Involvement of neutrophil gelatinase-associated lipocalin and osteopontin in renal tubular regeneration and interstitial fibrosis after cisplatin-induced renal failure

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Involvement of neutrophil gelatinase-associated lipocalin and osteopontin in renal tubular regeneration and interstitial fibrosis after cisplatin-induced renal failure Emi Kashiwagi a,b,∗ , Yutaka Tonomura a , Chiaki Kondo a , Koichi Masuno a , Kae Fujisawa a , Noriko Tsuchiya a , Shuuichi Matsushima a , Mikinori Torii a , Nobuo Takasu a , Takeshi Izawa b , Mitsuru Kuwamura b , Jyoji Yamate b a

Developmental Research Laboratories, Shionogi & Co., Ltd., 3-1-1 Futaba-cho, Toyonaka, Osaka 561-0825, Japan Laboratories of Veterinary Pathology, Life and Environmental Sciences, Osaka Prefecture University, Rinkuu Ourai Kita 1-58, Izumisano-shi, Osaka 598-8531, Japan b

a r t i c l e

i n f o

Article history: Received 17 February 2014 Accepted 29 April 2014 Keywords: Neutrophil gelatinase-associated lipocalin Osteopontin Cisplatin Renal failure

a b s t r a c t The kidney has a capacity to recover from ischemic or toxic insults that result in cell death, and timely tissue repair of affected renal tubules may arrest progression of injury, leading to regression of injury and paving the way for recovery. To investigate the roles of neutrophil gelatinase-associated lipocalin (NGAL/lcn2) and osteopontin (OPN/spp1) during renal regeneration, the expression patterns of NGAL and OPN in the cisplatin-induced rat renal failure model were examined. NGAL expression was increased from day 1 after injection; it was seen mainly in the completely regenerating proximal tubules of the cortico-medullary junction on days 3–35; however, the expression was not seen in abnormally dilated or atrophied renal tubules surrounded by fibrotic lesions. On the other hand, OPN expression was increased from day 5 and the increased expression developed exclusively in the abnormal renal tubules. NGAL expression level well correlated with the proliferating activity in the regenerating renal epithelial cells, whereas OPN significantly correlated with the ␣-smooth muscle actin-positive myofibroblast appearance, expression of transforming growth factor (TGF)-␤1, and the number of CD68-positive macrophages. Interestingly, rat renal epithelial cell line (NRK-52E) treated with TGF-␤1 decreased NGAL expression, but increased OPN expression in a dose-dependent manner. Because increases of TGF-␤1, myofibroblasts and macrophages contribute to progressive interstitial renal fibrosis, OPN may be involved in the pathogenesis of fibrosis; on the contrary, NGAL may play a role in tubular regeneration after injury. Expression analysis of NGAL and OPN would be useful to investigate the tubule damage in renal-toxicity. © 2014 Elsevier GmbH. All rights reserved.

1. Introduction In contrast to the heart or brain, the kidney has a capacity to recover from ischemic or toxic insults that result in cell death (Bonventre, 2003; Lieberthal, 2009). When the kidney recovers from injury, the surviving renal epithelial cells undergo the regulated process including epithelial cell spreading and migration to cover the exposed areas of the basement membrane, cell dedifferentiation, and proliferation to restore cell number, followed by differentiation (Bonventre, 2003; Lieberthal, 2009; Dankers et al., 2011). However, when the basal lamina is damaged, incomplete regeneration occurs. Incomplete regeneration gives rise to

∗ Corresponding author. Tel.: +81 072 4635334; fax: +81 072 4635346. E-mail address: [email protected] (E. Kashiwagi).

interstitial fibrosis around the affected renal tubules probably through epithelial-mesenchymal transition (EMT) (Yamate et al., 1995, 2000), ultimately culminating in chronic kidney disease (CKD) (Ferguson et al., 2008; Bonventre et al., 2010). Whereas timely tissue repair of affected renal tubules may arrest progression of injury, resulting in regression of injury and paving the way for recovery. Neutrophil gelatinase-associated lipocalin (NGAL/lcn2) was initially found as a 25-kDa protein bound to gelatinase in specific granules of neutrophils; thereafter, NGAL has been reported to be expressed in epithelial cells in lesions of inflammation and malignancy (Vaidya et al., 2008). Osteopontin (OPN/spp1), known as a 44 kDa bone phosphoprotein, is synthesized in bone and various epithelial tissues (Vaidya et al., 2008; Xie et al., 2001). OPN has diverse functions such as bone morphogenesis and tumorigenesis and accumulation/maintenance of inflammatory macrophages

http://dx.doi.org/10.1016/j.etp.2014.04.007 0940-2993/© 2014 Elsevier GmbH. All rights reserved.

