All ETDs from UAB

Advisory Committee Chair

Zina Moldoveanu

Advisory Committee Members

Jan Novak

Colin Reily

Document Type

Thesis

Date of Award

2022

Degree Name by School

Master of Science (MS) Heersink School of Medicine

Abstract

IgA nephropathy is an autoimmune disease characterized by glomerular IgA1- containing immunodeposits. These immunodeposits are enriched for aberrantly glycosylated IgA1 bound by IgG autoantibodies and contain complement component C3. Current research suggests a multi-hit disease pathogenesis: IgAN patients have elevated serum levels of IgA1 missing galactose in some hinge region O-glycans; this galactosedeficient IgA1 is recognized by IgG autoantibodies resulting in formation of circulating immune complexes; some of these complexes deposit in the glomeruli and induce kidney injury. As there is no disease-specific treatment, many patients progress to kidney failure. In this master’s thesis I sought to assess whether C3 is: i) associated with the circulating IgA1-IgG immune complexes in patients with IgAN, ii) present as C3, C3b, iC3b, and/or other protein fragments, and iii) attached to IgA, IgG, or both. To accomplish these aims, the following methods were employed: SEC was used to isolate high-molecular-mass IgA1-IgG immune complexes from sera of IgAN patients; IgA concentration was measured by ELISA; proliferation-stimulating activity of immune complexes was tested using cultured human primary mesangial cells; SDS-PAGE under non-reducing conditions that maintain intact thioester and disulfide bonds was combined with immunoblotting using antibodies specific for IgA, IgG, or C3. The results indicated that immune complexes from IgAN patients contained monomeric IgG as well as IgG of higher molecular mass, which appeared to contain covalently attached C3. Additionally, IgA that may also have covalently attached C3 was detected. Moreover, the amounts of C3, IgG, and IgA appear iv to be greater in immune complexes from patients with progressive disease compared to those with nonprogressive disease. Depletion of IgA from serum of IgAN patients by affinity chromatography removed IgA, as well as IgG and C3, from the immune complexes, thereby further confirming the association of IgA with IgG and C3. Using SDS-PAGE under reducing conditions, C3b and iC3b were detected in IgA-IgG immune complexes. Together, these results demonstrate that IgA-containing immune complexes in sera of IgAN patients consisted of IgA with IgG and complement C3, specifically C3b and iC3b. These findings enable further studies that will help to determine the role of complement C3 in IgAN pathogenesis.

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