Complement C3 Contributes to the Pathogenicity of Galactose-Deficient IgA1-Containing Immune Complexes in IgA Nephropathy

Background: IgA nephropathy (IgAN) is an autoimmune kidney disease wherein circulating immune complexes (IC) contain IgA1 with some O-glycans deficient in galactose (galactose-deficient IgA1; Gd-IgA1) bound by IgG autoantibodies specific for Gd-IgA1. Additional serum components, such as complement proteins, can be also associated with these IC. Some of these Gd-IgA1-containing IC deposit in the glomeruli and induce kidney injury. In this study, we assessed the role of complement C3 in the pathogenicity of Gd-IgA1-IgG IC. Methods: IC from native or IgA1-depleted sera of patients with IgAN were isolated by size-exclusion chromatography. Engineered IC were formed using recombinant polymeric Gd-IgA1 and recombinant IgG autoantibodies specific for Gd-IgA1 in C3-depleted or C3-repleted serum and isolated by size-exclusion chromatography. Biological activity of the isolated IC or engineered IC was determined based on their capacity to induce cellular proliferation of cultured primary human mesangial cells. IgA, Gd-IgA1, and IgA-IgG and IgA-C3 complexes were determined by ELISA. SDS-PAGE immunoblotting under non-reducing conditions was used to determine covalent association of C3 with IgA or IgG. Reducing conditions were used to determine C3 processing, i.e., presence of alpha-chain or its fragments indicative of C3, C3b, and iC3b, respectively. Results: IC >700 kDa from IgAN sera increased cellular proliferation of quiescent mesangial cells by 2-4-fold. These IC contained IgA, IgG, and C3; C3 was covalently associated with IgA and IgG. C3 molecular forms included C3, C3b, and iC3b. Removal of IgA1 from sera removed these stimulatory IC; the resultant preparations were devoid of IgA, IgG, and C3. To confirm the role of C3, we used engineered IC formed in C3-depleted or repleted serum. C3 was required for formation of large-molecular-mass engineered IC that stimulated mesangial cells to proliferate. Moreover, IgA and IgG formed covalent complexes with C3 in C3-repleted serum; these engineered IC contained C3, C3b, and iC3b. Conclusion: In summary, C3 has a central role in the formation of Gd-IgA1-containing IC with a nephritogenic capacity for IgAN. Funding: NIDDK Support - NIDDK Support, NIDDK Support, Private Foundation Support