Jabaudon 2015

AJRCCM Articles in Press. Published on 01-May-2015 as 10.1164/rccm.
201501-0020OC
Page 1 of 59
Soluble RAGE Predicts Impaired Alveolar Fluid Clearance in Acute Respiratory
Distress Syndrome
Matthieu Jabaudon1,2, Raiko Blondonnet1,2, Laurence Roszyk2,3, Damien Bouvier2,3, Jules
Audard1, Gael Clairefond2, Mathilde Fournier4, Geoffroy Marceau2,3, Pierre Déchelotte5,
Bruno Pereira6, Vincent Sapin2,3 and Jean-Michel Constantin1,2
1
CHU Clermont-Ferrand, Intensive Care Unit, Department of Anesthesiology, Critical Care
and Perioperative Medicine, Estaing University Hospital, Clermont-Ferrand, France
2
Clermont Université, Université d'Auvergne, EA 7281, R2D2, Clermont-Ferrand, France
3
CHU Clermont-Ferrand, Department of Medical Biochemistry and Molecular Biology,
Clermont-Ferrand, France
4
CHU Clermont-Ferrand, Department of Microbiology, Clermont-Ferrand, France
5
CHU Clermont-Ferrand, Department of Pathology, Estaing University Hospital, Clermont-
Ferrand, France
6
CHU Clermont-Ferrand, Department of Clinical Research and Innovation (DRCI),
Clermont-Ferrand, France
Corresponding author and address for reprints:
Matthieu Jabaudon, M.D., M.Sc.
Department of Anesthesiology, Critical Care and Perioperative Medicine, Intensive Care
Unit, Estaing University Hospital
Clermont Université, Université d'Auvergne, EA 7281, R2D2, Clermont-Ferrand, France
Copyright © 2015 by the American Thoracic Society

AJRCCM Articles in Press. Published on 01-May-2015 as 10.1164/rccm.201501-0020OC
Page 2 of 59
CHU Clermont-Ferrand, 1 Place Lucie Aubrac, 63003 Clermont-Ferrand Cedex 1, France
(Tel) +33 473 750 501 (Fax) +33 473 750 500
(Mail) mjabaudon@chu-clermontferrand.fr
AUTHORS CONTRIBUTIONS TO THE STUDY
MJ takes responsibility for the content of the manuscript. MJ was involved in the conception,
hypotheses delineation, and design of the study, acquisition and analysis of the data, in writing
the article and in its revision prior to submission.
RB was involved in the design of the study, hypotheses delineation, acquisition and analysis
of the data, in writing the article and in its revision prior to submission.
LR was involved in the design of the study, acquisition and analysis of the data, and in the
revision of this article prior to submission.
DB was involved in the acquisition and analysis of the data, and in the revision of this article
prior to submission.
JA was involved in the design of the study, acquisition and analysis of the data, and in the
GC was involved in the design of the study, acquisition and analysis of the data.
MF was involved in the acquisition and analysis of the data, and in the revision of this article
GM was involved in the acquisition and analysis of the data, and in the revision of this article
PD was involved in the design of the study, acquisition and analysis of the data, and in the

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BP was involved in the hypotheses delineation and design of the study, analysis of the data, in
writing the article and in its revision prior to submission.
VS was involved in the conception, hypotheses delineation, and design of the study, analysis
JMC was involved in the conception, hypotheses delineation, and design of the study, analysis
Funding: This work was supported by grants from the Auvergne Regional Council
(“Programme Nouveau Chercheur de la Région Auvergne” 2013), the french Agence
Nationale de la Recherche and the Direction Générale de l’Offre de Soins (“Programme de
Recherche Translationnelle en Santé” ANR-13-PRTS-0010) and from Clermont-Ferrand
University Hospital (“Appel d’Offre Interne 2010”, CHU Clermont-Ferrand). The funders had
no influence in the study design, conduct, and analysis or in the preparation of this article.
Short Running Head: sRAGE predicts alveolar fluid clearance in ARDS.
Subject Code: 4.2 ALI/ARDS: Diagnosis & Clinical Issues
Body of Manuscript Word Count: 3053
AT A GLANCE COMMENTARY
Scientific Knowledge on the Subject
Soluble RAGE is a marker of alveolar type I cell injury and correlates with severity and
outcome in patients with ARDS. Efficient alveolar fluid clearance is a major determinant of

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lung injury resolution, but reliable biological markers of such a process have been
underinvestigated to date.
What This Study Adds to the Field
Elevated levels of sRAGE in plasma and bronchoalveolar fluid could predict alveolar fluid
clearance and lung injury severity in both a first replication of a translational mouse model of
direct lung epithelial injury and an observational prospective study of patients with ARDS,
thus reinforcing a role for sRAGE as a marker of lung injury and repair.
This article has an online data supplement, which is accessible from this issue's table of
content online at www.atsjournals.org

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ABSTRACT
Rationale: Levels of the soluble form of the receptor for advanced glycation end-products
(sRAGE) are elevated during acute respiratory distress syndrome (ARDS) and correlate with
severity and prognosis. Alveolar fluid clearance (AFC) is necessary for the resolution of lung
edema, but is impaired in the majority of ARDS patients. No reliable marker of this process
has been investigated to date.
Objectives: To verify whether sRAGE could predict AFC during ARDS.
Methods: Anesthetized CD-1 mice underwent orotracheal instillation of hydrochloric acid. At
specified time-points, lung injury was assessed by analysis of blood gases, alveolar
permeability, lung histology, AFC and plasma/bronchoalveolar fluid measurements of pro-
inflammatory cytokines and sRAGE. Plasma sRAGE and AFC rates were also prospectively
assessed in thirty patients with ARDS.
Measurements and Main Results: The rate of AFC was inversely correlated with sRAGE
levels in the plasma and the bronchoalveolar fluid of both acid-injured mice (Spearman’s rho
= -0.73 and -0.69, respectively, P<10-3), and plasma sRAGE correlated with AFC in patients
with ARDS (Spearman’s rho = -0.59, P<10-3). Similarly, sRAGE levels were significantly
associated with lung injury severity, and decreased over time in mice while AFC was restored
and lung injury resolved.
Conclusions: Our results indicate that sRAGE levels could be a reliable predictor of impaired
AFC during ARDS, and should stimulate further studies on the pathophysiologic implications
of RAGE axis in the mechanisms leading to edema resolution.
(Abstract word count: 228)

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Keywords: Acid aspiration, animal model, alveolar epithelium, lung injury resolution,
pulmonary edema.
ClinicalTrials.gov Identifier: NCT00811629.

