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Article
Integrated Pathway Analysis of Rat Urine Metabolic
Profiles and Kidney Transcriptomic Profiles To Elucidate
the Systems Toxicology of Model Nephrotoxicants
Ethan Yixun Xu, Ally Perlina, Heather Vu, Sean P. Troth,
Richard J. Brennan, Amy G. Aslamkhan, and Qiuwei Xu
Chem. Res. Toxicol., 2008, 21 (8), 1548-1561 DOI: 10.1021/tx800061w Publication Date (Web): 26 July 2008
Downloaded from http://pubs.acs.org on November 19, 2008

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1548 Chem. Res. Toxicol. 2008, 21, 15481561

Integrated Pathway Analysis of Rat Urine Metabolic Profiles and

Kidney Transcriptomic Profiles To Elucidate the Systems Toxicology
of Model Nephrotoxicants
Ethan Yixun Xu,*, Ally Perlina, Heather Vu, Sean P. Troth, Richard J. Brennan,
Amy G. Aslamkhan, and Qiuwei Xu*,
Department of Safety Assessment, Merck Research Laboratories, West Point, PennsylVania 19486, and
GeneGo Inc., St. Joseph, Michigan 49085

ReceiVed February 15, 2008

In this study, approximately 40 endogenous metabolites were identified and quantified by 1H NMR in
urine samples from male rats dosed with two proximal tubule toxicants, cisplatin and gentamicin. The
excreted amount of a majority of those metabolites in urine was found to be dose-dependent and exhibited
a strong correlation with histopathology scores of overall proximal tubule damage. MetaCore pathway
analysis software (GeneGo Inc.) was employed to identify nephrotoxicant-associated biochemical changes
via an integrated quantitative analysis of both urine metabolomic and kidney transcriptomic profiles.
Correlation analysis was applied to establish quantitative linkages between pairs of individual metabolite
and gene transcript profiles in both cisplatin and gentamicin studies. This analysis revealed that cisplatin
and gentamicin treatments were strongly linked to declines in mRNA transcripts for several luminal
membrane transporters that handle each of the respective elevated urinary metabolites, such as glucose,
amino acids, and monocarboxylic acids. The integrated pathway analysis performed on these studies
indicates that cisplatin- or gentamicin-induced renal Fanconi-like syndromes manifested by glucosuria,
hyperaminoaciduria, lactic aciduria, and ketonuria might be better explained by the reduction of functional
proximal tubule transporters rather than by the perturbation of metabolic pathways inside kidney cells.
Furthermore, this analysis suggests that renal transcription factors HNF1R, HNF1, and HIF-1 might be
the central mediators of drug-induced kidney injury and adaptive response pathways.

Introduction (1). The conventional diagnostic biomarkers for ARF, serum

creatinine and blood urea nitrogen (BUN), have been found to
The mammalian kidney plays a prominent role in the
be insensitive and nonspecific in clinical investigations. In recent
excretion of metabolic wastes and the reabsorption of water and
years, urinary protein biomarkers such as N-acetyl-D-glu-
nutrients to maintain physiological homeostasis. Nephrotoxic
cosaminidase (NAG), kidney injury molecule-1 (KIM-1), and
damage manifested as acute renal failure (ARF)1 or chronic renal
Tamm-Horsfall glycoprotein have achieved higher sensitivity
failure (CRF) is a common side effect of many clinical and
as noninvasive indicators of ARF (3, 4). Although gene and
investigational drugs (1). Antibiotics, angiotensin converting
protein expression could be affected by toxicants, mechanisti-
enzyme inhibitors (ACEIs), and nonsteroidal anti-inflammatory
cally relevant events such as the inhibition of enzyme and/or
drugs (NSAIDs) are three major classes of drugs that are prone
transporter activity might be completely unrelated to transcrip-
to induce ARF, which is a major cause of morbidity and
tional, translational, or post-translational regulations. Under these
mortality in hospitalized patients (2).
circumstances, transcriptomic and proteomic profiles of target
ARF is physiologically characterized by an abrupt decrease
organs and biofluids are unlikely to reveal the relevant mech-
in the glomerular filtration rate (GFR) with consequent azotemia
anisms of toxicity. Biofluid metabolomics may provide a
complementary approach that allows researchers to eliminate
* To whom correspondence should be addressed. (E.Y.X.) E-mail:
ethan_xu@merck.com. (Q.X.) E-mail:qiuwei_xu@merck.com. potentially toxic candidates early in the drug development

Merck Research Laboratories. process based on diagnostic and/or mechanistic biomarkers (5).

