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 肾盂（RP）是一种将尿液从肾脏输送到输尿管的平滑肌器官， 有规律的有节奏的收缩，这对尿液运输和膀胱充盈至关重要。重复性宫缩 起源于RP的最近端区域，其中专门的细胞，称为“非典型”平滑肌细胞 （ASMCs）"，已经被提议用作起搏细胞。然而，ASMC的鉴定一直是 不精确，因此关于它们的表型和功能的实验发现是有争议的。模糊度 由于缺乏ASMC的特异性标志物，因此无法理解ASMC在驾驶中的作用 蠕动收缩或RP中起搏器活动的机制。新技术现在可以识别 由异质细胞群组成的组织内的特定细胞类型。这项研究将使用菌株 用细胞特异性报告基因和光遗传学传感器对小鼠进行了研究，以阐明 RP产生蠕动收缩。我们的初步数据表明，特化细胞ASMCs表达 血小板衍生生长因子受体α（PDGFRα），并使用表达组蛋白2B- eGFP融合蛋白驱动的内源性PDGFRα+启动子，我们可以明确地识别这些细胞， 完整组织或组织的酶分散体中。我们假设PDGFRα+细胞是起搏细胞， 肾盂中的细胞。以下具体目标将得到解决：1。检验PDGFRα+ 是RP的主要起搏细胞; 2.探讨PDGFRα+细胞中Ca 2+信号的动力学; 3. 阐明起搏点产生和传播的具体机制。因为他们的专业 作为起搏细胞，PDGFRα+细胞可能具有专门的基因表达模式， 必需的离子电导和其他信号分子，以促进起搏器活动。这将是 通过分析从分离的FACS富集群体PDGFRα+细胞中的基因表达进行了研究。 小鼠报告品系的近端RP。小鼠表达遗传编码的Ca 2+指示剂（GECI）， PDGFRα +细胞将用于原位成像PDGFRα+细胞中的Ca 2+信号传导。钙离子激活的离子通道 动力学将被确定，并将评估在反相质子化中这种传导的后果。 初步数据显示，PDGFRα+细胞在原位表现出动态的Ca 2+信号传导，并表达Ca 2+激活的 氯离子通道，Ano 1，使其成为起搏器电导的主要候选者。该项目将作为一个 为今后的研究奠定基础，以了解影响RP功能的发育或病理问题。 OMB编号0925-0001/0002（2018年1月批准至2020年3月31日修订版）页码续页格式页码