Research Article Received: 25 November 2013,
Revised: 20 January 2014,
Accepted: 22 January 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jat.2999
Usefulness of urinary kidney injury molecule-1 (Kim-1) as a biomarker for cisplatin-induced sub-chronic kidney injury Ken-ichiro Nan-yaa,b*, Masatomo Kajiharac, Natsuki Kojimad and Masakuni Degawab ABSTRACT: We explored biomarkers suitable for monitoring sub-chronic kidney injury using the three rat models of cisplatin (CDDP)-induced kidney injury, which were designed to extend the current knowledge beyond the sub-acute exposure period. In the pilot study, a single intravenous administration of 1.5 mg kg–1 CDDP to rats was confirmed to result in no histopathological changes. Subsequently, CDDP was intravenously administered to rats at a dose of 1.5 mg kg–1 for 4 days at 24-h intervals (Experimental model 1) and for up to 10 weeks at weekly intervals (Experimental models 2 and 3), and the changes in blood and urine components, such as recently recommended urinary biomarkers (Kim-1, clusterin and so on) and traditional blood biomarkers (blood urea nitrogen and serum creatinine), were examined together with the histopathological changes in renal tissues during the development of the kidney injury in each model. In these experimental models, a significant increase in urinary Kim-1 was observed prior to the histopathological changes in renal tissues, and these changes were retained after the adverse histopathological changes. Significant changes in all of the other urinary biomarkers examined occurred along with the histopathological changes. In addition, the increase in urinary Kim-1 after weekly treatment with CDDP for 4 weeks was reduced in a time-dependent manner after cessation of the drug. The present findings indicate that urinary Kim-1 is the most useful biomarker for CDDP-induced rat sub-chronic kidney injury among the biomarkers examined. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Kim-1; urinary biomarkers; sub-chronic kidney injury; cisplatin; rat
Introduction The kidney is one of the representative organs damaged by drugs, including cisplatin (CDDP). Therefore, an early diagnosis of drug-induced kidney injury is important for adequately altering the protocol of drug therapy. The blood urea nitrogen (BUN) and serum creatinine (SCr) levels are commonly used as preclinical and clinical biomarkers for kidney injury. However, these biomarkers have severe limitations with regard to sensitivity and specificity. Increases in BUN and SCr occur only after substantial renal injury, generally after loss of two thirds of the nephrons’ functional capacity (Pfaller and Gstraunthaler, 1998; Hart, 2005; Marrer and Dieterle, 2010), and such abnormal changes were also often observed in those with extra-renal injury. Moreover, the levels of these biomarkers are affected by various physiological conditions (Hart, 2005; Proulx et al., 2005; Bonventre et al., 2010; Urbschat et al., 2011). Therefore, the discovery of new markers for diagnosing and monitoring kidney injury is necessary. Recently, the Predictive Safety Testing Consortium (PSTC) organized by the Critical Path Institute has proposed seven novel urinary biomarkers, including Kim-1, clusterin, albumin, total protein, β2-microglobulin, cystatin C and trefoil factor 3, on the basis of the results in rat acute kidney injury (AKI) models. The usefulness of these biomarkers for AKI in rats was further confirmed by the U.S. Food and Drug Administration (FDA), European Medicines Agency (EMA), Pharmaceuticals and Medical Devices Agency (PMDA, Japan) and several research
J. Appl. Toxicol. 2014
groups (Dieterle et al., 2010a, b; Yu et al., 2010; Vaidya et al., 2010; Gautier et al., 2010; Harpur et al., 2011; Hoffmann et al., 2010a; Tonomura et al., 2010; Sasaki et al., 2011; Hosohata et al., 2012; Vinken et al., 2012). Moreover, the urinary Kim-1, clusterin, cystatin C and/or neutrophil gelatinase-associated lipocalin (NGAL) levels are reported to be more sensitive than BUN and SCr as biomarkers of the kidney injury induced by repeated nephrotoxicant administration to rats (Hoffmann et al., 2010a, b; Rouse et al., 2011; Fuchs et al., 2012; Vinken et al., 2012). However, to the best of our knowledge, the relationships between the histopathological changes in renal tissue and the changes in recently recommended urinary biomarkers, including *Correspondence to: Ken-ichiro Nan-ya, Drug Discovery Research Laboratories, Kyowa Hakko Kirin Co., Ltd, 1188 Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka, 411-8731, Japan. E-mail: [email protected] a Drug Discovery Research Laboratories, Kyowa Hakko Kirin Co., Ltd, 1188 Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka, 411-8731, Japan b Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan c Development Research Laboratories, Kyowa Hakko Kirin Co., Ltd, 1188 Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka, 411-8731, Japan d Discovery and Development Research Promotion Laboratories, Kyowa Hakko Kirin Co., Ltd, 1188 Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka, 411-8731, Japan
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K. Nan-ya et al. Kim-1, clusterin and NGAL, during the development of nephrotoxicant-induced sub-chronic kidney injury are still largely unknown. Therefore, the present study was designed to extend the current knowledge beyond the sub-acute exposure period into longer term scenarios to examine more fully the time course of biomarker events. CDDP is an anticancer drug with potent therapeutic effects against numerous tumors. However, CDDP often induces kidney injury in both humans and experimental animals. This CDDPinduced nephrotoxicity, which is thought to occur through the induction of oxidative stress and apoptosis in the kidney (Kuhlmann et al., 1997; Arany and Safirstein, 2003), is the most important index for determining the protocol for CDDP therapy. In the present study, the previously proposed urinary components (albumin, clusterin, glucose, Kim-1, NGAL and osteopontin) and blood components (BUN, SCr and cystatin C) were selected as biomarkers for CDDP-induced sub-chronic kidney injury in rats, and the usefulness of these biomarkers was comparatively assessed. Furthermore, the relationships between the changes in the levels of these biomarkers and the histopathological changes in renal tissues were examined, and consequently, urinary Kim-1 was found to be the most useful biomarker for monitoring CDDP-induced sub-chronic kidney injury in rats.
Materials and Methods Reagents CDDP and physiological saline were purchased from Nippon Kayaku Co., Ltd. (Tokyo, Japan) and Otsuka Pharmaceutical Factory, Inc. (Tokushima, Japan), respectively.
Animal and Husbandry Five-week-old male Crj:CD(SD)IGS rats were purchased from Charles River Japan, Inc. (Kanagawa, Japan). The animals were
6 or 7 weeks old at the time of the first administration, and were kept for more than 7 days after they were received for quarantine and acclimation prior to the start of experiments. The animals were housed in stainless-steel hanger cages for rats (260 mm W × 360 mm D × 187 mm H: TOKIWA Phytochemical Co., Ltd, Tokyo, Japan). The animals were housed in a barrier system animal room, which was controlled to maintain a temperature range of 22 ± 3 °C, relative humidity range of 50 ± 20%, ventilation of 15 times per hour or more and illumination for 12 h. Radiation-sterilized chow for rats and mice, FR-2 10 kGy (Funabashi Farm Co., Ltd., Chiba, Japan), was provided to the animals ad libitum. The diet was used within 6 months after production. Tap water was provided to the animals ad libitum. However, water was provided ad libitum from plastic bottles when the animals were housed in metabolism cages. All experiments were conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals of Kyowa Hakko Kirin Co, Ltd (Shizuoka, Japan).
Treatment of Rats with CDDP The three experimental protocols are depicted in Fig. 1. The purpose of experimental model 1 was to identify the biomarkers suitable for monitoring the progression of CDDP-induced subchronic kidney injury. The experimental models 2 and 3 were designated to identify the biomarkers suitable for the early detection and diagnosis of CDDP-induced sub-chronic kidney injury. The first day and the first week of administration are defined as Day 1 and Week 1, respectively. Experimental model 1. CDDP, diluted in 0.9% physiological saline, was administered intravenously to rats at a dose of 1.5 mg kg–1 per injection (5 ml kg–1 via tail vein) for four consecutive days. Control group rats received the vehicle (0.9% physiological saline, 5 ml kg–1 via the tail vein). Urine samples were collected from each rat on Days 2, 3, 4, 5, 19 and 33 without fasting. The blood samples from individual rats were collected from the
Figure 1. Experimental models designated for development of cisplatin (CDDP)-induced kidney injury. The closed triangle represents the administration of CDDP or saline. The first day and the first week of administration are defined as Day 1 and Week 1, respectively. The just one week before the first administration is defined as Week 0. The symbols, such as U, B, and N, in parentheses mean urine collection, blood collection and necropsy, respectively.
