特异性阻断尿激酶受体结构域的短肽在原发性局灶节段肾小球硬化的保护机制研究

Focal segmental glomerulosclerosis (FSGS) is an important cause of steroid-resistant nephrotic syndrome in both children and adults. The etiology and pathophysiology of primary FSGS is still unclear. Recent studies have discovered that the urokinase plasminogen activator receptor, uPAR (CD87) and soluble urokinase receptor in circulation may play a crucial role in FSGS mainly through its ability to form signaling complexes with other transmembrane proteins such as integrins. Podocyte anchored to the glomerular basement membrane by a3β1, expressing a5β3 integrin in slit diaphragm complex between foot processes. Moreover, they showed that circulating suPAR activated podocyte β3 integrin, causing foot process effacement, proteinuria, and a histological picture consistent with FSGS. suPAR represents the soluble form of the urokinase type plasminogen activator receptor (uPAR), which is a glycosyl-phosphatidylinositol (GPI)-anchored three-domain membrane protein that can bind to several ligands. Our previous work revealed that suPAR level associated with progression and outcome of FSGS, and addressed the interaction between uPAR and integrin β1. Our hereby proposal will continue to study on mechanism of uPAR and integrin interaction for pathogenesis of podocyte injury. By in vitro and in vivo studies performed on model of Puromycin Aminonucleoside and Adriamycin induced nephropathy for podocyte injury. To accomplish the objective, we knock down the activity of PLAUR and ITGβ3 gene in podocytes. Using a high affinity uPAR peptide antagonist AE105 to specifically block the binding domain of uPAR to integrins, We will demonstrate that down regulate the binding ability of uPAR domain to integrins in podocytes could restore the of slit diaphragm proteins and cytoskeleton structure in vitro via ERK/AKT phosphorylation pathway. To quantitatively in vivo assess expression level of uPAR and interaction with integrins in podocyte, and cell migrations by using a high affinity uPAR peptide antagonist AE105 conjugated with DOTA and labeled with 64Cu (64Cu-DOTA-AE105), detected by micro PET performed in three knock-out animal FSGS models (PLAUR-/-,ITG β1-/-, ITGβ3-/-). The innovations of this proposal are that clarifying the interaction domain between uPAR and integrin β1/β3, and intend to explore the mechanism of uPAR in podocytopathy and Better understanding of the mechanisms of suPAR in podocytes injury may provide new clues for prevention and treatment of glomerulosclerosis.

原发性局灶节段性肾小球硬化(FSGS)是构成难治性原发性肾病综合征的主要类型，其发病机制尚未阐明。已发现患者血中的可溶性尿激酶受体(suPAR)通过与足细胞足突裂隙膜复合物的整合素β3结合、激活并通过细胞内信号转导导致足细胞病变而引起发病。但目前尚不清楚是suPAR是否还能通过结合足突在基底膜上的锚着蛋白整合素β1参与发病。本研究使用SiRNA敲低uPAR和整合素基因表达的细胞及使用基因敲除动物，以及高亲和力的小分子短肽阻断suPAR的功能结构域，包括使用核素标记的microPET活体示踪技术进一步观察在同一动物体内随疾病发生发展，suPAR表达，与整合素β3及β1的结合能力、对足突裂隙膜的组成蛋白及足细胞内信号转导的变化，为今后的治疗新思路提供重要依据。

研究背景：足细胞损伤是肾病综合征发病的中心环节，但机制尚未阐明。在原发性肾病综合征中，局灶节段性肾小球硬化（FSGS）患者的足细胞损伤既可表现出适应不良性反应——广泛足突消失，与微小病变肾病（MCD）一样；又会表现出FSGS特有的足细胞毁灭性反应：足细胞凋亡、脱落，节段性硬化形成并扩展。血液中的致病因子一直是国际上的研究重点，已发现患者血中的可溶性尿激酶受体(suPAR)可能是其中的主要致病因子。本课题拟探索其通过与足细胞足突间裂隙膜复合物结合以及通过结合足突在基底膜上的锚着蛋白，引发足细胞骨架改变、凋亡而导致发病；同时在各种引发FSGS的人类疾病中观测相应的证据。.研究内容：按计划完成的内容有：观察从肾病期到完全缓解期不同程度蛋白尿MCD患者足细胞足突的变化过程；观察suPAR作用在足细胞引起的细胞骨架及凋亡的作用及相应中和分子AE105的保护作用；进一步在各种FSGS患者（IgA肾病、膜性肾病伴发FSGS、肥胖相关性FSGS、Alport综合征、结节硬化性糖尿病肾病）的血、尿中，检测suPAR水平，为临床提供预测线索。除完成计划书的任务之外，还建立了足细胞微粒体（microparticles）的收集和观察技术，观察了足细胞被suPAR损伤后释放微粒体并被正常足细胞吞入的过程。.重要结果和关键数据：本研究发现①足突随尿蛋白的减少，开始出现朝着正常形态变化的趋势，但即使尿蛋白消失，足突形态仍没有完全回到正常；②原发及继发FSGS血清中的高浓度suPAR可以与足细胞整合素结合，导致足细胞的骨架蛋白发生紊乱并且凋亡增加；损伤的足细胞可以释放微粒体进而影响正常的足细胞；③在原发性及多种继发性FSGS患者的血、尿中，suPAR水平升高，但与尿蛋白量没有相关性。.科学意义：本项目针对suPAR在FSGS发病机制中的病理形态学作用及临床意义进行了研究，为预测病理类型提供了数据，为揭示FSGS等足细胞病的发病机制和选择潜在的治疗靶点提供了新思路。