P66Shc介导糖尿病肾病肾小管上皮细胞线粒体自噬改变的作用及分子机制研究

It has been demonstrated that renal tubulointerstitial lesions is the early and original features of diabetic nephropathy(DN) and is significantly associated with the progression of DN. Mitophagy induced by high glucose has been recently proposed to play a critical role in the pathogenesis of tubulointerstitial injury in DN. We have shown earlier that P66Shc is a master regulator of renal tubular cell injury through mediating mitochondrial ROS production and mitochondrial fragmentation. In addition, excess mitochondrial ROS and mitochondrial fission may stimulate mitophagy activity in the kidney. Whereas under diabetic ambience，the precise role of P66Shc in mitophagy is still unclear, and the cytopathic effects of excessive mitophagy has also not been clarified yet. To explore these processes, in this project we will focus on verifing the effect of P66Shc- mitochondrial ROS and P66Shc- mitochondrial fragmentation on the regulation of mitophagy in tubular cells exposed to high glucose and confirming the role of mitophagy on tubular cell injury, as detection methods by cell molecular biology and STZ induced diabetic model rats. The aim of this subject is to unveil a new mechanism of tubular cell damage mediated by P66Shc, and provide a novel and effective target in fighting DN.

肾小管上皮细胞损伤是糖尿病肾病（diabetic nephropathy,DN）的重要病理特征，与DN进展密切相关。最新研究提示线粒体自噬异常在高葡萄糖诱导肾小管上皮细胞损伤中起重要作用。我们前期研究发现：衔接蛋白P66Shc在调控高葡萄糖诱导的肾小管上皮细胞线粒体ROS生成及线粒体片段化中起重要作用，而细胞内线粒体ROS水平及线粒体片段化改变与线粒体自噬密切相关。但P66Shc介导肾小管上皮细胞线粒体自噬改变的具体调控机制及产生的病理效应尚未阐明。本项目采用细胞分子生物学技术，研究P66Shc-线粒体ROS、P66Shc-线粒体片段化对DN肾小管细胞线粒体自噬的调控作用与分子机制，并探讨线粒体自噬与DN肾小管细胞损伤的关系。确定P66Shc-线粒体ROS/线粒体片段化-线粒体自噬这一新通路在DN小管损伤中的关键作用，旨在阐明DN肾小管细胞损伤的新机制，并为延缓DN进展提供新的干预靶点。

项目背景：肾小管上皮细胞损伤是糖尿病肾病（diabetic nephropathy,DN）的重要病理特征，与DN进展密切相关。最新研究提示线粒体功能紊乱（比如线粒体自噬异常，线粒体异常片段化，线粒体ROS等）在高葡萄糖诱导肾小管上皮细胞损伤中起重要作用。但在DN状态下肾小管上皮细胞内线粒体自噬调控的分子机制及其产生的病理生理效应仍不清楚。本项目重点研究肾小管上皮细胞内线粒体自噬调控及意义。.研究内容：（1）DN肾小管上皮细胞内线粒体自噬/线粒体片段化的改变、调控及其对DN肾小管间质损伤的影响。(2) P66Shc因子在DN肾小管间质损伤中的病理生理效应及其自身表达调控，P66Shc调控线粒体片段化的具体分子机制。.重要结果：DN肾小管上皮细胞内线粒体自噬水平显著降低，外源性使用二甲双胍可能通过cAMPK-PINK1-Parkin通路激活线粒体自噬，抑制线粒体ROS水平，从而抑制炎症复合体NLRP3的激活，最终抑制炎症因子IL-1以及促凋亡蛋白Caspase1的释放，发挥肾脏保护效应。另一方面，外源性的使用线粒体靶向肽SS31可以通过抑制Dp1表达而抑制线粒体片段化从而减轻DN小鼠肾小管间质损伤。在P66Shc与线粒体质量控制方面，我们发现高葡萄糖干预下，P66Shc可以促进Mfn1-Bak交联作用引起线粒体片段化增加，进一步引起线粒体膜电位降低，细胞色素C释放，过量线粒体ROS生成及细胞凋亡，导致DN状态下肾小管间质损伤。在P66Shc表达调控方面，实验结果发现SIRT1可以通过修饰P66Shc基因启动子-508至-250位点的乙酰化水平而调控P66Shc的表达，外源性的抗氧化剂普罗布考干预可以通过SIRT1-AcH3信号通路而抑制P66Shc表达，从而发挥抗炎，抗氧化，抗凋亡的药理学效应。.科学意义：本课题研究了DN肾小管上皮细胞内线粒体自噬的调控，探讨了P66Shc调控线粒体分裂的分子机制，初步明确了线粒体片段化，线粒体自噬及线粒体ROS生成之间存在交错的网络调控关系，但应进一步深入探讨其分子机制。此外本研究发现靶向干预线粒体功能紊乱能有效的延缓糖尿病肾病肾小管间质损伤，这些实验结果能有效的加强基础研究成果向临床应用的转换。