Combined proteomic and metabonomic studies in

three genetic forms of the renal Fanconi syndrome

Annalisa Vilasi, Pedro R. Cutillas, Anthony D. Maher, Severine F. M. Zirah,
Giovambattista Capasso, Anthony W. G. Norden, Elaine Holmes, Jeremy K.
Nicholson and Robert J. Unwin
Am J Physiol Renal Physiol 293:F456-F467, 2007. First published 9 May 2007;
doi:10.1152/ajprenal.00095.2007

You might find this additional info useful...

Supplemental material for this article can be found at:

http://ajprenal.physiology.org/content/suppl/2008/07/31/00095.2007.DC1.html

This article cites 60 articles, 20 of which can be accessed free at:

http://ajprenal.physiology.org/content/293/2/F456.full.html#ref-list-1

This article has been cited by 7 other HighWire hosted articles, the first 5 are:
Enhanced Excretion of Vitamin D Binding Protein in Type 1 Diabetes: A Role in Vitamin
D Deficiency?

Downloaded from ajprenal.physiology.org on March 22, 2011

Kathryn M. Thrailkill, Chan-Hee Jo, Gael E. Cockrell, Cynthia S. Moreau and John L. Fowlkes
J Clin Endocrinol Metab, January, 5 2011; 96 (1): 142-149.
[Abstract] [Full Text] [PDF]

Biomarkers of Fabry Disease Nephropathy

Raphael Schiffmann, Stephen Waldek, Ariela Benigni and Christiane Auray-Blais
CJASN, February, 2010; 5 (2): 360-364.
[Abstract] [Full Text] [PDF]

Urinary proteome pattern in children with renal Fanconi syndrome

Jens Drube, Eric Schiffer, Harald Mischak, Markus J. Kemper, Thomas Neuhaus, Lars Pape,
Ralf Lichtinghagen and Jochen H. H. Ehrich
Nephrol. Dial. Transplant., July, 2009; 24 (7): 2161-2169.
[Abstract] [Full Text] [PDF]

Urinary proteome pattern in children with renal Fanconi syndrome

Jens Drube, Eric Schiffer, Harald Mischak, Markus J. Kemper, Thomas Neuhaus, Lars Pape,
Ralf Lichtinghagen and Jochen H. H. Ehrich
Nephrol. Dial. Transplant., February, 18 2009; (): .
[Abstract] [Full Text] [PDF]

Evaluation of the proximal tubular function in hereditary renal Fanconi syndrome

Leo Monnens and Elena Levtchenko
Nephrol. Dial. Transplant., September, 2008; 23 (9): 2719-2722.
[Full Text] [PDF]

Updated information and services including high resolution figures, can be found at:
http://ajprenal.physiology.org/content/293/2/F456.full.html

Additional material and information about AJP - Renal Physiology can be found at:
http://www.the-aps.org/publications/ajprenal

This infomation is current as of March 22, 2011.

AJP - Renal Physiology publishes original manuscripts on a broad range of subjects relating to the kidney, urinary tract, and their
respective cells and vasculature, as well as to the control of body fluid volume and composition. It is published 12 times a year
(monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2007 by the
American Physiological Society. ISSN: 0363-6127, ESSN: 1522-1466. Visit our website at http://www.the-aps.org/.
Am J Physiol Renal Physiol 293: F456–F467, 2007.
First published May 9, 2007; doi:10.1152/ajprenal.00095.2007.

TRANSLATIONAL PHYSIOLOGY

Combined proteomic and metabonomic studies in three genetic forms of the

renal Fanconi syndrome
Annalisa Vilasi,5,6* Pedro R. Cutillas,1* Anthony D. Maher,3* Severine F. M. Zirah,3
Giovambattista Capasso,6 Anthony W. G. Norden,4 Elaine Holmes,3 Jeremy K. Nicholson,3
and Robert J. Unwin2
1
Ludwig Institute for Cancer Research and Department of Biochemistry and Molecular Biology and 2Centre for Nephrology
and Department of Physiology, University College London, London; 3Department of Biomolecular Medicine, Division of
Surgery, Oncology, Reproductive Biology, and Anaesthetics, Faculty of Medicine, Imperial College London, London;
4
Clinical Biochemistry, Addenbrooke’s Hospital, Cambridge, United Kingdom; 5Chair of Nephrology, Second University of
Naples, Naples; and 6Center of Mass Spectrometry, Proteomics, and Bioinformatics, Institute of Food Science, Consiglio
Nazionale Delle Ricerche, Avellino, Italy

Downloaded from ajprenal.physiology.org on March 22, 2011

Submitted 23 February 2007; accepted in final form 8 May 2007

Vilasi A, Cutillas PR, Maher AD, Zirah SF, Capasso G, Norden
AW, Holmes E, Nicholson JK, Unwin RJ. Combined proteomic and
metabonomic studies in three genetic forms of the renal Fanconi syn-
drome. Am J Physiol Renal Physiol 293: F456–F467, 2007. First pub-
lished May 9, 2007; doi:10.1152/ajprenal.00095.2007.—The renal Fan-
coni syndrome is a defect of proximal tubular function causing amino-
aciduria and low-molecular-weight proteinuria. Dent’s disease and Lowe
syndrome are defined X-linked forms of Fanconi syndrome; there is also
an autosomal dominant idiopathic form (ADIF), phenotypically similar to
Dent’s disease though its gene defect is still unknown. To assess whether
their respective gene products are ultimately involved in a common
reabsorptive pathway for proteins and low-molecular-mass endogenous
metabolites, we compared renal Fanconi urinary proteomes and metabo-
nomes with normal (control) urine using mass spectrometry and
1
H-NMR spectroscopy, respectively. Urine from patients with low-
molecular-weight proteinuria secondary to ifosfamide treatment (tubular
proteinuria; TP) was also analyzed for comparison. All four of the
disorders studied had characteristic proteomic and metabonomic profiles.
Uromodulin was the most abundant protein in normal urine, whereas
Fanconi urine was dominated by albumin. 1H-NMR spectroscopic data
showed differences in the metabolic profiles of Fanconi urine vs. normal
urine, due mainly to aminoaciduria. There were differences in the urinary
metabolite and protein compositions between the three genetic forms of
Fanconi syndrome: cluster analysis grouped the Lowe and Dent’s urinary
proteomes and metabonomes together, whereas ADIF and TP clustered
together separately. Our findings demonstrate a distinctive “polypeptide
and metabolite fingerprint” that can characterize the renal Fanconi syn-
drome; they also suggest that more subtle and cause-specific differences
may exist between the different forms of Fanconi syndrome that might
provide novel insights into the underlying mechanisms and cellular
pathways affected.
kidney; urine; mass spectrometry; NMR
THERE IS INCREASING INTEREST in applying “-omics” technologies
to discover new biomarkers and to explore pathophysiological
processes (39). There is also greater use of these methods in
* A. Vilasi, P. R. Cutillas, and A. D. Maher contributed equally to this work.
Address for reprint requests and other correspondence: R. J. Unwin, Centre
for Nephrology and Dept. of Physiology, Royal Free and Univ. College
Medical School, Rowland Hill St., London NW3 2PF, UK (e-mail:
robert.unwin@ucl.ac.uk).
combination to obtain a multilayered description of diseases at
different levels of biomarker organization (17, 46). In the
present study, we have combined proteomic and metabonomic
methods to try and obtain deeper insights into the molecular
processes underlying the renal Fanconi syndrome (FS), a
disorder that can exist in several forms and can arise from
different genetic mutations.
Dent’s disease and Lowe syndrome are X-linked genetic
forms of the renal FS with impaired proximal tubular function.
An autosomal dominant idiopathic form (ADIF) has also been
described (5). The defect in the proximal tubule results in a
failure to reabsorb a variety of filtered substances, particularly
low-molecular-weight proteins (LMWP) and peptides, leading
to their increased excretion in urine with other substances like
glucose, uric acid, phosphate, and amino acids. Dent’s disease
typically presents with renal stones, nephrocalcinosis, and
reduced renal function; it is due to mutations in CLCN5 on the
X chromosome, which encodes the chloride channel ClC-5.
This channel is expressed mainly in kidney proximal tubular
cells, which can endocytose filtered polypeptides and proteins
(13, 45, 47, 57). This endocytic process is receptor mediated
(12, 19, 41) and involves the multiligand receptor proteins
megalin and cubilin. The role of ClC-5, together with a
V-ATPase proton pump, is thought to be to acidify the early
endosomes, which is necessary to dissociate the receptor-
ligand complex as part of the normal reabsorptive uptake of
proteins and polypeptides from tubular fluid, as well as the
regulated endocytosis of some plasma membrane transport
proteins (2). Disturbed endosomal acidification in proximal
tubular cells may underlie the impaired reabsorption and in-
creased urinary losses of solutes and LMWP that occur in FS
(32, 37). Lowe syndrome (also known as oculocerebrorenal
syndrome) is a rarer X-linked multisystem disorder that is
associated with mental retardation, lens cataracts, glaucoma,
muscular hypotonia, growth defects, and renal failure (with
LMWP); it is due to a mutation in OCRL1 (8), which encodes
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

