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.

该申请人提出了一项计划，为他从事肾脏学术基础科学研究的职业生涯做好准备功能，解决生理和病理条件下肾脏质子分泌的调节生理条件。该研究将在丹尼斯·布朗博士的实验室进行麻省总医院膜生物学和肾脏科项目。肾H+分泌主要由液泡介导质子泵 ATP 酶（V-ATP 酶），一种还能酸化某些细胞内细胞器的酶。然而，当在质膜上表达时，如在集合管 A 型闰细胞 (1C) 中，V-ATP 酶介导跨上皮 H+ 分泌。 H+ 分泌缺陷导致远端肾小管酸中毒 (dRTA)，与人类感音神经性耳聋有关。 dRTA 是由编码基因突变引起的 V-ATP 酶的 56 kDa B1 亚基亚型。我们假设另一种 V-ATPase B 亚基 B2 同工型可能作为替代后备，在功能上替代肾 H+ 分泌中的 B1 在某些情况下，因为我们现有的 B1 亚基敲除小鼠不会出现酸中毒，并且它们表达更多 1C顶膜中的B2亚基比正常小鼠少。理解B1和B2的方式组装成 V-ATP 酶复合物以及这些亚基在 V-ATP 酶靶向和运输中的作用过程可以通过在 B1- 细胞中的“异构体替代疗法”来建议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新颖的确定 V-ATP 酶 B1 和 B2 亚型在 H+ 中的相对作用的技术和动物模型肾上皮细胞的分泌和 V-ATP 酶运输。 B2的膜表达如何缺乏 B1 的小鼠中亚基增加，这种表达/运输过程是如何调节的？我们将检查来自独特 B1 敲除小鼠的 1C 中 V-ATPase mRNA 和蛋白质表达的调节酸碱条件不同。 1C 的 mRNA 将通过激光捕获显微切割分离，使用新的在 1C 中表达 EGFP 的转基因小鼠。 1C 中的补偿性 B2-V-ATPase 功能将通过以下方法进行检查 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苷酸环化酶 (sAC) 在 V- 膜插入中的作用包含 B1 和 B2 亚基的 ATPase 将使用多学科方法进行检查。