C16 ceramide

Whole-cell planar patch-clamp recordings were performed using cultured murine podocytes. The planar patch-clamp technology combined with a pressure control system (Port-a-Patch, Nanion Technologies) was applied as previously described [35 (link)]. Ion currents were recorded, filtered, and analyzed using an Axopatch 200B amplifier, an Axon Digidata 1550B low-noise data acquisition system and the pClamp10 software (Axon instruments). Seal resistance was higher than 1 GΩ. Internal solution contained 50 mM CsCl, 10 mM NaCl, 60 mM CsF, 20 mM EGTA, and 10 mM HEPES/CsOH. External solution contained 140 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 5 mM D-glucose monohydrate, and 10 mM HEPES/NaOH. Seal enhancing solution contained 80 mM NaCl, 3 mM KCl, 10 mM MgCl₂, 35 mM CaCl₂, and 10 mM HEPES/HCl. Podocytes were stimulated by administration of compounds into the internal and external solutions. Carbenoxolone, probenecid, carmofur, ceranib-1, and ceranib-2 were purchased from Sigma-Aldrich. D-erythro-MAPP, C-16 ceramide (dissolved in dimethylformamide to 0.5 mg/mL and diluted to 40 μM with internal solution before addition), sphingosine, and sphingosine-1-phosphate (dissolved in methanol to 1 mg/mL and diluted to 40 μM with internal solution before addition) were purchased from Cayman. Recombinant mouse adiponectin protein was purchased from Abcam.