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INTRODUCTION
Acute respiratory distress syndrome (ARDS) is a major cause of acute respiratory
failure and death in critically ill patients(1, 2). The reabsorption of pulmonary edema fluid
from the alveolar space is necessary for the resolution of ARDS, and the magnitude of
damage to the alveolar type (AT) I cell is a major determinant of the severity of ARDS(3). In
addition, an intact alveolar epithelial barrier with preserved alveolar fluid clearance (AFC) is
associated with better clinical outcomes in patients with ARDS(3). To date, few interventions
have proved beneficial in ARDS(4–6) and pharmacological approaches remain limited, with
deceiving clinical translation(7, 8). Biomarkers reflecting alveolar epithelial functions and
injury may therefore be useful in order to run mechanistic explorations and ultimately
develop innovative diagnostic and therapeutic approaches in ARDS patients(9).
The soluble form of the receptor for advanced glycation end-products (sRAGE) is a
recently described novel marker of AT I epithelial cell injury with both prognostic and
pathogenic value in patients with ARDS(10–12). A recent study suggests that alveolar
sRAGE levels and alveolar fluid clearance (AFC) rates could be reliable markers of lung
injury onset and resolution in a recent translational mouse model of direct epithelial lung
injury(13). In addition, in an ex vivo model of isolated perfused human lungs declined for
transplantation, the rate of AFC was inversely correlated with the level of sRAGE in alveolar
fluid, but not with perfusate sRAGE levels(14). Apart from one recent study in mice, in
which alveolar sRAGE correlated with AFC during the acute phase of acid-induced lung
injury(15), sRAGE has not been comprehensively assessed as a marker of AFC in both
animal and clinical studies on ARDS.
We specifically designed an experimental and a clinical study to prospectively
determine if sRAGE levels are associated with AFC rate in the setting of ARDS.
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Partial results of this study have already been presented as an abstract or oral
communication during the conference “Congrès National de la Société Française
d’Anesthésie et Réanimation” (2014) and during the annual congress of the European Society
of Intensive Care Medicine (2014)(16, 17).
METHODS
Additional details are provided in the online supplement.
Animal Studies
Our animal ethics committee approved protocols. CD-1 mice were anesthetized prior
to orotracheal instillation of hydrochloric acid(13). After a recovery period under humidified
oxygen, mice were transferred to stabulation.
Criteria for experimental ARDS were evaluated at baseline in injured and sham
animals, and at specified time-points (1, 2, 4 days) after acid instillation in injured mice(13,
18). Mice were tracheotomized and ventilated for 30 min (tidal volume=8-9 mL.kg-1, positive
end-expiratory pressure=6 cmH2O, respiratory rate=160 breaths.min-1 and FiO2=1), before
blood gas measurements. Intravenous human serum albumin (HSA) was administered 1 h
before sacrifice. The permeability index was calculated as the BAL fluid-to-plasma HSA
ratio(19).
BAL was performed(13) and systemic blood was drawn from cardiac puncture. BAL
and plasma interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-17, keratinocyte-derived
chemokine (KC), macrophage inflammatory protein (MIP)-2, sRAGE and total proteins were
measured. BAL cells were counted and differential cytology was performed. After sacrifice,

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lungs were removed, fixed, embedded, and slices were stained with hematoxylin and eosin. A
standardized histology score was calculated(18).
In separate animals, AFC was determined using previous in situ models(13, 20, 21). A
bovine serum albumin solution was instilled into the trachea; after 30-min ventilation,
proteins were measured in the instilled fluid to calculate AFC rate: Percent AFC over 30 min
= 100 x [1 - (initial/final total protein)]. In mice, the initial protein concentration was
estimated by the protein concentration of the BSA instillate. Animals were categorized into
groups of maximal (≥ 14%/30min) or submaximal (≥ 3%/30min, < 14%/30min) AFC(3, 22).
Human Study
Our institutional review board approved protocols.
Arterial and alveolar samples for sRAGE and AFC measurements were obtained from
30 patients enrolled within 24 h of ARDS onset(23) in a prospective observational study of
soluble forms and ligands of RAGE in ARDS.
Baseline lung computed tomography (CT)-scan was performed; nonfocal morphology
was noted for diffuse or patchy patterns(24), according to the “CT-scan ARDS study group”
criteria(25). Undiluted pulmonary edema fluid and plasma were collected simultaneously, at
baseline (H0) and 4 hours later (H4). Edema fluid total proteins and baseline sRAGE were
measured(22, 24). The AFC rate (in % per hour) was calculated: Percent AFC = 100 x [1 -
(initial (H0) /final (H4) total protein)](26). Patients were categorized into three groups:
maximal (≥ 14%/h), submaximal (≥ 3%/h, < 14%/h) or impaired AFC (< 3%/h)(3, 22).
Statistical analysis

