Mechanisms and Relevance of Sodium Transport Regulation by AMPK

AMPK 调节钠转运的机制和相关性

• 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
• 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' -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

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，对调节身体容量状态和血压至关重要。最近的工作已经确定，代谢传感器AMP激活的蛋白激酶（AMPK）抑制ENaC和其他重要的上皮转运蛋白的活性，这似乎提供了一个敏感的离子转运和细胞代谢状态之间的耦合机制，并有助于在缺血性组织损伤期间代谢耗竭的条件下保存细胞能量。AMPK通过增强的内吞作用和通道泛素化降低其在顶膜的表达来抑制ENaC，所述通道泛素化通过增加ENaC与Nedd 4 -2的相互作用来实现，Nedd 4 - 2是一种E3泛素连接酶，已经成为ENaC调节的中心会聚点。然而，这种调节的机制细节和AMPK在体内缺血诱导的ENaC和其他转运蛋白抑制中的作用尚不清楚。初步数据表明，AMPK直接磷酸化Nedd 4 -2在一个网站，似乎是关键的细胞Nedd 4 -2的稳定性。额外的初步数据表明，Rho-GEF信号蛋白<$1Pix通过削弱14-3-3 <$与Nedd 4 -2的结合并促进Nedd 4 -2对ENaC的抑制来抑制ENaC，其被AMPK磷酸化，并且是AMPK依赖性抑制ENaC所需的。因此，我们假设AMPK对Nedd 4 -2发挥两种作用以增强其与ENaC的相互作用和抑制ENaC：（1）AMPK直接磷酸化Nedd 4 -2，这增强了Nedd 4 -2的稳定性;（2）AMPK磷酸化<$1Pix，这通过与Nedd 4 -2竞争14-3-3 <$相互作用来增强Nedd 4 -2与ENaC的结合。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，并且在AMPK-1敲除小鼠体内肾脏中ENaC上调，主要是肾脏特异性AMPK功能丧失。因此，我们假设AMPK调节ENaC是相关的在体内和AMPK激活在体内肾缺血损伤后的Na+转运抑制中起重要作用。 本研究旨在探讨AMPK依赖性Nedd 4 -2对ENaC的调控机制及其在缺血性肾损伤中的作用，具体目的是：（1）研究AMPK对Nedd 4 -2的磷酸化在AMPK依赖性ENaC调控中的作用及Nedd 4 -2细胞的稳定性;（2）研究AMPK对<$1Pix的磷酸化在ENaC调控中的作用;和（3）使用AMPK-1基因敲除小鼠和野生型同窝出生小鼠，检测AMPK在体内ENaC下调中的作用，以响应急性缺血性肾损伤。这些研究将促进我们对AMPK对ENaC和其他Nedd 4 -2调节转运蛋白的Nedd 4 -2依赖性调节、AMPK在转运-代谢偶联中的作用以及缺血性肾损伤的病理生理学的理解。