Novel Ca receptor signaling pathways for control of renal ion transport

控制肾离子转运的新型 Ca 受体信号通路

• 批准号: 7687870
• 负责人: Richard Tyler Miller
• 金额: --
• 依托单位: LOUIS STOKES CLEVELAND VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7687870
• 关键词: Affect; Bartter Disease; Bicarbonates; Biochemical; Biotinylation; Blood; Blood Pressure; Body Fluids; Calcium; Calcium-Sensing Receptors; Cardiovascular system; Carrier Proteins; Cell Communication; Cell surface; Cells; Characteristics; Cloning; Complications of Diabetes Mellitus; Coupled; Cultured Cells; Data; Disease; Distal; Dominant-Negative Mutation; Edema; Enzymes; Epithelial Cells; Family; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Health; Heart Diseases; Hematological Disease; Hereditary Disease; Heterotrimeric GTP-Binding Proteins; Hypercalcemia; Hypertension; Hypotension; Intracellular Second Messenger; Ion Transport; Ions; Kidney; Kidney Diseases; Lead; Location; MDCK cell; Measurement; Measures; Membrane; Membrane Transport Proteins; Minor; Modeling; Mutation; Nephrons; Pathologic; Pathway interactions; Patients; Phenotype; Phosphoproteins; Phosphotransferases; Physiological; Population; Potassium Channel; Process; Proteins; Receptor Activation; Receptor Signaling; Regulation; Renal Glycosuria; Risk Factors; Running; Scaffolding Protein; Second Messenger Systems; Signal Pathway; Signal Transduction; Signaling Protein; Small Interfering RNA; Sodium Chloride; Stroke; Surface; System; Testing; Two-Hybrid System Techniques; Urine; Variant; Vascular Diseases; Veterans; Water; Xenopus oocyte; Yeasts; adverse outcome; apical membrane; base; basolateral membrane; blood pressure regulation; body volume; clinically significant; density; extracellular; improved; in vivo; inward rectifier potassium channel; kidney cell; loss of function mutation; monolayer; mutant; novel; protein transport; receptor; research study; response; yeast two hybrid system

DESCRIPTION (provided by applicant): The goal of this project is to define the mechanism by which the Ca-sensing receptor (CaR) regulates (inhibits) NaCl transport in the distal nephron, and consequently the mechanism by which it affects blood pressure. Elevated blood pressure is a risk factor for adverse outcomes in cardiovascular, renal diseases and diabetes, all major health problems in the veteran population. In contrast to other G protein-coupled receptors that act via the same G proteins (G1i, G1q, and G113) and that are also located in the distal nephron, the CaR inhibits reabsorption of Na, K, Cl, and water, and so must act via distinct mechasnisms. The overall hypotheses of this application is that the CaR reduces distal nephron ion transport by activating WNK kinases that reduce cell surface expression and activity of Kir4.1, a basolateral K channel, and ClC-Kb, a basolateal Cl channel. This hypothesis is based on preliminary data that demonstrate that: 1) the CaR interacts with Kir4.1; 2) that Kir4.1 interacts with ClC-Kb; 3) that the CaR reduces cell surface expression of Kir4.1; 4) that WNK1 reduces cell surface expression and activity of Kir4.1; 5) that WNK1 siRNA blocks the CaR-dependent reduction in Kir4.1 surface expression and activity; and that 6) all of these proteins are located on the basolateral membrane of the distal nephron. The project has three specific aims: Aim 1. Define the mechanism by which the CaR activates WNK1 and WNK4. Cell surface expression (biotinylation) and channel density (whole cell patch) will be used as measures of CaR signaling through the WNK kinases in HEK-293 cells. Signaling pathways will be analyzed by expressing acitvated and dominant negative protein constructs and siRNAs. Aim 2. Define the effects of the CaR, WNK kinases, and Kir4.1, on the cell surface expression and activity of ClC-Kb (with barttin). These studies will define the interactions of these proteins and will make use of yeast two-hybrid assays and HEK-293 cells for the basic biochemical and electrophysiologic analysis, and Xenopus oocytes for ion-specific measurement of the ClC-Kb and Kir4.1 channel activity measurements. Aim 3. Define the importance of the CaR, WNK kinases, Kir4.1, and ClC-Kb in the control of transepithelial ion transport in polarized renal epithelial cells. The apical membranes of MDCK cell monolayers will be permeabilized, so that the transport characteristics of the the basolateral membrane are measured and the effects of the CaR and WNK kinases on Kir4.1 and ClC-Kb activities can be determined. These studies will define a novel mechansism by which the CaR regulates distal nephron ion transport, will lead to improved understanding of distal nephroin ion transport and blood pressure control, and potentilly improved therapy. PUBLIC HEALTH RELEVANCE: High blood pressure is a risk factor for heart disease, vascular disease, stroke, progression of kidney disease, and many complications of diabetes, all major health probelems for the veteran population. So far, all forms of high and low blood pressure that run in families are caused by processes that affect the amount of salt the kidneys release into the urine or retain in the body. For that reason, understanding how the kidneys retain or release salt is important for understanding control of blood pressure. The goal of this project is to understand how one protein, the calcium receptor (CaR) that senses calcium in the blood and that is present in the kidneys in a region that is important for determining how much salt is retained or released, affects how the kidneys retain or release salt. Some genetic conditions that turn on the CaR and diseases where blood calcium levels are high and activate the CaR to cause loss of salt through the kidneys often resulting in low blood pressure. We found that the CaR uses a group of enzymes called WNK kinases to control two proteins that affect how kidneys retain or release salt. The specific purpose of this application is to determine exacly how the CaR activates the WNK kinases and then how they affect the ability of the kidney to retain or release salt. The first group of experiments will identify the signaling proteins the CaR uses to activate the WNK kinases. These experiments will use simple cultured cells into which the proteins being studied will be introduced. Mutant forms of these proteins that either block or increase their function will be used to determine if they are important. The second set of experiments will test the effects of the CaR and WNK kinases on two proteins that transport salt and will determine if the CaR and WNK kinases affect the amount of salt they can transport. The third set of experiments will make use cultured kidney cells that are grown in a way that allows them to retain or release salt in a manner that is similar to what happens in a kidney. The CaR, the two transport proteins, the WNK kinases, and forms of these proteins that block effects and stimulate their effects will be put into the kidney cells to determine if what was found in the previous experiments applies to kidney cells and the way they normally retain or release salt. This information will result in recognition of a new way to control the amount of salt in the body and hopefully new ways to treat high and low blood pressure.