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Categorical data were expressed as numbers and percentages, and quantitative data as
means and standard deviations (SD) or medians and interquartile ranges (IQR) as appropriate.
Analyses were performed using Kruskal-Wallis with Bonferroni tests for pairwise
comparisons between time-points and sham controls. Spearman coefficient was used to test
the correlations between sRAGE and continuous variables. Receiver-operating characteristic
(ROC) curves were computed to determine which parameter distinguished better nonfocal
from focal ARDS. Areas under the curve were calculated with 95% confidence intervals (CI).
Analyses were performed using Prism 6 (Graphpad Software, La Jolla, CA). A P<0.05 (two-
sided) was considered significant.
RESULTS
Study patients
Thirty patients with criteria for ARDS were enrolled between February 2011 and
January 2013. Main baseline characteristics and clinical outcomes are summarized in table 1.
Median (± SD) PaO2/FiO2 ratio and tidal volume were 108 (± 41) mmHg and 6.7 (± 0.9)
mL.kg-1 ideal body weight, respectively. Positive end-expiratory and inspiratory plateau
pressures were 13.5 (± 3.3) and 27.9 (± 3.4) cmH2O, respectively. First plasma samples for
the study were drawn a mean of 33 (± 39) h after intubation. CT-scan lung morphology was
recorded at baseline in all patients, and ARDS was characterized with nonfocal morphology
in 23 (77%) and focal loss of aeration in 7 (23%).
Animal model of lung injury
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Alveolar-capillary barrier permeability, as assessed by BAL total protein
concentration and the permeability index, showed a substantial increase at day 1 and day 2,
with return to normal levels by day 4 (Figure 1A and C) in injured animals, as compared with
injured mice on day 0 and with sham animals at the same timepoints. No difference was
observed between sham animals at all timepoint; therefore the results from sham and from
day 0 injured mice were mixed for analyses. Arterial oxygenation deteriorated by day 1 after
injury with gradual improvement by day 4 (Figure 1B). Mean arterial oxygen tension
(PaO2/FiO2 ratios) achieved clinical ARDS criteria on day 1 and day 2. Following acid
instillation the number of total leukocytes in BAL fluid increased significantly (Figure 1D),
and mononuclear cells remained predominant over time (around 90-92% of total leukocytes
at all time-points). Significant changes in both BAL (Figure 2) and plasma (Figure 3) levels
of mouse neutrophil chemokines MIP-2 and KC and pro-inflammatory mediators TNF-α, IL-
6 and IL-17 peaked on the first two days after injury.
Lung injury scores were significantly increased on days 1 and 2 (Figure 4A), and acid
aspiration caused substantial changes in lung architecture compared with sham mice and day
0 injured animals (Figure 4B-C). Over the first 48 h there were disrupted alveoli and the
presence of fluid and hemorrhage within the alveolar space (Figure 4D-E). The alveolar walls
were thickened with the presence of a neutrophilic and mononuclear infiltrate. On day 4 lung
neutrophil infiltration, alveolar structure and debris were less intense, but there remained a
cellular infiltrate (Figure 4F).
AFC rate (Figure 5A) showed a significant deterioration during the first 2 days after
injury. The lungs regained the ability to clear fluid on day 4. In parallel, BAL (Figure 5B) and
plasma (Figure 5C) levels of sRAGE were significantly upregulated at day 1-2, and decreased
near baseline by day 4.
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Elevated levels of sRAGE correlate with impaired AFC
When analyzed as a continuous variable, both plasma (Figure 5D) and BAL (Figure
5E) sRAGE baseline levels were inversely correlated with AFC in acid-injured animals
(Spearman’s rho = -0.73 (95% CI, -0.88 to -0.45) and -0.69 (95% CI, -0.86 to -0.38),
respectively, P<10-3 for both). After correction by murine albumin measurement, AFC
remained correlated with plasma and BAL sRAGE levels (Spearman’s rho = -0.73 (95% CI, -
0.88 to -0.4; P<10-4) and -0.54 (95% CI, -0.78 to -0.15; P=0.008), respectively). In lung-
injured mice, AFC was also moderately, but significantly, correlated, with histological lung
injury score, PaO2/FiO2 ratio, permeability index and plasma IL-6, but not with other markers
(Table E1, online supplement).
In patients with ARDS, baseline plasma sRAGE levels also correlated with net AFC
rates (Spearman’s rho = -0.6 (95% CI, -0.79 to -0.29), P=0.0004 <10-3)(Figure 6A). A total of
13 patients (43%) had impaired AFC, 15 patients (50%) had submaximal AFC and 2 patients
(7%) had maximal AFC (Figure 6B). Baseline plasma sRAGE levels increased significantly
with decreasing AFC rates (P=0.005). Alveolar sRAGE and lung injury score (Murray’s
score) also correlated with AFC rates in ARDS patients (Spearman’s rhos = -0.8 (95% CI, -
0.91 to -0.63), P<0.0001, and -0.5 (95% CI, -0.75 to -0.19), P=0.03, respectively).
Correlations between AFC and other clinical or biological markers of lung injury did not
reach significance (Table E1, online supplement).
Levels of sRAGE are associated with lung injury severity
In acid-injured animals, plasma and BAL sRAGE levels were significantly correlated
with PaO2/FiO2 ratio, histological lung injury score and permeability index (Figure E1, online
supplement).
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The ability of baseline plasma sRAGE levels from patients with ARDS to inform on
CT scan-lung morphology was determined. The area under the ROC curve when plasma
sRAGE was used to differentiate the presence from absence of nonfocal loss of aeration was
0.75 (95% CI, 0.52-0.98; P=0.04) for a cut-off value of 3029 pg.mL-1, with a sensitivity of
88% (95% CI, 67-97) and a specificity of 71% (95% CI, 29-96)(Figure E2A, online
supplement). Median plasma sRAGE levels were significantly higher in patients with
nonfocal (6234 pg.mL-1 [3607 to 7420]) than in those with focal lung damage (2550 pg.mL-1
[2323 to 6368])(P=0.02)(Figure E2B, online supplement). The correlation between plasma
sRAGE and lung injury severity, as assessed by PaO2/FiO2 ratio and Murray lung injury
score, was also significant in patients with ARDS (Figure E3, online supplement).
Baseline plasma sRAGE levels were higher in patients with severe ARDS (6399
pg.mL-1 [4654 to 8656]; n=17) than in those with moderate ARDS (2846 pg.mL-1 [2498 to
4156]; n=13, P=0.0001), but baseline BAL sRAGE levels were not statistically different
(53829 pg.mL-1 [13873 to 275929] in patients with severe ARDS versus 17478 pg.mL-1
[4134 to 325516] in those with moderate ARDS, P=0.4)(Figure E4, online supplement). In
addition, net AFC rates were lower in patients with severe ARDS (0.8 %/h [0 to 5.5]) than in
those with moderate ARDS (5 %/h [2.4 to 11.3]), but this difference did not reach
significance (P=0.06). Day-28 survivors (N=18) had lower baseline plasma and alveolar
sRAGE levels but higher AFC rates than non-survivors (N=12)(Figure E5, online
supplement).
DISCUSSION
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Our main goal was to determine whether levels of sRAGE, the soluble form of the
receptor for advanced glycation end-products, would serve as a marker of alveolar fluid
clearance in the setting of ARDS. In both a clinical study of ARDS patients and an
experimental model of acid-induced lung injury in mice, alveolar and plasma levels of
sRAGE significantly correlated with AFC rate. sRAGE was also a marker of lung injury
severity over time in mice, as alveolar-capillary barrier permeability, arterial oxygenation
impairment, lung injury scores and the extent of human lung damage on CT-scan were all
associated with sRAGE levels.
In our study, plasma levels of sRAGE were associated in both ARDS patients and
lung-injured animals with impaired AFC. In a prior study by Briot and colleagues in isolated
perfused human lungs, airspace sRAGE levels were inversely correlated with the rate of
AFC, supporting the theory that sRAGE is a relevant marker of alveolar epithelial injury(14).
In a recently published mouse model of acid-induced lung injury reproducing several features
of the pathophysiology of ARDS, alveolar levels of sRAGE were elevated on days 1 and 2
and seemed to correlate with a decrease in alveolar fluid clearance on the same days(13),
although this correlation was not demonstrated for alveolar sRAGE only during the acute
phase of acid-lung injury in mice(15). Measurement of AFC has not been routinely
performed in small-animal ARDS studies, but it adds insights into the function of the alveolar
epithelium. Moreover, a decrease in AFC is characteristic of human ARDS(3, 22). We aimed
at replicating the translational model of direct epithelial lung injury published by Patel and
colleagues, a model of both the onset of lung injury and its resolution. The acid aspiration
model has been described as potentially the most translatable model of direct ARDS, because
there is a clear clinical correlate(27). To date, it has been poorly used because of a narrow
dosing window between no injury and overwhelming injury leading to high mortality(28). In
our study, we reproduced this mouse model of ARDS that replicates several features of
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ARDS, and plasma measurements of pro-inflammatory mediators and sRAGE were included.
There was a marked increase in pro-inflammatory cytokines at days 1 and 2 in HCl-treated
mice, as previously reported(13), and alveolar IL-17 (expressed by a distinct type of T cells,
T helper 17 cells and certain other lymphocytes) was still significantly increased on day 4.
This novel finding could suggest an under investigated role for the IL-17-T helper 17 cell
pathway during ARDS resolution and stimulate further research(29, 30). The report of
sRAGE plasma levels in lung injured mice is rather novel(31) and further supports the value
of sRAGE as a surrogate marker of lung injury severity in general(10–12), and of AFC in
particular, in both the experimental and clinical settings of ARDS. Because of the evidence
that alveolar epithelial type I cells transport sodium and contribute to AFC(32, 33), a marker
of AT I cell injury could reflect intact AFC. Our results, combined with those from others,
support sRAGE as a marker of epithelial injury and barrier dysfunction clinically; they also
provide prospective validation of previously published research in experimental ARDS and in
patients following lung transplantation(10, 34). These data also support the hypothesis that a
marker of alveolar epithelial injury could have predictive value in the assessment of lung
dysfunction and recovery during ARDS(8, 13). Our study was not designed to provide
insights on the precise molecular mechanisms through which RAGE axis activation could
modulate transepithelial fluid clearance, and on the effects of RAGE modulation on AFC.
Although RAGE has the striking capacity to induce cellular attachment and spreading(35, 36)
and to enhance the adherence of epithelial cells to the collagen coated surfaces(37), the role
of RAGE in the alveolar epithelial cell proliferation/differentiation and the regulation of the
