ASPP2调控老年自发性高血压大鼠AngII诱导的肾小球足细胞自噬机制的研究

The injury and loss of glomerular podocytes play an important role in the pathogenesis of hypertensive nephrosclerosis. Previous studies indicate that the abnormality in autophagy pathway of glomerular podocytes is one of the main causes of podocyte lesions. As the level of hypertension intrarenal angiotensin II(AngII) increases, cell experiments show that AngII induces the autophagy of conditionally immortalized mouse podocytes(CIMPs). Our latest results demonstrated that ASPP2, competing with ATG16 to bind ATG5/ATG12, prevents the formation of ATG16/ATG5/ATG12, thereby inhibiting autophagy; we also discovered high ASPP2 expression in the renal tissue of spontaneously hypertensive rats (SHR). Therefore, we hypothesis that the high level of ASPP2 expression in glomerular podocytes during hypertension is likely to decrease AngII-induced autophagy through the above-mentioned pathway, which would maintain cell homeostasis and alleviat podocyte injury. This project uses SHR and CIMPs as its model, and adopts immunoprecipitation, gene transfection, electron microscopy, et al as its methods. It will evaluate the age-related changes in the expression and localization of ASPP2, changes in the level of AngII, and changes in autophagic activity in the renal tissue of SHR; it will also assess the effect of ASPP2 regulation of autophagy and the influence of AngII on ASPP2 function in CIMPs. The results will reveal the new mechanism of hypertensive renal injury.

肾小球足细胞损伤和丢失，在高血压肾硬化发病机制中起重要作用。研究结果显示，足细胞中自噬途径的异常是足细胞病变的主要原因之一；高血压时肾组织局部AngII水平增高，细胞实验显示AngII诱导肾小球足细胞(CIMPs)自噬。我们的结果：ASPP2可通过与ATG16竞争结合ATG5/ATG12，阻止ATG16/ATG5/ATG12复合体形成，抑制自噬；并发现自发性高血压大鼠（SHR）肾组织中ASPP2高表达。因此，我们假设：高血压时肾小球足细胞中ASPP2高表达，有可能通过这一途径下调AngII诱导的自噬，减轻足细胞损伤，维持细胞稳态。本课题以SHR、CIMPs为模型，采用免疫沉淀、基因转染、电镜等方法，揭示随增龄SHR肾组织ASPP2表达及定位的变化、AngII水平的变化、自噬活性的变化，CIMPs中ASPP2对自噬的调控、AngII对ASPP2的影响，课题结果将深入揭示高血压肾损伤新机制。

本课题研究结果显示与正常血压大鼠（WKY）比较，自发性高血压大鼠（SHR）随增龄肾皮质AngⅡ水平明显降低，肾小球AT1R表达明显增多，AT2R表达明显减少；肾小球ASPP2、p53蛋白表达明显减少；肾小球自噬相关蛋白nephrin、LC3BⅡ、Atg5、p62蛋白表达明显减少。与WKY大鼠随增龄肾小球足细胞自噬活性缓慢下降相比较，SHR随增龄肾小球足细胞自噬活性高于正常血压WKY大鼠。ACEI/ARB药物降压的同时，上调了衰老SHR大鼠肾小球自噬相关蛋白nephrin、LC3BⅡ、Atg5、p62蛋白水平的表达。进一步推论，随增龄SHR肾小球AT1R蛋白表达升高，AngⅡ与AT1R结合增强抑制ASPP2蛋白表达，进而上调衰老肾小球足细胞自噬活性，反之ARB竞争抑制AngⅡ与AT1R的结合，下调衰老肾小球足细胞自噬活性、减轻肾损伤。揭示了高血压肾损伤、ACEI/ARB肾脏保护作用的新机制、新靶点。. 在研究AKI发病机制，寻找新的早期、敏感、可靠的急性肾脏损伤（AKI）生物标志物的探索中，以甘油模拟横纹肌溶解致青年大鼠AKI、顺铂模拟顺铂肾毒性致青年大鼠AKI为模型，结果显示甘油、顺铂模型组大鼠SCr、BUN、Cys C显著升高的同时，电镜下观察到肾小管上皮细胞中自噬体出现，肾组织Atg5-Atg12、Beclin1、p62、LAMP-2、p53和ASPP1蛋白表达水平显著升高，iASPP蛋白表达水平显著降低。与青年大鼠比较，老年大鼠顺铂诱导12h后，与Sham组比较，青年与老年大鼠SCr无明显变化，电镜观察到自噬体出现，老年大鼠肾组织Beclin 1、p62、LAMP-2和p53蛋白表达水平显著升高，iASPP蛋白表达水平显著降低，并且老年大鼠肾组织Beclin 1、LAMP-2和p53蛋白变化时间显著早于青年大鼠。揭示AKI发生时，在SCr开始升高前，肾小管上皮细胞内自噬活性出现变化，自噬相关蛋白和ASPPs表达出现显著性变化，且这些变化在老年大鼠出现的更早、更明显。为进一步深入研究自噬相关蛋白和ASPPs作为AKI早期生物标志物提供了动物实验依据。