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Inhibition of SET Domain–Containing Methyltransferase 7/9 Ameliorates Renal Fibrosis

Kensuke Sasaki,* Shigehiro Doi,* Ayumu Nakashima,* Taisuke Irifuku,* Kyoko Yamada,* † ‡ Keiko Kokoroishi,* Toshinori Ueno,* Toshiki Doi,* Eisuke Hida, Koji Arihiro, Nobuoki Kohno,§ and Takao Masaki*

*Department of Nephrology, Hiroshima University Hospital, Hiroshima, Japan †Center for Integrated Medical Research, Hiroshima University Hospital, Hiroshima, Japan ‡Department of Pathology, Hiroshima University Hospital, Hiroshima, Japan; and §Department of Molecular and Internal Medicine, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan

ABSTRACT TGF-b1 activity results in of lysine4ofhistoneH3(H3K4)throughSETdomain–containing lysine methyltransferase 7/9 (SET7/9) induction, which is important for the transcriptional activation of fibrotic genes in vitro.However,in vivo studies utilizing an experimental model of renal fibrosis are required to develop thera- peutic interventions that target SET7/9. In this study, we investigated the signaling pathway of TGF-b1-induced SET7/9 expression and whether inhibition of SET7/9 suppresses renal fibrosis in unilateral ureteral obstruction (UUO) mice and kidney cell lines. Among the SET family, SET7/9 was upregulated on days 3 and 7 in UUO mice, and the upregulation was suppressed by TGF-b1 neutralizing antibody. TGF-b1 induced SET7/9 expression via Smad3 in normal rat kidney (NRK)-52E cells. In human kidney biopsy specimens from patients diagnosed with IgA nephropathy and membranous nephropathy, SET7/9 expression was positively correlated with the degree of interstitial fibrosis (r=0.59, P=0.001 in patients with IgA nephropathy; and r=0.58, P,0.05 in patients with membranous nephropathy). In addition, small interfering RNA-mediated knockdown of SET7/9 expression significantly attenuated renal fibrosis in UUO mice. Sinefungin, an inhibitor of SET7/9, also suppressed the expression of mesenchymal markers and extracellular matrix proteins and inhibited H3K4 mono-methylation (H3K4me1) in kidneys of UUO mice. Moreover, sinefungin had an inhibitory effect on TGF-b1-induced a-smooth muscle actin expression and H3K4me1 in both NRK-52E and NRK-49F cells. In conclusion, sinefungin, a SET7/9 inhibitor, ameliorates renal fibrosis by inhibiting H3K4me1 and may be a candidate therapeutic agent.

J Am Soc Nephrol 27: 203–215, 2016. doi: 10.1681/ASN.2014090850

CKD is estimated to occur in 13%–15% of the pop- fibroblasts, deposition of extracellular matrix ulation in developed countries1,2 and is thus recog- (ECM) proteins, and loss of peritubular capillar- nized as a worldwide health problem.3 Importantly, ies.9,10 Although it has been reported that several CKD is responsible for progression to kidney failure4 cytokines participate in the pathogenesis of renal fi- and is linked to substantial disease burden, as evi- brosis, TGF-b1 has been identified as the most denced by increased morbidity and mortality.5,6 In order to prevent the development of CKD, inhibitors of the renin-angiotensin-aldosterone system have Received September 3, 2014. Accepted April 12, 2015. been established as a pharmacological intervention Published online ahead of print. Publication date available at 7 for CKD patients in clinical settings. However, the www.jasn.org. beneficial effects are not drastic and many patients Correspondence: Assistant Professor Shigehiro Doi or Professor eventually require renal replacement therapy. Takao Masaki, Department of Nephrology, Hiroshima University Regardless of initial causes, renal fibrosis is a Hospital, 1-2-3 Kasumi, Minami-Ku, Hiroshima 734-8551, Japan. common pathway to ESKD.8 Pathologically, renal fi- Email: [email protected] or [email protected] brosis is characterized by an increase in interstitial Copyright © 2016 by the American Society of Nephrology