描述（由申请人提供）： 本项目的目标是确定钙敏感受体（CaR）调节（抑制）远端肾单位中NaCl转运的机制，从而确定其影响血压的机制。血压升高是心血管、肾脏疾病和糖尿病不良后果的危险因素，这些都是退伍军人群体的主要健康问题。与其他G蛋白偶联受体（G1 i，G1 q和G113）通过相同的G蛋白发挥作用，也位于远端肾单位相反，CaR抑制Na，K，Cl和水的重吸收，因此必须通过不同的机制发挥作用。本申请的总体假设是，CaR通过激活WNK激酶减少远端肾单位离子转运，WNK激酶减少细胞表面表达和Kir4.1（基底外侧K通道）和ClC-Kb（基底外侧Cl通道）的活性。该假设基于初步数据，所述初步数据证明：1）CaR与Kir4.1相互作用; 2）Kir4.1与ClC-Kb相互作用; 3）CaR降低Kir4.1的细胞表面表达; 4）WNK 1降低Kir4.1的细胞表面表达和活性; 5）WNK 1 siRNA阻断Kir4.1表面表达和活性的CaR依赖性降低;以及6）所有这些蛋白质都位于远端肾单位的基底外侧膜上。该项目有三个具体目标：目标1。定义CaR激活WNK 1和WNK 4的机制。细胞表面表达（生物素化）和通道密度（全细胞片）将用作通过HEK-293细胞中的WNK激酶的CaR信号传导的量度。将通过表达活化和显性阴性蛋白构建体和siRNA来分析信号传导途径。目标2.定义CaR、WNK激酶和Kir4.1对细胞表面表达和ClC-Kb活性的影响（与barttin一起）。这些研究将确定这些蛋白质的相互作用，并将利用酵母双杂交试验和HEK-293细胞进行基本的生物化学和电生理学分析，并利用非洲爪蟾卵母细胞进行ClC-Kb和Kir4.1通道活性测量的离子特异性测量。目标3。明确CaR、WNK激酶、Kir4.1和ClC-Kb在极化肾上皮细胞跨上皮离子转运控制中的重要性。MDCK细胞单层的顶膜将被透化，以便测量基底外侧膜的转运特征，并且可以确定CaR和WNK激酶对Kir4.1和ClC-Kb活性的影响。这些研究将确定一种新的机制，通过这种机制，CaR调节远端肾单位离子转运，将导致对远端肾离子转运和血压控制的更好理解，并可能改善治疗。 公共卫生关系： 高血压是心脏病、血管疾病、中风、肾病进展和糖尿病的许多并发症的危险因素，这些都是退伍军人的主要健康问题。到目前为止，家庭中所有形式的高血压和低血压都是由影响肾脏释放到尿液中或保留在体内的盐量的过程引起的。因此，了解肾脏如何保留或释放盐对于了解血压控制非常重要。该项目的目标是了解一种蛋白质，即钙受体（CaR），它可以感知血液中的钙，并且存在于肾脏中的一个区域，该区域对于确定保留或释放多少盐很重要，影响肾脏如何保留或释放盐。一些遗传条件，打开CaR和疾病，其中血钙水平高，激活CaR，导致盐通过肾脏损失，往往导致低血压。我们发现CaR使用一组称为WNK激酶的酶来控制两种影响肾脏保留或释放盐的蛋白质。本申请的具体目的是准确地确定CaR如何激活WNK激酶，然后它们如何影响肾脏保留或释放盐的能力。第一组实验将鉴定CaR用于激活WNK激酶的信号蛋白。这些实验将使用简单的培养细胞，将被研究的蛋白质引入其中。这些蛋白质的突变形式，无论是阻止或增加其功能，将用于确定它们是否重要。第二组实验将测试CaR和WNK激酶对两种转运盐的蛋白质的影响，并将确定CaR和WNK激酶是否影响它们可以转运的盐的量。第三组实验将使用培养的肾细胞，这些细胞的生长方式允许它们以类似于肾脏中发生的方式保留或释放盐。CaR，两种转运蛋白，WNK激酶，以及这些蛋白质的阻断作用和刺激作用的形式将被放入肾细胞中，以确定先前实验中发现的是否适用于肾细胞以及它们通常保留或释放盐的方式。这些信息将导致人们认识到一种控制体内盐量的新方法，并有望找到治疗高血压和低血压的新方法。