Role of piezo channels in intercalated cells

压电通道在嵌入细胞中的作用

• 批准号: 10443098
• 负责人: Thomas R Kleyman
• 金额: $ 61.79万
• 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/10443098
• 关键词: Actins; Adult; Affect; Aldosterone; Apical; Attention; Bicarbonates; Binding; Brush Border; Calcium; Caliber; Cations; Cell Differentiation process; Cell membrane; Cell physiology; Cells; Cilia; Coupled; Cytoskeleton; Development; 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; Phase; Piezo 1 ion channel; Piezo ion channels; Potassium; Radial; Reporting; Role; Stretching; Testing; Tubular formation; Urine; Variant; Work; absorption; base; 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; 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.

醛固酮敏感性远端肾单位（ASDN）的嵌入细胞（IC）分泌H+和HCO 3-。新兴 有证据表明，IC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。IC中的顶端BK通道被流动激活- 刺激细胞内Ca 2+浓度（[Ca 2 +]i）增加。快速的初始机械诱导的增加， [Ca2+]i反映基底外侧Ca 2+进入和内部Ca 2+储存的释放。Piezo 1，一个家庭的成员， 机械敏感性非选择性阳离子通道，沿着IC的基底外侧膜表达， ASDN中的主细胞（PC）。在初步研究中，我们发现Piezo 1抑制剂抑制了流动- 诱导皮质集合管（CCD）和FIKS中的[Ca 2 +]i反应，而激活剂增加 [Ca2+]i在以低流速灌注的CCD中。流动诱导的IC [Ca 2 +]i反应在CCD中基本上不存在 来自具有IC特异性Piezo 1基因缺失的小鼠。这些观察结果表明，Piezo 1介导流动- 诱导早期基底外侧Ca 2+进入ASDN上皮细胞，这是BK通道激活的关键因素。 基于这些观察，我们假设Piezo 1通道在ASDN中起机械传感器的作用 并通过促进基底外侧Ca 2+进入和继发性激活管腔Ca 2+进入途径来实现FIKS， IC。目的1中提出的实验将定义Piezo 1在脑中的定位和发育表达。 ASDN和响应于低K+（LK）或高K+（HK）的CCD中Piezo 1表达的细胞类型特异性变化 饮食或醛固酮。目标2中的研究将确定Piezo 1激活剂和抑制剂对基础和 流量刺激的[Ca 2 +]i，对从小鼠中分离的CCD中净跨上皮Na+和K+转运（JNa和JK）的影响 对照K+（CK）和HK喂养的小鼠，以及通过膜片钳评估的对IC BK通道活性的影响。研究 目的3中提出的将确定IC中Piezo 1的靶向缺失是否改变小鼠中的K+处理，影响 在微灌注的CCD中，流动诱导的[Ca 2 +]i和JNa和JK增加，并影响IC BK通道活性， 通过膜片钳评估。我们希望我们提出的研究结果将揭示新的和意想不到的 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。