MECHANISM OF RENAL ACID/BASE HOMEOSTASIS

大量证据表明，H+，K+-ATP酶在肾脏K+稳态和酸碱平衡中起着关键作用。 H ~+，K ~+-ATP酶对慢性低钾血症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）。 两个，也许更多，α-亚基亚型已被定位于哺乳动物的肾脏，但不确定性仍然存在于特定的α亚型参与节段性K+和酸碱平衡。 转运研究被迫依赖于H+，K+-ATP酶的“特异性”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+吸收组分。 由于H+，K+-ATP酶对哇巴因和Sch 28080表现出不同的敏感性，因此尚不确定转运的调节和对慢性代谢性酸中毒和低钾血症的明确反应是否是HK α 1，HK α 2，HK α 4或尚未鉴定的同种型功能调节的结果。本研究旨在阐明在分子和功能水平上调节集合管节段和培养的髓集合细胞中H+，K+-ATP酶的病理生理因素。 通过用反义HK α 1、HK α 2和HK α 4转染mOMCD 1和mIMCD-3细胞，我们将确定哪种亚型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H+，K+-ATP酶活性的分子当量，其特征在于III型，这是由慢性K+耗竭上调。 这种方法需要合成和筛选消减cDNA文库。 第三，我们将确定醛固酮或内皮素是否调节培养的mOMCD 1和mIMCD-3细胞中的H+，K+-ATP酶功能，如果是这样，我们将描绘负责的α，H+，K+-ATP酶亚型。最后，我们将确定的H+，K+-ATP酶的贡献，净酸分泌OMCDis灌注在体外代谢性骨坏死过程中没有K+耗竭。 然后在mOMCD 1细胞培养物中模拟伴有和不伴有低钾血症的代谢紊乱，以描述低钾血症或碱血症本身是否上调α H+，K+-ATP酶，如果是，则哪种α H+，K+-ATP酶亚型对每种条件有特异性反应。 这些研究将有助于阐明K+耗竭维持代谢平衡的途径。 H+，K+-ATP酶仍然是遗传性和获得性远端肾小管酸中毒结构和功能异常的候选基因。 为了更全面地了解这组疾病，阐明这一转运蛋白家族的分子调控的基础研究将是必要的，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.