C4255

Selenium content was measured in serum by atomic absorption spectrophotometer (AA6501, Shimadzu Ltd., Japan). Plasma samples were used for measuring of aspartate amino transferase (AST), alanine glutamyl transferase (ALT), and creatinine calorimetrically by diagnostic kits (MAK055, MAK052, and C4255, respectively) manufactured by Sigma-Aldrich.

Creatinine and albumin levels in baseline urine samples, which were previously frozen and stored at − 80 °C for 2.5 years, were measured using the Jaffe and bromocresol green reactions (Sigma-Aldrich, MAK124), respectively. Samples were read at a specific wavelength using the Biochrom EZ Read 400 Microplate Reader. Urinary neutrophil gelatinase-associated lipocalin (uNGAL) levels were measured in a subsample. Samples from fifty-five randomly selected participants with stable kidney function and samples from all participants in the other two subgroups (rapid decline in kidney function and patients with established renal dysfunction; both of which are defined below) were analysed. uNGAL levels were measured using enzyme-linked immunoassay (ELH-Lipocalin2, RayBiotech), according to the manufacturer’s instructions. Albumin and uNGAL levels are reported as ratios to urinary creatinine levels measured using the Jaffe method (Sigma-Aldrich, C4255) [14 (link)].

Mice (male, 8–10 weeks old) were randomized into 8 groups (n = 3/group): Control group, Creatinine group, APAP group, APAP + Creatinine group, APAP + N-acetyl-L-cysteine (NAC) group, APAP + Creatinine + NAC group, APAP + SP600125 group and APAP + Creatinine + SP600125 group. For the creatinine pre-treatment groups, the mice were injected intraperitoneally with creatinine (C4255, Sigma-Aldrich, United States ) at the dose of 16 mg/g for 1 week before APAP treatment. For the APAP-induced acute liver injury model, mice were fasted overnight and then administered with APAP (A7085, Sigma-Aldrich, United States ) intraperitoneally at the dose of 400 mg/kg. The other groups were intraperitoneally administrated with equal volume of 0.9% saline solution. In some experiments, mice were given 150 mg/kg of NAC (A7250, Sigma-Aldrich, United States ) 1 h before the APAP administration to minimize the effect of ROS. In some cases, the pharmacologic inhibitor SP600125 (S1460, Selleck, United States ) was used at a dose of 15 mg/kg 1 h prior to the APAP injection to inhibit the activation of JNK. Mice were finally intraperitoneal injected with 1% pentobarbital (50 mg/kg) for anaesthetization and sacrificed at 6 h for flow cytometric assay and 24 h for biochemical and histological detection.