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2012 40: 1049 originally published online 11 May 2012Toxicol PatholKristel Buyens and Ann Lampo

Petra Vinken, Sofie Starckx, Erio Barale-Thomas, Adriana Looszova, Manisha Sonee, Nick Goeminne, Loes Versmissen,Tissue Kim-1 and Urinary Clusterin as Early Indicators of Cisplatin-Induced Acute Kidney Injury in Rats

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Tissue Kim-1 and Urinary Clusterin as Early Indicators ofCisplatin-Induced Acute Kidney Injury in Rats

PETRA VINKEN1, SOFIE STARCKX

1, ERIO BARALE-THOMAS1, ADRIANA LOOSZOVA

1, MANISHA SONEE2, NICK GOEMINNE

1,

LOES VERSMISSEN1, KRISTEL BUYENS

1, AND ANN LAMPO1

1Drug Safety Sciences, Janssen Research & Development, a division of Janssen Pharmaceutica N.V., Beerse, Belgium2Drug Safety Sciences, Janssen Research and Development, L.L.C., Raritan, New Jersey, USA

ABSTRACT

The kidney is one of the main targets of drug toxicity, and early detection of renal damage is critical in preclinical drug development. A model of

cisplatin-induced nephrotoxicity in male Sprague Dawley rats treated for 1, 3, 5, 7, or 14 days at 1 mg/kg/day was used to monitor the spatial and

temporal expression of various indicators of kidney toxicity during the progression of acute kidney injury (AKI). As early as 1 day after cisplatin

treatment, positive kidney injury molecule-1 (Kim-1) immunostaining, observed in the outer medulla of the kidney, and changes in urinary clusterin

indicated the onset of proximal tubular injury in the absence of functional effects. After 3 days of treatment, Kim-1 protein levels in urine increased

more than 20-fold concomitant with a positive clusterin immunostaining and an increase in urinary osteopontin. Tubular basophilia was also noted,

while serum creatinine and blood urea nitrogen levels were elevated only after 5 days, together with tubular degeneration. In conclusion, tissue Kim-1

and urinary clusterin were the most sensitive biomarkers for detection of cisplatin-induced kidney damage. Thereafter, urinary Kim-1 and osteopon-

tin, as well as clusterin immunostaining accurately correlated with the histopathological findings. When AKI is suspected in preclinical rat studies,

Kim-1, clusterin, and osteopontin should be part of urinalysis and/or IHC can be performed.

Keywords: nephrotoxicity; biomarker; Kim-1; clusterin; rat; urinalysis; immunohistochemistry; cisplatin.

INTRODUCTION

Proximal tubular injury due to drug-induced nephrotoxicity

occurs more frequently than other nephrotoxic effects, reflecting

the high number of xenobiotics that are reabsorbed and excreted

within the proximal tubule. The evaluation of nephrotoxicity is

therefore important for determination of the safety of drug candi-

dates. In addition to several urinary tests (e.g., urine density, dip-

stick analysis, and microscopy of urine sediment), blood urea

nitrogen (BUN) and serum creatinine are the traditional clinical

pathology markers for detecting kidney injury. Neither markers

are, however, very specific nor very sensitive, as there is usually

a significant loss of renal function and renal mass before BUN and

creatinine increase. Furthermore, their levels are also affected by

a range of other parameters, as the synthesis and release of BUN

into the blood depends on protein intake, catabolism, and liver

function, whereas serum creatinine levels depend on muscle

mass, age, and dehydration status.

It is therefore clear that there is a need for more specific and

sensitive biomarkers not only in clinical settings but also in pre-

clinical development, allowing early detection of potential

nephrotoxic drug candidates. In recent years, collaborative

cross-pharma projects, also involving regulatory agencies,

have delivered new tools for improved assessment of nephro-

toxicity, but also for improved clinical care and increased diag-

nostic performance (Dieterle et al. 2008, 2010; Vaidya et al;

2010). The Predictive Safety Testing Consortium (PSTC),

organized by the Critical Path Institute (C-Path), qualified a

‘‘kidney biomarker panel,’’ including Kim-1, clusterin, albu-

min, total protein, b2-microglobulin, cystatin C, and trefoil fac-

tor 3 (TFF3) in urine that was approved by the FDA and

EMEA in the context of nonclinical drug development (FDA

2008; EMEA 2008). In addition, renal papillary antigen-1

(RPA-1) was qualified by the ILSI/HESI consortium as urinary

biomarker for the detection of renal collecting duct injury in

male rats (EMA 2010). Other urinary renal safety biomarkers

such as osteopontin, neutrophil gelatinase-associated lipocalin

(NGAL), and a-gluthathione-S-transferase (a-GST) have been

described in recent literature (Gautier et al. 2010; Haase et al.

2009; Yang et al. 2007) as associated with renal injury.