GeneGo Inc.
1
Abbreviations: ARF, acute renal failure; CRF, chronic renal failure;
One-dimensional 1H nuclear magnetic resonance (NMR) of
PCT, proximal convoluted tubule; PST, proximal straight tubule; GFR, biofluids has been used by analytical toxicologists as a targeted
glomerular filtration rate; BUN, blood urea nitrogen; ACE, angiotensin biochemical tool since the advent of modern high-field NMR
converting enzyme; ACEIs, angiotensin converting enzyme inhibitors; instrumentations (6). This type of biofluid NMR analysis
NSAIDs, nonsteroidal anti-inflammatory drugs; NMR, nuclear magnetic
resonance; MRI, magnetic resonance imaging; DSS-d6, 2,2-dimethyl-2- requires some prior knowledge of the chemicals to be quantified,
silapentane-5-sulfonate sodium; ANOVA, analysis of variance; IQR, with the expectation that measurements of a combination of
interquartile range; PCC, Pearson correlation coefficient; HNF1R, hepatocyte endogenous metabolites would reveal specific biochemical
nuclear factor 1R; HNF1, hepatocyte nuclear factor 1; HIF1, hypoxia-
inducible factor 1; IBABP, ileal bile acid binding protein; VEGF, vascular
targets. For example, early targeted NMR analyses have
endothelial growth factor; IGFBP-1, insulin growth factor binding protein suggested urinary trimethylamine (TMA), dimethylamine (DMA),
1; HO-1, heme oxygenase 1; MDR1, multidrug resistance 1; NAA, neutral and -hydroxybutyrate (in the absence of other ketone bodies)
amino acid; TMA, trimethylamine; DMA, dimethylamine; KIM-1, kidney as potential region-specific indicators of nephrotoxicity (7, 8).
injury molecule 1; NAG, N-acetyl-D-glucosaminidase; IACUC, Institutional
Animal Care and Use Committees; NIH, National Institutes of Health; PMI, NMR-based metabolomics, on the other hand, is a global
Project Management Institute. profiling of endogenous metabolites. It assumes no prior
10.1021/tx800061w CCC: $40.75 2008 American Chemical Society
Published on Web 07/26/2008
Integrated Pathway Analysis of Nephrotoxicity
knowledge about the chemicals being analyzed and provides
comprehensive data sets that enable a global evaluation of
systemic responses to toxicants (911). Two major data analysis
strategies are frequently applied to NMR metabolomics: mul-
tivariate pattern recognition (12) and metabolite quantification
(13, 14). Only the second strategy is relevant to this study, and
it aims at identifying and quantifying as many endogenous
metabolites as possible by searching a 1H NMR reference
spectra database. In spectral regions where peaks are well-
resolved, metabolites corresponding to the assigned protons can
be readily quantified by comparing the integrated peak area
values with that of the internal standard [e.g., 2,2-dimethyl-2-
silapentane-5-sulfonate sodium (DSS-d6)]. In spectral regions
with many overlapping peaks, deconvolution methods such as
singular value decomposition (14) can be employed to assist
the quantification process.
Given that the molecular mechanisms of toxicity for classic
nephrotoxicants such as cisplatin (15), gentamicin (16), and
D-serine (1719) remain elusive, microarray-based kidney
transcriptomic profiling has been applied to animal toxicity
studies of these toxicants (2023). These toxicogenomics studies
revealed several sets of gene-based transcriptional biomarkers
of proximal tubule injury. More recently, metabolomic profiling
of biofluids (mainly urine and blood samples) has been deployed
to identify clinically accessible and minimally invasive meta-
bolic biomarkers of drug-induced nephrotoxicities. 1H NMR
profiling of urine samples from gentamicin-treated rats identified
elevated levels of glucose and reduced levels of trimethylamine
N-oxide (TMAO), while HPLC-TOF-MS/MS profiling of the
same samples showed reduced xanthurenic acid and kynurenic
acid in association with gentamicin nephrotoxicity (24). Portilla
and co-workers applied 1H NMR to the analysis of urine samples
from cisplatin-treated mice and showed that the appearance of
glucose, amino acids, and Krebs cycle metabolites preceded the
rise in serum levels of traditional biomarkers creatinine and
BUN. Their biochemical studies, using separate colorimetric
enzyme assays, found that the administration of cisplatin led to
a time-dependent accumulation of nonesterified fatty acids and
triglycerides in serum, urine, and kidney samples (25).
The inherent complexity in the interpretation of metabolomic
profiles necessitates a knowledge-based systematic approach of
computational data integration (26). In the field of microarray
functional genomics, software tools such as gene set enrichment
analysis (GSEA), parametric analysis of gene set enrichment
(PAGE), and ErmineJ have gained widespread popularity for
interpreting genome-wide expression profiles (2729). Com-
mercially available pathway and network analysis tools such
as GeneGos MetaCore (30) and Ingenuity Pathway Analysis
(www.ingenuity.com) have enabled the visualization of cellular
components as networks of biochemical interactions. Various
one-step direct interactions between genes, proteins, and
metabolites can be combined to form multistep modules and
pathways, enabling the construction of intracellular, intercellular,
intraorgan, and interorgan interaction networks (31). To apply
these tools to the realm of metabolomics, small-molecule
metabolites must be represented as nodes in biological networks,
in the same way as for genes and proteins. Pathway enrichment
analysis of differentially expressed gene lists from transcriptomic
profiling alone will not detect post-transcriptional or nontran-
scriptional regulatory events. Because metabolites are substrates
of enzymes and transporters, which are protein products of post-
transcriptional processes, integration of metabolomic data into
pathway enrichment analysis is expected to overcome this
limitation to some extent.
Chem. Res. Toxicol., Vol. 21, No. 8, 2008 1549
Previous works applying integrated analysis of transcriptom-
ics, metabolomics, and proteomics to toxicity studies focused
on different aspects of hepatotoxicity (3236). Knowledge-based
pathway analyses in previous reports were qualitative and
presented in a schematic way. Only one report examined
transcript-metabolite correlation coefficients between several
genes and key regions of NMR spectra representing one or more
metabolites (34). No integrated analysis of transcriptomic,
proteomic, and metabolomic data has been reported in the study
of drug-induced nephrotoxicity.
In this study, quantitative pathway enrichment analysis was
applied to the knowledge-based interpretation of 1H NMR urine
metabolite profiles and kidney gene expression profiles from
rat toxicity studies with two model nephrotoxicants: cisplatin
and gentamicin. Transcript-metabolite correlation patterns were
further analyzed to identify perturbed biochemical pathways by
enrichment analysis. Our integrated pathway analysis provides
potentially new explanations for the biochemical phenotypes
and exploratory metabolic biomarkers observed in studies of
model nephrotoxicants.
Materials and Methods
Animal Studies of Cisplatin- and Gentamicin-Induced
Nephrotoxicity. Male Sprague-Dawley rats were purchased from
Charles River Laboratories (Raleigh, NC). Animals were 50-75
days old at study initiation. Each animal was identified by an
implanted microchip. All animal husbandry procedures were in
accordance with the Guide for the Care and Use of Laboratory
Animals [National Institutes of Health (NIH) Publication, Vol. 25,
No. 28, August 16, 1996], and all experimental procedures were
approved by Institutional Animal Care and Use Committees
(IACUC) of the facilities in which the studies were conducted. All
animals were housed in standard laboratory animal facilities and
were provided free access to water and measured amounts of food
[Project Management Institute (PMI) certified rodent diet, 22 g/day/
animal].
Cisplatin was administered by a single intraperitoneal injection
at 0 (n ) 10), 0.5 (n ) 10), 3.5 (n ) 10), and 7 (n ) 20) mg/kg
(mpk). Gentamicin was administered by daily intraperitoneal
injections at 0 (n ) 15), 20 (n ) 15), 80 (n ) 15), and 240 (n )
20) mg/kg/day (mkd). The vehicle used in both studies was 0.9%
sodium chloride. The animals were placed in metabolic cages, and
food was removed during urine collection. Water was available ad
libitum at all times. Urine samples from all surviving animals were
collected overnight (approximately 16 h) in containers placed on
dry ice for NMR analysis and biochemical measurements prior to
their necropsy on day 3 or 8 after cisplatin administration or on
day 3, 9, or 15 after gentamicin administration (Tables S1 and S2).
