Na+-H+ Transport in Renal Apical Membranes

肾尖膜中的 Na -H 运输

• 批准号: 6616968
• 负责人: EDWARD J WEINMAN
• 金额: $ 32.97万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6616968
• 关键词: apical membrane; confocal scanning microscopy; cyclic AMP; dietary mineral; fluorescent dye /probe; gene targeting; hormone regulation /control mechanism; immunocytochemistry; immunoprecipitation; intracellular transport; ion transport; laboratory mouse; nutrition related tag; parathyroid hormones; phosphorus; phosphorylation; protein binding; protein isoforms; protein localization; protein protein interaction; renal tubular transport; sodium hydrogen exchanger; tissue /cell culture; transcription factor; vitamin D; western blottings

DESCRIPTION (provided by applicant): Regulation of renal proximal tubule transporters, NHE3 (sodium hydrogen exchanger 3) and Npt2 (sodium phosphate cotransporter Ila) involve the PDZ proteins NHERF-1 and NHERF-2 (sodium hydrogen exchanger regulatory factors) that bind to these transporters and transduce hormonal signals that regulate transport activity. The current application explores the hypothesis that the NHERF proteins interact with one another and with other PDZ proteins to regulate the activity and/or cell surface expression of NHE3, Npt2 and other proteins. To this end, a NHERF-1 (-/-) mouse was developed that will prove extremely valuable in defining the functions of NHERF in the mammalian kidney and providing new insights into the defects in electrolyte metabolism associated with human disease. Specific Aim 1 is to define the structural basis for NHERF localization in cells and its impact on recognition of renal targets. Gel overlays and pulldowns assays will be used to define the regions required for NHERF-1 phosphorylation, dimerization and recognition of targets such as NHE3 and Npt2. Coexpression, coimmunoprecipitation and confocal microscopy will be used to investigate the link between NHERF phosphorylation, localization, target recognition and physiologic effects. Specific Aim 2 is to define the role of NHERF isoforms in signal complex regulation of NHE3. Acute and chronic hormonal regulation of NHE3 will be determined in wild-type (WT) and NHERF-1 (-/-) null mice using in vivo microperfusion, fluorescence measurements in tubule suspensions and in primary cell cultures, and 22Na+uptake in isolated brush border membrane vesicles (BBM). NHE3 trafficking in WT and NHERF-1 null mice will be studied using immunocytochemistry, cell surface biotinylation and western immunoblotting of subcellular organelles. Specific Aim 3 will define the role of NHERF-1 in the trafficking and activity of Npt2. Clearance and balance experiments as well as studies using cultured cells and isolated BBM will be used to examine the effects of cAMP, hormones such as PTH and vitamin D and alterations in the dietary intake of phosphorus on Npt2 distribution and activity in WT and NHERF-1 (-/-) animals.

描述（由申请人提供）：肾近端小管转运蛋白NHE3（氢交换钠3）和Npt2（磷酸钠共转运蛋白Ila）的调控涉及PDZ蛋白NHERF-1和NHERF-2（氢交换钠调节因子），它们与这些转运蛋白结合并转导调节转运活性的激素信号。目前的应用探索了NHERF蛋白相互作用以及与其他PDZ蛋白相互作用来调节NHE3、Npt2等蛋白的活性和/或细胞表面表达的假设。为此，研究人员开发了一种NHERF-1（-/-）小鼠，该小鼠将被证明在确定NHERF在哺乳动物肾脏中的功能方面极具价值，并为与人类疾病相关的电解质代谢缺陷提供新的见解。