Monoclonal antibodies against membrane proteins of the rat glomerulus. Immunochemical specificity and immunofluorescence distribution of the antigens

Abstract

Monoclonal antibodies (MAbs) were generated to detergent-solubilized glomerular extracts to identify new epithelial and endothelial membrane proteins and to study the possible role of the corresponding antigens in the formation of immune deposits. Triton X-114 extracts of isolated glomeruli were subjected to phase separation, and the resultant detergent and aqueous phases were used to immunize mice. Monoclonal antibodies were prepared by standard techniques, and hybridomas secreting antibodies (IgGs) that recognize glomerular cell surface antigens were selected by enzyme immunoassay (EIA) and indirect immunofluorescence. The IgGs of 13 MAbs selected for study recognized antigens of different molecular weights (45-350 kd) by immunoprecipitation and immunoblotting and had different distributions in the glomerulus and in other renal structures by immunofluorescence. Several proved to recognize known antigens--ie, podocalyxin (MAbs 1A, 5A, 11A, and 20A), gp330 (20B), and dipeptidylpeptidase IV (26C). Others recognized antigens not previously characterized that fell into four groups: 1) those that were detected mainly in glomeruli; 2) those present in both glomeruli and peritubular capillaries; 3) those present in both glomeruli and tubule epithelia; and 4) those detected in all these sites. The pattern of glomerular staining also varied, but most of the antigens appeared to be expressed on either the endothelium or the epithelium, or on both. 27A IgG was specific for podocytes and weakly precipitated a 103-kd protein. 7A and 13A IgG precipitated a 120-kd protein and stained glomeruli as well as the basal aspects of distal tubules. 23A IgG recognized a more-than 350-kd antigen that appeared to be specific for endothelial cells in rat kidney and in all other organs studied. 14A IgG precipitated a 150-kd protein and stained glomeruli, proximal tubule brush borders, and endothelial and epithelial cells in rat kidney and in several other organs. 4B and 9B IgG gave a granular cytoplasmic staining in all cells. When injected intravenously into rats, all of the MAbs except 4B and 9B rapidly bound to glomeruli, demonstrating that the respective antigens are exposed at the cell surface and represent potential targets for antibody-mediated immune injury. It is concluded that selective detergent extraction of glomeruli is a useful approach for generation of antibodies that recognize native, nondenatured membrane components of glomerular endothelial and epithelial cells.