Sirt3 promotes the autophagy of HK-2 human proximal tubular epithelial cells via the inhibition of Notch-1/Hes-1 signaling

Diabetic nephropathy (DN) is a predominant cause of end-stage renal disease. The impairment of the autophagy of human renal tubular epithelial cells (HK-2 cells) is involved in the pathogenic mechanisms of DN. Sirtuin (Sirt)3 regulates the scavenging of damaged organelles and maintains energy balance. The present study aimed to examine the protective effects of Sirt3 on HK-2 cells stimulated by high glucose (HG). HK-2 cells were cultured in normal glucose (NG), HG or hyperosmotic medium. The viability of the HK-2 cells was detected using a Cell Counting Kit-8 assay. The expression and localization of Sirt3 were detected via immunofluorescence. Following transfection with an overexpression plasmid, the expression levels of key components in the Notch homolog 1 (Notch-1)/hairy and enhancer of split-1 (Hes-1) pathway and those of the autophagy-related proteins, Beclin-1, LC-3II and p62, were measured by western blot analysis and reverse transcription-quantitative PCR (RT-qPCR). As the Notch-1/Hes-1 pathway was inhibited, the expression levels of Beclin-1, LC-3II and p62 were also examined at transcriptional and translational level. It was found that prolonged culture in HG medium markedly reduced cell viability compared with the cells cultured in NG or in NG + mannitol, an effect that was aggravated with the increasing duration of culture. HG was capable of inhibiting the expression levels of Beclin-1, LC-3II and Sirt3, and upregulating p62 and the Notch-1/Hes-1 pathway, as verified by western blot analysis and RT-qPCR. The results of immunofluorescence staining revealed that HG decreased Sirt3 expression. Sirt3 reversed the HG-induced inhibition of the expression of Beclin-1 and LC-3II and the upregulation of p62. Moreover, Sirt3 reversed the HG-induced inhibition of the Notch-1/Hes-1 signaling pathway. However, this autophagy-promoting effect of Sirt3 was counteracted by the Notch-1/Hes-1 pathway activator. On the whole, the present study demonstrated that Sirt3 promoted the autophagy of HK-2 cells, at least partly, via the downregulation of Notch-1/Hes-1.