The major goal of this research project is to elucidate the cellular mechanisms by which phosphate (Pi) is reabsorbed from the lumen of proximal tubules across the brush border membrane (BBM). The investigations are based on the general hypothesis that: (A). Na+-gradient-dependent uptake of Pi at luminal BBM is of higher capacity in early proximal convoluted tubules (PCT) and is determined mainly by dietary phosphorus (P) intake, whereas in late proximal straight segments - pars recta (PR) it is of lower capacity and is regulated mainly by hormonal stimuli; (B). that long-term """"""""adaptive"""""""" stimuli regulate Pi transport across BBM by changing rate or mode of biogenesis of the Na-Pi cotransporter (Na-Pi-COT) of BBM, whereas short-term """"""""rapid"""""""" stimuli act by the changing balance between Na-Pi-COT within BBM and Na-P-COT in cytoplasmic depot vesicles - recycling of Na-Pi-COT. Specific objectives of the planned research include: 1). To isolate the BBM vesicles (BBMV) containing Na+-Pi-COT specifically from early proximal segments (PCT) and from the late proximal segments (PR). 2). To determine intramembranous localization of Na-Pi-COT within BBM, and the major biochemical components involved in its function and regulation. 3). To elucidate the biogenesis of the Na-Pi-COT in BBM from PCT and PR. To determine incorporation of amino acid and monosaccharide precursors into Na-P-COT of BBM; these studies will be conducted on renal preparations in vitro and in vivo. 4). To determine the cellular mechanism of action of a) long-term adaptive stimuli (dietary Pi intake, glucocorticoids, thyroxine) and b) rapid modulatory stimuli (PTH, calcitonin, acute Pi load) on biogenesis and recycling of Na-Pi-COT. We will examine whether long-term stimuli modify the rate of synthesis or the mode of post-translational modification of Na-Pi-COT, whereas, rapid stimuli modulate the balance between active Na-Pi-COT inserted into BBM and nonfunctional Na-Pi-COT located in cytoplasmic vesicles (membrane recycling). These regulatory mechanisms will be examined separately in BBM from PCT and BBM from PR. 5). To determine the biochemical nature, intramembranous BBM localization, and axial nephron localization of the Na-Pi-COT defect occurring in the murine model of X-linked hypophosphatemic rickets (""""""""Hyp-mice"""""""").
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