WNK-SPAK signaling in the Distal Nephron

WNK-SPAK激酶是钾依赖性信号传导系统的组成部分，称为“钾依赖性信号传导系统”。 开关”，其调节远端中噻嗪敏感性氯化钠协同转运蛋白（NCC）的活性。 曲小管（DCT）和形状的结构，整个远端肾单位，以维持钠和钾 平衡各种膳食钾摄入量。钾摄入量低，在现代饮食中很常见， 以增加钠吸收和增加血容量为代价， 压力血浆K+的减少导致细胞内Cl-的减少，Cl-刺激WNK/SPAK 这一过程可能通过激活NCC和DCT的肥大/增生途径来完成。分子的细节 信号通路仍然大多是未知的或有争议的，特别是肾脏特异性 WNK 1和Cab 39（MO 25）的同种型，Cab 39是一种激酶接头蛋白。Na+如何传输信号以增加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数，挑战主流观点， 以创新的，逐步的多学科方法推动该领域的边界，结合分子生物学， 遗传学、基因组学、细胞生物学、成像、新型小鼠模型中最先进的生理表型分析， 并且，在创新的体外系统中，目标1：新发表的和初步的数据挑战了 认为KS-WNK 1是WNK 4的抑制剂，并表明它可以增强WNK 4-SPAK信号传导， DCT。我们通过在DCT中特异性靶向KS-WNK 1基因敲除并进行分子- 生理表型分析;表征KS-WNK 1转录本和蛋白质，我们的初步数据 表明与先前所认为不同;并机械地定义KS-WNK 1如何调节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，Cab 39/Cab 39 l， 增强DCT中的WNK-SPAK信令。在这里，我们设计了一种新的小鼠模型， 在该领域的路障，测试这一想法在哺乳动物系统的第一次，并已开发出一个新的系统， 为了从机制上理解衔接蛋白如何与WNK 4和SPAK相互作用，并调节它们的功能， 功能目的3：我们对我们的DCT特异性组成型活性SPAK小鼠的RNAseq分析，这是足够的。 激活DCT肥大和增生，确定了一个DCT特异性转录因子网络。我们 假设这些Spalt样转录因子-3（Sall 3）中的一个是转录的主调节因子， 定义DCT的表观遗传指纹并在其扩展时维护DCT谱系的网络。测试 基于这一新的想法，我们将定义Sall 3是如何响应于SPAK和/或NCC的激活而被诱导的; 使用一种创新的新方法，在DCT中特异性靶向Sall 3敲除对重塑反应的影响 成像方法;并确定Sall 3是否抑制DCT特异性基因网络的基因甲基化。在一起， 我们希望这些研究能产生重大影响，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.