P66Shc在DKD小管细胞中表达调控机制及其对线粒体动力学的影响

Diabetic kidney disease (DKD) is a main cause of end-stage renal disease, and tubular cell injury is one of the early pathological characteristics of DKD. Cellular oxidative injury induced by reactive oxygen species (ROS) overproduction is known to play an key role in the pathogenesis of DKD, thereby investigations of the mechanism of ROS overproduction in diabetic tubular cell injury has been a hot spot of current renal research area. P66shc is a master regulator of cell cycle and oxidative stress through mediating mitochondrial ROS production. We have shown earlier that p66shc expression was significantly increased in the renal tubular cells of DKD,and is critically involved in the tubular oxidative injury of DKD. Whereas under diabetic ambience，the regulatory mechanisms of p66shc gene expression, protein modification and mitochondrial translocation are still unclear, and the precise role of p66shc in mitochondrial dynamics has also not been clarified yet. To explore these processes, the aim of the project is to: 1. Investigate the impact of histone acetylation, DNA methylation within p66shc promoter region and miRNA-197 on its transcriptional or post-transcriptional regulation; 2. Identify the role of PKCδon p66shc phosphorylation and mitochondrial translocation; 3. Examine the effect of p66shc on mitochondrial dynamic changes in renal tubule cells of DKD. The long-term goal is to unveil a new mechanism of tubular oxidative injury mediated by mitochondria in DKD, and provide a novel therapeutic target for the prevention and treatment of DKD.

糖尿病肾病（DKD）是引起ESRD的主要病因，而小管细胞损伤是DKD早期的病理特征之一。ROS过量蓄积导致细胞氧化损伤是DKD肾脏病理变化与发病机制中的关键环节，故研究ROS形成机制是当前肾脏病研究领域的热点之一。p66Shc是介导线粒体产生ROS、参与调控细胞氧化应激的关键蛋白。申请者早期发现：DKD小管细胞p66Shc基因、蛋白表达异常增高，与小管细胞氧化损伤密切相关。但迄今，在DKD状态下p66Shc基因表达调控、蛋白修饰、线粒体转位等机制尚不清；其对线粒体动力学变化的调节作用尚未明。本项目采用细胞分子生物学技术，通过观察组蛋白乙酰化、DNA甲基化、miR-197对P66shc基因表达调控作用与分子机制；研究PKCδ对p66Shc磷酸化、线粒体转位的影响；探讨p66Shc对线粒体动力学变化的调节作用等，旨在阐明DKD小管细胞线粒体介导的氧化损伤新机制，为防治DKD进展提供新的靶分子。

糖尿病肾病是引起ESRD的主要病因，而ROS过量蓄积导致细胞氧化损伤是肾脏病理变化与发病机制中的关键环节，但机制尚不十分清楚。本项目本项目主要围绕以下4个科学问题展开研究：①p66Shc是否可作为糖尿病肾病患者肾脏氧化损伤潜在的生物标记物；②PKCδ是否参与了p66Shc活化和线粒体转位介导高糖诱导的小管细胞氧化损伤；③p66Shc通路介导的线粒体动力学改变促进糖尿病小管细胞损伤和凋亡的分子机制；④普罗布考是否通过抑制p66Shc表达改善糖尿病肾病的氧化损伤。结果：首次证明了PKCδ调控氧化应激蛋白p66Shc激活与线粒体转位的关系，证明高糖通过激活PKCδ调控p66Shc表达与介导线粒体转位，诱导ROS产生参与DN氧化小管损伤机制。证明了p66Shc介导糖尿病状态下小管上皮细胞线粒体动力学变化，激活线粒体凋亡通路等。发现糖尿病肾病（DN）患者异常表达与激活的p66Shc与DN小管细胞病理损伤密切相关，p6Shc可作为DN进展的预测指标与治疗靶点。首次证明抗氧化剂普罗布考通过通过AMPK-SIRT1-AcH3途径及表观遗传学机制抑制p66Shc表达来改善DN的肾脏病氧化损伤与进展。项目阐明了DKD小管细胞线粒体介导的氧化损伤新机制，为防治DKD进展提供了新的靶分子。在本项目资助下，先后在Diabetes，JBC，Scientific Reports等知名刊物发表SCI论文12篇，培养研究生5人，参加国际交流2次。

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