Functional distinctions in cytosolic calcium regulation between cells of the glomerular filtration barrier

The importance of intracellular calcium ([Ca(2+)](i)) regulation in the glomerular filtration barrier (GFB) has recently been highlighted by mutations in the cation channel TRPC6, resulting in a renal-specific phenotype. We examined the effects of FFA, a tool that can activate TRPC6, on [Ca(2+)](i) in human conditionally immortalised glomerular endothelial cells (ciGEnC) and human podocytes (ciPod) that form the GFB. Changes in [Ca(2+)](i) stimulated by FFA were measured in Fura 2-AM loaded cells. In GEnC, cell activation by FFA was dependent on external Ca(2+), yet in ciPod it was not. Depletion of internal Ca(2+) stores with thapsigargin did not affect cell activation by FFA in ciGEnC, but inhibited it in ciPod in a nephrin-dependent manner, demonstrated using nephrin deficient (ND) ciPod in conjunction with nephrin rescue experiments. FFA induced [Ca(2+)](i) store release in ciPod, but not in ciGEnC or ND ciPod. In parallel, there were differences in the localisation of overexpressed TRPC6 between ciGEnC and ciPod. Furthermore, co-transfection of nephrin with TRPC6 in HEK293 cells reduced the FFA-induced increase in [Ca(2+)](i) and nephrin clustering altered TRPC6 distribution. In conclusion, cell activation by FFA in podocytes stimulates the opening of a Ca(2+) channel, probably TRPC6, in a nephrin-dependent manner with a different activation profile to GEnC.