Zeng Yan, Xiong Changbin, Chen Yinxiang, Yang Chunyun, Li Qiuyue
Department of Nephrology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China.
Exp Ther Med. 2023 Aug 22;26(4):473. doi: 10.3892/etm.2023.12172. eCollection 2023 Oct.
Rapamycin-insensitive companion of mTOR (Rictor) is a critical effector of mTOR protein complex 2 (mTORC2). The aim of the present study was to investigate the effect of Rictor in the mTORC2 signaling pathway in high glucose (HG)-induced diabetic podocyte injury by silencing the expression of Rictor. In the present study, mouse podocytes were treated with glucose (150 mM) and mannitol (200 mM), the gene was silenced using small interfering RNA (siRNA). Apoptosis was detected by flow cytometry, whereas podocyte cytoskeletal protein expression was detected by western blotting (WB) and immunofluorescence staining. The results demonstrated that, compared with that in the control group, the podocyte apoptotic rate was significantly increased in the mannitol group (negative group) and the groups that were treated with glucose (model groups). The podocyte apoptotic rate in the model + siRNA group was significantly decreased compared with that in the negative, model and the model glucose + siRNA negative control (NC) groups. WB indicated that the protein expression levels of podocalyxin and synaptopodin were reduced in the model and model + siRNA NC groups compared with those in the normal control and negative groups. Additionally, the protein expression levels of α-smooth muscle actin (α-SMA) and P-AKT/AKT were increased in the model and model + siRNA NC groups compared with the those in control and negative groups. Compared with those the model and model + siRNA NC groups, the protein expression levels of podocalyxin and synaptopodin were increased, whilst those of the α-SMA and P-AKT/AKT proteins were decreased, in the model + Rictor siRNA group. Results from immunofluorescence analysis were basically consistent with those of WB. Therefore, results of the present study suggest that silencing of the gene may reduce the damage to podocytes induced by HG, such that the Rictor/mTORC2 signaling pathway may be involved in the remodeling of podocyte actin cytoskeletal in diabetes.
雷帕霉素不敏感的mTOR伴侣蛋白（Rictor）是mTOR蛋白复合物2（mTORC2）的关键效应因子。本研究旨在通过沉默Rictor的表达，探讨其在高糖（HG）诱导的糖尿病足细胞损伤的mTORC2信号通路中的作用。在本研究中，小鼠足细胞用葡萄糖（150 mM）和甘露醇（200 mM）处理，使用小干扰RNA（siRNA）沉默该基因。通过流式细胞术检测细胞凋亡，而通过蛋白质印迹法（WB）和免疫荧光染色检测足细胞细胞骨架蛋白表达。结果表明，与对照组相比，甘露醇组（阴性组）和葡萄糖处理组（模型组）足细胞凋亡率显著增加。与阴性、模型及模型葡萄糖+siRNA阴性对照（NC）组相比，模型+siRNA组足细胞凋亡率显著降低。WB显示，与正常对照和阴性组相比，模型组和模型+siRNA NC组中足萼蛋白和突触足蛋白的蛋白表达水平降低。此外，与对照和阴性组相比，模型组和模型+siRNA NC组中α平滑肌肌动蛋白（α-SMA）和磷酸化AKT/AKT的蛋白表达水平增加。与模型组和模型+siRNA NC组相比，模型+Rictor siRNA组中足萼蛋白和突触足蛋白的蛋白表达水平增加，而α-SMA和磷酸化AKT/AKT蛋白水平降低。免疫荧光分析结果与WB结果基本一致。因此，本研究结果表明，沉默该基因可能减轻HG诱导的足细胞损伤，提示Rictor/mTORC2信号通路可能参与糖尿病足细胞肌动蛋白细胞骨架重塑。
