International Immunopharmacology

Therapeutic activity of the histone deacetylase inhibitor SB939 on renal fibrosis
Sun-Woo Kang a, Soung-Min Lee b, Joo-Yong Kim c, So-Yeon Kim b, Yeong-Hoon Kim a, Tae-Hee Kim a,
Mi-Seon Kang d, Won-Hee Jang e, Su-Kil Seo b,⁎
a Department of Nephrology, Busan Paik Hospital, College of Medicine, Inje University, Busan 614-735, South Korea
b Department of Microbiology and Immunology, College of Medicine, Inje University, Busan 614-735, South Korea
c Department of Orthopedic Surgery, Busan Korea Hospital, Busan 614-735, South Korea
d Department of Pathology, College of Medicine, Inje University, Busan 000-000, South Korea
e Department of Biochemistry, College of Medicine, Inje University, Busan 614-735, South Korea

a r t i c l e i n f o

Article history:
Received 9 August 2016
Received in revised form 8 November 2016
Accepted 11 November 2016 Available online xxxx

Keywords: Renal fibrosis TGF-beta Myofibroblast HDAC inhibitor

a b s t r a c t

Fibrosis is the final pathological outcome of many chronic kidney diseases and is quite common. Thus, develop- ment of effective anti-fibrotic agents is urgently needed. Although histone deacetylases (HDACs) have been re- ported to be involved in renal fibrosis, current HDAC inhibitors are unsatisfactory anti-fibrosis drugs. Therefore, more potentially relevant anti-renal fibrosis HDAC inhibitors are needed. We initially found that non-cytotoxic concentrations of SB939 (pracinostat) had strong anti-fibrotic activity, drastically decreasing TGF-β1-induced alpha smooth muscle actin (α-SMA) expression in the NRK renal fibroblast cell line. Similar anti-fibrotic activity of SB939 on epithelial-to-mesenchymal transition (EMT) was confirmed using the HK-2 human renal proximal tubular epithelial cell line. SB939 inhibited Smad-independent TGF-β signaling involving the MAPK and PI3K/AKT pathways. To evaluate in vivo anti-fibrotic activity, we administered SB939 in a unilat- eral ureteric obstruction (UUO) model. SB939 treatment markedly inhibited the accumulation of α-SMA and tis- sue injury. Inflammatory and pro-fibrotic cytokines in the obstructed kidney were also significantly decreased by SB939 treatment. Our results suggest that SB939 might be a promising therapeutic drug for preventing renal fibrosis.

1. Introduction

Fibrosis is a characteristic feature of a wide variety of chronic kidney diseases (CKD) and progresses to end-stage renal failure [1,2]. Deposi- tion of pathological extracellular matrix (ECM) in the interstitial space and within the walls of glomerular capillaries is a key driver of progres- sive organ dysfunction. Inflammation-mediated cellular events are cru- cial to maintain the persistence of ECM-producing myofibroblasts in the kidney [3]. Although multiple cell types produce ECM, resident fibro- blasts have been shown to be the primary source of ECM-producing myofibroblasts [4].
Transforming growth factor-β (TGF-β) is well known to have a crit-
ical role in promoting the fibrotic response. Increased levels of TGF-β are common in almost all types of CKD, both in animal models and in humans. In addition, TGF-β can stimulate myofibroblastic activation of mesangial cells, interstitial fibroblasts, and tubular epithelial cells,
Corresponding author at: Department of Microbiology and Immunology, College of Medicine, Inje University, Busan 614-735, South Korea.
E-mail address: [email protected] (S.-K. Seo).

which then become ECM-producing fibrogenic cells in vitro [5]. The Smad-dependent pathway has been recognized as a major pathway of TGF-β signaling in progressive renal fibrosis. However, it has been re- ported that Smad-independent TGF-β signaling through ERK1/2, p38 MAPK, PI3K/AKT, STAT3, and RhoA also contributes to renal fibrogenesis [5–10].
Histone deacetylases (HDACs) deacetylate both histone and non- histone proteins and have an essential role in the regulation of physio- logical and pathological gene expression [11]. Emerging evidence indi- cates that HDACs are associated with renal fibrogenesis [12,13]. Pharmacological inhibition of HDACs has been demonstrated to dimin- ish progression of renal fibrogenesis in vitro and in vivo. Trichostatin A (TSA), a pan-HDAC inhibitor (HDACi), has reported anti-fibrotic effects in a murine model of unilateral ureteric obstruction (UUO) [14,15]. TSA treatment also significantly inhibits TGF-β-induced fibroblast activation and tubular epithelial-to-mesenchymal transition (EMT) in vitro [14, 16]. In addition, MS-275, a selective class I inhibitor, ameliorates renal fibrosis in a UUO model and reduces activation of cultured renal intersti- tial fibroblasts [17]. These reports indicate that HDAC inhibition can be a useful treatment for renal fibrosis.

