with associated epithe- lial cell injury may
aggravate crystal adhesion and deposition.
ANIMAL MODELS OF NEPHROCALCINOSIS

A variety of animals, including mice, rabbits,

rats, and pigs, have been used to create
experimental models of nephro- calcinosis in
an attempt to reveal the mechanisms governing
this common yet complex condition. In rats,
hyperoxaluria induced by vitamin B6 depletion
or
administration
of
glycolic
acid,
glyoxylate, sodium oxalate, ammonium oxalate,
ethylene glycol, or hydroxy-L-proline has been
shown to lead to CaOx deposition in the
kidneys, and additional administration of
vitamin D or calcium chloride aggravates this
process.43,44 In most rat studies, ethylene
glycol was used to induce hyperoxaluria, and
it resulted in increased urinary excretion of
oxalate
within
2
days,
established
hyperoxaluria within
3 days, CaOx crystalluria within 2 weeks,
and CaOx nephrolithiasis within 46 weeks.45
Crystals appear first in the tubular lumen
and are associated with damage to the
epithelial cells lining the crystal-containing
renal tubules. Thereafter,
many
of
the
crystals
move
to
intercellular
and
intracellular locations and eventually into
the interstitium.46 Translocation into the
interstitium is associated with inflammation
that is, attraction of leukocytes, monocytes,
and macrophages that have been proposed to
remove the crystalline material.47 Following
the loss of surface epithe- lium, which
results in loosening of tight junctions, the
deposits become exposed to pelvic urine and
continue to grow as large papillary stones.48
In the rat, hyperoxaluria has been shown to
increase
urinary
levels
of
alkaline

39

phosphatase, -glutamyl transpeptidase, and Nacetyl--glucoseaminidase, probably reflecting

proximal tubular injury.49 The role of Tamm
Horsfall protein (THP)

in the rat CaOx nephrocalcinosis model is less

well
understood.
In
humans,
THP
(uromodulin), a potent inhibitor of CaOx
crystal aggregation, is consistently present
in the stone matrix; decreased urinary
excretion of THP has been demonstrated in
patients with CaOx or CaPi nephrolithiasis.50
However, in rats with CaOx stones, studies
have shown a decrease or an increase in THP
expression and production.51,52
Additional
molecules
are
involved
in
crystallization and the inflammatory cascade
of
experimentally
induced
nephrocalcinosis:
osteopontin,
various
inter-
inhibitorrelated macro- molecules, bikunin,
prothrombin,
and
heparan
sulfate
are
substantially
increased
in
rats
with
nephrocalcinosis,
as
determined
by
the
expression of their respective mRNAs.44
Even though most rats with experimentally
induced hyperoxaluria develop CaOx renal
deposits,
reliable
mouse
models
of
nephrocalcinosis seem to be more difficult to
generate. Mice with hyperoxaluria alone
either do not produce or maintain any CaOx
crystals deposits
or form only a few
crystals in their kidneys.53,54 Manipulations
of
the
gene
for
alanine
glyoxylate
aminotransferase
or
anion
transporter
Slc26a6 have produced hyperoxaluric mice.
The alanine glyoxylate aminotransferasenull
mice present with severe hyperoxaluria, but
develop only a few CaOx crystal deposits in
their
kidneys.55
Mice
lacking
anion
transporter Slc26a6 (the chlorideoxalate
exchanger in the proximal tubule) also
40
3543

