Participation of bone morphogenetic protein (BMP)-6 and osteopontin in cisplatin (CDDP)-induced rat renal fibrosis

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Participation of bone morphogenetic protein (BMP)-6 and osteopontin in cisplatin (CDDP)-induced rat renal fibrosis Ryo Yano a , Hossain M. Golbar a , Takeshi Izawa a , Osamu Sawamoto b , Mitsuru Kuwamura a , Jyoji Yamate a,∗ a Veterinary Pathology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku-Ourai-Kita, Izumisano City, Osaka 598-8531, Japan b Safety Evaluation, Preclinical Assessment, Otsuka Pharmaceutical Factory, Tokushima 722-8601, Japan

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Article history: Received 5 August 2014 Accepted 21 October 2014 Keywords: BMP-6 Cisplatin Osteopontin Renal fibrosis

a b s t r a c t The significance of bone morphogenetic protein (BMP)-6 and osteopontin (OPN) in renal fibrosis is poorly understood. We investigated the expression of BMP-6 and OPN in cisplatin (CDDP; 6 mg/kg, once, i.p.)induced renal fibrosis in F344 rats, and their effects on kidney fibroblast (NRK-49F), mesenchymal pericyte (MT-9) and renal epithelial cell (NRK-52E) lines. Histopathologically, the CDDP injection showed desquamation of renal tubular epithelial cells at the cortico-medullary junction on days 1–3 that followed insufficient regeneration on days 5–9 and progressive interstitial fibrosis by day 35. In addition to TGF␤1 (the most powerful fibrogenic factor), increase in mRNAs of BMP-6 and OPN was seen consistently after the injection. Immunohistochemically, BMP-6 was expressed both in the damaged renal epithelial cells and spindle-shaped myofibroblasts (expressing ␣-smooth muscle action [␣-SMA]) in the fibrotic areas; OPN expression was seen exclusively in the injured renal epithelial cells. Treatment of BMP-6 or OPN increased ␣-SMA mRNA in MT-9 cells, similar to effects of TGF-␤1 on MT-9 and NRK-49F cells. Interestingly, TGF-␤1 addition increased BMP-6 and OPN mRNAs in NRK-52E cells. Collectively, it was found that BMP-6 and OPN considerably participate in progressive renal fibrosis through development of myofibroblasts, in relation with TGF-␤1. © 2014 Elsevier GmbH. All rights reserved.

1. Introduction Renal fibrosis is the serious pathological aspect to injury, being histopathologically characterized by excessive accumulation of extracellular matrices (ECMs) such as collagens (Bani-Hani et al., 2008). Regardless of etiology, sustained injury leads to interstitial fibrosis in the kidney, which is mediated via activation of multiple pathways, culminating in the end stage renal failure at the advanced stages (Satirapoj, 2010). Because of complex interaction of divergent mediators evoked by cell to cell or cell to matrix, the mechanisms behind the renal fibrosis remain largely undetermined. The ECMs are produced mainly by myofibroblasts which have been considered to originate from progenitor cells including resident fibroblasts, pericytes, bone marrow-derived fibrocytes or renal tubular epithelial cells via epithelial-mesenchymal-transition (EMT) (LeBleu et al., 2013). Myofibroblasts, characterized by ␣smooth muscle actin (␣-SMA) expression, may be developed in

∗ Corresponding authors. Tel.: +81 72 463 5334; fax: +81 72 463 5346. E-mail address: [email protected] (J. Yamate).

