Sirt1介导的线粒体自噬在白蛋白诱导NLRP3炎症小体活化及肾小管损伤中的作用

Renal tubular epithelial cells (RTEC) injury is one of critical events leading to renal interstitial fibrosis. Mitochondrial dysfunction-dependent NLRP3 inflammasome activation initiates RTEC inflammation and injury, mitophagy blocks NLRP3 inflammsome activation by inhibition of mitochondrial dysfunction. Our preliminary data showed that albumin-overload induced proteinuria and renal inflammasome activation in mice, which was prevented by SIRT1 activator Reservatrol. Overexpression of SIRT1 upregulated mitophagy related gene including PINK1 and Parkin expression, and blocked albumin-induced mitochondrial dysfunction in cultured renal proximal tubular epithelial cells. Promoter sequence analysis predicted the FoxO1 binding sites in PINK1 and Parkin promoter. Therefore, we hypothesized that SIRT1/FoxO1 activation increases PINK1 and Parkin expression, which inhibits mitochondrial dysfunction and induces mitophagy, further blocks mitochondrial dysfunction-depdendent NLRP3 inflammasome activation and prevents RTEC from inflammation and injury. To test this hypothesis, we firstly examine the interaction of FoxO1 and the promoters of PINK1 and Parkin gene by chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), and promoter luciferase report system. Furthermore, we detecte the effect of SIRT1 on the mitophagy, mitochondrial dysfunction, and NLRP3 inflammasome activation in RTEC condiational SIRT1 knockout mice and SIRT1 stable transfected renal proximal tubular epithelial cells. Finally, we examine the effect of PINK1 and Parkin on the SIRT1-induced mitophagy and -inhibited mitochondrial dysfunction. This study highlights a new role for SIRT1/FoxO1 in mitochondrial dysfunction-mediated NLRP3 inflammasome activation and RTEC inflammation and injury, reveals a new molecular mechanism underlying SIRT1-induced mitophagy, and could aid in the design of new therapies for the prevention of tubulointerstitial lesions in chonic kidney disease.

肾小管上皮细胞(RTEC)损伤是导致肾脏纤维化的中心环节之一。线粒体功能障碍诱导的炎症小体活化是启动RTEC炎症损伤的重要步骤，线粒体自噬可通过阻断线粒体功能障碍抑制炎症小体活化。前期预实验发现SIRT1激动剂白藜芦醇可减轻白蛋白负荷诱导的蛋白尿和肾组织炎症小体活化；过表达SIRT1可上调线粒体自噬相关基因PINK1和Parkin表达，阻断白蛋白诱导的线粒体功能障碍；启动子分析表明上述基因启动子中含有FoxO1结合位点。由此推测：SIRT1/FoxO1可通过诱导PINK1和Parkin表达，启动线粒体自噬，阻断线粒体功能障碍介导的炎症小体活化。本课题将通过染色质免疫共沉淀和凝胶电泳迁移率等检测FoxO1与上述基因启动子的相互作用；并应用肾小管上皮细胞条件性SIRT1基因敲除小鼠，结合过表达和RNAi等技术，探讨SIRT1对RTEC线粒体自噬、线粒体功能障碍和炎症小体活化的影响及其分子机制。

我们的前期研究发现，线粒体功能障碍是慢性肾脏病的早期事件，线粒体功能障碍诱导的炎症小体活化是启动慢性肾脏病肾组织损伤的重要步骤。但线粒体功能障碍介导肾损伤的机制以及线粒体功能调控的分子机制尚不清楚。我们首先观察了SIRT1、PGC-1α和NLRP3在慢性肾脏病患者肾活检组织中的表达，结果显示，SIRT1和PGC-1α的表达显著下降，而NLRP3炎症小体的活化显著增强，且SIRT1和PGC-1α的表达与蛋白尿和肾组织病理损伤呈负相关，而NLRP3炎症小体与与蛋白尿和肾组织病理损伤呈正相关。随后，我们进一步观察了线粒体在NLRP3炎症小体活化中的作用。在体外培养的肾小管上皮细胞和足细胞中，应用MnTBAP保护线粒体功能，可阻断NLRP3炎症小体活化，并减轻肾小管上皮细胞和足细胞损伤。应用Nlrp3或caspase1基因敲除小鼠制备白蛋白负荷模型，结果显示Nlrp3或caspase1基因敲除能够显著减轻线粒体功能障碍，提示NLRP3炎症小体活化可加重线粒体损伤。我们制备了肾小球足细胞SIRT1基因敲除小鼠，结果显示SIRT1基因敲除显著加重醛固酮诱导的足细胞损伤，线粒体功能障碍和NLRP3炎症小体活化进一步加重。过表达SIRT1或应用SIRT1激动剂白藜芦醇能够保护线粒体功能，抑制NLRP3炎症小体活化，并减轻肾小管上皮细胞和足细胞损伤。我们进一步探讨SIRT1保护线粒体功能的分子机制。在肾小球足细胞SIRT1基因敲除小鼠中，线粒体自噬被显著抑制。体外培养的肾小管上皮细胞和足细胞过表达SIRT1或应用白藜芦醇能显著促进线粒体自噬，保护线粒体功能。在肾小球足细胞SIRT1基因敲除小鼠肾组织、醛固酮刺激的肾小球足细胞以及白蛋白刺激的肾小管上皮细胞中，PINK1/Parkin和BNIP3表达显著下降；过表达SIRT1或应用SIRT1激动剂白藜芦醇能够明显上调PINK1/Parkin和BNIP3表达，促进线粒体自噬，从而保护线粒体功能。过表达BNIP3能够显著促进线粒体自噬，减轻线粒体损伤，从而抑制肾小球足细胞和肾小管上皮细胞凋亡。这些研究结果表明，慢性肾脏病时肾组织中SIRT1表达下降；过表达SIRT1或应用其激动剂可上调PINK1/Parkin和BNIP3表达，促进线粒体自噬，保护线粒体功能，从而抑制NLRP3炎症小体活化，减轻慢性肾脏病肾组织病理损伤。