Mechanisms and Relevance of Sodium Transport Regulation by AMPK

描述(由申请人提供)：上皮性Na+通道(ENaC)的正确功能，是远端肾单位重吸收Na+的限速步骤，对调节身体容量状态和血压至关重要。最近的研究表明，代谢感受器AMP激活的蛋白激酶(AMPK)抑制ENaC和其他重要的上皮运输蛋白的活性，这似乎在离子转运和细胞代谢状态之间提供了一种敏感的耦合机制，并在缺血组织损伤的代谢耗竭条件下帮助保存细胞能量。AMPK通过增强ENaC与Nedd4-2的相互作用，增强ENaC与Nedd4-2的相互作用，减少其在顶膜上的表达，从而抑制ENaC的表达。Ned4-2是一种E3泛素连接酶，已成为ENaC调节的中心聚合点。然而，这种调节的机制细节以及AMPK在体内缺血诱导的ENaC和其他运输蛋白的抑制中的作用尚不清楚。初步数据表明，AMPK直接在对细胞Nedd4-2稳定性至关重要的位置上磷酸化Nedd4-2。更多的初步数据表明，Rho-Global信号蛋白1Pix通过损害14-3-3与Nedd4-2的结合而抑制ENaC，并促进Nedd4-2对ENaC的抑制，被AMPK磷酸化，是依赖AMPK抑制ENaC所必需的。因此，我们推测AMPK对Nedd4-2有两种作用：(1)AMPK直接使Nedd4-2磷酸化，增强Nedd4-2的稳定性；(2)AMPK对1Pix进行磷酸化，通过与Nedd4-2竞争14-3-3相互作用，增强Nedd4-2与ENaC的结合。进一步的初步数据表明，AMPK有助于在极化的皮质集合管细胞中因化学缺血而急性抑制ENaC，并且在AMPK？1基因敲除小鼠的肾脏中ENaC表达上调，AMPK功能在很大程度上是肾脏特异性丧失的。因此，我们假设AMPK对ENaC的调节在体内是相关的，并且AMPK的激活在体内肾缺血损伤后抑制Na+转运中起重要作用。为了探讨AMPK依赖的ENaC通过Nedd4-2调节ENaC的机制并测试其在体内缺血性肾损伤中的作用，本项目的具体目标是：(1)检测AMPK依赖的ENaC调节和Nedd4-2细胞稳定性中的作用；(2)检测AMPK对ENaC的调节中的AMPK磷酸化在ENaC调节中的作用；(3)利用AMPK-e1基因敲除小鼠和野生型小鼠，研究AMPK在体内下调ENaC对急性缺血性肾损伤的反应中的作用。这些研究将促进我们理解AMPK对ENaC和其他Nedd4-2调节的转运蛋白的Nedd4-2依赖的调节，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机制。 公共卫生相关性：我们预计，拟议的研究不仅将增强我们对肾脏代谢感应激酶AMPK对上皮细胞Na+通道调节机制的具体了解，还可能为预防和管理肾脏缺血提出新的治疗策略，肾脏缺血是急性肾损伤的主要原因。急性肾损伤在缺血的背景下是一个主要的公共卫生问题 我们许多住院患者的发病率和死亡率都很高。这些研究还可能为细胞AMPK功能的一般调控提供新的见解，该功能可能影响多种细胞过程，包括生长、凋亡、炎症和代谢途径的控制，这与我们对几种公共健康状况的理解有关，包括癌症、糖尿病、心脏病和肥胖症。)

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. PUBLIC HEALTH RELEVANCE: We anticipate that the proposed studies will not only enhance our specific understanding of the mechanisms of epithelial Na+ channel regulation by the metabolic sensing kinase AMPK in the kidney, but could also suggest novel therapeutic strategies for the prevention and management of renal ischemia, a major cause of acute kidney injury. Acute kidney injury in the setting of ischemia is a major public health concern that causes excess morbidity and mortality in many of our hospitalized patients. These studies may also provide novel insights into the general regulation of cellular AMPK function, which may affect a variety of cell processes, including growth, apoptosis, inflammation, and the control of metabolic pathways with relevance to our understanding of several public health conditions, including cancer, diabetes, heart disease, and obesity. )