MECHANISM OF RENAL ACID/BASE HOMEOSTASIS

大量证据表明，H+，K+-ATPase在肾脏K+动态平衡和酸碱平衡中起着关键作用。H+，K+-ATPase对慢性低血钾和慢性酸中毒的调节反应定位于外髓和内髓集合管(OMCD和IMCD)。哺乳动物肾脏中有两种或更多的α-亚基，但关于特定的α亚基亚基参与节段性K+和酸碱动态平衡的不确定性仍然存在。运输研究被迫依赖H+，K+-ATPase的“特定”抑制剂(如SCH 28080)的作用，以确定可归因于该转运蛋白的碳酸氢盐和/或K+吸收的成分。由于H+，K+-ATPase对哇巴因和SCH 28080表现出不同的敏感性，尚不能确切地知道转运的调节以及对慢性代谢性酸中毒和低钾血症的良好反应是HKalpha1、HKalpha2、HKalpha4功能调节的结果，还是尚未确定的亚型。本研究旨在阐明在分子水平和功能水平上，在集合管节段的H+，K+-ATPase和培养的髓质集合胞中进行调节的病理生理因素。通过将反义HKalpah1、HKalpha2和HKalpha4导入mOMCD1和mIMCD-3细胞，我们将确定哪个异构体负责对慢性低钾血症的广泛接受的适应性反应。然后我们将定义被描述为III型的H+，K+-ATPase活性的分子等价物，该酶活性因慢性K+耗竭而上调。这一方法需要合成和筛选消减cDNA文库。第三，我们将确定在培养的mOMCD1和mIMCD-3细胞中，醛固酮或内皮素是否调节H+，K+-ATPase的功能，如果是这样的话，我们将描绘出与此相关的α，H+，K+-ATPase异构体。最后，我们将确定在没有K+耗竭的代谢性碱中毒期间，H+，K+-ATPase对体外灌流的OMCD中净酸分泌的贡献。然后，在培养的mOMCD1细胞中模拟代谢性碱中毒伴和不伴低钾血症，以描绘低钾血症或碱性血症本身是否上调αH+，K+-ATPase，如果是这样，哪个αH+，K+-ATPase亚型对每种情况有特定的反应。这些研究将有助于阐明K+耗竭维持代谢性碱中毒的途径。在遗传性和获得性远端肾小管酸中毒中，H+，K+-ATPase仍然是结构和功能异常的候选基因。为了更全面地了解这一组疾病，有必要进行基础研究，阐明这一家族转运蛋白的分子调控，以进一步了解这种疾病的病理生理学。

Abundant evidence has established a pivotal role for an H+, K+- ATPase in renal K+ homeostasis and acid-base balance. The regulatory response of the H+, K+-ATPases to chronic hypokalemia and chronic acidosis has been localized to the outer medullary and inner medullary collecting ducts (OMCD and IMCD). Two, and perhaps more, alpha-subunit isoforms have been localized to the mammalian kidney, but uncertainties remain with respect to participation of specific alpha isoforms in segmental K+ and acid-base homeostasis. Transport studies have been forced to rely on the effect of "specific" inhibitors of the H+, K+-ATPase (such as Sch 28080) to identify that component of bicarbonate and/or K+ absorption attributable to this transporter. Since the H+, K+-ATPases exhibit differing sensitivities to ouabain and Sch 28080, it is not known with certainty if modulations in transport and the well established response to chronic metabolic acidosis and hypokalemia are the result of modulation in function of HKalpha1, HKalpha2, HKalpha4, or yet to be identified isoforms. This study is designed to elucidate the pathophysiologic factors which regulate at both molecular and functional levels, at H+, K+-ATPases in collecting duct segments, and in medullary collecting cells in culture. By transfecting mOMCD1, and mIMCD-3 cells with anti-sense HKalpah1, HKalpha2, and HKalpha4, we will define which isoform is responsible for the well-accepted adaptive response to chronic hypokalemia. We will then define the molecular equivalent of the H+, K+-ATPase enzymatic activity characterized as type III, which is upregulated by chronic K+ depletion. This approach will required the synthesis and screening of a subtraction cDNA library. Thirdly, we will determine if aldosterone or endothelin regulate H+, K+-ATPase function in mOMCD1 and mIMCD-3 cells in culture, and if so, we will delineate the alpha, H+, K+-ATPase iosoform responsible. Finally, we will define the contribution of the H+, K+-ATPases to net acid secretion in the OMCDis perfused in vitro during metabolic alkalosis without K+ depletion. Metabolic alkalosis with and without hypokalemia will then be simulated in mOMCD1, cells in culture to delineate whether hypokalemia or alkalemia per se upregulates the alpha H+, K+-ATPase, and if so, which alpha H+, K+-ATPase isoform responds specifically to each condition. These studies will help to elucidate the means by which K+ depletion can maintain metabolic alkalosis. The H+, K+- ATPase remains a candidate gene for abnormal structure and function in inherited and acquired forms of distal renal tubular acidosis. To understand this group of disorders more completely, fundamental studies which elucidate the molecular regulation of this family of transporters will be necessary to further our understanding of the pathophysiology of this disorder.