TOXICOLOGICAL SCIENCES 124(1), 23–34 (2011)
Advance Access publication August 24, 2011
Genomic-Derived Markers for Early Detection of Calcineurin Inhibitor
Yuxia Cui,* Qihong Huang,† James Todd Auman,* Brian Knight,† Xidong Jin,† Kerry T. Blanchard,† Jeff Chou,‡
Supriya Jayadev,† and Richard S. Paules*,1
*Environmental Stress and Cancer Group, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park,
North Carolina 27709; †Boehringer Ingelheim Pharma, Inc., Ridgefield, Connecticut 06877; and ‡Biostatistics Branch, National Institute of Environmental
Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
1To whom correspondence should be addressed at Environmental Stress and Cancer Group, National Institute of Environmental Health Sciences, Mail Drop
D2-03, P.O. Box 12233, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709. Fax: (919) 316-4771. E-mail: email@example.com.
Received June 30, 2011; accepted August 7, 2011
Calcineurin inhibitor (CI) therapy has been associated with
chronic nephrotoxicity, which limits its long-term utility for
suppression of allograft rejection. In order to understand the
mechanisms of the toxicity, we analyzed gene expression changes
that underlie the development of CI immunosuppressant–mediated
nephrotoxicity in male Sprague-Dawley rats dosed daily with
cyclosporine (CsA; 2.5 or 25 mg/kg/day), FK506 (0.6 or 6 mg/kg/
day), or rapamycin (1 or 10 mg/kg/day) for 1, 7, 14, or 28 days.
A significant increase in blood urea nitrogen was observed in
animals treated with CsA (high) or FK506 (high) for 14 and 28 days.
Histopathological examination revealed tubular basophilia and
mineralization in animals given CsA (high) or FK506 (low and
high). We identified a group of genes whose expression in rat
kidney is correlated with CI-induced kidney injury. Among these
genes are two genes, Slc12a3 and kidney-specific Wnk1 (KS-
Wnk1), that are known to be involved in sodium transport in the
distal nephrons and could potentially be involved in the mechanism
of CI-induced nephrotoxicity. The downregulation of NCC (the
Na-Cl cotransporter coded by Slc12a3) in rat kidney following CI
treatment was confirmed by immunohistochemical staining, and
the downregulation of KS-Wnk1 was confirmed by quantitative
real-time-polymerase chain reaction (qRT-PCR). We hypothesize
that decreased expression of Slc12a3 and KS-Wnk1 could alter the
sodium chloride reabsorption in the distal tubules and contribute to
the prolonged activation of the renin-angiotensin system, a demon-
strated contributor to the development of CI-induced nephrotox-
icity in both animal models and clinical settings. Therefore, if
validated as biomarkers in humans, SLC12A3 and KS-WNK1
could potentially be useful in the early detection and reduction of
CI-related nephrotoxicity in immunosuppressed transplant patients
when monitoring the health of kidney xenographs in clinical
Key Words: nephrotoxicity; cyclosporine; FK506 (tacrolimus);
rapamycin; gene expression; biomarker; renin-angiotensin system.
Calcineurin inhibitors (CIs) cyclosporine (CsA) and FK506
are the backbone of current immunosuppressive therapy, widely
used in organ transplant patients and patients with autoimmune
disease (Meier-Kriesche et al., 2006). Both compounds
suppress a number of immune genes by inhibiting calcineurin.
Since the introduction of CsA in the early 1980’s, early
allograft survival rate has improved significantly, due to
reduced acute rejection (Mayer et al., 1997; Williams and
Haragsim, 2006). However, concerns have been raised about
adverse side effects, mainly nephrotoxicity, caused by long-
term use of CIs. CI therapy has been associated with acute
kidney dysfunction defined as decreased glomerular filtration
rate and renal blood flow and chronic structural changes, such
as stripped interstitial fibrosis, arteriolar hyalinosis, or severe
tubular microcalcification (Duvoux and Pageaux, 2011;
Kivela et al., 2011; Nankivell et al., 2004; Olyaei et al.,
2001; Solez et al., 1998; Williams and Haragsim, 2006).
These side effects may cause late allograft loss in renal
transplant recipients or kidney failure in nonrenal transplant
patients, limiting the long-term utility of CI drugs. Although
the combination of a low-dose CI and a mammalian target of
rapamycin (mTOR) inhibitor has been an effective approach
to reduce CI-related chronic nephrotoxicity, the new approach
itself has complications and adverse effects, such as synergistic
nephrotoxicity, posttransplant diabetes mellitus, delayed graft
function, and wound-healing problems, as well as CI-related
nephrotoxicity cannot be avoid completely (Campistol, 2010).
Although most acute toxicity caused by CI can be resolved by
reducing drug dose or complete withdrawal, the long-term
effects are irreversible and can only be diagnosed by histology.
Therefore, alternative diagnostic tools and biomarkers for early
detection of CI-related nephrotoxicity prior to the generation of
irreversible damage are needed.