F456 0363-6127/07 $8.00 Copyright © 2007 the American Physiological Society http://www.ajprenal.org
 ANALYSIS OF RENAL FANCONI URINE BY MS AND NMR
a phosphatidylinositol (PtdIns)-(4,5)-bisphosphate (P2) 5-phos-
phatase present in the trans-Golgi network and in early endo-
somes (48). This enzyme belongs to the a PIP2 5-phosphatase
type II group and hydrolyzes PtdIns(4,5)P2 to PtdIns(4)P (60).
Both its substrate and product are associated with clathrin-
mediated trafficking: the former predominantly at the plasma
membrane and the latter at the Golgi (11). PtdIns-(4,5)-
bisphosphate (P2) 5-phosphatase is the major hydrolyzing en-
zyme in normal human kidney proximal tubule cells and is
absent in cells grown from Lowe syndrome patients, in which
PtdIns(4,5)P2 accumulates (43, 59). How loss of PtdIns-(4,5)-
bisphosphate (P2) 5-phosphatase activity in Lowe syndrome
causes the eye, brain, and kidney defects that occur is still
unclear. As in Dent’s disease, receptor-mediated endocytosis is
affected, although a direct link between OCRL and defective
endocytosis has not so far been demonstrated. The gene defect
in ADIF, which is phenotypically very similar to Dent’s
disease, has not been defined so far, although it has been
mapped to a large segment of chromosome 15 (33). If the final
common pathway for these genetic forms of FS is receptor-
mediated endocytosis, then their gene products should interact
in some way and this might be reflected in their respective
urinary proteomes and/or metabonomes, which was the rea-
soning behind this exploratory study.
Thus we describe a qualitative and quantitative comparison
of the urinary proteomes and metabonomes of Lowe syndrome,
Dent’s disease, ADIF, tubular proteinuria (TP), and normal
subjects (with normal renal function) (35). Two different
comparative proteomic strategies using mass spectrometry
(MS) were adopted to enhance the number and confidence of
protein identification (31, 34, 58). Two-dimensional gel elec-
trophoresis (2DE) (36, 49) was used first to compare Dent’s,
Lowe, and normal urinary protein patterns. The identities of
separated proteins present in gel spots were determined by
peptide mass fingerprinting using matrix-assisted laser desorp-
tion ionization (MALDI) time-of-flight MS (16). In a parallel
investigation, the resulting peptide products of digested pro-
teins (with an internal standard; IS) were then sequenced and
quantified by online liquid chromatography tandem MS (LC-
ESI-MS/MS) (21).
We also used 1H-NMR-based metabonomics, a well-estab-
lished and highly reproducible analytic technique, which has
been used successfully in the diagnosis and monitoring of
various diseases and toxicities, and also for biomarker discov-
ery (6). Metabolic profiling in this way can facilitate interpret-
ing multiparametric responses in the complex metabolic pro-
files of biofluids to genetic or pathophysiological stimuli (40).
Altered metabolite excretion in urine is also a consequence of
genetic mutation in FS patients, and so 1H-NMR-based meta-
bonomics may complement proteomic methods to increase the
range of urinary compounds that can be analyzed.
The results of our metabonomic and proteomic studies were
found to be self-consistent and indicated that the molecular
composition of FS urine is very different from that of normal
subjects, as previously reported (19). However, we also found
differences in the protein and metabolite compositions between
the different forms of FS studied, which suggest that different
reabsorptive pathways and mechanisms are differentially af-
fected and that this analytic approach may help in elucidating
the underlying defect(s).
AJP-Renal Physiol • VOL
F457
MATERIALS AND METHODS
Subjects. We studied eight male patients with X-linked Dent’s
disease, all with previously characterized mutations of CLCN-5;
seven male patients and one female with Lowe syndrome, all char-
acterized by mutations in OCRL; and eight normal subjects (41). In
addition, three ADIF male patients and two male subjects with
ifosfamide-induced TP, but without full-blown FS, were also studied.
All patients were between 35 and 50 yr old.
Control urine designated as “normal” was collected from eight
male subjects with no history of renal disease and aged between 40
and 50 yr. Patients and normal volunteers gave informed consent for
the collection and analysis of their urine; 50 ml of early morning urine
were collected, rapidly frozen in liquid nitrogen, and stored at ⫺80°C
until analyzed.
Urine samples were concentrated and separated from organic salts
by solid-phase extraction (SPE) using a reverse-phase (RP) C18
POROS R2 20 (Applied Biosystem, Warrington, UK) as the station-
ary phase. The protein concentration of extracted proteins was deter-
mined using the standard Bradford assay (4). To determine both
qualitative and quantitative differences, two different analytic pro-
Downloaded from ajprenal.physiology.org on March 22, 2011
teomic strategies using MS were adopted (23, 42).
2D-gel separation of Lowe syndrome and Dent’s disease urinary
proteins followed by peptide mass fingerprinting. Equal amounts (150
␮g) of normal, Lowe, and Dent’s urinary proteins were loaded on
Immobiline DryStrip (pH ⫽ 3–10, 18 cm; Amersham GE Healthcare
Life Sciences) and focused for 24 h. The second dimension was
12.5% of acrylamide gels, which were subsequently stained using
Coomassie brilliant blue (CBB) G-250. Gel runs were in triplicate,
and individual (that is, not pooled) urine samples were analyzed. Gels
were scanned using a densitometer (Bio-Rad 800), and gel images
were analyzed using Melanie software, which allows spot matching
and quantitation. To compensate for image differences caused by
variations in experimental conditions, spot intensity was expressed as
% optical density (OD), defined as
冢 /兺 冣
N
%OD ⫽ OD ODi ⫻ 100 (1)
i⫽1
where N is the total spot number. To assess the correlation between
%OD values of two protein patterns, Student’s t-test was used to
determine whether the value of their Pearson correlation coefficient R
was statistically different from 0 at P ⬍ 0.05. All the spots that could
be visualized were excised from the gels, in-gel digested with trypsin
(Sequencing Grade Modified Trypsin, Porcine, Promega, Madison,
WI), and analyzed by MALDI-MS. MALDI mass spectra of 2DE
spots were acquired using an Ultraflex time-of-flight (TOF)/TOF
instrument (Bruker Daltonics, Bremen, Germany).
LC-ESI-MS/MS analysis of tryptic peptides of different forms of FS
urine proteins. Desalted proteins were also separated by 1D SDS-
PAGE. Forty micrograms of extracted proteins from individual sam-
ples of each subtype of FS and control were pooled. Pooled samples
were representative for each class. Gel lanes corresponding to total
urinary proteomes were cut into five sections of approximately equal
molecular weight range. Proteins present in these sections were in-gel
digested using standard protocols (18). Peptides derived from trypsin
digestion of normal, Dent’s, Lowe, ADIF, and TP urine proteins were
spiked with 2 pmol of a protein internal standard (IS; carbonic
anhydrase, which was added to the gel pieces just before in-gel
digestion), and separated by RP-HPLC. Peptides eluted from the C18
RP column were mass analyzed and sequenced online by a hybrid
quadrupole-time of flight (Q-Tof) mass spectrometer (Micromass)
equipped with a nanoelectrospray (nanoESI) ion source.
Ions were automatically selected for MS/MS by the automatic
switching program in MassLynx 4.0 when their intensity in the MS
survey scan was ⬎25 ion counts/s. MS/MS spectra were smoothed
293 • AUGUST 2007 • www.ajprenal.org
F458 ANALYSIS OF RENAL FANCONI URINE BY MS AND NMR

