Lei Jie, Zhao Lei, Zhang Yujing, Wu Yanfeng, Liu Yanbo
Department of Pediatrics, the First Hospital of Jilin University, Changchun, China.
Department of Pediatrics, the Second Affiliate Hospital of Harbin Medical University, Harbin, China.
Cell Physiol Biochem. 2018;45(6):2431-2443. doi: 10.1159/000488231. Epub 2018 Mar 15.
BACKGROUND/AIMS: The mechanisms by which high glucose (HG) results in podocyte damage remains unclear. We investigated the potential role of endoplasmic reticulum (ER) stress and mTOR signaling in HG injured podocyte.
In cultured mouse podocytes, cellular apoptosis was assessed using FITC-Annexin V and propidium iodide staining followed by flow cytometry analysis. Apoptosis-related proteins as well as the ER stress and the mTOR signals were evaluated using immunoblot assay.
Compared to normal glucose (NG) and osmotic mannitol (MN) control, the percentage of apoptotic cells was increased significantly in HG-treated podocytes. The levels of CHOP, Grp78, phospho-PERKThr982, and caspase-12 were increased significantly following HG treatment. The downstream effects of ER stress were obtained in HG-treated podocytes, showing upregulation of Bax, Bak and cytochrome c, and downregulation of Bcl-2. HG-induced increase of cytochrome c, Bax and active caspase-3 was prevented by both ER inhibitor sodium 4-phenylbultyrate (PBA) and CHOP siRNA (siCHOP). PBA and CHOP knockdown remarkably decreased HG-induced apoptosis. In addition, the levels of phospho-mTORSer2448 and phospho- p70S6kThr389 as well as phospho-AMPKα (a sensor of energy consumption) were increased significantly in HG-treated cells. Moreover, the Erk inhibitor U0126 prevented HG-induced mTOR activation. Increased phospho-AMPKα, CHOP and Grp78 as well as cellular apoptosis were prevented by mTOR inhibitor rapamycin in HG-treated podocytes.
Our data demonstrate that the activated mTOR by Erk1/2 results in energy consumption, which in turn leads to ER stress signaling and thus induces apoptosis in HG-treated podocytes.
背景/目的：高糖（HG）导致足细胞损伤的机制尚不清楚。我们研究了内质网（ER）应激和mTOR信号在HG损伤足细胞中的潜在作用。
在培养的小鼠足细胞中，使用FITC-Annexin V和碘化丙啶染色，然后进行流式细胞术分析来评估细胞凋亡。使用免疫印迹法评估凋亡相关蛋白以及ER应激和mTOR信号。
与正常葡萄糖（NG）和渗透性甘露醇（MN）对照相比，HG处理的足细胞中凋亡细胞的百分比显著增加。HG处理后，CHOP、Grp78、磷酸化-PERKThr982和半胱天冬酶-12的水平显著增加。在HG处理的足细胞中获得了ER应激的下游效应，表现为Bax、Bak和细胞色素c上调，Bcl-2下调。ER抑制剂4-苯基丁酸钠（PBA）和CHOP siRNA（siCHOP）均可阻止HG诱导的细胞色素c、Bax和活性半胱天冬酶-3增加。PBA和CHOP敲低显著降低了HG诱导的细胞凋亡。此外，HG处理的细胞中磷酸化-mTORSer2448、磷酸化-p70S6kThr389以及磷酸化-AMPKα（能量消耗传感器）的水平显著增加。此外，Erk抑制剂U0126可阻止HG诱导的mTOR激活。mTOR抑制剂雷帕霉素可阻止HG处理的足细胞中磷酸化-AMPKα、CHOP和Grp78增加以及细胞凋亡。
我们的数据表明，Erk1/2激活的mTOR导致能量消耗，进而导致ER应激信号传导，从而诱导HG处理的足细胞凋亡。