The mechanisms that underlie the toxicity caused by CI are
not completely understood, although several mechanisms have
Published by Oxford University Press on behalf of the Society of Toxicology 2011.
been proposed (Lustig et al., 1987; Nankivell et al., 2004;
Olyaei et al., 2001; Pallet and Legendre, 2010; Solez et al.,
1998; Williams and Haragsim, 2006). These include CI-
induced imbalance between renal vasoconstrictor factors and
vasodilator factors, activation of sympathetic system, and
activation of the renin-angiotensin system (RAS). Activation of
RAS following CI treatment has been demonstrated in both
animal models and clinical settings (Iijima et al., 2000; Lassila,
2002; Lee, 1997). RAS plays important roles in both acute
kidney dysfunction and the development of chronic nephro-
toxicity due to its ability to promote vasoconstriction and
transforming growth factor-b expression (Nakatani et al., 2003;
Shihab et al., 1997). A number of studies have also
demonstrated that CI-induced renal impairment was attenuated
by the blockage of renin-angiotensin in both rats and organ
transplant patients, supporting the critical role of RAS activation
in the development of CI-related nephrotoxicity (Langham et al.,
2001; Pichler et al., 1995; Sun et al., 2005). The CI activation of
RAS is mediated by CI-induced renin overexpression in the
kidney, although the mechanism is not clear.
The current study was undertaken in order to gain a better
understanding of the mechanisms underlying CI-induced
nephrotoxicity and to identify potential biomarkers for early
detection of toxicity. Nephrotoxicity was induced in rats
through ip injection of CsA (2.5 or 25 mg/kg/day) and FK506
(0.6 or 6 mg/kg/day). Rapamycin (Rapa), an immunosup-
pressive drug that does not cause the same nephrotoxicity as
CI, was administered to another set of animals (1 or 10 mg/kg/
day) to help distinguish CI-specific nephrotoxic effects from
immunosuppressive effects. Our dosing model generated
pathological changes and functional toxicity that resemble
typical CI-induced toxicity such as tubular mineralization
(after 7-day treatment) and increase in blood urea nitrogen
(BUN; after 14 and 28 days) in animals given CsA or FK506.
Necrosis was also observed in CsA-treated animals. By
anchoring the gene expression changes induced by CI drugs to
the nephrotoxicity elicited, the current study identified a gene
expression signature in rat kidney that quantitatively correlated
with the progression of kidney injury. The downregulation of
these genes precedes pathological and/or functional changes, and
these gene expression changes may be predictive of CI
immunosuppressant–mediated kidney injury in rats. In addition,
genes and pathways not previously associated with CI-related
injury were discovered, which provide new mechanistic insights
into this toxicity.
MATERIALS AND METHODS
Materials. CsA (Chemical Abstract Service registry number 59865-13-3),
FK506 (Chemical Abstract Service registry number 104987-11-3), and Rapa
(Chemical Abstract Service registry number 53123-88-9) were obtained from
LC Laboratories (Woburn, MA). Rat genome chips (RAE 230 2.0) and One-
Cycle Target Labeling and Controls Kit were the products of Affymetrix Inc.
(Santa Clara, CA).
Animal treatment. Male Sprague-Dawley VAF/Plus (Virus Antibody
Free) albino rats [Crl:CD(SD) BR; Charles River, Kingston, NY] approximately
5–7 weeks old were maintained on certified rodent chow (PMI Feeds, Inc.,
Brentwood, MO) ad libitum in individual stainless steel wire bottom cages
suspended on racks. The animals were kept under controlled conditions of 12 h
light-dark cycle, 72?F ± 5?F, and 50 ± 20% relative humidity. The animals were
acclimated to this environment for 4–7 days prior to the start of the study. Rats
were randomly assigned to treatment groups and dosed ip with olive oil vehicle
or CsA (2.5 and 25 mg/kg/day), FK506 (0.6 and 6 mg/kg/day), or Rapa (1 and 10
mg/kg/day) for 1, 7, 14, and 28 consecutive days (4 animals/group for most
treatments and 6/group for 28-day exposure). The high doses of CsA and FK506
were selected based on a review of literature that produced nephrotoxicity in rats
without lethality after the desired exposure period. For Rapa, a dose that was
higher than pharmacological doses was picked as the high dose. The low doses
(1/10 of the high dose) were selected to target the pharmacology doses that do not
produce apparent nephrotoxicity. Following treatment, blood was collected at the
terminal necropsy (24 h after 1, 7, 14, or 28 days of treatment) for clinical
chemistry analysis, and kidneys were examined macroscopically. Each kidney
was cut in half (one cross-sectional and the other longitudinal). One half of the
left kidney and one half of the right kidney were used for histopathological
evaluation, and the remaining portions of the kidney were snap frozen in liquid
nitrogen for RNA isolation. Experiments were performed according to the
guidelines established in the National Institutes of Health Guide for the Care and
Use of Laboratory Animals.
Histopathology. Rat kidney specimens from animals treated with CsA,
FK506, Rapa, or olive oil vehicle were collected at necropsy, fixed in 10% neutral
buffered formalin, paraffin embedded, sectioned at 5l thickness, and stained with
hematoxylin and eosin (H&E). Histopathologic examinations of the tissue
sections were conducted by veterinary pathologists and peer reviewed. Trichrome
stain was performed to evaluate interstitial fibrosis in the kidney sections.
mRNA gene expression measurements. Total RNA was isolated from rat
kidney using QIAGEN RNeasy kits (QIAGEN Inc., Valencia, CA). RNA
samples were processed for gene expression analysis by Icoria Inc. (Research
Triangle Park, NC). The GeneChip Rat Genome 230 2.0 Array (Affymetrix)
was used for gene expression profiling. The probe synthesis was performed
using One-Cycle Target Labeling protocol by Affymetrix. Chip hybridization,
washing, and staining were done according to the standard Affymetrix protocol.