Here, day 3 refers to the time point of 48 h after the single
cisplatin dose or that of 24 h after the second daily gentamicin
dose. The same referencing convention was applied to other time
points specified in this report. The 12 h light/dark cycles were
enforced in the animal room; lights usually went off at 6 p.m. and
came back on at 6 a.m. every day. Urine collections typically started
around 3 p.m. until about 7 a.m. the next morning, when the urine
samples on dry ice were thawed and volumes were measured by
weight. A 2 mL aliquot of each urine sample was submitted for
measurement of urinalysis parameters, and the remaining sample
was frozen for subsequent NMR metabolic profiling. Blood samples
were also collected for biochemical measurements at the time of
necropsy when kidney samples were harvested for mRNA profiling
with rat genome microarrays.
Necropsy and Histopathology. Rats were fasted overnight prior
to scheduled necropsies. They were anesthetized under isoflurane,
bled via the vena cava, exsanguinated, and necropsied. Livers and
kidneys from all animals were examined and sampled at necropsy.
Terminal body weights and weights of liver and kidneys were
recorded from all animals at scheduled necropsies. The left kidney
1550 Chem. Res. Toxicol., Vol. 21, No. 8, 2008
was cut into two cross-sections, and each half was sectioned into
approximately 2 mm slices and stored on dry ice for subsequent
microarray gene expression analysis. The right kidney was cut into
three cross-sections with the center section (about 5 mm) and
anterior lobe fixed in 10% neutrally buffered formalin for routine
histopathology. A severity scale of 0-5 was employed to grade
histomorphologic changes: 0 (no observable pathology), 1 (very
slight), 2 (slight), 3 (moderate), 4 (marked), and 5 (severe). In
addition to grading specific kidney changes, a histopathology grade
(0-5) was assigned by the pathologist reflecting overall proximal
tubular injury: 0 ) no observable pathology, 1 ) less than 10%, 2
) 10-35%, 3 ) 35-60%, 4 ) 60-85%, and 5 ) more than 85%
of the proximal tubules affected.
RNA Extraction and Transcriptomic Profiling. Merck standard
protocols of RNA extraction, transcriptomic profiling, data process-
ing, and quality control have previously been described (37, 38).
Briefly, total RNA was isolated from homogenized rat kidney tissues
using a combination of TRIzol RNA extraction (Invitrogen,
Carlsbad, CA) with the RNeasy RNA extraction kit (Qiagen,
Valencia, CA). Transcriptomic profiling was conducted using
custom rat genome microarrays consisting of about 22.5K 60-mer
oligonucleotide probes (Agilent, Palo Alto, CA). Cy3- and Cy5-
labeled cRNA samples prepared from compound-treated animals
and from individual vehicle controls were hybridized on the 22.5K
microarrays against an RNA mass-balanced pool made from
vehicle-dosed control animals. All cRNA hybridizations were
performed in duplicate, with fluor reversal (Cy3 or Cy5) in the
second hybridization. The expression ratios of each kidney RNA
sample to the control pool from the fluor-reversed hybridization
pairs were averaged to give a single log-ratio measurement of each
expressed gene in that sample.
Sample Preparation for NMR Analysis. The urine samples
were stored in a -70 C freezer until sample preparation. After
they were thawed on Eppendorf shakers at 10 C, they were loaded
onto a Tecan robotic system. The NMR sample preparation in deep
96 well plates on the Tecan included the following steps. A 500
L aliquot of each of the urine samples was mixed with 300 L of
0.2 M potassium phosphate (99.9% D2O) buffer at pH 7.0. Solutions
were mixed on an Eppendorf shaker at 1200 rpm for 20 min at 10
C and centrifuged at 4000 rpm (2700g) for 10 min at 10 C. A
640 L aliquot of supernatant was then mixed with 160 L of 0.2
M potassium phosphate (99.9% D2O) buffer containing 5 mM DSS-
d6 [2,2-dimethyl-2-silapentane-5-sulfonate sodium or sodium 3-(tri-
methylsilyl)-1-propanesulfonate]. Solutions were mixed again on
an Eppendorf shaker at 1200 rpm for 10 min at 10 C. A 700 L
aliquot of the mixed solution was then transferred to 5 mm NMR
tubes. Deuterium oxide was used as the magnetic field lock, and
DSS-d6 was used as an internal chemical shift reference (0 ppm)
and quantification reference (1 mM).
NMR Conditions and Metabolite Quantification. NMR analy-
ses were performed on a Varian UNITYINOVA 700 MHz NMR.
The probe temperature was set to 25 C (calibrated). The 1D proton
NMR spectral width was 10000 Hz, covering a range of -2.37 to
11.91 ppm. The acquisition time was 3 s, corresponding to a digital
resolution at acquisition of 0.33 Hz. Repetitive 128 scans were
accumulated for each sample analysis. The delay between scans
was 15 s, which allowed a full re-equilibration of all protons,
especially DSS-d6, in the samples between 90 pulses. The water
peak was suppressed using the wet1d pulse sequence (39), which
was based on a selective excitation of the water peak and gradient
dephasing. The selective excitation profile was based on the sinc
waveform, which covered a region of 50 Hz around the water peak.
Time domain data were multiplied with an exponential decay
function of 0.2 Hz. The data were zero filled to 64k data points
before Fourier transformation. The chemical shift was referenced
to the internal DSS-d6 peak at 0 ppm.
The sample analysis was automated under the control of a Varian
768AS automatic robot system and the VNMRJ 1.1D software
(Varian Inc., Palo Alto, CA). The samples were stored in Peltier
sample racks (Gilson, Middleton, WI) at 8 C before and after
analyses in the magnet. Each sample was preconditioned at 25 C
Xu et al.
for 15 min before gradient shimming. After the convergence of
the gradient shimming (i.e., less than 1.0 in VNMRJ software),
the water (i.e., HDO) peak was located for the setting of the
transmitter frequency. The proton 90 pulse was calibrated at a given
pulse power, and the power for water suppression in the wet1d
pulse sequence was optimized to produce a minimum HDO signal.
The metabolites were identified according to an in-house 1D
proton NMR reference spectral library. Their concentrations in urine
were calculated by peak integration of both endogenous metabolites
and internal reference DSS-d6. The total excretion amounts of
endogenous metabolites were expressed in micromoles over 16 h
of urine collection (sample volumes provided in Tables S3 and S4).
Statistical Analysis and Visualization. The calculations of
analysis of variance (ANOVA) p values and Pearson correlation
coefficients (PCCs) and the generation of box plots of metabolic
and gene expression profiles were conducted in the R programming
environment. Because of the longitudinal nature of the study
designs, a three-way mixed-model ANOVA was applied to both
urinary metabolite data sets with dose and time as fixed-effect
factors and animal identification as a random-effect factor. The
urinary metabolites were then ranked by their dose ANOVA p
values. Heat maps of metabolite profiles along with the histopa-
thology scores were generated with MATLAB. Prior to running
ANOVA or drawing heat maps, we applied a logarithmic data
transformation with an offset value of 1 (so that zero values
remained zero after the transformation). To improve the visualiza-
tion quality of metabolite heat maps, a data point was treated as an
outlier if it was beyond the inner fence, which is defined as [Q1 -
1.5 interquartile range (IQR), Q1 + 1.5 IQR], where Q1 and
Q3 are the first and third quartiles and IQR is the interquartile range
(Q3 - Q1). Outlier values were replaced with (Q1 - 1.5 IQR)
if they were on the low end or with (Q1 + 1.5 IQR) if they
were on the high end. Then, the values in each metabolite profile
across urine samples were normalized by a scaling factor of the
maximum histopathology score divided by the range of metabolite
amounts.
Integrated Pathway Enrichment Analysis with GeneGo
MetaCore. The cisplatin and gentamicin kidney transcriptomic data
sets were extracted from the Merck internal Resolver gene
expression database. A list of NCBI LocusLink IDs corresponding
to the genes on the rat genome microarray (8760 out of 10626 genes
on the microarray have LocusLink IDs) was provided by the Merck
Molecular Profiling group. After the cisplatin and gentamicin
microarray data were loaded into the Merck in-house version of
MetaCore (Version 4.3, Build 9311), 7095 of the 8760 gene IDs
were recognized by the software.
Because the Merck cisplatin and gentamicin kidney transcrip-
tomic data sets were generated from the two-color Agilent mi-
croarray platform (where the data are represented by log ratios of