Some of these novel biomarkers can also be useful in other

preclinical species than the rat, like Kim-1 mRNA, described as

a predictive biomarker for AKI in dogs at time points earlier

than serum creatinine (McDuffie et al. 2010). Furthermore, the

investigation of several of the above mentioned kidney biomar-

kers in dogs and nonhuman primates is an ongoing effort by the

PSTC.

Address correspondence to: Petra Vinken, Janssen Research &

Development, Drug Safety Sciences, Turnhoutseweg 30, B-2340 Beerse, Bel-

gium; e-mail: [email protected].

Abbreviations: AKI, acute kidney injury; a-GST, a-glutathione-S-

transferase; BUN, blood urea nitrogen; C, control group; C-Path, Critical

Path Institute; CDDP, cisplatin, or cis-diamminedichloroplatinum(II); EGF,

endothelial growth factor; ELISA, enzyme-linked immunosorbent assay;

EMEA, European Medicines Agency (EMA); FDA, U.S. Food and Drug

Administration; H&E, hematoxylin and eosin; HESI, Health and Environmen-

tal Sciences Institute; IHC, immunohistochemistry; ILSI, International Life

Sciences Institute; Kim-1, kidney injury molecule-1; NGAL, neutrophil

gelatinase-associated lipocalin/lipocalin-2; OSOM, outer stripe of the outer

medulla; PSTC, Predictive Safety Testing Consortium; RPA-1, renal papillary

antigen-1; SD, Sprague Dawley; TFF3, trefoil factor 3; VEGF, vascular

endothelial growth factor.

1049

Toxicologic Pathology, 40: 1049-1062, 2012

Copyright # 2012 by The Author(s)

ISSN: 0192-6233 print / 1533-1601 online

DOI: 10.1177/0192623312444765



The present study focuses on AKI in male Sprague Dawley

(SD) rats after administration of CDDP (cisplatin, or cis-diam-

minedichloroplatinum(II)), a well-established nephrotoxicant

that induces lesions in the renal proximal tubules and comprises

the spatio-temporal characterization of a set of urinary biomar-

kers compared with the traditional end points. Kim-1, clusterin,

NGAL, osteopontin, albumin, b2-microglobulin, a-GST, and

RPA-1 protein levels were analyzed in urine and/or kidney sam-

ples using a combination of ELISA-techniques, multiplex biolo-

gical assays, and immunohistochemical (IHC) staining.

MATERIALS AND METHODS

Statement of Ethical Approval

All animal experiments were performed in accordance with

the European (ETS No. 123; 2007) and Belgian (Belgian Law

1991) guidelines, and with the principles of euthanasia as stated

in the ‘‘AVMA Guidelines on Euthanasia’’ (AVMA 2007). The

experiment was approved by the Janssen Research & Develop-

ment Ethical Committee and conducted in an AAALAC-

approved facility.

Animals and Maintenance

Fifty male Sprague Dawley rats (Crl: CD (SD) IGS) of

approximately 5 weeks of age upon arrival were supplied by

Charles River Laboratories and acclimatized for 1 week before

the start of the experiment. They were group-housed (5 rats/

cage) in polysulphone cages with a wire-mesh roof under rou-

tine conditions of temperature, relative humidity, ventilation,

and illumination. A pelleted maintenance diet (Ssniff, Ger-

many) was available ad libitum with free and continuous access

to drinking water. Prior to blood sampling, during urine collec-

tion, and prior to terminal kill, animals had access to water but

were fasted (20 hr + 30 min).

Study Design

Prior to the start of treatment, the rats were randomly allo-

cated to each of the control or CDDP group. CDDP (Sigma-

Aldrich, Germany) was prepared in sterile saline at a concen-

tration of 1 mg/kg body weight and administered once daily via

the intraperitoneal route (ip) for 1, 3, 5, 7, or 14 days. Control

animals received sterile saline at the same volume dosage

according to the same schedule.

Urine and blood samples were collected once before start of

treatment, on days 1, 3, 5, 7, and 14. Urine samples were col-

lected on ice overnight during approximately 20 hr via

individual metabolic cages, aliquoted, and stored at approxi-

mately –80�C until analysis. Blood for clinical chemistry was

collected from fasted rats from the sublingual veins after iso-

flurane anesthesia. On days 1, 3, 5, 7, and 14, 5 treated and 5

control animals were euthanized. The study design is depicted

in Table 1.

Laboratory Tests

Urine samples were analyzed for the following parameters

using standard laboratory procedures: urine volume (measured

gravimetrically and corrected for specific gravity), specific

gravity (Urisys 2400, Roche Diagnostics, Switzerland), creati-

nine, total urinary protein, and urinary glucose (Hitachi Modu-

lar, Roche Diagnostics, Switzerland). a-GST and RPA-1 in

urine were evaluated via enzyme immunoassay with commer-

cial antibodies (Biotrin, Ireland). Commercially available Rat

ELISA kits for detection of b2-microglobulin and clusterin

were used (BioVendor Laboratory Medicine, Czech Republic).

