Division of Nephrology, Department of Internal Medicine and.
Division of Nephrology and Hypertension, Department of Internal Medicine, Molecular Medicine Program, University of Utah, Salt Lake City, Utah.
J Am Soc Nephrol. 2018 May;29(5):1449-1461. doi: 10.1681/ASN.2017101091. Epub 2018 Mar 30.
With No Lysine kinase (WNK) signaling regulates mammalian renal epithelial ion transport to maintain electrolyte and BP homeostasis. Our previous studies showed a conserved role for WNK in the regulation of transepithelial ion transport in the Malpighian tubule. Using assays and transgenic lines, we examined two potential WNK regulators, chloride ion and the scaffold protein mouse protein 25 (Mo25), in the stimulation of transepithelial ion flux., autophosphorylation of purified WNK decreased as chloride concentration increased. In conditions in which tubule intracellular chloride concentration decreased from 30 to 15 mM as measured using a transgenic sensor, WNK activity acutely increased. WNK activity in tubules also increased or decreased when bath potassium concentration decreased or increased, respectively. However, a mutation that reduces chloride sensitivity of WNK failed to alter transepithelial ion transport in 30 mM chloride. We, therefore, examined a role for Mo25. In kinase assays, Mo25 enhanced the activity of the WNK downstream kinase Fray, the fly homolog of mammalian Ste20-related proline/alanine-rich kinase (SPAK), and oxidative stress-responsive 1 protein (OSR1). Knockdown of in the Malpighian tubule decreased transepithelial ion flux under stimulated but not basal conditions. Finally, whereas overexpression of wild-type , with or without , did not affect transepithelial ion transport, overexpressed with chloride-insensitive increased ion flux. Cooperative interactions between chloride and Mo25 regulate WNK signaling in a transporting renal epithelium.
无赖氨酸激酶（WNK）信号调节哺乳动物肾上皮细胞离子转运，以维持电解质和血压的平衡。我们之前的研究表明，WNK 在调节肾单位中跨上皮离子转运方面发挥着保守作用。利用测定法和转基因系，我们研究了两个潜在的 WNK 调节剂，氯离子和支架蛋白小鼠蛋白 25（Mo25），在刺激跨上皮离子流中的作用。结果显示，当氯离子浓度从 30 毫摩尔增加到 15 毫摩尔时，纯化的 WNK 的自磷酸化减少。在使用转基传感器测量的情况下，当管腔内细胞内氯离子浓度从 30 毫摩尔降低到 15 毫摩尔时，WNK 活性急性增加。当浴液钾浓度分别降低或增加时，WNK 活性在管腔中也增加或减少。然而，一种降低 WNK 氯离子敏感性的突变未能改变 30 毫摩尔氯离子条件下的跨上皮离子转运。因此，我们研究了 Mo25 的作用。在激酶测定中，Mo25 增强了 WNK 下游激酶 Fray 的活性，Fray 是哺乳动物 Ste20 相关脯氨酸/丙氨酸丰富激酶（SPAK）和氧化应激反应 1 蛋白（OSR1）的果蝇同源物。在马氏管中敲低 Mo25 可降低刺激但不基础条件下的跨上皮离子流。最后，尽管过表达野生型 Mo25 及其突变体均不影响跨上皮离子转运，但过表达具有氯离子不敏感的 Mo25 增加了离子流。氯离子和 Mo25 之间的协同相互作用调节了运输肾上皮细胞中的 WNK 信号。