Fig. 1. Analysis of Dent’s disease, Lowe syndrome, and normal (control) urinary proteomes by 2-dimensional gel electrophoresis (2DE). Desalted proteins were
analyzed by 2DE using standard protocols, visualized by Coomassie brilliant blue (CBB), and optical densities (%OD) for each protein spot were obtained by
densitometry. Numbered spots returned significant “hits.” The identity of proteins determined by matrix-assisted laser desorption ionization time-of-flight mass
spectroscopy (MALDI-TOF-MS) is listed in Table 1.

Downloaded from ajprenal.physiology.org on March 22, 2011

(mean smooth at 2 channels twice) and centroided (at 80% top).
Mascot used uninterpreted MS/MS data to interrogate the NCBI
protein database restricted to mammalian entries. Allowed mass
accuracies were 100 ppm and 150 millimass units for parent and
fragment ions, respectively. Protein hits were accepted when they had
statistically significant Mascot scores (P ⬍ 0.05) and at least two
peptides matched the protein entry.
Quantification was derived by chromatographic peak integration as
described previously (1, 10, 20, 25). We used an in-house computer
program to extract peak areas and heights, which were taken as peptide
ion intensity readings. Ion intensities were normalized for each analyte
peptide ion to those of the IS; final values were normalized to the addition
of the total peak heights for each particular gel fraction.
Hierarchical “cluster analysis” of quantitative LC-MS/MS data was
performed using Cluster and Treeview (24) by complete linkage
clustering and the Pearson correlation (uncentered) similarity metric
method for both genes and arrays. Data were log2-converted before
cluster analysis.

Table 1. Proteins identified by peptide mass fingerprinting that returned significant “hits” and their corresponding spot
number reported in Fig. 1
NCBI Accession No. Protein Name Lowe Spot No. Dent Spot No. Control Spot No.

gi 1942629 ␣-1-Anti-trypsin 330b 374b 201b

gi 69991 ␣-1-B-glycoprotein 118 120
gi 4502005 ␣-2-HS-glycoprotein 216b 216 208b
gi 29170378 ␣-1-Acid glycoprotein 2 307 290
gi 11275302 Anti-TNF-␣ antibody light-chain FAB fragment 331b
gi 89574029 Mitochondrial ATP synthase, H⫹-transporting F1 253b
␤-subunit
gi 999513 Anti-thrombin 183 164 170
gi 90108664 Lipid-free human apolipoprotein A-I 423b 423 422b
gi 6573461 ␤-2-Glycoprotein-I 194 191
gi 4502067 ␣-1 Microglobulin 367b 374b 422
gi 553181
gi 15079348 Angiotensinogen 202 264 202b
gi 178779 Apolipoprotein A-IV 275b 309b 315
gi 3745750 X-ray crystal structure of C3D: a C3 fragment and 330b 331
ligand for complement receptor 2
gi 67782358 Complement factor B 60 61
gi 1827805 Complement factor D 419 419b
gi 758073 Complement factor H 263b 265b
gi 4504893 Kininogen 182 186 182b
gi 55959887 Peroxiredoxin 1 343b
gi 15988024 Pigment epithelium-derived factor 259 253 244
gi 6063691 Porin isoform 1 308
gi 230284 Retinol-binding protein 39 427b 373
gi 4557871 Transferrin 103 104 189
gi 28373620 Vitamin D-binding protein 219b 221 221b
gi 4699583 Zinc-␣-2-glycoprotein 309 310
gi 386789 Hemopexin 143 148b
gi 9857753 IgG1 heavy chain 231b 232b
gi 38044288 Gelsolin 75 76
gi 3212456 Albumin 155 261b 178
gi 223057 Fibrin 368 368b
gi 59850812 Uromodulin 81
%Optical density (OD) values of the proteins in bold are correlated in Fig. 2.

AJP-Renal Physiol • VOL 293 • AUGUST 2007 • www.ajprenal.org

 ANALYSIS OF RENAL FANCONI URINE BY MS AND NMR
1
H-NMR spectroscopy. In contrast to proteomic analysis, prepara-
tion of biofluids for metabonomics is more straightforward, requiring
only the addition of buffer to urine samples. Thawed samples were
centrifuged at 13,000 g for 10 min; 200-␮l aliquots were then added
to 96-well plates containing 200 ␮l H2O and 200-␮l solution of 0.2 M
phosphate buffer (pH 7.4), 1 mM sodium trimethylsilyl [2,2,3,3-
2
H4]propionate (TSP), and 3 mM NaN3 in 20% D2O. Spectra from
individual (not pooled) samples were acquired on a 600-MHz Bruker
DRX spectrometer fitted with a 5-mm flow injection probe connected
to a Bruker Efficient Sample Transferring (BEST) system (Bruker
Biospin, Karlsruhe, Germany) employing a Gilson 215 liquid handler
(Gilson, Middleton, WI). 1D 1H-NMR spectra were acquired with a
pulse sequence of the form d1 ⫺ ␲/2x ⫺ t1 ⫺ ␲/2x ⫺ tm ⫺ ␲/2x, where
␲/2x represents a 90° hard pulse along the x-axis, d1 is a relaxation
delay, t1 is a short delay (4 ␮s), and tm is the mixing time (100 ms).
The resonance of H2O (approximately ␦4.7) was selectively irradiated
during d1 and tm. For each sample, 128 transients were collected into
32k data points. Before Fourier transformation, the free-induction
decay (FID) was multiplied by an exponential line-broadening factor
of 1 Hz. Spectra were phased in XWINNMR (Bruker) and referenced
to the TSP resonance (␦0.00) using in-house software (MetaSpectra
F459
mal, Lowe, and Dent’s urinary proteins were loaded on IPG
strips. After electrophoretic separation and CBB G-250 stain-
ing, all visualized spots were excised from each of the three
gels displayed in Fig. 1, and the identities of these spots were
determined by MALDI-MS. Labeled spots shown in Fig. 1
correspond to those proteins identified that returned significant
“hits,” as listed in Table 1. Identification details and spectra are
described in more detail in Supplemental Information S2. Most
of the proteins identified are of plasma origin and are normally
reabsorbed by receptor-mediated endocytosis in the proximal
tubule.
Several proteins, such as complement factor D, zinc-␣-2-
glycoprotein, apolipoprotein IV, vitamin and prosthetic group
carriers like vitamin D binding protein, retinol binding protein,
transferrin hemopexin, and growth factors [such as pigment
epithelium-derived factor], were found in significantly higher
amounts in Lowe and Dent’s urine; they represented a larger
proportion of the urinary proteome compared with normal
urine. In contrast, uromodulin and kininogen, proteins of renal