Rat albumin, Kim-1, osteopontin, and NGAL were measured in

a multiplex assay, Rat Kidney Injury Panel 1, on a Sector Ima-

ger–type instrument (Meso Scale Discovery, United States).

Concentrations of urinary biomarkers were normalized to

urinary creatinine, which was shown not to change in this

study. Routine blood chemistry parameters, including BUN

and serum creatinine, were obtained utilizing a Hitachi-

Modular automated clinical chemistry analyzer and Roche

reagents (Roche Diagnostics, Switzerland). Creatinine clear-

ance was calculated from the creatinine concentration in the

collected urine sample, the urine flow rate (volume/min), and

the creatinine serum concentration, and corrected for body

weight.

Histology and IHC

On the day of necropsy, a complete physical examination

was performed and the body weight was recorded for all rats.

After anesthesia with isoflurane, they were euthanatized by

exsanguination via the carotid artery, and a necropsy was per-

formed where all macroscopic changes were recorded. The

brain and kidneys were weighed (the brain weights were used

to calculate the kidney to brain weight ratio) and sampled

together with all tissues showing macroscopic changes in

10% formalin.

Kidneys were processed routinely (trimming, embedding,

sectioning at 3–5 mm, and staining with hematoxylin and eosin

[H&E] histochemical stain).

TABLE 1.—Experimental design.

Treatment Rats: number per time point [total] Blood collection: days Urine collection: days Necropsy: days

Control 10 (5)a [25] (–1)b – 1 – 3 – 5 – 7 – 14 (–2)–(–1)b, 0–1, 2–3, 4–5, 6–7, 13–14 1 – 3 – 5 – 7 – 14

CDDP: 1 mg/kg/day ip 10 (5)a [25] (–1)b – 1 – 3 – 5 – 7 – 14 (–2)–(–1)b, 0–1, 2–3, 4–5, 6–7, 13–14 1 – 3 – 5 – 7 – 14

a N ¼ 10 for blood and urine collections on days 1, 3, 5, and 7; N ¼ 5 for histopathology on all time points and blood and urine samples on day 14.b Predose collection N ¼ 50.

1050 VINKEN ET AL. TOXICOLOGIC PATHOLOGY



Additional sections of kidney fixed by immersion in 10%buffered formalin for 48 hr and embedded in paraffin were

prepared on silane-coated glass slides, and manually immu-

nostained for Kim-1 or clusterin detection. Briefly, for the

anti-Kim-1 antibody, after routine deparaffination and rehy-

dration through gradient ethanol immersions, the slides were

exposed to peroxidase-blocking solution (Dako, Denmark),

then avidin and biotin blocking solutions (Dako, Denmark)

according to the manufacturer. Then, normal horse serum

(Vector Laboratories, USA) at 1/10 was applied for 30 min

at 4�C, and the primary antibody (TIM/KIM-1, ref. AF3689,

R&D Systems, USA) at 3 mg/ml was incubated for 2 hr at

4�C, a horse anti-goat biotinylated secondary antibody

(Vector Laboratories, USA) was incubated at 1/200 for 30

min at room temperature, an avidine-biotine complex (VEC-

TASTAIN Elite ABC Kit, Vector Laboratories, USA) was

applied. Diaminobenzidine (Liquid DABþ, Dako, Denmark)

was used as chromogen, Mayer’s hematoxylin as counter-

stain, then the slides were dehydrated and coverslipped with

a synthetic mounting media. The buffer used in all steps

was TBS (prepared in the laboratory). For the anti-

clusterin antibody (Clusterin [H-330], ref. sc-8354, Santa

Cruz Biotechnology Inc., USA), the protocol was similar

except that the blocking steps were preceded by a heat-

induced epitope retrieval step (microwave technique with

Target Retrieval, pH 9, Dako, Denmark: 3 min at 600 W,

then 2 � 5 min at 180 W), the primary anti-clusterin anti-

body was incubated for 1 hr at room temperature, and PBS

(Invitrogen, USA) was used as buffer. A secondary antibody

control (Burry 2011) was performed by eliminating the pri-

mary antibody (‘‘negative control’’).

Organ weight and gross and histopathology data were captured

with the PathData system (PDS Preclinical Data Systems, Inc.,

USA) for statistical analysis and reporting. A pathologist per-

formed the assessment of each animal and recorded the findings

with a qualitative scale of 5 grades (minimal, slight, moderate,

marked, severe) indicating either an increase in the number of cells

affected or in the distribution of the finding, or an increase in the

staining intensity, particularly for the IHC stains. The individual

findings and their grades were then compiled in Tables 6 to 8.

TABLE 2.—Urinalysis and clinical chemistry findings after CDDP treatment.

