REGULATION OF COLONIC H-K-ATPASE IN THE KIDNEY

描述（改编自申请人的摘要）：胃（g） 和结肠（c）H+/K+-ATP酶（HKA）亚型以低水平表达 在肾集合管（CD）细胞中，但这些HKA的功能作用 仍然不确定。 他们提出，cHKA特别发挥了关键作用， 在几种病理生理状态下肾脏HCO 3-、K+和Na+转运中。 我们的研究表明，cHKA mRNA和/或活性在一定条件下增加， 与CD（近端RTA）的HCO 3输送增加相关，或 缺钾（KD）。 有趣的是，垂体切除术（HPX）抑制了 在KD中cHKA表达增加，但在近端RTA中不增加。 他们 假设cHKA对酸碱平衡、K+和Na+稳态至关重要： cHKA的上调减弱了HCO 3-损失，在增加的情况下， HCO_3 ~-进入远端肾单位，加速K ~+和Na ~+重吸收。 为了阐明这一假设，我们建议检查分子和 cHKA的功能调节：a）三种模型与增加的 HCO 3-向远端小管的输送，但酸碱状态不同 平衡：乙酰唑胺（酸中毒）、氯耗竭（代谢紊乱），和 NaHCO 3负荷（正常）;和B）钾耗竭。 酸碱和 电解质的变化，在离体灌注CCD和OMCD中的HCO 3重吸收， cHKA的皮质、髓质和肾单位段mRNA和蛋白质将 在大鼠和小鼠（cHKA缺陷型转基因和 野生型）与KD或增加的HCO 3-递送到远端小管。 KD大鼠 将在HPX +激素替代中重复实验。 最后，他们 将研究cHKA的转录调控。 cHKA转基因小鼠 启动子-荧光素酶报告基因（PLR）构建体将在上文中进行研究 模型 结合上述（a）和（B）的数据，这些研究表明 在培养的肾细胞中研究的可能信号。 这些将是 用LR的缺失构建体瞬时转染，并在 体外条件，以确定启动子内的传感元件， cHKA cDNA。 深入了解cHKA的监管将大大提高我们的 了解与电解质相关的各种病理状况 和酸碱失衡

DESCRIPTION (Adapted from the Applicant's Abstract): 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.