Downloaded from ajprenal.physiology.org on March 22, 2011

v3.0, O Cloarec, Imperial College London, London, UK). origin that are not involved in receptor-mediated endocytosis,
1
H-NMR spectral processing and multivariate analysis. 1H-NMR were reduced or absent in FS urine. The findings in Dent’s
data acquired from complex biofluids like urine (and especially from urine confirm our earlier reported observations (19).
human blood plasma) are information rich. Each metabolite gives rise Just on visual inspection of the 2D gels, it is apparent that
to several peaks in the spectra, with each peak varying in multiplicity the proteomic patterns of the Dent’s and Lowe FS urine are
(singlets, doublets, triplets, etc.), and some peaks overlapping with
neighboring peaks. Analysis of such complex data sets is made
very similar (Fig. 1). The actual values of the gel spots’ %ODs
possible by the application of unbiased statistical (chemometric) (of the proteins shown in bold and in the same order as listed
techniques, such as principal components analysis (PCA) and orthog- in Table 1) for Dent’s vs. Lowe correlate significantly (P ⬍
onal projections to latent structures by partial least squares discrimi-
nant analysis (O-PLS-DA). An NMR spectrum is a plot of intensities
(y-axis) over frequency (x-axis), with each molecule giving rise to one
or more peaks with a specific frequency and intensity that are directly
proportional to their respective concentrations in the sample. For
chemometric analysis, we consider each frequency coordinate (each
point on the x-axis) as a variable having a value defined by the
intensity at that point. In general, it is a feature of NMR spectroscopy
that from one spectrum to the next a given metabolite will have peaks
at the same frequency, although there is still minor peak position
variation for some metabolites, because of differences in salt content
and/or pH from one sample to the next.
Before chemometric analysis, spectral data regions corresponding
to residual water (␦4.68 ⫺ ␦4.93) and urea (␦5.53 ⫺ ␦6.16) were
removed to eliminate variations associated with presaturation. The
data were then normalized such that the total integral of the remainder
of each spectrum was a constant. PCA and O-PLS-DA (14, 50)
models were constructed using MetaSpectra. Statistical TOtal Corre-
lation SpectroscopY (STOCSY) was performed on the data set to
detect statistical correlations between resonances within the spectra
(15) and to facilitate assignment. Visualizing these correlated reso-
nances was facilitated by back projecting the unit variance as a
colored heat map on the O-PLS coefficient plot.
Database searches. Raw data from mass spectra were submitted to
Mascot, a software search engine designed for high-throughput and
automated interpretation of mass spectrometric data derived from
polypeptides (44). This software was used to interrogate the NCBI
database, as described in Supplemental Information S1 (all supple-
mentary material is available in the online version of this article on the
AJP-Renal Physiology web site).

RESULTS

Analysis of urinary polypeptides of Dent’s disease and Lowe

Fig. 2. Correlation of %OD values of the proteins in bold in Table 1.
syndrome by 2D gel electrophoresis. We compared the protein A: control vs. Dent’s. B: Lowe vs. Dent’s. C: control vs. Lowe. R is the
patterns present in the urine of Lowe and Dent’s patients with Pearson correlation coefficient. Note the weak correlation in A and C compared
normal individuals by 2DE. Equal amounts (150 ␮g) of nor- with the strong (underlined) correlation in B.

AJP-Renal Physiol • VOL 293 • AUGUST 2007 • www.ajprenal.org

F460 ANALYSIS OF RENAL FANCONI URINE BY MS AND NMR

0.05) (see Fig. 2B) in contrast to their correlation with normal
urine (P ⬎ 0.05) (see Fig. 2, A and C).
Analysis of urinary polypeptides in four different forms of FS
by LC-ESI-MS/MS. To determine whether other forms of the
FS also have similar patterns of urinary proteins, we used a
label-free quantitative proteomics approach (peak area with
internal standard) that allows exhaustive quantitative cross-
comparison of an unlimited number of proteomes (20). This
strategy (Fig. 3A) involves separating pooled protein mixtures
in parallel by 1D-SDS-PAGE. After staining, gel lanes were
cut into five sections (fractions) of equal molecular weight,
regardless of stain intensity. Proteins present in these gel
sections were digested using trypsin, and the peptides produced
were sequenced and quantified by LC-MS/MS. To illustrate
how this analysis was done, see Fig. 3B, which illustrates some
examples of the peptides present in fraction 4 derived from
hemopexin, uromodulin, and carbonic anhydrase (which is the
IS added to gel pieces before in-gel digestion). Peptides were
selected for MS/MS and quantified as described in MATERIALS
AND METHODS. Figure 3B shows that hemopexin was more

Downloaded from ajprenal.physiology.org on March 22, 2011

Fig. 3. Scheme for the identification and quantification of urinary proteins by liquid chromatography tandem MS (LC-MS/MS). A: experimental design for the
quantitative analysis of Fanconi syndrome (FS) and control urinary proteins. B: examples of extracted ion chromatograms of the indicated proteins. The areas
in integrated chromatographic peaks were used to derive quantitative information. C: examples of proteins found in fraction 4.

AJP-Renal Physiol • VOL 293 • AUGUST 2007 • www.ajprenal.org

 ANALYSIS OF RENAL FANCONI URINE BY MS AND NMR
abundant in FS urine, whereas uromodulin was more abundant
in control urine. Nine peptide ions of the IS were identified. By
normalizing all protein-derived peptides to the IS peptide ions,
it was possible to use the signal intensities in LC-MS to derive
quantitative information from MS across samples (20). Figure
3C shows some examples of proteins that were quantified in
this way in fraction 4. Quantification details of proteins iden-
tified are listed in Supplementary Table A.
Comparative analysis of three forms of the FS, two cases of
TP, and control urinary proteomes confirmed our previous
findings (19, 21, 22) and the data presented before (Figs. 1 and
2), indicating that the proteomes in FS are quantitatively and
qualitatively different from control. A good example is shown
in Fig. 4, in which uromodulin is seen to be the most abundant
protein in control urine, whereas it is albumin in FS urine.
Other quantitative differences between control and FS urine are
apparent in Fig. 4.
The ratios of the amounts of FS urinary proteins relative to
the same protein in normal urine were similar among the
different forms of the FS, with no obvious differences in the
overall patterns by visual inspection (Fig. 4). However, an
unbiased and unsupervised (i.e., assumes no a priori knowl-
edge about the origin of the sample) analysis of the data (using
the “clustering” tools designed for analysis of gene microarray
data) (see above and legend to Fig. 5) clustered Lowe and
Dent’s urinary proteomes together, whereas ADIF and TP were
clustered separately and together (Fig. 5). All FS proteomes
clustered separately from control. These results suggest (and
confirm) that the composition of the Lowe and Dent’s pro-
Fig. 4. Comparison of the quantities of the most abundant proteins in FS and normal urine. Peptides derived from each identified protein were added. The
comparison shown here is of the 13 most abundant proteins present in control urine.
AJP-Renal Physiol • VOL
F461
teomes is very similar, yet subtly different from ADIF and TP
urinary proteomes, which are more similar to each other.
NMR analysis of FS and control urine. 1H-NMR analysis of
FS and control urine by NMR spectroscopy can detect and
analyze the chemical fraction in urine that cannot be detected
by standard MS -based proteomics. To complement the pro-
teomics data, we looked for differences among the metabolite
compositions in the different forms of FS and made compari-
sons with control. Nineteen urine samples in total were ana-
lyzed by 1H-NMR spectroscopy. The samples were obtained
from four patients with Dent’s disease, five with Lowe syn-
drome, three with ADIF, two with isolated TP, and five normal
subjects. A representative 1H-NMR spectrum from each of the
five subclasses is shown in Fig. 6. The analysis of such
complicated spectra is greatly facilitated by chemometric
methods, such as PCA and O-PLS-DA (51). All spectra were
initially subject to PCA, an unsupervised pattern recognition
method that considers each data point in the frequency dimen-
sion as an independent variable. PCA presents an overview of
Downloaded from ajprenal.physiology.org on March 22, 2011
the data that can reveal grouping of observations, trends, and
outliers in a data set. A PCA model was constructed on 18
samples from this data set (one was removed due to contami-
nation). Figure 7A shows that normal subjects were clearly
separated from FS patients. There was also clustering of the
metabolic profiles of urine from patients with Dent’s disease
and Lowe syndrome and patients with TP and ADIF. Within
the first three PCs (comprising 69.8% of the total variance in
the dataset), all five subclasses were mutually exclusive. To
eliminate the possibility that paracetamol metabolites were
293 • AUGUST 2007 • www.ajprenal.org
F462 ANALYSIS OF RENAL FANCONI URINE BY MS AND NMR