Day 1 Day 3 Day 5 Day 7 Day 14

Serum

Creatinine (mg/dL) C 0.22 + 0.02 0.28 + 0.05 0.24 + 0.01 0.25 + 0.01 0.23 + 0.01

CDDP 0.23 + 0.00 0.31 + 0.03 0.75 + 0.08* 0.59 + 0.11* 0.69 + 0.09*

BUN (mg/dL) C 17.1 + 1.7 20.3 + 1.3 15.9 + 0.7 16.4 + 0.6 14.8 + 0.7

CDDP 15.9 + 0.3 22.2 + 1.9 43.1 + 4.3* 51.3 + 9.1* 92.1 + 24.7*

Urine

Glucose (mg/mg Ucr) C 0.19 + 0.01 0.19 + 0.01 0.18 + 0.01 0.18 + 0.01 0.21 + 0.01

CDDP 0.18 + 0.01 0.44 + 0.25 20.81 + 3.1* 3.23 + 0.97* 0.87 + 0.34*

Protein (mg/mg Ucr) C 0.7 + 0.1 0.6 + 0.0 0.7 + 0.0 0.9 + 0.1 1.0 + 0.1

CDDP 0.8 + 0.2 0.8 + 0.1 4.2 + 0.4* 1.3 + 0.2 1.1 + 0.3

Urea Nitrogen (mg/mg Ucr) C 24.5 + 1.4 23.2 + 0.8 23.1 + 0.7 22.7 + 0.7 22.3 + 1.4

CDDP 23.2 + 0.9 25.4 + 0.8 26.8 + 0.7* 30.8 + 1.8* 54.4 + 10.8*

Creatinine clearance (ml/min/kg) C 8.7 + 0.55 8.7 + 0.55 8.1 + 0.29 7.5 + 0.35 8.7 + 0.69

CDDP 8.6 + 0.54 8.6 + 0.54 2.8 + 0.35* 3.4 + 0.43* 2.1 + 0.43*

Volume (ml) C 13.6 + 1.8 14.3 + 1.0 18.6 + 2.4 16.6 + 1.8 18.9 + 2.8

CDDP 20.7 + 4.2 16.8 + 1.7 22.2 + 1.9 20.3 + 5.2 13.2 + 1.2*

Data are expressed as means + SE. N ¼ 10, except on day 14 where N ¼ 5. Values significantly different from control are indicated as *p<.05, **<.01, ***p<.001.

Ucr: urinary creatinine.

TABLE 3.—Urinary biomarker reference ranges in untreated male Sprague Dawley rats.

Biomarker Unit Mean SE N 95% confidence interval

a-GST mg/mg Ucr 0.56 0.03 71 0.50–0.61

RPA-1 U/mg Ucr 0.85 0.07 71 0.71–0.98

Albumin mg/mg Ucr 22 1.1 71 19.9–24.3

Clusterina ng/mg Ucr 285 21 65 244–326

Clusterinb ng/mg Ucr 80 5.7 45 68.6–91.4

b2-microglobulin ng/mg Ucr 1,348 58 71 1,234–1,462

KIM-1 ng/mg Ucr 2.0 0.42 69 1.18–2.8

Osteopontin ng/mg Ucr 5.2 0.49 49 4.2–6.2

NGAL ng/mg Ucr 427 52 70 325–529

Ucr: urinary creatinine.aCalculated from predose collection samples.bCalculated from control rats during treatment.

Vol. 40, No. 7, 2012 KIM-1 AND CLUSTERIN IN ACUTE KIDNEY INJURY IN RATS 1051



Statistical Analysis

Routine biochemistry and biomarker data were expressed as

means + standard error (SE). The significance of differences

between the CDDP-dosed group and the control group was

assessed by the Mann-Whitney U-test for pairwise comparison

(Siegel and Castellan 1988) (two-tailed with correction for

ties). Fold-changes of serum and urinary biomarkers results

were expressed versus time matched controls. Kidney weights

were expressed as means + standard deviation, and analyzed

by the closed Dunn’s test (Dunn 1964). Values significantly

FIGURE 1.—Analysis of serum (creatinine, BUN) and urinary (glucose, total protein, albumin, RPA-1,a-GST, NGAL,b2-microglobulin, clusterin, Kim-1,

and osteopontin) biomarkers in male SD rats treated with CDDP (1 mg/kg) for 1, 3, 5, 7, and 14 days. Parameters were also analyzed prior to CDDP treat-

ment on day –1 (predose). All urinary parameters are normalized to urinary creatinine. Data are expressed as means+ SE. N¼ 50 on day –1, N¼ 10 on

days 1,3, 5, and 7, N¼ 5 on day 14. Values significantly different from control are indicated as *p < .05, **p < .01, and ***p < .001 at each sampling point.




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and ***p < .001.

