Effects of silencing TRPC6 gene on the podocyte autophagy and apoptosis induced by AngII based on siRNA technology

Abstract

Objective To research the effect of silencing transient receptor potential cation channel protein 6 (TRPC6) on autophagy and apoptosis induced by AngⅡ. Methods The small molecule interfering RNA (siRNA) were designed to transfect podocyte and made TRPC6 genes silencing. Flow cytometry, laser confocal, transmission electron microscopy and Western Blot technique were used to detect the different concentrations of siRNA TRPC6 genes after transfection, and optimized for best effect of TRPC6 gene silencing. The mouse glomerular podocytes were cultured in vitro and were setted up the control group, AngⅡ group, empty carrier group, silenting TRPC6 gene group and AngⅡ+ silenting TRPC6 gene group. The control group were cultured with 0.02% DMSO RPMI 1640 nutrient solution; AngⅡ group was joined AngⅡ (10-8 M) to stimulate podocyte; Empty carrier group was joined empty carrier to podocyte; In Silenting TRPC6 gene group, siRNA interference technology was used to silence TRPC6 gene; AngⅡ+ silenting TRPC6 gene group was joined AngⅡ (10-8 M) and silenced TRPC6 gene at the same time. After 12 h, 24 h and 48 h, foot cells of the groups were collected respectively, and detected podocyte apoptosis rates using flow cytometry instrument. TRPC6 and autophagy related protein expression were detected with Western Blot. The autophagy related protein distribution were observated with transmission electron microscopy and laser confocal electron microscope. Results The autophagy expressing quantity was very small in the control group. the apoptosis rate increased significantly in AngⅡ group. Silenting TRPC6 made the podocye apoptosis rate significantly decreasing, and had statistical significance to compared with the control group (P<0.05). Podocyte ultrastructure changed in the AngⅡ group, autophagy expression increased, cytoplasm in independent double membrane structured, cytoplasmic components and lysosome organelles such as double and multilayer membrane structure of autophagy. Silenting TRPC6 decreased the expression of autophagy, and was statistically significant compared with AngⅡ group (P<0.05). Under laser confocal, LC3-Ⅱ expression increased in AngⅡ, silenting TRPC6 gene made the LC3-Ⅱ expression stable, compared with the control group, it was statistically significant (P<0.01). Conclusion AngⅡ promoted podocyte apoptosis, silenting TRPC6 gene can effectively protect podocyte injury which induced by AngⅡ, play the role of protecting. Key words: Podocytes; Transient receptor potential channels; Angiotensin Ⅱ; Autophagy; Apoptosis