J Am Soc Nephrol 27: 203–215, 2016 ISSN : 1046-6673/2701-203 203 BASIC RESEARCH www.jasn.org important mediator, playing a central role in the development of proteins in a mouse model of renal fibrosis remain unclear. fibrosis.11,12 In addition to potentiating fibrosis, TGF-b1func- Therefore, we first examined the gene expression profiles of tionsasananti-inflammatory effector molecule,13 suggesting that genes encoding epigenetic modification enzymes with SET inhibition of TGF-b1 could lead to severe adverse effects by en- domains in UUO mice by quantitative real-time RT-PCR (qRT- hancing systemic inflammation.14 Therefore, novel strategies that PCR). As shown in Figure 1A, we found that SET7/9 gene expres- specifically inhibit the fibrotic action of TGF-b1 are required. sion increased remarkably on both day 3 and day 7 in UUO mice refers to gene regulatory mechanisms specifically compared with a non-operated normal control group. Notably, not due to changes in the primary nucleotide sequence of genes.15 SET mRNAs apart from SET7/9 did not significantly increase For example, the tails on the surface are on day 7. SETD8 increased modestly on day 1 and day 3; targets for histone post-translational modifications (HPTMs), SETD4 increased on day 1, and SETD5 was slightly upregulated including acetylation, methylation, phosphorylation, and ubiq- on day 3. In contrast, SETD3 was suppressed on day 1, day 3, and uitylation.16 HPTMs are implicated in the regulation of chroma- day 7, and SETD6 was downregulated on day 3 and day 7. Western tin structure, which is essential for determining genomic DNA blot analysis revealed that the protein levels of SET7/9 were accessibility.17 Notably, HPTMs are regulated by specific enzymes and form a that modulates patterns of gene ex- pression.18 A recent study showed that TGF-b1 increases the expression of lysine 4 of (H3K4) methyltransferase SET domain–containing lysine methyltransferase 7/9 (SET7/9), and that subsequent H3K4 methylation plays an important role in TGF-b1-induced promoter activation of fibrosis-associated genes.19 These findings led us to the hypothesis that inhibition of SET7/9 will suppress renal fibrosis through inhibition of H3K4 methylation in a mouse model of renal fibrosis. In this study, we show that TGF-b1indu- ces SET7/9 via the Smad3 pathway. We also demonstrate that SET7/9 is responsible for H3K4 methylation as well as renal fibrosis in unilateral ureteral obstruction (UUO) mice. Furthermore, renal expression of SET7/9 is correlated with fibrotic areas in re- nal biopsy samples that were diagnosed with immunoglobulin A nephropathy (IgAN) and membranous nephropathy (MN). Finally, the small molecule inhibitor of SET7/9, sine- fungin,20,21 inhibits H3K4 methylation and ameliorates renal fibrosis. These data suggest that sinefungin, also known as an antifungal agent, may be a candidate therapeutic agent Figure 1. SET7/9 is upregulated in the kidney after obstructive injury. (A) Expression for CKD patients. profiles of genes encoding epigenetic modification enzymes containing the SET domain in UUO mice. On day 1 (gray bars), day 3 (hatched bars), and day 7 (black bars) compared with non-operated normal control (white bars), UUO-induced mRNA levels were de- RESULTS termined by qRT-PCR. Data were analyzed by one-way ANOVA followed by Dunnett post hoc test based on non-operated normal controls for each SET primer (n=5 for each Increased Expression of the H3K4 group). (B) Elevation of SET7/9 protein in whole kidney extracts at day 7 after UUO compared with sham-operated control. Typical results of western blot analysis are shown Methyltransferase SET7/9 in a Mouse in the upper panel. Band intensity was normalized to glyceraldehyde 3-phosphate de- Model of Renal Fibrosis hydrogenase . Relative levels of SET7/9 expression are shown in the lower panel (n=5 for SET7/9 has been described as an epigenetic each group). (C) Immunohistochemical staining for SET7/9 demonstrating the localization †† modification enzyme that promotes ECM of SET7/9 protein in the kidneys. *P,0.05; **P,0.01; P,0.01. GAPDH, glyceraldehyde protein production in vitro;19 however, ex- 3-phosphate dehydrogenase; non-immune, control non-immune serum; Non-op CTL, pression of SET7/9 and the other SET family non-operated normal control; Sham CTL, sham-operated controls.

