DNA methyltransferase 1 knockdown reverses PTEN and VDR by mediating demethylation of promoter and protects against renal injuries in hepatitis B virus-associated glomerulonephritis

Abstract

Background: Aberrant DNA methylation patterns, including hypermethylation of key genes that inhibit fibrosis and inflammation, have been described in human kidney diseases. However, the role of DNA methyltransferase 1 (DNMT1) in hepatitis B virus-associated glomerulonephritis (HBV-GN) remains unclear. Methods: We explored the underlying mechanism by establishing HBV X protein (HBx) overexpressing renal tubular epithelial (HK-2) cells and human podocytes with DNMT1 knockdown. Using RNA-sequencing to determine the downstream targets of DNMT1 and evaluate its levels of promoter methylation. HBV transgenic mice were used to examine the effects of DNMT1 inhibitor on renal in vivo. Results: DNMT1 was significantly upregulated in the renal tissue of HBV-GN patients, accompanied by injuries of HK-2 cells and podocytes. HBx markedly upregulated DNMT1 and induced epithelial-mesenchymal transition (EMT) and inflammation in HK-2 cells and human podocytes. This increased DNMT1 expression was attenuated after DNMT1 knockdown, accompanied by restored HK-2 cells and podocyte injuries resulting from the activation of PI3K/Akt/mTOR and nuclear factor-kappa B (NF-κB) pathways. Hypermethylation of the phosphatase and tensin homolog (PTEN) promoter and vitamin D receptor (VDR) was induced in HBx-overexpressing HK-2 cells and podocytes, respectively, whereas DNMT1 knockdown effectively corrected these alterations. Furthermore, PTEN and VDR ablation resulted in marked EMT and inflammation induction in HBx-overexpressing HK-2 cells and human podocytes even with DNMT1 knockdown. Downregulation of the PI3K/Akt/mTOR-related pathway attenuated HBx-induced EMT and inflammation in HK-2 cells. Luciferase reporter assay revealed VDR as a direct target of the Snail family transcriptional repressor 1 (SNAI1) in HBx-overexpressing podocytes. DNA methylation inhibitor 5-azacytidine alleviated urinary protein and renal inflammation in HBV transgenic mice via PTEN-PI3K/Akt signaling and VDR signaling axis. Conclusions: Our study clarifies the potential epigenetic mechanisms underlying HBx-induced renal injuries in HBV-GN and the renoprotective effects of inhibiting DNMT1, which can provide important insights into the development of treatments for HBV-GN.