Please cite this article in press as: Kashiwagi E, et al. Involvement of neutrophil gelatinase-associated lipocalin and osteopontin in renal tubular regeneration and interstitial fibrosis after cisplatin-induced renal failure. Exp Toxicol Pathol (2014), http://dx.doi.org/10.1016/j.etp.2014.04.007

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(Vaidya et al., 2008). It has been reported that the expressions of NGAL and OPN are rapidly up-regulated after renal injury (Ferguson et al., 2008; Mishra et al., 2004; Mori and Nakao, 2007; Vaidya et al., 2008). However, the pathological roles of NGAL and OPN in the regenerating process after renal injury remain not fully understood. To shed some light behind the pathogenesis of renal regeneration, we investigated the expression pattern of NGAL and OPN in cisplatin (cis-dichlorodiammineplatinum, CDDP)-induced tubular injury in rats. CDDP is an anticancer drug with nephrotoxicity as a side effect (Zhang et al., 2009), which causes dose limitation in clinical usage. It has been experimentally known that CDDP injection to rats causes degeneration and necrosis in renal epithelial cells, especially in the S3 proximal tubules. The present study shows that NGAL may attribute to favorable regeneration to renal epithelial cells, whereas OPN may oppose the contribution of NGAL; that is, OPN could be related to interstitial fibrosis. 2. Materials and methods 2.1. In vivo study 2.1.1. Animals and CDDP-induced renal failure model The animal study protocol was carefully reviewed by the Institutional Animal Care and Use Committee (IACUC). Forty eight 5-week-old male F344/DuCrj rats (Charles River Japan, Hino, Shiga, Japan), weighing 70–90 g, were used after a one-week acclimatization period. They were housed in an animal room controlled at 23 ± 3 ◦ C, 45–65% humidity and with 12 h-light–dark cycle, and allowed free access to a standard commercial diet (CA-1, Clea Japan Inc., Tokyo, Japan) and water (sterilized city water via an automatic water supplying system). CDDP (Nippon Kayaku Co. Ltd., Tokyo, Japan) was injected intraperitoneally into 40 rats at a single dose of 6 mg/kg body weight. Four animals were sacrificed each on days 1, 3, 5, 7, 9, 12, 15, 20, 25 and 35 after CDDP dosing. The remaining eight rats were injected with equivalent volume of physiological saline and sacrificed on each day 0 (on first injection day) and 35, and served as controls. At necropsy, blood was collected from the posterior vena cava under intraperitoneal pentobarbital anesthesia, and then put into a vacuum blood-collecting tube containing heparin sodium. Plasma was obtained by centrifugation at 1500 × g for 15 min at 4 ◦ C, and plasma creatinine (Cre) and blood urea nitrogen (BUN) were measured using an automatic analyzer H7180 (Hitachi HighTechnologies Co., Tokyo, Japan). 2.1.2. Histopathology and immunohistochemistry Kidneys were removed and then fixed in 10% neutral buffered formalin or Methacarn. Fixed specimens were processed routinely and embedded in paraffin. Formalin-fixed, paraffin-embedded samples were cut at 3–4 ␮m thickness, and stained with hematoxylin & eosin (H&E) for morphological observations. Additionally, the formalin-fixed tissue sections were stained with the Azan–Mallory method for collagen deposition. For immunohistochemistry, specimens were cut at 4 ␮m thickness, deparaffined with xylene, re-hydrated with graded ethanol, and washed in water. These sections were immersed in methanol containing 3% H2 O2 to block endogenous peroxidase for 10 min at room temperature. Primary antibodies used were NGAL (1:300; Abcam, Cambridge, UK), OPN (1:3000; LSL Co. Ltd., Tokyo, Japan), proliferating cell nuclear antigen (PCNA) (1:100; Dako. Glostrup, Denmark), ␣-smooth muscle actin (␣-SMA) (Dako) and CD68 (ED1; 1:150; Serotec Ltd., Oxford, UK). Tissue sections were incubated with the primary antibody for 60 min at room temperature.

