Recombinant klotho

Primary pulmonary fibroblasts were isolated from unchallenged C57BL/6 mice, bleomycin-, and saline-treated mice as previously described [49 (link)]. Briefly, fresh mouse lung was cleared of blood by perfusion with ice-cold sterile PBS through the right ventricle and minced with a sterile scalpel blade into 1 mm³ fragments. Minced lung was suspended in 5 ml of digestion solution consisting of 5 mg/ml collagenase I (Sigma Aldrich) and 0.33 U/ml DNase I (Roche, Basel, Switzerland) in PBS and incubated with frequent agitation at 37 °C for 45 min. Cells were then filtered through a 70 μg strainer (Merck and Millipore, Darmstadt, Germany), centrifuged at 540 g for 5 min at 4 °C, and plated in tissue culture flasks in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Burlington, ON, USA) with 15% fetal bovine serum (FBS) (Gibco), 100 μg/ml streptomycin, 100 U/ml penicillin, 0.25 μg/ml amphotericin B, and 10 mmol/l HEPES (Sigma Aldrich) at 37°C in a humidified 5% CO₂ atmosphere. Cells were passaged after being harvested with trypsin-EDTA (Sigma Aldrich). Cells from passage 3 to 6 were used. For TGF-β treatment, 50 ng/ml recombinant klotho (R&D system, Minneapolis, MN, USA) was added to serum-free medium 12 h after serum withdrawal. After 12 h of KL pre-incubation, pulmonary fibroblasts were incubated with or without 5 ng/ml TGF-β (PeproTech, Rocky Hill, NJ, USA) for another 24 h.