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描述(由申请人提供):噻嗪敏感性NaCl协同转运蛋白(NCC)介导肾脏远端肾单位(DN)的盐重吸收,是血压设定点的关键决定因素。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WNK 1在远端肾单位中表达为具有相反功能的两种主要亚型。具有完整丝氨酸-苏氨酸激酶活性的WNK 1的“长”同种型(L-WNK 1)刺激NCC。相比之下,缺乏激酶结构域的短“肾特异性”WNK 1同种型(KS-WNK 1)通过拮抗L-WNK 1来抑制NCC。先前的工作表明,这些激酶活性和缺陷产物的平衡控制NCC活性,有效地发挥“同种型开关”的作用。然而,调节WNK 1亚型平衡的上游机制仍然不清楚。我们的初步数据表明,醛固酮是一种生理相关的刺激,通过WNK 1开关激活肾脏中NCC介导的盐重吸收。这一提议是由三个新的观察结果驱动的:(1)首先,在DN细胞系中,醛固酮增加总WNK 1蛋白表达,但对激酶活性L-WNK 1的影响比对激酶缺陷型KS-WNK 1的影响更强;这触发了增加NCC质膜丰度和磷酸化的下游信号传导事件。(2)第二,在DN中蛋白质水平富集的WNK 1同种型含有“PY基序”-与Nedd 4 -2结合的序列,Nedd 4 -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-WNK 1转录水平在DN中很高,但它是一种固有的不稳定蛋白,与L-WNK 1的比较研究表明稳态表达和蛋白质周转存在显著差异。基于这些发现,我们假设醛固酮通过Nedd 4 -2抑制增加远端肾单位中WNK 1亚型的总蛋白丰度,调节它们的比例以有利于增加L-WNK 1活性和NCC活化。为了严格测试这个模型,我们建议回答三个关于WNK 1亚型调控的问题,这些问题尚未完全解决。首先,醛固酮对Nedd 4 -2的抑制如何调节WNK 1蛋白表达和NCC激活?第二,为什么L-WNK 1和KS-WNK 1表现出不同的蛋白质周转率?第三,醛固酮如何影响体内WNK 1亚型转换?阐明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,完成所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。
英文摘要
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.
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Model Systems Core
Regulation of Renal WNK Signaling in Intercalated Cells
Regulation of Renal WNK Signaling in Intercalated Cells
Regulation of Renal WNK Signaling in Intercalated Cells
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