Li Qiuyue, Zeng Yan, Jiang Qing, Wu Cong, Zhou Jing
Nephrology Department, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China.
Exp Ther Med. 2019 Apr;17(4):2495-2502. doi: 10.3892/etm.2019.7236. Epub 2019 Feb 4.
Podocyte injury, which promotes progressive nephropathy, is considered a key factor in the progression of diabetic nephropathy. The mammalian target of rapamycin (mTOR) signaling cascade controls cell growth, survival and metabolism. The present study investigated the role of mTOR signaling in regulating high glucose (HG)-induced podocyte injury. MTT assay and flow cytometry assay results indicated that HG significantly increased podocyte viability and apoptosis. HG effects on podocytes were suppressed by mTOR complex 1 (mTORC1) inhibitor, rapamycin, and further suppressed by dual mTORC1 and mTORC2 inhibitor, KU0063794, when compared with podocytes that received mannitol treatment. In addition, western blot analysis revealed that the expression levels of Thr-389-phosphorylated p70S6 kinase (p-p70S6K) and phosphorylated Akt (p-Akt) were significantly increased by HG when compared with mannitol treatment. Notably, rapamycin significantly inhibited HG-induced p-p70S6K expression, but did not significantly impact p-Akt expression. However, KU0063794 significantly inhibited the HG-induced p-p70S6K and p-Akt expression levels. Furthermore, the expression of ezrin was significantly reduced by HG when compared with mannitol treatment; however, α-smooth muscle actin (α-SMA) expression was significantly increased. Immunofluorescence analysis on ezrin and α-SMA supported the results of western blot analysis. KU0063794, but not rapamycin, suppressed the effect of HG on the expression levels of ezrin and α-SMA. Thus, it was suggested that the increased activation of mTOR signaling mediated HG-induced podocyte injury. In addition, the present findings suggest that the mTORC1 and mTORC2 signaling pathways may be responsible for the cell viability and apoptosis, and that the mTORC2 pathway could be primarily responsible for the regulation of cytoskeleton-associated proteins.
足细胞损伤会促进进行性肾病，被认为是糖尿病肾病进展的关键因素。雷帕霉素的哺乳动物靶点（mTOR）信号级联控制细胞生长、存活和代谢。本研究调查了mTOR信号在调节高糖（HG）诱导的足细胞损伤中的作用。MTT法和流式细胞术检测结果表明，HG显著增加了足细胞活力和凋亡。与接受甘露醇处理的足细胞相比，mTOR复合物1（mTORC1）抑制剂雷帕霉素抑制了HG对足细胞的作用，而双mTORC1和mTORC2抑制剂KU0063794进一步抑制了该作用。此外，蛋白质印迹分析显示，与甘露醇处理相比，HG显著增加了苏氨酸389磷酸化的p70S6激酶（p-p70S6K）和磷酸化Akt（p-Akt）的表达水平。值得注意的是，雷帕霉素显著抑制HG诱导的p-p70S6K表达，但对p-Akt表达没有显著影响。然而，KU0063794显著抑制HG诱导的p-p70S6K和p-Akt表达水平。此外，与甘露醇处理相比，HG显著降低了埃兹蛋白的表达；然而，α平滑肌肌动蛋白（α-SMA）的表达显著增加。对埃兹蛋白和α-SMA的免疫荧光分析支持了蛋白质印迹分析的结果。KU0063794而非雷帕霉素抑制了HG对埃兹蛋白和α-SMA表达水平的影响。因此，提示mTOR信号激活增加介导了HG诱导的足细胞损伤。此外，本研究结果表明，mTORC1和mTORC2信号通路可能与细胞活力和凋亡有关，且mTORC2通路可能主要负责调节细胞骨架相关蛋白。
