Regulation of V-ATPase-mediated Renal Proton Secretion

V-ATP酶介导的肾质子分泌的调节

• 批准号: 7993809
• 负责人: Teodor G. Paunescu
• 金额: $ 5.4万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-15 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7993809
• 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

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.

申请人提出了一个计划，以准备他的职业生涯在学术基础科学研究肾 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。这项研究将在丹尼斯·布朗博士的实验室进行， 在膜生物学和肾脏单位，MGH程序。肾脏H+分泌主要由空泡介导， 质子泵ATP酶（V-ATP酶），一种也酸化某些细胞内细胞器的酶。然而，在这方面， 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+分泌缺陷引起远端肾小管酸中毒（dRTA）， 与人类感觉神经性耳聋有关。dRTA是由编码 V-ATP酶的56 kDa B1亚单位同种型。我们假设另一种V-ATP酶B亚基B2 同种型可能作为一种替代备份，在功能上取代B1在肾H+分泌下， 在某些条件下，因为我们现有的B1亚基敲除小鼠没有酸中毒， B2亚单位在顶膜的1C比正常小鼠。了解B1和B2 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 过程可能提出新的治疗策略，即在B1- 介导的H+分泌缺陷。本建议和培训计划的主要目标是使用新的 技术和动物模型，以确定V-ATP酶B1和B2亚型在H+中的相对作用 分泌和V-ATP酶运输。B2的膜表达如何 缺乏B1的小鼠中亚基的增加，以及这种表达/运输过程是如何调节的？我们将 研究在B1基因敲除小鼠的1C中V-ATP酶mRNA和蛋白表达的调节， 不同的酸碱条件。来自1C的mRNA将使用新的激光捕获显微切割技术分离。 在1C中表达EGFP的转基因小鼠。 pH比值成像。B1相互作用蛋白NHE-RF 1在V-ATP酶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- 将使用多学科方法检查含有B1和B2亚基的ATP酶。