Single-channel analysis of TRPC channels in the podocytes of freshly isolated Glomeruli

Abstract

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 membrane turnover. Dysfunction of podocytes is a major cause of glomerular kidney diseases. Gain-of-function mutations in the TRPC6 channel underlie a subset of familial forms of focal segmental glomerulosclerosis (FSGS). While growing evidence supports an important role of TRPC channels in podocytes, the regulation of these channels has yet to be investigated in freshly isolated glomeruli. Native settings in glomeruli provide, by all means, the most appropriate as well as one of the most challenging environments to study ion channel regulation. Thus, it is important to develop new methods that would better reflect the native settings of the podocytes. To address this question, we have established an experimental approach that allows studying podocytes in the freshly isolated decapsulated intact glomeruli. Here we describe the preparation of the rat glomeruli for patch-clamping, focusing on special conditions required for single-channel analysis of TRPC channels. Several tricks useful for cell-attached patch-clamping of the glomerular podocytes and solutions appropriate for registration of the TRPC channels are also provided. © 2013 Springer Science+Business Media, LLC.