Investigating the functional capacity of autoantibodies in primary membranous nephropathy

项目摘要/摘要 膜性肾病(MN)是一种自身免疫性肾病。 足细胞表达的肾小球蛋白导致足细胞亚致死性损伤，滤过屏障丧失， 最终导致肾病综合征。整个病程可能相当长，持续时间和 蛋白尿的程度是肾功能进行性下降和终末期肾病的危险因素。PLA2R 是80%的原发MN病例的主要目标，监测循环中的抗PLA2R抗体(PLA2R- AB)在跟踪免疫性疾病活动和允许更快速的治疗方面发挥了重要作用 决定。然而，尽管存在使用PLA2R-ab血清学的指导方针，肾病学家仍在继续 由于显著的变异性，对谁以及何时治疗免疫抑制有很大的模棱两可 在结果中。PLA2R-ab滴度相对较低或相对较低的患者可能发展为严重的肾病综合征 轻微的疾病，水平非常高，这一观察结果仍然无法解释。这项提议的目标是 确定与PLA2R-ab变量谱相关的疾病结局的新预测因子(它们在 亚类和表位特异性)，并更好地理解PLA2R-ab介导的功能后果 受伤。目的1描述两种可增加总PLA2R-ab滴度的新的检测方法的发展 临床缓解的可能性。一种方法是用简单的酶联免疫吸附试验来评估抗原表位的存在或不存在。 超出PLA2R的N端免疫优势表位。第二个将是C3c的衡量标准 由PLA2R-ab产生(补体激活)以总结所有免疫球蛋白亚类的功能效应 和抗原表位的特异性。将使用COX比例风险模型(生存分析)来检验两者之间的关联 在PLA2R-ab水平之间，C3c的生成和表位随着时间的推移而扩散到主要结果(成就 部分或完全缓解)，使用两个特征良好的MN队列。AIM 2将使用从 从MN患者的残肾活检中询问PLA2R-ab是否与 免疫沉淀物中反映了循环的形式。将PLA2R连接到 肾小球基底膜(GBM)也将被评估。了解致病的PLA2R-ab 与肾脏沉积物中的抗原一起积累，以及了解PLA2R如何结合到沉积物中 可能为治疗靶向的疾病途径提供新的见解。它的建议来自于 MN免疫损伤反应产生的细胞外基质含有异位的动物研究 这些元素通常不存在于健康的GBM中；这些元素在人类疾病中尚未得到广泛研究。在AIM 3、表达PLA2R的人足细胞将作为PLA2R-ab诱导的细胞损伤的体外模型 导致抗体介导的PLA2R从细胞释放的早期事件，其与常规和更多的结合 新的基质元素，并广泛地观察细胞外基质产生的变化。新知识 从这些研究中获得的成果将扩大我们对沉积物如何在人类MN中形成和持续存在的了解。

PROJECT SUMMARY / ABSTRACT Membranous nephropathy (MN) is an autoimmune kidney disease in which antibodies against intrinsic glomerular proteins expressed by the podocyte lead to sublethal podocyte cell injury, loss of the filtration barrier, and ultimately the nephrotic syndrome. The overall disease course can be quite protracted and the duration and extent of proteinuria are risks for progressive decline of kidney function and end-stage kidney disease. PLA2R is the major target in 80% of primary MN cases and the monitoring of circulating anti-PLA2R antibodies (PLA2R- ab) has been instrumental in following immunologic disease activity and allowing for more rapid treatment decisions. Yet despite the existence of guidelines for such use of PLA2R-ab serology, nephrologists continue to have substantial ambiguity about whom to treat with immunosuppression, and when, due to significant variability in outcome. Patients may develop severe nephrotic syndrome with relatively low titers of PLA2R-ab or relatively mild disease with very high levels, an observation which remains unexplained. The goal of this proposal is to identify novel predictors of disease outcome associated with the variable repertoire of PLA2R-ab (which differ in subclass and epitope specificity) and to better understand the functional consequences of PLA2R-ab-mediated injury. Aim 1 describes the development of two novel assays that may add to total PLA2R-ab titer in determining likelihood of clinical remission. One assay will be a simple ELISA to assess the absence or presence of epitope spreading beyond the N-terminal immunodominant epitope of PLA2R. The second will be a measure of C3c generation (complement activation) by PLA2R-ab to summate the functional effects of all the IgG subclasses and epitope specificities. Cox proportional hazards models (survival analysis) will be used to test the associations between PLA2R-ab levels, C3c generation and epitope spreading with time to the primary outcome (achievement of partial or complete remission), using two well-characterized MN cohorts. Aim 2 will use IgG eluted from remnant kidney biopsies from patients with MN to ask whether or not the repertoire of PLA2R-ab that is bound within the immune deposits mirrors the circulating forms. The manner by which PLA2R is attached within the glomerular basement membrane (GBM) will also be assessed. Understanding the pathogenic PLA2R-ab that accumulate with antigen in the kidney deposits, as well as knowing how PLA2R is incorporated into deposits may offer new insights into disease pathways that could be targeted therapeutically. It has been suggested from animal studies that the extracellular matrix produced in reaction to the immune injury in MN contains ectopic elements not usually present in healthy GBM; these have not been extensively studied in human disease. In Aim 3, PLA2R-expressing human podocytes will serve as an in vitro model of PLA2R-ab-induced cell injury to assess early events that lead to antibody-mediated release of PLA2R from the cell, its binding to conventional and more novel matrix elements, and to broadly look at changes in extracellular matrix production. The new knowledge gained from these studies will expand our knowledge of how deposits form and persist in human MN.