Pharmacological Modulation of Renal Cell Lines

mpkCCD cells (a contribution from Alain Vandewalle, Paris) were cultured in modified DM medium as previously described^{41 (link),42 (link)} and were seeded on semipermeable filters (Transwell 0.4-μm pore size, 4.67 cm2; Corning Costar). The cells were cultured for 5 days, following which they were serum-starved and hormone-deprived for 12 h. The culture medium was changed daily. Tolvaptan (LKT Laboratories) (10–200 μM), L-sulforaphane (Sigma-Aldrich) (10 μM), ML385 (Selleck) (50 μM), dDAVP (Sigma-Aldrich) (1 nM), GSK2606414 (Sigma-Aldrich) (5 μM), thapsigargin (Sigma-Aldrich) (1 μM), and bardoxolone methyl (Cayman Chemical) (1–50 nM), and mozavaptan (Cayman Chemical) (100 μM) were applied to the basolateral side of the mpkCCD cells. The H9C2 cells were cultured in DMEM (Nacalai Tesque) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. On reaching 70–80% confluence, cells were exposed to Tolvaptan (200 μM), L-sulforaphane (10 μM), and dDAVP (Sigma-Aldrich) (1 nM). The renal proximal tubule-derived HK-2 cells were cultured in modified DM medium with 10% fetal calf serum, 100 U/ml penicillin, and 0.1 mg/mL streptomycin. HK2 cells were treated with Tolvaptan (200 μM), L-sulforaphane (10 μM), and dDAVP (Sigma-Aldrich) (1 nM) at 90–95% confluence. All reagents were solved with dimethyl sulfoxide (DMSO).