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Kim-1 as a biomarker for sub-chronic kidney injury postcava of non-fasted rats under anesthesia with isoflurane on Days 5 and 33. Some of the rats were killed on Days 5 and 33, and the kidneys were removed and used for the histopathological analysis. Experimental model 2. The dose of CDDP used was the same as that described for Experimental model 1. CDDP was intravenously administered to rats (5 ml kg–1 via tail vein) for 2, 4, 6, 8 and 10 weeks at weekly intervals. Control group rats received the vehicle (5 ml kg–1 via the tail vein). The urine samples were collected for 16 h from 6 to 7 days after the last administration of CDDP from individual rats that had fasted. The blood samples of individual rats were collected from the postcava under anesthesia with isoflurane 1 week after the last CDDP administration. Some of the rats were killed at the indicated times, and the kidneys were removed and used for the histopathological analysis. Experimental model 3. The protocol used for CDDP administration was almost the same as that described in Experimental model 2. After the 4-week administration of CDDP, the treatment was ceased for 1, 3, 5 or 7 weeks, and thereafter, blood and urine samples were collected from individual rats, as described in Fig. 1. The kidneys were removed from the rats 7 weeks after the last administration of CDDP, and were used for histopathological analysis.
Analyses of Blood Biomarkers The serum was prepared from each blood sample by centrifugation for 10 min at 4 °C, and was stored at –80°C until analysis. The amounts of serum BUN, SCr and cystatin C were measured using an automatic analyzer (H7170S; Hitachi, Ltd., Tokyo, Japan).
Analyses of Urinary Biomarkers The rats were housed in metabolism cages, and the urine (16-h urine) was collected from each rat for 16 h from Day 6 to Day 7 after the administration of CDDP or saline at the indicated schedule (Fig. 1). Rats had access to water ad libitum throughout the urine collection. Urine samples were centrifuged at 400 g for 5 min, and the separated supernatant was used for the analyzes of urinary biomarkers. The amounts of creatinine and glucose were measured using an autoanalyzer (H7070; Hitachi, Ltd.). The amounts of albumin, Kim-1, NGAL, osteopontin [Kidney Injury Panel 1 (rat) assay kit] and clusterin (Rat clusterin assay kit) were measured using a Multi-Array® assay system (Meso Scale Discovery, Rockville, MD, USA). The levels of the urinary biomarkers were corrected by the urine volume.
Histopathological Assessment The kidneys were fixed in 10% neutral-buffered formalin solution. The fixed kidneys were embedded in paraffin and then sectioned. The sections were stained with hematoxylin-eosin solution according to the routine methods and then used for histopathological analyzes.
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Statistical Analysis The data are presented as the means ± standard deviation (SD). Differences between CDDP-treated versus time-matched salinetreated (control) animals were evaluated by Student’s t-test.
Results Experimental Model 1 In the CDDP-treated rats, there were no clinical signs of toxicity (external appearance, nutritional condition, posture and behavior) at any time points, although a decrease in the mean body weight gain from Day 1 to Day 33 compared with the saline-treated (control) rats was observed (mean body weight gain: saline; 261.5 g, CDDP; 169.8 g). No significant changes in the levels of BUN and SCr were observed in rats treated with CDDP for 2 days at 24-h intervals in the pilot study (data not shown). On the day after treatment with CDDP for 4 days at 24-h intervals (Day 5), the levels of BUN and SCr were significantly increased to approximately 1.9- and 2.2-fold over the corresponding control levels, respectively (Table 1). Such increases in BUN and SCr were also observed on Day 33 (1.6- and 1.5-fold increases compared with the saline-treated rats, respectively) (Table 1). Significant increases in the levels of urinary biomarkers such as albumin, clusterin, glucose, Kim-1 and osteopontin were observed on Day 5 (Table 2). However, no significant changes in the levels of any of these urinary biomarkers examined were found on Days 19 and 33. Interestingly, a significant increase (5.7-fold over the control) in the level of Kim-1 was observed even on Day 2, and the level was further increased in a timedependent fashion, at least up to Day 5. The increases appeared to be retained up to Days 19 and 33; however, no significant increases were found because of the large individual differences. Further increases in clusterin and osteopontin were also observed on Days 19 and 33. In addition, a transient and slight decrease in the level of urinary creatinine was also observed on Day 3. No histopathological changes in the renal tissues were observed in the rats treated with CDDP for 2 days at 24-h intervals in the pilot study (data not shown). On the day after Table 1. Cisplatin (CDDP)-induced changes in the levels of serum biomarkers in the rats examined in ‘Experimental model 1’ Day of blood collection Day 5 Day 33
Treatment
BUN (mg/dL)
Creatinine (mg/dL)
Saline CDDP Saline CDDP
15.6 ± 1.5 (6)a 29.7 ± 6.1* (6) 17.6 ± 0.8 (3) 28.4 ± 5.5 (3)
0.20 ± 0.01 (6) 0.43 ± 0.08* (6) 0.26 ± 0.01 (3) 0.40 ± 0.05* (3)
The ‘Days 5 and 33’ are the serum collection days shown in Fig. 1. a The numbers shown in parentheses represent the number of the animals examined. The data of each biomarker examined present the mean ± SD. * Statistically significant differences from the corresponding saline-treated groups: P < 0.05.
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Saline CDDP Saline CDDP Saline CDDP Saline CDDP Saline CDDP Saline CDDP Saline CDDP Saline CDDP
Treatment
Day 3 2.9 ± 0.6 (6) 2.9 ± 1.5 (6) 0.78 ± 0.33 (6) 0.77 ± 0.20 (6) 0.11 ± 0.08 (6) 0.07 ± 0.02 (6) 2.44 ± 0.31 (6) 1.76 ± 0.48* (6) 1.52 ± 0.11 (6) 1.13 ± 0.27* (6) 3.04 ± 0.25 (6) 25.42 ± 10.51** (6) 1.23 ± 0.43 (6) 1.26 ± 0.83 (6) 8.78 ± 1.47 (6) 8.82 ± 4.19 (6)
Day 2 4.6 ± 3.2 (6)a 4.2 ± 1.7 (6) 1.10 ± 0.82 (6) 0.83 ± 0.31 (6) 0.09 ± 0.04 (6) 0.06 ± 0.02 (6) 2.54 ± 0.53 (6) 2.39 ± 0.71 (6) 1.37 ± 0.69 (6) 1.41 ± 0.47 (6) 3.44 ± 1.34 (6) 19.64 ± 7.04*** (6) 1.49 ± 0.75 (6) 1.59 ± 0.77 (6) 11.46 ± 7.54 (6) 9.08 ± 3.65 (6) 2.9 ± 1.2 (6) 2.6 ± 1.3 (6) 0.83 ± 0.28 (6) 8.71 ± 9.80 (6) 0.09 ± 0.04 (6) 0.10 ± 0.06 (6) 2.54 ± 0.47 (6) 1.74 ± 0.72 (6) 1.63 ± 0.23 (6) 2.11 ± 0.85 (6) 3.05 ± 0.83 (6) 27.31 ± 13.94** (6) 1.24 ± 0.60 (6) 1.15 ± 0.70 (6) 9.72 ± 5.38 (6) 8.57 ± 4.24 (6)
Day 4 3.5 ± 0.5 (6) 5.2 ± 3.2 (6) 0.64 ± 0.35 (6) 37.90 ± 17.94* (6) 0.12 ± 0.10 (6) 0.57 ± 0.28* (6) 2.86 ± 0.08 (6) 2.03 ± 0.81 (6) 1.36 ± 0.56 (6) 31.19 ± 17.95* (6) 3.65 ± 0.06 (6) 59.61 ± 37.65** (6) 1.04 ± 0.93 (6) 2.01 ± 0.90 (6) 10.95 ± 3.57 (6) 20.50 ± 6.90** (6)
Day 5
4.5 ± 3.8 (3) 6.3 ± 4.3 (3) 5.94 ± 3.15 (3) 10.17 ± 6.89 (3) 0.14 ± 0.04 (3) 2.81 ± 2.32 (3) 6.58 ± 1.58 (3) 6.03 ± 0.53 (3) 2.09 ± 0.63 (3) 3.60 ± 2.21 (3) 2.61 ± 4.52 (3) 98.93 ± 66.80 (3) 2.17 ± 3.75 (3) 6.47 ± 4.23 (3) 10.26 ± 17.78 (3) 97.27 ± 91.81 (3)
Day 19
5.4 ± 3.2 (3) 6.8 ± 3.7 (3) 4.24 ± 2.88 (3) 7.40 ± 3.05 (3) 0.22 ± 0.06 (3) 3.07 ± 2.80 (3) 10.27 ± 2.30 (3) 8.53 ± 0.81 (3) 3.93 ± 3.28 (3) 1.71 ± 0.86 (3) 2.85 ± 4.94 (3) 61.40 ± 43.99 (3) 2.13 ± 3.70 (3) 6.58 ± 4.26 (3) 10.70 ± 18.54 (3) 81.75 ± 83.44 (3)
Day 33
The 16-h urine was collected from each rat at the indicated schedule (Fig. 1) and used for the assays of the indicated biomarkers, as described in ‘Materials and Methods’. The ‘Days’ described are identified with those in ‘Experimental model 1’ (Fig. 1). a The numbers shown in parentheses represent the number of the animals examined. The value of each biomarker presents the mean ± SD. *,**,*** Statistically significant differences from the corresponding saline-treated groups: * P < 0.05; ** P < 0.01; *** P < 0.001.