response to fibrogenic stimuli; in particular, transforming growth factor (TGF)-␤1 is the most powerful fibrogenic factor playing crucial roles in the activation of progenitor cells towards myofibroblasts and deposition of ECMs leading to progression of renal fibrosis (Bani-Hani et al., 2008). Besides TGF-␤1, bone morphogenetic protein (BMP)-6 and osteopontin (OPN) have been considered as possible fibrogenic factors in the fibrosis (Leung et al., 2013). BMP-6, a member of TGF superfamily, is a major regulator of myofibroblast progenitor cells in inflammatory lesions in the kidney; BMP-6 deficiency aggravates tubular damage and enhances interstitial fibrosis (Dendooven et al., 2011). OPN is a phosphorylated glycoprotein abundant in the bone tissues, and is responsible for osteogenesis and osteoclastogenesis via the attachment to osteoblasts or osteoclasts (Chen et al., 2014); OPN is somewhat expressed in normal renal tubules, and may play roles in renal fibroblast proliferation and ECM synthesis (Kramer et al., 2005). However, detailed roles of BMP-6 and OPN have not yet been decided. To know their roles in renal fibrogenesis, we investigated their expression patterns in cisplatin (CDDP; cis-diamminedichloroplatinum II)-induced renal fibrosis in rats in vivo (Yamamoto et al., 2012). Additionally, the potential

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2 Table 1 Antibodies used for immunohistochemistry. Antibody

Host

Clone

Dilution

Sources

Specificity

OPN BMP-6 ␣-SMA Ki-67

Rabbit Mouse Mouse Mouse

Polyclonal Morph-6.1 1A4 MIB-1

1:30,000 1:10,000 1:1000 1:400

LSL/Cosmo Bio, Tokyo, Japan Millipore, Billerica, Massachusetts, USA DakoCytomation, Glostrup, Denmark DakoCytomation

Renal epithelial cells Renal epithelial cells; myofibroblasts Myofibroblasts Proliferating cells

OPN—osteopontin; BMP—bone morphogenetic protein; ␣-SMA, ␣-smooth muscle actin.

of BMP-6 and OPN in modulating fibrogenic factors was analyzed by using kidney-constituting cell lines in vitro: renal interstitial fibroblastic cell line (NRK-49F), renal tubular epithelial cell line (NRK-52E), and immature mesenchymal cell line (MT-9) resembling pericytes (Yamate et al., 2007). The present study shows the participation of BMP-6 and OPN in the CDDP-induced renal fibrosis, in close association with TGF-␤1. 2. Materials and methods 2.1. Rat renal fibrosis Thirty-six male F344 rats (Charles River Japan, Hino, Shiga, Japan), 6-week-old and weighing 105–135 g, were used. They were housed in a room under controlled temperature (22 ± 3◦ C) with a 12-h light-dark cycle, and allowed free access to standard rodent chow and tap water. After one-week acclimatization, thirty-three rats were injected intraperitoneally by a single injection of CDDP (Nippon Kayaku Co. Ltd., Tokyo, Japan) at a dose of 6 mg/kg body weight (to LD50 7.7 mg/kg; Yamamoto et al., 2012); this dose is sufficient to induce renal failure model in rats consisting of tubular injury and subsequent fibrosis (Yuasa et al., 2013). They were sacrificed under deep isoflurane anesthesia on post-injection days 1, 3, 5, 7, 9, 12, 15, 20, 25 and 35; at each point, three or four rats were examined. The remaining three rats received intraperitoneal injection of phosphate buffered saline (PBS), and sacrificed immediately on day 0; they served as controls. Rats are well known to develop age-dependent chronic progressive nephropathy (Percy and Barthold, 2007); rats used at weeks 6 to 9 in this study are too young to develop spontaneous chronic progressive nephropathy. The experiments were performed in compliance with the institutional guidelines and with prior approval of protocol by the ethical committee of Osaka Prefecture University for the Care and Use of Laboratory Animals. 2.2. Serum biochemistry Blood samples were collected from abdominal aorta at necropsy and separated sera were subjected to biochemical assay. The serum levels of blood urea nitrogen (BUN) and creatinine were measured using an automated analyzer 7170 (Hitachi, Tokyo, Japan). 2.3. Histopathology and immunohistochemistry The renal tissues obtained at each examination point were fixed in 10% neutral buffered formalin and Zamboni’s solution (0.21% picric acid and 2% paraformaldehyde in 130 mM phosphate buffer, pH 7.4); tissue samples were processed routinely and embedded in paraffin. Formalin-fixed deparaffinized sections (3 ␮m in thick) were stained with hematoxylin and eosin (HE) for morphological observations and the Azan–Mallory method for collagen. Zamboni’s solution-fixed sections were used for immunohistochemical staining. Briefly, after heat-induced epitope retrieval, the sections were treated with 3% H2 O2 to quench endogenous peroxidase and then with 5% skimmed milk to inhibit nonspecific reactions. Tissue sections were incubated with each