expression or function of epithelial membrane channels (e.g., Na+-K+-ATPase, sodium
channels, aquaporins) remains underinvestigated(38). More importantly, whether the
modulation of RAGE axis could lead to enhanced AFC has not been explored to date.
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Our findings, along with those from others, reinforce the role of sRAGE as a
biomarker of epithelial function during ARDS. Its diagnostic and prognostic values have
been reported, and its correlation with severity is established in ARDS(10–12). The
availability of the measurement of plasma biomarker sRAGE could be of particular interest to
assess response to therapy during ARDS, in preclinical studies(39) and ultimately in
patients(40). The impact of mechanical ventilation (MV) settings on sRAGE levels has been
reported in ARDS patients, with prognostic values in those receiving higher tidal
volumes(11), and the effects of various strategies, including alveolar recruitment strategy and
lung imaging-based MV settings are under investigation by our team. Our results support
plasma sRAGE as a marker of AFC during ARDS, and could be useful to better tailor
respiratory interventions to the individual ARDS patient. Nevertheless, whether patients with
the most impaired AFC and with diffuse ARDS might better respond to alveolar recruitment
than others is uncertain(24, 41). In this perspective, the availability of biomarkers should at
least stimulate further research on assessing biomarker-guided respiratory settings(24, 41).
Our study has some potential limitations. First, we have not withdrawn a reference
alveolar fluid sample immediately after bovine albumin instillation for AFC measurement in
mice, as previously reported in order to be as representative of alveolar content as
possible(13). On the other hand, we chose to use another published procedure in which the
instilled albumin concentration serves as reference(20, 42), but we measured mouse albumin
in the final alveolar sample to demonstrate that an increase in total protein concentration
(which is what should happen if AFC is occurring) is not due to leakage of mouse plasma
proteins into the bronchoalveolar space. Second, although we could replicate most
recommended features of ARDS in our experimental model, we did not found an increase in
the absolute number of neutrophils in BAL fluid from HCl-treated mice(13, 18). In our study,
lung injured mice had increased total alveolar leukocytes, higher lung injury scores and
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higher concentrations of pro-inflammatory cytokines in BAL, suggesting that we were able to
observe an inflammatory response, in both the lung and the systemic circulation(18).
Moreover, we believe that the lack of alveolar neutrophilia should not call under question our
main findings, as it is frequent not to be able to observe every single feature of lung injury in
experimental models of ARDS(18). On the other hand, our findings could be consistent with
a growing body of evidence supporting a role for monocytes in ARDS(43). As they may also
reflect frequent differences between the findings from mice and human studies(44), we chose
a priori to also validate in the clinical setting our findings in mice on the association between
sRAGE levels and AFC. Also, we limited our evaluation in animals at day 4 after injury and
did not assess later time-points(13). The exploration of this later resolution period in such a
model is promising in order to investigate the pathological processes focusing on resolution
and repair(45). Finally, as with any biomarker study, a derivation cohort always demonstrates
better biomarker test characteristics than a validation cohort. Validating our results in an
independent cohort of patients remains therefore necessary.
In conclusion, in both a translational mouse model of direct lung epithelial injury and
a prospective observational study of patients with ARDS, both plasma and alveolar sRAGE
levels were associated with AFC and lung injury severity. Our findings should prompt further
studies on the pathophysiologic implications of RAGE axis in the mechanisms leading to
alveolar epithelial fluid clearance.
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ACKNOWLEDGMENTS
The authors wish to thank the nurses of the intensive care unit at Estaing University
hospital, and the technicians and staff from the department of Medical Biochemistry and
Molecular Biology, Estaing University Hospital, CHU Clermont-Ferrand, and from the
Université d’Auvergne in Clermont-Ferrand, France.
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FIGURE LEGENDS
FIGURE 1. A) Changes in alveolar–capillary barrier permeability as measured by
bronchoalveolar lavage (BAL) fluid total protein level (n=4-6 for each time-point). B)
Arterial oxygen tension (PaO2)/inspiratory oxygen fraction (FiO2)(n=4-6 for each time-
point). C) Permeability index calculated as the bronchoalveolar lavage (BAL) fluid-to-plasma
ratio of human serum albumin (HSA) concentration (n=4-6 for each time-point). D)
Bronchoalveolar lavage (BAL) total numbers of leucocytes (n=4-6 for each time-point).
Values are reported as means ± standard deviations. ** P<10-2; *** P<10-3; **** P<10-4
versus sham controls.
FIGURE 2. Measurement of bronchoalveolar lavage (BAL) levels of A) interleukin (IL)-6,
B) tumor necrosis factor (TNF)-α, C) interleukin (IL)-17, D) macrophage inflammatory
protein (MIP)-2 and E) keratinocyte-derived chemokine (KC) at baseline (day 0) and after
acid aspiration (n=4-6 for each time-point). Values are reported as means ± standard
deviations. * P<0.05; ** P<10-2; *** P<10-3; **** P<10-4 versus sham controls.
FIGURE 3. Measurement of plasma levels of A) interleukin (IL)-6, B) tumor necrosis factor
(TNF)-α, C) interleukin (IL)-17, D) macrophage inflammatory protein (MIP)-2 and E)
keratinocyte-derived chemokine (KC) at baseline (day 0) and after acid aspiration (n=4-6 for
each time-point). Values are reported as means ± standard deviations. * P<0.05; ** P<10-2;
*** P<10-3; **** P<10-4 versus sham controls.
FIGURE 4. A) Lung injury scoring shows a significant injury on days 1, 2 and 4 in injured
animals as compared to sham controls (n=6-8 for each time-point). Lung injury was assessed
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on a scale of 0–2 for each of the following criteria: i) neutrophils in the alveolar space, ii)
neutrophils in the interstitial space, iii) number of hyaline membranes, iv) amount of
proteinous debris, and v) extent of alveolar septal thickening.
The final injury score was derived from the following calculation:
Score=[20x(i)+14x(ii)+7x(iii)+7x(iv)+2x(v)]/(number of fieldsx100). Values are reported as
means ± standard deviations. * P<0.05; **** P<10-4 versus sham controls. B-F)
Representative hematoxylin and eosin (H&E)-stained sections at x20 original magnification
of sham and injured animals at all time-points after acid aspiration. B) Sham, C) Day 0,
injured, D) Day 1, injured, E) Day 2, injured, F) Day 4, injured. There is greater cellularity
consisting mainly of neutrophils (white arrows) on days 1 and 2 with more areas of
atelectasis as well as increased alveolar disruption with hyaline membranes (arrowheads),
proteinous debris and hemorrhage. Scale bars 50 µm.
FIGURE 5. A) Measurement of alveolar fluid clearance (AFC) rate as a marker of epithelial
function (n=4-6 for each time-point). Measurement of B) plasma and C) bronchoalveolar
lavage (BAL) levels of soluble receptor for advanced glycation end-products (sRAGE) as a
marker of type 1 alveolar epithelial injury (n=4-6 for each time-point). Values are reported as
means ± standard deviations. ** P<10-2; *** P<10-3; **** P<10-4 versus sham controls. D)
Plasma and E) BAL sRAGE baseline levels plotted against AFC rate show that sRAGE
inversely correlated with the AFC rate in acid-injured animals (Spearman’s rho = -0.73 (95%
CI, -0.88 to -0.45) and -0.69 (95% CI, -0.86 to -0.38), respectively, P<10-3). Each data-point
represents a separate animal. F) Plasma soluble receptor for advanced glycation end-products
(sRAGE) levels in lung-injured mice with categories of alveolar fluid clearance: submaximal
(≥ 3%/30min, < 14%/30min), or maximal (≥ 14%/30min). N = number of animals. Values are
reported as means ± standard deviations and are analyzed with Kruskal-Wallis test
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(nonparametric data). G) BAL soluble receptor for advanced glycation end-products
(sRAGE) levels in lung-injured mice with categories of alveolar fluid clearance: submaximal
(≥ 3%/30min, < 14%/30min), or maximal (≥ 14%/30min). N = number of animals. Values are
reported as means ± standard deviations and are analyzed with Kruskal-Wallis test
(nonparametric data).
FIGURE 6. A) Plasma soluble receptor for advanced glycation end-products (sRAGE)
baseline levels plotted against alveolar fluid clearance (AFC) rate show that sRAGE inversely
correlated with the AFC rate in patients with acute respiratory distress syndrome
(ARDS)(Spearman’s rho = -0.59 (95% CI, -0.79 to -0.28), P<10-3). Each data-point
represents a separate patient. B) Plasma soluble receptor for advanced glycation end-products
(sRAGE) baseline levels in patients with ARDS with three categories of alveolar fluid
clearance: impaired (< 3%/h), submaximal (≥ 3%/h, < 14%/h), or maximal (≥ 14%/h). N =
number of subjects. Values are reported as means ± standard deviations and are analyzed
with Kruskal-Wallis test (nonparametric data).
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TABLE
Male sex, n (%) 16 (53)
Age (years) 61 ± 15
Body mass index (kg.m-2) 25.9 ± 6.9
Sequential Organ Failure Assessment score 10.9 ± 3.3
Acute Physiology and Chronic Health Evaluation II score 25.5 ± 7.3
Coexisting conditions, n (%)
- Atherosclerosis 7 (23)
- Diabetes 7 (23)
- Hypertension 12 (40)
- Dyslipidemia 6 (20)
- Current smoking 6 (20)
- COPD 4 (13)
- Asthma 1 (3)
- Hematologic neoplasms 8 (27)
- Chronic renal failure 1 (3)
Cause of ARDS, n (%)
- Sepsis 27 (90)
- Pneumonia 21 (70)
- Aspiration 1 (3)
- Severe trauma 1 (3)
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Lung injury score 3.3 ± 0.5
Baseline arterial level of sRAGE (pg.mL-1) 5502 ± 2994
Baseline alveolar level of sRAGE (pg.mL-1) 154734 ± 217417
Baseline AFC (% per hour) 2.1 ± 8.9
Maximal AFC (≥ 14%/h), n (%) 2 (7)
Submaximal AFC (≥ 3%/h, < 14%/h), n (%) 15 (50)
Impaired AFC (< 3%/h), n (%) 13 (43)
Ventilator-free days 7±9
Survival on day 28, n (%) 18 (60)
Table 1. Baseline clinical characteristics of patients with acute respiratory distress
syndrome (ARDS)(N=30). Data are expressed as mean ± standard deviation, unless
otherwise indicated. The body-mass index is the weight in kilograms divided by the square of
the height in meters. COPD: chronic obstructive pulmonary disease. sRAGE: soluble form of
the receptor for advanced glycation end-products. AFC: alveolar fluid clearance. ARDS:
acute respiratory distress syndrome. Lung injury (or Murray) score can range from 0 to 4,
with higher values indicating more severe lung injury. Percentages may not exactly total
100% because of rounding.
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192x137mm (300 x 300 DPI)

