REGULATION OF RENAL H+/K+ EXCHANGE

哺乳动物肾脏的皮质集合小管（CCT）是 负责尿液pH和钾（K）的最终调节 浓度. 参与该调节的2种主要细胞类型是 钠转运主细胞与酸碱调节 闰细胞 最近的证据表明存在胃- 型酸转运蛋白（H-K ATP酶）。 这 一项提案的重点是功能确定的H-K的贡献， H-K ATP酶对酸碱平衡和钾稳态过程的调节作用 皮质集合小管的闰细胞，利用 细胞内pH和钙的荧光测量。 将通过三个相关的准备工作来实现六个具体目标。 H-K交换器的功能活性将通过以下方式进行监测： 测量K依赖性细胞内pH恢复的速率 施加酸负荷。 这种交换剂在钾重吸收中的作用 和H分泌和调节pH值，K和醛固酮将被检查。 H-K交换器的基本表征包括评估 交换器的功能相似性，以充分表征胃 H-K ATP酶，交换器的顶端/基底侧定位，以及 定义其在基础水平下对细胞内pH调节的贡献 条件 适合于选定目标的准备工作包括： 开放的皮质集合小管，分离的壁细胞，和分离的 灌注的皮质集合小管，装载细胞内pH 指示剂BCECF或细胞内Ca指示剂Fura-2。 此外，本发明还提供了一种方法， BCECF和PBFI的未酯化形式，K-特异性指示剂， 用于灌注小管研究。 H-K交换机制的功能表征和定位 这种机制可能有助于解释一些临床观察结果： 1.低血清钾与细胞外膜增生的关系; 2. 代谢性酸中毒与钾排泄减少关系 这会使血清K升高到一定程度， 心脏的活动。 这些研究的结果将提供 关于正常和病理K状态和意志的重要信息 影响我们对代谢异常时钾平衡的理解 和利尿剂治疗。

The cortical collecting tubule (CCT) of the mammalian kidney is responsible for the final regulation of urine pH and potassium (K) concentration. The 2 major cell types involved in this regulation are the Na-transporting principal cells and the acid-base regulating intercalated cells. recent evidence suggests the presence of a gastric- type acid transporter (H-K ATPase) in the renal collecting duct. This proposal focuses on the contribution of a functionally identified H-K exchanger (H-K ATPase) to the process of acid-base and K homeostasis in intercalated cells of the cortical collecting tubule, utilizing fluorescence measurements of intracellular pH and calcium. Six specific aims will be addressed using three related preparations. The functional activity of the H-K exchanger will be monitored by measuring the rate of K-dependent intracellular pH recovery in response to an imposed acid load. the role of this exchanger in K reabsorption and H secretion and regulation by pH, K and aldosterone will be examined. Basic characterization of the H-K exchanger includes assessing the functional similarity of the exchanger to the well-characterized gastric H-K ATPase, apical/basolateral localization of the exchanger, and defining its contribution to intracellular pH regulation under basal conditions. Preparations appropriate to selected aims include: split open cortical collecting tubules, isolated parietal cells, and isolated perfused cortical collecting tubules, loaded with the intracellular pH indicator, BCECF or the intracellular Ca indicator, Fura-2. In addition, the unesterified forms of BCECF and PBFI, a K-specific indicator, will be used for perfused tubule studies. Functional characterization of an H-K exchange mechanism and localization of this mechanism may help explain a number of clinical observations: 1. the association between low serum K and extracellular alkalosis, 2. the relationship between metabolic acidosis and reduced excretion of K which can elevate serum K to a point where it interferes with electrical activity of the heart. The results of these studies will provide important information on normal and pathological K states and will influence our understanding of K balance during metabolic abnormalities and diuretic therapy.