The gene chips were scanned with the Affymetrix GeneChip 3000 Scanner, and
the expression signals were detected and quantitated by the algorithms in MAS
5.0. All the expression data have been deposited to Gene Expression Omnibus
and is accessible via accession number GSE19366.
Gene expression analysis. Kidney gene expression data were prepro-
cessed by array-based systematic variation normalization (Chou et al., 2005).
‘‘Extracting gene expression Patterns and Identifying coexpressed Genes’’
(EPIG) was then performed on all treated and control groups (Chou et al.,
2007). EPIG analysis identified 4 gene expression patterns and 729 coexpressed
probes in rat kidney (signal:noise ratio > 2 and r > 0.8). Principle component
analysis (PCA) of all animals was performed in Partek Genomic Suite using the
18 probes in the center node of the hierarchical cluster.
Immunohistochemical staining. For both NCC (the protein coded by
Slc12a3) and renin, immunohistochemical staining was performed for kidney
samples from animals treated for 7 or 28 days. The rabbit anti-rat NCC
antibody (Rabbit Anti–Thiazide-sensitive NaCl Cotransporter Antibody,
AB3553) was purchased from Millipore, Billerica, MA. Staining was
performed using the Ventana Discovery XT Autostainer (protocol #79;
Ventana, Tucson, AZ). The primary antibody was applied at a 1:250 dilution,
followed by secondary probing with reagents from the anti-rabbit multimer
detection kit. The slides were counterstained with hematoxylin. The NCC
staining foci in the rat kidney cortex were counted under the microscope at
a 320 magnification. For each sample, 10 fields were counted and averages for
NCC staining foci were used to calculate the mean of all samples in each
treatment group. The results are presented as mean staining foci per field ± SE
CUI ET AL.
(n ¼ 4–6). All treatments were compared with time-matched controls.
ANOVA-protected t-test was used for significance tests. The rabbit anti-rat
renin polyclonal antibody was a kind gift from Dr Tadashi Inagami at
Vanderbilt University. The high specificity of this antibody was documented
previously, as part of the full characterization of the antibody (Naruse et al.,
1981). For immunohistochemical staining of rat renin, each slide was blocked
with 10% normal donkey serum for 20 min at room temperature. The primary
antibody was used at a 1:2500 dilution, whereas the secondary antibody was
diluted 1:500. The labeled secondary antibody was from the Vector Rabbit
Elite Kit. Each slide was counterstained with hematoxylin.
Quantitative RT-PCR. Rat kidney total RNA was analyzed for the
messenger RNA (mRNA) levels of rat Wnk1 and kidney-specific Wnk1 (KS-
Wnk1) using quantitative real-time-polymerase chain reaction (qRT-PCR). Rat
beta-actin (Actb) was used as an endogenous control. The sequences of the
oligonucleotide primers used for Wnk1 (forward primer 3#-ACTCCG-
GAATTGGCAGGA-5#, reverse primer 3#-AGTCGCAGATGACGCTTCG-
5#), KS-Wnk1 (forward primer 3#-GTTTTGCCTTTTCTGATGGAT-5#, re-
verse primer 3#-TCCTTCACTTCAGGAATTGCT-5#), and Actb (forward
primer, 3#-TAAGGCCAACCGTGAAAAGAT-5#, reverse primer 3#-TCCAT-
CACAATGCCAGTGGT-5#) were designed using Web Primer (http://seq.
QuantiTect SYBR Green RT-PCR Kit (Qiagen, Chatsworth, CA) following
the manufacturer’s instructions in an ABI Prism 7000 Sequence Detection
System (Applied Biosystems, Foster City, CA). Three to four biological
replicates of RNA were prepared for each treatment, and each biological replicate
was measured three times. Equal amounts of total RNA from all control samples
of the same time point were pooled together. All measurements were normalized
to rat Actb of the same sample, and fold change of each gene following treatment
was calculated by comparing the normalized gene expression level with that
observed in the untreated, time-matched pooled control. Final results are
presented as mean log2fold ± SE (n ¼ 4). ANOVA-protected t-test was
performed to identify treatment significance (p < 0.05).
qRT-PCRwas performed using
Gross Pathology and Serum Chemistry
All but six animals survived until the scheduled necropsy.
The early demise of the six rats was most likely related to
complications associated with the ip injections. The effects of
CsA, FK506, and Rapa on body weight (BW), BUN, and
serum creatinine are presented in Table 1. Significant reduction
of BW gain was produced by the administration of CsA
(25 mg/kg), FK506 (0.6 and 6 mg/kg), or Rapa (1 and 10 mg/kg)
for 7, 14, and 28 days, compared with time-matched controls.
CsA (25 mg/kg/day) and FK506 (6 mg/kg) treatment caused
prominent elevation of BUN following 14 and 28 days of drug
administration. Increase of BUN was also observed in the rats
from FK506 (0.6 mg/kg) and Rapa (1 and 10 mg/kg), albeit to
a lesser extent. No effect on serum creatinine levels was ob-
served associated with the treatment of any immunosuppressant.
Neither CsA nor FK506 treatment caused any change in
electrolytes in serum (data not shown). Interestingly, Rapa
(1 and 10 mg/kg) treatment caused significant hypocalcaemia
(~10% decrease in calcium), hypokalemia (~25% decrease in
potassium), and hypophosphatemia (~25% decrease in inorganic
phosphorous; data not shown), consistent with previously
reported hypokalemia (Johnson et al., 2001) and hypophospha-
temia (Schwarz et al., 2001) associated with Rapa treatment for
renal allografts in clinic.
