Both the gastric (g) and colonic (c) isoforms of H+/K+-ATPase (HKA) are expressed at low levels in renal collecting duct (CD) cells but the functional role(s) for these HKA remain uncertain. They propose that cHKA specifically plays a pivotal role in renal HCO3-, K+ and Na+ transport in several pathophysiologic states. Our studies show that cHKA mRNA and or activity is increased in conditions associated with increased HCO3- delivery to the CD (proximal RTA) or in potassium depletion (KD). Interestingly, hypophysectomy (HPX) suppresses increased expression of cHKA in KD but not in proximal RTA. They hypothesize that cHKA is vital to acid-base, K+ and Na+ homeostasis: Upregulation of cHKA blunts HCO3- loss in instances of increased delivery of HCO3- to the distal nephron and accelerates K+ and Na+ reabsorption in KD. To elucidate this hypothesis, we propose to examine the molecular and functional regulation of cHKA in: a) three models associated with increased delivery of HCO3- to the distal tubule but different states of acid-base balance: acetazolamide (acidosis), chloride depletion (alkalosis), and NaHCO3 loading (normal); and b) in potassium depletion. Acid-base and electrolyte changes, HCO3-reabsorption in isolated perfused CCD and OMCD, and cortical, medullary, and nephron-segment mRNA and protein for cHKA will be determined in rats and mice (both cHKA-deficient transgenic and wild-type) with KD or increased HCO3-delivery to distal tubules. In KD rats experiments will be repeated in HPX + hormonal replacement. Lastly, they will examine transcriptional regulation of cHKA. Mice transgenic for a cHKA promoter-luciferase reporter (PLR) construct will be studied in the above models. Coupled with the data from (a) and (b) above, these studies suggest possible signals for study in cultured renal cells. These will be transiently transfected with deleting constructs of LR and studied under in vitro conditions to determine the sensing elements within the promoter of cHKA cDNA. Insight into regulation of cHKA will significantly enhance our knowledge on a variety of pathologic conditions associated with electrolyte and acid-base abnormalities.
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