204 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 203–215, 2016 www.jasn.org BASIC RESEARCH elevated in UUO kidneys compared with sham-operated con- they progressively increased in the negative control siRNA- trols, which was consistent with our mRNA results (Figure injected UUO group. In contrast, a-SMA and collagen 1 1B). Immunohistochemical staining for SET7/9 was per- were significantly decreased in the SET7/9-siRNA group formed in order to identify SET7/9 tissue expression patterns. (Figure 4, C–E). In UUO kidneys, marked staining of SET7/9 was detected in the nuclei of tubular epithelial cells and interstitial cells SET7/9 Expression is Associated with the Degree of (Figure 1C). Fibrosis in Human Kidney In order to assess the clinical significance of SET7/9 SET7/9 Expression is Positively Regulated by TGF-b1in expression, we performed immunostaining for SET7/9 and UUO Mice and in Renal Cells correlated with Masson’s trichrome (MT) staining on biopsy TGF-b1 expression induced by UUO22 is known to play an samples. A total of 38 renal tissue specimens (24 men and 14 important role in the development of renal fibrosis. Therefore, women) were collected from IgAN (n=26) and MN (n=12) we examined the role of TGF-b1 in SET7/9 expression in vitro patients who underwent renal biopsy for the first time at and in vivo. Normal rat kidney (NRK)-52E cells, a rat kidney Hiroshima University Hospital from April, 2008 to December, tubular epithelial cell line, and NRK-49F, a rat kidney intersti- 2010. In this patient population, the average clinical values tial fibroblast cell line, were used. TGF-b1-induced SET7/9 were as follows: 47618 years of age, body mass index was protein expression was upregulated in a dose-dependent man- 22.5163.22 kg/m2, creatinine clearance was 89.40622.96 ner, and SET7/9 expression significantly increased after 2 hours mL/minute, eGFR was 71.32618.64 mL/minute/1.73 m2,and of stimulation in both cell lines compared with vehicle- urinary protein excretion was 1.5561.90 g/24 hours. The treated controls (Figure 2, A and B). Furthermore, admin- clinical characteristics of IgAN and MN patients in relation istration of neutralizing TGF-b1 antibody23 immediately to renal function are shown in Supplemental Table 1 and after UUO attenuated UUO-induced SET7/9 expression, as Supplemental Table 2. The staining area for SET7/9 increased detected by western blot analysis and immunohistochemistry with the decline of renal function. SET7/9 was positively cor- (Figure 2, C and D). related with MT-positive fibrotic areas in a correlation dia- gram (Spearman correlation coefficient: r=0.59, P=0.001 in SET7/9 Expression is Regulated by the IgAN patients, and r=0.58, P,0.05 in MN patients) (Figure 5, TGF-b1–Smad3-dependent Pathway AandB). TGF-b1 is recognized to stimulate several pathways though acti- vation of TGF-b receptors. Among those, we investigated whether Sinefungin Ameliorates Renal Fibrosis in Obstructive TGF-b1-induced phosphorylation of Smad3 (p-Smad3), which Nephropathy is known as a canonical pathway for TGF-b1 signaling,24 is re- In view of the need for developing new therapeutic agents for sponsible for SET7/9 expression. In order to downregulate the treatmentof renalfibrosis, we assessed the effect of a small Smad3, NRK-52E cells were transfected with small interfering molecule inhibitor for SET7/9, sinefungin, on the expression RNA (siRNA) oligonucleotides targeting Smad3 (si-Smad3) or of mesenchymal markers and ECM proteins in UUO mice. negative control siRNA (si-Neg). After TGF-b1 stimulation (30 We examined a-SMA and fibroblast-specificprotein-1(FSP- minutes for Smad3 or 24 hours for SET7/9), total protein from 1) as mesenchymal markers, and collagen 1, collagen 3, and transfected cells was analyzed by western blots with SET7/9, fibronectin as ECM proteins. Following the injection of Smad3, and p-Smad3 antibodies. Glyceraldehyde 3-phosphate sinefungin, UUO-induced mRNA of a-SMA, collagen 1 dehydrogenase was used as an internal control. As shown in and collagen 3 were markedly suppressed in the kidney, Figure 3, A and B, TGF-b1-induced SET7/9 mRNA and protein both at 3 days and 7 days after UUO (Figure 6A). Western levels were significantly reduced in NRK-52E cells transfected blot analysis also showed that sinefungin inhibited a-SMA with Smad3-siRNA. Likewise, Smad3-siRNA treatment signifi- protein expression (Figure 6B). Our immunohistochemical cantly inhibited the expression of Smad3 and TGF-b1-induced analysis revealed that staining for a-SMA,FSP-1,collagen1, p-Smad3 (Figure 3, C and D). collagen 3, and fibronectin in the kidney tissues increased at 3 days, with a further increase at 7 days, after UUO. In con- Knockdown of SET7/9 in Vivo Attenuates TGF-b1- trast, sinefungin injection ameliorated those both at 3 and 7 Induced Fibrogenesis in Obstructive Nephropathy days after UUO (Figure 6, C and D). However, injection of Next, we knocked down SET7/9 expression using siRNA in sinefungin did not affect UUO-induced TGF-b1mRNAex- UUO mice to evaluate whether SET7/9 is responsible for pression levels (Figure 6E). fibrogenesis in vivo. First, we confirmed whether SET7/9 was sufficiently knocked down by SET7/9-siRNA injection. In the Sinefungin Inhibits H3K4me1 Simultaneously with the SET7/9-siRNA group, UUO-induced SET7/9 mRNA and pro- Amelioration of Renal Fibrosis in Obstructive tein expression was significantly decreased (Figure 4, A and B). Nephropathy Expression of a-smooth muscle actin (a-SMA) and collagen 1 It has previously been reported that increased levels of were very low in the sham-operated control group, whereas H3K4 methylation promote transcriptional activation of

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Figure 2. TGF-b1 induces SET7/9 expression in renal cells, and injection of neutralizing TGF-b1 antibody reduces SET7/9 expression after UUO. NRK-52E cells and NRK-49F cells were treated with TGF-b1. Representative western blot analysis showing the levels of SET7/9 protein expression in TGF-b1-stimulated NRK-52E cells and NRK-49F cells at (A) various dosages (time; 24 hours) and (B) time points (TGF-b1; 10 ng/mL). Expression levels were compared with vehicle-treated control. Quantification is shown in the lower panel. Data were analyzed by one-way ANOVA followed by Dunnett post hoc test based on vehicle-treated controls (n=5 for each group). (C) Representative western blot analysis with anti-SET7/9 antibody. Quantification is shown in the lower panel. Data were analyzed by one-way ANOVA followed by Dunnett post hoc test based on UUO mice with control IgG1 injection (n=5 for each group). (D) Images of SET7/9 staining demonstrating the levels of SET7/9 expression by intraperitoneal injection of neutralizing TGF-b1 antibody (TGF-b1-Ab) at a dose of 1.5 mg/kg/48 hours compared with control

IgG1 atthesamedoseofTGF-b1-Ab. Original magnification, 3200. **P,0.01. Sham CTL, sham-operated controls; IgG1, UUO mice with control IgG1 injection; non-immune, control non-immune serum; TGF-b1-Ab, UUO mice with neutralizing TGF-b1 antibody injection.

