Alcohol promotes renal fibrosis by activating Nox-mediated DNA methylation of Smad7 / 中国药理学与毒理学杂志

ABSTRACT

OBJECTIVE Alcohol is mainly metabolized through liver and excreted by kidney in the body. Kidney damage has been considered as the secondary to liver injury and kidney dysfunction is common in hospitalized patients with severe alcoholic hepatitis. Both acute and chronic alcoholism accumulation can compromise kidney function, although alcoholic kidney disease has drawn much more attention recently,the methodology for establishing the in vivo and in vitro alcoholic renal fibrosis models are still lacking,and the underlying mechanisms are to be determined. METHODS and RESULTS Mice were feed with a liquid diet containing alcohol for 4 weeks, 8 weeks and 12 weeks respectively, results of Masson′s Trichrome staining showed that kidney fibrosis peaked in 8-week model group, which consistent with the results of albumin assay,Western blot,immunostaining and real-time PCR of collagen I and α-SMA.In vitro study also confirmed that ethanol upregulated the level of fibrotic index-es,including collagen I and α-SMA,in tubular epithelial cells(HK2 cells).Additionally,both in vivo and in vitro studies showed that Smad7 was decreased and Smad3 was highly activated. Then we further detected the underlying mechanisms by which alcohol induced the imbalance of Smad7 and Smad3. Results of Genome-wide methylation sequencing found DNA methylation of Smad7 in the alcoholic fibrosis kidney,which may be mainly mediated by DNA methyltransferase 1(DNMT1),because knock-down of DNMT1,but not DNMT2 and 3,largely restored Smad7 level in ethanol-treated HK2 cells.Con-sequently, we found that NADPH Oxidases (nox)-mediated oxidative stress is the major force upregu-lating DNMT1,since knockdown of Nox2 and 4 could both decrease DNMT1 while rebalancing Smad7/Smad3 axis, and thereby relieved ethanol-induced fibrotic response in HK2 cells. More importantly, intraperitoneal injection of apocynin,an inhibitor of NADPH oxidoreductase,attenuated renal fibrosis in alcoholic kidney fibrosis mouse model. CONCLUSION By establishing the novel in vivo and in vitro models,we found that through activating oxidative stress-induced DNA methylation of Smad7,alcohol induces renal fibrosis by breaking the balance between Smad7 and Smad3.Elimination of Nox-mediated oxidative stress may be a potential therapy for treatment of long-term alcohol abuse-induced kidney fibrosis.