Immortalized Renal Proximal Tubule Cell Culture

Immortalized renal proximal tubule (RPT) cells from WKY rats and SHRs were cultured at 37°C in 95% air and 5% CO₂. The RPT cells were grown in DMEM/F12 with insulin, transferrin, epidermal growth factor, and dexamethasone [10 (link),11 (link),16 (link)]. The RPT cells (95% confluence) were serum-deprived for 2 h, and then treated with vehicle, adiponectin (Biovision), Compound C (adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) antagonist, 10⁻⁷ mol/l), NG-nitro-l-arginine methyl ester (l-NAME; non-selective NOS inhibitor, 5 × 10⁻⁷ mol/l) (Sigma–Aldrich), alone, or in combination. The inhibitors were added 30 min before the addition of adiponectin. Then, the treated cells were extracted in cold cell lysis buffer, sonicated, kept on ice for 1 h, then centrifuged at the speed of 14000 ×g for 30 min to collect the supernatant.