Thereafter, sections were washed three times with phosphatebuffered saline (PBS) and incubated for 30 min with the secondary antibody (Histofine Simple Stain, Nichirei Co., Tokyo, Japan). Positive reactions were visualized with 3,3 -diaminobenzidine (DAB). Sections were lightly counterstained with hematoxylin. Double immunohistochemical staining for PCNA and NGAL or PCNA and OPN was performed with samples of day 7. Immunohistochemistry for NGAL or OPN was conducted as mentioned above. After visualization with DAB, specimens were treated with microwave for 5 min. Primary antibody for PCNA was applied and incubated for 60 min in the room temperature, and then histofine simple stain for alkaline phosphatase (Nichirei) was used with a chromogen new fuchsin (red in color). 2.1.3. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) The piece of kidneys was quickly removed following exsanguination. Then the samples were stored in RNAlater® at −80 ◦ C until use. Kidney samples were then homogenized in QIAzol Lysis Reagent (Qiagen,Valencia, CA, USA) with a TissueLyser (Qiagen), and total RNA was extracted using an RNeasy® Mini Kit (Qiagen). The RNA concentration was determined using a Nano-Drop ND1000 spectrophotometer (Labtech International, East Sussex, UK). Total RNA (5 ␮g) was converted to cDNA by using the High Capacity cDNA Reverse Transcription Reagents (Applied Biosystems) for RT-PCR with random primer according to the manufacturer’s instructions. The levels of various RNAs were evaluated using commercially available TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA) with optimized primer and probe concentrations. The ABI assays used in this study were as follows: lcn2 (Rn00590612 m1; NGAL), spp1 Rn01449972 m1; OPN) and transforming growth factor (TGF)-␤1 (Rn00572010 m1). Real-time PCR was performed with an ABI PRISM 7500 Fast System (Applied Biosystems, Weiterstadt, Germany) by using 1 ␮L of template cDNA and SYBR® Premix Ex TaqTM (Perfect Real Time; Takara, Shiga, Japan) as recommended in the manufacturer’s protocol. Amplification conditions were 95 ◦ C for 30 s, followed by 40 cycles at 95 ◦ C for 5 s and 60 ◦ C for 30 s. The resulting cycle threshold (Ct ) value was processed based on the comparative Ct method, where Gapdh was used as an endogenous reference gene to normalize the expression level of OPN, NGAL and TGF-␤1 genes. In the in vitro study, the primers and internal fluorescent TaqMan probes used were as follows for ␣-SMA, 5 -GACCCTGAAGTATCCGATAGAACA-3 and 5 -CACGCGAAGCTCGTTATAGAAG3 (for primer); 5 -FAM-TGCCAGATCTTTTCC-TAMRA-3 (for probe) and for E-cadherin, 5 -ACCGAGGGCATTCTGAAAACA3 and 5 -CACTGTCACGTGCAGAATGTACTG-3 (for primer); 5 -FAM-TGCTTGGCCTCAAAATCCAAGCCCT-TAMRA-3 (for probe). 2.2. In vitro studies 2.2.1. Cell line and cell culture NRK-52E cells (Dainippon-Sumitomo Pharma Co., Esaka, Japan), which were established from normal rat proximal renal tubules (Lash et al., 2002), were used. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS) (Nichirei) as the growth medium. The cultures were incubated at 37 ◦ C in a humidified 5% CO2 atmosphere, and cells were serially subcultured by treatment with a mixture of 0.1% trypsin and 0.02% ethylendiaminetetraacetic acid in PBS. 2.2.2. Real-time RT-PCR in NRK-52E cells NRK-52E cells were incubated with TGF-␤1, the major fibrogenic factor (Fan et al., 1999; Rhyu et al., 2005; Zeisberg and Kalluri,

Please cite this article in press as: Kashiwagi E, et al. Involvement of neutrophil gelatinase-associated lipocalin and osteopontin in renal tubular regeneration and interstitial fibrosis after cisplatin-induced renal failure. Exp Toxicol Pathol (2014), http://dx.doi.org/10.1016/j.etp.2014.04.007

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Fig. 1. Value of blood urea nitrogen (BUN; a) and creatinine (Cre; b) in cisplatin (CDDP)-induced renal failure rats. Values are means ± standard deviation (S.D.). *Significantly different from controls (day 0).

2004). After being seeded in 25 mm2 flasks coated with collagen type I (BD Biosciences, Franklin Lakes, NJ, USA), cells grown to sub-confluence in the growth medium were incubated with 1% FBScontaining DMEM for 24 h. After the incubation, TGF-␤1 (0, 1, 5, 10 and 20 ng/ml; R&D Systems, Minneapolis, MN, USA) was added to the culture. To evaluate the specificity of the effect of TGF-␤1, anti-TGF-␤1 antibody (5 and 20 ng/ml; R&D System) was added. After 48-h incubation, the cells were washed twice with PBS. Total RNA was extracted from the collected cells using disrupted with RLT buffer (Qiagen). After extracting total RNA, gene expressions of the OPN, NGAL, E-cadherin (for epithelial marker) and ␣-SMA (for myofibroblastic marker) were examined using real-time RT-PCR as described above.

2.3. Evaluation and statistical analyses The immunoreactivities for NGAL, OPN and ␣-SMA were assessed under ×40 magnitude in the cortico-medullary junction semiquantitatively; −, no change; ±, faintly positive staining; 1+, moderately positive staining; 2+, more clearly evident staining; 3+, markedly positive staining. Data obtained in the real-time RT-PCR were presented as mean ± standard deviation (S.D.). The number of CD68-positive cells was counted in five high-powered fields (×400) in the affected cortico-medullary junction. The number of PCNApositive cells was also counted in five high-powered fields (×400) in the cortico-medullary junction; this method was appropriate to evaluate the proliferative activity, because the relative evaluation

Fig. 2. Histopathological findings of kidneys of CDDP-injected rats. (a) Day 5 after CDDP dosing; epithelial cells in the cortico-medullary junction desquamate into tubular lumina, and some epithelial cells have a condensed hyperchromatic nucleus. (b) Day 35; renal tubules with abnormally dilated or atrophied renal tubules are seen, being surrounded by interstitial fibrosis; collagen deposition in the fibrosis is stained blue by Azan–Mallory method (inset). H&E stain.

