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）： 该项目的目的是定义远端肾单位调节NaCl转运的CA敏感受体（CAR）的机制，从而通过其影响血压的机制来调节NaCl转运。血压升高是心血管，肾脏疾病和糖尿病的不良后果的危险因素，这是退伍军人人口中的所有主要健康问题。与其他G蛋白偶联受体相反，该受体通过相同的G蛋白（G1i，G1Q和G113）起作用，并且也位于远端肾单位中，CAR抑制了Na，K，Cl，Cl和Water等的重吸收必须通过不同的机械主义行动。该应用的总体假设是，CAR通过激活WNK激酶来降低远端肾离子离子的转运，从而减少基底外侧K通道Kir4.1的细胞表面表达和活性，而Clc-kb是基底层CL通道。该假设基于初步数据，该数据表明：1）汽车与Kir4.1相互作用； 2）Kir4.1与CLC-KB相互作用； 3）汽车降低了Kir4.1的细胞表面表达； 4）WNK1降低了Kir4.1的细胞表面表达和活性； 5）WNK1 siRNA阻止了Kir4.1表面表达和活性的CAR依赖性降低； 6）所有这些蛋白质都位于近肾远端的基底外侧膜上。该项目具有三个特定的目标：目标1。定义汽车激活WNK1和WNK4的机制。细胞表面表达（生物素化）和通道密度（全细胞斑块）将用作通过HEK-293细胞中WNK激酶的CAR信号的度量。信号通路将通过表达促和显性的负蛋白构建体和siRNA来分析。 AIM 2。定义汽车，WNK激酶和Kir4.1的影响，对CLC-KB（带Barttin）的细胞表面表达和活性。这些研究将定义这些蛋白质的相互作用，并将利用酵母两杂化测定和HEK-293细胞进行基本的生化和电生理分析，以及用于CLC-KB和KIR4.1通道离子特异性测量的Xenopus卵母细胞活动测量。 AIM 3。定义汽车，WNK激酶，KIR4.1和CLC-KB在偏光肾上皮细胞中旋转离子转运中的重要性。 MDCK细胞单层的顶膜将被透化，以便测量基底外侧膜的运输特性，并可以确定汽车和WNK激酶对KIR4.1和CLC-KB活性的影响。这些研究将定义一种新型的机甲主义，该机车可以通过该机制调节远端肾离子离子运输，从而提高人们对远端肾癌离子运输和血压控制的理解，并改善了预防疗法。 公共卫生相关性： 高血压是心脏病，血管疾病，中风，肾脏疾病的进展以及许多糖尿病并发症的危险因素，这是退伍军人人群的所有主要健康概率。到目前为止，在家庭中运行的所有形式的高血压都是由影响肾脏释放到尿液或保留体内的盐量引起的。因此，了解肾脏如何保留或释放盐对于理解对血压的控制很重要。该项目的目的是了解一种蛋白质，一种感知血液中钙的钙受体（CAR）以及在肾脏中存在的肾脏中如何影响确定保留或释放多少盐的区域中肾脏保留或释放盐。一些遗传条件会打开汽车和疾病，其中血钙水平很高并激活汽车以通过肾脏导致盐分流失，通常会导致血压低。我们发现，该汽车使用称为WNK激酶的一组酶来控制两种影响肾脏保持或释放盐的蛋白质。本应用的具体目的是确定汽车如何激活WNK激酶，然后它们如何影响肾脏保留或释放盐的能力。第一组实验将识别汽车用来激活WNK激酶的信号传导蛋白。这些实验将使用简单的培养细胞，其中将引入所研究的蛋白质。这些蛋白质的突变形式将使用阻断或增加其功能来确定它们是否重要。第二组实验将测试汽车和WNK激酶对两种输送盐的蛋白质的影响，并确定汽车和WNK激酶是否影响它们可以运输的盐量。第三组实验将使用培养的肾细胞，这些肾细胞的生长方式使它们能够以类似于肾脏发生的方式保留或释放盐。汽车，两种转运蛋白，WNK激酶以及这些蛋白质的形式，这些蛋白会阻止效应并刺激其作用保留或释放盐。这些信息将导致认识到一种控制体内盐量的新方法，并希望有新的方法来治疗高血压和低血压。