primary antibody (Table 1) for 14 h at 4 ◦ C. Thereafter, the sections were allowed to react with HRP-conjugated secondary antibody (Histofine Simple Stain MAX PO, Nichirei, Tokyo, Japan) for 30 min. Positive reactions were visualized with 3,3 -diaminobenzidine (DAB). Sections were counterstained lightly with hematoxylin. For double labeling, after visualization of the first primary antibody (anti-OPN antibody), the sections were autoclaved and incubated with the second primary antibody (anti-Ki-67 antibody) for 1 h at room temperature followed by reaction with secondary antibody (Histofine Simple Stain AP, Nichirei) for 30 min and visualization of reaction with fuchsin chromogen (Dako North America, Inc., USA). Mirror image sections were stained with anti-BMP-6 and anti-␣-SMA antibodies for the assessment of co-expression. 2.4. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) Extraction of RNA was performed using an SV Total RNA Isolation System Kit (Promega, Madison, WI, USA) from corticomedullary renal tissues which were immersed in RNA stabilizing reagent (RNAlater® , Qiagen GmbH, Hilden, Germany) and stored at −80 ◦ C or from cultured cells as described below. Concentration of RNA was measured on a NanodropTM spectrophotometer (Thermo Scientific, WI, USA) and 1 ␮g of the RNA was reverse-transcribed to cDNA using SuperScript First Strand Synthesis SystemTM (Invitrogen Co., Carpinteria, CA, USA) and amplified with the SYBR Green Real-Time PCR Master Mix (Toyobo Co. Ltd., Osaka, Japan) with LineGeneTM real-time PCR system (BioFlux, Tokyo, Japan) for BMP6, OPN, TGF-␤1, ␣-SMA and ␤-actin (internal control gene), the oligonucleotide sequences used are shown in Table 2. The expression values of target genes were normalized to the expression values of ␤-actin. 2.5. Effects of BMP-6, OPN and TGF-ˇ1 on cultured cells For in vitro studies, normal rat renal fibroblast cell line (NRK49F) and normal rat renal epithelial cell line (NRK-52E) were obtained from RIKEN, Tsukuba, Japan (Lash et al., 2002; Nishi et al., 2011). MT-9 cell line has been used as immature mesenchymal cells, and has features resembling pericytes (Yamate et al., 2007). These cell lines were cultured in Eagle’s minimum essential medium (E-MEM; Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS), 0.03% l-glutamine and 7.5% NaHCO3 in an incubator at 37 ◦ C in a humidified atmosphere containing 5% CO2 . For the treatment with TGF-␤1, OPN or BMP-6, at the subconfluent cultures of MT-9, NRK-49F and NRK-52E, cells were washed with sterile PBS and cultured in E-MEM supplemented with 2% FBS for 24 h. Recombinant human TGF-␤1 (0, 1 or 5 ng/mL diluted in E-MEM supplemented with 2% FBS; R & D Systems, MN, USA), recombinant mouse OPN (0, 1 or 5 ␮g/mL diluted in the medium; R & D Systems) or recombinant BMP-6 (0, 30 or 100 ng/mL diluted in the medium; Abnova, Taipei, Taiwan) was added to cultured cells and incubated for 24 h. Doses of TGF-␤1, OPN and BMP-6 were determined, according to results obtained in preliminary experiments or reported previously (Yamate et al., 2007). All experiments were carried out three times.

