L sulforaphane

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).

Human dermal microvascular endothelial cells, HMEC-1 (purchased from ATCC, Cat. No. CRL3243™), were cultured in MCDB 131 Medium (Thermo Fisher Scientific) supplemented with FBS (10%, Thermo Fisher Scientific), L-glutamine (2 mM, Thermo Fisher Scientific), hydrocortisone (0.05 mg/ml, Sigma-Aldrich), Epidermal Growth Factor (5 ng/ml), 100 U/ml penicillin, 10 μg/ml streptomycin, and 250 ng/ml amphotericin B (Sigma-Aldrich). HMEC-1 were cultured under standard conditions (37°C, 5% CO₂) and passaged two times a week. In all experiments, cells between the second and tenth passages were used only when they reached full postplating confluency. Bardoxolone methyl (CDDO methyl ester, Cayman Chemical), dimethyl fumarate (Sigma-Aldrich), and L-sulforaphane (Sigma-Aldrich) initially diluted in DMSO were added to the culture medium at final concentrations of 100 nM, 300 nM, 500 nM, 1 μM, 3 μM, and 5 μM in triplicates, if not stated otherwise. Control cells were treated with 0.05% DMSO added to the culture medium. To check for acute toxicity, the cells were incubated with tested agents for 3 hours. Standard toxicity assessment time was set to 24 hours, according to the literature [30 (link)].

To assess Nrf2 activation as a pretreatment strategy, we used the Nrf2 activator l-sulforaphane (SFN) to induce the translocation of Nrf2 to the nucleus. ECFCs were incubated with different concentrations of l-sulforaphane (Sigma-Aldrich) for 4 hours unless otherwise specified, prior to exposure to H₂O₂.