TGF-b1-induced fibrotic gene expression.19 To identify (H3K4me1) in mouse kidney (Figure 7A). In contrast, the effect of sinefungin on SET7/9 during the development H3K4 di-methylation (H3K4me2) and H3K4 tri-methylation of renal fibrosis, we tested whether sinefungin inhibits () in the kidneys of UUO mice did not show a H3K4 methylation. Injection of sinefungin significantly significant change after the injection of sinefungin (Figure inhibited UUO-induced H3K4 mono-methylation 7, B and C).

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NRK-52E (Figure 8, A and B) and NRK-49F (Figure 9, A and B) cells. In contrast, sinefungin had no significant effect on H3K4me2 and H3K4me3 either in vivo in UUO kidneys or in vitro in epithelial cells (Figure 8, C and D) and fibroblasts (Figure 9, C and D). Fi- nally, we examined whether TGF-b1 alters access located at H3K4me1-regulated sites using immunoprecipitation (ChIP) assays. We found that TGF-b1 in- creased H3K4me1 levels at collagen 1 (Col1a1), connective tissue growth factor (CTGF) and plasminogen activator inhibitor-1 (PAI-1) promoters in NRK-52E cells, and that sinefungin inhibited H3K4me1 levels (Figure 8E).

DISCUSSION

In this study, we have demonstrated five major findings. First, SET7/9 plays an important role in renal fibrogenesis in UUO mice. Second, TGF-b1 induces SET7/9 expression via the Smad3 pathway. Third, inhibition of SET7/9 suppresses TGF-b1-induced fibrogenesis. Fourth, SET7/9 expression is associated with the degree of fibrosis in the human kidney. Finally, sinefungin ameliorates renal fibrosis in UUO mice and inhibits TGF-b1-induced a Figure 3. Knockdown of Smad3 in NRK-52E cells inhibits TGF-b1-induced SET7/9 -SMA expression as well as H3K4me1 in re- expression. NRK-52E cells were transfected with Smad3 siRNA (si-Smad3) or negative nal cells. Results from this study illustrate that control (si-Neg) oligonucleotides. (A) SET7/9 mRNA levels determined by qRT-PCR in sinefungin, known as an antifungal agent, transfected NRK-52E cells with or without TGF-b1 (10 ng/mL, 24 hours). (B) Western may be a candidate therapeutic agent for blot analysis using SET7/9, (C) Smad3, and (D) phosphorylated Smad3 (p-Smad3) CKD patients. Our studies also provide in- antibodies in transfected NRK-52E cells with or without TGF-b1 (10 ng/mL, 30 minutes sights into the mechanism by which SET7/9 or 24 hours). Total cell lysates were subjected to immunoblotting. Because p-Smad3 induces renal fibrogenesis in a mouse model content peaked at 30 minutes after TGF-b1 stimulation, this time point was used only of renal fibrosis. fi in p-Smad3 experiments. Quanti cation is shown in the lower panel (n=5 for each Among a number of epigenetic modifi- group). Data were analyzed by one-way ANOVA followed by the post hoc test using t cations, the inhibitory effects of DNA meth- test with Bonferroni correction. ##P,0.01. si-Neg, UUO mice with negative control ylation25 and micro RNA26 on renal fibrosis oligonucleotides injection; si-Smad3, UUO mice with Smad3-siRNA injection. have been investigated so far. In this study, we first confirmed that histone methyltrans- ferase SET7/9 was increased during the de- Sinefungin Inhibits TGF-b1-Induced a-SMA-positive velopment of renal fibrosis in UUO mice and patients with IgAN Myofibroblast Expression and TGF-b1-induced and MN. We also showed that knockdown of SET7/9 with H3K4me1 in Renal Cells siRNA ameliorated renal fibrosis in UUO mice. These results As shown in Figures 6 and 7, sinefungin inhibited H3K4me1 and suggest that increased SET7/9 contributes to renal fibrosis, and ameliorated renal fibrosis induced by UUO. However, these re- that SET7/9 is a novel and attractive target for the treatment of sults were from whole kidney lysates in UUO mice. Toclarify the CKD. Moreover, we have demonstrated that sinefungin ameli- direct effect of sinefungin on TGF-b1-induced H3K4 methyla- orates renal fibrosis in UUO mice. Sinefungin functions as an tion, we performed immunoblotting for H3K4 methylation in S-adenosylmethionine (SAM) analog and competes for SAM NRK-52E and NRK-49F cells. Pretreatment with sinefungin sig- binding, thereby inhibiting the SAM-dependent methyltransfer- nificantly reduced TGF-b1-induced a-SMA protein expression ase activity of SET7/9.20,21,27 Previous studies have described that and inhibited H3K4me1 in a dose-dependent manner in both sinefungin administrated to mice at a dose of 0.5–25 mg/kg

