Role of piezo channels in intercalated cells

压电通道在嵌入细胞中的作用

• 批准号: 10598161
• 负责人: Thomas R Kleyman
• 金额: $ 60.45万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10598161
• 关键词: Actins; Adult; Affect; Aldosterone; Apical; Attention; Bicarbonates; Binding; Brush Border; Calcium; Cations; Cell Differentiation process; Cell membrane; Cell physiology; Cells; Cilia; Coupled; Cytoskeleton; Development; Diameter; Diet; Dietary Potassium; Disease; Distal; Duct (organ) structure; Epithelial Cells; Equation; Event; Excretory function; Exhibits; Extracellular Matrix; Family; Genetic; Intake; Intercalated Cell; Ion Channel; Kidney; Life; Link; Liquid substance; Location; Maintenance; Mechanics; Mediating; Membrane; Molecular; Mus; Natural Immunity; Nephrons; PKD2 protein; Pathway interactions; Perfusion; Phase; Piezo 1 ion channel; Piezo ion channels; Potassium; Radial; Reporting; Role; Stretching; Testing; Tubular formation; Urine; Variant; Work; absorption; basolateral membrane; cell type; epithelial Na+ channel; experimental study; extracellular; inhibitor; large-conductance calcium-activated potassium channels; mechanical force; mechanotransduction; member; neonatal mice; new therapeutic target; novel; patch clamp; polycystic kidney disease 1 protein; postnatal; response; sensor; shear stress; transmission process; urinary

Intercalated cells (ICs) in the aldosterone-sensitive distal nephron (ASDN) secrete H+ and HCO3-. Emerging evidence has identified nontraditional roles for ICs, including absorption of filtered Na+ and Cl-, flow-induced K+ secretion (FIKS) and participation in innate immunity. Apical BK channels in ICs are activated by a flow- stimulated increase in intracellular Ca2+ concentration ([Ca2+]i). The rapid initial mechanoinduced increase in [Ca2+]i reflects basolateral Ca2+ entry and release of internal Ca2+ stores. Piezo1, a member of a family of mechanosensitive non-selective cation channels, is expressed along the basolateral membrane of ICs and principal cells (PCs) in the ASDN. In preliminary studies, we found that a Piezo1 inhibitor dampens both the flow- induced [Ca2+]i response in cortical collecting ducts (CCDs) as well as FIKS, whereas an activator increases [Ca2+]i in CCDs perfused at slow flow rates. The flow-induced IC [Ca2+]i response was largely absent in CCDs from mice with an IC-specific genetic deletion of Piezo1. These observations suggest that Piezo1 mediates flow- induced early basolateral Ca2+ entry into ASDN epithelial cells, a key factor in the activation of BK channels. Based on these observations, we hypothesize that Piezo1 channels function as mechanosensors in the ASDN and enable FIKS by facilitating basolateral Ca2+ entry and secondarily activating luminal Ca2+ entry pathways in ICs. Experiments proposed in Aim 1 will define the localization and developmental expression of Piezo1 in the ASDN, and cell type-specific changes in Piezo1 expression in CCDs in response to a low K+ (LK) or high K+ (HK) diet, or aldosterone. Studies in Aim 2 will determine the effects of Piezo1 activators and inhibitors on basal and flow-stimulated [Ca2+]i, effects on net transepithelial Na+ and K+ transport (JNa and JK) in CCDs isolated from control K+ (CK) and HK fed mice, and effects on IC BK channel activity as assessed by patch clamp. Studies proposed in Aim 3 will determine whether targeted deletion of Piezo1 in ICs alters K+ handling in mice, affects flow-induced increases in [Ca2+]i and JNa and JK in microperfused CCDs, and affects IC BK channel activity as assessed by patch clamp. We expect that the results of our proposed studies will uncover novel and unexpected pathways involved in urinary K+ excretion and identify potential targets for novel therapies to treat K+ imbalances.

醛固酮敏感的远端肾单位 (ASDN) 中的闰细胞 (IC) 分泌 H+ 和 HCO3-。新兴 有证据表明 IC 具有非传统作用，包括吸收过滤后的 Na+ 和 Cl-、流动诱导的 K+ 分泌（FIKS）和参与先天免疫。 IC 中的顶端 BK 通道由流动激活 刺激细胞内 Ca2+ 浓度 ([Ca2+]i) 增加。初始机械诱导的快速增加 [Ca2+]i 反映基底外侧 Ca2+ 进入和内部 Ca2+ 储存的释放。 Piezo1，家庭成员 机械敏感的非选择性阳离子通道，沿着 IC 的基底外侧膜表达， ASDN 中的主小区 (PC)。在初步研究中，我们发现 Piezo1 抑制剂会抑制流动- 诱导皮质集合管 (CCD) 和 FIKS 中的 [Ca2+]i 反应，而激活剂则增加 CCD 中的 [Ca2+]i 以低流速灌注。 CCD 中基本上不存在流动引起的 IC [Ca2+]i 响应 来自具有 IC 特异性 Piezo1 基因缺失的小鼠。这些观察结果表明 Piezo1 介导流动- 诱导基底外侧 Ca2+ 早期进入 ASDN 上皮细胞，这是激活 BK 通道的关键因素。 基于这些观察，我们假设 Piezo1 通道在 ASDN 中充当机械传感器 并通过促进基底外侧 Ca2+ 进入并二次激活管腔 Ca2+ 进入途径来实现 FIKS 集成电路。目标 1 中提出的实验将定义 Piezo1 在 ASDN 和 CCD 中 Piezo1 表达的细胞类型特异性变化以响应低 K+ (LK) 或高 K+ (HK) 饮食或醛固酮。目标 2 中的研究将确定 Piezo1 激活剂和抑制剂对基础和 流动刺激的 [Ca2+]i，对分离自 CCD 的净跨上皮 Na+ 和 K+ 转运（JNa 和 JK）的影响 对照 K+ (CK) 和 HK 喂养的小鼠，并通过膜片钳评估对 IC BK 通道活性的影响。研究 目标 3 中提出的目标将确定 IC 中 Piezo1 的定向删除是否会改变小鼠的 K+ 处理，影响 微灌注 CCD 中流动诱导的 [Ca2+]i 以及 JNa 和 JK 增加，并影响 IC BK 通道活性： 通过膜片钳评估。我们期望我们提出的研究结果将发现新颖且意想不到的结果 涉及尿 K+ 排泄的途径，并确定治疗 K+ 失衡的新疗法的潜在靶点。