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\begin{document}

\title{Flucloxacillin worsens while imipenem-cilastatin protects against
vancomycin induced kidney injury in a translational rat model}



\author[1]{Gwendolyn Pais}%
\author[1]{Sylwia Marianski}%
\author[1]{Kimberly Valdez}%
\author[1]{Renz Paulo Melicor}%
\author[2]{Jiajun Liu}%
\author[1]{Roxane Rohani}%
\author[1]{Jack Chang}%
\author[3]{Steven Tong}%
\author[4]{Joshua Davis}%
\author[1]{Marc Scheetz}%
\affil[1]{Midwestern University - Downers Grove Campus}%
\affil[2]{FDA}%
\affil[3]{The Royal Melbourne Hospital, at the Peter Doherty Institute for Infection and Immunity}%
\affil[4]{The University of Newcastle Hunter Medical Research Institute}%


\vspace{-1em}



  
  \date{May 20, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background and Purpose Vancomycin is one of the most common antibiotics
administered in the hospital setting, yet acute kidney injury is a major
limiting factor. Common combinations of antibiotics with vancomycin have
been reported to worsen and improve vancomycin-induced kidney injury. We
aimed to study the impact of flucloxacillin and imipenem-cilastatin on
kidney injury when combined with vancomycin in our translational rat
model. Experimental Approach Male Sprague-Dawley rats received
allometrically scaled (1) vancomycin (2) flucloxacillin, (3)
vancomycin+flucloxacillin, (4) vancomycin+imipenem-cilastatin, or (5)
saline for 4 days. Vancomycin was administered intravenously and
flucloxacillin or imipenem-cilastatin were administered
intraperitoneally. Kidney injury was evaluated via drug accumulation and
urinary biomarkers including urinary output, kidney injury molecule-1
(KIM-1), clusterin, and osteopontin. Relationships between vancomycin
accumulation in the kidney and urinary kidney injury biomarkers were
explored. Key Results Urinary output increased every study day for
vancomycin+flucloxacillin; whereas in the vancomycin group it was
elevated after the first dose only. In the vancomycin+flucloxacillin
group, urinary KIM-1/24h increased on all days compared to vancomycin.
In the vancomycin+imipenem-cilastatin group, urinary KIM-1/24h was
decreased on days 1 and 2 compared to vancomycin. Similar trends were
observed for clusterin. More vancomycin accumulated in the kidney with
vancomycin+flucloxacillin compared to vancomycin and
vancomycin+imipenem-cilastatin. The accumulation of vancomycin in the
kidney tissue correlated with increasing urinary KIM-1 (4-parameter Hill
Slope, R2=0.7985). Conclusion and Implications Vancomycin+flucloxacillin
caused more kidney injury compared to vancomycin alone and
vancomycin+imipenem-cilastatin in a translational rat model as
determined by multiple kidney injury biomarkers. The combination of
vancomycin+imipenem-cilastatin was nephroprotective.%
\end{abstract}%



\sloppy


\textbf{Title:} Flucloxacillin worsens while imipenem-cilastatin
protects against vancomycin induced kidney injury in a translational rat
model

\textbf{Running Title:} Flucloxacillin worsens while cilastatin protects
against vancomycin AKI

\textbf{Authors:} Gwendolyn M. Pais\textsuperscript{1-2}, Sylwia
Marianski\textsuperscript{1}, Kimberly Valdez\textsuperscript{1}, Renz
Paulo Melicor\textsuperscript{1}, Jiajun Liu\textsuperscript{1-2,8},
Roxane Rohani\textsuperscript{1-2,9}, Jack Chang\textsuperscript{1-3},
Steven Y. C. Tong\textsuperscript{4,5}, Joshua S
Davis\textsuperscript{6} and Marc H. Scheetz\textsuperscript{1-3,7}*

\textbf{Affiliations:}

\textsuperscript{1} Midwestern University- Downers Grove Campus,
Department of Pharmacy Practice, Downers Grove, IL, USA;

\textsuperscript{2} Midwestern University- Downers Grove Campus,
Pharmacometrics Center of Excellence, Downers Grove, IL, USA;

\textsuperscript{3} Northwestern Memorial Hospital, Department of
Pharmacy, Chicago, IL, USA;

\textsuperscript{4}Victorian Infectious Diseases Service, The Royal
Melbourne Hospital, at the Peter Doherty Institute for Infection and
Immunity, Melbourne, Australia

\textsuperscript{5}Department of Infectious Diseases, The University of
Melbourne at the Peter Doherty Institute for Infection and Immunity,
Melbourne, Australia

\textsuperscript{6}Hunter Medical Research Institute, University of
Newcastle, Newcastle, NSW, Australia

\textsuperscript{7} Midwestern University- Downers Grove Campus,
Department of Pharmacology, Downers Grove, IL, USA;

\textsuperscript{8} Present affiliation: Division of Pharmacometrics,
Office of Clinical Pharmacology, Office of Translational Sciences,
United States Food and Drug Administration, Silver Spring, MD, USA

\textsuperscript{9} Present affiliation: Cellular and Molecular
Pharmacology, Foundational Sciences and Humanities, Chicago Medical
School, Rosalind Franklin University of Medicine and Science, North
Chicago, IL, USA.

