sPLA2-IB 及PLA2R1在调节足细胞nephrin内吞中的机制研究

Podocytes are major cellular components of glomerular- filtration barrier. Nephrin is an important structural and signal molecule of podocyte. Nephrin can regulate the function of podocyte through endocytosis，such as cytoskeleton rearrangement. However, its specific mechanisms of the signal transduction remain unclear. PLA2R1 is expressed in podocytes in human podocyte. sPLA2-IB is one of the ligands of the PLA2R1. We have discovered that sPLA2-IB and PLA2R1 play a key role in podocyte injury through affectting cytoskeleton rearrangement . This project intends to study the role of sPLA2-IB and PLA2R1 in nephrin endocytosis and cytoskeleton rearrangement. For verify the hypothesis, we will study human podocyte, HEK293 cell and clinical specimen by rt-PCR, Western-blot, IF, plasmids transfection, siRNA transfection and special inhibitors. We will evaluate the effect of sPLA2-IB and PLA2R1 in the role of cytoskeleton rearrangement from the level of molecules, cells and tissue aspects and clear regulatory mechanism of sPLA2-IB and PLA2R1 in mediating nephrin endocytosis. This investigation will provide a theoretical evidence for seeking a new therapeutic target of podocyte disease.

Nephrin是足细胞连接相邻足突和信号转导的主要分子，可通过內吞调节足细胞功能，如细胞骨架重排。但足细胞nephrin内吞的调节机制未明。PLA2R1是人足细胞跨膜受体，sPLA2-IB是其配体。我们前期研究发现二者结合后能导致足细胞骨架排列紊乱，损伤加重。为此，我们提出假说：在sPLA2-IB刺激下，PLA2R1介导 nephrin 内吞导致足细胞骨架重排从而影响足细胞功能。为验证这一假说，我们将通过人足细胞、HEK293细胞并收集临床标本，采用rt-PCR、Western-blot、IF、质粒转染、RNA干扰、特异性抑制剂干扰等手段，从分子、细胞及组织水平等多方面探讨sPLA2-IB及PLA2R1在足细胞骨架重排及损伤中的作用，明确sPLA2-IB及PLA2R1在介导nephrin 内吞中的调控机制。为寻求防治足细胞病尤其是膜性肾病的新靶点提供理论依据。

足细胞是一种终末分化细胞，是肾小球滤过的关键屏障。Nephrin是足细胞连接相邻足突和信号转导的主要分子，可通过內吞调节足细胞功能，如细胞骨架重排。但足细胞nephrin内吞的调节机制未明。 PLA2R是人足细胞跨膜受体，sPLA2-IB是其配体。首先，我们发现，sPLA2-IB与原发性膜性肾病（IMN）的关系密切，与蛋白尿水平密切相关，在IMN肾组织肾小球中与PLA2R存在共定位。其次，体外研究研究发现，在sPLA2-IB刺激下，PLA2R介导nephrin内吞导致足细胞骨架重排从而影响足细胞功能。在肾小球足细胞中 PLA2R通过 PLCβ-PKCα 途径导致 nephrin 磷酸化降低及内吞增强、足细胞损伤加重。本课题基本达到了预定的研究目标。拓展研究还发现sPLA2-IB与PLA2R异常调控足细胞自噬，通过p38MAPK/mTOR/ULK1ser757通路降低足细胞自噬水平，导致足细胞损伤加重。本研究为寻求防治足细胞病尤其是膜性肾病的新靶点提供了理论依据。本项目发表SCI论文4篇，中文核心期刊论文4篇，均为第一标注。论文投稿参加本领域国家级会议交流5篇，其中壁报4篇，口头交流1篇，其中获优秀壁报2篇。培养博士生2人，硕士生4人。