Osteopontin (ng)
NGAL (μg)
Kim-1 (ng)
Glucose (g)
Creatinine (mg)
Clusterin (μg)
Albumin (mg)
Urine volume (ml)
Biomarkers (Amount/16-h urine)
Table 2. Cisplatin (CDDP)-induced changes in the levels of urinary biomarkers in the rats examined in ‘Experimental model 1’
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Kim-1 as a biomarker for sub-chronic kidney injury four consecutive CDDP treatments (Day 5), kidney injury, as indicated by degeneration (vacuolization, necrosis and desquamation) of the proximal tubular epithelial cells, urinary cast formation and interstitial inflammatory cell infiltration was observed (Fig. 2B). Furthermore, on Day 33, the progression of the lesions, such as the regeneration of proximal tubular epithelial cell, cystic dilation of the renal tubules, pronounced interstitial mononuclear cell infiltration and fibrosis of the interstitial tissue, was observed in the kidneys of the rats with elevated levels of urinary Kim-1, clusterin and osteopontin (Fig. 2C). Experimental Model 2 In the CDDP-treated rats, there were no clinical signs of toxicity (external appearance, nutritional condition, posture and behavior) and no changes in the mean body weight gains at any time points.
Significant increases in the levels of the blood biomarkers (BUN and SCr) were observed 7 days after weekly administration of CDDP for 10 weeks, but were not observed for the first 6 weeks (Table 3). The levels of BUN and SCr in the rats treated with CDDP for 10 weeks were about 3.6- and 2.7-fold higher than the corresponding control levels, respectively. The levels of biomarkers in the 16-h urine, which was collected from each rat for 16 h from 6 to 7 days after the administration of CDDP or saline at the indicated schedule (Fig. 1), are summarized in Table 4. The level of urinary Kim-1 was markedly increased even after the CDDP treatment for only 4 weeks, and the levels were further increased in a treatment period-dependent manner up to 10 weeks. The urinary excretion levels of Kim-1 at Week 5, 7, 9 and 11 in the CDDP-treated rats were 2.8-, 3.5-, 9.7- and 15.2-fold higher than the corresponding control levels, respectively. The urinary levels of osteopontin
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Figure 2. Representative photomicrographs of renal tissues from the rats treated with saline (A) or cisplatin (CDDP) at a dose of 1.5 mg kg per day (B and C) for 4 days at 24-h intervals. The analyzes were performed using the rats in ‘Experimental model 1’. The tissue sections were stained with hematoxylin-eosin. No clear changes are observed in the saline-treated rats on Day 5 (A). In the CDDP-treated rats, degeneration (vacuolization, necrosis and desquamation) of the proximal tubular epithelial cells, urinary cast formation and interstitial inflammatory cell infiltration are observed on Day 5 (B). On the Day 33 (C), further progression of the lesions, including regeneration of proximal tubular epithelial cells, cystic dilation of the renal tubules, pronounced interstitial mononuclear cell infiltration and fibrosis of the interstitial tissues, is observed. Scale bar: 100 μm.
Table 3. Cisplatin (CDDP)-induced changes in the levels of serum biomarkers in the rats examined in ‘Experimental model 2’ Treatment
Week of blood collection
Saline CDDP
11 3 5 7 11
Number of dosing 10 2 4 6 10
BUN (mg/dL) a
13.4 ± 0.8 (3) 14.4 ± 1.9 (3) 10.7 ± 0.9 (3) 16.5 ± 2.1 (3) 48.4 ± 12.8* (3)
Creatinine (mg/dL) 0.29 ± 0.05 (3) 0.26 ± 0.05 (3) 0.27 ± 0.04 (3) 0.32 ± 0.08 (3) 0.79 ± 0.14** (3)
Cystatin C (mg/L) 0.12 ± 0.01 0.15 ± 0.03 0.13 ± 0.02 0.14 ± 0.03 0.20 ± 0.03
(3) (3) (3) (3) (3)
Blood collections were performed at the indicated schedule shown in Fig. 1. a The numbers shown in parentheses represent the number of the animals examined. The value of each biomarker presents the mean ± SD. *,** Statistically significant differences from the corresponding saline-treated groups: * P < 0.05; ** P < 0.001.
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K. Nan-ya et al. Table 4. Cisplatin (CDDP)-induced changes in the levels of urinary biomarkers in the rats examined in ‘Experimental model 2’ Biomarkers Treatment (Amount/16-h urine) Urine volume (ml) Albumin (mg) Clusterin (μg) Creatinine (mg) Glucose (g) Kim-1 (ng) NGAL (μg) Osteopontin (ng)
Saline CDDP Saline CDDP Saline CDDP Saline CDDP Saline CDDP Saline CDDP Saline CDDP Saline CDDP
Week 3
Week 5
Week 7
Week 9
Week 11
6.7 ± 1.9 (3)a 6.4 ± 4.1 (3) 1.13 ± 0.35 (3) 1.15 ± 0.68 (3) 0.10 ± 0.03 (3) 0.12 ± 0.04 (3) 3.72 ± 0.93 (3) 3.52 ± 0.66 (3) 0.61 ± 0.34 (3) 0.68 ± 0.32 (3) 5.87 ± 0.81 (3) 7.96 ± 1.95 (3) 0.66 ± 0.22 (3) 0.99 ± 0.24 (3) 10.41 ± 4.51 (3) 10.34 ± 1.04 (3)
7.9 ± 0.3 (3) 5.7 ± 3.4 (3) 1.40 ± 0.15 (3) 0.96 ± 0.52 (3) 0.18 ± 0.03 (3) 0.18 ± 0.05 (3) 5.59 ± 0.69 (3) 4.46 ± 0.57* (3) 0.56 ± 0.43 (3) 0.41 ± 0.39 (3) 6.39 ± 0.81 (3) 17.66 ± 4.41* (3) 0.47 ± 0.06 (3) 1.10 ± 0.37 (3) 8.26 ± 2.75 (3) 9.06 ± 4.11 (3)
6.6 ± 2.8 (3) 6.3 ± 2.9 (3) 1.14 ± 0.44 (3) 0.99 ± 0.44 (3) 0.11 ± 0.02 (3) 0.27 ± 0.06* (3) 6.30 ± 0.77 (3) 5.55 ± 1.12 (3) 0.69 ± 0.06 (3) 1.06 ± 0.29 (3) 5.83 ± 2.08 (3) 20.58 ± 7.84* (3) 0.47 ± 0.21 (3) 1.01 ± 0.25 (3) 9.03 ± 3.30 (3) 10.74 ± 3.24 (3)
5.5 ± 1.6 (3) 8.4 ± 4.0 (3) 0.91 ± 0.23 (3) 1.45 ± 0.71 (3) 0.17 ± 0.06 (3) 0.25 ± 0.09 (3) 6.74 ± 1.82 (3) 6.55 ± 0.79 (3) 0.98 ± 0.11 (3) 1.05 ± 0.21 (3) 4.38 ± 0.65 (3) 42.63 ± 11.57* (3) 0.54 ± 0.08 (3) 1.81 ± 0.23* (3) 10.74 ± 3.21 (3) 20.43 ± 4.42* (3)
7.0 ± 1.2 (3) 16.7 ± 12.8 (3) 1.24 ± 0.14 (3) 2.94 ± 2.16 (3) 0.13 ± 0.03 (3) 0.32 ± 0.10* (3) 7.67 ± 0.89 (3) 6.78 ± 0.64 (3) 2.47 ± 1.75 (3) 1.34 ± 0.70 (3) 4.55 ± 2.02 (3) 69.25 ± 36.60* (3) 0.50 ± 0.11 (3) 3.11 ± 2.24 (3) 9.30 ± 1.91 (3) 61.19 ± 31.26* (3)
The 16-h urine was collected from each rat at the indicated schedule (Fig. 1) and used for the assays of the indicated biomarkers, as described in ‘Materials and Methods’. The ‘Days’ described are identified with those in ‘Experimental model 2’ (Fig. 1). a The numbers shown in parentheses represent the number of the animals examined. The data of each biomarker examined present the mean ± SD. * Statistically significant differences from the corresponding saline-treated groups: P < 0.05. were clearly increased at Weeks 9 and 11, whereas no significant increases after the shorter periods of treatment were observed. The levels of urinary clusterin and NGAL were increased at Weeks 7, 9 and/or 11. No clear increases in the levels of urinary albumin and glucose occurred throughout any of the CDDP treatment periods examined.
