Downregulation of the renal outer medullary K+ channel ROMK by the AMP-activated protein kinase
Downregulation of the renal outer medullary K+ channel ROMK by the AMP-activated protein kinase
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AMP 激活蛋白激酶下调肾外髓 K 通道 ROMK
DOI：
10.1007/s00424-012-1180-1
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Lang F
中科院分区：
文献类型：
--
作者：
Siraskar B;Huang DY;Pakladok T;Siraskar G;Sopjani M;Alesutan I;Kucherenko Y;Almilaji A;Devanathan V;Shumilina E;Föller M;Munoz C;Lang F
5‘-腺苷单磷酸激活的丝氨酸/苏氨酸蛋白激酶(AMPK)受到能量耗竭、胞浆钙活性升高、氧化应激和一氧化氮的刺激。AMPK参与调节上皮细胞Na+通道ENaC和电压门控性K+通道KCNE1/KCNQ1。其部分作用是通过PI3K途径减少PIP2的形成。本研究探讨了AMPK对肾外髓质钾通道ROMK的调节作用。为此，将编码ROMK的CRNA注射到异种卵母细胞中，并分别在有或没有成分活性的AMPKγR70Q(AMPKα1-HA+AMPKβ1-FLAG+AMPKγ1R70Q)或无活性的AMPKαK45R(AMPKα1K45R+AMPKβ1-FLAG+AMPKγ1-HA)的情况下，用双电极电压钳和单通道膜片钳测量电流。用化学发光法和免疫印迹法检测全肾组织中ROMK蛋白丰度。此外，还测定了AMPKα1缺陷小鼠(AMPK−/−)和野生型小鼠(AMPK+/+)在急性K+负荷(1110 mM KCl30 mM NaHCO3，4.7 mMNaC l和2.25μ/dl BSA)前后肾脏Na+和K+排泄的变化。结果表明，共表达AMPKγR70Q而不表达AMPKαK45R显著降低了表达AMPK R70Q而不表达AMPK K45R的异种卵母细胞的电流。注射磷脂酰肌醇PI(4，5)P可显著增加表达ROMK1的Xenopus卵母细胞的电流，而AMPKγR70Q的作用则相反。在对照条件下，在肾小球滤过率、尿流率、血清醛固酮、血浆Na+、K+浓度以及绝对和部分Na+、K+排泄量方面，ampk−/−和ampk+/+小鼠之间无显著差异。在急性K+负荷后，两种基因型的肾小球滤过率、尿流率、血清醛固酮、血浆Na+和K+浓度再次相似，但Ampk−/−的肾脏绝对和部分Na+和K+排泄量高于inampk+/+小鼠。根据K+负荷后的显微穿刺法，在Ampk−/−小鼠中，Na+向早期远端小管的输送增加，而K+向近端晚期和早期远端小管的输送增加。急性K+负荷对肾ROMK1蛋白表达的上调作用在ampk−/−组较inampk+/+组更为明显。总而言之，AMPK下调了ROMK的表达，这种影响损害了急性K+负荷后肾脏排泄K+的能力。
The 5′-adenosine monophosphate-activated serine/threonine protein kinase (AMPK) is stimulated by energy depletion, increase in cytosolic Ca2+activity, oxidative stress, and nitric oxide. AMPK participates in the regulation of the epithelial Na+channel ENaC and the voltage-gated K+channel KCNE1/KCNQ1. It is partially effective by decreasing PIP2formation through the PI3K pathway. The present study explored whether AMPK regulates the renal outer medullary K+channel ROMK. To this end, cRNA encoding ROMK was injected intoXenopusoocytes with and without additional injection of constitutively active AMPKγR70Q(AMPKα1-HA+AMPKβ1-Flag+AMPKγ1R70Q), or of inactive AMPKαK45R(AMPKα1K45R+AMPKβ1-Flag+AMPKγ1-HA), and the current determined utilizing two-electrode voltage-clamp and single channel patch clamp. ROMK protein abundance was measured utilizing chemiluminescence inXenopusoocytes and western blot in whole kidney tissue. Moreover, renal Na+and K+excretion were determined in AMPKα1-deficient mice (ampk−/−) and wild-type mice (ampk+/+) prior to and following an acute K+load (111 mM KCl, 30 mM NaHCO3, 4.7 mM NaCl, and 2.25 g/dl BSA) at a rate of 500 μl/h. As a result, coexpression of AMPKγR70Qbut not of AMPKαK45Rsignificantly decreased the current in ROMK1-expressingXenopusoocytes. Injection of phosphatidylinositol PI(4,5)P2significantly increased the current in ROMK1-expressingXenopusoocytes, an effect reversed in the presence of AMPKγR70Q. Under control conditions, no significant differences betweenampk−/−andampk+/+mice were observed in glomerular filtration rate (GFR), urinary flow rate, serum aldosterone, plasma Na+, and K+concentrations as well as absolute and fractional Na+and K+excretion. Following an acute K+load, GFR, urinary flow rate, serum aldosterone, plasma Na+, and K+concentration were again similar in both genotypes, but renal absolute and fractional Na+and K+excretion were higher inampk−/−than inampk+/+mice. According to micropuncture following a K+load, delivery of Na+to the early distal tubule but not delivery of K+to late proximal and early distal tubules was increased inampk−/−mice. The upregulation of renal ROMK1 protein expression by acute K+load was more pronounced inampk−/−than inampk+/+mice. In conclusion, AMPK downregulates ROMK, an effect compromising the ability of the kidney to excrete K+following an acute K+load.
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DOI：
--
发表时间：
2009
期刊：
Bulletin et memoires de l'Academie royale de medecine de Belgique
影响因子：
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DOI：
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发表时间：
2002-12-01
影响因子：
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DOI：
10.1152/ajprenal.00300.2001
发表时间：
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期刊：
American journal of physiology. Renal physiology
影响因子：
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