Role of BK-Beta 1 in flow-dependent K+ secretion in the CNT

BK-Beta 1 在 CNT 流依赖性 K 分泌中的作用

基本信息

批准号：
7569312
负责人：
STEVEN SANSOM
金额：
$ 26.58万
依托单位：
UNIVERSITY OF NEBRASKA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-02-01 至 2012-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7569312
关键词：
Address Adenoviruses Arginine Attenuated Bartter Disease Brain Calcium Calcium-Activated Potassium Channel Canis familiaris Cell model Cells Coupling Cultured Cells Cyclic GMP Cyclic GMP-Dependent Protein Kinases Distal Distal convoluted renal tubule structure Diuretics Duct (organ) structure Electrolytes Epithelial Cells Excretory function Gene Transfer Genes ITPR1 gene In Vitro Intercalated Cell KCNJ1 gene Kidney Knockout Mice Laboratory Finding Limb structure Liquid substance MDCK cell Mediating Mediator of activation protein Methods Micropuncture Modeling Molecular Mus NG-Nitroarginine Methyl Ester Nephrons Nitric Oxide Oryctolagus cuniculus Pathway interactions Plasma Play Potassium Potassium Channel Property Protein Isoforms Recycling Renal tubule structure Role Scheme Signal Transduction Smooth Muscle Stretching Thiazide Diuretics Thick Tissues Transduction Gene Transfection Tubular formation apical membrane base in vivo inhibitor/antagonist large-conductance calcium-activated potassium channels novel promoter research study response shear stress wasting

项目摘要

DESCRIPTION (provided by applicant): Elevated distal flow rates resulting from volume expansion or diuretic therapy increase the delivery of fluid and electrolytes to the distal nephron resulting in stimulation of K+ secretion, often reducing plasma [K+] to critically low levels. It is known that "K+ wasting" is due partially to high flow rates that stimulate K+ secretion in the distal nephron. However, the cellular mechanisms for flow-stimulated K+ secretion have never been understood. In this proposal, we plan to address this question using novel molecular knockdown and gene transduction strategies in combination with traditional in vivo and in vitro methods for analyzing K+ secretion and channel activity. In the mammalian kidney, K+ is secreted in the distal nephron, which includes the connecting tubules and principal cells (PC) of cortical collecting ducts (CCD) and is primarily mediated under basal conditions by inward rectifying K+ channels (Kir1.1). However, recent studies have shown that large Ca-activated K+ channels (BK) are the pathways for flow-stimulated K+ secretion. We found that the BK-B1 accessory subunit, known to heighten Ca sensitivity and confer cGMP-kinase activation of the pore-forming BK-a, was expressed specifically in the rabbit and mouse connecting tubules (CNT). Moreover, we found that BK-B1 mice (unlike the wild type controls) failed to increase K+ excretion in response to volume expansion (VE)-evoked increases in flow rate. Flow-dependent K+ secretion has been demonstrated in the isolated rabbit CNT suggesting an intrinsic mechanism, such as stretch or Ca-mediated activation of BK. However, when examined with in vivo micropuncture experiments, the relation for distal flow rate vs. rate of K+ secretion has an increased slope, suggesting that other, extrinsic mediators, are influencing K+ secretion from outside the CNT. 1 possible mediator is nitric oxide, which is released with increased flow rates in the thick ascending limb and has been implicated in the increase in flow-induced K excretion in dogs. Therefore, it is hypothesized that the BK-B1 subunit augments K+ secretion in response to flow rate in the mammalian connecting tubule by conferring Ca and/or NO/cGMP sensitivity to BK-B1. The hypothesis of this proposal is based on 3 recent key findings from this laboratory: 1. BK-B1 knockout mice do not demonstrate flow-dependent K+ secretion. 2. The BK-P1 subunit is expressed specifically in the apical membranes of mammalian connecting tubules. 3. The presence of the BK-B1 enhances the calcium sensitivity of BK and is necessary for activation of BK by cGMP-mediated pathways. We will examine this hypothesis with the following Specific Aims: 1. Use BK-B1 knockout mice to determine if the BK-01 subunit is necessary for flow-dependent n secretion. 2. Determine the localization of the BK-B1 subunit in the distal nephron. 3. Determine by gene transduction whether the BK-beta1 in the mouse connecting tubule is necessary for flow-induced K+ secretion. 4. Determine the role of NO in the flow-induced increase in K+ secretion in the mouse distal nephron. Knockout mice to determine if the BK-/31 subunit is necessary for flow-dependent K+ secretion.

描述（由申请人提供）：容量扩张或利尿疗法导致远端流速升高，增加了向远端肾单位输送液体和电解质，从而刺激 K+ 分泌，通常将血浆 [K+] 降低至极低水平。众所周知，“K+ 消耗”部分是由于高流速刺激远端肾单位 K+ 分泌。然而，流量刺激 K+ 分泌的细胞机制尚未被了解。在本提案中，我们计划使用新颖的分子敲低和基因转导策略，结合传统的体内和体外方法来分析 K+ 分泌和通道活性，来解决这个问题。在哺乳动物肾脏中，K+ 在远端肾单位中分泌，其中包括皮质集合管 (CCD) 的连接小管和主细胞 (PC)，并且主要在基础条件下通过内向整流 K+ 通道 (Kir1.1) 介导。然而，最近的研究表明，大的 Ca 激活 K+ 通道 (BK) 是流量刺激 K+ 分泌的途径。我们发现 BK-B1 辅助亚基在兔和小鼠连接小管 (CNT) 中特异性表达，该亚基已知可提高 Ca 敏感性并赋予成孔 BK-a 的 cGMP 激酶激活作用。此外，我们发现 BK-B1 小鼠（与野生型对照不同）未能响应体积膨胀 (VE) 引起的流速增加而增加 K+ 排泄。流依赖性 K+ 分泌已在分离的兔 CNT 中得到证实，这表明存在内在机制，例如拉伸或 Ca 介导的 BK 激活。然而，当通过体内微穿刺实验进行检查时，远端流速与 K+ 分泌速率的关系斜率增加，表明其他外在介质正在影响 CNT 外部的 K+ 分泌。一种可能的介质是一氧化氮，它随着厚升肢中流速的增加而释放，并且与狗中流速引起的钾排泄的增加有关。因此，推测 BK-B1 亚基通过赋予 BK-B1 Ca 和/或 NO/cGMP 敏感性，增强 K+ 分泌，以响应哺乳动物连接小管中的流速。该提议的假设基于该实验室最近的 3 个关键发现： 1. BK-B1 敲除小鼠不表现出流量依赖性 K+ 分泌。 2. BK-P1亚基在哺乳动物连接小管的顶膜中特异性表达。 3. BK-B1 的存在增强了 BK 的钙敏感性，并且是通过 cGMP 介导的途径激活 BK 所必需的。我们将通过以下具体目标来检验这一假设： 1. 使用 BK-B1 敲除小鼠来确定 BK-01 亚基是否是流依赖性 n 分泌所必需的。 2. 确定 BK-B1 亚基在远端肾单位中的定位。 3. 通过基因转导确定小鼠连接小管中的 BK-β1 是否是流诱导 K+ 分泌所必需的。 4. 确定 NO 在血流诱导的小鼠远端肾单位 K+ 分泌增加中的作用。敲除小鼠以确定 BK-/31 亚基是否是流量依赖性 K+ 分泌所必需的。