Pacemaker cells and mechanism in the renal pelvis

肾盂起搏细胞及其机制

• 批准号: 10116375
• 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/10116375
• 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

项目概要 肾盂 (RP) 是一种平滑肌器官，负责将尿液从肾脏输送到输尿管，产生 有规律的节律性收缩对于尿液输送和膀胱充盈至关重要。传播宫缩 起源于 RP 的最近端区域，其中有称为“非典型”平滑肌细胞的特殊细胞 (ASMCs)”，已被提议作为起搏细胞。然而，ASMCs 的鉴定尚未得到证实。 不精确，因此关于其表型和功能的实验结果存在争议。歧义 其原因是缺乏 ASMC 的特异性标记物，并且阻碍了对 ASMC 在驱动中的作用的理解。 蠕动收缩或 RP 中起搏器活动的机制。更新的技术现在可以识别 组织内由异质细胞群组成的特定细胞类型。本研究将使用菌株 带有细胞特异性报告基因和光遗传学传感器的小鼠，以阐明起搏器和反应细胞如何 RP 产生蠕动收缩。我们的初步数据表明，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 eGFP 融合蛋白驱动内源性 PDGFRα+ 启动子，我们可以明确地识别这些细胞 完整的组织或组织的酶分散体。我们假设 PDGFRα+ 细胞是起搏器 肾盂中的细胞。将解决以下具体目标： 1. 检验 PDGFRα+ 的假设 是 RP 的主要起搏细胞； 2. 研究PDGFRα+细胞中Ca2+信号传导的动态； 3. 阐明起搏器产生和传播的具体机制。由于他们的专业 作为起搏细胞，PDGFRα+细胞可能具有编码的特殊基因表达模式 重要的离子电导和其他信号分子以促进起搏器活动。这将是 通过分析从 FACS 富集的群体中分离出的 PDGFRα+ 细胞的基因表达来进行研究 小鼠报告品系的近端 RP。表达基因编码 Ca2+ 指示剂 (GECI) 的小鼠 PDGFRα+细胞将用于对PDGFRα+细胞中的Ca2+信号传导进行原位成像。 Ca2+ 激活的离子通道 将确定动力学并评估 RP 蠕动中的这种电导的后果。 初步数据表明，PDGFRα+ 细胞在原位表现出动态 Ca2+ 信号传导，并表达 Ca2+ 激活的 Cl- 通道，Ano1，使其成为起搏器电导的主要候选者。该项目将作为 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（修订版 01/18 批准至 03/31/2020） 页面延续格式页面