Histopathological evaluation of stained kidney sections
revealed adverse histopathological findings associated with
CsA and FK506. Although the current study adopted the
dosing regimen reported previously (Gillum et al., 1988), we
did not observe arteriolopathy and interstitial fibrosis as
reported in that study. Three drug-related findings observed
in CsA-treated kidneys were tubular basophilia, tubular
mineralization, and tubular necrosis (Figs. 1 and 2). Proximal
and distal tubular epithelial basophilia (minimal to mild, three
animals in total) and tubular mineralization (minimal, one
animal) were present at low incidence in the control animals.
Effects of CsA, FK506, and Rapa on BW, Serum BUN, and Serum Creatinine (Cr)
Time (days)Olive oil, 4 ml/kgCsA, 2.5 mg/kgCsA, 25 mg/kgFK506, 0.6 mg/kgFK506, 6 mg/kgRapa, 1 mg/kgRapa, 10 mg/kg
246 ± 5
313 ± 8
371 ± 6
456 ± 7
245 ± 4
295 ± 8
364 ± 12
448 ± 19
243 ± 4
274 ± 6*
313 ± 10*
243 ± 4
270 ± 3*
303 ± 6*
351 ± 8*
245 ± 3
250 ± 6*
289 ± 7*
344 ± 14*
248 ± 17
264 ± 6*
279 ± 8*
286 ± 10*
251 ± 3
254 ± 4*
271 ± 5*
294 ± 9*
12 ± 1
13 ± 1
16 ± 1
18 ± 2
15 ± 1
18 ± 1
18 ± 2
32 ± 2*
44 ± 4*
11 ± 1
17 ± 2
20 ± 1
26 ± 2
11 ± 1
21 ± 2*
28 ± 2*
47 ± 18*
13 ± 2
16 ± 1
22 ± 2*
25 ± 7
12 ± 1
17 ± 1
19 ± 3
20 ± 1
0.7 ± 0.1
*Significantly different from time-matched controls, p < 0.05 (analyzed by ANOVA, followed by Dunnett’s test).
GENOMIC MARKERS OF CI-RELATED NEPHROTOXICITY
The degree of severity and incidence of tubular basophilia were
substantially increased in the high-dose-CsA and low- and
high-dose-FK506 group animals following 14 and 28 days of
treatment, compared with earlier treatment (Table 2). Tubular
mineralization was seen in collecting ducts within the cortico-
medullary region of the kidney (Fig. 2). Tubular mineralization
was present in high-dose-CsA–treated (7, 14, and 28 days),
low- (14 and 28 days) and high-dose-FK506–treated (14 and
28 days) kidneys (Table 2). The degree of severity and
incidence of tubular mineralization increased with prolonged
treatment. Animals in the high-dose-CsA group had slightly
more severe mineralization compared with those animals that
received FK506 treatment. Animals treated with CsA exhibited
minimal tubular epithelial cell necrosis within proximal
convoluted tubules (two in the low-dose group and seven in
the high-dose group). Besides drug-related histopathological
changes, renal capsulitis was observed and considered related
to ip injection. The capsulitis was characterized by the
infiltration of capsule by neutrophils and macrophages with
increased number of fibroblasts. This finding was absent in the
control group, presented in low incidence (one to two rats) in
the CsA and FK506 treatment groups. Interestingly, higher
incidence (three to five rats) of capsulitis was observed in the
low- and high-dose Rapa groups.
Based on the traditional indicators of kidney injury, we
classified animals into three groups: a group with nephrotox-
icity (‘‘toxicity’’ group, usually with both tubular lesions and
significantly elevated BUN comparing with time-matched
control), a group without nephrotoxicity (‘‘no toxicity’’ group,
with neither major changes in histopathology nor significantly
elevated BUN comparing with time-matched control) and
a group with mild nephrotoxicity (‘‘intermediate toxicity’’
group, with either tubular lesions or significantly elevated
BUN; Supplementary Table 1). This classification was in-
corporated into subsequent gene expression analyses in order to
build relationships between gene expression changes and CI-
related kidney injury.
Gene Expression Changes Associated with CI-Induced
An unsupervised clustering of all treated animals using
differentially expressed genes from all treatment groups
(twofold or more, p < 0.01) resulted in four major clusters:
days. Histopathological evidence of tubular basophilia (H&E, 3200). (A) Control. (B) CsA. Tubular cell necrosis within the outer cortex ([) and basophilia of
tubular epithelial cells lining the proximal convoluted tubules and DCTs (*). (C) FK506. Basophilia of tubular epithelial cells lining the proximal convoluted
tubules and DCTs (*). (D) Rapa. Chronic interstitial nephritis, characterized by neutrophil and macrophage infiltrates mixed with hemorrhage and edema fluid (#).
This finding is related to nephritis/capsulitis induced by ip injection.
