Aquaporin-2 (AQP2) Regulation by AMP-activated kinase(AMPK) in the Kidney Collec

描述(申请人提供)：水通道蛋白-2(AQP2)表达于肾脏集合管上皮主细胞，是维持体内总容量平衡所必需的。精氨酸加压素(AVP；或抗利尿激素)由下丘脑/脑下垂体分泌，是对血清渗透压升高或血压下降的正常反应。在主细胞中，AVP通过血管加压素受体II(V2R)发挥作用，导致cAMP/PKA、AQP2磷酸化、含AQP2囊泡的顶膜插入以及从尿液中将水重新吸收到间质中。AQP2的转运和亚细胞定位是由AQP2 C末端几个Ser残基的磷酸化状态和泛素化状态介导的。事实上，对AQP2调节的研究已经揭示了肾源性尿崩症(NDI)、SIADH和心力衰竭等疾病的发病机制。我们的初步数据显示，代谢应激(例如，在缺血期间)上调的代谢感受器AMP激活的激酶(AMPK)可以防止PKA介导的AQP2在体外肾脏切片中的急性心尖积聚。此外，显性负性AMPK可加速AQP2介导的卵母细胞在低渗休克时的肿胀，提示抑制AMPK活性可刺激AQP2功能。我们推测，AMPK通过直接磷酸化或通过调节PKA介导的AQP2磷酸化，诱导AQP2在细胞内积聚，并在代谢应激期间促进AQP2泛素化和降解。我们的目的是研究AMPK依赖的磷酸化调控AQP2转运和功能的机制，并研究AMPK在促进AQP2的泛素化、细胞质重新分布和降解方面的作用。我们将通过质谱法确定哪些AQP2残基被AMPK磷酸化，以及这些残基在卵母细胞和mpkCCDc14细胞中对这一水通道的亚细胞定位和活性的作用。我们还将确定AMPK是否增加了AQP2在肾脏切片和mpkCCDc14细胞中的泛素化和降解。最后，我们将确定AMPK抑制是否能阻止AQP2在化学缺血后在肾脏切片和mpkCCDc14细胞中的重新分布和/或降解。这项建议提出了一种新的潜在机制，即在缺血期间发生的代谢应激调节AQP2。我们的研究可能有助于更好地理解体内水分平衡如何与心脏和肾脏等重要器官的新陈代谢和病理相关联。 公共卫生相关性：水通道蛋白-2(AQP2)是一种在肾上皮细胞中表达的水通道，对维持全身容量的动态平衡是必不可少的。对AQP2的研究揭示了肾源性尿崩症、抗利尿激素释放不当综合征和心力衰竭的发病机制。我们推测，在急性肾缺血期间，AMPK被缺血激活，调节AQP2的含量和功能。我们的研究应该有助于更好地理解体内水平衡如何与新陈代谢和肾脏病理相关联。此外，它可能为治疗重要的身体水分平衡障碍，如肾源性尿崩症提供了洞察力。

DESCRIPTION (provided by applicant): Aquaporin-2 (AQP2) expressed in kidney collecting duct epithelial principal cells is essential for the maintenance of total body volume homeostasis. The hormone arginine vasopressin (AVP; or antidiuretic hormone) is secreted from the hypothalamus/pituitary as a normal response to increased serum osmolality or decreased blood pressure. In principal cells, AVP acts via the vasopressin receptor type II (V2R), leading to a cascade involving cAMP/PKA, AQP2 phosphorylation, apical membrane insertion of AQP2-containing vesicles and water reabsorption from the urine into the interstitium. AQP2 trafficking and subcellular localization are mediated by the phosphorylation status of several Ser residues in the AQP2 C-terminus as well as by ubiquitination status. Indeed, research on AQP2 regulation has shed light on the pathogenesis of diseases such as nephrogenic diabetes insipidus (NDI), SIADH and heart failure. Our preliminary data show that the metabolic sensor AMP-activated kinase (AMPK), which is upregulated by metabolic stress (e.g., during ischemia), prevents acute PKA-mediated AQP2 apical accumulation ex vivo in kidney slices. In addition, dominant-negative AMPK accelerates AQP2-mediated oocyte swelling upon hypotonic shock, suggesting that inhibition of AMPK activity stimulates AQP2 function. We hypothesize that AMPK induces intracellular accumulation of AQP2, either via direct phosphorylation or by modulating PKA-mediated AQP2 phosphorylation, and promotes AQP2 ubiquitination and degradation during periods of metabolic stress. Our aims are to examine the mechanisms by which AMPK-dependent phosphorylation regulates AQP2 trafficking and function and to examine the role of AMPK in enhancing the ubiquitination, cytoplasmic redistribution, and degradation of AQP2. We will determine which AQP2 residues are phosphorylated by AMPK by mass spectrometry and the role of those residues on the subcellular localization and activity of this water channel in oocytes and mpkCCDc14 cells. We will also determine whether AMPK increases AQP2 ubiquitination and degradation in kidney slices and in mpkCCDc14 cells. Finally, we will determine whether AMPK inhibition prevents AQP2 redistribution and/or degradation in kidney slices and mpkCCDc14 cells following chemical ischemia. This proposal addresses a novel potential mechanism by which metabolic stress, as occurs during ischemia, regulates AQP2. Our research will likely contribute to a better understanding of how body water homeostasis may be coupled to metabolism and pathology of vital organs such as heart and kidney. PUBLIC HEALTH RELEVANCE: Aquaporin-2 (AQP2), a water channel expressed in kidney epithelium, is essential for the maintenance of total body volume homeostasis. Research on AQP2 has shed light on the pathogenesis of nephrogenic diabetes insipidus, the syndrome of inappropriate antidiuretic hormone release, and heart failure. We hypothesize that AMPK, which is activated by ischemia, regulates AQP2 abundance and function during acute kidney ischemia. Our research should contribute to a better understanding of how body water homeostasis may be coupled to metabolism and kidney pathology. In addition, it may provide insight into the treatment of important body water balance disorders like nephrogenic diabetes insipidus.