#2053 FTO-mediated m6A demethylation inhibits the senescence of renal endothelial cells in diabetic nephropathy

Abstract Background and Aims Diabetic nephropathy (DN) is one of the common microvascular complications of diabetes mellitus, and has become the leading cause of end-stage renal disease. The stress-induced senescence of Endothelial cell is an important pathological basis for the occurrence and development of DN. FTO is a demethylase that can specifically remove the m6A modification on RNA, thereby affecting mRNA metabolism and regulating non-coding RNA. The role of FTO in DN in regulating endothelial cell senescence through m6A modification has not yet been studied. Methods The levels of m6A and m6A methylation-related proteins were assessed by immunohistochemistry in renal tissue samples from DN patients. In the kidney tissues of db/db mouse, m6A methylation related proteins were co-stained with endothelial cell markers by immunofluorescence. Human umbilical vein endothelial cells (HUVEC) with high glucose and high lipid (HG+PA) stimulation were used to simulate the establishment of an in vitro diabetic model. In this vitro HUVEC model, the senescence level was evaluated by SA-β-gal staining. And the expression of senescence biomarkers such as p21, p16, and lambin1 was evaluated by qRT-PCR and WB. Furthermore, the methylation level of HUVEC cells was detected by dot blot. The expression of m6A methylation related proteins was examinated by qRT-PCR, WB, and immunohistochemistry. Finally, FTO knockdown or FTO overexpression in HUVEC cell lines were constructed in vitro, and the effects of FTO on the senescence of HUVEC cells under HG+PA stimulation were analyzed by qRT-PCR, WB, and immunofluorescence staining. Results Immunohistochemical staining of kidney tissue of DN patients showed that m6A methylation level tends to be up-regulated, while the expression of FTO is down-regulated, with no significant changes in the expression of METT3, METTL14. The immunofluorescence staining of kidney tissues in db/db mice showed that FTO was co-located with CD31, a marker of renal vascular endothelial cells, and FTO also was co-located with p16.This finding indicates that the downregulation of FTO expression is related to the senescence of endothelial cells. Under the stimulation of HG+PA, the expression levels of SA-β-gal, p21, and p16 increased, and the expression of lambin1 decreased in HUVEC cells. In addition, dot blot results showed that m6A methylation levels were significantly up-regulated, which suggested that the increase of m6A methylation level is associated with the significant downregulation of FTO. In vitro, both qRT-PCR and WB results showed that FTO knockdown promoted the senescence of HUVEC cells and FTO overexpression significantly reversed induced cell senescence under diabetic condition. Conclusion This study showed that the expression of FTO in endothelial cells of DN was inhibited, leading to the upregulation of m6A methylation level, which promoted the senescence of endothelial cells. In vitro experiments showed that overexpression of FTO can inhibit the expression of senescence genes and reduce endothelial cell damage. Therefore, FTO can be used as a marker to inhibit endothelial cell senescence and alleviate DN progression.