The role of membrane protein proteolysis in membranous nephropathy

膜性肾病（MN）是一种肾小球自身免疫性疾病，由自身抗体介导，该抗体靶向肾小球足细胞表达的抗原。2009年，PLA2R1被鉴定为首个足细胞抗原，70%的MN患者对其产生自身抗体。2014年，我们在10%的MN患者中发现THSD7A作为自身免疫的第二个靶点。在MN中，自身抗体与位于足细胞足突的跨膜抗原结合。因此，足突消退和上皮下免疫沉积形成，这两者都是MN的形态学特征。患者发展为肾病综合征，以蛋白尿超过3.5 g/天、水肿和高脂血症为特征。在MN中足突消退和上皮下免疫沉积的机制尚不清楚，但最有可能涉及足细胞膜蛋白的蛋白水解，如细胞粘附蛋白、细胞-细胞接触蛋白和作为抗原的足细胞膜蛋白。根据其他系统的描述，PLA2R1和THSD7A都经历蛋白水解，导致（至少在THSD7A的情况下）产生可溶性的细胞外蛋白片段和小的细胞内片段，我们可以在足细胞中检测到。我们确定了在足细胞足突中表达的膜锚定蛋白酶A崩解素和金属蛋白酶（ADAM） 10。ADAM10蛋白水解活性导致Notch、l -选择素、E-cadherin、VE-cadherin、CXCL16和L1粘附分子等多种膜结合底物脱落，从而调节细胞迁移和增殖、神经突和轴突引导以及免疫等许多生物过程。一些已知的ADAM10靶点与足细胞存活、粘附和信号传导相关，因此ADAM10在足细胞中发挥重要作用。为了研究ADAM10是否参与了MN足细胞膜蛋白的脱落，我们制造了足细胞特异性ADAM10缺失的小鼠。这些小鼠没有表现出明显的发育表型，直到成年都很健康。在这一建议中，我们假设ADAM10参与足突消退和上皮下抗体/抗原沉积的发展，并将从四个方面解决这一假设。我们将研究导致MN足细胞ADAM10上调和激活的因素（目的1），我们开发的thsd7a特异性MN模型中ADAM10在上皮下抗体/抗原沉积中的病理生理作用（目的2），在阿霉素肾炎和蛋白质过载模型中ADAM10在足突消除中的病理生理作用（目的3），我们将致力于通过肾小球膜制剂和体内生物素化肾小球蛋白的细胞表面蛋白质组学来鉴定新的足细胞特异性ADAM10靶点（目标4）。这些研究将阐明ADAM10在膜性肾病中足细胞膜蛋白水解的作用。

Membranous nephropathy (MN) is an autoimmune disease of the glomerulus, mediated by autoantibodies that target antigens expressed by glomerular podocytes. 2009, PLA2R1 was identified as the first podocyte antigen, to which 70% of patients developed autoantibodies in MN. 2014 we identified THSD7A as the second target to autoimmunity in 10% of patients with MN. In MN autoantibodies bind to the transmembrane antigen situated at podocyte foot processes. As a consequence, foot processes efface and subepithelial immune deposits develop, both morphological hallmarks of MN. Patients develop a nephrotic syndrome, characterized by proteinuria over 3.5 g/day, edema, and hyperlipidemia. The mechanisms of foot process effacement and sub epithelial immune deposition are not clear in MN, but most likely involve the proteolysis of podocyte membrane proteins such as cell adhesion, cell-cell contact proteins, and of podocyte membrane proteins serving as antigen(s). It has been described from other systems that both PLA2R1 and THSD7A undergo proteolysis, resulting (at least in the case of THSD7A) in the generation of a soluble extracellular protein fragment and a small intracellular fragment, which we can detect in podocytes. We identified the membrane-anchored protease A Disintegrin And Metalloproteinase (ADAM) 10 to be expressed at podocyte foot processes. ADAM10 proteolytic activity results in the shedding of numerous membrane-bound substrates such as Notch, L-Selectin, E-cadherin, VE-cadherin, CXCL16, and L1 adhesion molecule and thereby regulates many biological processes such as cell migration and proliferation, neurite and axon guidance, and immunity. Some known ADAM10 targets are relevant for podocyte survival, adhesion and signaling, therefore favoring an important role for ADAM10 in podocytes. In order to investigate, whether ADAM10 is involved in the shedding of podocyte membrane proteins in MN, we generated mice with podocyte-specific ADAM10 deficiency. These mice exhibited no obvious developmental phenotype and are healthy until adulthood. In this proposal we hypothesize that ADAM10 is involved in the development of foot process effacement and subepithelial antibody/antigen deposition and will address this hypothesis in four aims. We will investigate the factors resulting in ADAM10 up regulation and activation in podocytes in MN (aim 1), the pathophysiologic role of ADAM10 for subepithelial antibody/antigen deposition in the THSD7A-specific MN model developed by us (aim 2), the pathophysiologic role of ADAM10 for foot process effacement in the well established model of adriamycin nephritis and protein overload model (aim 3), and we will aim at identifying new podocyte-specific ADAM10 targets by cell surface proteomics of glomerular membrane preparations and of in vivo biotinylated glomerular proteins (aim 4). These investigations will clarify the role of ADAM10 for podocyte membrane proteolysis in membranous nephropathy.