The long term objective of these studies is to characterize endocytosis in renal epithelia in order to better understand how endocytosis participates in renal disease processes. Endocytosis is an important function in cell biology, and in kidney epithelia plays a particularly important role as it participates in the functions of excretion and resorption. Renal ion transport is partially regulated by endocytic insertion and retrieval of transporters, and the signals regulating this endocytic traffic are themselves regulated by endocytosis of hormones. Small macromolecules in the tubule filtrate are reclaimed via furious rates of apical endocytosis in the proximal tubule which is consequently particularly sensitive to the contents of the tubule filtrate, with tubular damage resulting from uptake of filtered aminoglycoside antibiotics and from the uptake of excessive or toxic proteins during proteinuria. Studies of fibroblasts have established that quantitative fluorescence microscopy may be used to characterize the pH, fusion accessibility and the kinetics of ligand accumulation and sorting in endosomes. The intracellular endocytic kinetics have elucidated mechanisms of endocytic sorting and measurements of endosome pH have established the role of endosome acidification in the processes of lysosomal hydrolysis, virus and toxin penetration into the cytosol and intracellular drug sequestration. Although these processes are critical to normal and pathological function of renal epithelia, comparable studies have not yet been conducted in polarized epithelial cells due to technical difficulties. With the development of improved optical instrumentation, appropriate digital image processing techniques and transfected model cell systems, these studies of epithelial cells are now feasible. Specifically, the proposed studies will first address the regulation of endosome pH in model renal epithelia. A complete and specific characterization of endosomes on the recycling, degradative and transcytotic pathways derived from the apical and basolateral plasma membrane domains will be conducted using fluorescence ratio confocal microscopy of living cells. The mechanisms of endocytic sorting will also be addressed, using similar techniques of quantitative confocal microscopy. Particular types of endosomes can be functionally identified on the basis of their contents and the kinetics with which different ligands accumulate in, and efflux from these endosomes can elucidate specific steps of endocytic sorting. Biologically relevant factors regulating or perturbing endosome acidification and endocytic trafficking will also be studied. These studies address fundamental processes of endocytosis which have proven to be critical to proper cell function in non-polarized cells, but have not been studied in epithelial cells. The information provided by this research will be valuable to both epithelial cell biologists and also to renal biologists seeking to understand physiological and pathological renal cellular processes.
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