\textbf{* Corresponding author and reprint requests:} Marc H. Scheetz,
PharmD, MSc; Professor of Pharmacy Practice and Pharmacology; Midwestern
University; 555 31\textsuperscript{st} St., Downers Grove, IL 60515,
Phone: 630-515-6116; Fax: 630-515-6958; Email: mschee@midwestern.edu

\textbf{Word count:} 1880 \textbf{Abstract word count (250 max):} 251

ACKNOWLEDGEMENTS

The present study was funded in part by a MWU Intramural Grant (MS).
Control and vancomycin alone animals were carried forward (as described
in methods) from studies supported in part by the National Institute of
Allergy and Infectious Diseases under award number R21-AI149026 (authors
MS, GP). The content is solely the responsibility of the authors and
does not necessarily represent the official views of the National
Institutes of Health. Disclaimer from author J. Liu: The opinions
expressed in this article are those of the author and should not be
interpreted as the position of the United States Food and Drug
Administration.

The authors thank Kristi Kozierowski for providing the flucloxacillin .
The authors thank the staff of the MWU Vivarium and the Core Facility.
While this study followed our previously published approaches, the
research and analysis plan for this manuscript was not preregistered.

CONFLICT OF INTEREST STATEMENT

MS reports ongoing research contracts with Nevakar and SuperTrans
Medical as well as having filed patent US10688195B2. All other authors
have no other related conflicts of interest to declare.

\textbf{Data availability statement:} Data available on request from the
authors: The data that support the findings of this study are available
from the corresponding author upon reasonable request. Data sharing will
be subject to standard Data Use Agreements from Midwestern University.



\textbf{Abstract}

\textbf{Background and Purpose}

Vancomycin is one of the most common antibiotics administered in the
hospital setting, yet acute kidney injury is a major limiting factor.
Common combinations of antibiotics with vancomycin have been reported to
worsen and improve vancomycin-induced kidney injury. We aimed to study
the impact of flucloxacillin and imipenem-cilastatin on kidney injury
when combined with vancomycin in our translational rat model.

\textbf{Experimental Approach}

Male Sprague-Dawley rats received allometrically scaled (1) vancomycin
(2) flucloxacillin, (3) vancomycin+flucloxacillin, (4)
vancomycin+imipenem-cilastatin, or (5) saline for 4 days. Vancomycin was
administered intravenously and flucloxacillin or imipenem-cilastatin
were administered intraperitoneally. Kidney injury was evaluated via
drug accumulation and urinary biomarkers including urinary output,
kidney injury molecule-1 (KIM-1), clusterin, and osteopontin.
Relationships between vancomycin accumulation in the kidney and urinary
kidney injury biomarkers were explored.

\textbf{Key Results}

Urinary output increased every study day for vancomycin+flucloxacillin;
whereas in the vancomycin group it was elevated after the first dose
only. In the vancomycin+flucloxacillin group, urinary KIM-1/24h
increased on all days compared to vancomycin. In the
vancomycin+imipenem-cilastatin group, urinary KIM-1/24h was decreased on
days 1 and 2 compared to vancomycin. Similar trends were observed for
clusterin. More vancomycin accumulated in the kidney with
vancomycin+flucloxacillin compared to vancomycin and
vancomycin+imipenem-cilastatin. The accumulation of vancomycin in the
kidney tissue correlated with increasing urinary KIM-1 (4-parameter Hill
Slope, R\textsuperscript{2}=0.7985).

\textbf{Conclusion and Implications}

Vancomycin+flucloxacillin~caused more kidney injury compared to
vancomycin~alone and vancomycin+imipenem-cilastatin in a translational
rat model as determined by multiple kidney injury biomarkers. The
combination of vancomycin+imipenem-cilastatin was nephroprotective.

\textbf{Keywords:}

Vancomycin, flucloxacillin, imipenem-cilastatin, Acute Kidney Injury,
drug induced kidney injury, pre-clinical, rodent, nephrotoxicity,
biomarkers

\textbf{What is already known:}

In a prospective clinical study, combination of flucloxacillin and
vancomycin increased acute kidney injury as demonstrated by elevated
serum creatinine levels.

Imipenem-cilastatin has decreased vancomycin-associated nephrotoxicity
in pre-clinical studies.

