LUMINAL ACIDIFICATION BY THE MEDULLARY COLLECTING DUCT

描述(改编自申请人摘要)：功能数据 在本申请中提出的H+/K+-ATPase在 外髓内带在尿酸中的作用 正常(K-完全)和K-受限时的集合管(OMCDi) 情况。结构证据表明，H+/K+-ATPase是 在OMCDi的嵌合细胞和主细胞中均有表达， 内髓集合管(IMCD)细胞。进一步的证据表明 H+/K+-ATPase存在一个以上的亚基。 肾脏。因此，申请人假设：1)肾脏H+/K+-ATPase是 一个主要的质子泵，负责腔内酸化 2)肾H+/K+-ATPase活性，生化定义为 α亚基至少有两种异构体；3)肾脏H+/K+-ATPase是 负责由OMCDI的主细胞和由 IMCD细胞的IMCD以及集合的间质细胞 风管。 因此，申请人制定了以下三个具体目标：1) 详细描述管腔酸化的机理和与之相关的 OMCDi中的心尖K通道。一系列微灌流和膜片钳技术 提出了实验，将详细检查的机制， OMCDI中的管腔酸化和存在于 分别测定OMCDI根尖膜的α和 OMCDi中存在H+/K+-ATPase的β亚型。这一战略已经 鉴定了一种新的肾脏H，OMCDI-ATPase，提出了一种新的同功酶 OMCDi的H+/K+-ATPaseα和α亚基亚基异构体；3)确定 H+/K+-ATPaseα和β亚基mRNAs的细胞分布 肾脏中的蛋白质。利用原位杂交进行的一系列研究 免疫组织化学检测H+/K+-ATPase的分布 肾脏内的亚单位信使核糖核酸和蛋白质。具体地说，本地化 在OMCDi的主细胞和间质细胞以及在IMCD细胞中 将对IMCD进行审查。 这一综合的功能和结构方法产生了重要的 关于H+/K+-ATPase基本作用机制的新信息， 肾脏中存在的催化亚基的异构体，以及它们的 分发。因此，这些研究代表了一种系统性的检查。 为肾脏H+/K+-ATPase的研究开辟了重要的新领域。 特别是，这些实验应该提供对基本原理的洞察 H+/K+-ATPase一般的质子分泌机制，因此， 应该具有重大的影响，而不仅仅是由 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.