Na+-H+ Transport in Renal Apical Membranes

肾尖膜中的 Na -H 运输

• 批准号: 6751960
• 负责人: EDWARD J WEINMAN
• 金额: $ 35.64万
• 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/6751960
• 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）：肾近曲小管转运蛋白 NHE3（钠氢交换器 3）和 Npt2（磷酸钠协同转运蛋白 IIa）的调节涉及与结合的 PDZ 蛋白 NHERF-1 和 NHERF-2（钠氢交换器调节因子）这些转运蛋白并转导调节转运活动的激素信号。当前的应用探索了这样的假设：NHERF 蛋白彼此相互作用以及与其他 PDZ 蛋白相互作用，以调节 NHE3、Npt2 和其他蛋白的活性和/或细胞表面表达。为此，我们开发了一种 NHERF-1 (-/-) 小鼠，该小鼠对于确定哺乳动物肾脏中 NHERF 的功能以及为与人类疾病相关的电解质代谢缺陷提供新的见解将具有极其重要的价值。 具体目标 1 是确定 NHERF 在细胞中定位的结构基础及其对肾脏靶标识别的影响。凝胶覆盖和下拉分析将用于定义 NHERF-1 磷酸化、二聚化和识别 NHE3 和 Npt2 等靶标所需的区域。 共表达、免疫共沉淀和共聚焦显微镜将用于研究 NHERF 磷酸化、定位、靶标识别和生理效应之间的联系。 具体目标 2 是确定 NHERF 同工型在 NHE3 信号复合体调节中的作用。 NHE3 的急性和慢性激素调节将在野生型 (WT) 和 NHERF-1 (-/-) 无效小鼠中使用体内微灌注、肾小管悬浮液和原代细胞培养物中的荧光测量以及分离的 22Na+ 摄取来确定刷状缘膜囊泡（BBM）。将使用免疫细胞化学、细胞表面生物素化和亚细胞细胞器的蛋白质免疫印迹来研究 WT 和 NHERF-1 缺失小鼠中的 NHE3 运输。 具体目标 3 将定义 NHERF-1 在 Npt2 贩运和活动中的作用。清除和平衡实验以及使用培养细胞和分离的 BBM 的研究将用于检查 cAMP、PTH 和维生素 D 等激素的影响以及膳食磷摄入量的变化对 WT 和 NHERF-1 中 Npt2 分布和活性的影响（-/-） 动物。