Histopathology of kidney from control male rats and rats treated with CsA (25 mg/kg/day), FK506 (6 mg/kg/day), and Rapa (10 mg/kg/day) for 28
CUI ET AL.
two clusters from the earlier time point segregated by time (the
1-day treatment cluster and the 7-day treatment cluster) and
two clusters from later time point segregated by treatment (the
CI-treated cluster and the Rapa-treated cluster; Supplementary
figure 1). Clusters that segregated based on treatment correlated
with animals showing changes in traditional indicators of
kidney injury, demonstrating that a different set of biological
events were taking place in the injured animals (Supplementary
EPIG is a software that utilizes the underlying structure of
gene expression data to extract patterns and identify coex-
pressed genes that are responsive to experimental conditions
(Chou et al., 2007). It was used in order to extract kidney genes
that drive the separation of CI-treated animals from control and
Rapa-treated animals. One pattern contained 98 probes
(Supplementary table 3) that were downregulated by CI drugs
(CsA high, FK506 low and high) at later time points, a pattern
that was consistent with the progression of histopathological
changes and elevated BUN levels. Hierarchical clustering of
these 98 probes revealed a subgroup (the center node
containing 18 probes; Supplementary table 3) that was
inhibited by CI drugs at the 1-day time point (Fig. 3A).
Because no histopathological changes and/or kidney dysfunc-
tion was observed at 1 day, these genes may be predictors of
CI-induced kidney injury. Indeed, PCA of all samples using the
18 center node probes resulted in two main groupings (Figs. 3B
and 3C). Group I contained all of the animals with CI-related
nephrotoxicity (toxicity group and intermediate toxicity group),
whereas group II contained animals with no toxicity along with
two animals that had Rapa-induced toxicity. Group I
also contained CI-treated animals from the earlier time point
(7 days) that showed no toxicity, indicating the capacity of
early prediction. The two animals with non–CI-related
nephrotoxicity (caused by Rapa treatment) did not fall into
the same grouping as animals with CI-related kidney injury,
suggesting that this gene set shows specificity for correlation
with CI-induced nephrotoxicity.
Calbindin-1(Calb1) and epidermal growth factor (Egf) were
among the genes that appear to be predictive of CI-induced
kidney injury in rats, and both are well known to be involved
in the CI-related nephrotoxicity (Aicher et al., 1997; Lee
et al., 2002; Nijenhuis et al., 2004; Sairanen et al., 2008).
These studies support the current results and suggest that the
coexpression of these genes may play important roles in the
mechanism of CI-related nephrotoxicity. Therefore, addi-
tional experiments were carried out to understand the
functional relationship between other genes in this node and
nephrotoxicity. Slc12a3 and Wnk1 were the initial focus
days. Histopathological evidence of tubular mineralization (H&E, 3200). (A) Control. (B) CsA. Mineralization of collecting ducts within the corticomedullary
region (þ). (C) FK506. Mineralization of collecting ducts within the corticomedullary region (þ). (D) Rapa.
Histopathology of kidney from control male rats and rats treated with CsA (25 mg/kg/day), FK506 (6 mg/kg/day), and Rapa (10 mg/kg/day) for 28
GENOMIC MARKERS OF CI-RELATED NEPHROTOXICITY
because these are recognized to have important roles in
normal renal function.
NCC Expression Was Inhibited by CI Treatment
Slc12a3 encodes a sodium chloride cotransporter that is
exclusively expressed in the kidney distal convoluted tubule
(DCT). It is one of the major transporters responsible for
sodium reabsorption in distal tubules of the kidney (Yang
et al., 2005). Mutations in NCCT (human homolog of rat
NCC) cause Gitelman’s syndrome in humans (Naesens et al.,
2004; Simon et al., 1996). NCC has not been associated with
CI-induced nephrotoxicity, although decreased expression of
NCC in rat kidney following CsA treatment has been
reported in one study (Lim et al., 2007). In order to
determine if NCC protein changes correlated with mRNA
changes, immunohistochemical staining of NCC was per-
formed on kidney samples from animals treated for 7 and 28
days. NCC protein expression was significantly decreased in
a dose-dependent manner following CI treatment (CsA high,
FK506 low and high) for both time points (Figs. 4A–E).
Furthermore, the number of NCC staining foci in the kidney
cortex was also significantly decreased (Fig. 4A). This
suggests that NCC expression is not only decreased but also
absent in some parts of the DCT, which may significantly
reduce the reabsorption of sodium chloride. Treatment with
Rapa did not decrease NCC expression in rat kidney at any
time point (Figs. 4A and 4F).
KS-Wnk1 Expression Was Decreased by CI Treatment
WNK1, a member of the WNK (with-no-lysine [K]) family, is
an essential regulator of ion transport in the kidney (McCormick
et al., 2008; Xu et al., 2000). In humans, mutations in WNK1
cause familial hyperkalemic hypertension (Wilson et al., 2001).
The Wnk1 gene has two products in the kidney, a full-length
product (Wnk1) that is widely expressed as well as a kidney-
specific truncated product (KS-Wnk1) that lacks the kinase
domain and is predominantly expressed in the DCT and the
connecting tubules (CNT). In order to determine which form of
Wnk1 was downregulated by CI drugs, two pairs of primers were
used in qRT-PCR to specifically target different transcripts.
Consistent with Affymetrix gene expression data, KS-Wnk1 was
significantly downregulated by high-dose CI drug treatment at
both 7 and 28 days (Fig. 5). In contrast, expression of full-length
Wnk1 remained unchanged following CI treatment.