\textbf{What this study adds:}

\begin{itemize}
\tightlist
\item
  Increased kidney injury was confirmed when flucloxacillin was added to
  vancomycin as determined by multiple kidney injury biomarkers in a
  translational rat model.
\item
  There is increased accumulation of vancomycin in kidney tissue that
  could account for the synergistic nephrotoxicity of the
  vancomycin+flucloxacillin combination compared to vancomycin alone.
\item
  Imipenem-cilastatin provided some protection against vancomycin
  induced kidney injury.
\end{itemize}

Clinical significance

Antibiotic combinations can worsen or protect against vancomycin induced
kidney injury. Efforts should be made to advance kidney protecting
agents to clinical practice and to better understand the compounded
toxicity of co-nephrotoxins.



\textbf{1 INTRODUCTION}

Vancomycin is one of the most widely prescribed antibiotics in the
hospital setting (Baggs, Fridkin, Pollack, Srinivasan, \& Jernigan,
2016). It remains a first line therapeutic option for infections caused
by methicillin-resistant \emph{Staphylococcus aureus} (C. Liu et al.,
2011; Rybak et al., 2020), enterococci and other resistant Gram positive
bacteria. Kidney injury is a common adverse effect (Im et al., 2017)
with rates varying from 5\% to 43\% depending on the population treated
and concomitant medications (van Hal, Paterson, \& Lodise, 2013). Less
is known about how other medications increase and decrease kidney
injury. Recently, a clinical trial (Combination Antibiotics for
Methicillin Resistant Staphylococcus aureus, CAMERA2), aimed to evaluate
the combination therapy of a beta-lactam (primarily flucloxacillin) with
vancomycin for efficacy (Tong et al., 2020). However, the trial was
stopped early as more patients in the combination therapy group
developed kidney injury as defined by increased serum creatinine when
compared to standard therapy (Tong et al., 2020). Further, CAMERA2
failed to identify an efficacy benefit with combination therapy, only
that toxicity was increased among the those that received vancomycin and
flucloxacillin in combination. Even less is known about agents that can
prevent kidney injury due to vancomycin. Imipenem-cilastatin/relebactam
has been shown to decrease serum creatinine of mice that received
vancomycin (He, Souza, Matvekas, Crass, \& Pai, 2021) with similar
findings reported in retrospective clinical studies (Hakeam, AlAnazi,
Mansour, AlFudail, \& AlMarzouq, 2019).

Clinical studies for evaluating acute kidney injury (AKI) with
vancomycin have primarily focused on assessment of serum creatinine and
various clinical classification schemes based on serum creatinine and
urinary output (He et al., 2021; Nakamura, Kokuryo, Hashimoto, \& Inui,
1999). While serum creatinine is a commonly used surrogate of both
kidney injury and function, it is slow to change after an injury event
and is neither highly sensitive nor specific for meaningful injury and
functional changes. Animal studies offer the ability to study newer
biomarkers that are more sensitive and specific for detecting injury and
to assess drug accumulation in the kidney. For vancomycin induced kidney
injury, kidney injury molecule-1 (KIM-1) has been demonstrated as the
most sensitive and specific urinary biomarker for prediction of
histopathologic damage (Pais et al., 2019). KIM-1 has also been shown as
highly correlated with functional changes (i.e. glomerular filtration
changes) due to vancomycin (Chang et al., 2022; Pais, Chang, Liu, \&
Scheetz, 2022).

Since the findings of nephrotoxicity as classified by serum creatinine
in CAMERA2 were unexpected, the purpose of this study was to assess the
biologic plausibility of increased kidney injury caused by the addition
of flucloxacillin to vancomycin by employing sensitive urinary
biomarkers such as KIM-1, urinary output, and vancomycin accumulation in
the kidney in our translational rat model (Avedissian, Pais, O'Donnell,
et al., 2019; Pais, Liu, Avedissian, et al., 2020). Simultaneously, we
sought to determine if kidney injury was lessened with the addition of
imipenem/cilastatin in the same rat model. To address these questions,
we studied kidney injury in Sprague Dawley rats : administered saline,
vancomycin alone, vancomycin plus flucloxacillin, or vancomycin plus
imipenem-cilastatin.

\textbf{2 METHODS}

\textbf{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:\emph{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 (\selectlanguage{ngerman}± 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.

\textbf{2.2 Characteristics of animal cohorts}

In keeping with the 3Rs principle to reduce the number of animals used
in research, this study carried forward animal data from saline control
and vancomycin only treated rats treated in the exact manner as
described herein. Specifically, the saline group comprised rats with
identical experimental protocols, i.e. n=8 rats from a study published
in abstract form (''ACCP Abstract Booklet,'' 2021) and n=3 new animals,
n=11 total. Similarly, the vancomycin group included n=6 rats from the
same study published in abstract form (''ACCP Abstract Booklet,'' 2021)
and n=6 new animals (total n=12). The vancomycin+flucloxacillin group
started out with the minimum group size n=6. Due to the severe
nephrotoxicity, two rats had to be euthanized on day 3 prior to dosing
(i.e, they received two doses of the intervention). Hence, an additional
n=4 rats were included to account for early termination due to
nephrotoxicity. All animals provided data and were included in the study
(total n=10). Flucloxacillin and imipenem-cilastatin groups were new and
have the minimum sample size of n=6 per group.

