Davies Matthew, Fraser Scott A, Galic Sandra, Choy Suet-Wan, Katerelos Marina, Gleich Kurt, Kemp Bruce E, Mount Peter F, Power David A
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.
Kidney Laboratory, Institute for Breathing and Sleep, University of Melbourne, Heidelberg, Victoria, Australia;
Am J Physiol Renal Physiol. 2014 Jul 1;307(1):F96-F106. doi: 10.1152/ajprenal.00524.2013. Epub 2014 May 7.
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.
肾小管对盐的重吸收增强在肥胖相关高血压的发病机制中起重要作用，但其机制仍不清楚。为了确定肾脏中盐转运蛋白调节的变化，给C57BL/6小鼠喂食40%脂肪饮食[高脂肪饮食（HFD）]或12%脂肪饮食（对照饮食）14周。与喂食对照饮食的小鼠相比，喂食HFD的小鼠体重、血压、血清胰岛素和瘦素水平显著升高。当我们检测Na(+)转运蛋白表达时，全肾中Na(+)-K(+)-2Cl(-)共转运蛋白（NKCC2）无变化，皮质中减少，Na(+)-Cl(-)共转运蛋白（NCC）、α-上皮Na(+)通道（ENaC）和γ-ENaC无变化，β-ENaC减少。NCC的磷酸化未改变。NKCC2在S126位点的激活磷酸化增加了2.5倍。喂食HFD小鼠肾脏中STE-20/SPS1相关富含脯氨酸-丙氨酸蛋白激酶（SPAK）/氧化应激反应1激酶（OSR1）的激活增加，皮质中NKCC2在T96/T101位点的磷酸化增强明显。利尿实验证实了体内NKCC2活性增加。喂食HFD的小鼠肾皮质中AMP激活的蛋白激酶（AMPK）的激活磷酸化减少。在体外，AMPK的激活导致AMPK(+/+)小鼠胚胎成纤维细胞（MEF）中磷酸化SPAK/磷酸化OSR1减少，但在AMPK(-/-) MEF中未见效果，表明是一种AMPK介导的效应。用低NaCl溶液激活无赖氨酸激酶/SPAK/OSR1途径在AMPK(-/-) MEF中引起的磷酸化SPAK/磷酸化OSR1升高比在AMPK(+/+) MEF中更大，这与AMPK对SPAK/OSR1磷酸化的负调节作用一致。总之，本研究确定NKCC2在S126位点的磷酸化增加是肥胖中Na(+)重吸收增强的一种迄今未被认识的介质，并确定了AMPK在调节SPAK/OSR1活性中的新作用。
