Single-Channel Analysis of TRPC Channels in the Podocytes of Freshly Isolated Glomeruli
One of the most important functions of the kidney is the filtration of the blood that takes place in the glomeruli. Glomerular epithelial cells (podocytes) have several functions, including regulation of the filtration process and glomerular basement memb
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Introduction One of the main functions of the kidney is blood filtration that is tightly controlled by renal glomeruli. Glomerular podocytes are the central components of the renal filtration barrier. Podocytes are very dynamic cells which, at least in part, play a role in the pathogenesis of most proteinuric glomerulopathies (1, 2). Transient receptor potential canonical (TRPC) channels belong to the larger superfamily of the TRP channels. TRPC channels play an important role in the pathogenesis of renal and cardiovascular diseases (3–6). Several members of the TRPC family, including TRPC6, are essential components of the podocyte slit diaphragm, where they are integrated into a signaling complex that interacts
Nikita Gamper (ed.), Ion Channels: Methods and Protocols, Methods in Molecular Biology, vol. 998, DOI 10.1007/978-1-62703-351-0_28, © Springer Science+Business Media, LLC 2013
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Daria V. Ilatovskaya and Alexander Staruschenko
with a number of podocyte structural proteins, including nephrin, podocin, alpha-actinin-4, and calcineurin (7–10). It was demonstrated that TRPC6 protein is the genetic impetus for an autosomal dominant form of focal segmental glomerulosclerosis (FSGS) (11–13). The known TRPC6 gain-of-function mutations cause an increase in calcium current in podocytes. A number of studies provided new insights into the role of TRPC channels, and particularly TRPC6, in the functionality of the glomeruli and highlighted their role in mediating concomitant diseases (11, 12, 14). Most of these studies were conducted on the cultured podocytes (15–17) or in the recombinant systems (18, 19) which, although being quite sustainable, cannot provide the comprehensive setting for understanding the regulation of the channels under physiological conditions. The lack of such studies has not been a result of their unimportance, but rather resulted from the fact that electrophysiological recording of endogenous ion channels in their native surrounding is a complicated procedure requiring a combination of pure isolation of glomeruli, separation of endogenous currents, electrophysiological skills, etc. Gloy et al., who have developed a technique allowing to measure membrane voltages and ion conductances of podocytes in isolated glomeruli (20), stated later describing the approach they developed that “it is important to develop new methods that better reflect the in vivo situation of podocytes, but studying podocytes function in situ is methodologically very difficult” (2). Taking into account the emerging need to comprehend the functionality of ion channels of the podocytes, we have attempted to perform single-channel analysis of endogenous TRPC-like ion channels in the podocytes of isolated decapsulated glomeruli on the basis of the technique suggested by Gloy and colleagues (2, 20). The method described here consists of several steps including removal and perfusion of the rat kidney, isolation of the glomeruli fraction by sequentially pushing the kidney cortex through the steel sieves of different mesh
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