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Fig. 3. Immunohistochemistry (a–d) and mRNA expression (e) of neutrophil gelatinase-associated lipocalin (NGAL). (a) Control kidney; positive reactions are faintly observed in the proximal tubules of the cortico-medullary junction. (b) Day 1 after CDDP dosing; strong positive reaction for NGAL is observed in the proximal tubules of the affected cortico-medullary junction (arrowheads). (c) Day 7; NGAL expression is observed in the regenerating epithelial cells; some of these renal tubules show co-expression of NGAL and proliferating cell nuclear antigen (PCNA) (inset; arrowheads). (d) Day 20; expression of NGAL is restricted to renal tubules that nearly complete regeneration (arrows), but renal epithelial cells abnormally dilated renal tubules do not express NGAL (arrowhead). Immunohistochemical staining counterstained with hematoxylin. (e) NGAL expression is significantly increased on days 1, 5, 7, 9, 15, 20 and 25 after CDDP injection. Relative levels are determined by the standard 2(−Ct) method, normalized to Gapdh. *Significantly different from controls (day 0).

could not be used because of desquamation of nearly all the renal tubule epithelial cells on day 3. Differences between control and treated groups were evaluated by the ANOVA. The correlation between NGAL or OPN scoring and the number of PCNA-positive cell, ␣-SMA scoring, mRNA expression of TGF-␤1 or the number of CD68-positive cells were carried out by Spearman’s correlation coefficient test. Values of p < 0.05 were considered significant. 3. Results 3.1. In vivo study 3.1.1. CDDP-induced renal lesions Blood biochemically, BUN (Fig. 1a) and Cre (Fig. 1b) values showed the maximum with a significantly increase on day 5, and thereafter their values gradually decreased until day 35. Significant

increase of BUN value was transient only on day 5 (Fig. 1a), whereas that of Cre was seen on days 7, 25 and 35 (Fig. 1b). These findings indicated that renal failure due to CDDP injection was successfully induced in the present study. Histopathologically, there were no changes in the control animals both on days 0 and 35. In injured kidneys on day 1, the renal tubules, particularly the proximal tubules in the cortico-medullary junction, showed nuclear alterations, such as dispersed, segmented or condensed heterochromatin; some epithelial cells underwent apoptosis, demonstrable by the terminal deoxyribonucleotidemediated dUTP nick and labeling method. On day 3, epithelial cells in the affected renal tubules were desquamated into tubular lumina; some cells had swollen cytoplasm and nuclear lysis, indicative of necrosis. On day 5 when BUN and Cre values began to increase, necrosis or apoptosis of the renal epithelial cells was more apparent, and these lesions extended to the cortex (Fig. 2a).

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Fig. 4. Immunohistochemistry (a and b) and mRNA expression (c) of osteopontin (OPN). (a) Day 7 after CDDP dosing; OPN expression is noted in the regenerating epithelial calls; a few epithelial cells show co-expression of OPN and PCNA (inset, arrowheads). (b) Day 35; interstitial fibrosis is observed around the abnormally dilated or atrophied renal tubules showing OPN expression (arrows). Immunohistochemical staining counterstained with hematoxylin. (c) OPN expression is significantly increased on days 5, 7, 9, 12, 15, 20, 25 and 35 after CDDP injection. Relative levels are determined by the standard 2(−Ct) method, normalized to Gapdh. *Significantly different from controls (day 0).

In addition to these changes, on day 7, the affected tubules were slightly dilated and lined by flattened or cuboidal regenerating epithelial cells. Infiltration of mononuclear cells was also observed around the tubules and within their lumina. From days 9 to 15, renal tubules with variously sized lumina were seen in the affected cortico-medullary junction; these tubules were rimmed by flattened, polygonal or cuboidal regenerating epithelial cells. In the interstitium around the affected renal tubules, fibrosis began to be seen. Interstitial fibrosis was confirmed by the Azan–Mallory method, in which fibrotic lesion was stained blue (Fig. 2b inset). On days 20, 25 and 35, interstitial fibrosis gradually progressed, being accompanied by variously dilated renal tubules or atrophied renal tubules with thickened basement membrane (Fig. 2b). 3.1.2. Immunohistochemical and mRNA expressions for NGAL and OPN The immunohistochemical expression of NGAL was faintly observed in the proximal tubules in the cortico-medullary junction of the controls (Fig. 3a). On day 1, the intensity was increased in the proximal tubules of the affected cortico-medullary junction (Fig. 3b). On day 3, however, NGAL expression was fainter than that on day 1. On day 5 onwards, the expression of NGAL was noted in the regenerating epithelial cells of the affected tubules (Fig. 3c), and on day 7 onwards, the intensity was consistently seen in the renal tubules in the affected cortico-medullary junction; however, the expression was not seen in abnormally dilated or atrophied renal tubules (Fig. 3d). In agreement with immunohistochemical expression for NGAL, mRNA of NGAL was significantly increased on day 1 and decreased to control level on day 3 when renal epithelial cells were desquamated; on day 5, that of NGAL was again increased significantly showing the maximum, and thereafter the increased

