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

Abstract

TGF-β1 activity results in methylation of lysine 4 of histone H3 (H3K4) through SET domain-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 therapeutic interventions that target SET7/9. In this study, we investigated the signaling pathway of TGF-β1-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-β1 neutralizing antibody. TGF-β1 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-β1-induced α-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.

Keywords: TGF-β; cell signaling; obstructive nephropathy; renal fibrosis.

Figure 1.

SET7/9 is upregulated in the kidney after obstructive injury. (A) Expression 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 determined 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 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 in the upper panel. Band intensity was normalized to glyceraldehyde 3-phosphate dehydrogenase . Relative levels of SET7/9 expression are shown in the lower panel (n=5 for each group). (C) Immunohistochemical staining for SET7/9 demonstrating the localization of SET7/9 protein in the kidneys. *P<0.05; **P<0.01; ^††P<0.01. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; non-immune, control non-immune serum; Non-op CTL, non-operated normal control; Sham CTL, sham-operated controls.

Figure 2.

TGF-β1 induces SET7/9 expression in renal cells, and injection of neutralizing TGF-β1 antibody reduces SET7/9 expression after UUO. NRK-52E cells and NRK-49F cells were treated with TGF-β1. Representative western blot analysis showing the levels of SET7/9 protein expression in TGF-β1-stimulated NRK-52E cells and NRK-49F cells at (A) various dosages (time; 24 hours) and (B) time points (TGF-β1; 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-β1 antibody (TGF-β1-Ab) at a dose of 1.5 mg/kg/48 hours compared with control IgG₁ at the same dose of TGF-β1-Ab. Original magnification, ×200. **P<0.01. Sham CTL, sham-operated controls; IgG1_, UUO mice with control IgG₁ injection; non-immune, control non-immune serum; TGF-β1-Ab, UUO mice with neutralizing TGF-β1 antibody injection.

Figure 3.

Knockdown of Smad3 in NRK-52E cells inhibits TGF-β1-induced SET7/9 expression. NRK-52E cells were transfected with Smad3 siRNA (si-Smad3) or negative control (si-Neg) oligonucleotides. (A) SET7/9 mRNA levels determined by qRT-PCR in transfected NRK-52E cells with or without TGF-β1 (10 ng/mL, 24 hours). (B) Western blot analysis using SET7/9, (C) Smad3, and (D) phosphorylated Smad3 (p-Smad3) antibodies in transfected NRK-52E cells with or without TGF-β1 (10 ng/mL, 30 minutes or 24 hours). Total cell lysates were subjected to immunoblotting. Because p-Smad3 content peaked at 30 minutes after TGF-β1 stimulation, this time point was used only in p-Smad3 experiments. Quantification is shown in the lower panel (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<0.01. si-Neg, UUO mice with negative control oligonucleotides injection; si-Smad3, UUO mice with Smad3-siRNA injection.

Figure 4.

Knockdown of SET7/9 inhibits UUO-induced fibroblast activation in UUO mice. SET7/9-siRNA injection was performed at a dose of 7 mg/kg via the right ureter. (A) Expression level of SET7/9 mRNA determined by qRT-PCR. Gene expression was normalized to internal control 18S rRNA (n=5 for each group). (B) Representative western blot analysis showing the levels of SET7/9 expression. Quantification is shown in the lower panel (n=5 for each group). (C) Expression levels of α-SMA, and (D) collagen 1 mRNA determined by qRT-PCR. Gene expression was normalized to internal control 18S rRNA (n=5 for each group). (E) Typical results of western blot analysis showing the levels of α-SMA expression. Quantification is shown in the lower panel (n=5 for each group). Data were analyzed by one-way ANOVA followed by Dunnett post hoc test based on UUO mice with negative control oligonucleotides (si-Neg) injection. **P<0.01. Sham CTL, sham-operated controls; si-Neg, UUO mice with negative control oligonucleotides injection; si-SET7/9, UUO mice with SET7/9-siRNA injection.

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 magnification, ×200. The Japanese GFR equation based on serum creatinine was used as an eGFR. eGFR (mL/minute/1.73 m²)=194×Scr^−1.094×Age^−0.287×0.739 (if female). Age, years old; Scr, serum creatinine (mg/dL); non-immune, control non-immune serum.

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 α-SMA and FSP-1 as mesenchymal markers, and collagen 1, collagen 3, and fibronectin as ECM proteins. (A) α-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 demonstrating the levels of α-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 α-SMA, FSP-1, collagen 1, collagen 3, and fibronectin in UUO mice with or without sinefungin injection. (D) Quantification of FSP-1, collagen 1, collagen 3, and fibronectin expression by immunohistochemical staining. (n=5 for each group). (E) TGF-β1 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.

Figure 7.

Sinefungin inhibits UUO-induced upregulation of H3K4me1 in UUO mice concomitantly with the amelioration of renal fibrosis in obstructive nephropathy. The same protein lysates as in Figure 6 were used. (A) Representative western blot analysis showing the levels of H3K4me1, (B) H3K4me2, and (C) H3K4me3 protein expression in UUO mice with or without sinefungin injection (10 mg/kg). Quantification is shown in the right panel (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<0.01. Non-op CTL, non-operated normal controls; Sine, sinefungin; Veh, vehicle.

Figure 8.

Sinefungin ameliorates the TGF-β1-induced increase of α-SMA and inhibits the upregulation of histone H3K4 mono-methylation in renal epithelial cells. Pretreatment of sinefungin (0.5 or 1.0 μg/mL) was conducted 60 minutes before TGF-β1 (10 ng/mL) stimulation. (A) A typical western blot for α-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, and PAI-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.

Figure 9.

Sinefungin ameliorates the TGF-β1-induced increase of α-SMA and inhibits the upregulation of H3K4me1 in renal fibroblast cells. Pretreatment of sinefungin (0.5 or 1.0 μg/mL) was carried out 60 minutes before TGF-β1 (10 ng/mL) stimulation. (A) A typical western blot for α-SMA expression in NRK-49F cells. Quantification is shown in 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. Quantification is shown in the lower panel (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<0.01. Sine, sinefungin.