base 10), the NMR urine metabolite data set was converted to log
ratios over the vehicle group at the earliest time point of each study
to facilitate the integrated pathway analysis and the calculation of
PCCs between the expression profile of a gene and the concentration
profile of a metabolite. Metabolite quantities below the detection
limit were set to a very small value (0.00001) before being used to
calculate adjusted log ratios of base 10. Metabolite names were
converted into GeneGo ChemID by querying the GeneGo MetaBase
relational database before loading the NMR-adjusted log ratio data
into MetaCore.
Integrated pathway enrichment analysis was performed by using
the knowledge-based canonical pathways and endogenous metabolic
pathways in MetaCore. Ranking of relevant integrated pathways
was based on hypergeometric p values (40).
Results and Discussion
Mixed-Model ANOVA Ranking of Urinary Metabolites.
The statistical significance of urinary metabolites was ranked
according to the dose p values from mixed-model ANOVA
analyses. We implemented a novel heat map visualization
Integrated Pathway Analysis of Nephrotoxicity
method in MATLAB to display the patterns of correlation
between the urine metabolite profiles and the corresponding
histopathology scores of overall proximal tubule damage for
the same set of dosed rats (Figure 1A,B). Thirty-eight of the
43 metabolites in the cisplatin study and 33 of the 38 metabolites
in the gentamicin study were found to have significant dose p
values (p < 0.05). Thirty-one significant metabolites were
common between the two studies, suggesting that the biochemi-
cal manifestations of toxicity for cisplatin and gentamicin should
be very similar. The most noteworthy dose-dependent increasing
urine metabolites in both studies were glucose, lactate, acetoac-
etate, creatine, 3-hydroxybutyrate, alanine, valine, and isoleucine
(Figure 1A,B). Two of the seven significant metabolites specific
to the cisplatin study, succinate and fumarate, showed similar
dose-dependent decreases that correlated strongly with the
increase of histopathology scores (Figure 1A). Taurine showed
a significant dose-dependent decrease only in the gentamicin
study (Figure 1B).
Histomorphological Findings. Kidney weights were in-
creased in cispatin-treated rats on day 8 at 3.5 (by 31% of body
weight) and 7 mpk (by 92%) and in gentamicin-treated rats on
day 9 at doses g80 mkd (by 20-51%) and at all dose levels
on day 15 (by 17-118%). The increase in kidney weights
correlated with pallor and/or enlargement of the kidneys
observed grossly and with histomorphological changes as
described below. No gross or organ weight changes were
observed in the liver.
Histomorphological changes (slight to severe) were observed
only in kidney tissues from those animals exposed to 3.5 and 7
mpk of cisplatin or to 80 and 240 mkd of gentamicin (Figure 1
and Tables S5-S8). The general progression of histomorpho-
logical changes in the kidney consisted of degeneration (includ-
ing hyaline droplet formation) and necrosis of renal tubular
epithelium at early time points with subsequent inflammation,
tubular dilation, casts, and tubular regeneration occurring at later
time points.
On day 3, cisplatin-treated rats (3.5 and 7 mpk) had very
slight degeneration and necrosis of the tubular epithelium
primarily involving the outer stripe of the medulla. Day 8
histomorphological findings at 3.5 and 7 mpk include marked
to severe tubular degeneration and necrosis involving the cortex
and outer stripe of the medulla as well as tubular dilation,
regeneration, proteinaceous casts, and inflammation.
Very slight degeneration and necrosis of cortical tubules were
observed in only one out of five gentamicin-treated rats on day
3 at 80 and 240 mkd, respectively. On day 9, degeneration and
necrosis involving the cortex and outer stripe of the medulla
were observed in four out of five rats at 80 mkd (very slight to
slight) and five out of five rats at 240 mkd (marked to severe).
Physical signs including decreased activity, decreased food
consumption, and body weight loss (by 3-16%) prompted early
termination of the remaining 240 mkd gentamicin group on day
12 rather than on day 15. The terminated animals had very slight
to moderate tubular degeneration and necrosis. At scheduled
necropsy (day 15), very slight tubular degeneration was observed
at 20 mkd, and very slight to slight degeneration and necrosis
were observed at 80 mkd. Additional histomorphological
findings include tubular dilation, regeneration, proteinaceous
casts, and inflammation observed at 240 and 80 mkd on days
12 and 15, respectively.
Serum Biochemical Changes. Significant increases in serum
creatinine were only observed in animals dosed with 7 mpk of
cisplatin on day 8 and in animals dosed with 240 mkd of
gentamicin after day 9, suggesting that serum creatinine is a
Chem. Res. Toxicol., Vol. 21, No. 8, 2008 1551
relatively insensitive biomarker for both nephrotoxicants (data
not shown). On the other hand, a 2-fold increase of BUN was
observed in animals dosed with 3.5 and 7 mpk on day 3 of
cisplatin treatment and a more than 20-fold increase observed
at 7 mpk on day 8. No change of BUN levels was observed on
day 3 at any doses of gentamicin treatment; only the treatment
at the highest dose (240 mkd) led to a 6-fold increase of BUN
after day 9 (data not shown). These results suggest that urine
NMR metabolic profiling could potentially overcome the
limitations of traditional serum biochemistry biomarkers in some
toxicity studies.
In agreement with a previous study of cisplatin toxicity in
mice (25), rat serum glucose levels were elevated in our cisplatin
study. Moreover, there was a 2.5-fold increase on day 3 and a
1.5-fold increase on day 8 after injection of cisplatin at 7mpk
when compared to vehicle-treated rats (Table S9). However,
this drug-induced hyperglycemic effect was absent in the
gentamicin study (Table S10), suggesting that nephrotoxicant-
induced glucosuria was unlikely due to increased plasma
glucose, as in the case of diabetes mellitus. Nevertheless, it has
been reported that cisplatin-induced hyperglycemia in rats is
secondary to glucose intolerance, impaired insulin response, and
abnormal glucagon response to a glucose stimulus (4143).
Portilla et al. showed that the appearance of hyperglycemia and
glucosuria preceded the rise of classic ARF biomarkers such
as BUN and serum creatinine after intraperitoneal injection of
mice with cisplatin at the dose of 20 mg/kg (25). Therefore,
serum and urine glucose measurements alone may not be used
as a specific biomarker for cisplatin-induced nephrotoxicity, but
they are an indispensable component of the endogenous
metabolic profiles, indicative of potential renal toxicity with this
agent.
Pathway Enrichment Analysis of Kidney Transcript
and Urine Metabolite Profiles. Urine metabolic profiles from
animals dosed with proximal tubule toxicants are functional
readouts of the kidney dysfunction. Therefore, causal inferences
on the potential mechanisms of toxicity should benefit from the
integration of kidney transcriptomic profiling data into the
pathway analysis. To make this comparison, enrichment patterns
of canonical pathways and biological processes were analyzed
by building networks connecting the measured kidney protein-
encoding transcripts and urine metabolites after loading the
metabolomic and transcriptomic data sets into MetaCore. The
top 10 enriched canonical metabolic pathways in both cisplatin
and gentamicin studies were related to the metabolism and
transport of sugars and amino acids (Tables 1 and 2). The use
of both hypergeometric p values from transcriptomic and
metabolomic analyses for the ranking of enriched canonical
pathways appears to provide a more objective measure of their
mechanistic relevance.
Cisplatin- and Gentamicin-Induced Glucosuria Correlates
with the mRNA Decrease of Sodium-Dependent Glucose
Transporters SLC5A1 and SLC5A2. One of the most
significantly enriched canonical pathways in the context of
cisplatin or gentamicin nephrotoxicity was glycolysis and
glucose transport (Figure 2). In both studies, strong negative
correlations were apparent between the decrease of kidney
transcript levels of sodium-dependent luminal glucose trans-
porter SLC5A1 or SLC5A2 and the increase of urine glucose
with the corresponding PCCs ranging from -0.804 to -0.959
(Figure 3A,B and Table 3). As a rule of thumb in statistics,
absolute values of PCC larger than 0.6 are generally viewed as
significant. The magnitudes of those negative correlations were
1552 Chem. Res. Toxicol., Vol. 21, No. 8, 2008 Xu et al.