SB939 (pracinostat) is a new orally active hydroxamate-based HDACi currently in phase II clinical trials. It potently inhibits class I, II and IV HDACs with excellent pharmacokinetic properties [18,19]. Com- pared with other oral HDACis, SB939 shows an increased oral bioavail- ability and half-life. In cancer models, SB939 accumulates in tumor tissue and induces sustainable inhibition of histone deacetylation, which results in a more enhanced antitumor efficacy than other oral HDAC inhibitors [18].
In the present study, we demonstrate the anti-fibrotic effects of SB939 in vivo and in vitro. Our findings suggest that SB939 may be a po- tential therapeutic intervention for preventing and treating renal fibrosis.

2. Materials and methods

2.1. Cell culture and treatment

NRK-49F rat renal interstitial fibroblasts cells were cultured in DMEM (Hyclone, Logan, Utah, USA) containing 10% fetal bovine serum (Hyclone), 100 U/ml penicillin (Hyclone), and 100 μg/ml streptomycin (Hyclone). HK-2 human renal proximal tubular epithelial cells were cul- tured in RPMI 1640 medium (Hyclone) containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. NRK-49F or HK2 cells (2 × 105 cells/3 ml) were cultured in a 60-mm culture dish (Corning Costar, Cambridge, MA, USA) to sub-confluence. The cells were serum starved for 24 h and then treated with 10 ng/ml TGF-β1 (R&D systems, Minneapolis, MN, USA) with or without SB939 (Selleckchem, Houston, TX, USA) at the indicated concentrations for the indicated periods.

2.2. Western blot analysis

Cells and kidneys were homogenized in a lysis buffer (G-Biosciences, St. Louis, MO, USA) containing protease inhibitor (Sigma Aldrich, St. Louis, MO, USA), 5 mM DTT and 5 mM EDTA. Samples were separated on a 15% SDS-PAGE gel and transferred to a nitrocellulose membrane (Whatman, Piscataway, NJ, USA). Antibody reactions were performed using anti-SMA (1:200; Abcam, Cambridge, MA, USA), anti-acetyl-α-tu- bulin (1:15,000; Sigma Aldrich), anti-phospho-SMAD2 (1:1000; Cell Signaling Technology), anti-phospho-SMAD3 (1:1000; Cell Signaling Technology), anti-phospho-ERK (1:1000; Cell Signaling Technology), anti-phospho-AKT (1:1000; Cell Signaling Technology), anti-acetyl-His- tone H3 (1:1000; Cell Signaling Technology), and anti-β-actin (1:1000; Cell Signaling Technology) antibodies. Membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated overnight at 4 °C on a rocker with primary antibodies. Membranes were incubated with HRP-conjugated secondary antibodies and visualized with en- hanced chemiluminescence (ECL; Pierce, Rockford, CA). Images were captured using a LAS3000 Image Analyzer (FujiFilm, Tokyo, Japan).

2.3. Real-time polymerase chain reaction

Total RNA was isolated from kidney tissues or cells using TRIzol® Re- agent (Invitrogen Corporation, Carlsbad, CA, USA) according to the manufacturer’s protocol. 5 μg of RNA, treated with DNase I (Roche Life Science, Indianapolis, USA), were reverse-transcribed with oligo-dT and the product was subject to polymerase chain reaction (PCR) utiliz- ing primers designed from Massachusetts General Hospital (MGH) PrimerBank (http://pga.mgh.harvard.edu/primerbank). Real-time PCR was performed using SYBR-Green Supermix and the iCycler Detection System (Bio-Rad Corp., Hercules, CA, USA) according to the manufacturer’s instructions. Relative gene expression levels were nor- malized to GAPDH expression, and expressed relative to the untreated
sample utilizing the formula 2−△△CT [20]. PCR was performed using sense/antisense primers of the following sequences: human α-SMA,
5′-GTC CCC ATC TAT GAG GGC TAT-3′ and 5′-GCA TTT GCG GTG GAC

