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Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Department of Medicine, University of Southern California/University Kidney Research Organization, Kidney Research Center, Division of Nephrology and Hypertension, Keck School of Medicine of University of Southern California, Los Angeles, California.
Am J Physiol Renal Physiol. 2016 Nov 1;311(5):F890-F900. doi: 10.1152/ajprenal.00308.2016. Epub 2016 Aug 17.
Aquaporin-2 (AQP2) is essential to maintain body water homeostasis. AQP2 traffics from intracellular vesicles to the apical membrane of kidney collecting duct principal cells in response to vasopressin [arginine vasopressin (AVP)], a hormone released with low intravascular volume, which causes decreased kidney perfusion. Decreased kidney perfusion activates AMP-activated kinase (AMPK), a metabolic sensor that inhibits the activity of several transport proteins. We hypothesized that AMPK activation also inhibits AQP2 function. These putative AMPK effects could protect interstitial ionic gradients required for urinary concentration during metabolic stress when low intravascular volume induces AVP release. Here we found that short-term AMPK activation by treatment with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR; 75 min) in kidney tissue prevented baseline AQP2 apical accumulation in principal cells, but did not prevent AQP2 apical accumulation in response to the AVP analog desmopressin (dDAVP). Prolonged AMPK activation prevented AQP2 cell membrane accumulation in response to forskolin in mouse collecting duct mpkCCD cells. Moreover, AMPK inhibition accelerated hypotonic lysis of Xenopus oocytes expressing AQP2. We performed phosphorylation assays to elucidate the mechanism by which AMPK regulates AQP2. Although AMPK weakly phosphorylated immunoprecipitated AQP2 in vitro, no direct AMPK phosphorylation of the AQP2 COOH-terminus was detected by mass spectrometry. AMPK promoted Ser-261 phosphorylation and antagonized dDAVP-dependent phosphorylation of other AQP2 COOH-terminal sites in cells. Our findings suggest an increasing, time-dependent antagonism of AMPK on AQP2 regulation with AICAR-dependent inhibition of cAMP-dependent apical accumulation and AVP-dependent phosphorylation of AQP2. This inhibition likely occurs via a mechanism that does not involve direct AQP2 phosphorylation by AMPK.
水通道蛋白2（AQP2）对于维持机体水平衡至关重要。在血管加压素[精氨酸血管加压素（AVP）]的作用下，AQP2从细胞内囊泡转运至肾集合管主细胞的顶端膜，AVP是一种在血管内容量降低时释放的激素，可导致肾脏灌注减少。肾脏灌注减少会激活AMP激活的蛋白激酶（AMPK），这是一种代谢传感器，可抑制多种转运蛋白的活性。我们推测AMPK激活也会抑制AQP2的功能。当低血管内容量诱导AVP释放时，这些假定的AMPK效应可能会保护代谢应激期间尿液浓缩所需的间质离子梯度。在此我们发现，在肾组织中用5-氨基咪唑-4-甲酰胺-1-β-D-呋喃核糖苷（AICAR；75分钟）进行短期AMPK激活可阻止主细胞中基线AQP2顶端积累，但不能阻止对AVP类似物去氨加压素（dDAVP）的反应中AQP2顶端积累。在小鼠集合管mpkCCD细胞中，长时间的AMPK激活可阻止对福斯可林的反应中AQP2细胞膜积累。此外，AMPK抑制加速了表达AQP2的非洲爪蟾卵母细胞的低渗裂解。我们进行了磷酸化测定以阐明AMPK调节AQP2的机制。尽管AMPK在体外对免疫沉淀的AQP2进行了弱磷酸化，但质谱未检测到AQP2羧基末端的直接AMPK磷酸化。AMPK促进细胞中Ser-261磷酸化，并拮抗其他AQP2羧基末端位点的dDAVP依赖性磷酸化。我们的研究结果表明，AMPK对AQP2调节的拮抗作用呈时间依赖性增加，伴有AICAR依赖性抑制cAMP依赖性顶端积累和AQP2的AVP依赖性磷酸化。这种抑制可能通过一种不涉及AMPK直接磷酸化AQP2的机制发生。
