Inorganic phosphate (Pi) is an important constituent of bone and many important intracellular metabolites. The proximal tubule is the principal site where Pi is reabsorbed in the kidney. The initial and possibly rate- limiting step in the process is the coupled transport of Pi with Na+ across the luminal brush border membrane (BBM). The capacity of the BBM for Pi transport is altered by changes in the dietary Pi intake and by various hormones such as parathyroid hormone. While other factors may contribute to the regulation of renal Pi reabsorption, a specific defect in the adaptive mechanism of the Na+/Pi cotransport system has serious consequences for Pi homeostasis.
The specific aim of this proposal is to determine if membrane recycling is an important part of the mechanism whereby fast-acting regulators influence the renal Na+/Pi contransport system. This is part of a long-term goal aimed at understanding the regulation of renal Pi transport at the cellular level. A cultured renal epithelial OK cell line will be used as a model for the proximal tubule cell, and parathyroid hormone (inhibitory) and acute Pi deprivation (stimulatory) will be used as examples of fact acting regulators of Na+/Pi cotransport. One series of experiments will determine if these regulators change the rate of endocytosis or exocytosis, measured with fluid-phase markers. Other studies will determine if the normal response of Pi transport to these regulators is disrupted when endocytosis or exocytosis are altered by other maneuvers. Additional studies will iodinate proteins either in the plasma membrane or the endosomal membrane for direct assay of membrane recycling. At various times after attachment of 124 I, the cells will be disrupted and fractionated on a percoll density gradient in order to follow the shift of membrane proteins between the plasma membrane and endosomal fractions within the same gradient. Possible effects of parathyroid hormone and acute Pi deprivation on the rate of this intracellular shift will be determined. Specific labels for the Na+/Pi cotransporter will be used as part of these studies. A separate series of experiments will isolate endosomal membrane and, if so, to compare its characteristics with the BBM transport system. Taken together, these studies will determine if membrane recycling is an important part of the mechanism by which parathyroid hormone and acute Pi deprivation alter cell uptake of Pi.
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