Cisplatin

Male C57BL/6 mice aged 8 to 10 weeks were purchased from Orient Bio (Seongnam, Korea). All mouse experiments were performed in accordance with the animal protocols approved by the Institutional Animal Care and Use Committee of Jeju National University. Mice were intraperitoneally injected with cisplatin (a single dose of 30 mg/kg body weight, R&D Systems, Minneapolis, MN, USA) to induce nephrotoxicity or with 0.9% saline (control). Spermidine (1 to 10 mg/kg body weight; Sigma, St. Louis, MO, USA) was dissolved in distilled water (vehicle) and was administered orally at 24 and 1 hour before cisplatin injection. To use a small interfering RNA (siRNA) targeting ODC in vivo, ODC siSTABLE siRNA (siODC, 50 µg in 1 ml phosphate buffered saline [PBS]) modified from siGENOME (Dharmacon, Lafayette, CA, USA) or siSTABLE non-targeting siRNA (siControl, 50 µg in 1 ml PBS) was injected within 10 seconds into tail veins at 48 and 24 hours before cisplatin injection, as previously described [19 (link)20 (link)]. When the mice were anesthetized, the kidneys were either fixed in 4% paraformaldehyde for histological studies or snap-frozen in liquid nitrogen for biochemical studies.

Human kidney 2 (HK2), a proximal tubular cell line derived from normal kidneys, was obtained from the American Type Culture Collection (Rockville, MD, USA) and maintained in keratinocyte-serum-free medium containing 5 ng/ml human recombinant epidermal growth factor and 40 µg/ml bovine pituitary extract (Life Technologies, Grand Island, NY, USA) at 37℃ in an atmosphere of 5% CO₂. Cells were grown until 90% confluence on a 6-well tissue culture plate and then starved for 18 hours. After that, the cells were treated with 400 µM cisplatin (Sigma, St. Louis, MO, USA) in phosphate buffered saline (control) for 8 hours. The cells were also treated with 1 µM PJ34 hydrochloride (a potent PARP1 inhibitor, R&D Systems, Minneapolis, MN, USA) in phosphate buffered saline (vehicle) at 2 hours before treatment with cisplatin.