develop severe hyperoxaluria, but only those

with increased urinary calcium excretion
develop CaOx crystal deposits and bladder
calculi.44 Thus, it seems that experimental
induction
of produce
hyperoxaluria
alone is
not
sufficient
to
in
and additional
factorsnephrocalcinosis
suchmale
as mice,
concomitant
hypercalciuria
and
gender
appear to be critical factors for crystal
deposition. Mouse models with a disrupted/
sodium phosphate cotransporter gene (Npt2a
) exhibit increased urinary excretion of
phosphorus and hypophosphatemia that leads to
increased serum 1,25(OH)2 vitamin D levels,
increased expression of intestinal calcium
channels, intestinal calcium hyperabsorption,
hypercalcemia, and hypercalciuria.56 Using this
model, CaOx crystal deposits were successfully
produced in male Npt2a-null mice.44
Unlike CaOx stone mouse models, deposition
of CaPi crystals in mouse kidneys seems
to occur more easily;
crucially,
this
depends
on
THP
and
osteopontin. THP KO mice exhibit microcrystal
formation in their renal papillae, consisting
mainly
of
CaPi
(apatite)
and
located
interstitially or in the basement membrane
zone.57,58 Hypercalciuria and hyperoxaluria
induced by the administration of vitamin D3
and ethylene glycol, respectively, lead to
significant CaOx crystal deposition in the
kidneys in 76% of the THP KO mice, whereas no
crystal deposition can be detected in the
kidneys of wild-type mice.
Spontaneous interstitial deposits of CaPi
within the renal papillae were detected in a
small proportion of osteopontin KO mice.53
Lack of both osteopontin and THP proteins
caused interstitial renal crystallization in
39% of the double- null mice, associated with
elevated concentrations of urinary phosphate
and CaPi supersaturation.53
Kidney International (2015) 88,

Manipulations of the gene for Npt2a have

been shown to produce both tubular and
interstitial CaPi crystal deposition in male
and female Npt2a KO mice of different
ages.59 Moreover, sodiumhydrogen exchanger
regulator factor-1 NHERF-1null mice, which
also have very low expression of Npt2a,
develop
hypercalciuria,
hyperphosphaturia,
hyper- magnesuria, and uricosuria; however,
they show only a few interstitial CaPi
deposits in their renal papillae at 4854
weeks of age. By 72 months of age a
significant increase in interstitial CaPi
deposits is observed in these mice.45
Cortical and medullary nephrocalcinosis is
often detected in critically ill and lowbirth-weight preterm infants who were treated
especially (though not only) with furosemide
or vitamin D, and in children with inherited
renal tubulopathies with hypercalciuria, such
as Bartter syndrome. The mechan- ism by which
furosemide can cause nephrocalcinosis has been
attributed to hypercalciuria; however, a
recent rat model of CaPi (rather than the
more usual CaOx) nephrocalcinosis has been
described in which animals on a chloride-only
or sodium- and chloride-depleted diet, and
given furosemide, develop nephrocalcinosis.
Furosemide given alone on a control diet had
no effect, but rats on a sodium chloride
depleted
diet
without
furosemide
also
developed
significant
nephrocalcinosis.60
However, it is not clear whether CaPi
deposition in this model is mainly cortical
or medullary or both. An earlier rat model of
presumed CaPi nephrocalcinosis described by
Buck et al.61 involved regular intraperitoneal
injection of calcium gluconate, but the
calcium deposition was typically more cortical
than medullary. Thus, up to now, there are no
good animal models of medullary nephrocalcinosis due to CaPi deposition.
In conclusion, development of animal models
of
nephrocalcinosis
is
a
difficult
undertaking, and the models reproduce poorly
the
features
and
associations
seen
in
humans, confirming the complex interplay of
genetic and metabolic risk factors leading to
increased
urinary
excretion
of
calcium,
phosphate,
or
oxalate,
as
well
as
abnormalities
in
molecular
epithelial
transport
of
crystallization
inhibitors
required to trigger a process that can be
considered as nephrocalcinosis.
A PROPOSED MECHANISTIC EXPLANATION OF RENAL
CALCIFICATION IN NEPHROCALCINOSIS

Although
genetic
studies
have
provided
valuable insights into
the renal tubular pathways that regulate
calcium, phosphate, and oxalate handling, and
that predispose to metabolic disturbances
that may result in nephrocalcinosis, such as
increased
urinary
excretion
of
calcium,
oxalate, phosphate, or decreased excretion of
citrate, they do not provide a complete
explanation
of
the
complexity
of
the
mechanisms leading to renal calcification that
can be interstitial, intratubular,
or both.
Indeed, additional intratubular factors are
required to explain crystal retention and
adhesion to the tubular epithelium: decreased
urine volume, urinary supersaturation, the
presence of insufficient concentrations of
crystal
inhibitors
such
as
citrate,
magnesium, and various proteins (such as THP,
osteopontin,
bikunin,
urinary
prothrombin
fragment 1),

