ROS promote hyper-methylation of NDRG2 promoters in a DNMTS-dependent manner: Contributes to the progression of renal fibrosis

Abstract

Renal fibrosis is the common histopathological feature of chronic kidney diseases (CKD), and there is increasing evidence that epigenetic regulation is involved in the occurrence and progression of renal fibrosis. N-myc downstream-regulated gene 2 (NDRG2) is significantly down-regulated in renal fibrosis, the mechanism of which remains unclear. Previous studies have confirmed that the inhibition of NDRG2 expression in tumor cells is related to hyper-methylation, mainly regulated by DNA methyltransferases (DNMTS). Herein, we explored the expression of NDRG2 and its epigenetic regulatory mechanism in renal fibrosis. The results showed that the expression of NDRG2 was significantly inhibited in vivo and in vitro, while the overexpression of NDRG2 effectively alleviated renal fibrosis. Meanwhile, we found that the expression of DNMT1/3A/3B was significantly increased in hypoxia-induced HK2 cells and Unilateral Ureteral Obstruction (UUO) mice accompanied by hyper-methylation of the NDGR2 promoter. Methyltransferase inhibitor (5-AZA-dC) corrected the abnormal expression of DNMT1/3A/3B, reduced the methylation level of NDRG2 promoter and restored the expression of NDRG2. The upstream events that mediate changes in NDRG2 methylation were further explored. Reactive oxygen species (ROS) are important epigenetic regulators and have been shown to play a key role in renal injury due to various causes. Accordingly, we further explored whether ROS could induce DNA-epigenetic changes of the expression of NDRG2 and then participated in the development of renal fibrosis. Our results showed that mitochondria-targeted antioxidants (Mito-TEMPO) could reverse the epigenetic inhibition of NDRG2 in a DNMT-sensitive manner, showing strong ability of DNA demethylation, exhibiting epigenetic regulation and anti-fibrosis effects similar to 5-AZA-dC. More importantly, the anti-fibrotic effects of 5-AZA-dC and Mito-TEMPO were eliminated in HK2 cells with NDRG2 knockdown. These findings highlight that targeting ROS-mediated hyper-methylation of NDRG2 promoter is a potentially effective therapeutic strategy for renal fibrosis, which will provide new insights into the treatment of CKD.