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Renal glomeruli are an important site for deposition of immune complexes that cause inflammation and subsequent tissue damage. The glomerulus serves as a size and charge barrier for circulating macromolecules and presents a highly negatively charged (anionic) surface to the circulation (1). The role of the anionic charges on the binding, formation, and persistance of immune complexes in glomeruli has only recently begun to be examined. Gallo et al. (2) showed that immune complexes prepared with cationized antigen and cationized antigen alone bound to the anionic sites in the glomerular basement membrane within 1 h of administration, but later time points were not examined. Oite et al. (3) showed the binding of cationized antigen (Ab~.n) 1 to the glomerulus and the formation of subepithelial deposits when antibody was administered subsequent to the binding of the antigen. Furthermore, antibodies to DNA eluted from glomeruli of NZB/W and MRL/1 mice were more cationic than those in circulation (4), suggesting that cationic antibodies may preferentially lead to deposition of immune complexes in glomeruli.The proportion of naturally formed highly cationic antibodies is small and difficult to isolate in sufficient quantities to investigate their role in immune complex deposition. For these reasons, chemical cationization of antibodies was performed and antigen-binding ability was preserved. Immune complexes prepared with these cationized antibodies (AgAbED) demonstrated the important contribution of antibody charge to the glomerular deposition of immune complexes. The injected large-latticed AgAbED formed extensive subendothelial electron-dense deposits in mice. Materials and MethodsPreparation of Antibodies and Antigen. Rabbit immunoglobulin G (IgG) was purified from rabbit fraction II (Miles Laboratories, Elkhart, IN) by gel filtration over Sephadex G-200 column (Pharmacia Fine Chemicals, Pharmacia Inc., Piscataway, N J). Rabbit antibodies to human serum albumin (HSA) were isolated by affinity chromatography, and the monomeric fractions obtained by gel filtration were trace labeled with 125I, all by previously described

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This study was conducted to determine whether multivalent, precipitating antigens are required for formation of subepithelial electron-dense immune deposits in glomeruli. 2-nitro-4-azidophenyl (NAP) was conjugated with variable density to human serum albumin (HSA) to yield nonprecipitating (NAP3.1 X HSA and NAP11.4 X HSA) and precipitating (NAP19.7 X HSA) antigens with antibodies to the hapten. These antigen preparations were cationized with ethylene diamine to enhance deposition in renal glomeruli due to interaction with the fixed negative charges in the glomerular capillary wall. Following injection into the left renal artery of rats these antigens alone persisted in the glomeruli for a relatively short time by immunofluorescence microscopy. When antibodies to NAP were injected intravenously after the antigen injection, the nonprecipitating antigens and antibodies were detectable in the glomeruli by immunofluorescence microscopy up to 8 h, comparable to antigen alone. Electron-dense deposits were not formed in these glomeruli. In contrast, when the precipitating antigen was injected and followed by antibodies to the hapten, antigen and antibody were detected by immunofluorescence microscopy through 96 h. In these specimens electron-dense deposits were present from 40 min through 96 h and after 24 h the deposits were present only in the subepithelial area. The same results were obtained when the nonprecipitating hapten-carrier conjugates were followed with antibodies to the carrier molecule. These data indicate that the persistence of immune deposits by immunofluorescence microscopy and the formation of electron-dense deposits in the subepithelial area require a precipitating antigen-antibody system.

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