Regulation of V-ATPase-mediated Renal Proton Secretion

V-ATP酶介导的肾质子分泌的调节

基本信息

批准号：
7537222
负责人：
Teodor G. Paunescu
金额：
$ 13.47万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-01-01 至 2010-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7537222
关键词：
ATP phosphohydrolase Acid-Base Imbalance Acids Address Adenylate Cyclase Animal Model Back Basic Science Bicarbonates Cell membrane Cells Complex Cyclic AMP Cyclic AMP-Dependent Protein Kinases Defect Distal renal tubular acidosis Type 1 Duct (organ) structure Enzymes Epithelial Cells Fluorescence Genes Goals Holoenzymes Human Hybrids Image Intercalated Cell Kidney Knockout Mice Laboratories Measures Mediating Membrane Membrane Biology Messenger RNA Mus Mutation Organelles Physiological Process Protein Isoforms Proteins Proton Pump Protons Recovery Regulation Relative (related person)Renal function Replacement Therapy Research Role Sensorineural Hearing Loss Techniques Training Programs Transgenic Mice apical membrane base career in vivo interdisciplinary approach knockout animal laser capture microdissection novel programs protein expression response trafficking treatment strategy vacuolar H+-ATPase

项目摘要

This applicant proposes a program to prepare him for a career in academic basic science studying renal function, addressing the regulation of proton secretion in the kidney under physiological and patho- physiological conditions. The research will be conducted in the laboratory of Dr. Dennis Brown in the Program in Membrane Biology and Renal Unit, MGH. Renal H+ secretion is mainly mediated by the vacuolar proton-pumping ATPase (V-ATPase), an enzyme that also acidifies some intracellular organelles. However, when expressed on the plasma membrane, as in collecting duct A-type intercalated cells (1C),the V-ATPase mediates transepithelial H+ secretion. Defects in H+ secretion cause distal renal tubular acidosis (dRTA), associated with sensorineural deafness in humans. dRTA is caused by mutations in the gene encoding the 56 kDa B1 subunit isoform of the V-ATPase. We hypothesize that an alternative V-ATPase B subunit, the B2 isoform might serve as a replacement back-up that functionally replaces the B1 in renal H+ secretion under some conditions, because our available B1 subunit knockout mice are not acidotic and they express more B2 subunit in the apical membrane of 1C than do normal mice. Understanding the ways in which B1 and B2 assemble into V-ATPase complexes and the role of these subunits in V-ATPase targeting and trafficking processes could suggest novel treatment strategies by "isoform replacement therapy" in cells in which B1- mediated H+ secretion is defective. The main goal of this proposal and training program is use novel techniques and animal models to determine the relative role of the V-ATPase B1 and B2 isoforms in H+ secretion and V-ATPase trafficking in renal epithelial cells. How does the membrane expression of the B2 subunit increase in mice lacking B1, and how is this expression/trafficking process regulated? We will examine regulation of V-ATPase mRNA and protein expression in 1C from unique B1-knockout mice under different acid-base conditions. mRNA from 1C will be isolated by laser capture microdissection using new transgenic mice that express EGFP in 1C.Compensatory B2-V-ATPase function in 1C will be examined by pH ratio imaging. The role of the B1-interacting protein NHE-RF1 in V-ATPase trafficking will be addressed, and a role for the bicarbonate-stimulated soluble adenylate cyclase (sAC) in the membrane insertion of V- ATPases containing the B1 and B2 subunit will be examined using a multidisciplinary approach.

该申请人提出了一项计划，以准备他为肾脏研究的学术基础科学生涯做好准备功能，解决生理和病情下肾脏中质子分泌的调节生理条件。该研究将在丹尼斯·布朗博士的实验室中进行 MGH膜生物学和肾单位的计划。肾脏H+分泌主要由液泡介导质子泵式ATPase（V-ATPase），一种酶，也酸化了一些细胞内细胞器。然而，当在质膜上表达时，如收集导管A型插入细胞（1C）时，V-ATPase 介导旋转的H+分泌。 H+分泌的缺陷导致肾远端肾小管酸中毒（DRTA），与人类的感觉性耳聋有关。 DRTA是由编码基因突变引起的 V-ATPase的56 kDa B1亚基同工型。我们假设替代V-ATPase B亚基B2 同工型可以用作替代功能替代肾脏H+分泌B1的替代备份。某些条件，因为我们可用的B1亚基敲除小鼠不是酸性的，它们表达了更多 1C的顶膜中的B2亚基比正常小鼠的B2亚基。了解B1和B2的方式组装成V-ATPase复合物以及这些亚基在V-ATPase靶向和贩运中的作用过程可以通过在B1-的细胞中“同工型替代疗法”提出新的治疗策略。介导的H+分泌有缺陷。该提案和培训计划的主要目标是使用小说确定V-ATPase B1和B2同工型在H+的技术和动物模型肾上皮细胞中的分泌和V-ATPase运输。 B2的膜表达如何缺乏B1的小鼠的亚基增加，该表达/运输过程如何受到调节？我们将检查来自独特的B1敲除小鼠在1C中的V-ATPase mRNA和蛋白表达的调节不同的酸碱条件。使用新的，将通过激光捕获显微解剖来隔离1C的mRNA 在1C.com中表达EGFP的转基因小鼠将通过1C中的B2-V-ATPase功能检查 pH比成像。 B1相互作用蛋白NHE-RF1在V-ATPase运输中的作用将得到解决，以及碳酸氢盐刺激的可溶性腺酸环化酶（SAC）在V-的膜插入中的作用将使用多学科方法检查包含B1和B2亚基的ATPases。