AAT GGA-3′; rat α-SMA, 5′-ATC CGA TAG AAC ACG GCA TC-3′ and 5′- AGA AGA GGA AGC AGC AGT GG-3′; mouse α-SMA, 5′-GTC CCA GAC ATC AGG GAG TA-3′ and 5′-TCG GAT ACT TCA GCG TCA GG-3′; human collagen I, 5′-CAA TGC TGC CCT TTC TGC TCC TTT-3′ and 5′-CAC TTG GGT GTT TGA GCA TTG CCT-3′; mouse TNF-α, 5′-CCA CAC CGT CAG CCG ATT TG-3′ and 5′-CAC CCA TTC CCT TCA CAG AGC-3′; and mouse IL-6, 5′-CAT GTT CTC TGC GAA ATC GTG G-3′ and 5′-AAC GCA CTA GGT TTG CCG AGT A-3′; mouse TGF-β, 5′-CGC CAT CTA TGA GAA AAC C-3′ and 5′-GTA ACG CCA GGA ATT GT-3′; mouse IL-13, 5′-CCT GGC TCT TGC TTG CCT T-3′ and 5′-GGT CTT GTG TGA TGT TGC TCA-3′.

2.4. Cell viability analysis

NRK-49F cells were harvested, washed in binding buffer (10 mM Hepes-NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) and stained with PE-conjugated Annexin V (eBioscience, San Diego, CA, USA) and 7- AAD (eBioscience) for 15 min at room temperature. Apoptosis was de- termined as a percentage of both early apoptotic cells (Annexin V+/7- AAD−) and late apoptotic cells (Annexin V+/7-AAD+). Fluorescence was measured using a FACSCanto II flow cytometer (BD Biosciences, San Diego, CA, USA), and data were analyzed using FlowJo 9.6.4 soft- ware (Treestar, San Carlos, CA, USA).

2.5. Cell proliferation assay

NRK-49F cells (3 × 104 cells/200 μl) were plated in a 96-well flat bot- tom plate (Corning Costar) and treated with SB939 at the indicated con- centrations for 48 h. Cells were pulsed with [3H] thymidine (1 μCi/well; GE Healthcare, Piscataway, USA) during the last 15 h of incubation. The incorporated radioactivity was counted with a liquid scintillation coun- ter (PerkinElmer Wallac, Waltham, Massachusetts, USA).

2.6. Unilateral ureteral obstruction (UUO) model

Female C57BL/6 (B6) mice were purchased from Charles River (Tokyo, Japan) and used at 8–10 weeks of age. All mice were housed in autoclaved microisolator cages. All of the animal procedures were ap- proved by the Institutional Animal Care and Use Committee of Inje Uni- versity College of Medicine. The left ureter of B6 mice was exposed and permanently ligated twice with 4–0 nylon sutures. The contralateral kidney was used as a control. To examine the effects of SB939 on renal fibrosis after UUO injury, recipients were administered 75 mg/kg of SB939 i.p. (0.2 ml) once daily from day 3 to day 10. SB939 was dissolved in Cremophor EL and ethanol (1:2) and brought to a final concentration of 7.5 mg/ml in PBS.

2.7. Histologic analysis

Kidney tissues were fixed in a 10% formalin solution (Sigma Aldrich) and embedded in paraffin. Paraffin-embedded tissues were cut into 5- μm-thick sections and stained with H&E for histological examination. For assessment of renal fibrosis, Masson’s trichrome staining was per- formed according to the manufacturer’s protocol (Sigma Aldrich). Tissue morphology was assessed using a NanoZoomer 2.0 RS (Hamama- tsu, Shizuoka, Japan).

2.8. Statistical analysis

The Student’s t-test was used for statistical analysis of real-time PCR data. A p-value b 0.05 was defined as statistically significant. The data are presented as the mean ± standard error of the mean (SEM).