and the integrity of tubular epithelial

cells. A number of experimental and clinical
observations strongly suggest that crystals
do not adhere to the normally differentiated
tubular epithelium, but rather attach to
dedifferentiated/regenerating epithelial cells
that
express
multiple
crystal-binding
molecules such as sialic acidcontaining
proteins and/or phospholipids, phosphatidyl
serine, nucleolin-related protein, annexin II,
osteopontin, and hyaluronan on the luminal
surface of the tubular epithelium.6265 The
causal role of aberrant epithelial tissue
crystal adhesion has been demonstrated in
renal cell lines in vitro, in animal models, in
kidney transplant patients, and in neonates
in whom osteopontin and hyaluronan expression
are
closely
associated
with
nephrocalcinosis.6668 Although these factors
may
account
for
intratubular
crystal
formation
and
retention,
the
specific
pathogenic mechanisms leading to interstitial
crystal formation and deposition, including
the development of Randalls plaque, are still
unclear.1
However,
translocation
of
intratubular crystals and/
or de novo
interstitial calcification have been proposed.
Many factors have been put forward to
account for
intratubular crystal formation and retention,
but the specific pathogenic mechanisms
resulting in interstitial crystal formation, which must underlie
nephrocalcinosis in patients, have been
elusive, despite the many clinical and
genetic associations, and they are still the
subject of ongoing debate. Dysregulation of
calcium homeostasis locally within the renal
interstitium, and probably also systemically,
may have a key role in the pathogenesis of
nephrocalcinosis. Indeed, studying a rare
genetic disorder, the enamelrenal syndrome
mentioned earlier that presents with
abnormal enamel formation, impaired tooth
eruption, and nephrocalcinosis, has recently
delineated some of these processes. The
causative gene FAM20A was implicated in
this systemic disorder of calcium
homeostasis, and its protein product FAM20A
is locally secreted in low abundance in
saliva and blood. This association raises
the possibility that de novo interstitial
calcification,
rather
than
intratubular
crystallization,
is biologically plausible
in nephrocalcinosis and that it may be
primarily owing to increased calcium
transport and flux across the renal tubular
epithelium (Figure 2) that is normally
prevented from depositing in the
interstitium, and thus may resemble in some
way the process of abnormal metastatic
tissue calcification as the initiating event.
Indeed, a recent study of nephrocalcinosis in
mice has shown the importance of genes known
to be associated with ectopic mineralization,69 and the FAM20 family of secreted
protein kinases have as substrates the
phosphatonin-like peptides MEPE and DMP1
that can inhibit both bone and tooth
mineraliza- tion,70,71 and that have also been
detected in the kidney72 along with FAM20A28
(see earlier). Thus, we suggest that a complex
dysregulation between calcification
inhibitors and promoters, with similarities
to the process of vascular calcification,
may have a pivotal role in the development of
nephrocalcinosis. In this context, it is
currently thought that fetuin-A, matrix Gla
protein, osteoprotegerin, and pyrophos- phates

all

act

in

local

or

systemic manner
to prevent
41

Kidney International (2015) 88, 3543

Plasma Ca2+

Interstitium

Cells

Ca2+

Tubules

Ca2+

Proximal tubule

TAL
RP

Ca

2+

TL

Ca2+

Distal tubule

Fetuin-A, matrix Gla

protein,
osteoprotegerin,
pyrophosphates

Figure 2 | Simplified scheme of a proposed mechanism for

interstitial calcification in nephrocalcinosis. RP, Randalls
plaque; TAL, thick ascending limb of loop of Henle; TL,
thin (descending and ascending) limbs of loop of Henle.

calcification of the vasculature,31 and that

nephrocalcinosis
could
represent
another
process of unwanted calcification in which
the same or similar calcification inhibitors
can have a role locally in the renal
interstitium.
Over the past decade, a number of largescale epidemio- logical studies have provided
evidence for an association between kidney
stones and systemic conditions such as the
metabolic
syndrome,
hypertension,
chronic
kidney disease, and cardiovascular disease.
Our own recent study found that the abdominal
aorta is significantly more calcified in
calcium stone formers when compared with
healthy adults,72 suggesting that vascular
calcification may be an underlying mechanism
explaining reported associations between nephrolithiasis
and
cardiovascular
disease.
However, there is no study to date addressing
this issue in patients with nephrocalcinosis,
although renal interstitial calcification is
frequently
associated
with
disorders
of
extrarenal
organs
and
systems,
perhaps
pointing the way to a common underlying
process of abnormal calcium balance.
DISCLOSURE

All the authors declared no competing interests.

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