Downloaded from ajprenal.physiology.org on March 22, 2011

Fig. 5. Patterns of FS and normal urinary proteins. LC-
MS/MS analysis led to the identification of 130 proteins
in 5 fractions. Some of these proteins were present in
more than one fraction. The data were analyzed by
average hierarchical cluster analysis: complete linkage
clustering was performed, as for genes and arrays, using
a Pearson correlation (uncentered) similarity metric
method (see text); green for downregulation, red for
upregulation, and black for no change. Note that the
control urinary proteome clustered separately from the
four FS proteomes; that Dent’s and Lowe clustered
together, and separated from ADIF and TP, which clus-
tered together.

AJP-Renal Physiol • VOL 293 • AUGUST 2007 • www.ajprenal.org

 ANALYSIS OF RENAL FANCONI URINE BY MS AND NMR
Fig. 6. Representative 1H-NMR spectra from each subtype of FS and controls.
Spectra were Fourier transformed with line broadening corresponding to 1 Hz
and referenced to an internal standard (TSP, ␦0). Units on the ordinate are
arbitrary.
influencing the separation, PCA models were also constructed
for restricted regions of the spectra known predominantly to
contain resonances from amino acids (i.e., ␦0.5 to ␦2.0). Again,
control subjects were clearly separated from FS patients and,
within FS subtypes, Lowe and Dent’s patients separated from
ADIF and TP (data not shown).
Further chemometric analysis was carried out on the 1H-
NMR spectra using O-PLS-DA (50). This is a supervised
statistical approach that takes into account the class of sample
being analyzed. Constructing O-PLS-DA models from full-
resolution urinary 1H-NMR spectra allows identification of the
metabolites most significant in discriminating between two or
more classes of samples to a level of accuracy unobtainable
with bucketed spectra (29). Figure 7, B–E, presents O-PLS
coefficient plots for four pairwise models from subclasses or
groups of subclasses. These plots have been color-coded to
display the relative significance of each variable to the discrim-
ination, with red-orange colors being most significant, and
blue-green colors being least significant. The sign and intensity
of each variable indicate its covariance with the respective
sample class. In Fig. 7B, data from Dent’s and Lowe patients
were grouped together and compared with controls. The
O-PLS coefficient plot identified several resonances significant
in discriminating the two classes, such as a doublet at ␦1.48,
and multiplets at ␦1.71 and ␦1.91, in Dent’s and Lowe patients,
and singlets at ␦2.34 and ␦4.44. To facilitate assignment of
these, STOCSY analysis (15) was performed on the maxima of
each group of highly significant data points (corresponding to
the apex of an NMR signal). This allowed identification of
several potential biomarkers for each FS subtype. Controls
exhibited higher levels of N-methylnicotinic acid and a mole-
cule with a spectral pattern consisting of a singlet at ␦2.35 and
an apparent doublet of doublets centered at ␦7.25, the latter
being p-cresol sulfate. Dent’s and Lowe patients had higher
levels of the neutral amino acids alanine and glycine and the
basic amino acid lysine. Urinary profiles of Dent’s and Lowe
patients were directly compared to identify specific differences
between these two types of FS. Figure 7C shows the coefficient
AJP-Renal Physiol • VOL
F463
plot from the O-PLS-DA model, highlighting resonances more
significant in the discrimination. Assignment tables combined
with STOCSY revealed these to be hippurate and creatinine in
Dent’s disease, while relatively higher concentrations of ala-
nine, glycine, lysine, and lactate were more likely to be found
in urine from patients with Lowe syndrome. However, it
should be noted that the cross-validation parameter Q2 is low
(0.11), limiting its interpretability, attributable to the small
sample size and the relatively small difference between these
two disease classes.
An O-PLS-DA model was also constructed to compare urine
from patients with ADIF with controls. Figure 7D shows the
O-PLS coefficient plot for this model, showing many highly
significant (red) positive regions. STOCSY analysis and as-
signment tables showed these belong mainly to glucose, along
with the neutral branched-chain amino acids valine and leucine
compared with controls. Visual inspection of the data also
showed ADIF patients had considerably lower levels of citrate
in their urine compared with controls; but this was not detected
Downloaded from ajprenal.physiology.org on March 22, 2011
by O-PLS analysis due to the significant peak-positional vari-
ation in the citrate resonances. Finally, ADIF patients were
directly compared with the grouped Dent’s and Lowe patients’
urine. The O-PLS coefficient plot, shown in Fig. 7E, showed
specific and significant differences in the metabolic profiles:
urine spectra from ADIF patients were significantly higher in
glucose, valine and, to a lesser extent, leucine, while lysine was
more significant in discriminating Dent’s and Lowe patients as
a group.
DISCUSSION
Applying -omics science to physiology and clinical medi-
cine faces several practical problems, some of which are still
technical, but a critical one (similar to that in molecular
genetics) is how well defined or characterized (“phenotyped”)
are the animal models or patient groups under study. Only if
robust subclasses are defined can valid comparisons be made
between normal, disease and disease variants, and potential
biomarkers identified. Moreover, data analysis strategies are
also needed that emphasize differences in patterns, rather than
in discrete measurements of single variables, as the greater the
number of parameters defining a disease, the greater the reli-
ability of any diagnostic test. Therefore, the aims of this study
were twofold: 1) to provide and validate an innovative and
more comprehensive analysis and characterization of urine
composition by combining proteomic and metabonomic tech-
niques; and 2) to test the hypothesis that in defined FS, urine
profiling in this way might provide mechanistic insights, or
clues, as to the underlying defects and pathophysiology, as well
as a means to refine the different FS phenotypes.
Several qualitative and quantitative differences in the pro-
teomic and metabonomic profiles of the different genetic forms
of FS patients compared with normal subjects were found
using MS and NMR. A preliminary study was conducted on
Dent’s disease and Lowe syndrome urinary proteins with two
different proteomic methods, 2DE and LC-MS/MS. This was
done because the physiochemical heterogeneity of proteins
(which is the basis of their diverse biological functions) makes
a single analytic approach inadequate for a comprehensive
analysis of all proteins in a given sample (28). As an alternative
to 2DE, we used liquid chromatography for protein or proteo-
293 • AUGUST 2007 • www.ajprenal.org
F464 ANALYSIS OF RENAL FANCONI URINE BY MS AND NMR

Downloaded from ajprenal.physiology.org on March 22, 2011

Fig. 7. Principal components analysis (PCA) and orthogonal projections to latent structures by partial least squares discriminant analysis (O-PLS-DA) output
from 1H-NMR data from FS patients’ urine. A: 3D PCA scores plot for the first 3 principal components. Each data point represents 1 NMR spectrum (hence
1 patient) of reduced dimensionality, and the closer 2 data points are to each other in PC “space,” the more similar the corresponding metabolic profile. Numbers
in brackets represent the percentage of the variance explained in each component; key ⫽ blue for Dent’s; red for TP; green for ADIF; magenta for Lowe; black
for control. B–E: O-PLS coefficients plotted as a function of chemical shift color-coded according to significance in differentiating between pairs of groups.
Numbers in brackets are the cross-validation parameter Q2Yhat. B: Dent’s and Lowe (as a group) vs. normal (Q2Yhat ⫽ 0.74). C: Dent’s vs. Lowe (Q2Yhat ⫽
0.11). D: ADIF vs. normal (Q2Yhat ⫽ 0.91). E: ADIF vs. Dent’s and Lowe (as a group) (Q2Yhat ⫽ 0.87).

