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.