Figure 1. Heat map of rat urine metabolite amount in micromoles (volume times concentration, as determined by NMR, normalized to the scale
between 0 and 5) shows positive or negative correlations with kidney histopathology scores of overall proximal tubular damage (represented by the
vertical bar labeled Histo on the left, with 0 indicating no damage and 5 indicating maximal damage) after treatment with (A) cisplatin or (B)
gentamicin. The rows in the heatmaps are samples (named by dose, time, and replicate animal number) sorted by their corresponding histopathology
scores in ascending order. The columns from left to right are urine metabolites sorted by their corresponding ANOVA dose p values in ascending
order with a black vertical line separating the significant metabolites (p < 0.05, indicated by a red horizontal underline) from the insignificant
metabolites (indicated by a green horizontal underline).
mainly driven by the effects at the highest doses of cisplatin (7 SLC5A1 and SLC5A2 genes encode two sodium-dependent
mpk) and gentamicin (240 mkd). glucose transporters on the luminal membrane of renal tubular
Integrated Pathway Analysis of Nephrotoxicity Chem. Res. Toxicol., Vol. 21, No. 8, 2008 1553

Table 1. Top 10 Enriched Endogenous Metabolic Pathways that gentamicin significantly reduced SLC5A1-dependent glu-
Ranked by Hypergeometric p Values in the Cisplatin Studya cose transport and down-regulated mRNA and protein levels
metabolomic transcriptomic of SLC5A1 in cultured LLCPK1 cells as well as in mouse
pathway name max (-Log P) max (-Log P) kidney tissues (47).
TCA cycle and tricarboxylic 6.577 4.356 Interestingly, the urine glucose to kidney SLC5A1 transcript
acids transport correlation analysis displayed patterns that corresponded to the
glycine pathway 2.465 21.322
Ala, Gly, and Cys metabolism 21.302 5.777 dosing scheme difference (Figure 3A,B). In the current studies,
and transport cisplatin was administered to the animals as a single dose,
branched-chain amino acid 20.017 8.124 whereas gentamicin was dosed on a daily regimen. The elevation
metabolism of urinary glucose at the highest dose was similar between day
pyruvate metabolism and 18.622 6.795
transport
3 and day 8 in the cisplatin study, whereas the decrease of
glucosylceramide pathways and 2.421 18.119 SLC5A1 transcript was more significant on day 3 than on day
transport 8 (Figure 3A). It is conceivable that some animals might be
D-glucuronic acid pathway 18.050 11.236 able to recover or offset the cisplatin toxicity under a single-
Ala, Ser, Cys, Met, His, Pro, 17.947 4.858 dose regimen; therefore, changes of some endogenous metabo-
Gly, Glu, and Gln metabolism
and transport lites were more obvious on day 3 than on day 8. On the other
glycolysis, gluconeogenesis, and 8.527 16.538 hand, the daily dosing scheme used in the gentamicin study
glucose transport most likely produced cumulative effects on the phenotypic
1-palmitoyl-sn-glycero-3-phosphocholine 2.610 16.538 readouts, as the elevation of urine glucose and the decrease of
pathway
kidney SLC5A1 were both more dramatic on day 9 than on
a
Prior to running integrated pathway enrichment analysis, minimal day 3 (Figure 3B). This analysis is further supported by well-
absolute-value log ratio of 0.01 was applied to filter the 7095 documented evidence suggesting that aminoglycosides, includ-
MetaCore-recognizable kidney genes in the data set. Only statistically
significant urine metabolites with ANOVA dose p values less than 0.05
ing gentamicin, specifically accumulate in epithelial cells of
entered into the analysis. renal proximal tubule (48, 49).
Cisplatin- and Gentamicin-Induced Glucosuria Is Un-
Table 2. Top 10 Enriched Endogenous Metabolic Pathways likely Associated with Flux Alterations in the Glycolysis
Ranked by Hypergeometric p Values in the Gentamicin
Studya
Pathway. In biochemical pathway analyses of metabolomics
data, drug-induced synchronized perturbation of endogenous
metabolomic transcriptomic metabolites that are either in different metabolic pathways or
pathway name max (-Log P) max (-Log P)
produced by different enzymatic reactions usually supports
TCA cycle and tricarboxylic 7.540 4.202 transporter-related mechanisms rather than direct modulations
acids transport
Ala, Gly, and Cys metabolism 21.695 4.934 of metabolic fluxes. For example, urinary profiles of lactate and
and transport alanine were strikingly similar to each other, showing PCCs of
branched-chain amino acid 20.756 8.780 0.99 in the cisplatin study and 0.836 in the gentamicin study
metabolism (data not shown). However, pyruvate-to-alanine and pyruvate-
glycine pathway 2.597 19.271
Ala, Ser, Cys, Met, His, Pro, 18.603 4.429
to-lactate conversions are two disparate biochemical reactions.
Gly, Glu, and Gln metabolism The former is a transamination that requires only pyridoxyl
and transport phosphate (PLP), while the latter depends on the oxidation of
1-palmitoyl-sn-glycero-3-phosphocholine 2.744 17.996 NADH generated from glycolysis. Steady-state lactate rather
pathway than alanine concentration will be affected more significantly
glucosylceramide pathways and 2.553 17.457
transport by any inhibition of the glycolysis pathway. Therefore, similar
O-hexanoyl-(L)-carnitine 2.568 17.283 fluxes through the two reactions strongly support the working
pathway hypothesis that the down-regulation of proximal tubule SLC5A1
glucose pathway 18.050 2.511 and SLC5A2 transporters, rather than the inhibition of the
glycolysis, gluconeogenesis, and 8.946 15.175
glucose transport
glycolysis pathway, is a relevant biochemical explanation of
the glucosuria phenotype.
a
Prior to running integrated pathway enrichment analysis, similar
statistical filters were applied to the data set as described in the footnote Cisplatin- and Gentamicin-Induced Reduction of Hepa-
of Table 1. Because animal-to-animal response variation is too large on tocyte Nuclear Factor 1r (HNF1r) Transcript Correlates
day 15, only day 3 and day 9 data were used in this analysis. with the Transcriptional Down-Regulation of Both SLC5A1
and SLC5A2. Pontoglio et al. have shown that targeted gene
epithelial cells (44). Energetically unfavorable uptake of glucose deletion of the transcription factor HNF1R results in hepatic
from glomerular filtrate into tubular cells is provided by the dysfunction and renal Fanconi-like syndromes manifested by
concerted action of SLC5A1 and SLC5A2 in different sections glucosuria and hyperaminoaciduria (50). In a follow-up study,
along renal proximal tubules (45). After glucose is taken up they further demonstrated that HNF1R directly controls the
into the proximal tubule cells, the process of reabsorption is expression of mouse SLC5A2 gene in renal proximal tubule
not complete until glucose is transferred across the basolateral cells (51). On the other hand, Takamoto et al. reported that
membrane into the capillary space in an energetically favorable gentamicin reduced glucose reabsorption in mouse kidney
manner by the GLUT family of facilitative glucose transporters through the down-regulation of SLC5A1 expression and function
(46). In stark contrast to the dose-dependent decrease of (47). To test the hypothesis that the nephrotoxicant-induced
SLC5A1 and SLC5A2 transcripts, the mRNA levels of GLUT2 mRNA decrease of SLC5A1 and SLC5A2 is a downstream
and GLUT9 increased after cisplatin or gentamicin treatments event of the reduced expression of HNF1R, the correlation
(Figure S1). The strong negative correlations between SLC5A1 patterns of the three genes were examined in our toxicity studies.
or SLC5A2 mRNA and urinary glucose shown in both studies Significant positive correlations were detected between the dose-
(Figure 3A,B) were consistent with a previous report showing dependent decrease of kidney HNF1R transcript and that of the
1554 Chem. Res. Toxicol., Vol. 21, No. 8, 2008 Xu et al.