lytic product separation of FS and normal urine. With this
strategy, polypeptide detection is less discriminatory of the
physicochemical properties of the analyte. For example, pro-
teins such as megalin, apolipoprotein A-II, apolipoprotein
AJP-Renal Physiol • VOL
C-III, ␤-2-microglobulin precursor, cystatin C, cystatin M
precursor, immunoglobulins, insulin-like growth factor bind-
ing protein 6, pro-epidermal growth factor precursor, and
osteopontin were not detected by 2DE.
293 • AUGUST 2007 • www.ajprenal.org
 ANALYSIS OF RENAL FANCONI URINE BY MS AND NMR
Our results show that the urine protein patterns of patients
with Dent’s disease and Lowe syndrome are very similar. We
found that the plasma proteins normally reabsorbed from the
glomerular filtrate along the first part of the proximal tubule by
receptor-mediated endocytosis represent a larger proportion of
the FS urinary proteome compared with normal urine. These
results indicate that tubular reabsorption of plasma-derived
proteins is similarly reduced in Dent’s disease and Lowe
syndrome. In contrast to normal urine, proteins of renal origin
are decreased in the urine of patients with these forms of FS.
Thus the FS urinary proteome is dominated by proteins of
plasma origin, and proteins derived from the kidney itself (and
are present in normal urine) make up a smaller proportion of
the FS urinary proteome. These findings confirm our original
observations in Dent’s disease only (19) and indicate that in
this respect Lowe syndrome and Dent’s disease are indistin-
guishable.
1
H-NMR offers a robust and information-rich analytic ap-
proach that can complement proteomic data (38). By combin-
ing this highly reproducible form of spectroscopy with the
chemometric methods of PCA and O-PLS-DA, a more general
comparison of the different sources of FS urine and normal
urine can be made, and potential biomarkers for each type of
FS identified. Consistent with the proteomic findings, the
metabonomic analysis showed that the metabolic profiles of
urine from Dent’s and Lowe patients were very similar and
quite different from normal urine. The differences were due
mainly to increased amounts of the amino acids lysine (basic)
and alanine (neutral) in the urine of FS patients, and decreased
amounts of p-cresol sulfate and N-methyl nicotinic acid. This is
in keeping with early descriptions of Lowe syndrome, in which
basic and neutral (but not branch-chained) aminoaciduria was
a recognized feature (9). The decrease in N-methyl nicotinic
acid excretion suggests cation transporter dysfunction in the
proximal tubule (7, 52), while the decreased p-cresol sulfate, a
clostridial metabolite, could be due to altered metabolism by
gut microflora (56). It is perhaps relevant here that some forms
of FS are associated with different effects on amino acid
transport in the kidney and intestine (3), which may influence
gut microflora metabolism. Indeed, ClC-5 has been detected in
the rat intestine (53), but its function there, or its presence in
humans, is unknown. Alternatively, it may be that altered renal
function in FS patients results in inefficient clearing of p-cresol
sulfate, a well-known uremic toxin (54, 55). However, whether
this is a nonspecific reflection of impaired renal function or is
more specific to tubular dysfunction in FS is unclear at present.
In both Dent’s disease and Lowe syndrome, urinary megalin,
the receptor thought to be largely responsible for protein and
polypeptide reabsorption in the proximal tubule, is reduced or
absent in urine (41), which is again in keeping with our finding
of almost identical urinary proteomes and metabonomes, and is
thus a common underlying defect in receptor-mediated endo-
cytosis. The products of the gene defects in Dent’s disease and
Lowe syndrome could be closely involved in the same reab-
sorptive pathway, and one that may be common to other forms
of FS. To explore this concept, we analyzed the urine compo-
sition of a phenotypically similar autosomal dominant idio-
pathic form of FS (ADIF) and compared it with Dent’s disease,
Lowe syndrome, ifosfamide-induced TP, and normal subjects.
ADIF is another form of inherited FS, clinically similar to
Dent’s disease (5), but its underlying gene defect is autosomal
AJP-Renal Physiol • VOL
F465
and still unknown (33). Although proteomic analysis, as for
Dent’s disease and Lowe syndrome, confirmed that the ADIF
urinary proteome is different from control and broadly similar
to Dent’s and Lowe proteomes (with no clearly distinct pro-
teins or peptides), the urinary metabonome of ADIF showed
several qualitative differences in composition, with glucose
and valine (neutral) identified as potentially distinguishing
biomarkers for this form of FS (Fig. 7D). By cluster analysis
the ADIF and TP urinary proteomes appeared to be quite
similar, yet different from Dent’s and Lowe proteomes (Fig. 5).
An earlier 1H-NMR study of urine from patients treated with
ifosfamide reported glycosuria and amino aciduria, with con-
comitant decreases in hippurate and citrate (26, 27). We ob-
served similar metabolite excretion patterns for ifosfamide-
induced TP, except there was no apparent increase in histidine
in the urine of our patients. In grouping ADIF and TP together,
and Dent’s and Lowe together, both proteomic and metabo-
nomic analyses were consistent. The discriminators in ADIF
were glucose and valine, while in Dent’s and Lowe syndrome
Downloaded from ajprenal.physiology.org on March 22, 2011
it was lysine.
Although no specific mechanism(s) has been be identified by
this combined analysis, and our interpretation is both limited
and somewhat speculative, taking the proteomic and metabo-
nomic data together, the results do suggest that the molecular
defects and pathways affected in ADIF may be different from
those shared by Dent’s disease and Lowe syndrome, and more
similar to those occurring in ifosfamide-induced TP. Moreover,
our results do confirm our original proteomic findings in Dent’s
disease and show that the urinary protein and metabolite
patterns in Dent’s disease and Lowe syndrome are almost
identical, qualitatively and quantitatively, and it is likely that
their respective gene products are closely involved in the same
proximal tubular reabsorptive pathways. Indeed, the recent and
confirmed report that a significant number of adult patients
with a Dent’s-like (predominantly renal) phenotype had muta-
tions of OCRL1, and not CLCN5 (30), is consistent with our
findings and this interpretation.
Finally, in this small study we have demonstrated how
urinary proteomics and metabonomics can be combined effec-
tively to provide a more complete analysis of urine and that
