The studies proposed in this application are divided into three sections depending on the technique used. They are all designed to answer fundamental questions concerning the physiology and pathophysiology of distal urinary acidification. The first set of studies uses clearance techniques and will attempt to use the ability to lower the urine pC02 as an index of distal acidification. We believe that the titration of nonbicarbonate buffer consumes C02 and in the absence of bicarbonate buffer lowers the final urine pC02 to a value below that of blood. We also believe that this consumption of C02 can be disclosed in the presence of urinary bicarbonate by the simultaneous infusion of carbonic anhydrase. These studies will also examine the role of aldosterone on both cortical and medullary collecting duct acidification. The second group of studies uses the turtle bladder as a probe. It will attempt to examine the role of transepithelial voltage on carbonic anhydrase-independent acidification in this membrane. We will also attempt to dissect the different transport properties of the granular and mitochondrial rich cells in this membrane. The relationship of voltage to the backleak produced by the antifungal antibiotic amphotericin B will be examined, as will the effect of amiloride on bicarbonate permeability. Finally, the effect of vanadate, a well known inhibitor of proton secretion, on bicarbonate secretion will be examined. The third section of these studies deals with the role of proton ATPase in collecting duct acidification. These studies will test the hypothesis that proton ATPase can be measured in the mammalian collecting tubule and that its activity correlates with the state of acidification in this nephron segment. These studies will make use of the technique of enzyme analysis in in vitro nephron fragments. Taken as a whole, these studies are designed to continue our long-term examination of the forces which control distal urinary acidification under normal and abnormal circumstances.