Ddavp

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).

ODS was induced according to an adapted protocol [98 (link)]. Briefly, an osmotic minipump (Model 1007D, Charles River Laboratories, Germany) filled with desmopressin (dDAVP, 10 µg/ml, Sigma Aldrich, USA) was implanted subcutaneously caudal to the shoulder blade (day 0). Standard chow was switched to low-sodium liquid diet (EF15710-10 EF R/M AIN 76A, Ssniff, Germany), fed ad libitum during hyponatremia. At day 6, i.p. injection of sodium chloride solution (1 M, 1 ml per 100 g body weight) was used to increase serum sodium levels close to normonatremia. After sodium correction, food was switched back to standard pellet chow. At days 0 and 6, blood sodium levels were measured. All animals were monitored daily and brain tissue was harvested after 3 (n = 4), 4 (n = 3), 6 (n = 4), 7 (n = 3), 13 (n = 5), 14 (n = 2) and 21 (n = 6) days post correction (dpc). Untreated age-matched rats were used as healthy controls (n = 5).

Cells were stimulated for 15 min with dDAVP (1 nM or 1 μM, Sigma) or 25 μM forskolin (Sigma) dissolved in DMSO in the presence of 3-isobutyl-1-methylxanthine (IBMX). Controls were DMSO + IBMX. Total cAMP levels were measured using a commercially available cAMP enzyme immunoassay kit (Sigma).