In Vitro Evidence That Galactose-Deficient IgA1 Enables Formation of Pathogenic Immune Complexes in IgAN with IgG Autoantibodies and Complement C3

Background: IgA nephropathy (IgAN) is a chronic kidney disease with glomerular immune-complex deposits consisting of aberrantly glycosylated IgA1 (galactose-deficient IgA1; Gd-IgA1), IgG autoantibodies specific for Gd-IgA1, and C3. These immune complexes are thought to originate from the circulation. Here, we isolated and characterized IgA1-containing immune complexes from sera of patients with IgAN and assessed whether supplementation with Gd-IgA1 resulted in formation of additional immune complexes. Methods: Immune complexes from native or Gd-IgA1-supplemented sera of IgAN patients were isolated by size-exclusion chromatography as fractions with apparent molecular mass (Mr) ranging from 0.7 MDa to ~4 MDa. Recombinant polymeric Gd-IgA1 was produced in glycoengineered Expi293F cells. Biological activity of the complexes was determined by their capacity to induce proliferation of primary human mesangial cells. Polyclonal IgG specific for human C3 and monoclonal antibody 3E7 [binds C3b, iC3b, and C3(H2O) but not C3 with intact thioester bond] were used for capture in an ELISA to detect C3 complexes with IgA or IgG. 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 or C3b and iC3b. Results: Immune complexes from IgAN sera that stimulated cellular proliferation of quiescent mesangial cells contained IgA, IgG, and C3. C3 was covalently attached to IgA or IgG, likely by the thioester bond. Complexes of the high Mr (3-4 MDa) and medium Mr (~2-3 MDa) contained C3 [likely as C3(H2O)] whereas the complexes of low Mr (~0.7-2 MDa) also had C3b and iC3b. Supplementation with Gd-IgA1 resulted in formation of additional complexes predominantly of medium and low Mr. These complexes contained Gd-IgA1 with IgG and covalently attached C3, as did the native complexes. Conclusion: In conclusion, recombinant polymeric Gd-IgA1 added to IgAN-patient serum formed additional pathogenic immune complexes consisting of IgA1, IgG, and C3. Thus, an excess of IgG autoantibodies is available to form new pathogenic immune complexes during disease activity with episodic overproduction of Gd-IgA1. Funding: NIDDK Support - NIDDK Support, NIDDK Support, Private Foundation Support