RESULTS

Urinalysis and Serum Chemistry

After administration of CDDP at 1 mg/kg/day ip daily for up

to 14 days, the levels of nephrotoxicity biomarkers were

analyzed. Routine urinalysis and serum chemistry data are

shown in Table 2. Traditional measurements for detection of

renal damage were statistically (p < .05) increased from day 5

onward. Serum creatinine was increased by 2- to 3-fold on day

5, 7, and 14; and BUN showed a gradual increase from 3-fold

on day 5 up to 6-fold by day 14. Glucose was detected in urine

from day 5 onward (150-fold increase on day 5, 18-fold on day

7, and 5-fold on day 14, relative to controls), with no biologically

relevant alterations observed in serum glucose. Additionally on

day 5 and thereafter, creatinine clearance in CDDP-treated rats

was decreased to 24–40% of that in control rats, signaling

decreased renal functionality.

Analysis of Urinary Biomarkers

In control animals, urinary biomarker concentrations were

consistent with the predose data throughout the study, except

for RPA-1 and clusterin (Table 3). RPA-1 levels showed a gra-

dual increase in control rats during the 14-day period, whereas

clusterin levels in the control group were consistently lower

during the treatment period than for predose values (Figure 1).

Following repeated dosing with CDDP, the urinary concen-

trations of several biomarkers exhibited a time-related

increase, apparent from day 1 with an increase in clusterin,

urinary albumin, and NGAL. Clusterin in urine was increased

2- to 3-fold (p < .05) on day 1 and day 3 and 13-fold (p <

.001) on day 5 as compared with concurrent controls. There-

after clusterin levels declined but were still significantly

increased compared with control. Urinary albumin showed an

early and significant (p < .05) 3-fold increase in the CDDP

group on day 1, and although there was no measurable change

on day 3, remarkable increases of 73-, 29-, and 15-fold on day

5, 7, and 14, respectively, were observed. Increased urinary

protein levels were detected on day 5 only. Urinary NGAL was

increased in the CDDP dose group on day 1 (1.9-fold, p < .05)

and on days 5, 7, and 14 (up to 5.5-fold, p < .05) but did not

show a statistically significant increase on day 3.

The CDDP treated rats exhibited a marked elevation of urin-

ary Kim-1 in a time-dependent manner starting at day 3. Kim-1

exhibited a 22-, 60-, 57-, and 79-fold increase (p < .001)

compared with the control group on day 3, 5, 7, and 14, respec-

tively. An increase in urine osteopontin concentrations in urine

was detected after 3 days of treatment (3-fold increase, not sta-

tistically significant) and peaked on day 7 (18-fold increase, p <

.001) in the CDDP group, although the values remained mark-

edly elevated until the end of the study.

Urinary a-GST displayed a significant increase on day 5

(10-fold; p < .001) and day 7 (2-fold increase, p < .001) but

returned to baseline values comparable with control rats by day

14. Urinary b2-microglobulin was also increased on day 5 (2-

fold, p < .001) and on day 14 (2-fold, p < .01), but was compa-

rable with control levels on day 7.

No relevant treatment-related changes were observed for

RPA-1.

Body Weights, Organ Weights, Necropsy Findings

A toxicologically significant decrease in final body weights

in the groups dosed for 7 or 14 days with CDDP resulted in an

apparent increase in kidney to body weights (Table 4); as other

weight parameters were not modified, kidney weight change

was not considered toxicologically relevant. Pale discolora-

tions were noted in the kidney (Table 5), particularly at the cor-

ticomedullary junction, with an increasing incidence from day

5 to day 14. These findings are consistent with the histopatho-

logical changes seen in these groups (tubular dilatation, accu-

mulation of cell debris, and hyaline casts).

Histopathology Findings

No noteworthy findings were recorded in the control group

at any time point.

Microscopic examination of H&E slides showed typical

changes associated with CDDP administration (Yao et al.

2007) as shown in Figures 2 and 4. Table 6 presents the changes

recorded in relevant pathological processes. Tubular basophilia

with apoptosis in the pars recta of the proximal tubules (S3)

close to the outer stripe of the outer medulla (OSOM), of a min-

imal severity, was seen after 3 days of treatment. With

increased dosing duration, other degenerative changes were

noted: tubular giant cells, crowded basophilic nuclei with pro-

minent nucleoli and megalokaryosis, thickened basement

membrane in basophilic/degenerative tubules; tubular necrosis;

hyalin casts and cell debris/exfoliation in the lumen, tubular

dilatation. The incidence and severity of the changes increased

over time, and the distribution extended from the OSOM to the

medullary rays (straight tubules, then upper collecting ducts),

the cortex (proximal, S2 tubules), and the papilla (loop of

Henle, lower collecting ducts).

A minimal positive Kim-1 staining in the brush border of

cells of the pars recta was detected in the control animals.

In the treated animals, an increase of the specific staining

was seen first in the OSOM at day 1, as summarized in

Tables 7 and 8 and shown in Figures 3, 5, and 6. Its inten-

sity and extent increased with the treatment duration, in

close correlation with the histopathological changes. On day

1, the apical cytoplasm of OSOM tubular cells was stained

TABLE 5.—Macroscopic findings in CDDP-treated groups.

