Characterization and Control of the Renal WNK1 Signaling Pathway

描述（由申请人提供）：噻嗪类药物敏感的NaCl共转运体（NCC）介导肾脏远端肾元（DN）的盐重吸收，是血压设定值的关键决定因素。无赖氨酸（WNK）激酶调节NCC膜运输、磷酸化状态和活性。该家族的一个成员WNK1在远端肾元中作为两类功能相反的亚型表达。具有完整丝氨酸-苏氨酸激酶活性（L-WNK1）的WNK1“长”亚型刺激NCC。相反，缺乏激酶结构域的短“肾特异性”WNK1亚型（KS-WNK1）通过拮抗L-WNK1抑制NCC。先前的研究表明，这些激酶活性和缺陷产物的平衡控制着NCC的活性，有效地发挥着“异构体开关”的作用。然而，调控WNK1同工异构体平衡的上游机制仍然不清楚。我们的初步数据表明，醛固酮是一种生理上相关的刺激，通过WNK1开关激活ncc介导的肾脏盐重吸收。这一建议是由三个新的观察结果驱动的：(1)首先，在DN细胞系中，醛固酮增加了WNK1蛋白的总表达，但对激酶活性的L-WNK1的影响强于激酶缺陷的KS-WNK1；这触发下游信号事件，增加NCC质膜丰度和磷酸化。(2)其次，在DN蛋白水平上富集的WNK1亚型含有“PY基序”-与Nedd4-2结合的序列，Nedd4-2是一种E3泛素连接酶，其活性被醛固酮抑制。(3)第三，虽然KS-WNK1在DN中转录水平较高，但它是一种固有的不稳定蛋白，与L-WNK1的比较研究表明，在稳态表达和蛋白周转方面存在显著差异。基于这些发现，我们假设醛固酮通过抑制Nedd4-2增加了远端肾元中WNK1亚型的总蛋白丰度，调节了它们的比例，有利于增加L-WNK1活性和NCC激活。为了严格测试该模型，我们提出回答三个关于WNK1亚型调控的问题，这些问题尚未完全解决。首先，醛固酮抑制Nedd4-2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，体内醛固酮是如何影响WNK1亚型开关的？回答这些问题将为醛固酮作用、NCC调节和血压稳态的分子基础提供新的见解。因此，完成提出的目标将提高我们对原发性高血压发病机制的理解，并突出其治疗的新策略。

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.