level was gradually decreased, but there was a significant increase on days 7, 9, 15, 20, and 25, as compared with controls (Fig. 3e). In the controls, the immunohistochemical expression of OPN was not observed in the renal tubules of the cortico-medullary junction. In the affected cortico-medullary junction, OPN expression began to be seen on day 5; the positive reaction was observed in the regenerating renal tubules. On day 7 onwards, the expression was mainly seen in the variously dilated renal tubules (Fig. 4a); interestingly, on days 9–35 when interstitial fibrosis was developing, OPN-positive cells were noted in the abnormally dilated or atrophied renal tubules surrounded by fibrosis (Fig. 4b); the expression site of OPN (Fig. 4b) was different from that of NGAL (Fig. 3d). In agreement with immunohistochemical expression of OPN in the affected cortico-medullary junction, mRNA of OPN began to be significantly increased on day 5 and thereafter the level showed a gradual increase with a significant change (Fig. 4c). 3.1.3. Double immunohistochemical staining for NGAL or OPN and PCNA As mentioned above, immunohistochemical expressions of NGAL and OPN were seen in regenerating renal tubules in the affected cortico-medullary junction. To investigate whether regenerating epithelial cells could express NGAL or OPN, the double immunohistochemical staining for NGAL and PCNA or OPN and PCNA was performed using samples obtained on day 7, because on day 7 the number of PCNA-positive epithelial cells was the greatest (Fig. 5a) and histopathologically regenerating tubules after injury was the most prominent. Co-expression of PCNA and NGAL (Fig. 3c inset) or OPN (Fig. 4a inset) was observed. The percentage of NGAL and PCNA double positive cells among NGAL positive cells was larger than the percentage

Please cite this article in press as: Kashiwagi E, et al. Involvement of neutrophil gelatinase-associated lipocalin and osteopontin in renal tubular regeneration and interstitial fibrosis after cisplatin-induced renal failure. Exp Toxicol Pathol (2014), http://dx.doi.org/10.1016/j.etp.2014.04.007

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The number of PCNA positive cells Fig. 5. The number of PCNA-positive cells (a) and correlation graph between the number of PCNA-positive cells and NGAL expression (b). (a) The number of PCNA-positive cells is significantly increased from day 1 to 12; *significantly different from controls (day 0). (b) NGAL expression significantly correlates with the number of PCNA-positive cells; r = 0.7557, p = 0.0071.

of OPN and PCNA double positive cells among OPN positive cells (NGAL = 35.93% vs. OPN = 15.13%). 3.1.4. Correlation between the NGAL or OPN scoring and the number of PCNA-positive cells The number of PCNA-positive cells in the affected corticomedullary junction was significantly increased on days 1, 3, 5, 7, 9 and 12, indicating high proliferating activity of epithelial cells after renal tubular injury by CDDP; then, the number was gradually decreased (Fig. 5a). The number of PCNA-positive cells significantly correlated with the NGAL scoring (Pearson correlation coefficient, 0.7557; p = 0.0071) (Fig. 5b). On the other hand, there was no correlation between the OPN scoring and the number of PCNA-positive cells (Pearson correlation coefficient, −0.2924; p = 0.3829). 3.1.5. Correlation between the NGAL or OPN scoring and the ˛-SMA scoring Myofibroblasts capable of producing collagens are characterized by ␣-SMA expression by the immunohistochemistry (Yamate et al., 2005). ␣-SMA-positive cells were observed mainly around the abnormally dilated renal tubules (Fig. 6a). The expression of ␣-SMA was significantly increased on day 7 onwards, peaking on day 20 (Fig. 6b). The consistent increase from day 7 was corresponding to development of interstitial fibrosis in the affected cortico-medullary junction. The ␣-SMA scoring significantly correlated with OPN scoring (Pearson correlation coefficient, 0.8721; p = 0.0004) (Fig. 6b), whereas the relationship between the ␣-SMA scoring and NGAL scoring was not correlated (Pearson correlation coefficient, 0.4060; p = 0.2153).

3.1.6. Correlation between the NGAL or OPN scoring and the mRNA expression of TGF-ˇ1 The mRNA expression of TGF-␤1 was gradually increased as the day passed after injection and significant increase was noted on days 5, 9, 15, 20, 25 and 35 (Fig. 7a). The mRNA expression of TGF-␤1 significantly correlated with the OPN scoring (Pearson correlation coefficient, 0.8163; p = 0.0022) (Fig. 7b); in the contrast, the relationship between the mRNA expression of TGF-␤1 and NGAL scoring was not correlated (Pearson correlation coefficient, −0.5091; p = 0.1098). 3.1.7. Correlation between the NGAL or OPN scoring and the number of CD68-positive cells Infiltrating macrophages are known to play important roles in renal fibrosis by producing fibrogenic factors such as TGF-␤1 (Persy et al., 2003; Yamate et al., 2000). The CD68-positive cells were mainly observed around the dilated renal tubules (Fig. 8b). The number of CD68-positive macrophages was significantly increased from days 7 to 35 (Fig. 8c). The number of CD68-positive cells significantly correlated with the OPN scoring (Pearson correlation coefficient, 0.9491; p < 0.01) (Fig. 8b). On the other hand, the relationship between the number of CD68-positive cells and NGAL scoring was not correlated (Pearson correlation coefficient, 0.5185; p = 0.1022). 3.2. In vitro study: mRNA expression of NGAL and OPN in NRK-52E cells treated with TGF-ˇ1 EMT may play an important role in the progression of renal fibrosis; TGF-␤1 is known to be the major factor to induce the EMT

