Quantichrom urea and creatinine assay kits

Human kallistatin levels in the serum were determined by enzyme-linked immunosorbent assay (ELISA) specific for human kallistatin [22 (link)]. Blood urea nitrogen (BUN) and serum creatinine were measured with QuantiChrom Urea and Creatinine Assay Kits (BioAssay Systems, Hayward, CA, USA), respectively. Serum TNF-α levels were determined by a mouse TNF-α ELISA kit (EMD Millipore, Billerica, MA, USA). IL-6 levels in serum were measured using a mouse IL-6 ELISA kit (eBioscience, San Diego, CA, USA). Serum HMGB1 levels were determined by a mouse HMGB1 ELISA kit (Antibodies-online Inc., Atlanta, GA, USA). The levels of lung malondialdehyde (MDA) were determined as previously described [27 (link)]. Serum alanine transaminase (ALT) levels were measured using an ALT colorimetric assay kit (Cayman Chemical Company, Michigan, MI, USA).

All mouse studies were approved by the Animal Ethics Committee of The University of Western Australia in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (NH&MRC, 8^th Edition, 2013). Studies were performed using adult male C57BL/6J mice weighing approximately 20 g. Mice were treated with 1746c27 (40 mg/kg/day intraperitoneally) for 7 days (day 1–7), followed by no treatment on days 8–15. On day 15, mice were anesthetized with intraperitoneal injection of pentobarbitone sodium (240 mg/kg), and their terminal blood collected. Serum was extracted and used to measure kidney and liver toxicity. Urea and creatinine concentration was assessed using QuantiChrom Urea and Creatinine assay kits, respectively (BioAssay Systems). Aspartate transaminase (AST) and alanine transaminase (ALT) concentration were measured using EnzyChrom Aspartate Transaminase and Alanine Transaminase assay kits, respectively (BioAssay Systems). All assays were performed as per manufacturer’s instructions, using a spectrophotometer (PowerWave XS).

The collected blood samples were allowed to clot for 2 h at room temperature and centrifuged at 2000 × g for 20 min to obtain the serum. The following kits were used to determine the levels of the bone turnover markers: the QuantiChrom Calcium Assay Kit and ALP Assay Kit (BioAssay Systems, Hayward, CA); the TRAP 5b Enzyme Immunoassay (EIA) Kit and RatLaps EIA Kit for CTX (Immunodiagnostic Systems, Fountain Hills, AZ); and the Osteocalcin EIA Kit (Biomedical Technologies, Stoughton, MA). TNF-α and IL-1β levels were quantified using their respective commercially available ELISA kits (R&D Systems). ALT, AST, BUN, and creatinine levels were quantified using EnzyChrom ALT and AST Assay Kits and the QuantiChrom Urea and Creatinine Assay Kits (BioAssay Systems).