The expression and activity of many membrane transport proteins are inhibited under conditions of metabolic stress, thereby limiting the dissipation of ionic gradients and preserving the energy required to maintain them. However, the mechanisms involved in this inhibition are unclear. We have established the AMP-activated kinase (AMPK), a metabolic sensor and key regulator of cellular energy homeostasis, as an important potential link between cellular metabolism and ion transport activity. We recently found that AMPK inhibits the epithelial Na+ channel (ENaC), the rate-limiting pathway for renal salt reabsorption and a major regulator of total body volume status and blood pressure, by decreasing channel expression at the plasma membrane. However, AMPK does not bind or phosphorylate ENaC In vitro, suggesting that AMPK inhibits ENaC indirectly through other signaling pathways. Several lines of evidence suggest that Nedd4-2, a ubiquitin-protein ligase that promotes ENaC internalization and degradation, plays a central role in the regulation of ENaC by AMPK. AMPK-dependent ENaC inhibition is prevented in oocytes expressing an ENaC mutant that does not bind Nedd4-2, or in oocytes co-expressing ENaC and dominant-negative or constitutively active Nedd4-2 mutants. Moreover, AMPK phosphorylates Nedd4-2, suggesting a possible mechanism for AMPK-dependent modulation of Nedd4-2 and thus ENaC activity. Our central hypothesis is that AMPK plays a crucial role in the coupling of epithelial transport to cellular metabolic status through its regulation of important transport proteins such as ENaC. We further propose that ENaC inhibition by AMPK occurs via AMPK phosphorylation-dependent modulation of Nedd4-2 function.
The specific aims of this project are to: (1) determine the mechanism of AMPK-dependent inhibition of ENaC expression at the plasma membrane by measuring ENaC half-life, endocytosis and delivery rates at the plasma membrane as a function of AMPK activation; (2) examine the role of AMPK-dependent Nedd4-2 phosphorylation in the regulation of ENaC; and (3) determine the role of AMPK in the inhibition of ENaC in response to chemically induced metabolic stress. Results obtained from these studies should provide specific mechanistic insights into how ENaC is regulated by AMPK and broader insights into the coupling of ion transport to cellular metabolism. They should also promote our understanding of the consequences and pathogenic details of ischemic tissue injury, and the pathophysiology of common diseases, such as hypertension and cystic fibrosis, which are all relevant public health concerns. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK075048-01A1
Application #
7209155
Study Section
Special Emphasis Panel (ZRG1-RUS-E (02))
Program Officer
Ketchum, Christian J
Project Start
2007-02-01
Project End
2012-01-31
Budget Start
2007-02-01
Budget End
2008-01-31
Support Year
1
Fiscal Year
2007
Total Cost
$263,046
Indirect Cost
Name
University of Pittsburgh
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Ho, Pei-Yin; Li, Hui; Pavlov, Tengis S et al. (2018) ?1Pix exchange factor stabilizes the ubiquitin ligase Nedd4-2 and plays a critical role in ENaC regulation by AMPK in kidney epithelial cells. J Biol Chem 293:11612-11624
Pavlov, Tengis S; Levchenko, Vladislav; Ilatovskaya, Daria V et al. (2017) Lack of Effects of Metformin and AICAR Chronic Infusion on the Development of Hypertension in Dahl Salt-Sensitive Rats. Front Physiol 8:227
Rajani, Roshan; Pastor-Soler, Nuria M; Hallows, Kenneth R (2017) Role of AMP-activated protein kinase in kidney tubular transport, metabolism, and disease. Curr Opin Nephrol Hypertens 26:375-383
Christensen, Michael; Jensen, Jonas B; Jakobsen, Steen et al. (2016) Renoprotective Effects of Metformin are Independent of Organic Cation Transporters 1 &2 and AMP-activated Protein Kinase in the Kidney. Sci Rep 6:35952
Al-Bataineh, Mohammad M; Li, Hui; Ohmi, Kazuhiro et al. (2016) Activation of the metabolic sensor AMP-activated protein kinase inhibits aquaporin-2 function in kidney principal cells. Am J Physiol Renal Physiol 311:F890-F900
Al-Bataineh, Mohammad M; Alzamora, Rodrigo; Ohmi, Kazuhiro et al. (2016) Aurora kinase A activates the vacuolar H+-ATPase (V-ATPase) in kidney carcinoma cells. Am J Physiol Renal Physiol 310:F1216-28
Jiang, Chang; Veon, William; Li, Hui et al. (2015) Epithelial morphological reversion drives Profilin-1-induced elevation of p27(kip1) in mesenchymal triple-negative human breast cancer cells through AMP-activated protein kinase activation. Cell Cycle 14:2914-23
Pastor-Soler, NĂºria M; Sutton, Timothy A; Mang, Henry E et al. (2015) Muc1 is protective during kidney ischemia-reperfusion injury. Am J Physiol Renal Physiol 308:F1452-62
Li, Hui; Satriano, Joseph; Thomas, Joanna L et al. (2015) Interactions between HIF-1? and AMPK in the regulation of cellular hypoxia adaptation in chronic kidney disease. Am J Physiol Renal Physiol 309:F414-28
Roy, Ankita; Al-Qusairi, Lama; Donnelly, Bridget F et al. (2015) Alternatively spliced proline-rich cassettes link WNK1 to aldosterone action. J Clin Invest 125:3433-48

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