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. )

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Special Emphasis Panel (ZRG1)
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Ketchum, Christian J
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University of Pittsburgh
Internal Medicine/Medicine
Schools of Medicine
United States
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