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Table 2 Primers used for real-time RT-PCR. Gene name

Oligonucleotide sequences

BMP-6

Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

BMP-6 (in vitro) OPN TGF-␤1 ␣-SMA ␤-actin

5 -CCTTCATGGTGGCCTTCTTC-3 5 -GTAGCCTTTGGGTGCGATG-3 5 -GAGCTTTACGTGAGCTTCCAGGAC-3 5 -GGCATTCATGTGTGCATTGAGAG-3 5 -GTGGTTTGCTTTTGCCTGTTC-3 5 -TGGCTTTCATTGGAGTTGCTT-3 5 -CTTCAGCTCCACAGAGAAGAACTGC-3 5 -CACGATCATGTTGGACAACTGCTCC-3 5 -GCCATCAGGAACCTCGAGAA-3 5 -GGAGCATCATCACCAGCAAAG-3 5 -TAAAGACCTCTATGCCAACAC-3 5 -CTCCTGCTTGCTGATCCACAT-3

BMP—bone morphogenetic protein; OPN—osteopontin; TGF-␤1—transforming growth factor-␤1; ␣-SMA, ␣-smooth muscle actin.

2.6. Statistical analysis Quantitative data represents mean ± standard deviation (SD), and statistical analyses were performed using Dunnett’s test or Mann–Whitney’s U test. A P value <0.05 was considered significant. 3. Results 3.1. Histopathological findings in CDDP-induced rat renal lesions In control kidneys, no significant alteration in histological architecture was seen (Fig. 1A). On day 1 after CDDP injection, renal proximal tubular epithelial cells, especially at the S3 segment in

the cortico-medullary junction, underwent swelling with nuclear degeneration. On day 3, epithelial desquamation was seen in the affected renal tubules (Fig. 1B). On day 5, inflammatory cell infiltration began to be seen around the affected renal tubules, with gradual increase. In addition to inflammation, on day 7, the affected tubules were variously dilated, being lined by cuboidal or flattened epithelial cells with basophilic cytoplasm indicating regeneration; fibrosis around the affected tubules began to develop (Fig. 1C). On days 9, 12, and 15, the fibrotic areas consisting of spindleshaped myofibroblasts in the interstitium around the damaged renal tubules were gradually formed; the myofibroblasts reacted to ␣-SMA as mentioned below. On days 25 and 35, the extensive fibrosis was developed, being entrapping dilated or atrophied

Fig. 1. Histopathology of control (A) and CDDP-induced renal lesions (B–D) in rats. At the cortico-medullary junction of control rat, no significant changes are seen (A). After CDDP injection, the renal tubular epithelial cells at the cortico-medullary junction shows swelling and desquamation on day 3 (B), and dilated or atrophied renal tubules lined by flattened to cuboidal epithelial cells are seen accompanied by fibrosis and macrophage infiltration (arrows) on day 7 (C). Severe collagen deposition including variously dilated and atrophied renal tubules are seen in the fibrotic lesions on day 35 (D). HE. Bar: 100 ␮m (A–C), 50 ␮m (D).

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Fig. 2. Renal interstitial fibrosis in CDDP-treated rats on day 25. Collagen deposition blue by the Azan–Mallory stain is seen in the fibrotic areas around the affected renal tubules (A), and the fibrotic lesions consist of ␣-smooth muscle actin (␣-SMA)-positive myofibroblasts (B). The Azan–Mallory stain (A) and immunohistochemistry with ␣-SMA antibody, counterstained with hematoxylin (B). Bar: 200 ␮m (A), 100 ␮m (B).

