Smart patch of podocytes

足细胞智能斑块

• 批准号: 10284970
• 负责人: ALEXANDER STARUSCHENKO
• 金额: $ 18.69万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-23 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10284970
• 关键词: Animals; Apoptosis; Area; Astrocytes; Behavior; CD2-associated protein; Calcium; Calcium Channel; Calcium Signaling; Cells; Characteristics; Chronic Kidney Failure; Communication; Complex; Development; Diabetic Nephropathy; Disease; Electrophysiology (science); Elements; Endothelial Cells; Epithelial Cells; Focal Segmental Glomerulosclerosis; Foot Process; Functional disorder; Funding Opportunities; Gatekeeping; Genetic; Genetic Transcription; Goals; Hematological Disease; Human; Imaging Techniques; Injury; Intervention; Ion Channel; Kidney; Kidney Diseases; Knowledge; Left; Lesion; Link; Maintenance; Maps; Measurement; Measures; Mediating; Methods; Molecular; Mutation; NPHS2 protein; Nephrotic Syndrome; Neurons; Outcome; Pathogenesis; Pathologic; Pathology; Pathway interactions; Permeability; Physiological; Physiology; Play; Process; Property; Protocols documentation; Recording of previous events; Regulation; Renal function; Renal glomerular disease; Reporting; Research; Rodent; Role; Scanning; Series; Signal Transduction; Structural Protein; Structure; Techniques; Technology; Testing; Therapeutic; Urologic Diseases; alpha Actinin; cell motility; cell transformation; glomerular basement membrane; glomerular endothelium; glomerular filtration; innovation; insight; nephrin; novel; novel strategies; patch clamp; podocyte; prognostic; protein complex; receptor; scanning ion conductance microscopy; slit diaphragm; tool

Project Summary The glomerular filtration barrier (GFB) is comprised of the fenestrated glomerular endothelium, the glomerular basement membrane, and the podocyte. Physiologic permeability of the GFB depends on the slit diaphragms formed by the foot processes of the podocytes. Podocyte damage is a hallmark of glomerular disease, such as focal segmental glomerulosclerosis, minimal change disease, and more common diabetic kidney disease. Therefore, the podocyte has become a central focus for novel interventions in many renal diseases due to its vital role in the regulation of glomerular permeability and maintenance of glomerular structure through interactions with other glomerular parenchymal cells. Mutations in several podocyte structural proteins such as nephrin, podocin, alpha-actinin 4, CD2-associated protein, and transient receptor potential canonical channel 6 (TRPC6) were identified and have emerged to provide critical insight into the pathogenesis of glomeruli injury. The discovery of the calcium channel TRPC6 in podocyte foot processes as part of the slit diaphragm protein complex suggests that these structural podocyte elements are functional Ca2+ compartments. This opens the question about the role of intracellular Ca2+ signaling mediated by ion channels influx in foot processes for mechanisms and pathways in normal and pathological conditions and, thereby, the maintenance of a proper GFB. However, the activity of any channels in the foot processes of podocytes has never been reported due to technical challenges. We propose to establish and test a novel approach to study the contributions of ion channels localized in the foot processes of podocyte of freshly isolated glomeruli toward the GFB control or development of glomerular injury. We will do this by combining our recently developed tools: 1) vibrodissociation for fast isolation of rodent and human glomeruli; 2) scanning ion conductance microscopy (SICM) technique; 3) and smart SICM topography oriented patch-clamp with the ultimate goal to develop a protocol allowing electrophysiological recordings of ion channels activity in the podocyte foot processes. The potential outcome will be critically important for the understanding of podocyte physiology and pathophysiology in FSGS, MCD, and DKD. We have demonstrated our capabilities to apply many cutting-edge techniques to study renal function at the genetic, molecular, cellular, and whole animal levels. We feel strongly that our technical and topical expertise will lead to the development of innovative tool allowing to study the podocyte foot processes on functional level. The Specific Aim of this proposal is to develop a novel approach allowing to measure ion channel activity in the podocyte foot processes and perform initial characterization of native endogenous channels in normal and pathological state.

项目概要 肾小球滤过屏障（GFB）由有孔的肾小球内皮、肾小球 基底膜和足细胞。 GFB 的生理渗透性取决于狭缝隔膜 由足细胞的足突形成。足细胞损伤是肾小球疾病的一个标志，例如 局灶节段性肾小球硬化症、微小病变以及更常见的糖尿病肾病。 因此，足细胞因其独特的功能而成为许多肾脏疾病新干预措施的中心焦点。 通过调节肾小球通透性和维持肾小球结构发挥重要作用 与其他肾小球实质细胞的相互作用。几种足细胞结构蛋白的突变，例如 去氧肾上腺素、podocin、α-肌动蛋白 4、CD2 相关蛋白和瞬时受体电位规范通道 6 (TRPC6) 的发现和出现为肾小球的发病机制提供了重要的见解 受伤。足细胞足突中钙通道 TRPC6 的发现，作为裂隙隔膜的一部分 蛋白质复合物表明这些结构性足细胞元件是功能性 Ca2+ 区室。这 提出了足突中离子通道流入介导的细胞内 Ca2+ 信号传导的作用问题 正常和病理条件下的机制和途径，从而维持适当的 GFB。然而，由于足细胞足突中任何通道的活性从未被报道过 技术挑战。我们建议建立并测试一种新方法来研究离子的贡献 位于新鲜分离的肾小球足细胞足突中的通道朝向 GFB 控制或 肾小球损伤的发展。我们将通过结合我们最近开发的工具来做到这一点：1）振动解离 用于快速分离啮齿动物和人类肾小球； 2）扫描离子电导显微镜（SICM）技术； 3） 和智能 SICM 形貌导向膜片钳，最终目标是开发一种协议，允许 足细胞足突中离子通道活动的电生理记录。潜在的结果 对于理解 FSGS、MCD、足细胞生理学和病理生理学至关重要 和 DKD。我们已经展示了应用许多尖端技术来研究肾功能的能力 在遗传、分子、细胞和整个动物水平上。我们强烈地感觉到我们的技术和主题 专业知识将导致创新工具的开发，允许研究足细胞足部过程 功能级别。该提案的具体目标是开发一种新方法，允许测量离子 足细胞足突中的通道活动并执行天然内源性的初步表征 正常和病理状态下的通道。