Day of treatment 1 3 5 7 14

Number of animals 5 5 5 5 5

Pale kidney 0 0 1 3 5

No animal from the concurrent control groups showed this macroscopic change.




(Figure 3B); in the later time points, all the cytoplasm and

the luminal debris were stained. Conversely, the loops of

Henle and collecting ducts in the papilla showed a positive

staining of luminal debris first, and a cytoplasmic staining

in later time points.

The clusterin staining followed a pattern similar to the Kim-

1 staining, except that there was no positive staining at day 1,

the staining was generally less intense, mainly luminal and

rarely cytoplasmic (and in only 1 animal at day 3), and less

areas were positively stained in the cortex and medulla,

although more areas were stained in the papilla (Figure 7).

The staining similarities between Kim-1 and clusterin were

exemplified in 2 animals: both markers correctly detected that

the severity of the findings of 1 animal dosed for 5 days was

lower than the mean of its group, while it was higher in 1 ani-

mal dosed for 3 days (however, the clusterin staining was less

intense than the Kim-1).

Overall, there was a close correlation between the kidney to

body weight increase (after dosing for 7 and 14 days), necropsy

findings (discoloration after dosing for 5, 7, and 14 days), and

microscopy (tubular and ductular degeneration to necrosis), in

relation to dose duration.

DISCUSSION

The purpose of this 2-week study was to evaluate the time

course of both newly qualified and traditional nephrotoxicity

biomarkers after repeated CDDP dosing in parallel with histo-

pathology assessments, in a well-characterized model of

nephrotoxicity in Sprague Dawley rats. Due to the number of

time points, and to reduce the number of animals included in

the study, it was decided to only use male rats. The animals

were dosed daily at 1 mg/kg/day for 1, 3, 5, 7, or 14 consecutive

days. The simultaneous collection of serum, urine, and kidney

samples allowed us to relate the response of biomarkers in

serum or urine to renal histopathology and IHC of specific

biomarkers.

Histologically, administration of CDDP resulted in the

expected kidney changes, namely degeneration and necrosis

FIGURE 2.—H&E stain. A (control) and B (CDDP, 1 day): no finding. C (CDDP, 3 days): basophilic tubules (Þ). D (CDDP, 7 days): tubular dila-

tion (Ł) and tubular cell debris. (¤). E (CDDP, 7 days): hyaline casts in collecting ducts (�). F (14 days): marked changes. 1: cortex; 2: OSOM;

3: inner stripe of the outer medulla; 4: pelvis; 5: inner medulla. Original magnification: 5�, bar ¼ 250 mm.




of the S3 segment of the renal proximal tubules (Yao et al.

2007), and confirmed the validity of our experiment.

We interpreted the minimally positive immunostaining seen

in control animals as a limited specific expression of Kim-1 in

untreated animals, rather than a background or nonspecific

staining, because it was limited to the brush border of cells in

the pars recta, very different from an edge effect seen in the

subcapsular area, and there was no background staining in the

negative control in our experiment. Moreover, this result is in

line with previous reports of low levels of Kim-1 mRNA (van

Timmeren et al. 2006) and low kidney Kim-1 protein expres-

sion (de Borst et al. 2007) in control rats.

The increase in specific Kim-1 IHC staining allowed detec-

tion of the kidney damage as early as 24 hours after the first

CDDP dose, and showed a large magnitude of change over time

with the progression of toxicity. Changes in urinary Kim-1 par-

alleled renal Kim-1 staining although with a slight time lag,

with elevated levels by day 3 of CDDP administration, as

Kim-1 expression on the apical membrane of the proximal

tubule is required before Kim-1 can be shed in the lumen and

detected in urine. All these data are consistent with other

reports of Kim-1 as a highly sensitive marker of acute drug-

induced kidney injury (Vaidya et al. 2010; Tonomura et al.

2010) and a useful biomarker for monitoring toxicity progres-

sion following repeated dosing up to 90 days in a subchronic

setting (Hoffmann, Fuchs, et al. 2010).

Clusterin immunostaining was as specific but less notice-

able than Kim-1, in contrast with urinalysis, where clusterin

was more sensitive than Kim-1 for early detection, although the

response magnitude was smaller and the biomarker’s progres-

sion differed from that of Kim-1. Levels peaked at day 5 but did

not increase further with progressing toxicity at subsequent

time points, although still remained elevated compared with

controls. Clusterin upregulation has been described with prox-

imal tubule and collecting duct degeneration and regeneration

(Harpur et al. 2011). However, the more limited clusterin

FIGURE 3.—Kim-1 stain. A (control): specific cytoplasmic staining, apical, in pars recta (S3) in the OSOM (Þ); insert: similar area in a negative

control at higher magnification. B (CDDP, 1 day): slight cytoplasmic staining in the same area (Ð). C (CDDP, 3 days) and D (CDDP, 7 days): the

staining extends in the medullary rays (¤) with an increased intensity. E (CDDP, 7 days): the staining reaches the collecting ducts in the medulla

(�). F (CDDP, 14 days): medullary hyalin casts are stained (�); in the cortex, the only areas preserved are around glomeruli (not shown). Original

magnification: 5�, bar ¼ 250 mm. Respective images from Figures 2 and 3 are taken from serial sections.




immunostaining, compared to both the urine clusterin data and

the Kim-1 immunostaining, suggests that the antibody and the

immunostaining method lacked sensitivity.

