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目标方面取得了巨大进展。 尿素转运蛋白的调节。下一个资助期的总体目标是调查是否 加压素增加顶端质膜 UT-A1 的积累，以确定加压素- 刺激增加 UT-A1 积累的信号通路，并研究检索、周转和 UT-A1 的降解。尿素和水的渗透率经常一起变化，但也有这样的情况： 它们在 IMCD 中相互独立地受到监管。我们提出的研究将提供见解 一个有趣的生物学问题：加压素如何通过单一受体 V2- 发挥作用？ 受体，在单个肾单位节段中独立于水渗透性调节尿素渗透性， 终末 IMCD 并且实际上可能在同一细胞内独立？假设 I. 加压素 快速增加顶端质膜中 UT-A1 的积累。具体目标 1. 确定是否 加压素通过 V2-加压素增加顶端质膜中 UT-A1 的积累 受体。理由：我们的初步数据表明，急性加压素给药会增加剂量 质膜中的 UT-A1。具体目标 2. 确定哪条环 AMP 通路调节 UT-A1 在质膜中积累。理由：我们的初步数据表明，Epac 的激活是一种 新型 cAMP 依赖性但 PKA 独立途径，增加 IMCD 血浆中 UT-A1 的积累 膜，但不改变 AQP2。这些数据表明加压素能够独立地 至少部分地通过激活不同的 cAMP 依赖性信号传导来调节尿素和水的结果 IMCD 细胞中的途径。假设二。加压素调节 UT-A1 的恢复和/或降解。 具体目标 3. 确定 UT-A1 在顶端质膜和细胞中的功能半衰期 负责 UT-A1 检索和/或降解的系统。理由：我们在 UT-A1 中的初步数据- MDCK细胞显示：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显着增加尿素通量（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 UT-A1 从质膜的回收，并靶向其在蛋白酶体中降解。