\textbf{2.3 Sample collection}

Blood samples (0.125 mL, maximum 16 samples/animal) were drawn from a
jugular vein catheter into EDTA-containing tubes, centrifuged at 3000
rpm for 10 min, (O'Donnell et al., 2017; Rhodes et al., 2016) and the
resultant plasma was stored at -80\textsuperscript{o}C for batch
analysis. Blood was sampled daily under the \#3151 protocol and each day
except Day 3 under the \#3080 protocol. Blood sampling was carried out
for a pharmacokinetic study that will ultimately be detailed elsewhere.
Urine was collected continuously, aliquoted every 24 h, centrifuged
(500xg, 10 minutes at 4°C), and supernatant was stored at
-80\textsuperscript{o}C until batch analysis. At the end of the
protocol, animals were anesthetized with ketamine/xylazine (100/10 mg
kg-1, by intraperitoneal injection) and euthanized by exsanguination
through the right atrium while under anesthesia. Kidneys were obtained
at euthanasia, rinsed in cold saline, flash frozen and stored at -80\selectlanguage{english}0C
for vancomycin assay.

\textbf{2.4 Urinary biomarkers}

Urine samples were analyzed using MILLIPLEX\selectlanguage{ngerman}® MAP Rat Kidney Toxicity
Magnetic Bead Panel 1 (EMD Millipore Corporation, Charles, MO) according
to the manufacturer's protocol (Pais, Liu, Avedissian, et al., 2020).
MILLIPLEX® Analyst v5.1 Flex software was used for calibration curve and
analyte concentration determination.

\textbf{2.5 Estimation of vancomycin in the kidney}

Vancomycin concentrations in the kidney were analyzed by LC-MS/MS
similar to our previous report (Avedissian et al., 2019) with minor
noted exceptions. Samples were diluted 20x and atenolol was used as the
internal standard with transitions (m/z) of 267.2 \selectlanguage{english}-145.0 (quantifier)
and 267.2 \selectlanguage{english}-190.0 (qualifier). Briefly, whole kidneys were thawed on ice
and homogenized using a Polytron System PT 10-35 GT (Fisher Scientific,
Hampton, NH) in a 1:3 (w:v) ratio of kidney : water. The homogenate was
centrifuged, and the resultant supernatant was used in the analyses.
Calibration curves were prepared using control rat kidney tissue
homogenates from the saline-treated group of rats. Vancomycin-
containing kidney homogenates were diluted 20 x with control kidney
tissue homogenate. Kidney concentrations are expressed as vancomycin
(\selectlanguage{greek}µ\selectlanguage{english}g)/ kidney (g) of wet kidney tissue. Assays were linear between 1.0
and 60 mg/L. Precision was 3-6\% for all measurements, including intra-
and inter assay measurements. Greater than 80.27\% accuracy was observed
in all standards tested, with an overall mean assay accuracy of 102\%
and all concentrations above the lower limit of quantification were
between 85 and 115\%.

\textbf{2.6 Materials}

Treatments were clinical grade vancomycin (Fresenius Kabi, Lake Zurich,
IL), flucloxacillin (Juno Pharmaceuticals Pty Ltd, South Yarra, VIC
Australia), imipenem-cilastatin (Novation LLC, Italy Novaplus/Novation
Mfd. Fresenius Kabi) and normal saline (Veterinary 0.9\% Sodium Chloride
Injection USP, Abbott Laboratories, North Chicago, IL).

For LC-MS/MS, analytical grade vancomycin hydrochloride (Lot\#LRAC0718),
and atenolol (Lot\#BCBT5643) were obtained from Sigma Aldrich (St.
Louis, MO), LC-MS/MS grade acetonitrile and methanol were obtained from
VWR International, Radnor, PA, formic acid was obtained from Fisher
Scientific, Waltham, MA and frozen, non-medicated, non-immunized, pooled
Sprague--Dawley rat EDTA plasma was obtained from BioreclamationIVT,
Westbury, NY, USA. Control rat kidney homogenate was prepared from
saline-treated rat kidneys.

