Disorders of extracellular inorganic phosphate (Pi) concentration and impairment in Pi reabsorption are common clinical problems. Aging, diabetes mellitus, malignancy, alcoholism, transplantation, AIDS, and several therapeutic drugs are well known to cause or to be associated with hypophosphatemia or hyperphosphatemia, mainly by affecting renal tubular Pi transport. Disorders of phosphate metabolism have severe consequences on organ function including the cardiovascular system and bone mineralization. The kidney plays a critical role in the regulation of Pi homeostasis. The evidence to date indicates that regulation of the overall renal tubular Pi transport by dietary, hormonal, or metabolic factors occurs at the level of the proximal tubular apical brush border membrane (BBM) Na/Pi cotransport system (NaPi-IIa protein). Studies in our laboratory have determined that alterations in renal lipid composition, including cholesterol and glycosphingolipids play an important role in the regulation of renal Na/Pi cotransport activity and BBM membrane microdomains (or lipid rafts). The mechanisms how lipids modulate Na/Pi transport activity and NaPi-Iia protein expression and trafficking however are not well known and need to be elucidated. Our hypotheses are that perturbations in membrane cholesterol and glycosphingolipid composition regulate Na/Pi transport activity by coordinated cellular and molecular mechanisms that include I) modulating the formation of cholesterol and glycosphingolipid enriched lipid microdomains or lipid rafts that results in increased dynamic partitioning of the Na/Pi cotransporter protein in the lipid microdomains, II) modulating the lateral diffusion or mobile fraction and/or the clustering or aggregation and/or the conformational change of the Na/Pi transport proteins at the level of the apical membrane, and lII) modulating the vesicular trafficking of the Na/Pi transport protein, including endocytosis from and/or exocytosis to the apical membrane in response to alterations in extracellular Pi or PTH.
The specific aims of this proposal are: 1) To determine if alterations in cholesterol and/or glycosphingolipid composition modulate a) the dynamics of cholesterol and glycosphingolipid enriched membrane microdomains (lipid rafts), b) the partitioning of the Na/Pi cotransporter proteins in the cholesterol and glycosphingolipid enriched membrane microdomains, and e) the function or transport activity of Na/Pi cotransporter proteins in these domains; 2) To determine if alterations in cholesterol or glycosphingolipid composition modulates a) the lateral diffusion and mobility, or b) the clustering and aggregation, or e) the conformational state of Na/Pi protein molecules; 3) To determine if alterations in cholesterol or glycosphingolipid composition modulates a) the apical membrane expression of Na/Pi protein and b) the acute trafficking of Na/Pi protein in response to alterations in extracellular Pi or PTH and c) the role of the actin cytoskeleton in regulation of NaPi trafficking.
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