Isolation and characterization of circulating immune complexes from rats with experimental membranous nephropathy

Circulating immune complexes (CIC) were isolated from serum from controls and rats with active Heymann nephritis (n = 31) by two methods. CIC detected by the fluid phase Clq binding assay were precipitated from serum using Clq and polyethylene glycol. CIC were also isolated by sequential chromatography with anion exchange and lectin affinity supports. The isolated material was analyzed by PAGE and immunoblotting. The immune complex material isolated by both methods from rats with Heymann nephritis contained the same 60/65-kDa tubular Ag. By immunoblotting, the 60/65-kDa tubular Ag-bound antibodies from rats with active Heymann nephritis, but not antibodies to gp330. Antibody bound to the 60/65-kDa tubular protein in the CIC was isolated. This antibody bound to a similar Ag in glomerular eluates from rats with active Heymann nephritis when tested by immunoblotting. These observations suggest that glomerular immune deposits and CIC in rats with Heymann nephritis contain the same tubular Ag. The 60/65-kDa Ag was isolated from CIC by HPLC using anion exchange and hydrophobic interaction columns. Rats immunized with this Ag developed Heymann nephritis. These studies suggest that CIC contribute to the development of glomerular subepithelial immune deposits in this model of membranous nephropathy. These studies do not exclude the participation of other Ag-antibody systems in Heymann nephritis, including gp330. This report describes methods for isolation and characterization of Ag-antibody components of CIC that might be useful to studies of other immune complex-mediated diseases.