Figure 2. Dynamic metabolite and transcript changes in the canonical pathways of glycolysis and glucose transport after treatment with cisplatin
or gentamicin. Urine metabolites and kidney transcripts measured in the studies are indicated by a filled circle on the upper right-hand corner of
each network object; a red dot in the circle indicates up-regulation, whereas a blue dot indicates down-regulation from the vehicle control group.
If the dot has mixed colors, it indicates differential regulation patterns across dose and time groups. Small-molecule metabolites are represented by
purple hexagons, and reactions are represented by gray rectangles. The gray rectangle highlighted by a red oval indicates the luminal sodium-
dependent glucose transport reactions captured in the MetaCore knowledgebase that led to the discovery of negative correlations between urinary
glucose and kidney transcripts SLC5A1 and SLC5A2. Gene/protein objects are represented by other various shapes and colors depending on their
functional annotations. Note that objects with extracellular region in their names are simply duplicates of their intracellular counterparts used in
the knowledge-based canonical pathway representation by the MetaCore software; a urine NMR profile by itself is not able to distinguish intracellular
vs extracellular metabolites. Green arrows represent activation, red arrows represent inhibition, and gray arrows represent other types of unspecified
interactions (e.g., molecular transport or complex component binding).

two luminal glucose transporters after cisplatin or gentamicin
treatment (Figure 3A,B and Table 4), suggesting that both
SLC5A1 and SLC5A2 may be target genes under the control
of HNF1R. These results are not in complete agreement with a
published study by Pontoglio et al., where they showed that
HNF1R directly controls the transcription of SLC5A2 but not
that of SLC5A1 in mice (51). Whether the discrepancy is due
to the species difference between mouse and rat remains unclear,
and further investigation is warranted.
Cisplatin- and Gentamicin-Induced Increase of Hypoxia-
Inducible Factor HIF1r Transcript Showed Different Cor-
relation Patterns with the Transcriptional Up-Regulation
of Two Renal GLUT Isoforms. It has been well-documented
that oxygen tensions in kidney tissues are comparatively low
despite high blood flow (52) and that many GLUT genes are
hypoxia responsive (53, 54). The relevance of hypoxia to drug-
induced ARF was highlighted by a report showing that
transgenes of the hypoxia-inducible transcription factor HIF1R
were significantly activated in rat kidneys during cisplatin
treatment (55). Therefore, the observation of the dose-dependent
renal mRNA increase of GLUT2 and GLUT9 after cisplatin or
gentamicin administration (Figure S1) led to an interesting
hypothesis that HIF1R might be responsible for those transcrip-
tional up-regulation events. Indeed, dose- and time-dependent
elevation of renal HIF1R mRNA levels was observed in our
cisplatin and gentamicin studies (Figure S1). Positive correla-
tions between HIF1R and GLUT2 mRNA levels were detected
in both studies (Table 4), whereas HIF1R and GLUT9 mRNA
profiles were found to be correlated only in the gentamicin study
(Table 4).
Hypoxia-inducible factor 1 (HIF1) is a heterodimeric basic
helix-loop-helix (bHLH) protein consisting of two subunits:
HIF1R, which contains an oxygen-dependent degradation
(ODD) domain that is modified at two key prolines by
hydroxylation, and HIF1, which is constitutively present in
cells (56). When cells experience hypoxia, HIF1R is stabilized
by the lack of proline hydroxylation and dimerizes with HIF1
in the nucleus to form transcriptionally active HIF1 to activate
the expression of many target genes such as GLUT1, GLUT3,
and insulin growth factor binding protein 1 (IGFBP-1) (57).
Intriguingly, our observation that cisplatin or gentamicin treat-
ment up-regulated renal HIF1R at the mRNA level (Figure S1)
revealed an adaptive response mechanism in addition to the
hypoxia-induced HIF1R protein stabilization reported in a
cisplatin toxicity study by Tanaka et al. (55).
Integrated Pathway Analysis of Nephrotoxicity Chem. Res. Toxicol., Vol. 21, No. 8, 2008 1555

Figure 3. (A and B) The correlation patterns of urine glucose profiles (represented by adjusted log ratios of base 10 as described in the Materials
and Methods) and kidney mRNA profiles of the transcription factor HNF1R with the mRNA profiles of two luminal sodium-dependent glucose
transporters SLC5A1 and SLC5A2 (all renal mRNA profiles are represented by log ratios of base 10) in (A) the cisplatin study and (B) the gentamicin
study. (C and D) The negative correlation of urinary profiles of lactate, acetoacetate, and 3-hydroxybutyrate with the kidney mRNA profiles of the
monocarboxylate transporter SLC16A7 in (C) the cisplatin study and (D) the gentamicin study. In all box plots, a red color indicates positive
median log ratios, and a blue color indicates negative median log ratios.

Because proximal tubular GLUTs are facilitative transporters
mediating either efflux or influx of glucose depending on its
concentration gradient across the basolateral membrane, up-
regulation of GLUT2 gene expression by increasing amount of
functional HIF1 (Figure S1) might serve as a compensatory
mechanism to sustain nutrient delivery to those tubular cells
injured by nephrotoxicants. When the expression of SLC5A1
and SLC5A2 is reduced and the sodium gradient functionally
collapses across the luminal membrane, increased expression
of GLUTs on the basolateral membrane could provide an
alternative means for the nephrotoxicant-injured tubular cells
to obtain glucose from the blood side. In further support of an
adaptive role for HIF1 in renal injury, it has been reported that
HIF1 directly activates the expression of many genes involved
in tissue remodeling and/or protective responses to renal toxicity,
such as vascular endothelial growth factor (VEGF), heme
oxygenase 1 (HO-1), IGFBP-1, vimentin, multidrug resistance
1 (MDR1), and MDR2, all of which were found to be
up-regulated in previous toxicogenomics studies of nephrotoxi-
cants (20, 21, 23, 58).
Cisplatin- and Gentamicin-Induced Hyperaminoaciduria
Correlates with the mRNA Decrease of the Sodium-Depend-
ent Renal Amino Acid Transporter SLC6A18. The elevation
of urinary amino acids has also been well-documented in the
studies of classic nephrotoxicants such as cisplatin, gentamicin,
and D-serine (8, 17, 59). The integrated pathway analysis may
also provide insights into the underlying biochemical processes
that led to this phenotypic readout. The significant urinary loss
of neutral amino acids (NAAs) such as leucine, isoleucine, and
valine (Figure 1) is reminiscent of Hartnup disorder, where loss-
of-function mutations were identified for a majority of cases in
the sodium-dependent system B amino acid transporter SLC6A19
(60, 61).
Although the rat SLC6A19 gene was not included on the rat
genome microarray (August 2004 version) used in this study,
kidney expression profiles of a related system B transporter
1556 Chem. Res. Toxicol., Vol. 21, No. 8, 2008 Xu et al.

