PTEN/YAP介导的SR-A反馈调节在脂质肾脏损伤中的作用

Dyslipidemia promotes progression of chronic kidney disease (CKD) by causing inflammatory, oxidative and ER stress. Our previous study found that scavenger recepor A (SR-A), which was up-regulated by the increased level of cytolipids, accelerated progressive nephropathy in high fat diet (HDF) mice by increasing the lipid-uptaking and inflammation. However, the exact mechanism of this postive feedback ragulation remains unclear. Recently, our preliminary data suggested that partailly deletion of PTEN markedly deteriorated the glomerulosclerosis by increaseing the podocyte apoptosis in lipid-induced nephropathy mice model. And this processe was attributed to the activation of Hippo-YAP pathway. Based on our novel observations and the several line of studies, which reported that YAP signaling pathway has emerged as a major regulator of cell proliferation and survival in metazoans, we argue that PTEN/YAP plays a critical role in the progressive nephropathy induced by HFD via its involvement in the up-regulation of SR-A. We now propose an integrated in vitro and in vivo approach to test the hypothesis that the inhibiting PTEN will increase SR-A expression in ox-LDL treated podocytes, and to identify the specific conponent and the specific phosporylation site which PTEN combined with. In vivo, with the new constructed podocyte specific PTEN knockin mice model, we will observer the effect of overexpression of PTEN in podocytes on the lipid-induced glomerulosclerosis. The successful completion of this proposal will provide a new insight into the pathogenesis of the lipid-induced nephropathy from a the stand point of PTEN/YAP pathway. It will lead to development of novel therapeutic strategies in the prevention of progression of CKD.

清道夫受体（SR）A可介导细胞摄取脂质，由于不受细胞内胆固醇的负反馈调节，在脂质肾脏损伤中起关键作用，但机制不明。我们新近研究发现：敲除PTEN可促进高脂饮食小鼠肾小球硬化和YAP的活化，在体外上调核转录因子YAP可使ox-LDL刺激的足细胞SR-A表达增加，我们推测PTEN/YAP通过上调足细胞SR-A表达参与脂质肾脏损伤。本研究拟①进一步观察高脂饮食对小鼠肾脏组织中PTEN、YAP活性和SR-A表达的影响；②明确调控PTEN表达对ox-LDL刺激的足细胞YAP活化、调控YPA对SR-A表达的影响，阐明PTEN确切的靶蛋白及精确作用位点；③制作足细胞特异性条件表达PTEN的小鼠模型，证实PTEN在高脂饮食小鼠中潜在的肾脏保护作用。本项目以探讨SR-A反馈调节新机制为切入点，深入揭示PTEN/YAP信号通路在高脂诱导的肾小球硬化中的作用，有可能在SR-A的上游获得干预脂质肾脏损伤的新靶标。

清道夫受体(SR)A 是细胞表面识别和摄取氧化低密度脂蛋白(ox-LDL)的重要受体，由于不受细胞内胆固醇的负反馈调节，在脂质肾脏损伤中起关键作用，但机制不明。我们前期研究发现：敲除PTEN可促进高脂饮食小鼠肾小球硬化和核转录因子YAP的活化，在体外上调YAP可使ox-LDL刺激的足细胞 SR-A表达增加，我们推测PTEN/YAP通过上调足细胞SR-A表达促进足细胞损伤、凋亡，参与脂质肾脏损伤。本项目通过实验研究得出：①动物实验证实过表达PTEN对高脂饮食诱导的脂质肾脏损伤的保护作用。构建足细胞特异性 PTEN敲进（PPKI）转基因小鼠，HFD诱导转基因小鼠肾损伤。HFD组小鼠的血肌酐、尿白蛋白排泄水平上升，足细胞SR -A表达及肾内脂质含量明显增加，系膜基质增生，足突融合增加及基底膜增厚；HFD+PPKI组小鼠上述肾损伤指标均减轻。HFD+PPKI组小鼠血胆固醇明显下降，PAS染色显示基质增生改善，油红O染色发现肾脂质积聚减少，免疫荧光发现SR-A表达下降，P-YAP表达下调，蛋白电泳及免疫组化显示CTGF、FN、COL-IV 的表达均有下降。②高脂饮食小鼠足细胞中P-YAP明显增加，而足细胞敲进PTEN能明显降低P-YAP的表达水平。③体外实验证实PTEN/YAP调控 SR⁃A表达进而调控脂质摄取，导致足细胞损伤。体外ox-LDL刺激能上调足细胞SR-A表达、磷酸化p-YAP表达增加；上调PTEN的表达可抑制ox-LDL诱导的p-YAP表达增加，敲除PTEN或抑制PTEN磷酸酶活性均能进一步上调ox-LDL诱导的p-YAP的表达。敲除YAP可使ox-LDL诱导的SR-A表达下降。④PTEN 在Ser127位点去磷酸化P-YAP。在ox-LDL刺激的足细胞损伤模型中，通过CO-IP、PLA（邻位连接技术）发现 PTEN与P-YAP蛋白发生相互作用、且是以直接接触方式：GST-pulldown证明，PTEN 在Ser127位点去磷酸化P-YAP。本项目以探讨 SR-A 反馈调节新机制为切入点， 阐明PTEN/Yap-SR-A信号通路介导足细胞损伤在高脂诱导的肾小球硬化中的作用机制，有望在SR-A的上游获得干预脂质肾脏损伤的新靶标。本研究的科学意义在于研究结果可以加深对脂质肾毒性的理论认识、在加深对CKD进展机制理论认识、 获得防治CKD进展方面有重要的应用前景。