Abstract IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world. Unfortunately, many patients progress to kidney failure. IgAN is defined by the characteristic IgA1 mesangial deposits. Based on our published data, we proposed a hypothesis that describes sequential steps in the pathogenesis of IgAN. This multi-hit process is recognized by other researchers in the field as a blueprint for the development of disease-specific biomarkers and treatments. In the past funding period, we showed that most IgAN patients produce pathogenic IgA1-containing immune complexes. We determined that these complexes consist of polymeric IgA1 with some hinge-region O-glycans deficient in galactose (Gd-IgA1) and devoid of sialic acid (asialo-Gd-IgA1). These IgA1 glycoforms are recognized by IgG autoantibodies, resulting in the formation of circulating immune complexes, some of which deposit in glomeruli. We have determined that IgG in glomerular immunodeposits of IgAN patients is specific for asialo-Gd-IgA1. Using our in vitro and in vivo models of IgAN, we demonstrated that i) large-molecular-mass Gd-IgA1-IgG immune complexes are pathogenic; ii) complement C3 is covalently attached to IgA1 and IgG in these complexes; iii) C3 is required for the biological activity of those complexes in vitro and in vivo; and iv) these IgG-Gd-IgA1-C3 complexes activate multiple signaling pathways in mesangial cells. Despite progress in the identification of genetic and biochemical factors involved in IgAN pathogenesis, there are still many gaps in our knowledge. To address these gaps, we will use biospecimens from IgAN patients of opposing clinical phenotypes, i.e., with progressive vs. non-progressive disease, to determine the factors that are associated with disease progression. Based on our pilot experiments, we hypothesize that IgA1 immune complexes of IgAN patients with progressive disease have greater biological activity than do the complexes of patients with non-progressive IgAN. We further hypothesize that variances in the composition of these complexes determine differences in biological activity. For the circulating IgA1 immune complexes from IgAN patients with progressive vs. non-progressive disease, we will determine their size and biological activity (to induce cellular proliferation, signaling, and cytokine production in mesangial cells; Aim 1). We will determine molecular O-glycophenotypes of IgA1 in the circulating immune complexes of IgAN patients with progressive vs. non-progressive disease (Aim 2) and determine the impact of C3 processing and the presence of complement-regulating proteins on the activity of IgA1 immune complexes (Aim 3). To accomplish these goals, we have assembled a multidisciplinary team that will leverage our access to well-characterized clinical samples and our innovative in vitro and in vivo models of IgAN. Relevance: The results from these studies will provide molecular-level details concerning factors that determine ...