Mechanisms and Relevance of Sodium Transport Regulation by AMPK

描述（由申请人提供）：上皮Na+通道（ENaC）的正常功能是远端肾元Na+重吸收的限速步骤，对调节机体容量状态和血压至关重要。最近的研究表明，代谢传感器amp活化蛋白激酶（AMPK）抑制ENaC和其他重要上皮转运蛋白的活性，这似乎提供了离子转运和细胞代谢状态之间的敏感耦合机制，并有助于在缺血性组织损伤期间代谢耗损的条件下保存细胞能量。AMPK通过增加ENaC与Nedd4-2（一种E3泛素连接酶，已成为ENaC调节的中心汇聚点）的相互作用，通过增强内吞作用和通道泛素化，降低ENaC在根尖膜上的表达，从而抑制ENaC。然而，这种调控的机制细节以及AMPK在缺血诱导的ENaC和其他转运蛋白体内抑制中的作用尚不清楚。初步数据表明，AMPK在一个位点直接磷酸化Nedd4-2，该位点似乎对细胞Nedd4-2的稳定性至关重要。另外的初步数据表明，Rho-GEF信号蛋白¿1Pix通过破坏14-3-3¿与Nedd4-2的结合并促进Nedd4-2对ENaC的抑制来抑制ENaC，该蛋白被AMPK磷酸化，并且是AMPK依赖性ENaC抑制所必需的。因此，我们假设AMPK对Nedd4-2发挥两种作用，增强其与ENaC的相互作用和抑制作用：(1)AMPK直接磷酸化Nedd4-2，增强Nedd4-2的稳定性；(2) AMPK磷酸化¿1Pix，通过与Nedd4-2竞争14-3-3¿相互作用，增强了Nedd4-2与ENaC的结合。进一步的初步数据表明，AMPK有助于极化皮质集管细胞化学缺血时ENaC的急性抑制，AMPK-¿1基因敲除小鼠体内肾脏中ENaC的表达上调，AMPK功能在很大程度上特异性丧失。因此，我们假设AMPK对ENaC的调节在体内是相关的，AMPK的激活在体内肾缺血损伤后的Na+转运抑制中起重要作用。为了评估AMPK依赖的Nedd4-2调控ENaC的机制，并测试其在体内缺血性肾损伤中的作用，本项目的具体目的是：(1)研究AMPK磷酸化Nedd4-2在AMPK依赖的ENaC和Nedd4-2细胞稳定性调控中的作用；(2)研究AMPK磷酸化¿1Pix在ENaC调控中的作用；(3)利用AMPK-¿1基因敲除小鼠和野生型幼崽，研究AMPK在急性缺血性肾损伤时下调ENaC的体内作用。本研究将促进我们对AMPK依赖nedd4 -2调控ENaC及其他nedd4 -2调控的转运蛋白，AMPK在转运-代谢耦合中的作用，以及缺血性肾损伤的病理生理的理解。

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.