Table 3. Correlation Coefficients between Urinary or gentamicin treatment. The mechanistic relevance of the
Metabolite Profiles and Kidney Gene Expression Profiles in sodium-dependent glucose and amino acid transporters in
the Cisplatin and Gentamicin Studiesa cisplatin nephrotoxicity is further supported by the dose-
metabolite/gene SLC5A1 SLC5A2 SLC6A18 SLC16A7 dependent increase of fractional sodium excretions in urinalyses
cisplatin study from both cisplatin and gentamicin studies (data not shown)
glucose -0.814 -0.804 and a 23Na live-animal magnetic resonance imaging (MRI) study
leucine -0.970 showing that cisplatin treatment significantly reduced the renal
valine -0.983 sodium concentration gradient from cortex to medulla (Haiying
lactate -0.866
acetoacetate -0.837 Liu, Merck Imaging Group, personal communication).
3-hydroxybutyrate -0.779 Functional Roles of HNF1r and HNF1 in Mediating
gentamicin study Nephrotoxicant-Induced Hyperaminoaciduria through the
glucose -0.848 -0.959 Transcriptional Regulation of Collectrin. It has been reported
leucine -0.976 that SLC6A18, SLC6A19, and SLC6A20 proteins all coimmu-
valine -0.735
lactate -0.979
noprecipitate with collectrin, a homologue of angiotensin-
acetoacetate -0.902 converting enzyme 2 (ACE2) (67). Targeted deletion of the
3-hydroxybutyrate -0.821 collectrin gene in mice was reported to cause a massive urinary
a
Those metabolite-transcript PCCs without known
loss of NAAs leading to the formation of urinary amino acid
substrate-transporter relationships were not calculated and were left crystals (67, 68). Other reports showed that collectrin is a target
blank in this table. gene downstream of the transcription factors HNF1R and
HNF1 (6971). In light of the dose-dependent decrease of
Table 4. Correlation Coefficients between the Kidney collectrin mRNA in both cisplatin and gentamicin studies
Expression Profiles of Several Transcription Factors (TF) (Figure 4C,D), the transcript correlation patterns of collectrin
and Their Putative Target Genes (TG) in the Cisplatin
and the Gentamicin Studiesa
with HNF1R and HNF1 were further examined to assess the
reported transcriptional hierarchy in renal proximal tubule cells.
TF/TG SLC5A1 SLC5A2 GLUT2 GLUT9 collectrin Significant positive correlations were found between collectrin
cisplatin study and HNF1R in both studies, whereas the collectrin-HNF1
HNF1A 0.813 0.739 0.780 correlation was only significant in the gentamicin study (Table
HNF1B 0.434
HIF1A 0.769 0.005 4A,B). Like the similarity of glucosuria found in these neph-
rotoxicant-treated rats (Figure 1) and in HNF1R knockout mice
gentamicin study
HNF1A 0.900 0.937 0.883
(50), the hyperaminoaciduria profiles found in our cisplatin and
HNF1B 0.895 gentamicin studies also share some similarity with those found
HIF1A 0.411 0.813 in collectrin knockout mice. The positive HNF1R-collectrin and
a HNF1-collectrin correlations revealed in our nephrotoxicant
PCCs between those gene pairs without a putative TFTG
relationship were not calculated and were left blank in this table. studies are consistent with earlier studies reporting that the
collectrin gene is under the direct transcriptional control of both
SLC6A18 (62) were measured in our transcriptomic experi- HNF1R (70) and HNF1 (71).
ments. Very strong negative correlations between the mRNA Cisplatin- and Gentamicin-Induced Lactic Aciduria
decrease of SLC6A18 and the increase of urinary leucine and and Ketonuria Correlate with the mRNA Decrease of
valine were observed in both studies (Figure 4A,B and Table Renal Monocarboxylate Transporter SLC16A7. The cor-
3). These results suggest that the transcriptional down-regulation relation analysis performed on the cisplatin and gentamicin
of system B NAA transporters might play a major role in studies also revealed a very strong negative correlation between
mediating cisplatin- or gentamicin-induced hyperaminoaciduria. the decrease of the renal mRNA level of monocarboxylate
SLC6A18 is an orphan amino acid transporter mainly localized transporter SLC16A7 and the elevation of urinary lactate or
on the luminal membrane of the S2 and S3 segments of renal ketone bodies such as acetoacetate and 3-hydroxybutyrate
proximal tubule (62, 63), whereas SLC6A19 is mainly localized (Figure 3C,D). SLC16A7 is a proton-coupled transporter that
in the S1 segment (60). Urinalysis of SLC6A18 knockout mice catalyzes the proton-coupled transport of many monocarboxy-
in comparison to wild-type mice showed significant loss of lates such as lactate, pyruvate, and ketone bodies across the
glycine (64) and other NAAs (65). The striking negative plasma membrane (72). The absolute values of PCCs between
correlations found in our cisplatin and gentamicin studies (Figure lactate and SLC16A7 were found to be higher than PCCs
4A,B and Table 3) indicate that SLC6A18 might function as a between ketone bodies and SLC16A7 (Table 3), supporting the
sodium-dependent reabsorption transporter of NAAs such as literature reports that lactate is one of the major substrates
leucine, valine, and isoleucine. transported by SLC16A7.
Portilla et al. speculated that cisplatin-induced neutral ami- These findings suggest that the scope of nephrotoxicant-
noaciduria might be explained by the reduction of reabsorption induced renal Fanconi-like syndromes could be expanded to
by several system A or system L amino acid transporters (25). include lactic aciduria and ketonuria. Nephrotoxicant-induced
This model appears to be inconsistent with the energetics of lactic aciduria has been previously reported in 1H NMR studies
renal NAA reabsorption, which is known to be driven by the of mercuric chloride (73), p-aminophenol (74), sodium chromate
sodium gradient across the luminal membrane (66). Most of (8), hexachloro-1,3-butadiene (8), thioacetamide (75), and
the system A and system L NAA transporters are localized on ethionine (76). Furthermore, ketonuria (urinary elevation of
the basolateral membrane of proximal tubule where they mediate acetoacetate and 3-hydroxybutyrate) was reported to occur in
the energetically favorable step of NAA reabsorption (62). rats following mercuric chloride treatment (73). While the
Importantly, we provided convincing evidence that a luminal urinary increase of these monocarboxylates after cisplatin or
sodium-dependent NAA transporter SLC6A18 may in fact be gentamicin treatment could be attributed to causes other than
linked to the hyperaminoaciduria that occurred after cisplatin renal cell injury (77, 78), the strong negative correlations
Integrated Pathway Analysis of Nephrotoxicity Chem. Res. Toxicol., Vol. 21, No. 8, 2008 1557

Figure 4. (A and B) Negative correlation of urine leucine and valine profiles with the kidney mRNA profiles of the sodium-dependent amino acid
transporter SLC6A18 in (A) the cisplatin study and (B) the gentamicin study. (C and D) The positive kidney mRNA correlations of the transcription
factors HNF1R and HNF1 with collectrin in (C) the cisplatin study and (D) the gentamicin study.
between the renal monocarboxylate transporter SLC16A7 and transcriptional down-regulation of many families of renal
the urinary rise of lactate, acetoacetate, and 3-hydroxybutyrate transporter genes in association with high severity of tubular
(Figures 3C,D) suggest that the decrease of proximal tubule damage (58). Because of the presence of tubular cell necrosis
transporter reabsorption may be more relevant than metabolic (21, 79) and apoptosis (55, 80) under those treatment conditions,
flux perturbations. This mechanism is further supported by a they could not rule out the possibility that the observed reduction
previous kidney microarray study of six nephrotoxicants that in the expression of the membrane transporters might result from
identified SLC16A7 as one of the significantly down-regulated the loss of tubular epithelial cells. However, they still observed
genes (58). Therefore, our integrated pathway analysis seems
reduced expression of a subset of transporters in association
to bridge the gap between toxicogenomic and metabolomic
with mild severity of proximal tubule toxicity without histo-
findings for the elucidation of the connections between acces-
sible biofluid phenotypic readouts and their underlying bio- pathological indication of tubular cell death (58). In our cisplatin
chemical processes. and gentamicin studies, two lines of evidence were presented
Transcriptional Down-Regulation of Luminal Sodium- in favor of the transcriptional down-regulation of luminal
Dependent Transporters Plays an Important Role in Me- sodium-dependent transporters over the loss of epithelial cells
diating Nephrotoxicant-Induced Renal Fanconi-Like Syn- to explain the observed renal Fanconi-like syndromes despite
dromes. In the kidney transcriptomic profiling of several other the potentially confounding contribution of mRNA degradation
proximal tubule toxicants, Thukral et al. observed pronounced resulting from tubular cell death.
1558 Chem. Res. Toxicol., Vol. 21, No. 8, 2008 Xu et al.

