线粒体自噬在糖尿病肾小管损伤和间质纤维化中的作用和分子机制

Diabetic kidney disease (DKD) is the main cause of end stage renal disease (ESRD). It is suggested that tubular damage and interstitial fibrosis are the early and original features of ESRD and are closely associated to the progress of DKD. Increased Mitochondrial ROS can lead to cellular oxidative damage, which is very important to the pathogenesis of DKD. Mitophagy has been recently proposed to play a critical role in regulating intracellular oxidative damage through removal of the damaged mitochondria with decreasing ROS production. We found that mitochondria morphological change and dysfunction are contributed to ROS overproduction and apoptosis of tubule cells in DKD. However, whether mitophagy involved in this process and by which factor regulation of the mitophagy are still unknown. In this project we will focus on studing the relationship between mitophagy and ROS production under DKD condition, verify the effect of PINK-Parkin, Nix/Bnip3 and autophagy regulation protein Atg7 on the mitophagy of tubular cells exposed to high glucose and Angiotensin II，confirm the role of mitophagy on tubular damage and interstitial fibrosis, as detection methods by cell molecular biology and Agt7 condition knockout mouse. On the other hand, we also study the role of mitophagy-ROS-NLRP3 pathway in the tubular cell injury and fibrosis of DKD. The aim of the subject is to illustrate a new mechanism of tubular cell damage mediated by mitochondrial oxidative damage as well as provide a new theory in the pathogenesis and therapy targets of DKD.

糖尿病肾病（DKD）是引起ESRD的主要病因，小管损伤和间质纤维化是其早期和原始特征，与本病进展密切相关。线粒体ROS增多，导致氧化损伤在DKD发病中起核心作用。最新发现，线粒体自噬是清除病变线粒体，降低ROS生成、调节细胞氧化损伤的关键。我们以往证明：DKD小管细胞线粒体形态和功能异常、ROS增加与小管凋亡相关。但线粒体自噬在其中的作用与调控机制尚不明。本项目采用细胞分子生物学技术与转基因动物，观察DKD小管细胞线粒体自噬与ROS的关系；研究PINK-Parkin、Nix/Bnip3、Atg7对DKD(高糖、Ang II)小管细胞线粒体自噬的影响；探讨线粒体自噬在DKD小管损伤和间质纤维化中的作用与分子机制；确立线粒体自噬-ROS-NLRP3这一新信号通路在DKD小管损伤及间质纤维化中的关键作用，旨在阐明DKD小管细胞线粒体介导的氧化损伤新机制，为DKD发病机制研究提供新思路与防治靶点。

糖尿病肾病（DKD）是引起 ESRD 的主要病因，发病机制复杂，目前尚不完全清楚。近年认为：肾小管损伤是DKD的早期和原始特征，较肾小球相比，其与肾功能恶化更为密切。在本项目的资助下，我们围绕线粒体自噬在DKD小管损伤及间质纤维化中的作用与机制展开了系列研究，主要包括观察DKD状态下小管细胞mitophagy及小管损伤、间质纤维化情况，分析mitophagy与小管凋亡、间质纤维化的关系；探讨高糖对小管细胞mitophagy的影响及相关信号通路与分子机制；分析高糖诱导的小管细胞mitophagy变化与ROS形成、线粒体片段化的关系；体内研究mitophagy对DKD小管损伤和间质纤维化的影响。重点探讨线粒体靶向抗氧化剂MitoQ对DKD状态下小管细胞线粒体自噬及损伤的影响与机制。结果发现2型DKD患者肾组织、STZ诱导的1型DKD小鼠模型和db/db 2型DKD小鼠模型肾组织小管及高糖诱导的肾小管上皮细胞中均存在线粒体自噬障碍，并与小管损伤、间质纤维化呈现负相关，线粒体自噬抑制剂（3-MA）可加重小管细胞线粒体损伤与凋亡，表现为线粒体膜电位下降、ROS生成和片段化增加等。线粒体自噬相关调控信号PINK/Parkin、Nix、ATG7等可能参与调节高糖诱导的小管细胞线粒体自噬。研究表明， MitQ可明显减轻DKD状态下肾小管上皮细胞凋亡和损伤，并可回复高糖诱导的HK-2细胞Nrf2表达、核转位及活性，促进PINK1转录增加，Parkin磷酸化，回复小管细胞线粒体自噬障碍，改善线粒体形态和功能异常以及细胞凋亡,提示Nrf2/PINK1是参与MitQ对DKD肾小管保护的重要信号分子。另外，db/db小鼠肾组织中NLRP3/IL1β炎症体表达增加，mitoQ可抑制高糖状态下肾小管细胞炎症体的表达。通过项目资助，我们尚发现，小分子GTPase酶Rab1b可能通过C/EBP-b–PGC-1a减轻DKD肾小管线粒体异常与损伤；同时， P66Shc可能是DKD患者小管氧化损伤的一个新型生物标记物。这些研究有望为DKD发病机制提供新的理论基础与防治分子靶点。在本项目资助下，先后在Diabetes、Redox Biology等发表SCI论文11篇，中华肾脏病杂志6篇，获得第十一届湖南医学科技奖一等奖和湖南省医学优秀学术论文奖一等奖，培养研究生6名，参与国外交流2次。