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已经局限于外髓和内髓集管（OMCD和IMCD）。两种，也许更多，α -亚基亚型已经定位于哺乳动物肾脏，但关于特定α -亚型参与K+和酸碱稳态的不确定性仍然存在。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，以确定归因于该转运体的碳酸氢盐成分和/或K+吸收。由于H+， K+- atp酶对瓦巴因和Sch 28080表现出不同的敏感性，因此尚不确定运输中的调节以及对慢性代谢性酸中毒和低钾血症的既定反应是否是HKalpha1， HKalpha2， HKalpha4功能调节的结果，或者尚未确定的亚型。本研究旨在阐明在分子和功能水平上调控收集管段H+， K+- atp酶和培养的髓质收集细胞的病理生理因素。通过将反义hkalpa1、HKalpha2和HKalpha4转染mOMCD1和mIMCD-3细胞，我们将确定哪一种异构体对慢性低钾血症的适应性反应负责。然后，我们将定义H+， K+- atp酶活性的分子等价物，其特征为III型，这是由慢性K+耗尽而上调的。这种方法需要一个减法cDNA文库的合成和筛选。第三，我们将确定醛固酮或内皮素是否在培养的mOMCD1和mIMCD-3细胞中调节H+， K+- atp酶的功能，如果是这样，我们将描述α， H+， K+- atp酶的作用。最后，我们将确定H+， K+- atp酶在体外代谢性碱中毒过程中对OMCDis净酸分泌的贡献。然后在培养的mOMCD1细胞中模拟有或没有低钾血症的代谢性碱中毒，以描绘低钾血症或碱血症本身是否上调α H+， K+- atp酶，如果是，哪种α H+， K+- atp酶异构体对每种情况有特异性反应。这些研究将有助于阐明钾离子耗竭维持代谢性碱中毒的途径。H+， K+- atp酶仍然是遗传和获得性远端肾小管酸中毒中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对于进一步了解这一疾病的病理生理学是必要的。

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.