Girdin蛋白下调经Akt-GSK3β途径损伤线粒体致足细胞凋亡

Podocyte injury is a common feature of chronic kidney disease and is a central initiating factor contributed to the progression of this kind of diseases. Apoptosis is one major approach of podocyte injury. It is known that mitochondrion damage can lead to cell apoptosis. Until now, very little was known about the molecular mechanisms that were believed to be involved in mitochondrion damage. It was reported that over-expression of cytoskeleton-associated protein Girdin can lead to elevated activity of Akt and then protect podocyte against apoptosis, whereas low-expression of Girdin lead to increased apoptosis. It was also known in liver cancer cells that high level of Girdin could activate Akt followed by a decreased activity of GSK3β, which is the downstream molecular of Akt. In our previous research, we found that Girdin in podocyte was decreased in adriamycin-induced podocyte injury model. We also found that the number of deformed mitochondria increased when Girdin was knocked out in some tumor cell lines. The above clues suggested that downregulation of Girdin can lead to podocyte apoptosis by damaging mitochondria through the way of Akt/GSK3β. In this study, we will explore the corresponding molecular mechanism by analyzing the expression of Girdin, the variation of the shape and function of mitochondria, and the ratio of apoptotic podocyte and further by altering the level of Girdin in podocyte. The results of this study will provide a new understanding for the pathogenesis of podocyte injury.

足细胞损伤是慢性肾脏病的共同特征及病程进展的中心始动因素，细胞凋亡是其重要途径,线粒体损伤则是导致足细胞凋亡的主要因素之一，但目前对线粒体损伤的分子机制所知有限。已知足细胞中过表达细胞骨架相关蛋白Girdin会引起Akt活性升高，保护细胞免于凋亡，Girdin下降则效应相反；对肝癌细胞系的研究发现，过表达Girdin在活化Akt的基础上致其下游GSK3β活性下降。由于申请人前期发现阿霉素大鼠足细胞Girdin表达下降，同时在肿瘤细胞中发现Girdin下降可致线粒体损伤，因此我们认为Girdin下降通过Akt/GSK3β途径引起线粒体损伤致足细胞凋亡。本研究将通过培养的足细胞和动物模型分析Girdin表达情况、足细胞线粒体形态和功能变化及足细胞凋亡间的关系，进而改变足细胞Girdin水平来验证其通过Akt/GSK3β途径损伤线粒体的机制。本研究结果能为深入了解足细胞损伤的分子机制提供证据。

慢性肾脏病是中国发病率高、严重威胁患者生活质量和身体健康的一大类疾病。足细胞损伤是慢性肾脏病的共同特征及病程进展的中心始动因素，细胞凋亡是其重要途径,阐明导致足细胞凋亡的机制是指导临床治疗和预后的重要手段。Girdin是骨架蛋白分子，也是细胞内重要的信号转导分子，它位于蛋白酪氨酸激酶与 G 蛋白两条信号通路的交叉点，多种生长因子包括IGF-1、VEGF 以及 EGF 等可通过 PI3K-Akt-Girdin 通路调节细胞的生长、移行等。有证据表明，PI3K/Akt信号系统可以通过mTOR的活化调节自噬。同时，Girdin也参与自噬的调节过程。本研究在对培养的足细胞经阿霉素刺激后其超微结构的观察中发现，随着阿霉素作用时间的延长，足细胞的自噬水平明显升高。因此，本研究拟通过对Akt、mTOR、Girdin、LC3B等蛋白表达情况及相互之间关系的分析，探讨足细胞调亡的发生机制。研究结果显示，随着阿霉素作用时间的延长，细胞早期凋亡和晚期凋亡的发生率均显著升高。同时，随着药物作用时间的延长，足细胞中Girdin的表达水平逐渐升高，Akt和mTOR表达在各组中无显著差别，而pAkt虽在阿霉素短时作用后无明显变化，但随着作用时间的延长，其表达明显增加，而p-mTOR呈现先增加后下降的趋势，Pro-Caspase、Active-Caspase及Caspase在药物长时间作用后表达量显著增加，而在对照组、6h组、12h组均未见表达。同时，自噬相关蛋白LC3B-Ⅱ随药物作用时间延长表达量增加。综上提示，在阿霉素所致足细胞损伤过程中，Girdin表达上调引起Akt和mTOR磷酸化水平的升高，同时自噬水平显著升高最终引起细胞凋亡的增加。本实验为了解足细胞损伤时发生凋亡的分子机制提供了新的证据，对于进一步了解肾脏病的发生有指导意义。