Please cite this article in press as: Kashiwagi E, et al. Involvement of neutrophil gelatinase-associated lipocalin and osteopontin in renal tubular regeneration and interstitial fibrosis after cisplatin-induced renal failure. Exp Toxicol Pathol (2014), http://dx.doi.org/10.1016/j.etp.2014.04.007

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Fig. 6. The immunohistochemistry for ␣-smooth muscle actin (␣-SMA) (a), the immunohistochemical expression of ␣-SMA (b) and correlation graph between ␣-SMA expression and OPN expression (c). (a) ␣-SMA-positive cells are observed mainly around the abnormally dilated renal tubules from on day 7. Immunohistochemical staining counterstained with hematoxylin. Bar = 100 ␮m. (b) The expression of ␣-SMA is significantly increased from days 7 to 35; *significantly different from controls (day 0). (c) OPN expression significantly correlates with ␣-SMA expression; r = 0.8721, p = 0.0004.

(Zeisberg and Kalluri, 2004). In fact, we previously demonstrated that the addition of TGF-␤1 to NRK-52E increased mRNA expression of ␣-SMA, whereas decreased mRNA expression of E-cadherin (a marker for epithelial cells) (Yamamoto et al., 2010; data not shown) was seen; this indicates the EMT of NRK-52E cells. By using this cell line, we investigated effects of TGF-␤1 on OPN and NGAL expressions. The mRNA expression of NGAL showed a dose-dependent decrease with significant change at 5 and 10 ng/ml of TGF␤1 (Fig. 9a); by neutralization with anti-TGF-␤1 antibody, the decreased level recovered to control level (0 ng/ml). On the other hand, the expression of OPN mRNA was increased in a dosedependent manner by addition with TGF-␤1; the mRNA level showed a significant increase at 5 and 10 ng/ml, and the increased level was reduced to control level by the addition with anti-TGF-␤1 antibody (Fig. 9b).

4. Discussion 4.1. CDDP-induced renal lesions After injury, renal tubular epithelial cells can regenerate; the level of regeneration may be dependent on the degree of damage (including complete or incomplete basement membrane damage) or microenvironmental conditions evoked by cell–cell and cell–matrix interactions (Bonventre, 2003). The expressions of NGAL and OPN are known to elevate after renal injury (Bonventre et al., 2010; Ferguson et al., 2008; Vaidya et al., 2008); however, their roles in the tubular regeneration remain not clarified. Therefore, we investigated the expression patterns of NGAL and OPN in regenerating renal tubules of the CDDP-induced rat renal failure model. In the present CDDP rat model, the values of BUN and Cre significantly increased on day 5 and then decreased

Please cite this article in press as: Kashiwagi E, et al. Involvement of neutrophil gelatinase-associated lipocalin and osteopontin in renal tubular regeneration and interstitial fibrosis after cisplatin-induced renal failure. Exp Toxicol Pathol (2014), http://dx.doi.org/10.1016/j.etp.2014.04.007

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mRNA expression of TGF-β1

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Immunoreactivity score of OPN Fig. 7. The mRNA expression of transforming growth factor (TGF)-␤1 (a) and correlation graph of TGF-␤1 expression and OPN expression (b). (a) TGF-␤1 is significantly increased on days 5, 9, 15, 20, 25 and 35. Relative levels are determined by the standard 2(−Ct) method, normalized to Gapdh. *Significantly different from controls (day 0). (b) OPN expression corresponds to TGF-␤1 expression; r = 0.8163, p = 0.0022.

on day 7 onwards, indicating transient renal failure. After renal injury, the affected renal tubules began to undergo regeneration as early as day 1 and showed the peak on day 7, as indicated by the PCNA immunohistochemistry; on day 9 onwards, interstitial fibrosis gradually developed around the affected renal tubules, particularly the variously dilated or atrophied renal tubules, as demonstrated by ␣-SMA immunohistochemistry for developing myofibroblasts and the Azan–Mallory staining for collagen deposition. It is well known that TGF-␤1, which might be produced by CD68-positive macrophages, is responsible for renal fibrosis (Persy et al., 2003; Yamate et al., 2000). In this study, gradual increases of TGF-␤1 expression and CD68-positve macrophage number were confirmed. 4.2. Expression and possible roles of NGAL Immunoexpression for NGAL is faintly seen in the proximal renal tubules of the controls. It is known that NGAL is constitutively expressed in the rat kidney (Mishra et al., 2004) and has a protective role against bacterium through siderophore-chelating property (Viau et al., 2010; Yang et al., 2003). Interestingly, the present study showed that the expression for NGAL was markedly increased as early as day 1 in the affected epithelial cells in the proximal tubules of the cortico-medullary junction. The affected epithelial cells began to be desquamated on day 3; therefore, the expression of NGAL was transiently decreased, in addition to decreased number of PCNA-positive cells on day 3. After that, the affected epithelial