No histopathological changes in the kidneys were observed in the rats treated with saline (Fig. 3A) or CDDP for 4 weeks (Fig. 3B) at weekly intervals. In the kidneys of the rats treated with CDDP for 6 weeks at weekly intervals, mild degeneration (vacuolization, necrosis and desquamation) of the proximal tubular epithelial cells and interstitial inflammatory cell infiltration were observed
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Figure 3. Representative photomicrographs of renal tissues from rats treated with saline (A) or cisplatin (CDDP) at a dose of 1.5 mg kg per week (B–D) for 4, 6 and 10 weeks at weekly intervals. The analyzes were performed using the rats in ‘Experimental model 2’. The tissue sections were stained with hematoxylin-eosin. One week after the 4-week treatment with CDDP (B), no clear changes are observed in the rat renal tissues. In the renal tissues 1 week after the 6-week treatment with CDDP (C), mild degeneration (vacuolization, necrosis and desquamation) of the proximal tubular epithelial cells and interstitial inflammatory cell infiltration are observed. Further progression of the lesions is observed in the rats 1 week after the 10-week treatment with CDDP (D). Scale bar: 100 μm.
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Kim-1 as a biomarker for sub-chronic kidney injury (Fig. 3C). Furthermore, the 10-week treatment with CDDP resulted in progression of the lesions (Fig. 3D). Experimental Model 3 In the CDDP-treated rats, there were no clinical signs of toxicity (external appearance, nutritional condition, posture and behavior) and no changes in the mean body weight gains at any time points. A significant increase in the urinary Kim-1 level occurred even after the weekly treatment with CDDP for 4 weeks (Fig. 4), whereas histopathologically adverse changes were observed after the treatment for not less than 6 weeks, as described in ‘Experimental model 2’. In addition, the kidneys showed no lesions 7 weeks after weekly administration of CDDP for 4 weeks (data not shown). In this experiment using the rats treated with CDDP for 4 weeks (assumed to be no histopathological adverse changes), the time-dependent effects of the drug cessation on the urinary excretion level of Kim-1 were examined. The urinary Kim-1 level was decreased in a time-dependent manner from 3 to 7 weeks after cessation (Fig. 4). No significant changes in the levels of other urinary biomarkers, such as albumin, clusterin, glucose, NGAL and osteopontin, occurred throughout the study period (data not shown).
Discussion Sub-chronic kidney injury induced by repeated administration of CDDP at a low dose has been reported previously (Fenoglio et al., 2005; González et al., 2005). Therefore, using three experimental models with slight modifications to these models, we searched for candidate biomarkers for the early detection and monitoring of low-dose (1.5 mg kg–1) CDDP-induced kidney injury. Single administration of this dose of CDDP did not cause kidney injury. On Day 5 (following day after treatment with CDDP for 4 days) (Experimental model 1), the kidneys showed proximal tubular
Figure 4. Changes in the levels of urinary kidney injury molecule-1 (Kim-1) after cisplatin (CDDP) cessation. The analyzes were performed using the rats in ‘Experimental model 3’. The 16-h urine was collected from each rat and used for determining amounts of Kim-1, as described in ‘Materials and Methods’. Closed circles represent the individual rats (five rats at each time point examined). The bar represents the mean value of each experimental group. ‘Week 0’ means just 1 week before , the first administration (Fig. 1). * **Statistically significant differences from the corresponding saline-treated groups: *P < 0.05; **P < 0.01.
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injury. The kidney lesions subsequently deteriorated even after drug withdrawal, and the lesion on Day 33 was representative of sub-chronic kidney injury. The levels of BUN and SCr increased on Day 5, but the levels then remained unchanged up to Day 33, in spite of progression of the lesions. In contrast, the urinary Kim-1 excretion level was increased even on Day 2 and further increased in a time-dependent fashion up to Day 33. Increases in the levels of clusterin and osteopontin were also observed on Days 19 and 33, when the lesion had progressed. Therefore, these three urinary biomarkers are considered to be more useful to monitor the progression of CDDP-induced kidney injury than BUN and SCr. In addition, the early and dramatic increases in urinary Kim-1 are consistent with the findings of other reports indicating that Kim-1 is a sensitive biomarker of CDDP-induced AKI (Vaidya et al., 2006, 2010; Tonomura et al., 2010; Sasaki et al., 2011). In Experimental model 2, the histopathological examinations revealed no kidney lesions for up to 4 weeks of administration of CDDP. Mild kidney injury was observed after 6-weeks administration, and after 10-weeks administration, the progression of lesions was observed. An increase in the level of urinary Kim-1 was observed in a time-dependent fashion from 4 weeks, although changes in the levels of BUN and SCr were observed for the first time after 10 weeks of administration. Thus, the changes in the urinary Kim-1 level were observed prior to those in other blood biomarkers and before histopathological changes were observed, again suggesting that Kim-1 is a useful biomarker for the early detection of CDDP-induced sub-chronic kidney injury. The lesion at an initiation stage of CDDP-induced kidney injury is usually transient, and the damaged proximal tubular epithelial cells can be repaired (Pinches et al., 2012). The subchronic kidney injury seems to develop through repeated cellular damage and repair. In fact, in the present study, interstitial fibrosis and mononuclear cell infiltration in the kidneys were observed in both Experimental models 1 and 2. Kim-1 is a phosphatidylserine receptor expressed on renal epithelial cells that recognizes apoptotic cells and plays an active role in the clearance of apoptotic cells (Ichimura et al., 2008). Dramatic increases in the levels of Kim-1 mRNA and protein occurred in the injured kidneys, although the levels were extremely low in the normal kidney. During the development of kidney injury, the ectodomain of Kim-1 is cleaved by matrix metalloproteases to the soluble form of Kim-1, which can then enter into the urine (Ichimura et al., 1998). Therefore, urinary Kim-1 appears to be a sensitive and early diagnostic indicator for the kidney injury by nephrotoxicants, including drugs and environmental toxicants (Prozialeck et al., 2007; Vaidya et al., 2008; Zhou et al., 2008; Swain et al., 2011). Kim-1 expression is primarily localized in proliferating, dedifferentiated, vimentin-expressing epithelial cells of the S3 segment of the renal proximal tubule (Ichimura et al., 1998). The level of Kim-1 expression in tubular epithelial cells was previously reported to correlate with the levels of tissue osteopontin and α-smooth muscle actin expression, and Kim-1 colocalized with these two markers of tubulointerstitial damage (Kuehn et al., 2002; Kramer et al., 2009). In the clinical setting, Kim-1 is induced on the apical side of dilated tubules in fibrotic areas (van Timmeren et al., 2007; Huo et al., 2010). Therefore, the Kim-1 expression in the proximal tubules might be related to the regeneration process and development of interstitial fibrosis.
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K. Nan-ya et al. The level of urinary clusterin is increased in the urine after AKI in rats, dogs and primates (Witzgall et al., 1994; Rosenberg and Silkensen, 1995; Chevalier, 1996; Davis et al., 2004; Kharasch et al., 2006; Dieterle et al., 2010b; Vinken et al., 2012). Clusterin, like Kim-1, is also expressed on the dedifferentiated tubular cells after injury (Harding et al., 1991; Correa-Rotter et al., 1998). The physiological function of osteopontin in the kidney is related to the accumulation of monocytes and macrophages, as well as the regeneration of cells after injury. Upregulation of osteopontin has been reported in rats with various kidney diseases (Xie et al., 2001; Fuchs and Hewitt, 2011; Fuchs et al., 2012). In the present study, the increases in the levels of clusterin and osteopontin were observed only in the rats with kidney lesions. These increases were later than that of urinary Kim-1, which was observed prior to histopathological adverse changes, indicating that Kim-1 was a more sensitive biomarker for CDDPinduced sub-chronic kidney injury than osteopontin and clusterin. Furthermore, Kim-1 can be translated well from animals to human, as the highly conserved structure and functionality of Kim-1 have been demonstrated (Ichimura et al., 1998). Accordingly, the urinary Kim-1 level is expected to be a valuable biomarker for the early diagnosis and monitoring of drug-induced kidney injury in humans. The increase in urinary Kim-1 in the rats without histopathological changes recovered to the control level in a time-dependent fashion after the cessation of CDDP, as shown in ‘Experiment model 3’, indicating that measuring the urinary Kim-1 level can allow for an adequate protocol for CDDP therapy to be developed and modified as needed. In contrast, other urinary biomarkers examined in the present study, such as albumin, glucose and NGAL, were not considered to be appropriate for monitoring the CDDP-induced sub-chronic kidney injury. The transient increases in the levels of urinary albumin and glucose observed in ‘Experimental model 1’ may reflect transient damage of renal proximal tubules, but the exact cause remains unclear. As there are many nephrotoxicants that induce different types of kidney injury, it is difficult to comprehensively monitor kidney injury using a single biomarker. Hence, further studies on the basis of histopathological changes are necessary for identifying adequate biomarker(s) for each type of chemical-induced kidney injury. In conclusion, we herein demonstrated that the urinary Kim-1 level is a useful biomarker for the early detection and monitoring of CDDP-induced sub-chronic kidney injury in rats. Acknowledgements The authors thank Ms Noriko Onoda for valuable technical support, Ms Tomomi Yoneshige for assistance with the clinical pathology studies, and Ms Toyoko Kashiwagi and Ms Hiroko Sanada for preparation of the pathology specimens.
Conflict of Interest The Authors did not report any conflict of interest.