renal tubules (Fig. 1D); thickening of basement membrane around atrophied renal tubules and infiltration of mononuclear cells were also seen in the fibrotic areas. The fibrotic areas were stained blue by the Azan–Mallory method, indicating collagen deposition (Fig. 2A), and immunohistochemically, ␣-SMA-labeled myofibroblasts were seen around the affected renal tubules (Fig. 2B), in agreement with development of fibrotic lesions. On day 3 onwards, the values of BUN and creatinine were significantly increased with the peak on day 5, indicative of renal failure due to the CDDP injection. 3.2. mRNA expressions of BMP-6, OPN and TGF-ˇ1 Although there was no significant change on days 9, 15, and 35, mRNA level of BMP-6 showed a consistent increase on day 3 onwards (Fig. 3A). OPN mRNA began to be significantly increased on day 7 and the increased level retained until the end of day 35, although the level did not exhibit a significant change on day 25 (Fig. 3B). On the other hand, TGF-␤1 mRNA was significantly increased only on day 7, but there was tendency to increase on day 3 onwards (Fig. 3C). Among BMP-6, OPN and TGF-␤1, the expression level of OPN mRNA was the greatest (32–48 fold to controls), in contrast to BMP-6 (2–4 folds to controls) and TGF-␤1 (4–6 fold to controls). 3.3. Immunohistochemical findings In control kidneys, podocytes in glomeruli reacted to BMP-6 (Fig. 4A) and its reactivity was not changed in CDDP-treated rats

Fig. 3. Real-time RT-PCR analyses for bone morphogenetic protein (BMP)-6, osteopontin (OPN) and transforming growth factor (TGF)-␤1 in CDDP-induced renal fibrosis in rats. Compared to control (Cont), the transcripts of mRNA for BMP-6 (A), OPN (B) and TGF-␤1 (C) are increased in CDDP-induced renal fibrosis. Dunnett’s test; ∗ P < 0.05.

after the injection (Fig. 4B). Interestingly, although there were no immunopositive cells in the renal tubules or interstitium of the control, a few interstitial cells reacting to BMP-6 began to be seen in CDDP-treated rats on day 3. On days 5 and 7, in CDDP-treated rats, BMP-6 expression was seen in interstitial spindle-shaped cells around the affected renal tubules, and damaged renal epithelial cells also showed a faint reaction to BMP-6. The reactivity of BMP-6 became much greater in the affected renal epithelial cells of dilated or atrophied tubules; additionally, interstitial spindle-shaped cells in the fibrotic areas reacted strongly to BMP-6 on days 9 (Fig. 4C) and 12. The reactivity of BMP-6 seen on days 9 and 12 retained

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Fig. 4. Expression of BMP-6 in control and CDDP-induced renal fibrosis in rats. BMP-6 expression is seen in glomerular podocytes in control (A). Besides podocytes, on day 35, epithelial cells (arrows) of the affected renal tubules and interstitial myofibroblasts react to BMP-6 (B). Epithelial cells (arrows) of variously dilated renal tubules and peritubular mesenchymal cells (arrowheads) in the interstitial fibrosis show positive reactivity for BMP-6 on days 9 (C) and 35 (D). Immunohistochemistry with BMP-6 antibody, counterstained with hematoxylin. Bar: 100 ␮m.

until day 35 (Fig. 4D). To identify the BMP-6-positive spindle cells in the fibrotic areas, the mirror sections were stained with antibodies for BMP-6 or ␣-SMA. BMP-6-positive interstitial spindle-shaped cells (Fig. 5A) almost corresponded to ␣-SMA-positive myofibroblasts; however, ␣-SMA reactivity was not seen in renal epithelial cells, whereas there were some BMP-6-positive epithelial cells of the affected tubules (Fig. 5A).

A few OPN-positive cells were seen in renal epithelial cells in controls, although the reactivity was very faint. In CDDP-injected rats, OPN expression began to be clearly seen in some injured renal epithelial cells in the cortico-medullary junction as early as day 3. On day 5, OPN expression was seen in epithelial cells of abnormally dilated or atrophied renal tubules; afterwards, the cuboidal or flattened epithelial cells lining the abnormal tubules

Fig. 5. Co-expression between BMP-6 and ␣-SMA in CDDP-induced renal fibrosis in rat on day 25 using mirror sections. BMP-6 reactivity is seen both in renal epithelial cells (arrowheads) and interstitial spindle-shaped cells (arrows) (A); the BMP-positive spindle-shaped cells (arrows) correspond to the ␣-SMA-expressing myofibroblasts (arrows) (B). Immunohistochemistry, counterstained with hematoxylin. Bar: 50 ␮m.

