Pacemaker cells and mechanism in the renal pelvis

肾盂起搏细胞及其机制

• 批准号: 10397176
• 负责人: KENTON M SANDERS
• 金额: $ 47.77万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10397176
• 关键词: Address; Affect; Alpha Cell; Anatomy; Animal Model; Automobile Driving; Biological Markers; Bladder; Cells; Chimeric Proteins; Data; Defect; Development; Diabetes Mellitus; Distal; Electrophysiology (science); Exhibits; Fertilization; Functional disorder; Future; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Generations; Heart; Histones; Hydronephrosis; Image; In Situ; Inflammation; Intervention; Investigation; Ion Channel; Kidney; Kidney Failure; Location; Molecular; Monitor; Morphology; Mouse Strains; Mus; Muscle; Obstruction; Organ; Outcome Study; PDGFRB gene; Pacemakers; Pathologic; Periodicity; Peristalsis; Phenotype; Physiological; Platelet-Derived Growth Factor alpha Receptor; Population; Population Heterogeneity; Pump; Reagent; Regulatory Pathway; Renal pelvis; Reporter; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Smooth Muscle; Smooth Muscle Myocytes; Techniques; Testing; Therapeutic Intervention; Tissue Model; Tissues; Transgenic Mice; Ureter; Urinary tract; Urine; Visceral; cell type; clinical investigation; follow-up; interstitial; interstitial cell; kidney cell; new technology; nodal myocyte; novel; optogenetics; pre-clinical; preclinical study; prevent; promoter; receptor; renal damage; sensor; targeted treatment; therapeutic evaluation

Project Summary The renal pelvis (RP), a smooth muscle organ that transports urine from the kidney to the ureter, generates regular rhythmic contractions that are vital for urine transport and bladder filling. Propagating contractions originate in the most proximal region of the RP, where specialized cells, called “atypical” smooth muscle cells (ASMCs)”, have been proposed to serve as the pacemaker cells. However, identification of ASMCs has been imprecise, and thus experimental findings regarding their phenotype and functions are controversial. Ambiguities arise from the lack of specific markers for ASMCs and have prevented understanding the role of ASMC in driving peristaltic contractions or the mechanism of pacemaker activity in the RP. Newer technologies can now identify specific cell-types within tissues composed of heterogeneous populations of cells. This study will employ strains of mice with cell-specific reporters and optogenetic sensors to clarify how the pacemaker and responder cells of the RP generate peristaltic contractions. Our preliminary data show that the specialized cells, ASMCs, express platelet-derived-growth-factor-receptor-alpha (PDGFRα), and using transgenic mice that express a histone 2B- eGFP fusion protein driven off the endogenous PDGFRα+ promoter, we can identify these cells unequivocally in intact tissues or in enzymatic dispersions of the tissues. We hypothesize that PDGFRα+ cells are pacemaker cells in the renal pelvis. The following Specific Aims will be addressed: 1. Test the hypothesis that PDGFRα+ are the primary pacemaker cells of the RP; 2. Investigate the dynamics of Ca2+ signaling in PDGFRα+ cells; 3. Elucidate the specific mechanisms of pacemaker generation and propagation. Because of their specialized function as pacemaker cells, PDGFRα+ cells are likely to have specialized gene expression patterns that encode essential ionic conductances and other signaling molecules to facilitate pacemaker activity. This will be investigated by analysis of gene expression in FACS-enriched populations PDGFRα+ cells isolated from the proximal RP of the reporter strain of mice. Mice expressing a genetically encoded Ca2+ indicator (GECI) in PDGFRα + cells will be used to image Ca2+ signaling in PDGFRα+cells in situ. The ion channels activated by Ca2+ dynamics will be determined and the consequences of this conductance in RP peristalsis will be evaluated. Preliminary data show that PDGFRα+ cells exhibit dynamic Ca2+ signaling in situ and express the Ca2+-activated- Cl- channel, Ano1, making this a prime candidate for the pacemaker conductance. This project will serve as a basis for future studies to understand developmental or pathological problems affecting RP function. OMB No. 0925-0001/0002 (Rev. 01/18 Approved Through 03/31/2020) Page Continuation Format Page

项目摘要 肾盆(RP)是一种将尿液从肾脏输送到输尿管的平滑肌肉器官，它产生 有规律的有节奏的收缩，对尿液运输和膀胱充盈至关重要。传播性宫缩 起源于RP的最近端，在那里有特殊的细胞，称为“非典型”的平滑肌细胞 (ASMC)“，已被提议用作起搏细胞。然而，ASMC的鉴定一直是 因此，关于它们的表型和功能的实验结果是有争议的。模棱两可 由于缺乏ASMC的特定标志物，阻碍了对ASMC在驾驶中的作用的了解 RP的蠕动收缩或起搏活动的机制。较新的技术现在可以识别 由不同种类的细胞组成的组织内的特定细胞类型。这项研究将使用菌株 具有细胞特异性报告和光遗传传感器的小鼠，以阐明起搏器和反应细胞如何 RP产生蠕动收缩。我们的初步数据显示，特化细胞，ASMC，表达 血小板衍生生长因子受体-α(PDGFRα)，并使用表达组蛋白2B的转基因小鼠- 从内源性PDGFRα+启动子驱动的绿色荧光蛋白融合蛋白中，我们可以明确地在 完整的组织或酶分散的组织。我们假设PDGFRα+细胞是起搏器 肾盆中的细胞。将解决以下具体目标：1.检验假设：PDGFRα+ 研究PDGFRα+细胞内钙信号的动态变化； 阐明起搏器产生和繁殖的具体机制。因为他们的专业 作为起搏细胞，PDGFRα+细胞可能具有编码 促进起搏器活动的基本离子电导和其他信号分子。这将是 用流式细胞仪分析从人脐血中分离的PDGFRα+细胞的基因表达 报告品系小鼠的近端RP。表达基因编码的钙指示剂(Geci)的小鼠 PDGFRα+细胞将被用于原位成像PDGFRα+细胞中的钙信号。钙离子激活的离子通道 将确定动力学，并将评估这种电导在RP蠕动中的后果。 初步数据显示，PDGFRα+细胞在原位表现出动态的钙信号，并表达钙激活的... CL-通道，Ano1，使其成为起搏器电导的主要候选者。这个项目将作为一个 为未来研究了解影响RP功能的发育或病理问题奠定基础。 OMB编号0925-0001/0002(01/18修订版批准至2020年3月31日)页面续格式页面