肾小球滤过膜蛋白Perlecan下调在子痫前期肾病发生中的作用和机制

Kidney is one of the target organs which was damaged earliest and seriously in preeclampsia, thus will increase the risk of suffering from future terminal renal disease. Perlecan is the important component of glomerular filtration membrane, participating in the maintaining of charge barrier and having the effect of promoting angiogenesis and inhibiting autophagy. According to previous research, we have identified the protein perlecan and found that the concentration decreases in the urine of preeclampsia patients using proteomic technology. Meantime, other studies indicated that perlecan were also lower expressed in the renal tissues of various renal diseases. However right now there is no report about the change of perlecan and its effect on the kidney of preeclampsia. Based on the positive relations between preeclampsia and excessive autophagy, insufficient angiogenesis, we propose the following hypothesis: the decrease of perlecan in renal tissue of preeclampsia will cause the component change of glomerular filtration membrane,activation of mTOR signaling pathway and inhibition of VEGF pathway. Then the glomerular endothelial cells will appear autophagy death and the vascular angiogenesis to repair the endothelial damage will decrease, therefore leading to proteinuria and renal dysfunction. This project plans to build the preeclampsia mouse model and perlecan knockout mouse model to detect the concentration changes of perlecan and its degrading enzyme, VEGF, Ras, pMEK1, PI3K, Akt, mTOR and autophagy proteins using glomerular separation, electron microscopy observation, RT-PCR and Western Blot technologies with the purpose of verifying the down-regulation of perlecan will induce the development of preeclampsia nephropathy and providing related experimental evidences for the preventive and therapeutic schemes.

肾脏是子痫前期受损最早且严重的靶器官，增加罹患终末肾病的风险。Perlecan作为肾小球滤过膜重要组分，参与电荷屏障维持，具有促进血管生成和抑制自噬作用。前期研究我们利用蛋白组学鉴定出Perlecan并发现其在子痫前期患者尿液含量下降；研究发现不同肾病肾组织中也存在Perlecan表达下降，但子痫前期肾脏该蛋白如何变化及作用机制无相关报道。依据子痫前期与自噬过度、血管生成减少的正相关性，我们提出假说：子痫前期肾脏Perlecan含量减少，滤过膜成分改变； mTOR信号通路激活，内皮细胞自噬性死亡，VEGF信号通路抑制，血管生成减少，内皮损伤修复不力，共同作用肾脏引起损伤。本项目拟构建子痫前期小鼠和基因敲除小鼠模型，采用肾小球分离和电镜、分子生物等技术检测Perlecan及降解酶、VEGF和mTOR通路变化，证明Perlecan下调可以引起子痫前期肾病发生发展，解释发病机制，指导疾病防治。

肾脏是子痫前期受损最早且严重的靶器官，增加罹患终末肾病的风险。Perlecan作为肾小球滤过膜重要组分，参与电荷屏障维持，具有促进血管生成和抑制自噬作用。前期研究我们利用蛋白组学鉴定出Perlecan并发现其在子痫前期患者尿液含量下降；研究发现不同肾病肾组织中也存在Perlecan表达下降，但子痫前期肾脏该蛋白如何变化及作用机制无相关报道。依据子痫前期与自噬过度、血管生成减少的正相关性，我们推测Perlecan在子痫前期肾脏损伤中起着一定的作用。为探讨Perlecan在子痫前期肾脏中的变化及其作用机制，我们成功构建子痫前期动物模型和HSPG2肾小球特异性敲低小鼠，发现与正常孕鼠相比，子痫前期小鼠肾小球基底膜负电荷基本消失，电荷屏障破坏，肾脏组织中Perlecan蛋白含量减少，Hpa表达增加且活性增强，mTOR信号通路抑制，通路相关蛋白PI3K、p-AKT、p-mTOR表达下降，自噬相关标志物LC3-I/LC3-II、Beclin-1蛋白表达含量增加，血管内皮细胞自噬过度，同时VEGF信号通路被抑制，通路相关因子Ras、Raf-1、p-MEK1、p-ERK1/2含量减少，血管生成减少，内皮损伤修复不力，从而引起肾脏损伤，对HSPG2肾小球特异性敲低小鼠肾小球血管内皮细胞的实验验证了以上结果，同时进一步对子痫前期小鼠给予低分子肝素治疗后，发现Hpa减少，从而减少对Perlecan蛋白的降解，一方面减少了肾小球电荷屏障的损伤，另一方面通过调节mTOR和VEGF信号通路减少血管内皮损伤，从而缓解蛋白尿改善肾功能。由此可见，子痫前期肾脏组织中Perlecan减少可能通过引起肾小球滤过屏障损伤和血管内皮细胞受损导致肾脏损害，低分子肝素治疗通过减少对Perlecan蛋白的降解缓解肾脏损伤，从而指导疾病防治。