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inhibited the development of various fungi, viruses, and para- sites.28,29 In this study, we administrated sinefungin at a dose of 10 mg/kg/day, which resulted in the suppression of renal fibrosis as well as H4K4me1. Taken together, SET7/9 upregulation is involved in the progression of renal fibrosis, and sinefungin sup- presses renal fibrosis through SET7/9 inhibition. We have demonstrated for the first time that inhibition of SET7/9 activity ameliorates renal fibrosis as well as decreasing H3K4me1 levels both in vivo and in vitro. Renal fibrosis is due to transcriptional activation of fibrotic genes. Recent studies have shown that methylated H3K4 correlated with transcriptionally competent chromatin, resulting in increased gene expres- sion.30,31 Importantly, a previous study has reported that tran- scriptional activation of fibrosis-associated genes is positively regulated by H3K4me1/2/3 levels.19 In this study, we identified that UUO increases H3K4me1 levels, but not H3K4me2 and H3K4me3, indicating that H3K4me1 mainly contributes to the progression of renal fibrosis in vivo. In renal cell lines, we found that TGF-b1 induces H3K4me1, but not H3K4me2 and H3K4me3. Conversely, one recent study showed that SET7/9- induced methylation of H3K4 enhanced TGF-b1expressionin the liver of bile duct ligation rats.32 However, we did not observe any inhibitory effect of sinefungin on TGF-b1productionin UUO mice. These findings suggest that sinefungin suppresses renal fibrosis through inhibiting H3K4me1, but does not reduce TGF-b1production. We have described that UUO-induced SET7/9 expression is inhibited by TGF-b1-neutralizing antibody, indicating that TGF- b1 leads to SET7/9 expression in a mouse model of renal fibrosis. We also showed that TGF-b1-induced phosphorylation of Smad3 is responsible for SET7/9 expression. TGF-b1–Smad3 signaling is a canonical pathway, and p-Smad3 serves as a component of transcriptional factors that promote renal fibrosis.24 Notably, our results show that TGF-b1 also increases H3K4me1 levels at fi- brotic gene promoters, such as Col1a1, CTGF,andPAI-1.19 Taken together, TGF-b1–Smad3 signaling plays an important role in the increased transcriptional activity of fibrosis-associated genes through SET7/9 production and subsequent H3K4me1. TGF-b1 is not only known as a major mediator in the progression of renal fibrosis, but also functions as an anti- fl Figure 4. Knockdown of SET7/9 inhibits UUO-induced fibroblast ac- in ammatory cytokine, suggesting that systemic inhibition of tivation in UUO mice. SET7/9-siRNA injection was performed at a dose of TGF-b1 signaling may lead to systemic inflammation.14 In 7 mg/kg via the right ureter. (A) Expression level of SET7/9 mRNA fact, Tgfb1-knockout mice exhibited a lethal postnatal inflam- determined by qRT-PCR. Gene expression was normalized to internal matory phenotype, whereas the systematic effect of long-term control 18S rRNA (n=5 for each group). (B) Representative western blot inhibition of TGF-b1 signaling in humans remains unclear.33 analysis showing the levels of SET7/9 expression. Quantification is Among the histone modifications, H3K4me3 is reported to par- showninthelowerpanel(n=5 for each group). (C) Expression levels of ticipate in an increased expression of forkhead box p3, resulting a-SMA, and (D) collagen 1 mRNA determined by qRT-PCR. Gene ex- in regulatory T cell generation.34 In this study, we did not ob- pression was normalized to internal control 18S rRNA (n=5 for each serve any inhibitory effect of sinefungin on H3K4me3, implying group). (E) Typical results of western blot analysis showing the levels of that sinefungin does not disturb the immunosuppressive func- a-SMA expression. Quantification is shown in the lower panel (n=5 for each group). Data were analyzed by one-way ANOVA followed by tion of regulatory T cells. In addition, a recent study has reported Dunnett post hoc test based on UUO mice with negative control oli- that TGF-b1 suppresses IL-2 production from T cells through gonucleotides (si-Neg) injection. **P,0.01. Sham CTL, sham-operated , but not H3K4me1.35 These findings raise the possi- controls; si-Neg, UUO mice with negative control oligonucleotides in- bility that inhibition of H3K4me1 is a novel and attractive ther- jection; si-SET7/9, UUO mice with SET7/9-siRNA injection. apeutic strategy for treating renal fibrosis.

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Figure 5. Correlation between the expression of SET7/9 and the degree of fibrosis in IgAN and MN patients. (A) Representative images of SET7/9 staining and MT staining demonstrating strong SET7/9 staining with the decline of renal function in IgAN and (B) MN patients. Vertical columns show images from the same person. Scatter diagram of bivariate correlations in the right panel demonstrating a positive correlation between SET7/9 and MT staining. Spearman’s correlation coefficient test was used. r, Spearman correlation coefficient; r=0.59, P=0.001 in IgAN patients, and r=0.58, P,0.05 in MN patients. Non-immune, control non-immune serum. Original magnifi- 2 cation, 3200. The Japanese GFR equation based on serum creatinine was used as an eGFR. eGFR (mL/minute/1.73 m2)=1943Scr 1.094 2 3Age 0.28730.739 (if female). Age, years old; Scr, serum creatinine (mg/dL); non-immune, control non-immune serum.

