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

描述（由申请人提供）：在肾收集管上皮主细胞中表达的水通道蛋白-2 （AQP2）对于维持总体容量稳态至关重要。精氨酸抗利尿激素（AVP或抗利尿激素）是当血清渗透压升高或血压降低时，下丘脑/垂体分泌的一种正常反应。在主细胞中，AVP通过抗利尿激素受体II型（V2R）起作用，导致包括cAMP/PKA、AQP2磷酸化、含AQP2的囊泡顶膜插入和尿中水重吸收到间质在内的级联反应。AQP2转运和亚细胞定位是由AQP2 c端几个丝氨酸残基的磷酸化状态以及泛素化状态介导的。事实上，对AQP2调控的研究揭示了肾源性尿崩症（NDI）、SIADH和心力衰竭等疾病的发病机制。我们的初步数据表明，代谢传感器amp活化激酶（AMPK）在代谢应激（例如，在缺血期间）下上调，可防止肾片中pka介导的急性AQP2顶端积累。此外，低渗休克时，显性阴性AMPK可加速AQP2介导的卵母细胞肿胀，表明抑制AMPK活性可刺激AQP2功能。我们假设AMPK通过直接磷酸化或调节pka介导的AQP2磷酸化诱导AQP2在细胞内的积累，并在代谢应激期间促进AQP2的泛素化和降解。我们的目的是研究AMPK依赖性磷酸化调节AQP2运输和功能的机制，并研究AMPK在增强AQP2泛素化、细胞质再分布和降解中的作用。我们将通过质谱法确定AMPK磷酸化了哪些AQP2残基，以及这些残基在卵母细胞和mpkCCDc14细胞中亚细胞定位和该水通道活性中的作用。我们还将确定AMPK是否会增加肾切片和mpkCCDc14细胞中AQP2的泛素化和降解。最后，我们将确定AMPK抑制是否会阻止肾切片和mpkCCDc14细胞在化学缺血后AQP2的再分布和/或降解。这一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代谢和病理相关联。

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.