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183x195mm (300 x 300 DPI)

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228x271mm (300 x 300 DPI)

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160x95mm (300 x 300 DPI)

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270x389mm (300 x 300 DPI)

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232x330mm (300 x 300 DPI)

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Jabaudon et al. sRAGE predicts alveolar fluid clearance in ARDS. Online Data Supplement
Online Data Supplement
Soluble RAGE Predicts Impaired Alveolar Fluid Clearance in Acute Respiratory
Distress Syndrome
Matthieu Jabaudon, Raiko Blondonnet, Laurence Roszyk, Damien Bouvier, Jules Audard,
Gael Clairefond, Mathilde Fournier, Geoffroy Marceau, Pierre Déchelotte, Bruno Pereira,
Vincent Sapin and Jean-Michel Constantin
METHODS
Animal Studies
Our institutional animal care and ethics committee approved study protocols (Comité
d’Ethique en Matière d’Expérimentation Animale d’Auvergne, approval number CE 67-12).
Male CD-1 mice (Janvier Labs, Saint-Berthevin, France), aged 10-12 weeks and
weighing 25-30 g, were anesthetized by intraperitoneal injection of xylazine (10 mg.kg-1) and
ketamine (100 mg.kg-1), and given an intraperitoneal fluid bolus of 10 µL.g-1 0.9% isotonic
saline as pre-emptive fluid resuscitation. Mice were suspended vertically from their incisors
on a custom-made mount for orotracheal instillation, as described previously(E1). A fine
catheter was guided 1 cm below the vocal cords, and 75 µL of an iso-osmolar (to mouse
plasma, i.e. 322 mOsm.L-1) solution of 0.1 M hydrochloric acid (pH 1.0) was instilled. For
the next 4 h, during which time animals exhibited significant respiratory depression/distress,
mice were kept in a transparent recovery box under humidified supplemental oxygen

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(inspiratory oxygen fraction (FiO2) reduced gradually from 1.0 to 0.21). During this period,
animals were carefully monitored and body temperature was maintained using external heat
sources, after which they were transferred to individually ventilated cages with air and free
access to food and water.
Criteria for experimental ARDS were evaluated as recommended by the American
Thoracic Society(E2) at baseline in injured and sham animals, and at specified time-points (1,
2 and 4 days) after acid instillation in injured mice(E1). In brief, mice were anesthetized,
tracheotomized and ventilated using an appropriate ventilator (TOPOTM Small Animal
Ventilator, Kent Scientific, Torrington, CT). After an initial lung recruitment maneuver (30
cmH2O for 5 s), animals were ventilated for 30 min (tidal volume 8-9 mL.kg-1, positive end-
expiratory pressure 6 cmH2O, respiratory rate 160 breaths.min-1 and FiO2 1) to standardize
the volume history of the lungs. At the end of ventilation, blood gases were measured and
mice were sacrificed by anesthetic overdose with intraperitoneal pentobarbital (150 mg.kg-1).
Acid-injured animals were compared with otherwise sham mice, receiving just saline tracheal
instillation, surgical preparation and 30-min ventilation. All mice received 10 mg.kg-1 of
human serum albumin (HSA) dissolved in 100 µL of saline intravenously, 1 h before
euthanasia, for measurement of the lung permeability index, presented as percentage. This
permeability index was defined as the ratio of HSA in bronchoalveolar lavage (BAL) fluid to
that in plasma collected at the end of the experiments. The HSA concentration was measured
by enzyme linked immunosorbent assay (ELISA) using a human albumin ELISA Kit (R&D
Systems, Minneapolis, MN). The lower limit of detection was 5 ng.mL-1.
BAL was performed with 750 µL of saline as described previously(E1) and systemic
blood was drawn from cardiac puncture; the samples were centrifuged at 240xg. Protein
levels in BAL fluid were quantified with a colorimetric method (Pierce Biotechnology,