3. Results

3.1. SB939 strongly inhibits TGF-β1 induced fibrosis in NRK cells

Because HDACis are potent inducers of apoptosis and growth inhibi- tion in a variety of transformed cells [21], we initially determined the optimal concentrations of SB939 to produce anti-fibrotic effects without nonspecific cytotoxic effects. NRK rat kidney fibroblast cells were treat- ed with various concentrations of SB939. SB939 did not cause cell death until its concentration reached 500 nM (Fig. 1A). The proliferation of NRK cells was not affected until the SB939 concentration reached
100 nM and was significantly inhibited at SB939 concentrations
≥ 200 nM (Fig. 1B). Based on these results, we determined the proper concentrations of SB939 for further experimentation were within 100 nM.
To assess whether SB939 has anti-fibrotic properties, we analyzed α-SMA expression, which is a reliable marker of fibrosis after TGF-β-in- duced fibroblast activation. Treatment with TGF-β markedly induced the expression of α-SMA protein. Treatment of NRF-49F cells with 100 nM SB939 strongly inhibited α-SMA expression induced by TGF-β (Fig. 1C). Real-time PCR analysis confirmed significant inhibition of α- SMA mRNA expression by SB939 treatment at 100 nM (Fig. 1D). These data indicate that SB939 has strong anti-fibrotic effects on TGF-β-in- duced renal myofibroblasts.
We next tested the effects of TSA, pan-HDACi which has been the most studied for anti-fibrosis, in our experimental system. We observed a distinct inhibitory effect of treatment with 100 nM TSA for 48 h on

TGF-β-induced α-SMA expression in NRK cells (Fig. 2A). However, the treatment (100 nM for 48 h) also significantly inhibited cell prolifera- tion and induced cell death (Fig. 2B), indicating that TSA has a narrow concentration spectrum between anti-fibrotic activity and cellular tox- icity. Taken together, these data indicate that SB939 has less cytotoxicity than TSA on anti-renal fibrosis.

3.2. SB939 inhibits Smad-independent TGF-β signaling

Next, we determined the molecular mechanism of SB939 inhibition of α-SMA in TGF-β-treated NRK-49F cells. Although application of 100 nM of SB939 showed a strong inhibitory effect on α-SMA ex- pression, TGF-β-induced phosphorylation of Smad2 and Smad3 were not significantly inhibited (Fig. 3A and B). We further investi- gated Smad-independent TGF-β signaling pathways involved in fibrogenesis. We found that phosphorylation of ERK, p38, and AKT were markedly inhibited by SB939 treatment (Fig. 3C-E). These results indicate that the blockade of the TGF-β-induced fibrotic response by SB939 may be associated with inhibition of Smad-inde- pendent TGF-β signaling pathways such as MAPK and PI3K/AKT pathways.

3.3. SB939 strongly inhibits TGF-β1-induced EMT in HK-2 cells

The transformation of renal tubular epithelial cells into mesenchy- mal cells (EMT) contributes to the development of fibrosis in chronic renal failure [22]. To investigate whether SB939 inhibits EMT, we

Fig. 1. SB939 strongly inhibits TGF-β1-induced fibrosis in NRK-49F cells. (A, B) Determining the concentration of SB939 for anti-fibrotic effect assessment. NRK-49F cells were treated with SB939 at the indicated concentrations for 48 h. (A) Cell death and (B) proliferation were measured as described in Materials and methods. The data are representative of three independent experiments (n = 3 per group). (C, D) Anti-fibrotic effect of SB939. Serum-starved NRK-49F cells were exposed to recombinant human TGF-β1 (10 ng/ml) and treated with SB939 at the indicated concentrations for 48 h. (C) α-SMA was detected using western blotting. Representative blots from one of three independent experiments are shown. (D) α-SMA was quantified using real-time PCR. The data are representative of three independent experiments (n = 3 per group). *p b 0.05, ***p b 0.001 (vehicle treatment group vs. SB939 treatment group). The data represent the mean ± SEM.

