WNK-SPAK signaling in the Distal Nephron

WNK-SPAK是钾依赖的信号系统的重要组成部分，被称为 Switch，调节远端硫氮敏感的氯化钠协同转运体(NCC)的活性。 曲管(DCT)，形成整个远端肾单位的结构以维持钠和钾 平衡饮食中广泛的钾摄入量。低钾消耗，在现代饮食中很常见， 以增加钠吸收和增加血液为代价，按下保存钾的途径 压力。血浆K+减少，导致细胞内氯离子减少，从而刺激WNK/SPAK 途径，导致NCC的激活和DCT的肥大/增生。该基因的分子细节 信号通路大多仍不清楚或有争议，特别是肾脏特异性的具体作用 WNK1和CAB39(MO25)的异构体，它是一种激酶适配蛋白。Na+如何转运信号以增加DCT质量 仍然是另一个巨大的谜团。在这里，我们解决这些紧迫的未知问题，挑战主流观点，以及 以创新、循序渐进的多学科方法推动该领域的边界，结合分子 遗传学，基因组学，细胞生物学，成像，最新的小鼠模型的生理表型， 和，在创新的体外系统中的机械目标1：新公布的初步数据挑战 认为KS-WNK1是WNK4的一种抑制因子，提示KS-WNK1增强WNK4-SPAK信号转导。 DCT。我们通过在DCT中专门针对KS-WNK1基因敲除并执行一个分子来测试这一想法- 生理表型分析；鉴定KS-WNK1转录本和蛋白，这是我们的初步数据 表明与之前认为的不同；并从机械上定义KS-WNK1如何调制NCC 功能。目的2：最近的数据，包括我们的初步数据，表明激酶接头蛋白，Cab39/Cab39l， 增强DCT中的WNK-SPAK信号。在这里，我们设计了一种新的老鼠模型，克服了 在野外设置路障，首次在哺乳动物系统中测试这一想法，并开发了一种新系统 从机制上了解接头蛋白如何与WNK4和SPAK相互作用，并调节它们的 功能。目标3：我们对DCT特异性成分活性Spak小鼠的RNAseq分析，这是足够的 为了激活DCT肥大和增生，鉴定了一组DCT特异性转录因子网络。我们 假设其中一个Spalt样转录因子-3(Sall3)是转录的主要调节因子 定义DCT的表观遗传指纹并在DCT扩展时维护DCT谱系的网络。为了测试 这个新的想法，我们将定义Sall3是如何响应Spak和/或NCC的激活而诱导的；建立 使用创新的新技术在DCT中特定靶向Sall3基因敲除对重塑反应的影响 成像方法；并确定Sall3是否抑制DCT特异性基因网络的基因甲基化。一起， 我们预计这些研究将产生重大影响，推动该领域向前发展。

WNK-SPAK kinases are integral components of a potassium-dependent signaling system, coined the “potassium switch,” that adjusts the activity of the thiazide-sensitive sodium chloride cotransporter (NCC) in the distal convoluted tubule (DCT) and shapes the structure of the entire distal nephron to maintain sodium and potassium balance over a wide range of dietary potassium intakes. Low potassium consumption, common in modern diets, presses the pathway to conserve potassium at the expense of increasing sodium absorption and increasing blood pressure. A decrease in plasma K+, leads to a decrease in intracellular Cl- which stimulates the WNK/SPAK pathway, leading to activation of NCC and hypertrophy/hyperplasia of the DCT. The molecular details of the signaling pathway are still mostly unknown or are controversial, especially the specific role of a kidney-specific isoform of WNK1 and Cab39 (MO25), a kinase adaptor protein. How Na+ transport signals to increase DCT mass remains another great mystery. Here, we tackle these pressing unknowns, challenging prevailing views, and pushing the boundaries of the field with an innovative, stepwise multidisciplinary approach, combining molecular genetics, genomics, cellular biology, imaging, state-of-the-art physiological phenotyping in novel mouse models, and, mechanistically in innovative in vitro systems Aim 1: Newly published and preliminary data challenges the view that KS-WNK1 is an inhibitor of WNK4 and suggests that it acts to enhance WNK4-SPAK signaling in the DCT. We test this idea by specifically targeting KS-WNK1 gene knockout in the DCT and performing a molecule- to-physiology phenotyping analysis; characterize the KS-WNK1 transcript and protein, which our preliminary data indicate are different than previously believed; and define mechanistically how KS-WNK1 modulates NCC function. Aim 2: Recent data, including our preliminary data, indicate that kinase adaptor proteins, Cab39/Cab39l, enhance WNK-SPAK signaling in the DCT. Here, we have engineered a new mouse model, overcoming a roadblock in the field, to test this idea in a mammalian system for the first time, and have developed a new system to understand mechanistically how the adaptor proteins interact with WNK4 and SPAK, and modulate their function. Aim 3: Our RNAseq analysis on our DCT-specific constitutively active SPAK mouse, which is sufficient to activate DCT hypertrophy and hyperplasia, identified a network of DCT-specific transcription factors. We hypothesize that one of these Spalt like transcription factor-3 (Sall3) is the master regulator of the transcription network that defines the epigenetic fingerprint of the DCT and maintains the DCT lineage as it expands. To test this novel idea, we will define how Sall3 is induced in response to activation of SPAK and/or NCC; establish the impact of specifically targeting Sall3 knockout in the DCT on the remodeling response using a innovative new imaging approach; and determine if Sall3 represses gene methylation of DCT-specific gene networks. Together, we expect these studies to have a major impact and push the field forward.