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Figure 6. Sinefungin ameliorates the UUO-induced increase in mesenchymal markers and deposition of ECM proteins. Mice were treated daily with sinefungin at a dose of 10 mg/kg by intraperitoneal injection. We examined a-SMA and FSP-1 as mesenchymal markers, and collagen 1, collagen 3, and fibronectin as ECM proteins. (A) a-SMA, collagen 1, and collagen 3 mRNA levels determined by qRT-PCR in UUO mice with or without sinefungin injection (n=5 for each group). (B) Representative western blot analysis dem- onstrating the levels of a-SMA protein expression in UUO mice with or without sinefungin injection. Quantification is shown in the lower panel (n=5 for each group). (C) Typical immnohistochemistry of a-SMA,FSP-1,collagen1,collagen3,andfibronectin in UUO mice with or without sinefungin injection. (D) Quantification of FSP-1, collagen 1, collagen 3, and fibronectin expression by

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identified inhibition of SET7/9 as a thera- peutic target for kidney fibrosis, and sug- gest that sinefungin may be a candidate therapeutic agent for CKD patients.

CONCISE METHODS

Animals Male C57BL/6J mice (8 weeks of age) were purchased from Charles River Laboratories Japan (Yokohama, Japan). All animal experi- ments were approved by the Institutional An- imal Care and Use Committee at Hiroshima University (Hiroshima, Japan) and were per- formed in accordance with the National Insti- tutes of Health Guidelines on the Use of Laboratory Animals. UUO, a well characterized mouse model of renal fibrosis, was performed under general anesthesia as previously de- scribed.36 Mice were killed on day1, day 3, or day 7 after UUO and renal tissues were harvested.

Drug and siRNA Administration In Vivo Mice were administered with neutralizing anti- TGF-b1 antibody (1D11, 1.5 mg/kg; R&D Sys- Figure 7. Sinefungin inhibits UUO-induced upregulation of H3K4me1 in UUO mice tems, Minneapolis, MN), or normal mouse concomitantly with the amelioration of renal fibrosis in obstructive nephropathy. The IgG (11711, 1.5 mg/kg; R&D Systems) imme- same protein lysates as in Figure 6 were used. (A) Representative western blot analysis 1 showing the levels of H3K4me1, (B) H3K4me2, and (C) H3K4me3 protein expression in diately after UUO by intraperitoneal injection. UUO mice with or without sinefungin injection (10 mg/kg). Quantification is shown in Thesametreatmentswererepeatedevery48 the right panel (n=5 for each group). Data were analyzed by one-way ANOVA fol- hours until mice were killed as previously de- lowed by the post hoc test using t test with Bonferroni correction. ##P,0.01. Non-op scribed.37 siRNA (In Vivo Pre-designed SET7/9- CTL, non-operated normal controls; Sine, sinefungin; Veh, vehicle. siRNA and In Vivo Negative Control #1 siRNA; Ambion, Carlsbad, CA) and Invivofectamine 2.0 reagent (Invitrogen, Carlsbad, CA) complex In summary, SET7/9 expression is regulated by the TGF- (0.7 mg/mL) was prepared according to the manufacturer’s instruc- b1–Smad3 pathway, leading to transcriptional activation of tions. Immediately after right ureteral obstruction, 50 mL of SET7/9- fibrotic genes through increased H3K4me1. We confirmed siRNA solution (7 mg/kg) was injected retrogradely once into the the actual expression of SET7/9 in renal biopsy samples right kidney via the ureter.38,39 Sinefungin (Sigma-Aldrich, St Louis, from patients who were diagnosed with IgAN and MN, and MO) was prepared as a suspension in distilled water and 0.9% NaCl showed that SET7/9 expression correlated with fibrotic areas. solution, and administered intraperitoneally (0.1 mL per mouse) at a Inhibition of SET7/9 not only suppressed H3K4me1 levels but dose of 10 mg/kg per day immediately after UUO. The control group also ameliorated renal fibrosis in a mouse model of renal fi- was administered an equal volume of vehicle (0.1 mL of distilled brosis. Furthermore, a small molecular inhibitor of SET7/9, water and 0.9% NaCl solution) intraperitoneally. The same treat- sinefungin, also showed decreased H3K4me1 levels as well as ments were repeated every 24 hours until mice were killed. We se- suppressed fibrogenesis in vivo and in vitro. In conclusion, we lected the dose of sinefungin based on described studies.28,29

immunohistochemical staining. (n=5 for each group). (E) TGF-b1 mRNA levels in UUO mice with or without sinefungin injection quantified by qRT-PCR (n=5 for each group). Data were analyzed by one-way ANOVA followed by the post hoc test using t test with Bonferroni correction. #P,0.05; ##P,0.01. Non-immune, control non-immune serum; Non-op CTL, non-operated normal controls; Sine, sinefungin; Veh, vehicle.