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Fig. 7. Real-time RT-PCR analyses for the expression of ␣-SMA in MT-9 cells (pericytes). Treatment of MT-9 cells with BMP-6 (A), OPN (B) or TGF-␤1 (C) increases dose-dependently ␣-SMA mRNAs. Mann–Whitney’s U test; ∗ P < 0.05.

Fig. 6. OPN expression in CDDP-induced renal fibrosis in rats. Epithelial cells of the affected renal tubules react to OPN (arrows) at the cortico-medullary junction on days 15 (A) and 35 (B) in varying degrees. Double immunohistochemical staining for OPN (brown) with Ki-67 (red); some OPN-positive epithelia cells show nuclear staining to Ki-67 (arrows) (C), indicating regenerating capacity of the affected renal tubules. Immunohistochemistry, counterstained with hematoxylin. Bar: 100 ␮m (A, C), 50 ␮m (B). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

reacted strongly to OPN (Fig. 6A and B). Double immunohistochemical staining for OPN with Ki-67 showed that OPN-positive renal epithelial cells reacted to Ki-67, indicating active regenerating cells (Fig. 6C). 3.4. Effects of BMP-6, OPN and TGF-ˇ1 on kidney-constituting mesenchymal cell lines (MT-9 and NRK-49F) MT-9 cells and NRK-49F cells are regarded as pericytes and interstitial fibroblasts, respectively (Yamate et al., 2007; Ponnusamy et al., 2014). The effects of BMP-6, OPN and TGF-␤1 on the expression of ␣-SMA were investigated in MT-9 cells and

NRK-49F cells by using the RT-PCR. The dose-dependent increase of ␣-SMA mRNA was seen in MT-9 cells by treatment with BMP-6 (Fig. 7A), OPN (Fig. 7B) and TGF-␤1 (Fig. 7C). Similarly, treatment of NRK-49F cells with TGF-␤1 showed increased expression of ␣-SMA mRNA (Fig. 8A); additionally, NRK-49F cells treated with BMP-6 increased expression of OPN mRNA (Fig. 8B).

3.5. Effects of BMP-6, OPN and TGF-ˇ1 expression in renal tubular epithelial cell line RNK-52E cells The effects of BMP-6, OPN and TGF-␤1 on NRK-52E cells were analyzed at mRNA levels. TGF-␤1 treatment showed a dose-dependent increase of BMP-6 mRNA in NRK-52E cells (Fig. 9A). Similarly, TGF-␤1 treatment increased OPN mRNA expression in NRK-52E cells (Fig. 9B). By contrast, treatment with BMP-6 to NRK-52E cells significantly decreased TGF-␤1 mRNA expression (Fig. 9C).

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Fig. 8. Real-time RT-PCR analyses for the expression of ␣-SMA and OPN mRNA in NRK-49F (renal interstitial fibroblasts). Treatment of NRK-49F cells with TGF-␤1 (A) and BMP-6 (B) shows a dose-dependent increment of ␣-SMA and OPN mRNAs. Mann–Whitney’s U test; ∗ P < 0.05.