Fig. 2. Assessment of TSA on anti-fibrotic effect and cellular toxicity. (A) The analysis of TSA on α-SMA expression was performed under the same condition of Fig. 1C. Serum-starved NRK- 49F cells were exposed to recombinant human TGF-β1 (10 ng/ml) and treated with TSA at the indicated concentrations for 48 h. α-SMA was detected using western blotting. Representative blots from one of three independent experiments are shown. (B) The analysis of TSA on cell death and proliferation was performed under the same condition of Fig. 1A. NRK-49F cells were treated with TSA at the indicated concentrations for 48 h. Cell death and proliferation were measured as described in Materials and methods. The data are representative of three independent experiments (n = 3 per group). *p b 0.05 (vehicle treatment group vs. TSA-treatment group). The data represent the mean ± SEM.

performed an EMT assay using a human renal proximal tubular epithe- lial cell line, HK2. Conversion of fibroblast morphology by TGF-β treat- ment was not observed in the presence of SB939 (Fig. 4A). SB939
strongly inhibited TGF-β-induced expression of α-SMA and collagen I (Fig. 4B). These data indicate that SB939 has a strong inhibitory effect on TGF-β-induced EMT.

Fig. 3. The anti-fibrotic effect of SB939 is associated with inhibition of ERK phosphorylation. Serum-starved NRK-49F cells were exposed to recombinant human TGF-β1 (10 ng/ml) and treated with the indicated concentrations of SB939. (A, B) p-smad2 and 3 were detected using western blotting at 24 h. (C-E) p-ERK, p-p38, and p-AKT were detected at 30 min. Representative blots from one of three independent experiments are shown. A densitometric analysis was performed to determine p-Smad2/total Smad2, p-Smad3/total Smad3, p- ERK/total ERK, p-p38/total p38, and p-AKT/total AKT ratios and plotted in a graph. The data are combined from three independent experiments (n = 3 per group). **p b 0.01, *** p b 0.001 (vehicle treatment group vs. SB939 treatment group). The data represent the mean ± SEM.

Fig. 4. SB939 strongly inhibits TGF-β1-induced EMT. Serum-starved HK2 cells were exposed to recombinant human TGF-β1 and treated with SB939 (100 nM). (A) Morphologic examination. After 3 days, cells were observed using light microscopy. (B) α-SMA and collagen I was quantified using real-time PCR. The relative expression is presented as a fold change relative to the control group, which is normalized to a value of 1. Untreated cells were used as a control. The data are representative of three independent experiments (n = 3 per group). *p b 0.05, **p b 0.01 (vehicle treatment group vs. SB939 treatment group). The data represent the mean ± SEM.

3.4. Antifibrotic effects of SB939 on a UUO model

To determine the effects of SB939 on renal fibrosis in vivo, we used a UUO model. We first investigated whether HDAC activity is induced in obstructed kidneys. The levels of acetyl-histone H3 and acetyl-α-tubu- lin in obstructed kidneys were clearly reduced compared with sham kidneys (Fig. 5A), indicating that HDAC activity in kidney is increased during the development of fibrosis. We found that SB939 treatment completely prevented the increase in α-SMA expression caused by

UUO (Fig. 5B). We next performed masson’s trichrome staining for assessing interstitial collagen fibrils in UUO mice. Tissue injury and col- lagen deposition were also remarkably decreased in obstructed kidneys treated with SB939 (Fig. 5C). Finally, we found that increased expres- sion of inflammatory cytokines (TNF-α and IL-6) and pro-fibrotic cyto- kines (TGF-β and IL-13) in obstructed kidneys were significantly reduced by SB939 treatment (Fig. 6). Taken together, these data indicate that blocking HDAC activity in vivo through treatment with SB939 may strongly inhibit renal inflammation and fibrosis.

Fig. 5. SB939 treatment markedly inhibits renal fibrosis in an UUO model. The right ureter of C57BL/6 mice was exposed and permanently ligated twice with 4–0 nylon sutures. Vehicle (n = 4) or SB939 (n = 4) was administered i.p. at a dosage of 75 mg/kg daily for 8 days (days 3–10). At day 11, both kidneys were excised from mice. (A) Enzyme activity of HDACs. Tissue extracts were prepared and subjected to western blotting with antibodies specific for the acetylated forms of histone H3 and α-tubulin. The contralateral kidney (sham) from vehicle- treated mice was used as a control. (B) α-SMA was detected using western blotting. Representative blots from one of three independent experiments are shown. (C) Histological examination: H&E staining (upper) and Masson’s trichrome staining (lower). Representative images from one of three independent experiments are shown.

