The overall objective of this application is to determine the mechanisms of renal phosphate (Pi) regulation. The specific objective of this proposal is to determine the role of intrarenal dopamine production and of to other catecholamine in the regulation of Pi reabsorption. The hypothesis to be tested is that dopamine is an intrarenal paracrine substance synthesized in proximal tubules that specifically decreases Pi reabsorption during selective increases in dietary phosphate intake. An increase in renal dopamine synthesis is postulated to due to increased pyridoxal phosphate levels or to increased L-DOPA uptake by proximal tubule cells. Pyridoxal phosphate is a necessary cofactor for the conversion of DOPA to dopamine by DOPA decarboxylase. The effect of dietary Pi intake on renal dopamine production will be determined by measuring urinary catecholamines and their stable metabolites in metabolic balance studies. Dopamine formation form L- DOPA will also be measured in proximal tubule suspensions and microdissected rat proximal tubule subsegments prepared from rats fed different dietary Pi intakes. L-DOPA uptake by proximal tubules will be determined in rates fed different dietary Pi intakes using in vivo microperfusion and proximal tubule cell suspension. The role of changes in alkaline phosphatase in proximal tubule cells ad a mechanism for local increases in pyridoxal phosphate cofactor will be tested by measurement of these enzymes in proximal tubules harvested from the rats fed different Pi intakes. Studies using a specific inhibitor of alkaline phosphatase will determine the link between alkaline phosphatase, pyridoxal phosphate, and renal dopamine synthesis. The nephron sites, the specificity, and the cellular mechanisms of the inhibition of Pi reabsorption by dopamine and the possible interaction with PTH will be addressed by micropuncture studies and by using isolated renal brush border membranes. The second hypothesis to be tested is that the neural stimulation of renal alpha- and beta-adrenergic receptor activities increases renal Pi reabsorption during the need for Pi conservation. The possible role of stimulation of renal alpha- and beta-adrenoreceptors on Pi reabsorption will be explored. The possibility that stimulation of renal alpha-adrenoreceptors blunts the inhibition of Pi reabsorption by PTH in the proximal convoluted ut not the proximal straight tubule by attenuating PTH-stimulated adenylate cyclase activity will be addressed by a combination of micropuncture renal brush border membrane vesicles, and microdissected rat proximal tubule subsegment methodologies. The possibility that stimulation o renal beta- adrenoreceptors blunts the inhibition of Pi reabsorption by PTH in the proximal straight tubule not the proximal convoluted tubule by cAMP- independent mechanisms, will also be evaluated using similar methods as for the study of alpha-adrenoreceptors. These studies have the potential to provide crucial information for the understanding of the regulation of Pi homeostasis which impacts on the pathogenesis of urolithiasis.
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