The aim of this Program Project is to investigate the cellular biology of membrane function and dysfunction in renal epithelial and glomerular cells. The strategy will be to use convergent, advanced techniques in ultrastructural immunochemistry, freeze- fracture, molecular biology, biophysics and biochemistry to explore interrelated problems in different cell types. Specifically, membrane recycling and the effect of microfilament modulation on vasopressin-induced water flow will be examined. We will investigate the role of clathrin coated pits and vesicles in the recycling of membrane water channels, and attempt to isolate, purify and characterize the water channel protein(s). The interactive role of microfilaments in transepithelial water flow will be studied in intact cells and correlations made with in vitro systems which will evaluate purified actin and actin-associated protein interactions and their ability to undergo gel-sol transformation in the presence and absence of osmotic gradients. Membrane recycling of a defined protein, the H+ATPase, will be examined in proton secreting cells, taking advantage of a monoclonal antibody against H+ATPase. The relationship of H+ATPase molecules to other characteristic membrane features, including transport by unique nonclathrin coated vesicles, will be examined. These studies will be complemented by studies on cellular dysfunction of renal cells. The cellular and molecular biology of antigens involved in the pathogenesis of Heymann nephritis will be examined to determined how shedding versus endocytosis of a nephritogenic antigen, gp330, is achieved in glomerular epithelial cells. Glomerular mesangial cell function will be probed by determining if mediators of inflammation activate a calcium response in the mesangial cell, which in turn promotes contraction, proliferation, and prostanoid production that can be related to similar phenomona in diabetic nephropathy. By probing a number of general cell biological processes intimately involved with the dynamic regulation of membrane function, we hope to interactions can be defined at the molecular level. All of the projects involved in this research proposal share this common theme. It is likely that a number of problems in cell biology which are key to normal and abnormal cell function will be answered, enhancing our understanding of renal function and disease.
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