Regulation of Renal WNK Signaling in Intercalated Cells

肾脏在维持钾的动态平衡中起主要作用。对醛固酮敏感的末端 肾单位(ASDN)通过将尿钾排泄率与尿钾浓度的变化相匹配来调节钾平衡。 胞外[K]。间质细胞在这一过程中起着重要的作用。在这些细胞内，顶端较大 电导BK通道在切应力作用下开放，以促进流动诱导的K分泌(FIKS)，a 高血钾激活的过程。尽管ICs介导K从肾小管周跨细胞移动 间质进入小管腔，它们的独特之处在于它们的基底外侧K进入步骤不是由 Na/K-ATPase。相反，目前的证据表明，这一过程需要布美他尼敏感。 Na-K-2Cl-共转运蛋白，NKCC1(Slc12a2)。NKCC1通过直接磷酸化激活，这是一个过程 由WNK-Spak/OSR1通路介导。与这条通路在Fiks中的作用一致，我们发现 WNK1和Spak的激酶活性形式在高钾血症时在ICs中被刺激，可能被激活 基底外侧NKCC1和心尖BK通道。然而，这与当前的范式相矛盾，目前的范式认为 当血液中的钾含量较高时，WNK激酶应该关闭。这个项目的总体目标是 应用是确定NKCC1和WNK-Spak/OSR1通路在FIKS中的重要性，并测试 一种新的机制来解释这些蛋白是如何在ASDN的ICs中被特异性激活的 高钾血症。为了实现这一目标，我们将使用各种实验方法，包括整体 动物研究，离体灌流管的运输测量，荧光细胞分选和分离 ICS来源于全肾，并在细胞培养模型中进行体外研究。从这些信息中获得的信息 研究将促进我们对ASDN应答的分子机制的了解 钾胁迫。

The kidneys play a primary role in the maintenance of potassium homeostasis. The aldosterone sensitive distal nephron (ASDN) regulates potassium balance by matching the rate of urinary K+ excretion with changes in extracellular [K+]. Intercalated cells (ICs) play an important role in this process. Within these cells, apical large conductance BK channels open in response to shear stress to facilitate flow-induced K+ secretion (FIKS), a process activated by hyperkalemia. Though ICs mediate the transcellular movement of K+ from the peritubular interstitium into the tubule lumen, they are unique in that their basolateral K+ entry step is not carried out by the Na+/K+-ATPase. Instead, current evidence suggests that this process requires a bumetanide-sensitive Na+-K+-2Cl- cotransporter, NKCC1 (SLC12A2). NKCC1 is activated via direct phosphorylation, a process mediated by the WNK-SPAK/OSR1 pathway. Consistent with a role for this pathway in FIKS, we find that the kinase active forms of WNK1 and SPAK are stimulated in ICs during hyperkalemia, likely to activate basolateral NKCC1 and apical BK channels. This, however, contradicts the current paradigm, which contends that WNK kinases should be switched off when potassium levels are high in the blood. The overall goal of this application is to determine the importance of NKCC1 and the WNK-SPAK/OSR1 pathway in FIKS, and to test a novel mechanism that explains how these proteins are specifically activated in ICs of the ASDN during hyperkalemia. To accomplish this objective, we will use a variety experimental approaches, including whole animal studies, transport measurements in isolated perfused tubules, fluorescent cell sorting and isolation of ICs derived from whole kidney, and in vitro studies in cell culture models. The information gained from these studies will advance our knowledge of the molecular mechanisms underlying the ASDN’s response to potassium stress.