Pd123319

Opossum kidney (OK) proximal tubular cells (American Type Culture Collection, Manassas, VA, USA) were cultured at 37°C in an atmosphere of 95% air/5% CO₂ in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 mg/ml). Once confluent, the OK cells were treated with azilsartan (1.0 × 10⁻⁶ mol/l), candesartan (3.0 × 10⁻⁷ mol/l), or vehicle for 24 h, and simultaneously with angiotensin II (1.0 × 10⁻¹¹ mol/l).
To evaluate the contributions of different protein-degradation routes, the effect of azilsartan on NHE3 was examined in the presence of inhibitors of lysosomal (leupeptin 0.5 μg/ml) (Wako) or proteasomal (lactacystin 10 μmol/l) (Wako) pathways of degradation. To assess the involvement of the angiotensin II type-2 receptor (AT2R), the AT2R antagonist PD123319 (10 μmol/l; (Wako) was used.

Cell culture dishes and multi-well plates were purchased from Becton Dickinson Biosciences (Bedford, MA, USA), Thermo Scientific (Waltham, MA, USA), and TrueLine (Nippon Genetics Co. Ltd.; Tokyo, Japan). Dulbecco’s modified Eagle’s medium (DMEM) and alpha modified Eagle’s medium (αMEM) were purchased from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan) and ICN Biomedicals (Aurora, OH, USA), respectively, and fetal bovine serum (FBS) was from Nichirei Bioscience Inc. (Tokyo, Japan). Angiotensin II was from Peptide Institute Inc. (Osaka, Japan). Losartan potassium salt and PD123319 were purchased from Fujifilm Wako Pure Chemical (Osaka, Japan) and Abcam (Cambridge, UK), respectively. MIA was obtained from Sigma-Aldrich (St. Louis, MO, USA).