Osteopontin showed a similar time course as urine Kim-1

but seemed somewhat less sensitive for early detection. Osteo-

pontin in urine indicated kidney damage on day 3, which is

comparable in time scale with H&E staining. Similarly to

Kim-1, osteopontin gradually increased with the duration of

dosing and its levels in urine correlated with the degree of kid-

ney damage.

Urinary albumin, NGAL, a-GST, and b2-microglobulin did

not increase consistently prior to day 5 and hence did not show

a greater sensitivity compared with the standard clinical pathol-

ogy parameters. However, the magnitude of increase in these

FIGURE 4.—OSOM, H&E stain. A (control) and B (CDDP, 1 day): no finding. C (CDDP, 3 days): early basophilic tubules (Þ) with slightly darker

tubules and crowed nuclei; insert: basophilic, regenerating tubule with thickened basement membrane. D (CDDP, 7 days): degenerative tubular

cells (Ð) with apoptotic bodies; hyaline casts (�); insert: mitosis of an exfoliated giant tubular epithelial cell. E (CDDP, 7 days): exfoliating cells

(Ł) with tubular dilation, elongating, regenerating tubular cells and inconspicuous inflammation; insert: megalokaryosis of epithelial cells, with

prominent nucleoli. F (CDDP, 14 days): similar, more severe changes; insert: exfoliation of tubular cells. Original magnification: 20�, bar ¼100 mm.

TABLE 6.—Summary of histopathological observations in kidney from CDDP-treated groups (H&E staining).



Tubular basophilia – þ þ þþ þþþApoptosis – þ þþ þþ þþDegenerative/necrotic changes, with debris and hyaline casts – – þþ þþþ þþþþTubular dilatation – – þþ þþþ þþþGiant cells – – þ þþ þþþ

–, no change or change similar to concurrent control groups; þ, minimal; þþ, slight; þþþ, moderate; þþþþ, marked.




novel biomarkers was larger than for serum creatinine or BUN,

and their correlation with specific kidney areas is said to be

higher (Dieterle et al. 2008).

In contrast to Kim-1 and osteopontin, the concentrations of

other urinary markers of toxicity, including glucose, total pro-

tein, albumin, and a-GST, displayed peak levels in urine gen-

erally on day 5 but declined thereafter, despite ongoing and

progressive damage to the kidneys as evidenced by micro-

scopy. The reason for this discrepancy between expanding kid-

ney damage and regressing markers of toxicity is unclear. It

may be postulated, however, that due to an overload state at the

level of the remaining intact proximal tubular cells, cell signal-

ing pathways and gene transcription become altered and as a

result the expression and activity of renal transporters is modu-

lated to compensate for the glucose and protein loss (Mather

and Pollock 2011; Baines and Brunskill 2010). For a-GST,

which can be found in proximal tubular cells, this regression

could be related to the abnormal physiology of cells which are

either deeply damaged or contributing to tubular regeneration

(see Figures 4 and 5); in contrast, Kim-1 secretion would be

very robust as long as the cell is not dead. The temporal expres-

sion of all the parameters is summarized in Table 9.

Urinary biomarkers were also measured prior to start of

treatment to establish a preliminary reference range and to

assess the physiological and biological variation of the new

urinary biomarkers. Despite known differences in immunoas-

say results due to differences in sensitivity and specificity of

antibodies and differences in standardization, the normal range

obtained for the different biomarkers was generally in line with

other published data (Hoffmann, Adler, et al. 2010; Ozer et al.

2010; Zhou et al. 2008). It should be noted that predose sam-

ples were collected under suboptimal conditions because the

rats were not allowed time to acclimatize to the metabolism

cages. As a consequence, urinary clusterin values were remark-

ably higher in control animals during the predose collection

compared with the results obtained during the dosing period.

FIGURE 5.—OSOM, Kim-1 stain. A (control): inconspicuous staining of some deep tubules (Þ); insert: staining of apical cytoplasm and brush

border. B (CDDP, 1 day): the staining is more intense and more basal (Ð), and extends in the medullary rays (¤); insert: cytoplasm almost com-

pletely stained. C (CDDP, 3 days): more intense staining; insert: cytoplasm completely stained in a basophilic tubule, with thickened basement

membrane. D (CDDP, 5 days): most of the tubular cells are stained, but also the cell debris and hyaline casts in the tubular lumen (Ł); insert:

flattened, regenerating cells are also stained. E (CDDP, 7 days): the full cytoplasm is stained; note the staining of exfoliated tubular cells (�).