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Figure 8. Sinefungin ameliorates the TGF-b1-induced increase of a-SMA and inhibits the upregulation of histone H3K4 mono- methylation in renal epithelial cells. Pretreatment of sinefungin (0.5 or 1.0 mg/mL) was conducted 60 minutes before TGF-b1(10ng/mL) stimulation. (A) A typical western blot for a-SMA in NRK-52E cells. Quantification is shown in the lower panel (n=5 for each group). (B) Representative western blot analysis of the expression of histone H3K4me1, (C) H3K4me2, and (D) H3K4me3 expression in NRK-52E cells. Quantification is shown in the lower panel (n=5 for each group). (E) Representative ChIP assay analysis of the expression of binding of H3K4me1 protein to Col1a1, CTGF,andPAI-1 promoters in NRK-52E cells. ChIP assays were performed with H3K4me1 antibody. Immunoprecipitated DNA and input DNA were subjected to qRT-PCR. Results were normalized to input DNA (n=5 for each group). Data were analyzed by one-way ANOVA followed by the post hoc test using t test with Bonferroni correction. #P,0.05; ##P,0.01. Sine, sinefungin.

212 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 203–215, 2016 www.jasn.org BASIC RESEARCH

according to the manufacturer’s instructions. Af- ter 6 hours, the transfected cells were washed, and fresh medium containing 0% FBS was added. The next day, cells were treated with or without TGF-b1, and processed for mRNA or protein extraction at the indicated time periods.

RNA Extraction and Quantitative Real-time RT-PCR RNA extraction and qRT-PCR were per- formed as previously described.40 qRT-PCR was performed using an ABI 7500 fast real- time PCR system (Applied Biosystems, Fos- ter City, CA), and expression of genes related to epigenetic modification of UUO mice was compared between the groups. Specific oligonucleotide primers and probes for a-SMA (assay ID: Mm00725412_s1), colla- gen 1 (assay ID: Mm00801666_g1), colla- gen 3 (assay ID: Mm01254476_m1), SETDB1 (assay ID: Mm00616964_m1), SETD2 (as- say ID: Mm01250225_m1), SETD3 (assay ID: Mm01730314_gH), SETD4 (as- say ID: Mm00520991_m1), SETD5 (as- say ID: Mm00712606_m1), SETD6 (assay ID: Mm01243947_g1), SET7/9 (assay ID: Mm00499823_m1), SETD8 (assay ID: Mm03031474_g1), and 18S rRNA (endogenous control) were obtained as TaqMan gene ex- pression assays (Applied Biosystems). The mRNA levels were normalized for the level of Figure 9. Sinefungin ameliorates the TGF-b1-induced increase of a-SMA and inhibits 18S rRNA. the upregulation of H3K4me1 in renal fibroblast cells. Pretreatment of sinefungin (0.5 or 1.0 mg/mL) was carried out 60 minutes before TGF-b1 (10 ng/mL) stimulation. (A) A typical western blot for a-SMA expression in NRK-49F cells. Quantification is shown in Western Blot Analysis Renal tissues or cells grown in six-well dishes the lower panel (n=5 for each group). (B) Representative western blot analysis of the expression of H3K4me1, (C) H3K4me2, and (D) H3K4me3 expression in NRK-49F cells. were lysed in 2% SDS sample buffer and son- – Quantification is shown in the lower panel (n=5 for each group). Data were analyzed icated for 10 30 seconds using Taitec ultrasonic by one-way ANOVA followed by the post hoc test using t test with Bonferroni cor- homogenizer VP-050 at 20% power. Immuno- rection. ##P,0.01. Sine, sinefungin. blotting was performed as previously de- scribed.41 Primary antibodies used in this study were anti-SET7/9 (Cell Signaling Technology, Cell Culture Danvers, MA), anti-p-Smad3 (Cell Signaling Technology), anti- NRK-52E and NRK-49F cells were obtained from the American Type Smad3 (Cell Signaling Technology), anti-H3K4me1 (Cell Signal- Culture Collection (Manassas, VA).ThesecellsweremaintainedinDMEM ing Technology), anti-H3K4me2 (Cell Signaling Technology), containing 5% FBS and penicillin/streptomycin. All cells were washed and anti-H3K4me3 (Cell Signaling Technology), anti-histone H3 (Cell growth was arrested for 24 hours in DMEM containing 0% FBS prior to Signaling Technology), anti-a-SMA antibody (Sigma-Aldrich), anti- each stimulation. Preincubation of sinefungin was carried out 60 minutes glyceraldehyde 3-phosphate dehydrogenase (Sigma-Aldrich), anti- before TGF-b1 (R&D Systems) stimulation. NRK-52E and NRK-49F b-actin (Sigma-Aldrich), and anti-a-tubulin (Sigma-Aldrich). cells were treated with TGF-b1 at the indicated dosage levels and times. Secondary antibodies used in this study were horseradish peroxidase- conjugated goat anti-rabbit immunoglobulin G antibody (Dako, siRNA Transfection In Vitro Glostrup, Denmark) or goat anti-mouse immunoglobulin G antibody NRK-52Ecellswereplatedinsix-wellculturedishesandweretransfected (Dako). Signals were detected using the SuperSignal West Dura or Pico 6 hours later (30% confluent) with Smad3 Silencer Select siRNA system (Thermo Fisher, Rockford, IL). The intensity of each band was (si-Smad3, 12.5 nM; Invitrogen) or Silencer Select Negative Control #1 determined using ImageJ software (version 1.46r; National Institutes of siRNA (si-Neg; Invitrogen) using lipofectamine 2000 (Invitrogen) Health, Bethesda, MD).