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Rockwood, IL). BAL and plasma levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α,
IL-17, keratinocyte-derived chemokine (KC), macrophage inflammatory protein (MIP)-2
were determined in duplicate using the Bio-Plex 200 System, which is based on Luminex
xMAP Technology (Bio-Rad, Hercules, CA, USA). In the present study, we screened BAL
and plasma samples using the Mouse Cytokine 4-plex panel and MIP-2 SET (Bio-Rad,
Marnes-la-Coquette, France). BAL and plasma levels of soluble receptor for advanced
glycation end-products (RAGE) were determined with ELISA (R&D Systems, Minneapolis,
MN). BAL cell counts were obtained using a hemocytometer, with differential cytology
performed on DiffQuik-stained samples prepared by cytospin (Thermo Fisher Scientific, St.
Leon-Rot, Germany).
In a separate series of experiments, alveolar fluid clearance (AFC) was determined at
baseline and at days 1, 2 and 4 after acid instillation (and in untreated animals), using a
modification of previously described established in situ models(E1, E3, E4). Once the mouse
was stable on the ventilator, it was briefly disconnected to permit instillation of 300 µL of 5%
BSA/saline (322 mOsm/L, iso-osmotic to mouse plasma) into the dependent (left) lung via
the tracheal cannula, which was then flushed with 100 µL of air before reconnection to the
ventilator. Thirty minutes after instillation, a surgical pneumothorax was induced through
blunt dissection of the diaphragm to maximize the recovery of the remaining instillate from
the lungs. After 30-min ventilation, instilled fluid was aspirated to measure protein content
and calculate AFC rate: Percent AFC over 30 min = 100 x [1 - (initial protein/final total
protein)]. In mice, the initial protein concentration was the protein concentration of the BSA
instillate. Measurement of AFC assumes that the concentration of instilled protein is not
altered significantly by the presence of excess alveolar fluid and/or protein.

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AFC was also calculated with correction for excess murine alveolar protein, as
previously described(E4). Acid instillation may cause significant damage to the broncho-
alveolar-capillary barrier, which may result in significant leakage of murine serum proteins
into the alveolar space, resulting in overestimation of AFC rates if based solely upon total
protein levels in AFC aspirates. To correct for epithelial leakage, the amount of murine
albumin was measured in duplicate in the fluid aspirated from the lungs at the end of the 30-
min ventilation period using specific ELISA (Abcam, Cambridge, UK). Final AFC values
were corrected by a factor of (100-% murine albumin present in the aspirate)/100. All
samples had a coefficient of variation less than 10%. Animals were further categorized into
three groups of AFC: maximal (≥ 14%/30min), submaximal (≥ 3%/30min, < 14%/30min) or
impaired (< 3%/30min).
In a separate series of experiments, acid injured mice (at each time-point) and
untreated animals were sacrificed and their both lungs removed, fixed with alcoholic acetified
formalin and embedded with paraffin. Slices at 10-µm thickness were subsequently stained
with hematoxylin and eosin (Sigma-Aldrich Ltd). The histology injury score was derived
from the following calculation: Score = [20x(i) + 14x(ii) + 7x(iii) + 7x(iv) + 2x(v)] / (number
of fields x 100)(table E2)(E2).
Human Study
Samples for plasma sRAGE and AFC measurements were obtained from patients enrolled in
a prospective observational study of soluble forms and ligands of RAGE in ARDS
(clinicaltrials.gov number: NCT01270295). Thirty consecutive patients under mechanical
ventilation with acute lung injury/ARDS were identified based on the Berlin definition(E5).
and included within 24 hours of disease onset in the adult general intensive care unit (ICU) at

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Estaing University Hospital, Clermont-Ferrand, France. Patients were ineligible if: they were
pregnant; < 18 years old; they had a history of acute exacerbation of diabetes, dialysis for
end-stage kidney disease, Alzheimer’s disease, amyloidosis or evolutive solid neoplasm.
Clinical and biological data were collected prospectively. All patients were
mechanically ventilated at baseline and were cared for by the ICU staff. Clinical data were
recorded and included hemodynamic parameters, respiratory and ventilatory parameters,
multiorgan system function, and medication administered. All patients were followed until
death, 28 days or ICU discharge, whichever occurred first. The Acute Physiology And
Chronic Health Evaluation II (APACHE II), Sequential Organ Failure Assessment (SOFA)
and the lung injury scores were calculated at baseline(E6–E8). Intensive care management of
patients included in the study was conducted using our ICU-based standard protocols. Thus,
mechanical ventilation strategy (including weaning strategy), sepsis management, and the use
of sedative agents were based on currently available guidelines(E9, E10). A lung-protective
ventilation strategy was applied; a tidal volume of 6 mL.kg-1 (ideal) body weight and a
pressure plateau of <30 cm H2O were targeted in all mechanically ventilated patients(E9,
E10).
According to our institutional protocol, a baseline lung computed tomography (CT)-
scan was performed, as previously described(E11). On day 0, two physicians transferred
ARDS patients to the Department of Radiology of our institution, as previously described by
our group(E11). During CT-scan image acquisition, particular attention was paid to avoid any
change in patient positioning. Electrocardiogram, pulse oximetry, and systemic arterial
pressure were continuously assessed throughout the CT-scan procedure. The lowest value of
hemoglobin oxygen saturation allowed during the imaging exam was 85%(E12). Lung CT-
scanning was performed in the supine position from the apex to the diaphragm, using a

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multislice helical Multidetector CT-scanner (Aquilion 64, Toshiba, Japan). Without iodine
contrast medium injection, 1-mm thick contiguous CT sections were acquired using a low-
dose protocol. All images were observed and photographed at a window width of 1600
Hounsfield units (HU) and a level of -700 HU. The exposures were taken at 120 Kv and 250
mA. Two independent senior radiologists at our institution, blinded to clinical and biological
data, interpreted CT-scans. Nonfocal lung morphology was noted for diffuse or patchy
patterns, according to the “CT-scan ARDS study group” criteria (E12). One of the
investigators (JMC) adjudicated disagreements (n=2) between radiologists. The strength of
agreement for the characterization of focal versus nonfocal ARDS in our study was very good
between the two observers using the inter-rater agreement Kappa statistic (Κ=0.87, 95% CI
0.74 to 0.99).
Arterial blood samples were collected from patients at baseline. Levels of sRAGE
were measured in thawed samples using a commercially available sandwich enzyme
immunoassay kit (Human sRAGE Quantikine ELISA Kit, R&D Systems, Minneapolis, MN),
according to the manufacturer’s instructions. All steps were undertaken at room temperature.
Briefly, 96-well Costar EIA plates (Corning Life Sciences, Lowell, MA) were coated with the
capture antibody. Plates were blocked with phosphate-buffered saline (PBS) containing 1%
bovine serum albumin, and incubated with 100-µL samples of plasma/edema fluid or
standards followed by biotinylated detection antibody. After subsequent incubation with
streptavidin-conjugated horseradish peroxidase, colorimetric detection was done using
tetramethylbenzidine as the substrate. Plasma/edema levels of the measured marker were
determined by a computer software regression calculation based on the optical density values
at 450 nm and 540 nm read with a microtiter plate reader (Rainbow, Tecan, Maennedorf,
Switzerland); readings at 540 nm were subtracted from those at 450 nm for correction for
optical imperfections in the plate. All measurements were conducted in duplicate. The intra-

