LUMINAL ACIDIFICATION BY THE MEDULLARY COLLECTING DUCT

描述（改编自申请人的摘要）：功能数据 表明H+/K+-ATP酶在这一应用中起着主要作用。 外髓内条在尿酸化中的作用 正常（K-充盈）和K-限制期间的集合管（OMCDi） 情节 结构证据表明H+/K+-ATP酶是 存在于OMCDi的闰细胞和主细胞中， 内髓集合管（IMCD）细胞。 进一步的证据表明 H+/K+-ATP酶的一个以上的亚基存在于细胞中， 肾 因此，申请人假设：1）肾脏H+/K+-ATP酶是 一个主要的质子泵，负责管腔酸化的 OMCDi; 2）肾脏H+/K+-ATP酶活性，生化定义，代表在 a亚基的至少两种同种型;和3）肾H+/K+-ATP酶是 负责由OMCDi的主细胞和由 IMCD的IMCD细胞以及收集的闰细胞 管道。 因此，申请人制定了以下三个具体目标：1） 详细描述了管腔酸化的机制和相关的 OMCDi中的顶端K通道。 一系列微灌流和膜片钳技术 实验提出，将详细研究的机制， OMCDi中的管腔酸化和存在于 OMCDi的顶膜，分别; 2）以确定α和 OMCDi中存在的H+/K+-ATP酶的β亚型。 这一策略 确定了一种新的肾脏H，OMCDi-ATPase a亚型， OMCDi的H+/K+-ATP酶α和α亚基同种型; 3）确定 H+/K+-ATP酶α和β亚基mRNA的细胞分布， 肾脏中的蛋白质。 一系列利用原位杂交的研究 免疫组化检测H+/K+-ATP酶的分布 亚基mRNA和蛋白质。 具体来说，本地化 在OMCDi的主细胞和闰间细胞中， 将对IMCD进行审查。 这种功能和结构一体化的方法产生了重要的 关于H+/K+-ATP酶基本作用机制的新信息， 存在于肾脏中的催化亚基的同种型，以及它们的 分布 因此，这些研究代表了一个系统的检查， 肾脏H+/K+-ATP酶的研究开辟了重要的新领域。 特别是，这些实验应该提供洞察的基本 H+/K+-ATP酶的质子分泌机制，因此， 应该有显着的影响以外的管腔酸化的 OMCDi。

DESCRIPTION (Adapted from the Applicant's Abstract): The functional data presented in this application indicate that an H+/K+-ATPase plays a major role in urinary acidification by the inner stripe of the outer medullary collecting duct (OMCDi) during normal (K-replete) as well as K-restricted circumstances. The structural evidence indicates that an H+/K+-ATPase is present in both intercalated cells and principal cells of the OMCDi and in inner medullary collecting duct (IMCD) cells. Further evidence suggests that more than one (a subunit) isoform of H+/K+-ATPase is present in the kidney. Accordingly, the applicant hypothesizes: 1) renal H+/K+-ATPase is a major proton pump that is responsible for luminal acidification by the OMCDi; 2) renal H+/K+-ATPase activity, defined biochemically, represents at least two isoforms for the a subunit; and 3) renal H+/K+-ATPase is responsible for luminal acidification by principal cells of the OMCDi and by IMCD cells of the IMCD as well as by intercalated cells of the collecting duct. Thus, the applicant formulates the following three Specific Aims: 1) to characterize in detail the mechanism of luminal acidification and associated apical K channels in the OMCDi. A series of microperfusion and patch-clamp experiments are proposed that will examine in detail the mechanism of luminal acidification in the OMCDi and the ion channels present at the apical membrane of the OMCDi, respectively; 2) to determine the alpha and beta isoforms of H+/K+-ATPase present in the OMCDi. A strategy that has identified a novel renal H,OMCDi-ATPase a isoform is proposed to determine H+/K+-ATPase alpha and alpha subunit isoforms of the OMCDi; 3) to determine the cellular distribution of H+/K+-ATPase alpha and beta subunit mRNAs and proteins in the kidney. A series of studies utilizing in situ hybridization and immunohistochemistry will examine the distribution of H+/K+-ATPase subunit mRNA and protein within the kidney. Specifically, the localization in principal cells and intercalated cells of the OMCDi and in IMCD cells of the IMCD will be examined. This integrative functional and structural approach has yielded important new information regarding the basic mechanism of operation of H+/K+-ATPase, the isoforms of the catalytic subunit present in the kidney, and their distribution. Accordingly, these studies represent a systemic examination of renal H+/K+-ATPase and have opened important new areas of investigation. In particular, these experiments should provide insight into the fundamental mechanism of proton secretion by H+/K+-ATPase in general and, as such, should have significant implications beyond luminal acidification by the OMCDi.