ALTERED ANIONIC GBM COMPONENTS IN MONOCLONAL-ANTIBODY AGAINST SLIT DIAPHRAGM-INJECTED PROTEINURIC RATS

Background. We previously reported that monoclonal antibody (mAb) 5-1- 6 bound to renal filtration slits induces massive proteinuria without causing ultrastructural changes in the glomerulus. This study evaluate d the underlying mechanisms of the increase in glomerular permeability . Methods. The distribution of endogenous albumin and IgG in the glome rular basement membrane (GBM) was studied in in situ drip-fixed glomer uli of Munich-Wistar rats by use of immunogold immunocytochemistry in the presence and absence of mAb 5-1-6. The density of foot process gly cocalyx components was estimated by labeling with Limax fluvus lectin- or Helix pomatia lectin-goId complexes. Anionic sites in the GEM were examined by labeling with cationic gold at pH 2.0 or 7.4. Carboxyl gr oups, which also furnish an anionic charge to the GEM, were examined b y specific biotinylation and colloidal gold probe methods. In addition , the infusion-staining of anionic sites was performed by use of ruthe nium red in both Munich-Wistar and Wistar rats. Results. The urinary e xcretion of albumin and IgG was increased markedly in the treated rats , indicating a non-selective barrier defect. In the control rats, albu min and IgG molecules were mainly located along the inner half of the GEM, and to a lesser degree in the lamina rara externa. In the treated rats, the albumin and IgG moieties were more equally distributed thro ughout the width of the GEM. Newly appearing, small dense peaks at the outer side of the GEM were evident, indicating a barrier function of outer zone of the GEM and/or epithelial cell layer. No intergroup diff erences in the density of lectin binding sites on foot processes were seen. The reduction in the number of ruthenium red-positive anionic si tes and cationic gold (pH 2.0)-labeled anionic sites in the lamina rar a externa was significant in the treated rats at day 3, indicating a p ossible alteration of charged proteoglycan in the lamina rara externa. No such changes were seen with cationic gold (pH 7.4)-labeled anionic sites in the GEM. The density of labeled carboxyl groups was signific antly reduced in the treated rats relative to the controls. Conclusion s. These results show that the injection of mAb 5-1-6 induced a pertur bation of the charge- and probably the size-selective glomerular filtr ation barrier. The observed reduction in the levels of various negativ ely charged substances resulted in massive proteinuria, implying that alteration of target antigens can affect the integrity of the GEM cons tituents maintaining the normal barrier function.