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）： 该申请人提出了一项计划，以准备他为肾功能的学术基础科学职业做好准备，并针对生理和病情生理条件下肾脏中质子分泌的调节。该研究将在MGH的膜生物学和肾单位的计划中在丹尼斯·布朗博士的实验室进行。肾脏H+分泌主要是由液泡质子泵化ATPase（V-ATPase）介导的，该酶也酸化了一些细胞内细胞器。然而，当在质膜上表达时，如收集导管A型插入细胞（IC）时，V-ATPase介导了跨性别的H+分泌。 H+分泌的缺陷引起肾小管酸中毒（DRTA），与人类的感觉性耳聋有关。 DRTA是由编码V-ATPase 56 kDa B1亚基同工型的基因中的突变引起的。我们假设是替代V-ATPase B亚基，B2同工型可能是替代备份，在某些情况下在功能上替代肾脏H+分泌的B1，因为在某些条件下，我们可用的B1亚基敲除小鼠不是酸性小鼠，并且它们在IC中比正常小鼠的APICAL MEMBRANE中表达更多的B2亚基。了解B1和B2在V-ATPase复合物中组装成V-ATPase复合物的方式以及这些亚基在V-ATPase靶向和运输过程中的作用可以通过B1-介导的H+分泌有缺陷的细胞中的“同工替代疗法”在细胞中提出新的治疗策略。该提案和培训计划的主要目标是使用新颖的技术和动物模型来确定V-ATPase B1和B2同工型在H+分泌中的相对作用以及在肾上皮细胞中V-ATPase运输中的相对作用。 B2亚基的膜表达如何增加缺乏B1的小鼠，该表达/运输过程如何调节？我们将检查来自不同酸碱条件下独特的B1-敲除小鼠IC中V-ATPase mRNA和蛋白表达的调节。来自IC的mRNA将通过激光捕获显微解剖分离，使用在IC中表达EGFP的新转基因小鼠。 IC中的代偿性B2-V-ATPase功能将通过pH比成像检查。将解决B1相互作用蛋白NHE-RF1在V-ATPase运输中的作用，并使用多学科方法在包含B1和B2亚基的V-ATPases膜中插入碳酸氢盐刺激的可溶性腺酸环化酶（SAC）的作用。