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: 7162612
• 负责人: Teodor G. Paunescu
• 金额: $ 13.3万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 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/7162612
• 关键词: ATP phosphohydrolase; Acid-Base Imbalance; Acids; Address; Adenylate Cyclase; Animal Model; Back; Basic Science; Bicarbonates; Cell membrane; Cells; Complex; Condition; 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; vacuolar H+-ATPase

DESCRIPTION (provided by applicant): 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 (IC), 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 IC 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 IC from unique B1-knockout mice under different acid-base conditions. mRNA from IC will be isolated by laser capture microdissection using new transgenic mice that express EGFP in IC. Compensatory B2-V-ATPase function in IC 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.

描述（由申请人提供）： 该申请人提出了一个计划，为他在学术基础科学研究肾功能，解决生理和病理生理条件下肾脏质子分泌的调节职业生涯做准备。该研究将在MGH膜生物学和肾脏部门的Dennis Brown博士实验室进行。肾脏H+分泌主要由液泡质子泵ATP酶（V-ATP酶）介导，V-ATP酶也可酸化某些细胞内细胞器。然而，当在质膜上表达时，如在集合管A型闰细胞（IC）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），与人类的感音神经性耳聋有关。dRTA是由编码V-ATP酶56 kDa B1亚基同种型的基因突变引起的。我们假设，一个替代的V-ATP酶B亚基，B2亚型可能作为一个替代备份，功能上取代B1在肾脏H+分泌在某些条件下，因为我们现有的B1亚基敲除小鼠是不酸中毒，他们表达更多的B2亚基在顶膜IC比正常小鼠。了解B1和B2组装成V-ATP酶复合物的方式以及这些亚基在V-ATP酶靶向和运输过程中的作用，可以通过B1介导的H+分泌缺陷的细胞中的“同种型替代疗法”提出新的治疗策略。本研究的主要目的是利用新的技术和动物模型来确定V-ATPase B1和B2亚型在肾上皮细胞H+分泌和V-ATPase运输中的相对作用。在缺乏B1的小鼠中，B2亚基的膜表达是如何增加的，这种表达/运输过程是如何调节的？我们将研究在不同的酸碱条件下，从独特的B1基因敲除小鼠IC中V-ATP酶mRNA和蛋白表达的调节。将使用在IC中表达EGFP的新转基因小鼠通过激光捕获显微切割分离IC的mRNA。将通过pH比率成像检查IC中的代偿性B2-V-ATP酶功能。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，碳酸氢盐刺激的可溶性腺苷酸环化酶（sAC）在含有B1和B2亚基的V-ATP酶的膜插入中的作用将使用多学科方法进行研究。