Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand.
Sci Rep. 2020 Apr 3;10(1):5843. doi: 10.1038/s41598-020-62665-w.
Mitochondrial dysfunction has been thought to play roles in the pathogenesis of diabetic nephropathy (DN). However, precise mechanisms underlying mitochondrial dysfunction in DN remained unclear. Herein, mitochondria were isolated from renal tubular cells after exposure to normal glucose (5.5 mM glucose), high glucose (25 mM glucose), or osmotic control (5.5 mM glucose + 19.5 mM mannitol) for 96 h. Comparative proteomic analysis revealed six differentially expressed proteins among groups that were subsequently identified by tandem mass spectrometry (nanoLC-ESI-ETD MS/MS) and confirmed by Western blotting. Several various types of post-translational modifications (PTMs) were identified in all of these identified proteins. Interestingly, phosphorylation and oxidation were most abundant in mitochondrial proteins whose levels were exclusively increased in high glucose condition. The high glucose-induced increases in phosphorylation and oxidation of mitochondrial proteins were successfully confirmed by various assays including MS/MS analyses. Moreover, high glucose also increased levels of phosphorylated ezrin, intracellular ATP and ROS, all of which could be abolished by a p38 MAPK inhibitor (SB239063), implicating a role of p38 MAPK-mediated phosphorylation in high glucose-induced mitochondrial dysfunction. These data indicate that phosphorylation and oxidation of mitochondrial proteins are, at least in part, involved in mitochondrial dysfunction in renal tubular cells during DN.
线粒体功能障碍被认为在糖尿病肾病 (DN) 的发病机制中起作用。然而，DN 中线粒体功能障碍的确切机制仍不清楚。在此，将肾管状细胞在正常葡萄糖（5.5mmol/L 葡萄糖）、高葡萄糖（25mmol/L 葡萄糖）或渗透控制（5.5mmol/L 葡萄糖+19.5mmol/L 甘露醇）条件下孵育 96 小时后分离出线粒体。比较蛋白质组学分析显示，在这三组中存在 6 种差异表达蛋白，随后通过串联质谱（nanoLC-ESI-ETD MS/MS）鉴定，并通过 Western blot 进行验证。在所有这些鉴定出的蛋白中都发现了几种不同类型的翻译后修饰（PTMs）。有趣的是，在仅在高葡萄糖条件下增加的线粒体蛋白中，磷酸化和氧化最为丰富。通过包括 MS/MS 分析在内的各种检测方法成功地证实了高葡萄糖诱导的线粒体蛋白磷酸化和氧化增加。此外，高葡萄糖还增加了磷酸化 ezrin、细胞内 ATP 和 ROS 的水平，这些都可以被 p38 MAPK 抑制剂（SB239063）消除，表明 p38 MAPK 介导的磷酸化在高葡萄糖诱导的线粒体功能障碍中起作用。这些数据表明，线粒体蛋白的磷酸化和氧化至少部分参与了 DN 期间肾小管细胞中线粒体功能障碍。
