The role of membrane protein proteolysis in membranous nephropathy

膜蛋白水解在膜性肾病中的作用

• 批准号: 417738345
• 负责人: Professorin Dr. Catherine Meyer-Schwesinger
• 金额: --
• 依托单位: Institut für Zelluläre und Integrative Physiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/417738345?language=en
• 关键词: role; membrane; protein; proteolysis; membranous

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.

膜性肾病 (MN) 是一种肾小球自身免疫性疾病，由针对肾小球足细胞表达的抗原的自身抗体介导。 2009年，PLA2R1被确定为第一个足细胞抗原，70%的MN患者产生自身抗体。 2014 年，我们将 THSD7A 确定为 10% MN 患者的第二个自身免疫靶标。在 MN 中，自身抗体与位于足细胞足突的跨膜抗原结合。结果，足突消失和上皮下免疫沉积物形成，这都是 MN 的形态特征。患者出现肾病综合征，其特征是蛋白尿超过3.5克/天、水肿和高脂血症。 MN 中足突消失和上皮下免疫沉积的机制尚不清楚，但很可能涉及足细胞膜蛋白（如细胞粘附、细胞间接触蛋白）和作为抗原的足细胞膜蛋白的蛋白水解。据其他系统描述，PLA2R1 和 THSD7A 都会经历蛋白水解，导致（至少在 THSD7A 的情况下）生成可溶性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它们。我们鉴定出膜锚定蛋白酶 A 解整合素和金属蛋白酶 (ADAM) 10 在足细胞足突处表达。 ADAM10 蛋白水解活性导致许多膜结合底物（例如 Notch、L-选择蛋白、E-钙粘蛋白、VE-钙粘蛋白、CXCL16 和 L1 粘附分子）脱落，从而调节许多生物过程，例如细胞迁移和增殖、神经突和轴突引导和免疫。一些已知的 ADAM10 靶点与足细胞存活、粘附和信号传导相关，因此有利于 ADAM10 在足细胞中发挥重要作用。为了研究 ADAM10 是否参与 MN 中足细胞膜蛋白的脱落，我们培育了足细胞特异性 ADAM10 缺陷的小鼠。这些小鼠没有表现出明显的发育表型，并且在成年之前都保持健康。在本提案中，我们假设 ADAM10 参与足突消失和上皮下抗体/抗原沉积的发展，并将通过四个目标解决这一假设。我们将研究导致 ADAM10 在 MN 足细胞中上调和激活的因素（目标 1），ADAM10 在我们开发的 THSD7A 特异性 MN 模型中对上皮下抗体/抗原沉积的病理生理作用（目标 2），病理生理作用ADAM10 在成熟的阿霉素肾炎模型和蛋白质超载模型（目标 3）中用于足突消失，我们的目标是确定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确定新的足细胞特异性 ADAM10 靶点（目标 4）。这些研究将阐明 ADAM10 在膜性肾病足细胞膜蛋白水解中的作用。