Fig. 6. SB939 treatment significantly suppresses tissue cytokines associated with inflammation and fibrosis. Kidney tissues were obtained as described in Fig. 5. Cytokine mRNA levels were determined using real-time PCR. The relative expression is presented as a fold change relative to the control group from the kidney tissue of naive mice, which is normalized to a value of 1. The contralateral kidney from vehicle-treated mice was used as a sham. The data are representative of three independent experiments (n = 4 mice per group). *p b 0.05, **p b 0.01, *** p b 0.001 (vehicle treatment group vs. SB939 treatment group). The data represent the mean ± SEM.

4. Discussion

Although fibrogenesis is recognized as a primary cause of morbidity and mortality in most chronic kidney diseases, few treatments are avail- able that specifically target fibrogenesis. Recently, HDAC inhibition has been reported to significantly reduce fibrogenesis in several experimen- tal systems, demonstrating that HDAC inhibitors might be used as anti- fibrotic drugs. Among HDAC inhibitors, the pan-HDAC inhibitor TSA has been primarily studied in renal fibrosis. Treatment with TSA suppresses TGF-β1-induced EMT in human renal epithelial cells by inducing Id2 and BMP-7 expression [16]. Anti-fibrotic effects of TSA have also been shown in TGF-β-activated fibroblasts and the UUO model [14]. Molecu- lar mechanisms underlying the anti-fibrotic effects of TSA involve inhibiting Smad-independent pathways, rather than Smad-dependent pathways, in TGF-β signaling [7]. Similar to TSA, we confirmed that SB939 has strong anti-fibrotic effects and inhibits Smad-independent TGF-β signaling. Therefore, we compared the anti-fibrotic activity of these drugs in NRK cells. We observed a distinct inhibitory effect of treatment with 100 nM TSA for 48 h on TGF-β-induced α-SMA expres- sion in NRK cells (Fig. 2). However, the treatment (100 nM for 48 h) also significantly inhibited cell proliferation and induced cell death. Our data indicate that TSA has a narrow concentration spectrum between anti-fi- brotic activity and cellular toxicity. Previous studies have reported that application of TSA at 100 nM for 24 h has a non-cytotoxic effect on mouse splenocytes in vitro [23] and suppresses TGF-β1-induced apo- ptosis of RTECs [24]. In contrast, TSA treatment has been reported to im- pair intrinsic proliferation of fibroblasts even at 25 nM for 48 h [25], similar to our data. The discrepancy between these data might be due to differences in treatment period and cell type used. Nevertheless, TSA might cause non-specific cytotoxicity even at concentrations that produce anti-fibrotic effects. On the other hand, SB939 drastically inhibited α-SMA expression at 100 nM without cell growth inhibition or death (Fig. 1). Based on our results, SB939 might be a more appropri- ate HDAC inhibitor for anti-renal fibrosis than TSA. Further studies are needed to dissect the differences in anti-fibrotic effects and cellular cy- totoxicity between these HDAC inhibitors.
Many distinct triggers are involved in the development of progres- sive fibrotic disease. A feature common to all fibrotic diseases is the gen- eration of ECM-producing myofibroblasts. It is now clear that the innate and adaptive immune responses are essential to the differentiation and activation of fibroblasts [26]. Acute inflammatory reactions play an im- portant role in triggering fibrosis in the kidney. Damaged epithelial and endothelial cells release a variety of cytokines such as TNF-α and IL-6, which activate innate immune cells. Then, activated innate immune cells such as macrophages produce TGF-β [3]. In the UUO model, admin- istration of SB939 significantly reduced expression of pro-inflammatory cytokines in obstructed kidneys, indicating that SB939 might directly in- hibit obstruction-mediated acute inflammation. Moreover, SB939

treatment clearly inhibits IL-13 production (Fig. 6). Numerous studies have suggested that the type 2 cytokine response is a key driver of pro- gressive fibrosis [27]. Among type 2 cytokines, IL-13 has emerged as a dominant mediator of fibrotic tissue remodeling in several models of fi- brosis. Thus, the anti-fibrotic effects of SB939 may result from not only inhibiting TGF-β-induced fibrogenesis but also from regulating innate and adaptive inflammatory responses.
In summary, our data clearly demonstrate the anti-renal fibrotic ef- fects of SB939 in vivo and in vitro, suggesting that SB939 might be a po- tential therapeutic intervention for preventing and treating renal fibrosis.

Conflict of interest
The authors declare that there are no conflicts of interest.

Acknowledgments
This work was supported by the 2015 Inje University research grant (SWK).

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