Sequence of events in the glomerular capillary wall at the onset of proteinuria in passive Heymann nephritis

Proteinuria in passive Heymann nephritis (PHN) results from complement-medi ated glomerular in jury, since complement depletion with cobra venom factor (CVF) prevents proteinuria. However, there are no comprehensive morphologi cal studies identifying the sites of injury leading to onset of proteinuria . To address this issue, we attempted to locate sites of injury involved in the onset of proteinuria in PHN. PHN was induced in intact Munich-Wistar r ats (PHN-rats, examined at days 3, 5, and 7) and in complement-depleted rat s (CVF treated, PHN-CVF-rats, examined at days 3 and 5). The distribution o f endogenous albumin in the glomerular basement membrane (GBM) was studied in in situ drip-fixed glomeruli using immunogold immunocytochemistry, and g lomerular anionic sites were visualized by polyethyleneimine staining. In a ddition, the ultrastructural localization of an epitope recognized by a pro teinuria-inducing monoclonal antibody (called 5-1-6) directed against the s lit diaphragm was examined. Significant proteinuria was seen in intact PHN- rats, starting at day 5. The intensity of gold labeling for endogenous albu min was significantly increased at the outermost site of the GEM (GBM inter facing foot process and the filtration slit, designated area O) at day 3 in both PI-IN-rats and PHN-CVF-rats in comparison to untreated controls. At d ay 5, labeling for albumin in area O was decreased in PHN-rats, but not in PHN-CVF-rats, where it was then higher; in PI-IN-rats, some areas between e pithelial cells and subepithelial deposits were almost free of albumin labe ling at day 7. There was no evidence of epithelial cell detachment in any g roup at day 5, but on day 7 limited focal detachment was seen exclusively i n PHN-rats. In proteinuric rats, amorphous material that stained for albumi n could be seen in the urinary space, without any exocytosis of labeling by glomerular epithelial cells. A significant reduction of intensity of stain ing for anionic sites was seen in parallel in both groups, but only in the regions of the lamina rara externa adjacent to subepithelial deposits. This local loss of charge might contribute to enhanced permeability to albumin in both PHN- and PHN-CVF-rats. Changes in the appearance of the filtration slits and in the density and distribution of antigen recognised by monoclon al antibody 5-1-6 were similar in PHN- and PHN-CVF-rats at day 5. Complemen t depletion prevented neither the reduction in anionic sites of the GEM nor the changes in the slit diaphragm observed. These data suggest that albumi n leakage between the epithelial cell and the GEM (area O) could occur in P HN-rats, perhaps as a result of epithelial foot-process changes. This may b e the final link in the chain of events responsible for the onset of protei nuria in PHN.