2.1 Experimental design and animals
This preclinical model replicates aspects of human vancomycin-induced kidney injury (Scheetz et al., 2021). This study was performed at Midwestern University in Downers Grove, Illinois with the approval of the Institutional Animal Care and Use Committee (IACUC protocol #s 3080 and 3151). All experiments were conducted in compliance with the Guide (”National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals. Guide for the Care and Use of Laboratory Animals. 8th edition. Washington (DC): National Academies Press (US); 2011. Available from:https://www.ncbi.nlm.nih.gov/books/NBK54050/ doi: 10.17226/12910,”)
The animal model employed for this study has been previously described (Pais et al., 2020). Male Sprague-Dawley rats (n = 45; age, approximately 9 to 10 weeks; mean (± SD) weight, 289 (± 6.3) g, Envigo, Indianapolis, IN, USA) were housed in a light- and temperature-controlled room and were allowed free access to food and water for the duration of the study. Animals underwent double jugular catheterization 72 h prior to protocol initiation. Ketamine xylazine 100/10 mg kg-1 was employed as a surgical anesthetic. Animals were monitored 4-6 h post-surgery for signs of pain and rescue analgesia (buprenorphine 0.02 mg kg-1) was administered subcutaneously as needed. A pre-planned protocol required that animals displaying overt signs of discomfort (severe lethargy, lack of response to stimuli, marked weight loss [i.e., greater than 15% of starting weight during the treatment period] or a significant decrease in food and water intake) were euthanized.
Following recovery from surgery, animals were transferred to metabolic cages (Nalgene, Rochester, NY) for 24-h urine collection, 24 h pre-study (day 0) and on days 1-4. Rats were assigned to one of five treatment groups vancomycin 150 mg kg-1/day (n=12), flucloxacillin 90 mg kg-1/day (n=6), vancomycin+flucloxacillin (n=10), vancomycin+ imipenem-cilastatin 90 mg kg-1/day (n=6) or saline controls (n=11). See below for rationale for numbers in each group. FDA approved fixed doses were converted to weight-based doses using a standardized patient weight of 70 kg (https://www.fda.gov/media/72309/download). Vancomycin 150 mg kg-1/day was selected to allometrically scale the human dose (25 mg kg-1/day) for the rat (i.e., 25 mg kg-1 x 6.2 [rat factor] = 155 mg kg-1). Similarly, flucloxacillin 90 mg kg-1/day and imipenem-cilastatin 90 mg kg-1/day were allometrically scaled based on FDA approved human doses. Conduct of the animal protocol was not blinded as this was considered impractical; however, samples were analyzed in a blinded fashion and used objective measurements of biomarkers. Rats received the same treatment daily for 4 days. Vancomycin was administered intravenously, flucloxacillin and imipenem-cilastatin were given intraperitoneally.
A minimum sample size of n=6 per group was used based on our previous experiments where differences in total excreted urinary KIM-1 were discernable and corresponded to the “gold-standard” of histopathological change on day 3 (Pais, Liu, Avedissian, et al., 2020). Two human clinical trials CAMERA 2 and PROVIDE did not generate any signal for sex-based differences (Legg et al., 2022; Lodise et al., 2020); therefore, sex as a biological variable was not incorporated in this particular study.