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Revised: 20 January 2014,
Accepted: 22 January 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jat.2999
Usefulness of urinary kidney injury molecule-1 (Kim-1) as a biomarker for cisplatin-induced sub-chronic kidney injury Ken-ichiro Nan-yaa,b*, Masatomo Kajiharac, Natsuki Kojimad and Masakuni Degawab ABSTRACT: We explored biomarkers suitable for monitoring sub-chronic kidney injury using the three rat models of cisplatin (CDDP)-induced kidney injury, which were designed to extend the current knowledge beyond the sub-acute exposure period. In the pilot study, a single intravenous administration of 1.5 mg kg–1 CDDP to rats was confirmed to result in no histopathological changes. Subsequently, CDDP was intravenously administered to rats at a dose of 1.5 mg kg–1 for 4 days at 24-h intervals (Experimental model 1) and for up to 10 weeks at weekly intervals (Experimental models 2 and 3), and the changes in blood and urine components, such as recently recommended urinary biomarkers (Kim-1, clusterin and so on) and traditional blood biomarkers (blood urea nitrogen and serum creatinine), were examined together with the histopathological changes in renal tissues during the development of the kidney injury in each model. In these experimental models, a significant increase in urinary Kim-1 was observed prior to the histopathological changes in renal tissues, and these changes were retained after the adverse histopathological changes. Significant changes in all of the other urinary biomarkers examined occurred along with the histopathological changes. In addition, the increase in urinary Kim-1 after weekly treatment with CDDP for 4 weeks was reduced in a time-dependent manner after cessation of the drug. The present findings indicate that urinary Kim-1 is the most useful biomarker for CDDP-induced rat sub-chronic kidney injury among the biomarkers examined. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Kim-1; urinary biomarkers; sub-chronic kidney injury; cisplatin; rat
Introduction The kidney is one of the representative organs damaged by drugs, including cisplatin (CDDP). Therefore, an early diagnosis of drug-induced kidney injury is important for adequately altering the protocol of drug therapy. The blood urea nitrogen (BUN) and serum creatinine (SCr) levels are commonly used as preclinical and clinical biomarkers for kidney injury. However, these biomarkers have severe limitations with regard to sensitivity and specificity. Increases in BUN and SCr occur only after substantial renal injury, generally after loss of two thirds of the nephrons’ functional capacity (Pfaller and Gstraunthaler, 1998; Hart, 2005; Marrer and Dieterle, 2010), and such abnormal changes were also often observed in those with extra-renal injury. Moreover, the levels of these biomarkers are affected by various physiological conditions (Hart, 2005; Proulx et al., 2005; Bonventre et al., 2010; Urbschat et al., 2011). Therefore, the discovery of new markers for diagnosing and monitoring kidney injury is necessary. Recently, the Predictive Safety Testing Consortium (PSTC) organized by the Critical Path Institute has proposed seven novel urinary biomarkers, including Kim-1, clusterin, albumin, total protein, β2-microglobulin, cystatin C and trefoil factor 3, on the basis of the results in rat acute kidney injury (AKI) models. The usefulness of these biomarkers for AKI in rats was further confirmed by the U.S. Food and Drug Administration (FDA), European Medicines Agency (EMA), Pharmaceuticals and Medical Devices Agency (PMDA, Japan) and several research
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groups (Dieterle et al., 2010a, b; Yu et al., 2010; Vaidya et al., 2010; Gautier et al., 2010; Harpur et al., 2011; Hoffmann et al., 2010a; Tonomura et al., 2010; Sasaki et al., 2011; Hosohata et al., 2012; Vinken et al., 2012). Moreover, the urinary Kim-1, clusterin, cystatin C and/or neutrophil gelatinase-associated lipocalin (NGAL) levels are reported to be more sensitive than BUN and SCr as biomarkers of the kidney injury induced by repeated nephrotoxicant administration to rats (Hoffmann et al., 2010a, b; Rouse et al., 2011; Fuchs et al., 2012; Vinken et al., 2012). However, to the best of our knowledge, the relationships between the histopathological changes in renal tissue and the changes in recently recommended urinary biomarkers, including *Correspondence to: Ken-ichiro Nan-ya, Drug Discovery Research Laboratories, Kyowa Hakko Kirin Co., Ltd, 1188 Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka, 411-8731, Japan. E-mail: [email protected] a Drug Discovery Research Laboratories, Kyowa Hakko Kirin Co., Ltd, 1188 Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka, 411-8731, Japan b Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan c Development Research Laboratories, Kyowa Hakko Kirin Co., Ltd, 1188 Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka, 411-8731, Japan d Discovery and Development Research Promotion Laboratories, Kyowa Hakko Kirin Co., Ltd, 1188 Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka, 411-8731, Japan
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K. Nan-ya et al. Kim-1, clusterin and NGAL, during the development of nephrotoxicant-induced sub-chronic kidney injury are still largely unknown. Therefore, the present study was designed to extend the current knowledge beyond the sub-acute exposure period into longer term scenarios to examine more fully the time course of biomarker events. CDDP is an anticancer drug with potent therapeutic effects against numerous tumors. However, CDDP often induces kidney injury in both humans and experimental animals. This CDDPinduced nephrotoxicity, which is thought to occur through the induction of oxidative stress and apoptosis in the kidney (Kuhlmann et al., 1997; Arany and Safirstein, 2003), is the most important index for determining the protocol for CDDP therapy. In the present study, the previously proposed urinary components (albumin, clusterin, glucose, Kim-1, NGAL and osteopontin) and blood components (BUN, SCr and cystatin C) were selected as biomarkers for CDDP-induced sub-chronic kidney injury in rats, and the usefulness of these biomarkers was comparatively assessed. Furthermore, the relationships between the changes in the levels of these biomarkers and the histopathological changes in renal tissues were examined, and consequently, urinary Kim-1 was found to be the most useful biomarker for monitoring CDDP-induced sub-chronic kidney injury in rats.
Materials and Methods Reagents CDDP and physiological saline were purchased from Nippon Kayaku Co., Ltd. (Tokyo, Japan) and Otsuka Pharmaceutical Factory, Inc. (Tokushima, Japan), respectively.
Animal and Husbandry Five-week-old male Crj:CD(SD)IGS rats were purchased from Charles River Japan, Inc. (Kanagawa, Japan). The animals were
6 or 7 weeks old at the time of the first administration, and were kept for more than 7 days after they were received for quarantine and acclimation prior to the start of experiments. The animals were housed in stainless-steel hanger cages for rats (260 mm W × 360 mm D × 187 mm H: TOKIWA Phytochemical Co., Ltd, Tokyo, Japan). The animals were housed in a barrier system animal room, which was controlled to maintain a temperature range of 22 ± 3 °C, relative humidity range of 50 ± 20%, ventilation of 15 times per hour or more and illumination for 12 h. Radiation-sterilized chow for rats and mice, FR-2 10 kGy (Funabashi Farm Co., Ltd., Chiba, Japan), was provided to the animals ad libitum. The diet was used within 6 months after production. Tap water was provided to the animals ad libitum. However, water was provided ad libitum from plastic bottles when the animals were housed in metabolism cages. All experiments were conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals of Kyowa Hakko Kirin Co, Ltd (Shizuoka, Japan).
Treatment of Rats with CDDP The three experimental protocols are depicted in Fig. 1. The purpose of experimental model 1 was to identify the biomarkers suitable for monitoring the progression of CDDP-induced subchronic kidney injury. The experimental models 2 and 3 were designated to identify the biomarkers suitable for the early detection and diagnosis of CDDP-induced sub-chronic kidney injury. The first day and the first week of administration are defined as Day 1 and Week 1, respectively. Experimental model 1. CDDP, diluted in 0.9% physiological saline, was administered intravenously to rats at a dose of 1.5 mg kg–1 per injection (5 ml kg–1 via tail vein) for four consecutive days. Control group rats received the vehicle (0.9% physiological saline, 5 ml kg–1 via the tail vein). Urine samples were collected from each rat on Days 2, 3, 4, 5, 19 and 33 without fasting. The blood samples from individual rats were collected from the
Figure 1. Experimental models designated for development of cisplatin (CDDP)-induced kidney injury. The closed triangle represents the administration of CDDP or saline. The first day and the first week of administration are defined as Day 1 and Week 1, respectively. The just one week before the first administration is defined as Week 0. The symbols, such as U, B, and N, in parentheses mean urine collection, blood collection and necropsy, respectively.
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Kim-1 as a biomarker for sub-chronic kidney injury postcava of non-fasted rats under anesthesia with isoflurane on Days 5 and 33. Some of the rats were killed on Days 5 and 33, and the kidneys were removed and used for the histopathological analysis. Experimental model 2. The dose of CDDP used was the same as that described for Experimental model 1. CDDP was intravenously administered to rats (5 ml kg–1 via tail vein) for 2, 4, 6, 8 and 10 weeks at weekly intervals. Control group rats received the vehicle (5 ml kg–1 via the tail vein). The urine samples were collected for 16 h from 6 to 7 days after the last administration of CDDP from individual rats that had fasted. The blood samples of individual rats were collected from the postcava under anesthesia with isoflurane 1 week after the last CDDP administration. Some of the rats were killed at the indicated times, and the kidneys were removed and used for the histopathological analysis. Experimental model 3. The protocol used for CDDP administration was almost the same as that described in Experimental model 2. After the 4-week administration of CDDP, the treatment was ceased for 1, 3, 5 or 7 weeks, and thereafter, blood and urine samples were collected from individual rats, as described in Fig. 1. The kidneys were removed from the rats 7 weeks after the last administration of CDDP, and were used for histopathological analysis.