cells began to regenerate on days 5 and 7; the regenerating epithelial cells revealed more increased expression for NGAL. The pattern of immunohistochemical expression of NGAL in the affected renal tubules well corresponded to NGAL mRNA expression by the RTPCR. Positive correlation was observed between NGAL scoring and the number of PCNA-positive regenerating cells, and the double immunohistochemistry confirmed that NGAL positive cells exhibited nuclear PCNA reactivity. More interestingly, on day 9 and onwards when the interstitial fibrosis was progressing, NGAL was expressed in epithelial cells of the affected proximal renal tubules which appeared to undergo complete regeneration. On the other hand, the NGAL expression was not seen in irregularly dilated or abnormally atrophied renal tubules; these renal tubules were considered to be at abnormal aspects, because interstitial fibrosis developed around such renal tubules. Interstitial fibrosis is formed by extracellular matrix deposition produced by myofibroblasts; the myofibroblasts may be developed partly through the EMT in which epithelial cells at abnormal conditions could transform into mesenchymal myofibroblasts under influence of TGF-␤1 (Iwano et al., 2002; Yamate et al., 2005), although detailed molecular mechanisms were not fully understood. In the present study, the ␣-SMA expression, the marker for myofibroblasts, began to be increased on day 7 and thereafter the number was gradually increased. The addition of TGF-␤1 to NRK-52E cells increased ␣-SMA mRNA (Lieberthal, 2009), whereas the treated cells decreased NGAL mRNA in a dose-dependent manner. The findings that NGAL expression was not seen in epithelial

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Fig. 8. Immunohistochemistry (a and b), the number of CD68-positive cells (c) and correlation graph between the number of CD68-positive cells and OPN expression (d). (a) Control; few CD68-positive cells are observed. (b) Day 7 after CDDP dosing; CD68- positive macrophages are noted around the dilated renal tubules (arrowheads). Immunohistochemical staining counterstained with hematoxylin. (c) The number of CD68-positive cells is significantly increased from days 7 to 35; *significantly different from controls (day 0). (d) OPN expression significantly correlates to the number of CD68-positive cells; r = 0.9491, p = 8.2541E−06.

cells of abnormally dilated or atrophied renal tubules might be supported by the in vitro study; likely, locally produced TGF-␤1 could suppress NGAL expression. In fact, there was no correlation between ␣-SMA scoring and NGAL expression. Taking these findings together, NGAL might be involved in favorable regeneration of the affected renal epithelial cells. 4.3. Expression and possible roles of OPN OPN is a protein originally isolated from bone. In the kidney, OPN is constitutively produced by renal tubular epithelial cells, and play roles in the inhibition of calcium oxalate crystal formation (Verstrepen et al., 2001; Xie et al., 2001). On days 1 and 3 when epithelial injury and desquamation took place in the affected cortico-medullary junction, increased OPN expression was not

shown by both immunohistochemistry and mRNA analysis. At early stages in rat renal failure models induced by CDDP, OPN seems not to participate in regenerating process. More interestingly, the immunohistochemical expression of OPN was localized exclusively in epithelial cells of abnormally dilated or atrophied renal tubules which were surrounded by fibrotic lesions. Although there were some epithelial cells coexpressing OPN and PCNA, the percentage of OPN/PCNA double positive cells among OPN positive cells was much smaller than that of NGAL (OPN = 15.13% vs. NGAL = 35.93%). These findings suggested that OPN was not always essential in epithelial proliferation in the affected renal tubules. It is interesting to note that a significant correlation existed between the OPN and ␣-SMA scoring or mRNA expression of TGF-␤1. OPN might be involved in the progressive interstitial

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a mRNA expression of NGAL

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§

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*

0.4 0.2 0

TGF-β1 (ng/ml)

0

1

5

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10

10

anti TGF-β1 (ng/ml)

0

0

0

0

5

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*: p<0.05 vs. control (TGF-β1 0 ng/ml)

mRNA expression of OPN

b

: p<0.05 vs. TGF-β1 10ng/ml 8

*

7 6 5

*

4

§

3 2 1

§

0

TGF-β1 (ng/ml)

0

1

5

10

10

10

anti TGF-β1 (ng/ml)

0

0

0

0

5

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*: p<0.05 vs. control (TGF-β1 0 ng/ml) : p<0.05 vs. TGF-β1 10ng/ml Fig. 9. mRNA expression of NGAL (a) and OPN (b) of TGF-␤1 treated NRK-52E. (a) NGAL expression is significantly decreased in a dose-dependent manner by treatment of TGF-␤1. (b) OPN expression is significantly increased dose-dependently by treatment of TGF-␤1. These changes are significantly suppressed by the addition of anti-TGF-␤1 antibody. *Significantly different from controls (TGF-␤1 0 ng/ml); significantly different from TGF-␤1 10 ng/ml.

