MiR-34a alleviates podocyte injury in mice with diabetic nephropathy by targeted downregulation of Notch signaling pathway

Abstract

To explore the effects of miR-34a on injury and apoptosis of podocytes in diabetic nephropathy (DN) and the role of Notch signaling pathway in mediating its effects. The expression of miR-34a in podocytes exposed to high glucose (30 mmol/L) was detected using RT-PCR. A podocyte line with miR-34a overexpression was constructed, and the miRNA-target relationship between miR-34a and Notch 1 was verified with luciferase assay. The effects of overexpression of Notch 1 and both miR-34a and Notch 1 on podocyte survival and apoptosis were evaluated using CCK-8 and flow cytometry and by detecting apoptosis-related proteins using Western blotting. In a DN mouse model established by high-fat diet and streptozotocin, the effect of tail vein injection of agomir-34a and agomir-NC on pathology and apoptosis in the renal tissues were observed with HE staining and TUNEL staining, and the renal expressions of apoptosis-related proteins and Notch 1 protein were detected with Western blotting. High glucose exposure significantly lowered miR-34a expression in cultured human podocytes (P < 0.05). The expression of Notch 1 was significantly lowered in miR-34a-overexpressing podocytes as compared with the cells with miR-NC transfection (P < 0.05). Luciferase assay confirmed the mRNA-target relationship between miR-34a and Notch 1 (P < 0.05). MiR-34a overexpression obviously promoted podocyte survival (P < 0.05), reduced Notch 1 expression, and lowered apoptosis rate and the protein expressions of caspase-3, caspase-9 and Bax/Bcl-2 levels in the cells (P < 0.05), while the reverse changes were observed in Notch 1-overexpressing podocytes (P < 0.05). In DN mouse models, treatment with miR-34a obviously alleviated renal pathologies. Compared with that in the control group, the expression level of miR-34a in the renal tissues was significantly lowered in DN model group (P < 0.05) and increased in miR-34a group (P < 0.05). The mice in the model group showed significantly higher apoptosis index of the renal tissues with increased expressions of caspase-3, caspase-9 and Notch 1 (P < 0.05), which were lowered by treatment with miR-34a (P < 0.05). MiR-34a is capable of improving podocyte injury and apoptosis in DN mice by targeted downregulation of Notch 1.