\textbf{2.7 Data and Statistical analysis}

The data and statistical analysis comply with the recommendations on
experimental design and analysis in pharmacology (Curtis et al., 2018).
Urinary output is expressed as mean \selectlanguage{ngerman}± SD. All available urine samples
were analyzed. Urinary biomarkers were standardized for urinary volume
by calculating the total amount excreted in each 24-h period.
Differences between groups were assessed using mixed-effects, restricted
maximum likelihood estimation regression, with repeated measures
occurring over days; measures were repeated at the level of the
individual rat (STATA 17.0 BE, StataCorp LLC, College Station, Texas).
The primary analysis reports the simple effects from joint tests of drug
treatment group within each level of treatment day. Margins were
calculated for a full factorial of the variables, i.e. main effects for
each variable and interactions. Referent groups were pre-treatment
baseline values and saline as a treatment, except where noted.
Secondarily, to remain agnostic to outcome variable relationship over
time (and assess treatment groups over time), locally weighted
scatterplot smoothing (LOWESS) trendlines with 95\% confidence intervals
were generated. LOWESS graphs were produced in R version 4.2.2 using
ggplot2 (''H. Wickham. ggplot2: Elegant Graphics for Data Analysis.
Springer-Verlag New York, 2016.,''). No data were excluded as outliers.
Vancomycin kidney concentrations were expressed as median (interquartile
range) and compared by the non-parametric Kruskal Wallis test. Multiple
Hill-type models were explored to describe the relationship between
vancomycin kidney concentrations and urinary KIM-1. All tests were
two-tailed, a p\textless{}0.05 was required for statistical
significance. Graphs were generated using GraphPad version 9.3.1 unless
specified otherwise.



\textbf{3 RESULTS}

\textbf{3.1 Weight loss}

On average (SD), rats weighed 289 g (6.3 g). Mean weight loss post
catheterization between day -3 and day 1 was similar between control,
flucloxacillin, vancomycin, vancomycin+flucloxacillin, vancomycin+
imipenem-cilastatin (10.2 ± 6.1 g, 12.4 ± 3.7 g, 6.7 ± 7.0 g, 11.0 ± 5.1
g, 7.6. ± 4.8 g) animals.

\textbf{3.2 Urinary output and urinary biomarkers}

Mean total urine output increased on Day 1 following the first dose in
the vancomycin-treated groups. Vancomycin + flucloxacillin resulted in
higher urinary volume when compared with baseline and saline-treated
animals on days 3 and 4 days. Urinary output increased on every study
day for vancomycin+flucloxacillin, whereas in the vancomycin group it
was elevated after the first dose only. Mean urinary output is
significantly increased with vancomycin+flucloxacillin compared to
vancomycin and vancomycin+ imipenem-cilastatin following the 3rd dose
(FIG 1a). There was a gradual increase in the mean total urine output in
the flucloxacillin-treated animals compared with baseline and
saline-treated animals on Days 2 and 3.

In the vancomycin group, absolute amount of urinary KIM-1 over 24 h was
significantly increased from Day 1 compared to the saline and
flucloxacillin control groups (Table 1, FIG 1b ). While in the
vancomycin+flucloxacillin group, KIM-1 was significantly increased
following the 3\textsuperscript{rd} day compared to vancomycin. In the
vancomycin+ imipenem-cilastatin group, KIM-1 was significantly decreased
on Days 1 and 2 compared to vancomycin. Similar trends were observed in
the vancomycin+flucloxacillin group for clusterin and osteopontin (FIG
1c,d). Locally weighted scatterplot smoothing (LOWESS) trendlines with
95\% confidence intervals visually confirmed similar findings for all
comparisons (FIG1 supplemental).

\textbf{3.3 Vancomycin concentration in the kidney}

Vancomycin concentration in rat kidney was significantly elevated after
treatment with vancomycin alone or in combination (FIG 2). A
relationship was identified between vancomycin concentration in the
kidney tissue and KIM-1 level in the urine (FIG 3).

\textbf{4 DISCUSSION}

In this study, the combination of vancomycin+flucloxacillin~caused more
kidney damage compared to vancomycin~alone and to vancomycin+
imipenem-cilastatin as determined by multiple kidney injury biomarkers
and accumulation of vancomycin in the kidney.~Imipenem-cilastatin was
protective for vancomycin induced kidney injury as shown by lower KIM-1.
Thus, this translational rat study reproduces the increased clinical
toxicity seen in the CAMERA 2 trial (Legg et al., 2022) with combination
therapy (vancomycin+flucloxacillin) which resulted in early termination
of the trial. Secondly, these data accord with in pre-clinical models
(Becerir, Tokgün, \& Yuksel, 2021; He et al., 2021; Hori et al., 2017;
Humanes et al., 2015; Im et al., 2017; Kusama et al., 1998; Nakamura et
al., 1999; Toyoguchi, Takahashi, Hosoya, Nakagawa, \& Watanabe, 1997)
that demonstrate protection against kidney injury by cilastatin. It is
also notable that previous work in our translational rat models have
consistently demonstrated that piperacillin-tazobactam does not worsen
vancomycin kidney injury and may in fact be protective (Avedissian,
Pais, Liu, Rhodes, \& Scheetz, 2019; Chang et al., 2022; Pais, Liu,
Avedissian, et al., 2020; Pais, Liu, Zepcan, et al., 2020). Thus, while
the exact mechanism is not yet clear, differences in toxicity exist
based on the specific type of penicillin used.