4. Discussion 4.1. Renal fibrosis induced by CDDP and expression of BMP-6, OPN and TGF-ˇ1 Renal fibrosis is a sequel of progressive diseases characterized by excessive deposition of ECMs produced mainly by myofibroblasts. CDDP injection to rats could induce renal epithelial damage and subsequent interstitial fibrosis as reported previously (Yamamoto et al., 2012; Yuasa et al., 2013). The consistently increased levels of BUN and creatinine were indicative of persistent renal failure by CDDP. Tubular epithelial cell damage by CDDP underwent incomplete regeneration due to disordered molecular events, culminating in progressive interstitial fibrosis (Yamamoto et al., 2010). The myofibroblast development and excessive ECM deposition were confirmed in the fibrotic areas of the present CDDP-treated rat kidneys (Fig. 2). TGF-␤1 is well known to be an inducer of myofibroblasts from the progenitors, and its expression is increased in organs undergoing fibrogenesis (Golbar et al., 2011; Golbar et al., 2013; Juniantito et al., 2013; Kang et al., 2007; Liu, 2006). TGF-␤1 mRNA expression was increased in the present CDDP-induced kidney lesions. Very interestingly, besides increased TGF-␤1 expression, BMP-6 and OPN mRNAs were markedly and persistently increased in the present CDDP-treated kidney. The increase of BMP-6 and OPN mRNAs was greater and more consistent than that of TGF-␤1; particularly, increase in OPN mRNA was much greatest among these factors. Along with TGF-␤1, these findings indicated the involvement of BMP-6 and OPN in the pathogenesis of CDDP-induced rat renal fibrosis. 4.2. Immunohistochemistry for OPN and BMP-6 in renal fibrosis Because BMP-6 and OPN mRNAs were increased consistently with advancement of renal fibrosis, we employed

Fig. 9. Real-time RT-PCR analyses for the expression of BMP-6, OPN and TGF␤1 in NRK-52E (renal tubular epithelial cells). Treatment with TGF-␤1 shows a dose-dependent increase of BMP-6 (A) and OPN (B) mRNA expression. By contract, treatment with BMP-6 decreases TGF-␤1 mRNA expression (C). Mann–Whitney’s U test; ∗ P < 0.05.

immunohistochemical staining with antibodies against BMP-6 and OPN to identify the producing cells. Although renal epithelial cells and interstitial cells did not react to BMP-6, in control rat kidneys, podocytes in the glomeruli strongly reacted to BMP-6 and the reactivity retained even in CDDP-injected rats. To our knowledge, this is the first demonstration of BMP-6-immunoreactivity of podocytes in rats; BMP-6 immunohistochemistry would be used to identify podocytes in glomerular pathology. In CDDPinjected rats, BMP-6 reactivity was seen in renal epithelial cells of the dilated or atrophied tubules and spindle-shaped cells in the fibrotic areas; the reactivity was in agreement with BMP-6 mRNA expression. By using mirror image sections, the peritubular spindle-shaped cells expressed both BMP-6 and ␣-SMA. Because ␣-SMA is a canonical marker for myofibroblasts (Henderson et al., 2013), it was found that, in addition to damaged renal epithelial cells, myofibroblasts are an important source of BMP-6 in rat renal fibrosis. OPN is used as a biomarker for renal injury; its expression is related to the extent of renal damage (Kashiwagi et al., 2014; Wadey et al., 2014). OPN immune-reactivity may be seen in renal tubules, particularly in the loop of Henley, in normal rats (Wadey et al., 2014); however, the reactivity of OPN was faint and limited in the present control rats. In CDDP-injected rats, OPN