RAS Was Activated following CI Treatment
It is believed that the CI-induced activation of RAS is
mediated by renin overexpression in the kidney. Immunostaining
Rat Kidney Histopathology Following CsA, FK506, and Rapa Treatments
Number of incidences (average scorea)
1 Day 7 Days14 Days 28 Days
Olive oil 4 ml/kg Basophilia, tubular
1/4 (2.0) 1/4 (1.0)1/5b(1.0)
CsA 2.5 mg/kg2/4 (1.0)2/4 (1.0)1/5b(1.0)
CsA 25 mg/kg1/4 (1.0) 4/4 (2.0)
FK506 0.6 mg/kg6/6 (2.0)
FK506 6 mg/kg 4/4 (1.0)3/3b(1.3)
Rapa 1 mg/kg1/4 (1.0) 1/4 (1.0)2/6 (2.5)
Rapa 10 mg/kg 1/4 (1.0)3/6 (1.3)
aAverage score: 1, minimal; 2, mild; 3, moderate; 4, marked.
bOne animal failed to survive the treatment.
cTwo animals failed to survive the treatment.
CUI ET AL.
for renin on kidney samples from animals treated by CI for 7
or 28 days showed an increase in numbers of staining foci in
the kidney cortex and elongated expression of renin along the
afferent arterioles, suggesting that CI treatment resulted in
the recruitment of renin-containing cells (Figs. 6C–E, 7-day
data not shown; Iijima et al., 2000; Tufromcreddie et al.,
1993). Renin staining in the kidney juxtaglomerular cells of
CI-treated animals is less intense than in the control or Rapa-
treated animals, probably due to increased renin release from
these cells, which supports the activation of RAS following
CI treatment. Several studies have previously demonstrated
that sodium depletion can promote CsA- or FK506-induced
tubulointerstitial injury in rat and mouse kidney (Andoh
et al., 1995, 1997; Elzinga et al., 1993; Shihab et al., 1997).
In addition, the enhancing effect of low sodium on chronic
structural changes has been attributed to the activation of the
RAS by low-sodium diet. In the current study, the NCC
protein expression decrease may result in a reduction in
sodium reabsorption in the DCT and subsequently contribute
to the prolonged overexpression of renin. Interestingly,
microarray data indicated a strong correlation between renin
mRNA level induction and Slc12a3 mRNA level decrease
(Fig. 7). This suggests that reduced NCC expression may
contribute to the overexpression of renin and the stimulation
Over the past few years, considerable effort has been
devoted to searching for protein markers associated with
kidney disease and drug-induced nephrotoxicity (Perco et al.,
2006; Thukral et al., 2005; Wang et al., 2008). Among the
candidate markers reported, kidney injury molecule-1 (Kim1)
and clusterin (Clu) are two of the most promising renal injury
markers and have been associated with many forms of renal
toxicity and diseases (Abulezz, 2008; Zhang et al., 2008). Both
Kim1 and Clu were upregulated in injured animals in the
current study (Fig. 8). We compared the expression of Kim1
and Clu with Slc12a3 and KS-Wnk1, as measured by the
Affymetrix GeneChip for each individual treated animal
relative to levels in control animals. We found that Slc12a3
and KS-Wnk1 were strongly correlated with Kim1 and Clu
and that Slc12a3 and KS-Wnk1 appear to be more specific to
CI-related injuries. Both Slc12a3 and KS-Wnk1 were not
changed in Rapa-treated animals, whereas both Kim1 and Clu
were upregulated in later time point Rapa-treated animals
(Fig. 8). This probably is due to Slc12a3 and KS-Wnk1
downregulation being associated with the mechanism of CI-
area). Each vertical bar represents an experiment animal, and each horizontal bar represents a probe on the array. Probes used for clustering were identified by
EPIG. Color scale is indicated in the bottom. Fold change of each gene was calculated by comparing with time-matched controls. (B, C) PCA of rat kidney gene
expression. Each sphere in the graph represents an experimental animal, and all experimental animals were mapped based on the expression of 18 probes in the
center node of (A) (boxed area). Animals in (B) are colored by the degree of nephrotoxicity. Animals in (C) are colored based on the treatment and sized by the
duration of treatment.
A kidney gene expression signature correlates with CI-induced nephrotoxicity in the rat. (A) A gene expression signature identified by EPIG (boxed
GENOMIC MARKERS OF CI-RELATED NEPHROTOXICITY
In the current study, a rat model was used to study the effects
of CI treatment on global gene expression of kidney, in order to
understand the mechanisms underlying CI-related nephrotox-
icity and to search for biomarkers of toxicity. CI treatment
induced microcalcification and isometric vacuolization in rat
kidney, which resemble the pathological findings in CI toxicity
in human patients (Jennette et al., 2007). Although we did not
observe arteriolopathy and interstitial fibrosis, gene expression
analysis revealed significant upregulation of genes involved in
tissue repair, extracellular matrix/fibrosis, and immune and
inflammation following 28 days of treatment with high-dose
CsA and FK506, all of which have been reported to be
activated prior to development of tubulointerstitial damage in
humans (Vitalone et al., 2010; Supplementary table 2).
Therefore, both pathological and gene expressional findings
suggest the current model is suitable for the purpose of the
Biomarkers for Early Detection of CI-Related Nephrotoxicity
CI-related chronic nephrotoxicity is the Achilles’ heel of the
current immunosuppressive regimens (Naesens et al., 2009).
Although the histological lesions caused by CI drugs are well
defined, the histological diagnosis remains problematic due to
difficulties in the differential diagnosis because other injurious
processes in renal transplant patients can cause similar
pathological changes (Naesens et al., 2009). Using a genome-
wide approach, this study identified a group of genes that are
specifically regulated by CI immunosuppressants. These genes
include both upregulated genes, such as renin and Klks3,
and downregulated genes, such as Slc12a3 and KS-Wnk1.