fibrosis. As mentioned above, fibrotic lesions may be induced partly through the EMT (Iwano et al., 2002; Yamate et al., 2005). In in vitro study, the addition of TGF-␤1 to NRK-52E cells decreased E-cadherin and increased ␣-SMA; furthermore, TGF-␤1-treated NRK-52E cells showed a dose-dependent increase of OPN. OPN has been reported to relate with the infiltration of macrophages in the cyclosporine-induced renal failure or obstructive uropathy models of mice (Mazzali et al., 2002; Ophascharoensuk et al., 1999). Macrophage infiltration is involved in the renal fibrosis probably through their producing fibrogenic factors such as TGF-␤1 (Persy et al., 2003; Yamate et al., 2000). Since the OPN scoring correlated with the number of CD68-positive macrophages in the present CDDP-induced rat renal failure model, OPN might also play an important role in the infiltration/accumulation of macrophages leading to progressive fibrosis. There might be another possibility that OPN produced by renal epithelial cells could induce the EMT via autocrine or paracrine fashion, because the present immunohistochemistry revealed that OPN expression was markedly increased

in renal epithelial cells of abnormally dilated and atrophied renal tubules surrounded by fibrotic lesion, and the in vitro studies showed that the addition of TGF-␤1 to NRK-52E cells increased OPN mRNA expression. In conclusion, the present study showed that NGAL could play important roles in favorable regeneration of renal epithelial cells after injury, because there was significant correlation between immunohistochemical expression of NGAL and PCNApositive regenerating renal epithelial cells. On the other hand, OPN was expressed exclusively in abnormal renal epithelial cells in dilated or atrophied tubules, and there was a significant correlation between the OPN-positive epithelial cell scoring and ␣-SMA scoring, the CD68-positive macrophage number or TGF-␤1 mRNA expression. In addition, we showed in vitro that up-regulation of OPN mRNA in NRK-52E cells was induced by TGF-␤1, whereas TGF-␤1-treated NRK-52E cells reduced NGAL expression. Collectively, in CDDP-induced renal failure, NGAL may be a favorable factor for complete regeneration of affected renal epithelial cells,

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whereas OPN could oppose the complete regeneration under TGF␤1 influence; therefore, OPN might induce progressive fibrosis. The expression analysis of NGAL and OPN would become useful tools to investigate the pathogenesis of renal-toxicity. Additionally, it is interesting to pursue the mechanisms of imbalance between NGAL and OPN in this CDDP rat model, which would give us therapeutic insights for the progressive interstitial fibrosis. Acknowledgments This study was supported in part by Grant-in-Aids for Scientific Research (B) (No. 22380173), and for Challenging Exploratory Research (No. 23658265) from Japanese Society for the Promotion of Science (JSPS). References Bonventre JV. Dedifferentiation and proliferation of surviving epithelial cells in acute renal failure. J Am Soc Nephrol 2003;14:S55–61. Bonventre JV, Vaidya VS, Schmouder R, Feig P, Dieterle F. Next-generation biomarkers for detecting kidney toxicity. Nat Biotechnol 2010;28:436–40. Fan JM, Ng YY, Hill PA, Nikolic-Paterson DJ, Mu W, Atkins RC, et al. Transforming growth factor-␤ regulates tubular epithelial-myofibroblast transdifferentiation in vitro. Kidney Int 1999;56:1455–67. Ferguson MA, Vaidya VS, Bonventre JV. Biomarkers of nephrotoxic acute kidney injury. Toxicology 2008;245:182–93. Dankers PYW, Boomker JM, Meijer EW, Popa ER, Van Luyn MJA. From kidney development to drug delivery and tissue engineering strategies in renal regenerative medicine. J Control Release 2011;152:177–85. Iwano M, Plieth D, Danoff TM, Xue C, Okada H, Neilson EG. Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest 2002;110:341–50. Lash LH, Putt DA, Matherly LH. Protection of NRK-52E cells, a rat renal proximal tubular cell line, from chemical-induced apoptosis by overexpression of a mitochondrial glutathione transporter. J Pharmacol Exp Ther 2002;303:476–86. Lieberthal W. Mechanisms of renal regeneration and repair after acute kidney injury. US Nephrol 2009;4:60–3. Mazzali M, Hughes J, Dantas M, Liaw L, Steitz S, Alpers CE, et al. Effects of cyclosporine in osteopontin null mice. Kidney Int 2002;62:78–85. Mishra J, Mori K, Ma Q, Kelly C, Yang J, Mitsnefes M, et al. Amelioration of ischemic acute renal injury by neutrophil gelatinase-associated lipocalin. J Am Soc Nephrol 2004;15:3073–82.

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Please cite this article in press as: Kashiwagi E, et al. Involvement of neutrophil gelatinase-associated lipocalin and osteopontin in renal tubular regeneration and interstitial fibrosis after cisplatin-induced renal failure. Exp Toxicol Pathol (2014), http://dx.doi.org/10.1016/j.etp.2014.04.007