The glycoprotein laminin is a major component of all basement membranes, but its ultrastructural distribution is uncertain. The structural modifications that occur within basement membranes during disease are also largely unknown. The goals of this project are to (1) determine the normal ultrastructural distribution of laminin within renal glomerular basement membranes (GBM). A new post-embedding method of immunocytochemistry will be used. Conjugates of affinity purified sheep or rabbit anti-laminin IgG (Alpha L) and biotin will be intravenously injected into rats and kidneys will be fixed in situ. Ultrathin sections of Lowicryl and epoxy-embedded tissue will then be stained with a particulate tracer, avidin-colloidal gold. Biotin conjugates of monoclonal Alpha L against pepsin and elastase resistant laminin fragments will also be injected and localized. These results will then be compared with those obtained with the enzymatic tracer, horseradish peroxidase (HRP)-Alpha L. These techniques will also be extended to localize within the GBM affinity purified and monoclonal anti-type IV collagen IgG. (2) To trace GBM development and turnover, Alpha L-biotin and Alpha L-HRP will be injected into newborn rats and kidneys will be examined at intervals from 1 hr to 18 months thereafter. Double label experiments will also be conducted to determine whether laminin is added only to new capillary loops or to existing GBM as well. (3) A cross-reacting, laminin-like molecule in rat kidney extracts will be immunopurified, examined by rotary shadowing, tested as an autoantigen in rats, and used for further ultrastructural studies. (4) In an accelerated model of glomerulonephritis, rats that have been preimmunized against rabbit Alpha L will receive injections of Alpha L. The resulting proteinuria and ultrastructural changes will be correlated with the titles of rat anti-Alpha L. This experiment differs significantly from previous work because it utilizes antibodies solely against laminin to create pathological changes. (5) Nephrotoxic nephritis (NTN) will be induced in rats by the injection of rabbit anti-kidney IgG. The distribution of laminin during the heterologous and autologous phases of NTN will then be determined by injecting Alpha L, Alpha L-biotin, and AlphaL-HRP. These will serve as specific GBM probes that can be visualized by immunofluorescent and immunoelectron microscopy. Of particular interest will be whether infiltrating neutrophils, monocytes, and lymphocytes affect the distribution of GBM laminin. This research will provide new information on the fundamental structure of basement membranes during health and disease.
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