The overall focus of this project continues to be the molecular mechanisms of anion transport in the proximal tubule. Previous work on this project demonstrated the operation of at least three distinct anion exchangers in renal microvillus membrane vesicles (ie. Cl-formate, Cl- oxalate and oxalate SO4=CO3= exchangers), and support a novel model by which Cl-absorption in the proximal tubule involves uphill Cl uptake across the luminal membrane by exchange with formate and oxalate in parallel with organic anion recycling. Microperfusion studies in the proximal tubule carried out in collaboration with project #1 strongly supported this model. Studies are now proposed to test the hypothesis that activities of luminal membrane anion exchangers are appropriately regulated to permit independent regulation of proximal tubule NaHCO3 and NaCI reabsorption. Specifically, it will be determined whether luminal membrane anion exchangers are regulated in response to adaptive states and hormones known to modulate proximal tubule Na+-H+ exchange. Importantly, findings in isolated microvillus membrane vesicles will be correlated with results obtained in project #1 at the level of the microperfused proximal tubule in situ. In a related series of studies, affinity chromatography has been used to identify disulfonic stilbene-binding proteins with properties of anion transporters. In particular, a 71kD stilbene-binding protein with properties of the renal basolateral membrane Na+-HCO3-cotransport system was characterized. Monoclonal antibodies were generated that revealed that this protein is kidney-specific and is expressed on the basolateral membrane of multiple nephron segments where Na+-HCO3- cotransport activity has been described. Studies are now proposed to directly test the hypothesis that this 71kD stilbene-binding protein is in fact the renal basolateral membrane Na+-HCO3-cotransporter. The principal approach will be to isolate and clone a cDNA encoding this protein, and then to use this cDNA to demonstrate functional expression of Na+-HCO3- cotransporter is verified, studies will be performed to isolate cDNA encoding other isoforms of the Na+HCO3-cotransporter. Specific antisera will be generated for use in determining the cell and membrane sites of expression of different isoforms of the Na+-HCO3-cotransporter in the kidney and other regulation of renal C1- and HCO3-transport, and is therefore of relevance for understanding clinical disorders of NaCl and acid-base balance.
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