Analyses of Blood Biomarkers The serum was prepared from each blood sample by centrifugation for 10 min at 4 °C, and was stored at –80°C until analysis. The amounts of serum BUN, SCr and cystatin C were measured using an automatic analyzer (H7170S; Hitachi, Ltd., Tokyo, Japan).
Analyses of Urinary Biomarkers The rats were housed in metabolism cages, and the urine (16-h urine) was collected from each rat for 16 h from Day 6 to Day 7 after the administration of CDDP or saline at the indicated schedule (Fig. 1). Rats had access to water ad libitum throughout the urine collection. Urine samples were centrifuged at 400 g for 5 min, and the separated supernatant was used for the analyzes of urinary biomarkers. The amounts of creatinine and glucose were measured using an autoanalyzer (H7070; Hitachi, Ltd.). The amounts of albumin, Kim-1, NGAL, osteopontin [Kidney Injury Panel 1 (rat) assay kit] and clusterin (Rat clusterin assay kit) were measured using a Multi-Array® assay system (Meso Scale Discovery, Rockville, MD, USA). The levels of the urinary biomarkers were corrected by the urine volume.
Histopathological Assessment The kidneys were fixed in 10% neutral-buffered formalin solution. The fixed kidneys were embedded in paraffin and then sectioned. The sections were stained with hematoxylin-eosin solution according to the routine methods and then used for histopathological analyzes.
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Statistical Analysis The data are presented as the means ± standard deviation (SD). Differences between CDDP-treated versus time-matched salinetreated (control) animals were evaluated by Student’s t-test.
Results Experimental Model 1 In the CDDP-treated rats, there were no clinical signs of toxicity (external appearance, nutritional condition, posture and behavior) at any time points, although a decrease in the mean body weight gain from Day 1 to Day 33 compared with the saline-treated (control) rats was observed (mean body weight gain: saline; 261.5 g, CDDP; 169.8 g). No significant changes in the levels of BUN and SCr were observed in rats treated with CDDP for 2 days at 24-h intervals in the pilot study (data not shown). On the day after treatment with CDDP for 4 days at 24-h intervals (Day 5), the levels of BUN and SCr were significantly increased to approximately 1.9- and 2.2-fold over the corresponding control levels, respectively (Table 1). Such increases in BUN and SCr were also observed on Day 33 (1.6- and 1.5-fold increases compared with the saline-treated rats, respectively) (Table 1). Significant increases in the levels of urinary biomarkers such as albumin, clusterin, glucose, Kim-1 and osteopontin were observed on Day 5 (Table 2). However, no significant changes in the levels of any of these urinary biomarkers examined were found on Days 19 and 33. Interestingly, a significant increase (5.7-fold over the control) in the level of Kim-1 was observed even on Day 2, and the level was further increased in a timedependent fashion, at least up to Day 5. The increases appeared to be retained up to Days 19 and 33; however, no significant increases were found because of the large individual differences. Further increases in clusterin and osteopontin were also observed on Days 19 and 33. In addition, a transient and slight decrease in the level of urinary creatinine was also observed on Day 3. No histopathological changes in the renal tissues were observed in the rats treated with CDDP for 2 days at 24-h intervals in the pilot study (data not shown). On the day after Table 1. Cisplatin (CDDP)-induced changes in the levels of serum biomarkers in the rats examined in ‘Experimental model 1’ Day of blood collection Day 5 Day 33
Treatment
BUN (mg/dL)
Creatinine (mg/dL)
Saline CDDP Saline CDDP
15.6 ± 1.5 (6)a 29.7 ± 6.1* (6) 17.6 ± 0.8 (3) 28.4 ± 5.5 (3)
0.20 ± 0.01 (6) 0.43 ± 0.08* (6) 0.26 ± 0.01 (3) 0.40 ± 0.05* (3)
The ‘Days 5 and 33’ are the serum collection days shown in Fig. 1. a The numbers shown in parentheses represent the number of the animals examined. The data of each biomarker examined present the mean ± SD. * Statistically significant differences from the corresponding saline-treated groups: P < 0.05.
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Saline CDDP Saline CDDP Saline CDDP Saline CDDP Saline CDDP Saline CDDP Saline CDDP Saline CDDP
Treatment
Day 3 2.9 ± 0.6 (6) 2.9 ± 1.5 (6) 0.78 ± 0.33 (6) 0.77 ± 0.20 (6) 0.11 ± 0.08 (6) 0.07 ± 0.02 (6) 2.44 ± 0.31 (6) 1.76 ± 0.48* (6) 1.52 ± 0.11 (6) 1.13 ± 0.27* (6) 3.04 ± 0.25 (6) 25.42 ± 10.51** (6) 1.23 ± 0.43 (6) 1.26 ± 0.83 (6) 8.78 ± 1.47 (6) 8.82 ± 4.19 (6)
Day 2 4.6 ± 3.2 (6)a 4.2 ± 1.7 (6) 1.10 ± 0.82 (6) 0.83 ± 0.31 (6) 0.09 ± 0.04 (6) 0.06 ± 0.02 (6) 2.54 ± 0.53 (6) 2.39 ± 0.71 (6) 1.37 ± 0.69 (6) 1.41 ± 0.47 (6) 3.44 ± 1.34 (6) 19.64 ± 7.04*** (6) 1.49 ± 0.75 (6) 1.59 ± 0.77 (6) 11.46 ± 7.54 (6) 9.08 ± 3.65 (6) 2.9 ± 1.2 (6) 2.6 ± 1.3 (6) 0.83 ± 0.28 (6) 8.71 ± 9.80 (6) 0.09 ± 0.04 (6) 0.10 ± 0.06 (6) 2.54 ± 0.47 (6) 1.74 ± 0.72 (6) 1.63 ± 0.23 (6) 2.11 ± 0.85 (6) 3.05 ± 0.83 (6) 27.31 ± 13.94** (6) 1.24 ± 0.60 (6) 1.15 ± 0.70 (6) 9.72 ± 5.38 (6) 8.57 ± 4.24 (6)
Day 4 3.5 ± 0.5 (6) 5.2 ± 3.2 (6) 0.64 ± 0.35 (6) 37.90 ± 17.94* (6) 0.12 ± 0.10 (6) 0.57 ± 0.28* (6) 2.86 ± 0.08 (6) 2.03 ± 0.81 (6) 1.36 ± 0.56 (6) 31.19 ± 17.95* (6) 3.65 ± 0.06 (6) 59.61 ± 37.65** (6) 1.04 ± 0.93 (6) 2.01 ± 0.90 (6) 10.95 ± 3.57 (6) 20.50 ± 6.90** (6)
Day 5
4.5 ± 3.8 (3) 6.3 ± 4.3 (3) 5.94 ± 3.15 (3) 10.17 ± 6.89 (3) 0.14 ± 0.04 (3) 2.81 ± 2.32 (3) 6.58 ± 1.58 (3) 6.03 ± 0.53 (3) 2.09 ± 0.63 (3) 3.60 ± 2.21 (3) 2.61 ± 4.52 (3) 98.93 ± 66.80 (3) 2.17 ± 3.75 (3) 6.47 ± 4.23 (3) 10.26 ± 17.78 (3) 97.27 ± 91.81 (3)
Day 19
5.4 ± 3.2 (3) 6.8 ± 3.7 (3) 4.24 ± 2.88 (3) 7.40 ± 3.05 (3) 0.22 ± 0.06 (3) 3.07 ± 2.80 (3) 10.27 ± 2.30 (3) 8.53 ± 0.81 (3) 3.93 ± 3.28 (3) 1.71 ± 0.86 (3) 2.85 ± 4.94 (3) 61.40 ± 43.99 (3) 2.13 ± 3.70 (3) 6.58 ± 4.26 (3) 10.70 ± 18.54 (3) 81.75 ± 83.44 (3)
Day 33
The 16-h urine was collected from each rat at the indicated schedule (Fig. 1) and used for the assays of the indicated biomarkers, as described in ‘Materials and Methods’. The ‘Days’ described are identified with those in ‘Experimental model 1’ (Fig. 1). a The numbers shown in parentheses represent the number of the animals examined. The value of each biomarker presents the mean ± SD. *,**,*** Statistically significant differences from the corresponding saline-treated groups: * P < 0.05; ** P < 0.01; *** P < 0.001.
