Thursday, December 26, 2024
12/26/2024
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.
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