A number of the identified genes have previously been associated
with CI-related nephrotoxicity, such as renin, Calb1, and Egf
(Nakatani et al., 2003; Nijenhuis et al., 2004; Yang et al.,
2002); however, a longitudinal assessment of the relationship
between expressionand the
nephrotoxicity has been lacking. By gene expression profiling
of rat kidney samples collected over a 4-week period of time, we
days). The number of stained foci per field was counted at 310 magnification. Results are presented as mean stain foci per field ± SE (n ¼ 4–6). *p < 0.05. (B–F)
Representative images of NCC immunohistochemical staining in kidney cortex of rats treated for 28 days with vehicle alone (B), high-dose CsA (C), low-dose
FK506 (D), high-dose FK506 (E), or high-dose Rapa (F) (3200).
NCC expression in rat kidney cortex following CI or Rapa treatment. (A) Quantitative assessment of NCC staining foci in rat kidney cortex (7 and 28
after 7 and 28 days. (A) Gene expression following 7 days of treatment.
(B) Gene expression following 28 days of treatment. Rat kidney total RNA was
isolated from animals treated with CsA, FK506, Rapa, or vehicle alone. Fold
change was computed by comparing with time-matched control group. Results
are presented in mean log2(fold change) ± SE (n ¼ 4). *p < 0.05 when
compared with 0 (no change in expression).
mRNA levels of Wnk1 (full length) and KS-Wnk1 in rat kidney
CUI ET AL.
were able to show that the regulation of these genes by CI is
time and dose dependent and is quantitatively correlated with
the progression of kidney injury. The transcriptional changes in
rat kidney had an earlier onset (1–7 days; shown in Fig. 3A)
than changes in BUN (14 days) and kidney histopathology (7–14
days). In fact, using only a subgroup of downregulated genes, it
was possible to identify not only all animals with significant CI-
related nephrotoxicity but also CI-treated animals with mild and
no overt toxicity (mostly animals from earlier time points),
demonstrating that these genes could be potential biomarkers for
early detection of CI-induced nephrotoxicity (Fig. 3B).
Over the past few years, considerable effort has been devoted
to searching for protein markers associated with kidney disease
and drug-induced nephrotoxicity (Perco et al., 2006; Thukral
et al., 2005; Wang et al., 2008). In one of the studies, Wang
et al. (2008) evaluated changes of a panel of 48 putative
genomic markers of nephrotoxicity in rats using a variety of
nephrotoxicants. In most cases, the expression of an individual
genetic marker in rat kidney was changed by multiple types of
nephrotoxicants. This suggests the necessity of using the profile
of multiple genetic markers for the detection of specific types of
kidney injury. In a recent study, increased KIM1 mRNA level
has been observed in biopsies of CI nephrotoxicity or interstitial
fibrosis and tubular atrophy patients (Nogare et al., 2010). Thus,
of rats treated for 28 days with vehicle alone (A), low-dose CsA (B), high-dose CsA (C), low-dose FK506 (D), high-dose FK506 (E), or high-dose Rapa (F)
(3400). Arrows demarcate areas of elongated renin expression along blood vessels.
Renin expression in rat kidney cortex following CI or Rapa treatment. (A–F) Representative images of renin immunohistochemical staining in kidneys
Slc12a3 in rat kidney. Affymetrix rat genome array (RAT 230 2.0) GeneChip
data. Fold change was calculated by comparing with time-matched control
group. Results are presented in mean log2(fold change) ± SE (n ¼ 4–6).
Renin mRNA levels are inversely correlated with the expression of
animals treated with CI or Rapa. mRNA levels of Kim1, Clu, Slc12a3, and KS-
Wnk1 in each individual treated animal was normalized to time-matched
untreated animals, respectively. For each individual animal, four mRNA level
values (on log scale) were listed (Clu, solid square; Kim1, empty square;
Slc12a3, solid circle; KS-Wnk1, empty triangle). Results were from Affymetrix
rat genome array (RAT 230 2.0) GeneChip data.
mRNA levels of potential genomic biomarkers in rat kidney from
GENOMIC MARKERS OF CI-RELATED NEPHROTOXICITY
in combination with some of the general kidney injury markers
such as KIM1, the mRNA levels of SLC12A3 and KS-WNK1
may serve as specific genomic markers for CI-related kidney
injury in humans. In fact, together, these genes successfully
separated animals with CI-related kidney injury from animals
with Rapa-related kidney injury (Fig. 3).
RAS Activation and Sodium Transport in the Distal Nephron
RAS activation following CI treatment is a consistent finding
in animal models (Lassila, 2002). CI can cause elevated plasma
renin activity by increasing the synthesis and excretion of renin
in kidney juxtaglomerular cells (Andoh et al., 1995; Lee, 1997;
Nakatani et al., 2003; Shihab et al., 1997; Stillman et al.,
1995). In humans, although CI therapy usually does not cause
significant increase in plasma renin activity, it activates the
intrarenal RAS, which plays an important role in the
development of CI-related interstitial fibrosis (Iijima et al.,
2000; Kobori et al., 2007; Shang et al., 2008). How CIs induce
renin synthesis remains unclear due to the complexity of the
feedback mechanism between the RAS and kidney hemody-
namics. On the other hand, it is well established that kidney
juxtaglomerular cells are the main source of renin and that
reduced sodium chloride concentration in the macula densa can
signal these cells to increase renin production (Harris, 1996;
Lorenz et al., 1991; Skott and Briggs, 1987).