Osteopontin (ng)
NGAL (μg)
Kim-1 (ng)
Glucose (g)
Creatinine (mg)
Clusterin (μg)
Albumin (mg)
Urine volume (ml)
Biomarkers (Amount/16-h urine)
Table 2. Cisplatin (CDDP)-induced changes in the levels of urinary biomarkers in the rats examined in ‘Experimental model 1’
K. Nan-ya et al.
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J. Appl. Toxicol. 2014
Kim-1 as a biomarker for sub-chronic kidney injury four consecutive CDDP treatments (Day 5), kidney injury, as indicated by degeneration (vacuolization, necrosis and desquamation) of the proximal tubular epithelial cells, urinary cast formation and interstitial inflammatory cell infiltration was observed (Fig. 2B). Furthermore, on Day 33, the progression of the lesions, such as the regeneration of proximal tubular epithelial cell, cystic dilation of the renal tubules, pronounced interstitial mononuclear cell infiltration and fibrosis of the interstitial tissue, was observed in the kidneys of the rats with elevated levels of urinary Kim-1, clusterin and osteopontin (Fig. 2C). Experimental Model 2 In the CDDP-treated rats, there were no clinical signs of toxicity (external appearance, nutritional condition, posture and behavior) and no changes in the mean body weight gains at any time points.
Significant increases in the levels of the blood biomarkers (BUN and SCr) were observed 7 days after weekly administration of CDDP for 10 weeks, but were not observed for the first 6 weeks (Table 3). The levels of BUN and SCr in the rats treated with CDDP for 10 weeks were about 3.6- and 2.7-fold higher than the corresponding control levels, respectively. The levels of biomarkers in the 16-h urine, which was collected from each rat for 16 h from 6 to 7 days after the administration of CDDP or saline at the indicated schedule (Fig. 1), are summarized in Table 4. The level of urinary Kim-1 was markedly increased even after the CDDP treatment for only 4 weeks, and the levels were further increased in a treatment period-dependent manner up to 10 weeks. The urinary excretion levels of Kim-1 at Week 5, 7, 9 and 11 in the CDDP-treated rats were 2.8-, 3.5-, 9.7- and 15.2-fold higher than the corresponding control levels, respectively. The urinary levels of osteopontin
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Figure 2. Representative photomicrographs of renal tissues from the rats treated with saline (A) or cisplatin (CDDP) at a dose of 1.5 mg kg per day (B and C) for 4 days at 24-h intervals. The analyzes were performed using the rats in ‘Experimental model 1’. The tissue sections were stained with hematoxylin-eosin. No clear changes are observed in the saline-treated rats on Day 5 (A). In the CDDP-treated rats, degeneration (vacuolization, necrosis and desquamation) of the proximal tubular epithelial cells, urinary cast formation and interstitial inflammatory cell infiltration are observed on Day 5 (B). On the Day 33 (C), further progression of the lesions, including regeneration of proximal tubular epithelial cells, cystic dilation of the renal tubules, pronounced interstitial mononuclear cell infiltration and fibrosis of the interstitial tissues, is observed. Scale bar: 100 μm.
Table 3. Cisplatin (CDDP)-induced changes in the levels of serum biomarkers in the rats examined in ‘Experimental model 2’ Treatment
Week of blood collection
Saline CDDP
11 3 5 7 11
Number of dosing 10 2 4 6 10
BUN (mg/dL) a
13.4 ± 0.8 (3) 14.4 ± 1.9 (3) 10.7 ± 0.9 (3) 16.5 ± 2.1 (3) 48.4 ± 12.8* (3)
Creatinine (mg/dL) 0.29 ± 0.05 (3) 0.26 ± 0.05 (3) 0.27 ± 0.04 (3) 0.32 ± 0.08 (3) 0.79 ± 0.14** (3)
Cystatin C (mg/L) 0.12 ± 0.01 0.15 ± 0.03 0.13 ± 0.02 0.14 ± 0.03 0.20 ± 0.03
(3) (3) (3) (3) (3)
Blood collections were performed at the indicated schedule shown in Fig. 1. a The numbers shown in parentheses represent the number of the animals examined. The value of each biomarker presents the mean ± SD. *,** Statistically significant differences from the corresponding saline-treated groups: * P < 0.05; ** P < 0.001.
J. Appl. Toxicol. 2014
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K. Nan-ya et al. Table 4. Cisplatin (CDDP)-induced changes in the levels of urinary biomarkers in the rats examined in ‘Experimental model 2’ Biomarkers Treatment (Amount/16-h urine) Urine volume (ml) Albumin (mg) Clusterin (μg) Creatinine (mg) Glucose (g) Kim-1 (ng) NGAL (μg) Osteopontin (ng)
Saline CDDP Saline CDDP Saline CDDP Saline CDDP Saline CDDP Saline CDDP Saline CDDP Saline CDDP
Week 3
Week 5
Week 7
Week 9
Week 11
6.7 ± 1.9 (3)a 6.4 ± 4.1 (3) 1.13 ± 0.35 (3) 1.15 ± 0.68 (3) 0.10 ± 0.03 (3) 0.12 ± 0.04 (3) 3.72 ± 0.93 (3) 3.52 ± 0.66 (3) 0.61 ± 0.34 (3) 0.68 ± 0.32 (3) 5.87 ± 0.81 (3) 7.96 ± 1.95 (3) 0.66 ± 0.22 (3) 0.99 ± 0.24 (3) 10.41 ± 4.51 (3) 10.34 ± 1.04 (3)
7.9 ± 0.3 (3) 5.7 ± 3.4 (3) 1.40 ± 0.15 (3) 0.96 ± 0.52 (3) 0.18 ± 0.03 (3) 0.18 ± 0.05 (3) 5.59 ± 0.69 (3) 4.46 ± 0.57* (3) 0.56 ± 0.43 (3) 0.41 ± 0.39 (3) 6.39 ± 0.81 (3) 17.66 ± 4.41* (3) 0.47 ± 0.06 (3) 1.10 ± 0.37 (3) 8.26 ± 2.75 (3) 9.06 ± 4.11 (3)
6.6 ± 2.8 (3) 6.3 ± 2.9 (3) 1.14 ± 0.44 (3) 0.99 ± 0.44 (3) 0.11 ± 0.02 (3) 0.27 ± 0.06* (3) 6.30 ± 0.77 (3) 5.55 ± 1.12 (3) 0.69 ± 0.06 (3) 1.06 ± 0.29 (3) 5.83 ± 2.08 (3) 20.58 ± 7.84* (3) 0.47 ± 0.21 (3) 1.01 ± 0.25 (3) 9.03 ± 3.30 (3) 10.74 ± 3.24 (3)
5.5 ± 1.6 (3) 8.4 ± 4.0 (3) 0.91 ± 0.23 (3) 1.45 ± 0.71 (3) 0.17 ± 0.06 (3) 0.25 ± 0.09 (3) 6.74 ± 1.82 (3) 6.55 ± 0.79 (3) 0.98 ± 0.11 (3) 1.05 ± 0.21 (3) 4.38 ± 0.65 (3) 42.63 ± 11.57* (3) 0.54 ± 0.08 (3) 1.81 ± 0.23* (3) 10.74 ± 3.21 (3) 20.43 ± 4.42* (3)
7.0 ± 1.2 (3) 16.7 ± 12.8 (3) 1.24 ± 0.14 (3) 2.94 ± 2.16 (3) 0.13 ± 0.03 (3) 0.32 ± 0.10* (3) 7.67 ± 0.89 (3) 6.78 ± 0.64 (3) 2.47 ± 1.75 (3) 1.34 ± 0.70 (3) 4.55 ± 2.02 (3) 69.25 ± 36.60* (3) 0.50 ± 0.11 (3) 3.11 ± 2.24 (3) 9.30 ± 1.91 (3) 61.19 ± 31.26* (3)
The 16-h urine was collected from each rat at the indicated schedule (Fig. 1) and used for the assays of the indicated biomarkers, as described in ‘Materials and Methods’. The ‘Days’ described are identified with those in ‘Experimental model 2’ (Fig. 1). a The numbers shown in parentheses represent the number of the animals examined. The data of each biomarker examined present the mean ± SD. * Statistically significant differences from the corresponding saline-treated groups: P < 0.05. were clearly increased at Weeks 9 and 11, whereas no significant increases after the shorter periods of treatment were observed. The levels of urinary clusterin and NGAL were increased at Weeks 7, 9 and/or 11. No clear increases in the levels of urinary albumin and glucose occurred throughout any of the CDDP treatment periods examined.
