PEA3：阻断线粒体功能障碍及足细胞损伤的新靶标

Glomerular epithelial cells, podocytes, are highly specialized cells that serve as a crucial component of the glomerular filtration barrier. It is becoming increasingly clear that podocyte injury leads to proteinuria and occurs in many glomerular diseases that finally progress to chronic kidney disease(CKD). Recently, a growing number of studies have shown that mitochondrial dysfunction (MtD) is associated with a range of renal diseases, including acute kidney injury after ischemia-reperfusion (IR) injury, renal Fanconi syndrome and some glomerulopathies. MtD represents a malfunction in biochemical processes, resulting from either its own mitochondrial DNA (mtDNA) or from nuclear DNA. Our previous studies have demonstrated that MtD is not just a key mediator, but is important in early stage processes of podocyte injury. Moreover, we found that overexpression of peroxisome proliferator activated receptor-γ coactivator 1α (PGC-1α), a major regulator of oxidative metabolism and mitochondrial biogenesis, protected against podocyte injury by blocking MtD. However, the underlying regulatory mechanisms of PGC-1α remain far from clear. In a series of preliminary experiments we found that the promoter of PGC-1α gene contains two binding sites for transcription factor Polyomavirus Enhancer Activator 3（PEA3）, which belongs to the PEA3 subfamily of EST transcription factors and is essential for certain organogenesis in mammals. Our preliminary experiments also showed that overexpression of PEA3 could promote PGC-1α expression and block MtD. Therefore, we hypothesize that PEA3 may block MtD to protect podocyte via regulating PGC-1α. To establish our main hypothesis, proposed studies are designed and divided into three sections. First, the molecular mechanism of PEA3 on MtD and podocyte injury will be tested via overexpression and RNAi suppression of PEA3. Second, it will be determined whether PEA3 binds to PGC-1α promoter by using independent Chromosome Immunoprecipitation （ ChIP ） PCR, Electrophoretic Mobility Shift Assay（EMSA）and luciferase reporter system assay. Finally, the effect of PGC-1α repression by specific siRNA will be observed on PEA3-reduced MtD and podocyte injury. These studies are expected to open a new avenue in the understanding of podocyte injury, which may guide us in therapeutic strategies for CKD.

足细胞损伤是慢性肾脏病(CKD)蛋白尿和肾小球硬化的重要病理基础。我们前期研究发现线粒体功能障碍(MtD)介导足细胞损伤，过氧化物酶体增殖物活化受体γ共激活因子-1α（PGC-1α）过表达可通过阻断MtD而减轻足细胞损伤，但PGC-1α表达调控机制尚不清楚。预实验发现PGC-1α启动子区含有两个转录因子PEA3结合位点，过表达PEA3可促进PGC-1α表达，抑制足细胞凋亡。由此推测，PEA3可通过促进PGC-1α表达而阻断MtD，从而保护足细胞。本课题将通过过表达和RNAi等技术，探讨PEA3对MtD及足细胞损伤的影响及其分子机制；在此基础上通过染色质免疫共沉淀、凝胶电泳迁移率和虫荧光素酶报告系统检测PEA3与PGC-1α启动子的相互作用，并通过RNAi特异性抑制PGC-1α，观察其对PEA3减轻MtD和足细胞损伤的影响，为足细胞损伤机制的研究提供新视点，对CKD新药研发具有指导意义。

足细胞损伤是慢性肾脏病（CKD）发生和发展的重要病理基础。对足细胞损伤的病理机制进行深入研究对我们认识CKD的病理机制及发现有效的临床干预靶点具有重要的意义。在本项目，我们提出PEA3可能通过调控PGC-1alpha在足细胞损伤中起到了重要的作用。本研究主要是明确：1）PEA3在足细胞损伤中的调节及作用及对线粒体功能的影响；2）PGC-1alpha在足细胞损伤中的作用及对线粒体功能的影响；3）PEA3对PGC-1alpha的调控作用。 我们的研究结果显示：1）：PEA3在足细胞损伤中起到了保护作用，不但抑制了细胞凋亡的发生，还改善了足细胞的线粒体功能；2）PGC-1alpha也在足细胞的病理损伤及其线粒体功能障碍过程起到了保护作用；3）经荧光素酶报告基因检测发现，PEA3可以直接和PGC-1alpha的启动子结合，进而促进了PGC-1alpha的表达。本研究显示PEA3/PGC-1alpha通路是干预足细胞损伤及CKD的有益靶点。