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.

醛固酮敏感远端肾单位(ASDN)中的间质细胞(ICs)分泌H+和HCO3-。新兴 有证据表明，ICs的非传统作用，包括吸收过滤的Na+和Cl-，流动诱导的K+ 分泌(FIKS)和参与先天免疫。ICs中的心尖BK通道由一种流激活- 刺激细胞内钙离子浓度([Ca2+]i)升高。最初由机械引起的快速增长 [Ca~(2+)]i反映内钙储备的基础侧钙进入和释放。PIEZO1，一个家庭的成员 机械敏感的非选择性阳离子通道，沿着ICs的基底膜和 ASDN中的主信元(PC)。在初步研究中，我们发现Piezo1抑制剂抑制了这两种流动- 在皮质集合管(CCDs)和FIks中诱导[Ca~(2+)]i反应，而激活剂则增加 慢流条件下灌流的CCDs中[Ca~(2+)]i。在CCDS中，流动诱导的IC[Ca~(2+)]i反应基本不存在 来自具有IC特异性Piezo1基因缺失的小鼠。这些观察表明，Piezo1介导流- 诱导基底外侧钙离子早期进入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中起机械传感器的作用 并通过促进基底外侧钙离子内流和继而激活管腔内钙内流通路来激活FIKS ICS.目标1中提出的实验将定义Piezo1在 低K+(LK)或高K+(HK)条件下CDs中Piezo1表达的细胞类型特异性变化 节食，或称醛固酮。AIM 2的研究将确定Piezo1激活剂和抑制剂对基础和 流刺激的CDs[Ca~(2+)]_i对Na~+和K~+跨上皮净转运的影响 对照组K+(CK)和HK饲喂小鼠，并用膜片钳方法观察对ICBK通道活动的影响。研究 在目标3中提出的将确定在ICs中靶向删除Piezo1是否改变小鼠对K+的处理，影响 流诱导微灌流的CCs内[Ca~(2+)]i、JNA和JK升高，并影响ICBK通道的活动，如 用膜片钳检测。我们期待我们提议的研究结果将揭示出新奇和意想不到的东西。 参与尿K+排泄的途径，并确定治疗K+失衡的新疗法的潜在靶点。