No histopathological changes in the kidneys were observed in the rats treated with saline (Fig. 3A) or CDDP for 4 weeks (Fig. 3B) at weekly intervals. In the kidneys of the rats treated with CDDP for 6 weeks at weekly intervals, mild degeneration (vacuolization, necrosis and desquamation) of the proximal tubular epithelial cells and interstitial inflammatory cell infiltration were observed
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Figure 3. Representative photomicrographs of renal tissues from rats treated with saline (A) or cisplatin (CDDP) at a dose of 1.5 mg kg per week (B–D) for 4, 6 and 10 weeks at weekly intervals. The analyzes were performed using the rats in ‘Experimental model 2’. The tissue sections were stained with hematoxylin-eosin. One week after the 4-week treatment with CDDP (B), no clear changes are observed in the rat renal tissues. In the renal tissues 1 week after the 6-week treatment with CDDP (C), mild degeneration (vacuolization, necrosis and desquamation) of the proximal tubular epithelial cells and interstitial inflammatory cell infiltration are observed. Further progression of the lesions is observed in the rats 1 week after the 10-week treatment with CDDP (D). Scale bar: 100 μm.
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J. Appl. Toxicol. 2014
Kim-1 as a biomarker for sub-chronic kidney injury (Fig. 3C). Furthermore, the 10-week treatment with CDDP resulted in progression of the lesions (Fig. 3D). Experimental Model 3 In the CDDP-treated rats, there were no clinical signs of toxicity (external appearance, nutritional condition, posture and behavior) and no changes in the mean body weight gains at any time points. A significant increase in the urinary Kim-1 level occurred even after the weekly treatment with CDDP for 4 weeks (Fig. 4), whereas histopathologically adverse changes were observed after the treatment for not less than 6 weeks, as described in ‘Experimental model 2’. In addition, the kidneys showed no lesions 7 weeks after weekly administration of CDDP for 4 weeks (data not shown). In this experiment using the rats treated with CDDP for 4 weeks (assumed to be no histopathological adverse changes), the time-dependent effects of the drug cessation on the urinary excretion level of Kim-1 were examined. The urinary Kim-1 level was decreased in a time-dependent manner from 3 to 7 weeks after cessation (Fig. 4). No significant changes in the levels of other urinary biomarkers, such as albumin, clusterin, glucose, NGAL and osteopontin, occurred throughout the study period (data not shown).
Discussion Sub-chronic kidney injury induced by repeated administration of CDDP at a low dose has been reported previously (Fenoglio et al., 2005; González et al., 2005). Therefore, using three experimental models with slight modifications to these models, we searched for candidate biomarkers for the early detection and monitoring of low-dose (1.5 mg kg–1) CDDP-induced kidney injury. Single administration of this dose of CDDP did not cause kidney injury. On Day 5 (following day after treatment with CDDP for 4 days) (Experimental model 1), the kidneys showed proximal tubular
Figure 4. Changes in the levels of urinary kidney injury molecule-1 (Kim-1) after cisplatin (CDDP) cessation. The analyzes were performed using the rats in ‘Experimental model 3’. The 16-h urine was collected from each rat and used for determining amounts of Kim-1, as described in ‘Materials and Methods’. Closed circles represent the individual rats (five rats at each time point examined). The bar represents the mean value of each experimental group. ‘Week 0’ means just 1 week before , the first administration (Fig. 1). * **Statistically significant differences from the corresponding saline-treated groups: *P < 0.05; **P < 0.01.
J. Appl. Toxicol. 2014
injury. The kidney lesions subsequently deteriorated even after drug withdrawal, and the lesion on Day 33 was representative of sub-chronic kidney injury. The levels of BUN and SCr increased on Day 5, but the levels then remained unchanged up to Day 33, in spite of progression of the lesions. In contrast, the urinary Kim-1 excretion level was increased even on Day 2 and further increased in a time-dependent fashion up to Day 33. Increases in the levels of clusterin and osteopontin were also observed on Days 19 and 33, when the lesion had progressed. Therefore, these three urinary biomarkers are considered to be more useful to monitor the progression of CDDP-induced kidney injury than BUN and SCr. In addition, the early and dramatic increases in urinary Kim-1 are consistent with the findings of other reports indicating that Kim-1 is a sensitive biomarker of CDDP-induced AKI (Vaidya et al., 2006, 2010; Tonomura et al., 2010; Sasaki et al., 2011). In Experimental model 2, the histopathological examinations revealed no kidney lesions for up to 4 weeks of administration of CDDP. Mild kidney injury was observed after 6-weeks administration, and after 10-weeks administration, the progression of lesions was observed. An increase in the level of urinary Kim-1 was observed in a time-dependent fashion from 4 weeks, although changes in the levels of BUN and SCr were observed for the first time after 10 weeks of administration. Thus, the changes in the urinary Kim-1 level were observed prior to those in other blood biomarkers and before histopathological changes were observed, again suggesting that Kim-1 is a useful biomarker for the early detection of CDDP-induced sub-chronic kidney injury. The lesion at an initiation stage of CDDP-induced kidney injury is usually transient, and the damaged proximal tubular epithelial cells can be repaired (Pinches et al., 2012). The subchronic kidney injury seems to develop through repeated cellular damage and repair. In fact, in the present study, interstitial fibrosis and mononuclear cell infiltration in the kidneys were observed in both Experimental models 1 and 2. Kim-1 is a phosphatidylserine receptor expressed on renal epithelial cells that recognizes apoptotic cells and plays an active role in the clearance of apoptotic cells (Ichimura et al., 2008). Dramatic increases in the levels of Kim-1 mRNA and protein occurred in the injured kidneys, although the levels were extremely low in the normal kidney. During the development of kidney injury, the ectodomain of Kim-1 is cleaved by matrix metalloproteases to the soluble form of Kim-1, which can then enter into the urine (Ichimura et al., 1998). Therefore, urinary Kim-1 appears to be a sensitive and early diagnostic indicator for the kidney injury by nephrotoxicants, including drugs and environmental toxicants (Prozialeck et al., 2007; Vaidya et al., 2008; Zhou et al., 2008; Swain et al., 2011). Kim-1 expression is primarily localized in proliferating, dedifferentiated, vimentin-expressing epithelial cells of the S3 segment of the renal proximal tubule (Ichimura et al., 1998). The level of Kim-1 expression in tubular epithelial cells was previously reported to correlate with the levels of tissue osteopontin and α-smooth muscle actin expression, and Kim-1 colocalized with these two markers of tubulointerstitial damage (Kuehn et al., 2002; Kramer et al., 2009). In the clinical setting, Kim-1 is induced on the apical side of dilated tubules in fibrotic areas (van Timmeren et al., 2007; Huo et al., 2010). Therefore, the Kim-1 expression in the proximal tubules might be related to the regeneration process and development of interstitial fibrosis.
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K. Nan-ya et al. The level of urinary clusterin is increased in the urine after AKI in rats, dogs and primates (Witzgall et al., 1994; Rosenberg and Silkensen, 1995; Chevalier, 1996; Davis et al., 2004; Kharasch et al., 2006; Dieterle et al., 2010b; Vinken et al., 2012). Clusterin, like Kim-1, is also expressed on the dedifferentiated tubular cells after injury (Harding et al., 1991; Correa-Rotter et al., 1998). The physiological function of osteopontin in the kidney is related to the accumulation of monocytes and macrophages, as well as the regeneration of cells after injury. Upregulation of osteopontin has been reported in rats with various kidney diseases (Xie et al., 2001; Fuchs and Hewitt, 2011; Fuchs et al., 2012). In the present study, the increases in the levels of clusterin and osteopontin were observed only in the rats with kidney lesions. These increases were later than that of urinary Kim-1, which was observed prior to histopathological adverse changes, indicating that Kim-1 was a more sensitive biomarker for CDDPinduced sub-chronic kidney injury than osteopontin and clusterin. Furthermore, Kim-1 can be translated well from animals to human, as the highly conserved structure and functionality of Kim-1 have been demonstrated (Ichimura et al., 1998). Accordingly, the urinary Kim-1 level is expected to be a valuable biomarker for the early diagnosis and monitoring of drug-induced kidney injury in humans. The increase in urinary Kim-1 in the rats without histopathological changes recovered to the control level in a time-dependent fashion after the cessation of CDDP, as shown in ‘Experiment model 3’, indicating that measuring the urinary Kim-1 level can allow for an adequate protocol for CDDP therapy to be developed and modified as needed. In contrast, other urinary biomarkers examined in the present study, such as albumin, glucose and NGAL, were not considered to be appropriate for monitoring the CDDP-induced sub-chronic kidney injury. The transient increases in the levels of urinary albumin and glucose observed in ‘Experimental model 1’ may reflect transient damage of renal proximal tubules, but the exact cause remains unclear. As there are many nephrotoxicants that induce different types of kidney injury, it is difficult to comprehensively monitor kidney injury using a single biomarker. Hence, further studies on the basis of histopathological changes are necessary for identifying adequate biomarker(s) for each type of chemical-induced kidney injury. In conclusion, we herein demonstrated that the urinary Kim-1 level is a useful biomarker for the early detection and monitoring of CDDP-induced sub-chronic kidney injury in rats. Acknowledgements The authors thank Ms Noriko Onoda for valuable technical support, Ms Tomomi Yoneshige for assistance with the clinical pathology studies, and Ms Toyoko Kashiwagi and Ms Hiroko Sanada for preparation of the pathology specimens.
Conflict of Interest The Authors did not report any conflict of interest.
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