F (CDDP, 14 days): note rare, unstained, and intact tubules (�). Original magnification: 20�, bar ¼ 100 mm. Respective images from Figures

4 and 5 are taken from serial sections.




We therefore believe that the results for clusterin obtained from

control animals during the study (ip saline) are more represen-

tative than those collected predose. Several urinary RPA-1 data

in control animals are published, showing either steady concen-

trations (Gautier et al. 2010) or a degree of variation at different

time points (Rouse et al. 2011), and in this study, RPA-1 also

showed a trend for a continuous increase over time in control

animals. In view of the observed variability we consider

RPA-1 a less robust marker in our experiment. Furthermore,

RPA-1 levels in treated rats did not differ from the controls and

this was in contrast to Kim-1 IHC staining in the papilla at day

5 (lumen only) and at days 7 and 14 (lumen and cytoplasm).

In conclusion, the advantage of the novel urinary biomar-

kers is that they provide a noninvasive and quick way for the

early assessment of kidney injury, and that they also allow

monitoring of the pathophysiological process in the same ani-

mal. This is an important feature in preclinical toxicity studies,

to enable the detection of potential issues related to kidney

toxicity earlier during the screening and development of new

drug candidates. Our experiment in male SD rats demonstrated

once more that traditional biomarkers, which are primarily

functional endpoints, fail to detect minimal to moderate kidney

injury. Tissue Kim-1 and urine clusterin were the most sensi-

tive parameters for detection of CDDP-induced proximal tubu-

lar toxicity before any histopathological alterations occurred.

Thereafter, levels of urinary Kim-1 and osteopontin, as well

as clusterin immunostaining accurately correlated with the pro-

gression of kidney injury. In preclinical rat studies, the combi-

natorial use of the above-described biomarkers with traditional

endpoints allows the early detection and monitoring of nephro-

toxicity, together with tissue Kim-1 for the precise localization

of the region of kidney injury.

ACKNOWLEDGMENTS

We thank the laboratory staff of the Drug Safety Sciences

department for the labour intensive tasks involved in this study,

Tim Erkens for his helpful suggestions, and Graham Bailey for

FIGURE 6.—Kim-1 stain. Cortex (A, B, C); papilla (E, F, G). A (control): no staining. B (CDDP, 7 days): staining in medullary rays. C (CDDP, 14

days): staining of most cortex, except the glomerules and the tubules closest to the glomerules. D (CDDP, 5 days): staining of hyaline casts; insert:

epithelial cells are not stained. E (CDDP, 7 days): hyaline cast staining in loops of Henle (Þ) and collecting ducts (Ð); insert: detail of stained

epithelial cells and cellular debris. F (CDDP, 14 days): stronger and more frequent staining, not only of hyaline casts but also cytoplasmic (�).

Original magnification: 20�, bar ¼ 100 mm.




FIGURE 7.—Clusterin (A, B, C) vs. Kim-1 (D, E, F) stain, same animal treated with CDDP for 14 days. Compare staining in similar areas of cortex

(A, D), OSOM (B, E), papilla (C, F): tubular cells (Ł), hyaline casts/debris (��Þ), cytoplasm in loop of Henle (¡). Original magnification:

20�, bar ¼ 100 mm.

TABLE 7.—Summary of Kim-1 staining in kidney from CDDP-treated groups.



Tubular staining in the OSOM þ þþ þþþ þþþ þþþþTubular staining in the medullary rays – þ þþ þþþ þþþþTubular staining in the cortex – – þ þþ þþþPapillary staining – – þ þþ þþþ

–, no staining or similar to concurrent control groups; þ, minimal; þþ, slight; þþþ, moderate; þþþþ, marked.

TABLE 8.—Summary of clusterin staining in kidney from CDDP-treated groups.



Tubular staining in the OSOM – – þ þ þþTubular staining in the medullary rays – þ þ þþ þþþTubular staining in the cortex – – þ þ þþPapillary staining – – þ þþþ þþþþ

–, no staining or similar to concurrent control groups; þ, minimal; þþ, slight; þþþ, moderate; þþþþ, marked.




the review of this article. We also thank the Renal Integrated

Safety Assessment (ISA) team and the Safety Biomarkers Cen-

ter of Excellence within Janssen Research and Development

for their support of this manuscript. Petra Vinken, Sofie

Starckx, and Erio Barale-Thomas contributed equally to this

work

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TABLE 9.—Temporal changes of relevant parameters.


Serum Creatinine – – þ þ þBUN – – þ þ þ

Urine Glucose – – þ + +Total Protein – – þ – –

Decreased Creatinine Clearance – – þ þ þa-GST – – þ + –

RPA-1 – – – – –

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