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Norris KC, Greene T, Kopple J, Lea J, Lewis J, Lipkowitz M, Miller P, anti-SET7/9 antibody (Abcam, Inc.). The signal intensity of FSP-1, Richardson A, Rostand S, Wang X, Appel LJ: Baseline predictors of renal fi disease progression in the African American Study of Hypertension and collagen1, collagen3, bronectin, SET7/9, and MT staining were – fi Kidney Disease. JAmSocNephrol17: 2928 2936, 2006 quanti ed using ImageJ software by examination of predetermined 5. Go AS, Chertow GM, Fan D, McCulloch CE, Hsu CY: Chronic kidney high (3200) or low (3100) power fields of the cortex (five fields) as disease and the risks of death, cardiovascular events, and hospitaliza- previously described.37 In the case of human kidney, the study was tion. NEnglJMed351: 1296–1305, 2004 approved by the Ethics Committee of Hiroshima University (H-895). 6. 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J Am Soc Nephrol 27: 203–215, 2016 SET7/9 and Renal Fibrosis 215 Supplemental Data (Sasaki,K. et al.)

Supplemental Data

Inhibition of SET Domain-Containing Lysine Methyltransferase 7/9 Ameliorates Renal Fibrosis

Kensuke Sasaki,1 Shigehiro Doi,1* Ayumu Nakashima,1 Taisuke Irifuku,1 Kyoko Yamada,1 Keiko Kokoroishi,1 Toshinori Ueno,1 Toshiki Doi,1 Eisuke Hida,2 Koji Arihiro,3 Nobuoki Kohno,4 and Takao Masaki1*

1Department of Nephrology, Hiroshima University Hospital, Hiroshima, Japan; 2Center for Integrated Medical Research, Hiroshima University Hospital, Hiroshima, Japan; 3Department of Pathology, Hiroshima University Hospital, Hiroshima, Japan; and 4Department of Molecular and Internal Medicine, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan

*CORRESPONDENCE TO: Shigehiro Doi, M.D., Ph.D. Assistant Professor Department of Nephrology, Hiroshima University Hospital 1-2-3 Kasumi, Minami-Ku, Hiroshima 734-8551, Japan E-mail: [email protected] TEL: +81-82-257-5960 FAX: +81-82-256-5560

Takao Masaki, M.D., Ph.D. Professor Department of Nephrology, Hiroshima University Hospital 1-2-3 Kasumi, Minami-Ku, Hiroshima 734-8551, Japan E-mail: [email protected] TEL: +81-82-257-1506 FAX: +81-82-257-1508

This Supplemental Data includes: Supplemental Table 1 - 2

1

Table 1. Clinical characteristics of IgAN patients eGFR, mL/min/1.73m2 83.0 - 118.0 64.9 - 80.3 40.2 - 64.0 (n = 8) (n = 6) (n = 12) Age, years 28 ± 10 34 ± 9 47 ± 10 Sex, M/F 5/3 4/2 10/2 BMI, kg/m2 21 ± 4.2 21 ± 1.5 24 ± 2.5 Ccr, mL/min 114.6 ± 18.43 82.27 ± 10.74 76.55 ± 19.77 eGFR, mL/min/1.73m2 99 ± 12 74 ± 5 54 ± 7 Urinary protein, g/24 h 0.19 ± 0.12 0.51 ± 0.42 0.99 ± 0.81 Renal fibrosis, % 4.4 ± 2.7 9.9 ± 1.1 15.2 ± 4.4 Values indicate means ± SD BMI, body mass index; Ccr, creatinine clearance; eGFR, estimated glomerular filtration rate

2

Table 2. Clinical characteristics of MN patients eGFR, mL/min/1.73m2 75.0 - 84.5 64.6 - 74.4 48.6 - 63.4 (n = 4) (n = 4) (n = 4) Age, years 66 ± 9 66 ± 7 73 ± 4 Sex, M/F 2/2 3/1 3/1 BMI, kg/m2 22 ± 4.3 23 ± 1.1 24 ± 1.2 Ccr, mL/min 103.9 ± 20.85 89.1 ± 13.06 74.1 ± 9.70 eGFR, mL/min/1.73m2 79 ± 3.5 71 ± 3.8 56 ± 5.4 Urinary protein, g/24 h 4.37 ± 3.15 2.86 ± 1.07 3.35 ± 1.31 Renal fibrosis, % 6.7 ± 1.5 8.2 ± 1.2 8.8 ± 0.5 Values indicate means ± SD BMI, body mass index; Ccr, creatinine clearance; eGFR, estimated glomerular filtration rate

3