LUMINAL ACIDIFICATION BY THE MEDULLARY COLLECTING DUCT

髓质收集管的管腔酸化

基本信息

批准号：
2749548
负责人：
Charles S Wingo
金额：
$ 15.57万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-08-01 至 2000-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2749548
关键词：
adenosinetriphosphatase enzyme structure immunocytochemistry in situ hybridization isozymes kidney function laboratory rabbit potassium channel renal medulla urine acidity voltage /patch clamp

项目摘要

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.

描述（改编自申请人的摘要）：功能数据本申请中提出的 H+/K+-ATP 酶在外髓内条纹在尿液酸化中的作用正常（K 充足）和 K 受限期间的集合管 (OMCDi) 情况。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。进一步的证据表明多于一种（亚基）H+/K+-ATPase 亚型存在于肾。因此，申请人假设：1)肾H+/K+-ATP酶是一个主要的质子泵，负责管腔酸化 OMDI; 2) 肾 H+/K+-ATP 酶活性，以生化定义，表示为 a 亚基至少有两种同工型； 3) 肾 H+/K+-ATP 酶是负责 OMCDi 主细胞的管腔酸化和 IMCD 的 IMCD 细胞以及集合的嵌入细胞管。因此，申请人制定了以下三个具体目标： 1) 详细描述管腔酸化和相关的机制 OMCDi 中的心尖 K 通道。微灌注和膜片钳系列提出的实验将详细检查其机制 OMCDi 中的管腔酸化和存在于分别为 OMCDi 的顶膜； 2）确定α和 OMCDi 中存在 H+/K+-ATP 酶的 beta 亚型。一个策略有鉴定出一种新型肾 H,OMCDi-ATPase a 亚型，建议确定 OMCDi 的 H+/K+-ATPase α 和 α 亚基亚型； 3）确定 H+/K+-ATPase α 和 β 亚基 mRNA 的细胞分布以及肾脏中的蛋白质。利用原位杂交的一系列研究免疫组织化学将检查 H+/K+-ATPase 的分布肾脏内的亚基 mRNA 和蛋白质。具体来说，本土化在 OMCDi 的主细胞和闰细胞以及 IMCD 细胞中 IMCD 将受到审查。这种综合功能和结构方法产生了重要的有关 H+/K+-ATP 酶基本运作机制的新信息，肾脏中存在的催化亚基的异构体及其分配。因此，这些研究代表了系统性检查肾 H+/K+-ATP 酶的研究，并开辟了重要的新研究领域。特别是，这些实验应该提供对基本原理的深入了解。 H+/K+-ATP酶分泌质子的一般机制，因此，除了管腔酸化之外，还应具有重大影响 OMCDi。