H2S通过诱导Klotho基因启动子去甲基化改善单侧输尿管梗阻（UUO）小鼠肾脏纤维化

H2S ameliorates renal fibrosis in unilateral ureteral obstruction （UUO） mice via inducing demethylation of Klotho promotor

目的探讨硫化氢(hydrogen sulfide,H 2S)改善单侧输尿管梗阻(unilateral ureteral obstruction,UUO)小鼠肾脏纤维化的作用机制。方法采用C57BL/6小鼠单侧输尿管结扎建立肾脏纤维化小鼠模型。随机分为假手术(Sham)组、UUO组和UUO+硫氢化钠(NaHS)组。ELISA法检测血清H 2S浓度;HE染色和Masson染色观察肾脏病理改变;免疫组织化学和Western blot检测Klotho和DNA甲基转移酶1(DNA methyltransferase 1,DNMT1)表达;RT-PCR检测双加氧酶(ten-eleven translocation,TET)表达;比色法检测TET活性;焦磷酸测序检测肾组织Klotho甲基化水平;羟甲基化DNA免疫共沉淀联合实时定量PCR法检测肾组织Klotho羟甲基化水平。结果与Sham组相比,UUO组血清H 2S浓度显著降低[(5.18±0.34)μmol/L vs.(4.23±0.21)μmol/L, P <0.05]。NaHS显著减轻UUO模型小鼠的肾小管间质纤维化( P <0.01),下调α平滑肌肌动蛋白( P <0.05)和纤连蛋白( P <0.05)表达,同时上调UUO小鼠肾组织Klotho表达( P <0.05),下调DNMT1表达( P <0.01),升高TET活性[(0.03±0.01) ng·min^-1·mg^-1 vs.(0.43±0.08) ng·min^-1·mg^-1 , P <0.05],降低 Klotho 基因启动子甲基化水平(11.83%±0.53% vs .7.39%±0.70%, P <0.01),升高 Klotho 基因启动子羟甲基化水平( P <0.05)。结论H 2S可通过增强TET活性,诱导 Klotho 基因启动子去甲基化,上调 Klotho 基因表达,从而减轻UUO小鼠肾脏纤维化。

Objective To explore the effect and possible mechanisms of hydrogen sulfide (H 2S) in alleviating renal fibrosis in mice with unilateral ureteral obstruction (UUO). Methods Renal fibrosis model was established by unilateral ureteral ligation in C57BL/6 mice.Animals were randomly divided into 3 groups: Sham, UUO, and UUO+sodium bisulfate (NaHS). Serum H 2S concentration was measured by ELISA.Renal histological changes were assessed by HE and Masson trichrome staining.Relative expression of proteins such as Klotho and DNA methyltransferase 1 (DNMT1) were determined by immunohistochemistry and Western blot.Relative level of mRNA of ten-eleven translocation (TET) was measured by RT-PCR.Colorimetry method was adopted to detect TET activity.The methylation and hydroxymethylation levels of renal Klotho gene were analyzed by bisulfite pyrosequencing and hydroxymethylated DNA immunoprecipitation-real time quantitative PCR (hMeDIP-qPCR). Results Compared with Sham group,the concentration of serum H 2S significantly decreased in UUO group[(5.18±0.34)μmol/L vs.(4.23±0.21)μmol/L, P <0.05].NaHS treatment could alleviate UUO-induced renal fibrosis,as evidenced by reduced areas of interstitial collagen deposition ( P <0.01) and decreased expression level of α-smooth muscle actin (α-SMA)( P < 0.05 ) and fibronectin( P <0.05).Furthermore,exogenous H 2S administration contributed to the upregulation of Klotho ( P <0.05),downregulation of DNMT1( P <0.01),increased activity of TET[(0.03±0.01) ng·min^-1·mg^-1 vs.(0.43±0.08) ng·min^-1·mg^-1 , P <0.05] and decreased methylation (11.83%±0.53% vs. 7.39%±0.70%, P <0.01) and increased hydroxymethylation ( P <0.05) in Klotho promoter region in kidney tissue following UUO. Conclusions H 2S can ameliorate UUO-induced renal fibrosis by increasing TET activity which can induce the hydroxymethylation of Klotho promotorand upregulate its expression.