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immune-expression began to be seen in injured renal tubules, and its expression was gradually increased in renal epithelial cells of the dilated or atrophied tubules; some of them showed dual labeling to OPN and Ki-67, suggesting regenerating epithelial cells (Regner et al., 2011). The immune-expression pattern of OPN almost corresponded to the mRNA expressions. In contrast to BMP-6 immunoreaction, OPN was not seen in spindle-shaped myofibroblasts in the fibrotic lesions. OPN might be produced exclusively in the affected renal tubules in CDDP-induced renal fibrosis. OPN is regarded as a factor capable of inducing macrophages (Xie et al., 2001); Knock-out of OPN gene in mice showed decrease in infiltrating macrophage number (Ophascharoensuk et al., 1999). In CDDP-induced rat renal fibrosis, because macrophage infiltration was observed (Yamamoto et al., 2012; Yamate et al., 1996), OPN produced by the affected renal tubular epithelial cells might be related to macrophage infiltration, in addition to induce myofibroblasts as mentioned below in in vitro experiments. 4.3. Effects of BMP-6, OPN and TGF-ˇ1 on kidney-constituting cell lines Because increased expression of BMP-6, OPN and TGF-␤1 mRNAs and immune-reactivity of BMP-6 and OPN were confirmed in CDDP-induced rat renal fibrosis, possible functions of these factors in renal fibrosis was analyzed by using kidney-constituting cell lines (NRK-49F, MT-9, and NRK-52E) in vitro. Myofibroblasts appearing in renal fibrosis may be derived from interstitial fibroblasts (NRK-49F), mesenchymal cells existing around blood vessels (so-called pericytes; MT-9), and renal epithelial cells (NRK52E) through EMT (Yuasa et al., 2013). Treatment of NRK-49F cells or MT-9 cells with TGF-␤1 significantly increased ␣-SMA in the in vitro experiment; previously, we showed increased expression of ␣-SMA in NRK-52E treated with TGF-␤1, suggestive of possible EMT (Yamamoto et al., 2010). Taken these findings together, it was considered that TGF-␤1 should be the major fibrogenic factor for development of myofibroblasts (expressing ␣-SMA) from various kidney-constituting cells. The present study also revealed that MT9 cells treated with BMP-6 and OPN increased ␣-SMA expression. BMP-6 and OPN were regarded as factors for the transdifferentiation of immature pericytes (MT-9) towards myofibroblasts. The pericytes may be an important cell having potential to differentiate into myofibroblasts (Yuasa et al., 2013). Immune-reactivity of BMP-6 was seen both in interstitial spindle-shaped myofibroblasts and renal epithelial cells of the damaged tubules, and OPN was expressed in the damaged renal epithelial cells. Furthermore, BMP6 stimulated OPN expression in NRK-49F cells. More intriguingly, the addition of TGF-␤1 to NRK-52E cells increased expressions of BMP-6 and OPN. BMP-6 and OPN, of which production might be stimulated by TGF-␤1, could participate in the myofibroblast development via autocrine and/or paracrine fashions. On the contrary, BMP-6 treatment significantly decreased TGF␤1 expression in NRK-52E. It has been reported that BMP-6 reduces TGF-␤1 activity by suppressing JNK and Smad2/3 signaling (Yan et al., 2009). BMP-6 may have biphasic capacity in CDDP-induced rat renal fibrosis: the promotion of myofibroblast development from mesenchymal progenitors such as the pericytes (MT-9) vs. the suppression of TGF-␤1 expression in NRK-52E renal epithelial cells. In conclusion, in addition to TGF-␤1 (the most powerful fibrogenic factor), the present study showed that BMP-6 and OPN mRNAs were markedly and consistently increased in CDDPinduced rat renal fibrosis. Immunohistochemically, BMP-6 was expressed in renal epithelial cells of the affected tubules and myofibroblasts in the fibrotic areas, and OPN expression was seen in the damaged renal epithelia cells. In in vitro experiments, BMP-6 and OPN treatments increased ␣-SMA expression (the most useful marker of myofibroblasts) in MT-9 cells (pericytes); the increased

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Please cite this article in press as: Yano R, et al. Participation of bone morphogenetic protein (BMP)-6 and osteopontin in cisplatin (CDDP)-induced rat renal fibrosis. Exp Toxicol Pathol (2014), http://dx.doi.org/10.1016/j.etp.2014.10.002

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Please cite this article in press as: Yano R, et al. Participation of bone morphogenetic protein (BMP)-6 and osteopontin in cisplatin (CDDP)-induced rat renal fibrosis. Exp Toxicol Pathol (2014), http://dx.doi.org/10.1016/j.etp.2014.10.002