Regulation of Renal WNK Signaling in Intercalated Cells

闰细胞中肾脏 WNK 信号传导的调节

基本信息

批准号：
10214600
负责人：
AROHAN R SUBRAMANYA
金额：
$ 37.54万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-23 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10214600
关键词：
Affect Aldosterone Animals Apical Blood Bumetanide CUL3 gene Cell Culture Techniques Cell Separation Cells Chlorides Complex Coupled Data Defect Dietary Potassium Disease Distal Distal convoluted renal tubule structure Ensure Equilibrium Excretory function Exhibits Goals Health Homeostasis Hypokalemia In Vitro Intercalated Cell Kidney Knockout Mice Knowledge Life Liquid substance Lysine Maintenance Measurement Measures Mediating Medical Modeling Molecular Movement Na(+)-K(+)-Exchanging ATPase Nephrons Pathway interactions Phosphorylation Phosphotransferases Physiological Plasma Play Potassium Process Protein Isoforms Proteins Pump Regulation Role Signal Pathway Signal Transduction Stress Surface System Testing Urine Work base basolateral membrane cost extracellular falls hyperkalemia large-conductance calcium-activated potassium channels novel response sensor shear stress sodium-potassium-chloride cotransporter 1 protein ubiquitin ligase urinary voltage

项目摘要

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.

孩子们在维持钾稳态中发挥了主要作用。醛固酮敏感的不同肾单位（ASDN）通过与尿k+排泄率与变化的变化来调节钾的平衡细胞外[K+]。插入的细胞（IC）在此过程中起着重要作用。在这些单元中，顶部大电导BK通道响应剪切应力开放，以促进流动诱导的K+分泌（FIKS），A 高钾血症激活的过程。尽管IC介导了k+的跨细胞运动在管腔腔中的间质，它们的独特之处在于它们的基底外侧K+进入步骤未通过 Na+/K+-ATPase。相反，当前的证据表明，此过程需要对bumetanide敏感 Na+-K+-2Cl- cotransporter，NKCC1（SLC12A2）。 NKCC1通过直接磷酸化激活，这是一个过程由WNK-SPAK/OSR1途径介导。与Fiks中这一途径的角色一致，我们发现在高钾血症期间，在IC中刺激了WNK1和SPAK的激酶活性形式，可能会激活基底外侧NKCC1和顶端BK通道。但是，这与当前的范式相矛盾，该范式认为当血液中钾水平较高时，应关闭WNK激酶。总体目标应用是确定NKCC1和WNK-Spak/OSR1途径在FIKS中的重要性，并测试一种新的机制，解释了如何在ASDN的IC中专门激活这些蛋白质高钾血症。为了实现这一目标，我们将使用一种多种实验方法，包括整个动物研究，孤立灌注小管中的转运测量，荧光细胞分类和分离 IC源自整个肾脏和细胞培养模型中的体外研究。从这些中获得的信息研究将促进我们对ASDN对反应的分子机制的了解钾胁迫。