The protective findings with cilastatin are notable. Cilastatin is a
dehydropeptidase-1 inhibitor (DHP-1), an enzyme produced by the proximal
tubules (Kahan, Kropp, Sundelof, \& Birnbaum, 1983). It is commercially
available in combination with imipenem, a broad spectrum carbapenem
beta-lactam antibiotic, with or without relebactam, a beta-lactamase
inhibitor, to prevent imipenem-induced nephrotoxicity.

In the present study, cilastatin was purchased in combination with
imipenem, to translationally represent the combination that is already
FDA approved and available in the hospital settings for treatment of
variety of infections. Additionally, it is worthwhile to note that we
specifically tested a low allometrically scaled dose of
imipenem-cilastatin. A dose of 90 mg kg-1/day in the rat equates to a
human dose of 14.5 mg kg-1/day of imipenem-cilastatin or 1 gram daily
for a 70 kg patient (''Guidance for Industry. Estimating the Maximum
Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult
Healthy Volunteers. U.S. Department of Health and Human Services. Food
and Drug Administration. Center for Drug Evaluation and Research (CDER).
,'' 2005), a dose that is 25\% of the maximum FDA approved dose
(''Primaxin Package insert.
https://www.accessdata.fda.gov/drugsatfda\_docs/label/2016/050587s074lbl.pdf.
Merck 12/2016.,''). Full dose ranging experiments with urinary
biomarkers are needed to provide fully quantitative results for
percentage of protection available. The results here build upon those in
the literature. He et al. (He et al., 2021) found lower SCr and BUN
values (as AKI biomarkers) in vancomycin+imipenem-cilastatin/relebactam
treated male C57BL/6J mice versus vancomycin treated animals and
reported that imipenem-cilastatin/relebactam maintained a
nephroprotective effect. This is consistent with absolute amount of
urinary biomarker KIM-1 in our studies with imipenem-cilastatin versus
vancomycin group. In their study, the investigators used 320 mg kg-1
imipenem-cilastatin/relebactam in mice (which is equivalent to 160 mg
kg-1 in the rat), at approximately half the cilastatin dose (45 mg kg-1
of cilastatin component) we were able to reproduce the nephroprotective
effect. Humanes et al. (Humanes et al., 2015) assessed effect of
cilastatin on vancomycin nephrotoxicity in cultured renal proximal
tubular epithelial cells. They reported that cilastatin protects against
proximal tubule apoptosis caused by vancomycin. Postulated mechanism of
nephroprotection by cilastatin involves megalin and/or p-glycoprotein.
Megalin is an endocytic receptor expressed on the apical surface of the
proximal tubule cells (Christensen et al., 1995) and mediates the renal
uptake of some antibiotics with consequent nephrotoxicity (Kuwahara et
al., 2016; Moestrup et al., 1995; Suzuki et al., 2013). Cilastatin
competitively binds to megalin and blocks uptake of vancomycin (Hori et
al., 2017). A number of studies in mice, rats and in primary porcine
proximal tubule cells have demonstrated that cilastatin can decrease
cellular uptake of vancomycin and vancomycin concentrations in the
kidney (Hori et al., 2017; Humanes et al., 2015; Kusama et al., 1998;
Toyoguchi et al., 1997). Hori et al (Hori et al., 2017), assessed
nephrotoxicity of vancomycin and other nephrotoxins in proximal tubule
epithelial cells and in megalin knock-out mice. Both studies
demonstrated that vancomycin binds to megalin and results in kidney
damage; while cilastatin binds megalin and reduces damage caused by
vancomycin. Others (Im et al., 2017) have suggested that the protection
mediated by cilastatin is a function of upregulating p-glycoprotein.
Thus, cilastatin may act both through megalin blockade as well as
p-glycoprotein upregulation. A study of vancomycin and cilastatin in
rabbits demonstrated that vancomycin clearance is higher in rats that
received cilastatin than those that received vancomycin alone (Toyoguchi
et al., 1997). Both p-glycoprotein upregulation and megalin inhibition
both theoretically will increase vancomycin clearance in the urine.
Finally, a Wistar albino rat study that used supratherapeutic vancomycin
doses between 500-1000 mg kg-1 (which allometrically translate to 80-160
mg kg-1/day in humans) demonstrated that cilastatin protects against
decreasing glomerular function (measured by inulin clearance) from
vancomycin (Nakamura et al., 1999). Kusama et al. demonstrated that
vancomycin accumulates less in kidneys of rats treated with cilastatin
(Kusama et al., 1998). Our study is the first to our knowledge to assess
the nephroprotective effect of imipenem-cilastatin in a translational
rat model using urinary biomarker KIM-1 that has been linked to human
outcomes (Scheetz et al., 2021). These results show consistent findings
across ex-vivo and in-vivo analyses of multiple species. A small
retrospective study in humans (total of 227 patients) found lower rates
of nephrotoxicity in patients that received imipenem-cilastatin in
combination with vancomycin as opposed to patients in the meropenem
(which contains no cilastatin) + vancomycin group (6.2\% vs 17.1\%
respectively) (Hakeam et al., 2019).

