Regulation of Rat Renal Inner Medullary Function

大鼠肾内髓功能的调节

• 批准号: 7903711
• 负责人: JEFF M. SANDS
• 金额: $ 10万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-20 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7903711
• 关键词: Acute; Apical; Biological; Brattleboro Rats; Canis familiaris; Carrier Proteins; Cell Line; Cell membrane; Cells; Centrifugation; Cloning; Complementary DNA; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Duct (organ) structure; Electron Microscopy; Forskolin; Funding; Genes; Goals; Half-Life; Kidney; MDCK cell; Measures; Mediating; Membrane; Nephrons; Pathway interactions; Permeability; Phosphorylation; Physiological; Proteasome Inhibitor; Protein Isoforms; Protein Kinase A Inhibitor; Proteins; Rattus; Regulation; Research Personnel; Retrieval; Signal Pathway; Silicon Dioxide; Suspension substance; Suspensions; System; Testing; Ubiquitination; Urea; Urine; V2 Receptors; Vasopressin Receptor; Vasopressins; Water; Work; activator 1 protein; analog; insight; multicatalytic endopeptidase complex; novel; polyclonal antibody; promoter; receptor; urea transporter

During the past decade, we and others have made tremendous progress in understanding the long-term regulation of urea transport proteins. The overall goals of the next funding period are to investigate whether vasopressin increases the apical plasma membrane accumulation of UT-A1, to determine the vasopressin- stimulated signaling pathway(s) that increase UT-A1 accumulation, and to study the retrieval, turnover, and degradation of UT-A1. Urea and water permeabilities often change together, but there are situations when they are regulated independently of one another in the IMCD. Our proposed studies will provide insights into an intriguing biological question: how does vasopressin, acting through a single receptor, the V2- receptor, regulate urea permeability independently of water permeability in a single nephron segment, the terminal IMCD and, indeed, probably independently within the same cells? HYPOTHESIS I. Vasopressin rapidly increases UT-A1 accumulation in the apical plasma membrane. Specific Aim 1. Determine whether vasopressin increases UT-A1 accumulation in the apical plasma membrane through the V2-vasopressin receptor. Rationale: our preliminary data show that acute vasopressin administration increases the amount of UT-A1 in the plasma membrane. Specific Aim 2. Determine which cyclic AMP pathway regulates UT-A1 accumulation in the plasma membrane. Rationale: our Preliminary Data show that activation of Epac, a novel cAMP-dependent but PKA-independent pathway, increases UT-A1 accumulation in the IMCD plasma membrane, but does not change AQP2. These data suggest that vasopressin's ability to independently regulate urea and water results, at least in part, from activation of different cAMP-dependent signaling pathways in IMCD cells. HYPOTHESIS II. Vasopressin regulates UT-A1 retrieval and/or degradation. Specific Aim 3. Determine the functional half-life of UT-A1 in the apical plasma membrane and the cellular systems responsible for UT-A1 retrieval and/or degradation. Rationale: our Preliminary Data in UT-A1- MDCK cells show that: 1) UT-A1 is a ubiquitinated protein in rat inner medulla and in UT-A1-MDCK cells; and 2) pre-treatment with a proteasome inhibitor produces a marked increase in urea flux (function), UT-A1 protein abundance, and the sizes of biotinylated UT-A1. These data suggest that ubiquitination may regulate retrieval of UT-A1 from the plasma membrane and target it for degradation in the proteasome.

在过去的十年中，我们和其他人在理解长期方面取得了巨大进步 调节尿素转运蛋白。下一个资金期的总体目标是调查是否 加压素增加了UT-A1的顶端质膜积累，以确定加压素 - 刺激的信号通路会增加UT-A1的积累，并研究检索，周转和 UT-A1的降解。尿素和水的渗透率通常会一起改变，但是有些情况 它们在IMCD中彼此独立地监管。我们提出的研究将提供见解 进入一个有趣的生物学问题：加压素如何通过单个受体作用，V2- 受体，独立于单个肾单位段中的水渗透性调节尿素渗透率， 终端IMCD，​​实际上可能独立于同一细胞内？假设I.加压素 迅速增加顶端质膜中的UT-A1积累。特定目标1。确定是否 加压素通过V2-vasopressin在顶端质膜中增加UT-A1的积累 受体。理由：我们的初步数据表明，急性加压素给药增加了数量 质膜中的UT-A1。特定目标2。确定哪种环状AMP途径调节UT-A1 质膜积聚。理由：我们的初步数据表明EPAC的激活 新型CAMP依赖但与PKA无关的途径，增加了IMCD等离子体中的UT-A1积累 膜，但不会更改AQP2。这些数据表明加压素独立的能力 至少部分地调节尿素和水结果，从不同的cAMP依赖性信号传导激活 IMCD细胞中的途径。假设II。加压素调节UT-A1检索和/或降解。 特定目标3。确定顶端质膜和细胞中UT-A1的功能半衰期 负责UT-A1检索和/或退化的系统。理由：UT-A1-中的初步数据 MDCK细胞表明：1）UT-A1是大鼠内髓质和UT-A1-MDCK细胞中的泛素化蛋白； 2）用蛋白酶体抑制剂预处理可产生明显增加的尿素通量（功能），UT-A1 蛋白质丰度和生物素化UT-A1的大小。这些数据表明泛素化可能 调节从质膜中检索UT-A1，并将其靶向蛋白酶体中的降解。