Smart patch of podocytes

项目摘要 肾小球滤过屏障(GFB)由有窗口的肾小球内皮细胞、肾小球 基底膜和足细胞。GFB的生理通透性取决于狭缝隔膜 足细胞的足突形成的。足细胞损伤是肾小球疾病的标志，例如 局灶性节段性肾小球硬化，微小病变，以及更常见的糖尿病肾病。 因此，足细胞已成为许多肾脏疾病新型干预措施的中心焦点，因为它 在调节肾小球通透性和维持肾小球结构中的重要作用 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，如 纽弗林、podocin、α-肌动蛋白4、CD2相关蛋白和瞬时受体电位典型通道6 (TRPC6)被发现并出现，为肾小球的发病机制提供了关键的见解 受伤。足细胞足突作为裂隙隔膜一部分的钙通道TRPC6的发现 蛋白质复合体表明，这些结构足细胞元件是功能性的钙隔间。这 揭示了离子通道内流在足突中介导的细胞内钙信号的作用 在正常和病理条件下的机制和途径，从而维持适当的 GFB。然而，足细胞足突中的任何通道的活动从未被报道过，因为 技术挑战。我们建议建立和测试一种新的方法来研究离子的贡献。 位于新鲜分离的肾小球足突的通道朝向GFB对照或 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实现这一点：1)振动解离 用于快速分离啮齿动物和人肾小球；2)扫描离子电导显微镜技术；3) 和智能SICM面向拓扑学的膜片钳，最终目标是开发一种允许 足细胞足突离子通道活动的电生理记录。潜在的结果 对于理解足细胞生理学和病理生理学在FSGS、MCD、 和DKD。我们已经证明了我们有能力应用许多尖端技术来研究肾功能 在遗传、分子、细胞和整个动物层面上。我们强烈地感觉到我们的技术和专题 专业知识将导致开发创新的工具，使其能够研究足细胞足部过程 职能层面。这项建议的具体目的是开发一种新的方法，允许测量离子 足细胞足突中的通道活动和执行本地内源性的初步特征 经络处于正常和病理状态。

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.