these analytic methods are consonant. By extending urinary-
omics in this way (and with the development of high-through-
put sampling methods), especially in well-defined and charac-
terized renal disorders, or by comparing clinically similar
disorders (as in FS), potential biomarkers and novel mechanis-
tic insights may be revealed.
GRANTS
A. Vilasi, P. R. Cutillas, and R. J. Unwin thank the St. Peter’s Trust for
Kidney, Bladder, and Prostate Research for financial support. A. D. Maher and
S. F. M. Zirah received financial support from the European Union FP6
MolPAGE project.
REFERENCES
1. Andersen JS, Wilkinson CJ, Mayor T, Mortensen P, Nigg EA, Mann
M. Proteomic characterization of the human centrosome by protein cor-
relation profiling. Nature 426: 570 –574, 2003.
2. Bachmann S, Schlichting U, Geist B, Mutig K, Petsch T, Bacic D,
Wagner CA, Kaissling B, Biber J, Murer H, Willnow TE. Kidney-
specific inactivation of the megalin gene impairs trafficking of renal
inorganic sodium phosphate cotransporter (NaPi-IIa). J Am Soc Nephrol
15: 892–900, 2004.
293 • AUGUST 2007 • www.ajprenal.org
F466 ANALYSIS OF RENAL FANCONI URINE BY MS AND NMR
3. Binder HJ. A comparison of intestinal and renal transport systems. Am J
Clin Nutr 23: 330 –335, 1970.
4. Bradford MM. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem 72: 248 –254, 1976.
5. Brenton DP, Isenberg DA, Cusworth DC, Garrod P, Krywawych S,
Stamp TC. The adult presenting idiopathic Fanconi syndrome. J Inherit
Metab Dis 4: 211–215, 1981.
6. Brindle JT, Antti H, Holmes E, Tranter G, Nicholson JK, Bethell HW,
Clarke S, Schofield PM, McKilligin E, Mosedale DE, Grainger DJ.
Rapid and noninvasive diagnosis of the presence and severity of coronary
heart disease using 1H-NMR-based metabonomics. Nat Med 8: 1439 –
1444, 2002.
7. Burckhardt G, Wolff NA. Structure of renal organic anion and cation
transporters. Am J Physiol Renal Physiol 278: F853–F866, 2000.
8. Charnas LR, Bernardini I, Rader D, Hoeg JM, Gahl WA. Clinical and
laboratory findings in the oculocerebrorenal syndrome of Lowe, with
special reference to growth and renal function. N Engl J Med 324:
1318 –1325, 1991.
9. Charnas LR, Bernardini I, Rader D, Hoeg JM, Gahl WA. Clinical and
laboratory findings in the oculocerebrorenal syndrome of Lowe, with
special reference to growth and renal function. N Engl J Med 324:
1318 –1325, 1991.
10. Chelius D, Bondarenko PV. Quantitative profiling of proteins in complex
mixtures using liquid chromatography and mass spectrometry. J Proteome
Res 1: 317–323, 2002.
11. Choudhury R, Diao A, Zhang F, Eisenberg E, Saint-Pol A, Williams
C, Konstantakopoulos A, Lucocq J, Johannes L, Rabouille C, Greene
LE, Lowe M. Lowe syndrome protein OCRL1 interacts with clathrin and
regulates protein trafficking between endosomes and the trans-Golgi
network. Mol Biol Cell 16: 3467–3479, 2005.
12. Christensen EI, Birn H. Megalin and cubilin: multifunctional endocytic
receptors. Nat Rev Mol Cell Biol 3: 256 –266, 2002.
13. Christensen EI, Moskaug JO, Vorum H, Jacobsen C, Gundersen TE,
Nykjaer A, Blomhoff R, Willnow TE, Moestrup SK. Evidence for an
essential role of megalin in transepithelial transport of retinol. J Am Soc
Nephrol 10: 685– 695, 1999.
14. Cloarec O, Dumas ME, Craig A, Barton RH, Trygg J, Hudson J,
Blancher C, Gauguier D, Lindon JC, Holmes E, Nicholson J. Statistical
total correlation spectroscopy: an exploratory approach for latent biomar-
ker identification from metabolic 1H NMR data sets. Anal Chem 77:
1282–1289, 2005.
15. Cloarec O, Dumas ME, Trygg J, Craig A, Barton RH, Lindon JC,
Nicholson JK, Holmes E. Evaluation of the orthogonal projection on
latent structure model limitations caused by chemical shift variability and
improved visualization of biomarker changes in 1H NMR spectroscopic
metabonomic studies. Anal Chem 77: 517–526, 2005.
16. Cotter RJ. Time-of-flight mass spectrometry for the structural analysis of
biological molecules. Anal Chem 64: 1027A–1039A, 1992.
17. Craig A, Sidaway J, Holmes E, Orton T, Jackson D, Rowlinson R,
Nickson J, Tonge R, Wilson I, Nicholson J. Systems toxicology: inte-
grated genomic, proteomic and metabonomic analysis of methapyrilene
induced hepatotoxicity in the rat. J Proteome Res 5: 1586 –1601, 2006.
18. Cutillas PR, Biber J, Marks J, Jacob R, Stieger B, Cramer R,
Waterfield M, Burlingame AL, Unwin RJ. Proteomic analysis of plasma
membrane vesicles isolated from the rat renal cortex. Proteomics 5:
101–112, 2005.
19. Cutillas PR, Chalkley RJ, Hansen KC, Cramer R, Norden AG,
Waterfield MD, Burlingame AL, Unwin RJ. The urinary proteome in
Fanconi syndrome implies specificity in the reabsorption of proteins by
renal proximal tubule cells. Am J Physiol Renal Physiol 287: F353–F364,
2004.
20. Cutillas PR, Geering B, Waterfield MD, Vanhaesebroeck B. Quantifi-
cation of gel-separated proteins and their phosphorylation sites by LC-MS
using unlabeled internal standards: analysis of phosphoprotein dynamics
in a B cell lymphoma cell line. Mol Cell Proteomics 4: 1038 –1051, 2005.
21. Cutillas PR, Norden AG, Cramer R, Burlingame AL, Unwin RJ.
Detection and analysis of urinary peptides by on-line liquid chromatog-
raphy and mass spectrometry: application to patients with renal Fanconi
syndrome. Clin Sci 104: 483– 490, 2003.
22. Cutillas PR, Norden AG, Cramer R, Burlingame AL, Unwin RJ.
Urinary proteomics of renal Fanconi syndrome. Contrib Nephrol 141:
155–169, 2004.
AJP-Renal Physiol • VOL 293 • AUGUST 2007 •
23. Edwards JJ, Anderson NG, Tollaksen SL, von Eschenbach AC,
Guevara J Jr. Proteins of human urine. II. Identification by two-dimen-
sional electrophoresis of a new candidate marker for prostatic cancer. Clin
Chem 28: 160 –163, 1982.
24. Eisen MB, Spellman PT, Brown PO, Botstein D. Cluster analysis and
display of genome-wide expression patterns. Proc Natl Acad Sci USA 95:
14863–14868, 1998.
25. Foster LJ, de Hoog CL, Zhang Y, Zhang Y, Xie X, Mootha VK, Mann
M. A mammalian organelle map by protein correlation profiling. Cell 125:
187–199, 2006.
26. Foxall PJ, Lenz EM, Lindon JC, Neild GH, Wilson ID, Nicholson JK.
Nuclear magnetic resonance and high-performance liquid chromatogra-
phy-nuclear magnetic resonance studies on the toxicity and metabolism of
ifosfamide. Ther Drug Monit 18: 498 –505, 1996.
27. Foxall PJ, Singer JM, Hartley JM, Neild GH, Lapsley M, Nicholson
JK, Souhami RL. Urinary proton magnetic resonance studies of early
ifosfamide-induced nephrotoxicity and encephalopathy. Clin Cancer Res
3: 1507–1518, 1997.
28. Gygi SP, Corthals GL, Zhang Y, Rochon Y, Aebersold R. Evaluation
of two-dimensional gel electrophoresis-based proteome analysis technol-
ogy. Proc Natl Acad Sci USA 97: 9390 –9395, 2000.
29. Holmes E, Cloarec O, Nicholson JK. Probing latent biomarker signa-
