Characterization and Control of the Renal WNK1 Signaling Pathway

肾脏 WNK1 信号通路的表征和控制

• 批准号: 8694696
• 负责人: AROHAN R SUBRAMANYA
• 金额: $ 33.41万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8694696
• 关键词: Address; Affect; Aldosterone; Antibodies; Binding; Biochemical; Blood Pressure; Cardiovascular system; Cell Line; Cell membrane; Cessation of life; Comparative Study; Congestive Heart Failure; Data; Disease; Distal; Employee Strikes; End stage renal failure; Epithelium; Equilibrium; Essential Hypertension; Event; Excretory function; Exhibits; Family member; Health; Heart Diseases; Hormones; Hypertension; Kidney; Knockout Mice; Knowledge; Light; Lysine; Measures; Mediating; Membrane Protein Traffic; Modeling; Molecular; Mus; N-terminal; Nephrons; Pathogenesis; Phosphorylation; Phosphotransferases; Populations at Risk; Protein Isoforms; Protein-Serine-Threonine Kinases; Proteins; Regulation; Role; SWI1; Signal Pathway; Signal Transduction; Sodium; Sodium Chloride; Stimulus; Stroke; Testing; Transcript; Ubiquitination; Work; base; blood pressure regulation; clinically relevant; improved; in vivo; insight; novel; protein degradation; protein expression; public health relevance; response; thiazide; trafficking; treatment strategy; ubiquitin-protein ligase; urinary

DESCRIPTION (provided by applicant): The thiazide-sensitive NaCl cotransporter (NCC) mediates salt reabsorption in the distal nephron (DN) of the kidney and is a key determinant of the blood pressure set point. With-No-Lysine (WNK) kinases regulate NCC membrane trafficking, phosphorylation status, and activity. One member of this family, WNK1, is expressed in the distal nephron as two major classes of isoforms with opposing functions. "Long" isoforms of WNK1 that possess intact serine-threonine kinase activity (L-WNK1) stimulate NCC. In contrast, short "kidney-specific" WNK1 isoforms that lack a kinase domain (KS-WNK1) inhibit NCC by antagonizing L-WNK1. Prior work indicates that the balance of these kinase-active and -defective products controls NCC activity, effectively functioning as an "isoform switch". The upstream mechanisms regulating WNK1 isoform balance, however, remain obscure. Our preliminary data suggest that aldosterone is a physiologically relevant stimulus that signals through the WNK1 switch to activate NCC-mediated salt reabsorption in the kidney. This proposal is driven by three novel observations: (1) First, in DN cell lines, aldosterone increases total WNK1 protein expression, but has a stronger effect on kinase active L-WNK1 than kinase defective KS-WNK1; this triggers downstream signaling events that increase NCC plasma membrane abundance and phosphorylation. (2) Second, WNK1 isoforms enriched at the protein level in the DN contain "PY motifs"- sequences which bind to Nedd4-2, an E3 ubiquitin ligase whose activity is suppressed by aldosterone. (3) Third, although KS-WNK1 transcript levels are high in the DN, it is an inherently unstable protein, and comparative studies with L-WNK1 indicate striking differences in steady state expression and protein turnover. Based on these findings, we hypothesize that aldosterone increases the total protein abundance of WNK1 isoforms in the distal nephron via Nedd4-2 inhibition, adjusting their ratio to favor increased L-WNK1 activity and NCC activation. To critically test this model, we propose to answer three questions about the regulation of WNK1 isoforms that remain incompletely addressed. First, how does the inhibition of Nedd4-2 by aldosterone regulate WNK1 protein expression and NCC activation? Second, why do L-WNK1 and KS-WNK1 exhibit different protein turnover rates? Third, how does aldosterone affect the WNK1 isoform switch in vivo? Answering these questions should provide novel insights into the molecular basis of aldosterone action, NCC regulation, and blood pressure homeostasis. Completion of the proposed aims will therefore improve our understanding of the pathogenesis of essential hypertension and highlight new strategies for its treatment.

描述（由申请人提供）：噻嗪类敏感的NaCl共转运蛋白（NCC）介导了肾脏远端肾单位（DN）中的盐重吸收，并且是血压设定点的关键决定因素。与非赖氨酸（WNK）激酶一起调节NCC膜运输，磷酸化状态和活性。该家族的一个成员WNK1在远端肾单位表示，是具有相反功能的两种主要同工型。具有完整的丝氨酸 - 硫代激酶活性（L-WNK1）的WNK1的“长”同工型刺激NCC。相比之下，缺乏激酶结构域（KS-WNK1）的简短“肾脏特异性” WNK1同工型通过拮抗L-WNK1抑制NCC。先前的工作表明，这些激酶活性和缺陷产品的平衡控制着NCC的活性，有效地充当了“同工型开关”。但是，调节WNK1同工型平衡的上游机制仍然晦涩。我们的初步数据表明，醛固酮是一种与生理相关的刺激，它通过WNK1开关发出信号，以激活肾脏中NCC介导的盐吸收。该建议是由三个新颖的观察结果驱动的：（1）首先，在DN细胞系中，醛固酮增加了总WNK1蛋白的表达，但对激酶活性L-WNK1的作用比激酶有缺陷的KS-WNK1更强。这会触发下游信号传导事件，从而增加NCC质膜丰度和磷酸化。 （2）第二，DN中富含蛋白质水平的WNK1同工型包含“ PY序列” - 与Nedd4-2结合的序列，该序列是NEDD4-2，这是一种E3泛素连接酶，其活性被醛固酮抑制。 （3）第三，尽管DN中的KS-WNK1转录水平很高，但它是一种固有的不稳定蛋白质，而使用L-WNK1的比较研究表明稳态表达和蛋白质更新方面存在明显的差异。基于这些发现，我们假设醛固酮通过NEDD4-2抑制增加了远端肾单位中WNK1同工型的总蛋白质丰度，从而调整其比率以有利于增加L-WNK1活性和NCC活化。为了批判性测试该模型，我们建议回答有关WNK1同工型调节的三个问题，这些问题尚未完全解决。首先，醛固酮对NEDD4-2的抑制如何调节WNK1蛋白表达和NCC激活？第二，为什么L-WNK1和KS-WNK1表现出不同的蛋白质周转率？第三，醛固酮在体内如何影响WNK1同工型开关？回答这些问题应提供对醛固酮作用，NCC调节和血压稳态的分子基础的新见解。因此，提出的目标的完成将提高我们对基本高血压的发病机理的理解，并突出其治疗的新策略。