#1323 Mesangioproliferative activity of IgA1-IgG immune complexes in IgA nephropathy requires complement C3 and can be inhibited by a broad-spectrum kinase inhibitor

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Abstract Background and Aims IgA nephropathy (IgAN) is an immune-complex (IC)-mediated kidney disease wherein ICs form in the circulation by aberrantly O-glycosylated IgA1 (galactose-deficient IgA1; Gd-IgA1) and IgG autoantibodies. Additional serum components, such as complement proteins, can also be associated with these ICs. Some of these circulating ICs enter glomerular mesangium and activate mesangial cells, leading to mesangioproliferative kidney injury. In this study, we assessed the role of complement C3 (C3) in the mesangioproliferative activity of Gd-IgA1-IgG ICs and tested the capacity of a broad-spectrum protein tyrosine kinase inhibitor, dasatinib, to block IC-mediated mesangioproliferative activity. Method ICs from native or IgA1-depleted sera of patients with IgAN were isolated by size-exclusion chromatography (SEC). Engineered ICs were formed using polymeric Gd-IgA1 (myeloma or recombinant protein) and recombinant IgG autoantibodies in C3-depleted or C3-repleted serum and then isolated by SEC. SDS-PAGE immunoblotting under non-reducing conditions was used to determine covalent association of C3 with IgA and IgG. IgA-IgG and IgA-C3 complexes were confirmed by ELISA. Reducing conditions were used to determine C3 processing, i.e., presence of alpha-chain or its fragments indicative of C3, C3b, and iC3b. Biological activity of the isolated ICs or engineered ICs was determined based on their capacity to induce cellular proliferation of cultured primary human mesangial cells, without or with dasatinib in the culture medium. In our passive mouse model of IgAN, complement C3 was depleted by intraperitoneal (i.p.) injection of cobra venom factor (CVF). C3 was ~99% depleted from the circulation 1 day after CVF injection, and this depletion was maintained for at least 5 days. One day after CVF injection, ICs were administered i.v. on three consecutive days to a group of nude mice and to a control group of mice without CVF treatment. Other control groups of mice received only CVF or vehicle (PBS). To test the effect of dasatinib, nude mice received an i.v. dose of engineered ICs (every other day x 3 doses) without or with dasatinib (30 mg/kg/d by gavage). Control mice did not receive any engineered ICs. Pathologic glomerular changes were evaluated by quantitative morphometry using H&E-stained sections of kidneys harvested one day after the last IC dose. Results ICs >700 kDa from IgAN sera increased cellular proliferation of quiescent mesangial cells by 2-4-fold. These ICs contained IgA, IgG, and C3; C3 was covalently associated with IgA and IgG. C3 molecular forms included C3, C3b, and iC3b. Depletion of total IgA1 from sera removed stimulatory ICs; the resultant preparations were devoid of IgA, IgG, and C3. To confirm the role of C3, we used engineered ICs formed in C3-depleted or C3-repleted serum. C3 was required for formation of large-molecular-mass engineered ICs that stimulated mesangial cells to proliferate. IgA and IgG formed covalent complexes with C3 in C3-repleted serum. These ICs contained C3, C3b, and iC3b, as observed for isolated ICs from IgAN patients. In our mouse model of IgAN, CVF-mediated C3 depletion prevented mesangioproliferative changes induced by ICs. Specifically, ICs increased glomerular cellularity compared to control (P = 0.0001) whereas CVF blocked that effect (P = 0.021). CVF alone did not alter glomerular cellularity. As Gd-IgA1-IgG-C3-containing ICs activated a broad range of protein-tyrosine kinases in cultured mesangial cells, we tested the effect of dasatinib on IC-induced mesangial-cell proliferation. Dasatinib inhibited cellular proliferation of cultured mesangial cells stimulated with isolated or engineered ICs. We confirmed these findings in the mouse model of IgAN, wherein engineered ICs increased glomerular cellularity (P < 0.0001 vs control mice) and dasatinib prevented this response (P < 0.0001 vs mice given only engineered ICs). Conclusion Complement C3 was required for Gd-IgA1-IgG ICs to stimulate proliferation of mesangial cells in culture and in an in vivo model of IgAN. Dasatinib blocked these stimulatory effects.

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