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 le...