The DCT is responsible for approximately 5–7% of the
reabsorption of filtered sodium and plays an important role in
electrolyte balance and ion homeostasis (Ecelbarger and Tiwari,
2006). In this study, NCC, one of the major sodium transporters
in the DCT, was found to be downregulated following treatment
of CsA and FK506. Loss-of-function mutations in NCCT cause
a human disease known as Gitelman’s syndrome, characterized
by hypomagnesemia, hypocalciuria, and increased renin activity.
Some of these features are also associated with CI use (Naesens
et al., 2004; Simon et al., 1996). The results of the current study
demonstrate that NCC membrane expression was significantly
decreased following CI treatment in a time- and dose-dependent
fashion, and renin was overexpressed, suggesting activation of
RAS. Based on the strong correlation between Slc12a3 decrease
and renin increase, we propose that decreased expression of
NCC in the DCT will result in reduced sodium reabsorption in
the distal nephrons and contribute to increased expression of
renin in rat kidney. In addition, the mechanistic relevance of
a decrease in NCC expression in distal tubules to the CI-induced
nephrotoxicity appears to be supported by the observed
phenotype of NCC null mice (Schultheis et al., 1998). NCC
null mice showed no significant change in sodium levels in
serum or urine but exhibited increased renin mRNA levels in
the kidney and were unable to adequately compensate blood
pressure in response to a sodium-depleted diet. Our
hypothesis is supported further by previous observations in
rats that low sodium intake can enhance chronic nephrotox-
icity caused by CIs due to the activation of the RAS on low-
sodium diet (Andoh et al., 1995, 1997; Elzinga et al., 1993;
Shihab et al., 1997).
However, factors other than NCC cannot be ruled out as
contributing to CI-induced activation of RAS. Reduced
expression of Na-K-2Cl cotransporter (NKCC2) in tubular
epithelial cells has also been associated with CI treatment–
induced change in renin expression (Naesens et al., 2009). The
regulation of NKCC2 by CI appears to be complex with
differing effects reported and variance based on exposure time
and/or tissue section (Lim et al., 2007). This complexity may
explain why we did not see significant reduction in the overall
mRNA level of NKCC2 in the current study. Interestingly, as
seen in both patients with Gitelman’s syndrome (NCCT
mutations) and patients with CI-related nephrotoxicity, poly-
uria, hypomagnesemia, and hyperreninemic hyperaldosteron-
ism are also features of type I Bartter’s syndrome (NKCC2
mutations; Naesens et al., 2004, 2009). This suggests that both
transporters may play a role in prolonged activation of RAS
induced by CI.
The activity of NCC is also regulated by a signaling pathway
involving the WNK kinase family, which is essential for blood
pressure regulation in humans (Wilson et al., 2001; Xu et al.,
2000). WNK1 affects NCC indirectly by inhibiting WNK4’s
ability to reduce NCC surface expression. KS-WNK1, the renal
isoform of WNK1, is expressed in epithelial cells predomi-
nately along the DCT and CNT of the kidney and is a negative
regulator of WNK1 (McCormick et al., 2008; Richardson
et al., 2008; Subramanya et al., 2006). Decrease in KS-WNK1
activates NCC in DCT by no longer inhibiting WNK1,
allowing WNK1 to inhibit WNK4. However, our results
suggest both KS-Wnk1 and NCC are downregulated by CI
treatment as confirmed by qRT-PCR and immunohistochem-
istry, respectively. The consequence of KS-Wnk1 down-
regulation remains unclear given the fact that WNK1 and
KS-WNK1 also regulate the activity of other transport proteins
along the distal nephron in addition to NCC. More studies are
needed to examine the effects of CI treatment on the WNK
The current study identified a group of genes with
expression changes quantitatively correlated with the pro-
gression of rat kidney injury. The downregulation of these
genes precedes pathological and/or functional changes; there-
fore, the expression pattern may be predictive of CI
immunosuppressant–mediated kidney injury in rats. Combin-
ing expression changes in these CI-specific kidney injury–
responsive genes with expression changes in general kidney
injury–associated genes such as KIM1 or clusterin may provide
a diagnostic signature or biomarker of CI-specific kidney
injury. Among the genes, Slc12a3 and KS-Wnk1, which are
important components of the sodium transport regulation
CUI ET AL.
network in the DCT, are potentially involved in the mechanism
of CI-related RAS activation. How the activity of NCC and
other transporters, under the regulation of the WNK kinase
network, contribute to renin overexpression is not clear.
However, our findings support the conclusion that sodium
transport may be important in the mechanism of CI-induced
chronic nephrotoxicity. Further study of transporter activities
and the WNK kinase signaling cascade following CI treatment
may provide for a more detailed understanding of the CI-
Supplementary data are available online at http://toxsci.
Intramural Research Program of the National Institutes of
Health; National Institute of Environmental Health Sciences;
Cooperative Research and Development Agreement between
National Institute of Environmental Health Sciences and
Boehringer Ingelheim Pharma., Inc. (CRADA Z01ES023026).
The authors would like to thank Drs John Pritchard, Richard
Irwin, and Scott Auerbach for their helpful comments.
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