Incidence of attributable acute kidney injury (AKI) caused by vancomycin
exceeds 10\% (Wunderink et al., 2012). Thus, it is essential for
clinicians to be aware of drugs (nephroprotectants and nephrotoxins)
that can potentiate or alleviate risk of vancomycin AKI. Our rat model
confirms that synergistic nephrotoxicity exists, such as observed in
CAMERA 2 trial (Legg et al., 2022; J. Liu et al., 2020) due to addition
of flucloxacillin to vancomycin; thus we recommend against use of such
combination in the hospital settings. Our animal model confirms that
imipenem-cilastatin should be explored as a nephroprotectant. However,
while cilastatin is available in a clinically approved preparation as
imipenem-cilastatin, promotion of antimicrobial resistance will preclude
regular use. Thus, the development of a -vancomycin-cilastatin
preparation would be desirable, and more information will be needed on
the benefits and risks of that combination.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles
for transparent reporting and scientific rigour of preclinical research
as stated in the BJP guidelines for Design \& Analysis and Animal
Experimentation, and as recommended by funding agencies, publishers and
other organizations engaged with supporting research.

AUTHOR CONTRIBUTIONS

MS and GP conceived and designed the study. GP, JL, RM, SM, KV were
involved in the acquisition of data. GP, MS analyzed the data. ST, JD,
JL, JC, MS interpreted the data;

GP, RR, MS drafted the manuscript. ST, JD, MS revised it critically for
important

intellectual content.

All authors have given final approval of the version to be published.
All authors agree to be accountable for all aspects of the work in
ensuring that questions related to the accuracy or integrity of any part
of the work are appropriately investigated and resolved.



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\textbf{FIGURE LEGENDS}

\textbf{FIG 1} Comparison of daily urinary biomarker measurements
between treatment groups and across dosing days. (a) Urinary output.
Values expressed as mean \selectlanguage{ngerman}± SD; * vancomycin vs saline, \#
vancomycin+flucloxacillin vs saline, @ vancomycin+flucloxacillin vs
vancomycin. Total amounts of urinary KIM-1 (b), clusterin (c), and OPN
(d) in saline (n=11), flucloxacillin (n=6), vancomycin (n=12),
vancomycin+flucloxacillin (n=10) and vancomycin+imipenem-cilastatin
(n=6)-treated rats. Values expressed as median (min-max); * vs
vancomycin. KIM-1, kidney injury molecule-1; OPN, osteopontin.

\textbf{FIG 2} Vancomycin concentration in the kidney tissue. Values
expressed as median and interquartile range.

\textbf{FIG 3} Correlation between urinary KIM-1 (ng/24 h) and
vancomycin in the kidney (\selectlanguage{greek}µ\selectlanguage{english}g/g).

\textbf{FIG 1} Comparison of daily urinary biomarker measurements
between treatment groups and across dosing days. (a) Urinary output.
Values expressed as mean \selectlanguage{ngerman}± SD; * vancomycin vs saline, \#
vancomycin+flucloxacillin vs saline, @ vancomycin+flucloxacillin vs
vancomycin. Total amounts of urinary KIM-1 (b), clusterin (c), and OPN
(d) in saline (n=11), flucloxacillin (n=6), vancomycin (n=12),
vancomycin+flucloxacillin (n=10) and vancomycin+imipenem-cilastatin
(n=6)-treated rats. Values expressed as median (min-max); * vs
vancomycin. KIM-1, kidney injury molecule-1; OPN, osteopontin.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{FIG 2} Vancomycin concentration in the kidney tissue. Values
expressed as median and interquartile range.

\textbf{FIG 3} Correlation between urinary KIM-1 (ng/24 h) and
vancomycin in the kidney (\selectlanguage{greek}µ\selectlanguage{english}g/g).