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assay coefficient of variation was 5.8%. Personnel responsible for performing assays had no
knowledge of the clinical data.
Undiluted pulmonary edema fluid samples were collected from patients at baseline
and 4 h later, as previously described(E13, E14). Briefly, a soft 14-Fr-gauge suction catheter
(PharmaPlast, Maersk Medical, Denmark) was advanced into a wedged position in a distal
bronchus via an endotracheal tube. Pulmonary edema fluid was collected in a suction trap by
gentle suction. All samples were centrifuged at 3000 rpm at 4°C for 10 min in a refrigerated
centrifuge. Supernatants were collected, and the total protein concentration in edema fluid
was measured by the biuret method(E13, E14).
On the basis of the observation that the rate of clearance of edema fluid from the
alveolar space is much faster than the rate of protein removal(E15), the net AFC rate was
calculated: Percent AFC = 100 x [1 - (initial edema protein/final edema total protein)]. Initial
and final samples were drawn at baseline and 4 h later, respectively. This method has been
validated in prior clinical and experimental studies(E13, E16–E19). Patients were further
categorized into three groups of AFC: maximal (≥ 14%/h), submaximal (≥ 3%/h, < 14%/h) or
impaired (< 3%/h)(E13, E16–E19). All samples had a coefficient of variation less than 10%.
Levels of sRAGE were also measured by duplicate ELISA in the pulmonary edema fluid
from patients with ARDS (Human sRAGE Quantikine ELISA Kit, R&D Systems,
Minneapolis, MN), according to the manufacturer’s instructions.
Protocols were approved by the Institutional Review Board of the University Hospital
of Clermont-Ferrand, France (Comité de Protection des Personnes Sud Est VI, approval
number AU870). All participants, or their next-of-kin, provided written consent to participate
in this study.

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Statistical analysis
Categorical data were expressed as numbers and percentages, and quantitative data as
mean and standard deviation (SD) or median and interquartile range (IQR) according to
statistical distribution. Statistical analyses of physiological parameters were carried out by
Kruskal-Wallis with Bonferroni tests for pairwise comparisons between each time-point and
sham controls (represented as day 0). Spearman correlation coefficient was used to test the
correlations between sRAGE levels and other continuous variables in injured animals or
patients. Receiver-operating characteristic (ROC) curves were computed to determine which
parameter distinguished better nonfocal from focal ARDS. Areas under the curve were
calculated and presented with 95% confidence intervals (CI)(e.g., for sensitivity and
specificity), and several indexes were proposed to establish the best threshold (Youden, Liu,
efficiency). All analyses were performed using Prism 6 (Graphpad Software, La Jolla, CA).
A P<0.05 (two-sided) was considered statistically significant.
A limited number of animals was used for baseline comparisons (n=3-4), and 4-6
animals were used in each group on days 1, 2 and 4 in order to detect a difference of 1
mg.mL-1 (SD=0.5) in BAL protein concentration and of 5 % per 30 min (SD =2.5) in AFC
rate on day 1 or day 2, when considering alpha and beta risks of 5% (bilateral) and 10%,
respectively. Statistical power of 90% was considered sufficient to allow multiple
comparisons between groups. The size of the clinical validating cohort was based on previous
calculations in critically ill patients, with the ability to detect a 1500 pg.mL-1 difference in
baseline sRAGE levels between ARDS (a priori estimation: 3356 ± 1780 pg.mL-1) and
mechanically-ventilated controls (a priori estimation: 525 ± 480 pg.mL-1), when considering
type I error alpha and statistical power of 5% (bilateral) and 80%, respectively.

Page 44 of 59
RESULTS
Levels of sRAGE are associated with lung injury severity
In acid-injured animals, plasma and BAL sRAGE levels were significantly correlated
with PaO2/FiO2 ratio (Spearman’s rho = -0.74 (95% CI, -0.89 to -0.46) and -0.66 (95% CI, -
0.85 to -0.33), respectively, P<10-3 for both), histological lung injury score (Spearman’s rho
= 0.74 (95% CI, 0.47 to 0.89) and 0.66 (95% CI, 0.32 to 0.85), respectively, P<10-3 for both)
and permeability index (Spearman’s rho = 0.73 (95% CI, 0.44 to 0.88) and 0.78 (95% CI,
0.53 to 0.90), respectively, P<10-4 for both)(Figure E1, online supplement).
The ability of baseline plasma sRAGE levels from patients with ARDS to
discriminate between the types of loss of aeration based on CT scan-lung morphology was
determined. The area under the ROC curve when plasma sRAGE was used to differentiate
the presence from absence of nonfocal loss of aeration was 0.75 (95% CI, 0.52-0.98; P=0.04)
for a cut-off value of 3029 pg.mL-1, with a sensitivity of 88% (95% CI, 67-97) and a
specificity of 71% (95% CI, 29-96)(Figure E2A, online supplement). Median plasma sRAGE
levels were significantly higher in patients with nonfocal (6234 pg.mL-1 [3607 to 7420]) than
in those with focal lung damage (2550 pg.mL-1 [2323 to 6368])(P=0.02)(Figure E2B, online
supplement). The correlation between plasma sRAGE and lung injury severity was also
significant in patients with ARDS, as assessed by PaO2/FiO2 ratio (Spearman’s rho = -0.65,
95% CI, -0.82 to -0.37, P<10-3) and Murray lung injury score (Spearman’s rho = 0.79, 95%
CI, 0.61 to 0.90, P<10-4)(Figure E3, online supplement).
Baseline plasma sRAGE levels were higher in patients with severe ARDS (6399
pg.mL-1 [4654 to 8656]; n=17) than in those with moderate ARDS (2846 pg.mL-1 [2498 to
4156]; n=13, P=0.0001), but baseline BAL sRAGE levels were not statistically different

Page 45 of 59
(53829 pg.mL-1 [13873 to 275929] in patients with severe ARDS versus 17478 pg.mL-1
[4134 to 325516] in those with moderate ARDS, P=0.4)(Figure E4, online supplement). In
addition, net AFC rates were lower in patients with severe ARDS (0.8 %/h [0 to 5.5]) than in
those with moderate ARDS (5 %/h [2.4 to 11.3]), but this difference did not reach
significance (P=0.06).
Baseline plasma and BAL sRAGE levels were moderately correlated with APACHE
2 score (Spearman’s rho = 0.37, 95% CI, -0.66 to 0.002, P=0.04 and -0.37, 95% CI, -0.65 to
0.009, P=0.04, respectively); but not with other severity scores (SAPS 2, SOFA score). There
was no significant correlation between AFC and APACHE 2 (Spearman’s rho = 0.12, 95%
CI, -0.27 to 0.47, P=0.5), SOFA (Spearman’s rho = 0.13, 95% CI, -0.26 to 0.48, P=0.5) or
SAPS 2 (Spearman’s rho = 0.14, 95% CI, -0.2 to 0.49, P=0.5) scores. There was no
correlation between the number of ventilatory-free days at day 28 and baseline plasma
(Spearman’s rho = 0.09, 95% CI, -0.29 to 0.45, P=0.6), alveolar (Spearman’s rho = 0.05, 95%
CI, -0.32 to 0.41, P=0.8) sRAGE or AFC (Spearman’s rho = 0.06, 95% CI, -0.32 to 0.42,
P=0.7). Day-28 survivors (N=18) had lower baseline plasma and alveolar sRAGE levels but
higher AFC rates than non-survivors (N=12)(Figure E5, online supplement).
10

