PEA3通过诱导线粒体自噬阻断NLRP3炎症小体活化和肾小管上皮细胞损伤

Renal tubular epithelial cells (RTEC) injury is one of the 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 previous study showed that aldosterone induced proteinuria and renal inflammasome activation in wild-type mice, which was prevented in PEA3 transgenic mice. Overexpression of PEA3 upregulated mitophagy related gene including PINK1 and Parkin and mitochondrial anti-oxidant gene MnSOD expression, and blocked aldosterone-induced mitochondrial dysfunction in cultured human renal tubular epithelial cells HK-2. Promoter analysis predicted the PEA3 binding sites in MnSOD, PINK1, and Parkin promoter. Therefore, we hypothesized that PEA3 increases MnSOD, PINK1, and Parkin expression, which inhibits mitochondrial oxidative stress and induces mitophagy, further blocks mitochondrial dysfunction-depdendent NLRP3 inflammasome activation and prevents RTEC from inflammation and injury. To test this hypothesis, we firstly examined the interaction of PEA3 and the promoters of MnSOD, PINK1, and Parkin gene by chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), and promoter luciferase report system. Furthermore, we detected the effect of PEA3 on the mitophagy, mitochondrial dysfunction, and NLRP3 inflammasome activation in PEA3 transgenic mice and PEA3 stable transfected HK-2 cells. Finally, we examined the effect of MnSOD, PINK1, and Parkin on the PEA3-induced mitophagy and -inhibited mitochondrial oxidative stress and mitochondrial dysfunction. This study highlights a new role for PEA3 in mitochondrial dysfunction-mediated NLRP3 inflammasome activation and RTEC inflammation and injury, reveals a new molecular mechanism underlying PEA3-induced mitophagy and anti-oxidant effects, 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PEA3转基因小鼠可减轻醛固酮诱导的蛋白尿和肾组织炎症小体活化；过表达PEA3可上调线粒体自噬相关基因PINK1和Parkin及线粒体抗氧化基因MnSOD，阻断醛固酮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PEA3结合位点。由此推测：PEA3可通过诱导MnSOD、PINK1和Parkin表达，抑制线粒体氧化应激，促进线粒体自噬，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PEA3与上述基因启动子的相互作用；并应用PEA3转基因小鼠，结合过表达和RNAi等技术，探讨PEA3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。

肾小管上皮细胞损伤是导致肾脏纤维化的中心环节之一。我们前期研究发现线粒体功能障碍在肾小管上皮细胞损伤和肾脏纤维化中发挥重要作用，PEA3能够阻断肾小管上皮细胞表型转化。本研究将探讨PEA3对线粒体功能的调控及其作用机制。我们研究发现PEA3在UUO小鼠模型、庆大霉素大鼠模型、阿霉素肾病模型、醛固酮灌注小鼠模型、顺铂诱导的急性肾损伤小鼠模型及人类慢性肾脏病肾组织中的表达均显著增加。体外培养的肾小管上皮细胞中，TGF-β1或白蛋白刺激可诱导肾小管上皮细胞表型转化，而PEA3预处理则阻断了TGF-β1或白蛋白诱导的细胞表型转化，并保护线粒体功能，恢复线粒体膜电位和线粒体DNA拷贝数，降低ROS产生。PEA3转基因小鼠明显降低醛固酮灌注导致的蛋白尿，减轻肾小管上皮细胞凋亡，同时保护线粒体功能。PEA3转基因小鼠肾皮质及PEA3稳定过表达的肾小管上皮细胞中MnSOD、PINK1和Parkin表达显著增加。这些研究结果提示PEA3可通过上调MnSOD、PINK1和Parkin表达保护线粒体功能，减轻肾损伤。我们发现PEA3过表达上调PGC1-α表达，敲低PGC1-α则阻断了PEA3对线粒体功能的保护作用。过表达PGC1-α可阻断醛固酮诱导的线粒体功能障碍。PGC1-α转基因小鼠亦保护线粒体功能，减轻醛固酮诱导肾损伤，并降低蛋白尿。NLRP3炎症小体在线粒体功能调控中起着重要作用。CKD患儿肾组织NLRP3表达显著增加，并与蛋白尿呈正相关。白蛋白呈剂量及时间依赖性地诱导NLRP3活化。NLRP3 siRNA阻断白蛋白诱导的肾小管损伤，减轻细胞凋亡及表型转化。体内实验显示，NLRP3基因敲除的白蛋白负荷小鼠肾小管损伤较野生型小鼠明显好转，肾小管细胞凋亡显著减少。应用MnTBAP保护线粒体功能可阻断白蛋白诱导的NLRP3炎症小体活化。NLRP3及caspase1基因敲除小鼠，给予白蛋白负荷后，与野生型白蛋白负荷组比较，线粒体功能障碍明显好转，肾小管细胞凋亡显著减轻。在稳定过表达PEA3的肾小管上皮细胞以及PEA3转基因小鼠中，白蛋白诱导的NLRP3炎症小体活化能够被明显阻断。这些研究结果表明，PEA3可通过上调PGC1-α、MnSOD、PINK1和Parkin表达，保护线粒体功能，阻断炎症小体活化，减轻肾小管上皮细胞损伤。