\textbf{Table 1:} \textbf{Contrast means versus vancomycin as referent
group}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\textbf{Dosing day} & \textbf{Treatment groups} & \textbf{Treatment
groups} & \textbf{Treatment groups} & \textbf{Treatment
groups}\tabularnewline
\midrule
\endhead
& \textbf{Saline} & \textbf{FLUCLOX} & \textbf{VAN+FLUCLOX} &
\textbf{VAN+CILASTATIN}\tabularnewline
Urinary KIM-1 (ng/24h), mean (95\% CI) & Urinary KIM-1 (ng/24h), mean
(95\% CI) & Urinary KIM-1 (ng/24h), mean (95\% CI) & Urinary KIM-1
(ng/24h), mean (95\% CI) & Urinary KIM-1 (ng/24h), mean (95\%
CI)\tabularnewline
Day 0 & -3.4 (-53.9 to 47.1) & -2.3 (-62.8 to 58.1) & -2.7 (-54.5 to
49.1) & -2.7 (-63.2 to 57.8)\tabularnewline
Day 1 & -115.9 (-166.4 to -65.4)\textbf{*} & -117.5 (-178.0 to
-57.0)\textbf{*} & 66.4 (14.6 to 118.2)\textbf{*} & -61.0 (-121.5 to
-0.5)\textbf{*}\tabularnewline
Day 2 & -146.7 (-197.2 to -96.2)\textbf{*} & -146.6 ( -207.1 to
-86.1)\textbf{*} & 58.1 (6.3 to 110.0)\textbf{*} & -106.5 (-167.0 to
-46.0)\textbf{*}\tabularnewline
Day 3 & -102.8 (-153.3 to -52.3)\textbf{*} & -101.1 (-161.6 to
-40.6)\textbf{*} & 126.2 (74.4 to 178.0)\textbf{*} & -54.5 (-115.0 to
6.0)\tabularnewline
Day 4 & -83.4 (-133.9 to -32.9)\textbf{*} & -82.5 (-143.0 to
-22.0)\textbf{*} & 140.5 (86.0 to 195.0)\textbf{*} & -31.1 (-91.6 to
29.4)\tabularnewline
Urinary clusterin (ng/24h), mean (95\% CI) & Urinary clusterin (ng/24h),
mean (95\% CI) & Urinary clusterin (ng/24h), mean (95\% CI) & Urinary
clusterin (ng/24h), mean (95\% CI) & Urinary clusterin (ng/24h), mean
(95\% CI)\tabularnewline
Day 0 & -1547 (-12812 to 9718) & -825 (-14319 to 12667) & 706 (-10848 to
12262) & -1334 (-14827 to 12159)\tabularnewline
Day 1 & -12517 (-23782 to -1253)\textbf{*} & -12273 (-25767 to 1220) &
13016 (1461 to 24571)\textbf{*} & -5105 (-18598 to 8388)\tabularnewline
Day 2 & -20360 (-31626 to -9096)\textbf{*} & -20062 (-33556 to
-6570)\textbf{*} & 11961 (406 to 23516)\textbf{*} & -10787 (-24280 to
2706)\tabularnewline
Day 3 & -15823 (-27088 to -4558)\textbf{*} & -14261 (-27755 to
-768)\textbf{*} & 19852 (8297 to 31407)\textbf{*} & -2543 (-16036 to
10950)\tabularnewline
Day 4 & -12319 (-23585 to -1055)\textbf{*} & -11041 (-24535 to 2452) &
34581 (22481 to 46682)\textbf{*} & 2177 (-11316 to 15671)\tabularnewline
Urinary OPN (ng/24h), mean (95\% CI) & Urinary OPN (ng/24h), mean (95\%
CI) & Urinary OPN (ng/24h), mean (95\% CI) & Urinary OPN (ng/24h), mean
(95\% CI) & Urinary OPN (ng/24h), mean (95\% CI)\tabularnewline
Day 0 & -0.02 (-0.77 to 0.73) & -0.18 (-1.08 to 0.73) & -0.00 (-0.78 to
0.77) & 0.13 (-0.77 to 1.04)\tabularnewline
Day 1 & -0.64 (-1.39 to 0.11) & -0.75 (-1.65 to 0.15) & 0.67 (-0.10 to
1.45) & 0.58 (-0.32 to 1.48)\tabularnewline
Day 2 & -0.99 (-1.74 to -0.24)\textbf{*} & -1.25 (-2.15 to
-0.35)\textbf{*} & 1.00 (0.23 to 1.77)\textbf{*} & -0.23 (-1.13 to
0.67)\tabularnewline
Day 3 & -0.28 (-1.03 to 0.47) & -0.40 (-1.30 to 0.50) & 2.01 (1.24 to
2.78)\textbf{*} & 0.64 (-0.26 to 1.54)\tabularnewline
Day 4 & -0.24 (-0.99 to 0.51) & -0.21 (-1.11 to 0.69) & 1.97 (1.15 to
2.78)\textbf{*} & 1.45 (0.55 to 2.35)\textbf{*}\tabularnewline
\bottomrule
\end{longtable}

Supplemental FIG 1. Trends in urinary biomarkers visualized using
locally weighted scatterplot smoothing (LOWESS) trendlines with 95\%
confidence intervals.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
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