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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.

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