Glomerulopathy induced by a single monoclonal autoantibody against GP330

Abstract

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.

Isolation of cDNAs encoding immunogenic regions of gp330, the autoantigen involved in Heymann nephritis

“…In addition, a substitution was found to be located within the putative internalization signal directing receptors to clathrin-coated pits. This substitution may result in a functionally different structure of the gp330 protein, and would be in accordance with reports showing that gp330 is not restricted to clathrin-associated coated pits [30,31]. Furthermore, a difference in the length of the cytoplasmic tail of gp330 was reported in their study, suggesting either a variable signal transduction or a membrane-adapted anchoring of gp330.…”

Autoimmunogenicity of the helix-loop-helix DNA-binding domain

“…The development of autoimmune reactivities to nuclear components eventually leads to the development of autoimmune-associated disease (Ravirajan et al, 2001). Clinical observations made in the experimental systems (Chen et al, 1994;Gormly et al, 1981;Luca et al, 1995) coincide with observations made in mice immunized with RT-P/DNA complex. All manifestations, as hair loss, splenomegaly, spleen and liver fibrosis, and proteinuria, correlated with the presence and levels of autoantibodies to DNA.…”

Treatment with Mycophenolate Mofetil Attenuates the Development of Heymann Nephritis

“…As a measure of the systemic effect of MMF on the humoral response, serum titers of anti-gp330 autoantibodies were determined, since gp330 is the major antigen in AHN and since injection of monoclonal antibodies against this antigen is capable of inducing proteinuria [14]. Although anti-gp330 antibody levels in the serum remained clearly lower in the MMF-treated rats with AHN throughout the experiment, no obvious differences in the IgG deposition in the glomeruli could be observed.…”

“…The gp330 was affinity purified from rat kidneys using monoclonal antibodies as described previously [14]and coated on microtiter plates at 1 µg/well in phosphate-buffered saline for 1 h at 37°C. Serum samples were incubated at dilutions of 1:500 and 1:1,000 in phosphate-buffered saline, 0.05% Tween 20, and 2% bovine serum albumin in duplicate.…”

“…The disease is characterized by the formation of subepithelial immune complexes in the glomerulus and heavy proteinuria. The immune complexes mainly consist of autoantibodies specific for a high molecular weight epithelial glycoprotein, gp330, that is expressed in the microvillar area of the brush border of the proximal tubule and on the glomerular epithelium [11, 12, 13, 14]. Circulating autoantibodies against gp330 derived from autoreactive B cells in the lymph nodes draining the site of immunization [15]bind to the glomerulus in situ.…”