Mechanisms and Relevance of Sodium Transport Regulation by AMPK

AMPK 调节钠转运的机制和相关性

基本信息

批准号：
8532882
负责人：
KENNETH R HALLOWS
金额：
$ 31.2万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-17 至 2016-07-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8532882
关键词：
5&apos -AMP-activated protein kinase AMP-activated protein kinase kinase Acute Acute Renal Failure with Renal Papillary Necrosis Affect Apoptosis Binding Blood Pressure Carrier Proteins Cell physiology Cells Chemicals Coupling Data Diabetes Mellitus Distal Down-Regulation Duct (organ) structure Endocytosis Epithelial Epithelium Fluid Balance Functional disorder Growth Heart Diseases Hormones Hypoxia In Vitro Inflammation Injury Ion Transport Ischemia Ischemic Preconditioning Kidney Knock-out Knockout Mice Malignant Neoplasms Mass Spectrum Analysis Measurement Metabolic Metabolic Pathway Metabolic stress Metabolism Modeling Morbidity - disease rate Mus Nephrons Obesity Oocytes Pathway interactions Patients Phospho-Specific Antibodies Phosphorylation Phosphorylation Site Phosphotransferases Play Prevention strategy Process Proteins Public Health Regulation Role Signaling Protein Site Sodium Stimulus Testing Tissues Ubiquitination Work apical membrane body volume cell growth regulation collecting tubule structure epithelial Na+ channel extracellular in vivo insight kidney cell mortality mutant novel novel therapeutics patch clamp protein protein interaction public health relevance renal ischemia response rho sensor ubiquitin-protein ligase

项目摘要

DESCRIPTION (provided by applicant): Proper function of the epithelial Na+ channel (ENaC), the rate-limiting step for Na+ reabsorption in the distal nephron, is critical to the regulation of body volume status and blood pressure. Recent work has established that the metabolic sensor AMP-activated protein kinase (AMPK) inhibits the activity of ENaC and other important epithelial transport proteins, which appears to provide a sensitive coupling mechanism between ion transport and cellular metabolic status and helps conserve cellular energy under conditions of metabolic depletion during ischemic tissue injury. AMPK inhibits ENaC by decreasing its expression at the apical membrane via enhanced endocytosis and channel ubiquitination through increased ENaC interaction with Nedd4-2, an E3 ubiquitin ligase that has emerged as a central convergence point for ENaC regulation. However, the mechanistic details of this regulation and role of AMPK in the ischemia-induced inhibition of ENaC and other transport proteins in vivo are unclear. Preliminary data indicate that AMPK directly phosphorylates Nedd4-2 at a site that appears to be critical for cellular Nedd4-2 stability. Additional preliminary data suggest that the Rho-GEF signaling protein ¿1Pix, which inhibits ENaC by impairing 14-3-3 ¿ binding to Nedd4-2 and promoting Nedd4-2 inhibition of ENaC, is phosphorylated by AMPK and required for the AMPK- dependent inhibition of ENaC. We thus hypothesize that AMPK exerts two effects on Nedd4-2 to enhance its interaction with and inhibition of ENaC: (1) direct Nedd4-2 phosphorylation by AMPK, which enhances Nedd4-2 stability; and (2) AMPK phosphorylation of ¿1Pix, which enhances Nedd4-2 association with ENaC by competing with Nedd4-2 for 14-3-3 ¿ interaction. Further preliminary data suggest that AMPK contributes to the acute inhibition of ENaC with chemical ischemia in polarized cortical collecting duct cells and that ENaC is upregulated in the kidney in vivo in AMPK- ¿1 knockout mice with largely kidney-specific loss of AMPK function. We thus hypothesize that AMPK regulation of ENaC is relevant in vivo and that AMPK activation plays an important role in Na+ transport inhibition following renal ischemic injury in vivo. To evaluate the mechanisms of AMPK-dependent ENaC regulation via Nedd4-2 and test its role in ischemic kidney injury in vivo, the Specific Aims of this project are to: (1) examine the role of Nedd4-2 phosphorylation by AMPK in the AMPK-dependent regulation of ENaC and Nedd4-2 cellular stability; (2) examine the role of AMPK phosphorylation of ¿1Pix in the regulation of ENaC; and (3) examine the role of AMPK in the down-regulation of ENaC in vivo in response to acute ischemic kidney injury using AMPK- ¿1 knockout mice and wild-type littermates. The proposed studies should promote our understanding of the Nedd4-2-dependent regulation of ENaC and other Nedd4-2-regulated transport proteins by AMPK, of the role of AMPK in transport-metabolism coupling, and the pathophysiology of ischemic renal injury.

描述（由申请人提供）：上皮 Na+ 通道（ENaC）的正常功能是远端肾单位 Na+ 重吸收的限速步骤，对于身体容量状态和血压的调节至关重要。代谢传感器 AMP 激活蛋白激酶 (AMPK) 抑制 ENaC 和其他重要上皮转运蛋白的活性，这似乎在离子转运和细胞代谢状态之间提供了敏感的耦合机制AMPK 通过增强内吞作用来减少 ENaC 在顶膜的表达，并通过增加 ENaC 与 Nedd4-2（一种已成为 E3 泛素连接酶）的相互作用来帮助保存细胞能量。然而，这种调节的机制细节以及 AMPK 在缺血诱导的 ENaC 抑制中的作用。初步数据表明，AMPK 在对细胞 Nedd4-2 稳定性至关重要的位点直接磷酸化 Nedd4-2。 1Pix，通过损害 14-3-3 ¿ 来抑制 ENaC与 Nedd4-2 结合并促进 Nedd4-2 对 ENaC 的抑制，被 AMPK 磷酸化，并且是 AMPK 依赖性抑制 ENaC 所必需的，因此我们发现 AMPK 对 Nedd4-2 发挥两种作用，以增强其与 ENaC 的相互作用并抑制 ENaC。：(1) AMPK 直接磷酸化 Nedd4-2，从而增强 Nedd4-2 的稳定性；(2) AMPK 磷酸化 ¿ 1Pix，通过与 Nedd4-2 竞争 14-3-3 ¿ 来增强 Nedd4-2 与 ENaC 的关联进一步的初步数据表明，AMPK 有助于急性抑制 ENaC 与极化皮质集合管细胞中的化学缺血，并且 ENaC 在 AMPK- 体内的肾脏中上调。 1 基因敲除小鼠的 AMPK 功能很大程度上是肾脏特异性的，因此我们观察到 AMPK 对 ENaC 的调节在体内是相关的，并且 AMPK 的激活在肾缺血损伤后的 Na+ 转运抑制中发挥着重要作用，以评估 AMPK 的机制。通过 Nedd4-2 依赖的 ENaC 调节并测试其在体内缺血性肾损伤中的作用，该项目的具体目标是：（1）检查 AMPK 磷酸化 Nedd4-2 的作用ENaC 和 Nedd4-2 细胞稳定性的 AMPK 依赖性调节；(2) 检查 ¿ 1Pix 在 ENaC 的调节中；(3) 使用 AMPK-¿ 检查 AMPK 在体内响应急性缺血性肾损伤时下调 ENaC 的作用1 基因敲除小鼠和野生型同窝小鼠的研究应促进我们对 AMPK 对 ENaC 和其他 Nedd4-2 调节的转运蛋白的 Nedd4-2 依赖性调节、AMPK 在转运代谢耦合中的作用的理解，以及缺血性肾损伤的病理生理学。