Tolvaptan

Animal studies were performed using 8-week-old C57BL/6 male mice (CLEA JAPAN) that had free access to food and water. The mice were subcutaneously injected with tolvaptan (LKT Laboratories) or the same amount of DMSO (Sigma-Aldrich) control solution for 2 days by osmotic minipumps (Alzet model 1003D) as previously described42 (link). The mice were then intraperitoneally injected with 300 μl of Wnt5a or PBS control. Urine samples were collected within 2 h after the administration of Wnt5a. Urine osmolality was measured with a Fiske One-ten Osmometer. Tissue samples for western blot and immunofluorescence were obtained 1 h after the administration of Wnt5a. The animal experiments were approved by The Animal Care and Use Committee of Tokyo Medical and Dental University.

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).

The animal studies were performed using 10-week-old male C57BL/6 mice that were housed individually in metabolic cages with free access to food and water to measure urine osmolality, urine output, and water intake. The mice were subcutaneously infused with tolvaptan (LKT Laboratories, Inc., St. Paul, MN, USA) or tolvaptan plus FMP-API-1 using osmotic minipumps (Alzet model 1003D; ALZA Corporation, Cupertino, CA, USA) as previously described^{35 (link)}. Urine osmolality was measured with a Fiske One-ten Osmometer (John Morris Scientific Pty Ltd., Chatswood, NSW, Australia). The protocols of the animal experiments were approved by the Animal Care and Use Committee of Tokyo Medical and Dental University.