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: 7019865
• 负责人: Teodor G. Paunescu
• 金额: $ 13.14万
• 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/7019865
• 关键词: acid base balance; adenylate cyclase; cyclic AMP; gene induction /repression; genetically modified animals; green fluorescent proteins; hydrogen transport; hydrogen transporting ATP synthase; kidney cell; kidney function; laboratory mouse; laboratory rat; laser capture microdissection; membrane activity; protein isoforms; protein transport; proteomics; small interfering RNA; sodium hydrogen exchanger; transcription factor

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）： 该申请人提出了一项计划，为他从事研究肾功能的学术基础科学研究职业做好准备，解决生理和病理生理条件下肾脏中质子分泌的调节问题。该研究将在麻省总医院膜生物学和肾脏科项目丹尼斯·布朗博士的实验室中进行。肾 H+ 分泌主要由液泡质子泵 ATP 酶 (V-ATP 酶) 介导，该酶还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 型嵌入细胞 (IC) 中，V-ATP 酶介导跨上皮 H+ 分泌。 H+ 分泌缺陷会导致远端肾小管酸中毒 (dRTA)，与人类感音神经性耳聋相关。 dRTA 是由编码 V-ATP 酶 56 kDa B1 亚基亚型的基因突变引起的。我们假设另一种 V-ATPase B 亚基 B2 亚型可能作为替代后备，在某些条件下在肾 H+ 分泌中功能性地替代 B1，因为我们现有的 B1 亚基敲除小鼠不会酸中毒，并且它们在 IC 顶膜中表达比正常小鼠更多的 B2 亚基。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，可以为 B1 介导的 H+ 分泌有缺陷的细胞中的“异构体替代疗法”提出新的治疗策略。该提案和培训计划的主要目标是使用新技术和动物模型来确定 V-ATP 酶 B1 和 B2 亚型在肾上皮细胞 H+ 分泌和 V-ATP 酶运输中的相对作用。在缺乏 B1 的小鼠中，B2 亚基的膜表达如何增加？这种表达/运输过程是如何调节的？我们将检查不同酸碱条件下独特的 B1 敲除小鼠 IC 中 V-ATPase mRNA 和蛋白质表达的调节。将使用在 IC 中表达 EGFP 的新型转基因小鼠，通过激光捕获显微切割分离来自 IC 的 mRNA。 IC 中的补偿性 B2-V-ATP 酶功能将通过 pH 比成像进行检查。将探讨 B1 相互作用蛋白 NHE-RF1 在 V-ATP 酶运输中的作用，并将使用多学科方法研究碳酸氢盐刺激的可溶性腺苷酸环化酶 (sAC) 在包含 B1 和 B2 亚基的 V-ATP 酶膜插入中的作用。