Monoclonal autoantibodies were obtained from Lewis rats with active Heymann nephritis. Two cloned rat/mouse hybridomas, 3D9B and 5B8C, that secreted rat IgG2a autoantibodies were selected for their ability to react with gp330 in ELISA and propagated further. Their specificity was confirmed by immunoprecipitation of crude antigens from a yolk sac carcinoma cell line expressing gp330. The size of the precipitated molecule was identical to that immunoprecipitated by previously described anti-gp330 antibodies. Indirect immuno-electron microscopy showed that 3D9B exclusively stained the intermicrovillous areas of the tubular brush border membrane, while 5B8C stained the full tubular microvillous membrane and the glomerular epithelial coated pits. Passive transfer of 3D9B did not induce Ig deposits or functional renal damage within 7 days. However, injection of 5B8C caused granular glomerular deposits within 1 h, subepithelial immune aggregates within 6 days and antibody deposition on the brush border within 7 days. Only ascites production of clone 5B8C in rats, but not in mice, caused subepithelial immune deposits and abnormal proteinuria. This study shows that a single monoclonal autoantibody to gp330 is able to induce a mild form of Heymann nephritis.
Abstract. In a time-study of active Heymann nephritis, the expression of agrin, the main heparan sulfate proteoglycan in the glomerular basement membrane, was analyzed in relation to deposition of IgG and complement in the glomerular capillary wall and the development of albuminuria. Binding of IgG autoantibodies to the glomerular capillary wall could be detected from 2 wk onward, followed by activation of complement after 6 wk. Progressive albuminuria developed from 6 wk onward to a level of 274 ± 68 mg/18 h at week 12. The staining intensity for the agrin core protein decreased slightly, and the staining intensity for the heparan sulfate stubs that were still attached to the core protein after heparitinase digestion remained normal. From week 6 onward, however, a progressive decrease was seen in the staining of two monoclonal antibodies (mAb) directed against different epitopes on the heparan sulfate polysaccharide side chain of agrin (to 35 and 30% of the control level, respectively, at week 12, both mAb P = 0.016). Moreover, albuminuria was inversely correlated with heparan sulfate staining as revealed by these antibodies (rs = -0.82 and rs = -0.75, respectively, both mAb P < 0.0001). This decrease in heparan sulfate staining was due to a progressive reduction of glomerular heparan sulfate content to 46 and 32% of control level at week 10 and week 12 of the disease, respectively, as measured biochemically. It is speculated that the observed decrease in glomerular heparan sulfate in active Heymann nephritis is due to complement-mediated cleavage of heparan sulfate, resulting in an increased permeability of the glomerular basement membrane to macromolecules.
SUMMARYActive Heymann nephritis is an organ-specific autoimmune disease of the rat kidney, characterized by the formation of immune complexes located subepithelially in the glomerulus. The T cell-mediated humoral immune response is directed to gp330, a large renal epithelial glycoprotein which is expressed both in the proximal tubule and on glomerular podocytes. In this study polyclonal rabbit antibodies raised against affinity-purified rat gp330 were used to screen a -gt11 expression library of the rat kidney. One cDNA clone that was recognized by the antibodies coded for a 2 . 7-kb protein that is not described in the sequence database of GenBank/EMBL. Two other groups of cDNA clones were identified that displayed similarity with several members of the low-density lipoprotein (LDL)-receptor gene family to which gp330 belongs. By comparison with the gp330-cDNA sequence, these two clones could be mapped to two remote areas on the extracellular domain of gp330. The antigenicity of these two areas is in accordance with their location in highly hydrophilic regions on the extracellular domain of gp330. The cDNA clones described in this study may represent two main immunodominant regions on rat gp330.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.