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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：
7754663
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/7754663
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.

这位申请人提出了一个计划，为他在学术基础科学研究肾脏的职业生涯做准备