Downloaded from ajprenal.physiology.org on March 22, 2011
tures and in vivo pathway activity in experimental disease states via
statistical total correlation spectroscopy (STOCSY) of biofluids: applica-
tion to HgCl2 toxicity. J Proteome Res 5: 1313–1320, 2006.
30. Hoopes RR Jr, Shrimpton AE, Knohl SJ, Hueber P, Hoppe B, Matyus
J, Simckes A, Tasic V, Toenshoff B, Suchy SF, Nussbaum RL,
Scheinman SJ. Dent Disease with mutations in OCRL1. Am J Hum Genet
76: 260 –267, 2005.
31. Knepper MA. Proteomics and the kidney. J Am Soc Nephrol 13: 1398 –
1408, 2002.
32. Leheste JR, Rolinski B, Vorum H, Hilpert J, Nykjaer A, Jacobsen C,
Aucouturier P, Moskaug JO, Otto A, Christensen EI, Willnow TE.
Megalin knockout mice as an animal model of low molecular weight
proteinuria. Am J Pathol 155: 1361–1370, 1999.
33. Lichter-Konecki U, Broman KW, Blau EB, Konecki DS. Genetic and
physical mapping of the locus for autosomal dominant renal Fanconi
syndrome, on chromosome 15q15.3. Am J Hum Genet 68: 264 –268, 2001.
34. Lin D, Tabb DL, Yates JR III. Large-scale protein identification using
mass spectrometry. Biochim Biophys Acta 1646: 1–10, 2003.
35. Mann M, Hendrickson RC, Pandey A. Analysis of proteins and pro-
teomes by mass spectrometry. Annu Rev Biochem 70: 437– 473, 2001.
36. Marshall T, Williams KM. Clinical analysis of human urinary proteins
using high resolution electrophoretic methods. Electrophoresis 19: 1752–
1770, 1998.
37. Marshansky V, Ausiello DA, Brown D. Physiological importance of
endosomal acidification: potential role in proximal tubulopathies. Curr
Opin Nephrol Hypertens 11: 527–537, 2002.
38. Moy FJ, Haraki K, Mobilio D, Walker G, Powers R, Tabei K, Tong H,
Siegel MM. MS/NMR: a structure-based approach for discovering protein
ligands and for drug design by coupling size exclusion chromatography,
mass spectrometry, and nuclear magnetic resonance spectroscopy. Anal
Chem 73: 571–581, 2001.
39. Nicholson JK, Connelly J, Lindon JC, Holmes E. Metabonomics: a
platform for studying drug toxicity and gene function. Nat Rev Drug
Discov 1: 153–161, 2002.
40. Nicholson JK, Lindon JC, Holmes E. ‘Metabonomics’: understanding
the metabolic responses of living systems to pathophysiological stimuli
via multivariate statistical analysis of biological NMR spectroscopic data.
Xenobiotica 29: 1181–1189, 1999.
41. Norden AG, Lapsley M, Igarashi T, Kelleher CL, Lee PJ, Matsuyama
T, Scheinman SJ, Shiraga H, Sundin DP, Thakker RV, Unwin RJ,
Verroust P, Moestrup SK. Urinary megalin deficiency implicates abnor-
mal tubular endocytic function in Fanconi syndrome. J Am Soc Nephrol
13: 125–133, 2002.
42. Pang JX, Ginanni N, Dongre AR, Hefta SA, Opitek GJ. Biomarker
discovery in urine by proteomics. J Proteome Res 1: 161–169, 2002.
43. Pendaries C, Tronchere H, Plantavid M, Payrastre B. Phosphoinositide
signaling disorders in human diseases. FEBS Lett 546: 25–31, 2003.
44. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS. Probability-based
protein identification by searching sequence databases using mass spec-
trometry data. Electrophoresis 20: 3551–3567, 1999.
www.ajprenal.org
 ANALYSIS OF RENAL FANCONI URINE BY MS AND NMR
45. Piwon N, Gunther W, Schwake M, Bosl MR, Jentsch TJ. ClC-5
Cl⫺-channel disruption impairs endocytosis in a mouse model for Dent’s
disease. Nature 408: 369 –373, 2000.
46. Rantalainen M, Cloarec O, Beckonert O, Wilson ID, Jackson D,
Tonge R, Rowlinson R, Rayner S, Nickson J, Wilkinson RW, Mills JD,
Trygg J, Nicholson JK, Holmes E. Statistically integrated metabonomic-
proteomic studies on a human prostate cancer xenograft model in mice. J
Proteome Res 5: 2642–2655, 2006.
47. Sousa MM, Norden AG, Jacobsen C, Willnow TE, Christensen EI,
Thakker RV, Verroust PJ, Moestrup SK, Saraiva MJ. Evidence for the
role of megalin in renal uptake of transthyretin. J Biol Chem 275:
38176 –38181, 2000.
48. Suchy SF, Olivos-Glander IM, Nussabaum RL. Lowe syndrome, a
deficiency of phosphatidylinositol 4,5-bisphosphate 5-phosphatase in the
Golgi apparatus. Hum Mol Genet 4: 2245–2250, 1995.
49. Tracy RP, Young DS, Hill HD, Cutsforth GW, Wilson DM. Two-
dimensional electrophoresis of urine specimens from patients with renal
disease. Appl Theor Electrophor 3: 55– 65, 1992.
50. Trygg J. O2-PLS for qualitative and quantitative analysis in multivariate
calibration. J Chemometrics 16: 283–293, 2002.
51. Trygg J, Johansson B, Eriksson O. Photoelectron spectra and magnetic
moments in UPdSn: theory. Phys Rev B Condensed Matter 49: 7165–
7169, 1994.
52. Ullrich KJ, Fritzsch G, Rumrich G, David C. Polysubstrates: substances
that interact with renal contraluminal PAH, sulfate, and NMeN transport:
sulfamoyl-, sulfonylurea-, thiazide- and benzeneamino-carboxylate (nico-
tinate) compounds. J Pharmacol Exp Ther 269: 684 – 692, 1994.
53. Vandewalle A, Cluzeaud F, Peng KC, Bens M, Luchow A, Gunther W,
Jentsch TJ. Tissue distribution and subcellular localization of the ClC-5
AJP-Renal Physiol • VOL 293 • AUGUST 2007 •
F467
chloride channel in rat intestinal cells. Am J Physiol Cell Physiol 280:
C373–C381, 2001.
54. Vanholder R, De SR, Glorieux G, Argiles A, Baurmeister U, Brunet P,
Clark W, Cohen G, De Deyn PP, Deppisch R, Scamps-Latscha B,
Henle T, Jorres A, Lemke HD, Massy ZA, Passlick-Deetjen J, Rodri-
guez M, Stegmayr B, Stenvinkel P, Tetta C, Wanner C, Zidek W.
Review on uremic toxins: classification, concentration, and interindividual
variability. Kidney Int 63: 1934 –1943, 2003.
55. Vanholder R, Schepers E, Meert N, Lameire N. What is uremia?
Retention versus oxidation. Blood Purif 24: 33–38, 2006.
56. Wang Y, Holmes E, Nicholson JK, Cloarec O, Chollet J, Tanner M,
Singer BH, Utzinger J. Metabonomic investigations in mice infected with
Schistosoma mansoni: an approach for biomarker identification. Proc Natl
Acad Sci USA 101: 12676 –12681, 2004.
57. Wrong OM, Norden AG, Feest TG. Dent’s disease; a familial
proximal renal tubular syndrome with low-molecular-weight protein-
uria, hypercalciuria, nephrocalcinosis, metabolic bone disease, pro-
gressive renal failure and a marked male predominance. QJM 87:
473– 493, 1994.
58. Yates JR III, Link AJ, Schieltz D. Direct analysis of proteins in
mixtures. Application to protein complexes. Methods Mol Biol 146:
17–26, 2000.
Downloaded from ajprenal.physiology.org on March 22, 2011
59. Zhang X, Hartz PA, Philip E, Racusen LC, Majerus PW. Cell lines
from kidney proximal tubules of a patient with Lowe syndrome lack
OCRL inositol polyphosphate 5-phosphatase and accumulate phospha-
tidylinositol 4,5-bisphosphate. J Biol Chem 273: 1574 –1582, 1998.
60. Zhang X, Majerus PW. Phosphatidylinositol signalling reactions. Semin
Cell Dev Biol 9: 153–160, 1998.
www.ajprenal.org