title: Novel mechanisms of Na+ retention in obesity: phosphorylation of NKCC2 and regulation of SPAK/OSR1 by AMPK.
abstract: Enhanced tubular reabsorption of salt is important in the pathogenesis of obesity-related hypertension, but the mechanisms remain poorly defined. To identify changes in the regulation of salt transporters in the kidney, C57BL/6 mice were fed a 40% fat diet [high-fat diet (HFD)] or a 12% fat diet (control diet) for 14 wk. Compared with control diet-fed mice, HFD-fed mice had significantly greater elevations in weight, blood pressure, and serum insulin and leptin levels. When we examined Na(+) transporter expression, Na(+)-K(+)-2Cl(-) cotransporter (NKCC2) was unchanged in whole kidney and reduced in the cortex, Na(+)-Cl(-) cotransporter (NCC) and α-epithelial Na(+) channel (ENaC) and γ-ENaC were unchanged, and β-ENaC was reduced. Phosphorylation of NCC was unaltered. Activating phosphorylation of NKCC2 at S126 was increased 2.5-fold. Activation of STE-20/SPS1-related proline-alanine-rich protein kinase (SPAK)/oxidative stress responsive 1 kinase (OSR1) was increased in kidneys from HFD-fed mice, and enhanced phosphorylation of NKCC2 at T96/T101 was evident in the cortex. Increased activity of NKCC2 in vivo was confirmed with diuretic experiments. HFD-fed mice had reduced activating phosphorylation of AMP-activated protein kinase (AMPK) in the renal cortex. In vitro, activation of AMPK led to a reduction in phospho-SPAK/phospho-OSR1 in AMPK(+/+) murine embryonic fibroblasts (MEFs), but no effect was seen in AMPK(-/-) MEFs, indicating an AMPK-mediated effect. Activation of the with no lysine kinase/SPAK/OSR1 pathway with low-NaCl solution invoked a greater elevation in phospho-SPAK/phospho-OSR1 in AMPK(-/-) MEFs than in AMPK(+/+) MEFs, consistent with a negative regulatory effect of AMPK on SPAK/OSR1 phosphorylation. In conclusion, this study identifies increased phosphorylation of NKCC2 on S126 as a hitherto-unrecognized mediator of enhanced Na(+) reabsorption in obesity and identifies a new role for AMPK in regulating the activity of SPAK/OSR1.
authors.affiliation: Kidney Laboratory, Institute for Breathing and Sleep, University of Melbourne, Heidelberg, Victoria, Australia; Department of Nephrology, University of Melbourne, Heidelberg, Victoria, Australia; Department of Medicine, University of Melbourne, Heidelberg, Victoria, Australia; and.
authors.forename: Matthew
authors.initials: M
authors.lastname: Davies
authors.affiliation: Kidney Laboratory, Institute for Breathing and Sleep, University of Melbourne, Heidelberg, Victoria, Australia;
authors.forename: Scott A
authors.initials: SA
authors.lastname: Fraser
authors.affiliation: St. Vincent's Institute, Fitzroy, Victoria, Australia.
authors.forename: Sandra
authors.initials: S
authors.lastname: Galic
authors.affiliation: Kidney Laboratory, Institute for Breathing and Sleep, University of Melbourne, Heidelberg, Victoria, Australia; Department of Nephrology, University of Melbourne, Heidelberg, Victoria, Australia; Department of Medicine, University of Melbourne, Heidelberg, Victoria, Australia; and.
authors.forename: Suet-Wan
authors.initials: SW
authors.lastname: Choy
authors.affiliation: Kidney Laboratory, Institute for Breathing and Sleep, University of Melbourne, Heidelberg, Victoria, Australia;
authors.forename: Marina
authors.initials: M
authors.lastname: Katerelos
authors.affiliation: Kidney Laboratory, Institute for Breathing and Sleep, University of Melbourne, Heidelberg, Victoria, Australia;
authors.forename: Kurt
authors.initials: K
authors.lastname: Gleich
authors.affiliation: St. Vincent's Institute, Fitzroy, Victoria, Australia.
authors.forename: Bruce E
authors.initials: BE
authors.lastname: Kemp
authors.affiliation: Kidney Laboratory, Institute for Breathing and Sleep, University of Melbourne, Heidelberg, Victoria, Australia; Department of Nephrology, University of Melbourne, Heidelberg, Victoria, Australia; Department of Medicine, University of Melbourne, Heidelberg, Victoria, Australia; and.
authors.forename: Peter F
authors.initials: PF
authors.lastname: Mount
authors.affiliation: Kidney Laboratory, Institute for Breathing and Sleep, University of Melbourne, Heidelberg, Victoria, Australia; Department of Nephrology, University of Melbourne, Heidelberg, Victoria, Australia; Department of Medicine, University of Melbourne, Heidelberg, Victoria, Australia; and David.Power@austin.org.au.
authors.forename: David A
authors.initials: DA
authors.lastname: Power
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000818:Animals; D053503:Epithelial Sodium Channels / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009765:Obesity / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017673:Sodium Chloride, Dietary / Q000378:metabolism*; D064466:Solute Carrier Family 12, Member 1 / Q000378:metabolism*
chemical_list: D053503:Epithelial Sodium Channels; D017673:Sodium Chloride, Dietary; D064466:Solute Carrier Family 12, Member 1; C486407:Stk39 protein, mouse; C534421:OXSR1 protein, mouse; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases
keywords: AMP-activated protein kinase; Na+-K+-2Cl− cotransporter; STE-20/SPS1-related proline-alanine-rich protein kinase; oxidative stress responsive 1 kinase