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：
10662286
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/10662286
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 K ATPase Kidney Knockout Mice Knowledge Life Liquid substance Lysine Maintenance Measurement Measures Mediating Medical Modeling Molecular Movement Nephrons Pathway interactions Phosphorylation Phosphotransferases Physiological Plasma Play Potassium Process Protein Isoforms Proteins Pump Regional Perfusion Regulation Role Signal Pathway Signal Transduction Stress Surface System Testing Tubular formation Urine Work basolateral membrane cost extracellular falls hyperkalemia large-conductance calcium-activated potassium channels novel response sensor shear stress sodium-potassium-chloride cotransporter 1 protein symporter 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.

肾脏在维持钾稳态中起主要作用，醛固酮敏感的远端。肾单位 (ASDN) 通过将尿 K+ 排泄率与钾的变化相匹配来调节钾平衡细胞外 [K+] 在此过程中发挥着重要作用，顶端较大。电导 BK 通道响应剪切应力而打开，以促进流动诱导的 K+ 分泌 (FIKS)，尽管 IC 介导 K+ 从肾小管周围的跨细胞运动，但高钾血症会激活该过程。间质进入肾小管腔，它们的独特之处在于它们的基底外侧 K+ 进入步骤不是由相反，目前的证据表明该过程需要布美他尼敏感。 Na+-K+-2Cl- 协同转运蛋白 NKCC1 (SLC12A2) 通过直接磷酸化过程被激活。由 WNK-SPAK/OSR1 通路介导，与该通路在 FIKS 中的作用一致，我们发现高钾血症期间 IC 中 WNK1 和 SPAK 的激酶活性形式受到刺激，可能会激活然而，这与当前的范式相矛盾。当血液中钾含量较高时，应关闭 WNK 激酶这是总体目标。应用的目的是确定 NKCC1 和 WNK-SPAK/OSR1 通路在 FIKS 中的重要性，并测试一种新的机制解释了这些蛋白质如何在 ASDN 的 IC 中被特异性激活为了实现这一目标，我们将使用各种实验方法，包括整体治疗。动物研究、分离灌注小管中的运输测量、荧光细胞分选和分离 IC 来自整个肾脏，并在细胞培养模型中进行体外研究。研究将增进我们对 ASDN 响应的分子机制的了解钾胁迫。