Figure 5. Schematic representation of the proximal tubule reabsorption processes perturbed in cisplatin- or gentamcin-induced renal Fanconi-like
syndromes that are manifested by the urinary elevation of glucose, NAAs, and monocarboxylates. The uptake of glucose, NAA, and monocarboxylate
into the epithelial cells is mediated by luminal sodium-dependent transporters such as SLC5A1, SLC5A2, SLC6A18, and SLC16A7. The sodium
electrochemical gradient across the luminal membrane is provided by the activity of the basolateral sodium/potassium ATPase. The entry of cisplatin
(denoted as C) or gentamicin (denoted as G) into the tubular epithelial cells results in the transcriptional down-regulation of HNF1R and
HNF1, which in turn leads to the reduction of mRNA levels of SLC5A1, SLC5A2, and collectrin. Cisplatin or gentamicin treatment also leads to
the reduction of SLC6A18 and SLC16A7 mRNA through unknown transcription factors. Both nephrotoxicants also induce hypoxia in renal tubular
cells, which leads to the transcriptional up-regulation and post-translational stabilization of hypoxia-inducible factor HIF1R. An increased amount
of HIF1 up-regulates the transcription of basolateral GLUT transporters as one of the adaptive responses to proximal tubule injury. Up-regulated
gene names are shown in a red color, while down-regulated ones are shown in a blue color. For schematic convenience, these regulated genes are
shown next to each other, although they are located on different chromosomes inside the nucleus.

First, the basolateral GLUT2 and GLUT9 transporters were
found to be transcriptionally up-regulated across all dose/time
conditions of cisplatin or gentamicin treatment (Figure S1).
GLUT1 and GLUT2 are two major isoforms of facilitative
bidirectional glucose transporters on the basolateral membrane
of renal tubular epithelial cells (45, 46, 81). There were large
animal-to-animal variations of renal GLUT1 mRNA levels in
both studies presumably due to its relatively low expression in
kidney (82, 83), but its transcriptional up-regulation was still
observed under a subset of dose/time conditions (data not
shown). GLUT9 is highly expressed in kidney and might also
contribute to renal glucose reabsorption, but its substrate
specificity is yet to be determined (81, 83). A combination of
in situ hybridization with immunohistochemistry has demon-
strated that GLUT2 is expressed along with SLC5A2 in the S1
cells [the proximal convoluted tubule (PCT) segment] of rat
kidney but not in S2 and S3 cells (84). On the other hand, renal
GLUT2 was found exclusively in the PCT segment (82, 85).
By contrast, GLUT1 was shown to be expressed at low levels
in S3 cells [the proximal straight tubule (PST) segment] of rat
kidney (82), where the predominant luminal glucose transporter
is SLC5A1 (84). Cytosolic mRNA degradation during necrosis
or apoptosis, if significantly affecting whole-kidney mRNA
quantification, is expected to be nondiscriminatory on genes
expressed in the same cells. Therefore, the mRNA decrease of
SLC5A1 and SLC5A2 (Figure 3A,B), concomitant with the
mRNA increase of GLUT1 (data not shown) and GLUT2
(Figure S1) in the same tubular epithelial cells clearly supports
the essential role of transcriptional regulations in mediating the
nephrotoxic effect of cisplatin and gentamicin in spite of the
potential complications caused by drug-induced tubular cell
death.
Second, luminal sodium-dependent bile acid transporter
SLC10A2 was also found to be transcriptionally up-regulated
after cisplatin or gentamicin treatment (Figure S2). In contrast
to the results reported by Huang et al. (21), renal SLC10A1
mRNA levels actually showed a dose-dependent decrease in
both studies (Figure S2). This discrepancy could be explained
by a potential gene annotation error from their 250-gene cDNA
microarray because only SLC10A2 is known to be expressed
on the luminal membrane of renal proximal tubule, whereas
SLC10A1 is mainly found in liver and pancreas (86). A similar
dose-dependent mRNA increase of SLC34A2 was also observed
in the current cisplatin and gentamicin studies (data not shown).
The dose-dependent mRNA increase of at least two functionally
unrelated luminal transporters in renal epithelial cells from
independent studies of disjoint sets of nephrotoxicants reaffirms
the importance of transcriptional regulation during the onset of
drug-induced nephrotoxicity.
Concluding Remarks
Both cisplatin and gentamicin induce renal Fanconi-like syn-
dromes manifested by glucosuria and hyperaminoaciduria (8, 25, 87),
but the causes of those biochemical phenotypes and the mechanisms
by which both drugs cause renal cell injury have not been fully
Integrated Pathway Analysis of Nephrotoxicity
elucidated (1). In this study, we applied integrated pathway analysis
and metabolite-transcript correlation analysis to define perturbed
biochemical pathways and molecular functions that may be relevant
to the mechanisms of nephrotoxicity. Our absolute quantification
of about 40 endogenous metabolites by 1H NMR in urine samples
from both studies identified unique biochemical patterns or
signatures that are more comprehensive than those documented in
previously published studies (24, 25). This analysis also demon-
strated the use of a set of indicative endogenous metabolites to
deepen our understanding of the biochemical alterations associated
with drug-induced nephrotoxicity. Furthermore, our metabolite-
transcript correlation analysis, in the context of relevant literature
knowledge in renal physiology, highlights the essential role of the
transcriptional down-regulation of luminal sodium-dependent
transporters SLC5A1, SLC5A2, SLC6A18, and SLC16A7 in
causing renal Fanconi-like syndromes observed in drug-induced
proximal tubule injury (Figure 5). The application of in silico
pathway and network visualization tools has allowed us to integrate
knowledge-based analysis and statistical analysis for the generation
of experimentally testable hypotheses in investigative toxicology.
As more types of biofluid and tissue samples become amenable to
metabolite profiling with ever-increasing accuracy of quantification,
it is imperative for us to raise pathway analysis software tools to
a new level with more powerful automated text mining and robust
statistical measures of relevance.
Acknowledgment. We thank Janet Kerr, Beatrice Sacre-
Salem, David Gerhold, Alema Galijatpovic, Holly Clouse,
and Carolann Beare for coordinating the toxicity studies of
cisplatin and gentamicin. We are grateful to Julie Bryant and
Yuri Nikolsky for coordinating and supporting this collabora-
tion between Merck and GeneGo. We appreciate efforts from
Merck colleagues in generating the clinical chemistry and
histopathology data for both studies. We also thank Huanying
Ge and Lifeng Tian for help with the box plots and the NMR
metabolite heatmaps. We express our gratitude to our
colleagues at the Gene Expression Laboratory (GEL) of
Merck Molecular Profiling for conducting the kidney tran-
scriptomics experiments. We also thank John Metz, Yudong
He, Olivia Fong, and Xiaodan Zhang for helping with the
metabolite and gene ID translations. Assistance in preparing
Figure 5 from Jill Williams and insightful discussions with
Haiying Liu, Jun Zhu, Zhidong Tu, and Yan Yu are gratefully
acknowledged. We are also grateful to Frank Sistare, Joe
Sina, and Bill Schaefer for their scientific guidance and
critical reading of the manuscript.
Supporting Information Available: Tables showing the
study designs, volumes of urine samples collected, and
histopathological and serum biochemical findings for the
cisplatin (Tables S1, S3, S5, S6, and S9) and gentamicin
(Tables S2, S4, S7, S8, and S10) studies. Box plots showing
different correlation patterns of GLUT2 and GLUT9 with
HIF1R (Figure S1) and different transcript profiles of
SLC10A1, SLC10A2, and ileal bile acid binding protein
(IBABP) (Figure S2) in rat kidneys after cisplatin or
gentamicin treatments. This material is available free of
charge via the Internet at http://pubs.acs.org.
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