The purpose of this renewal application is to continue studies on the ultrastructural distribution and biology of laminin within developing and mature renal glomerular and tubular basement membranes (GBM and TBM). First, the epitopes for nine different at anti-mouse laminin monoclonal antibodies will be identified using biochemical mapping techniques: purified polypeptides from proteolytic digests and separate laminin chains will be assayed for immunoreactivity by dot and electroblotting and rotary shadowing electron microscopy. Whether binding of these monoclonals affect the biological functions of laminin in vitro will also be tested in cell adhesion and matrix assembly experiments. These monoclonals will then be localized within the GBM, TBM, and non- renal basement membranes using pre- and post-embedding immunoelectron microscopy. Second, these and other polyclonal and monoclonal antibodies against collagen type IV, heparan sulfate proteoglycans, and fibronectin will be intravenously injected into newborn mice and rats to map the distribution of these components during assembly of the GBM and TBM by developing nephrons. Pulse- chase studies in vivo should also unambiguously identify the origin and fate of these molecules during GBM assembly. Double-label immunolocalization studies will also show whether these components co-localize to the same subcellular compartments in the secretory pathway within developing endothelial, mesangial, and glomerular and tubular epithelial cells. In addition, comparisions of basement membrane synthesized in transfilter cultures of nephrogenic mesenchyme and spinal cord with that synthesized in vivo will indicate how the endothelium contributes to GBM formation. Third, the process of kidney and intestinal epithelial basement membrane biosynthesis, assembly, and turnover with aging will be examined in mature mice and rats. These projects will provide detailed new information on the molecular architecture of basement membranes as well a mechanisms for their assembly during development and disassembly during turnover.
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