Page 46 of 59
REFERENCES
E1. Patel BV, Wilson MR, Takata M. Resolution of acute lung injury and inflammation: a
translational mouse model. Eur Respir J 2012;39:1162–1170.
E2. Matute-Bello G, Downey G, Moore BB, Groshong SD, Matthay MA, Slutsky AS,
Kuebler WM, Acute Lung Injury in Animals Study Group. An official American
Thoracic Society workshop report: features and measurements of experimental acute
lung injury in animals. Am J Respir Cell Mol Biol 2011;44:725–738.
E3. Aeffner F, Traylor ZP, Yu ENZ, Davis IC. Double-stranded RNA induces similar
pulmonary dysfunction to respiratory syncytial virus in BALB/c mice. Am J Physiol
Lung Cell Mol Physiol 2011;301:L99–L109.
E4. Wolk KE, Lazarowski ER, Traylor ZP, Yu ENZ, Jewell NA, Durbin RK, Durbin JE,
Davis IC. Influenza A virus inhibits alveolar fluid clearance in BALB/c mice. Am J
Respir Crit Care Med 2008;178:969–976.
E5. ARDS Definition Task Force, Ranieri VM, Rubenfeld GD, Thompson BT, Ferguson
ND, Caldwell E, Fan E, Camporota L, Slutsky AS. Acute respiratory distress syndrome:
the Berlin Definition. JAMA 2012;307:2526–2533.
E6. Gall J-RL, Loirat P, Alpcrovitch A. APACHE II-A Severity of Disease Classification
System. Crit Care Med 1986;14:754.
E7. Vincent JL, Moreno R, Takala J, Willatts S, De Mendonça A, Bruining H, Reinhart CK,
Suter PM, Thijs LG. The SOFA (Sepsis-related Organ Failure Assessment) score to
describe organ dysfunction/failure. On behalf of the Working Group on Sepsis-Related
Problems of the European Society of Intensive Care Medicine. Intensive Care Med
1996;22:707–710.
E8. Murray JF, Matthay MA, Luce JM, Flick MR. An expanded definition of the adult
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respiratory distress syndrome. Am Rev Respir Dis 1988;138:720–723.
E9. Dellinger RP, Levy MM, Carlet JM, Bion J, Parker MM, Jaeschke R, Reinhart K, Angus
DC, Brun-Buisson C, Beale R, Calandra T, Dhainaut J-F, Gerlach H, Harvey M, Marini
JJ, Marshall J, Ranieri M, Ramsay G, Sevransky J, Thompson BT, Townsend S, Vender
JS, Zimmerman JL, Vincent J-L, International Surviving Sepsis Campaign Guidelines
Committee, American Association of Critical-Care Nurses, American College of Chest
Physicians, American College of Emergency Physicians, Canadian Critical Care
Society, et al. Surviving Sepsis Campaign: international guidelines for management of
severe sepsis and septic shock: 2008. Crit Care Med 2008;36:296–327.
E10. Ventilation with lower tidal volumes as compared with traditional tidal volumes for
acute lung injury and the acute respiratory distress syndrome. The Acute Respiratory
Distress Syndrome Network. N Engl J Med 2000;342:1301–1308.
E11. Malbouisson LM, Muller JC, Constantin JM, Lu Q, Puybasset L, Rouby JJ, CT Scan
ARDS Study Group. Computed tomography assessment of positive end-expiratory
pressure-induced alveolar recruitment in patients with acute respiratory distress
syndrome. Am J Respir Crit Care Med 2001;163:1444–1450.
E12. Puybasset L, Cluzel P, Gusman P, Grenier P, Preteux F, Rouby JJ. Regional distribution
of gas and tissue in acute respiratory distress syndrome. I. Consequences for lung
morphology. CT Scan ARDS Study Group. Intensive Care Med 2000;26:857–869.
E13. Matthay MA, Wiener-Kronish JP. Intact epithelial barrier function is critical for the
resolution of alveolar edema in humans. Am Rev Respir Dis 1990;142:1250–1257.
E14. Constantin J-M, Cayot-Constantin S, Roszyk L, Futier E, Sapin V, Dastugue B, Bazin J-
E, Rouby J-J. Response to recruitment maneuver influences net alveolar fluid clearance
in acute respiratory distress syndrome. Anesthesiology 2007;106:944–951.
E15. Berthiaume Y, Staub NC, Matthay MA. Beta-adrenergic agonists increase lung liquid
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clearance in anesthetized sheep. J Clin Invest 1987;79:335–343.
E16. Verghese GM, Ware LB, Matthay BA, Matthay MA. Alveolar epithelial fluid transport
and the resolution of clinically severe hydrostatic pulmonary edema. J Appl Physiol
1999;87:1301–1312.
E17. Ware LB, Matthay MA. Alveolar fluid clearance is impaired in the majority of patients
with acute lung injury and the acute respiratory distress syndrome. Am J Respir Crit
Care Med 2001;163:1376–1383.
E18. Ware LB, Golden JA, Finkbeiner WE, Matthay MA. Alveolar epithelial fluid transport
capacity in reperfusion lung injury after lung transplantation. Am J Respir Crit Care
Med 1999;159:980–988.
E19. Sakuma T, Okaniwa G, Nakada T, Nishimura T, Fujimura S, Matthay MA. Alveolar
fluid clearance in the resected human lung. Am J Respir Crit Care Med 1994;150:305–
310.
13

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FIGURE LEGENDS
Figure E1. A) Plasma and B) Bronchoalveolar lavage (BAL) soluble receptor for advanced
glycation end-products (sRAGE) baseline levels plotted against arterial oxygen tension
(PaO2)/inspiratory oxygen fraction (FiO2) show that sRAGE inversely correlated with
oxygenation in acid-injured animals (Spearman’s rho = -0.74 (95% CI, -0.89 to -0.46) and -
0.66 (95% CI, -0.85 to -0.33), respectively, P<10-3). C) Plasma and D) BAL soluble receptor
for advanced glycation end-products (sRAGE) baseline levels plotted against histological
lung injury scoring show that sRAGE positively correlated with lung injury in acid-injured
animals (Spearman’s rho = 0.74 (95% CI, 0.47 to 0.89) and 0.66 (95% CI, 0.32 to 0.85),
respectively, P<10-3). E) Plasma and F) BAL soluble receptor for advanced glycation end-
products (sRAGE) baseline levels plotted against the permeability index (calculated as the
BAL fluid-to-plasma ratio of human serum albumin (HSA) concentration) show that sRAGE
positively correlated with alveolar-capillary barrier permeability (Spearman’s rho = 0.73
(95% CI, 0.44 to 0.88) and 0.78 (95% CI, 0.53 to 0.90), respectively, P<10-4). Each data-
point represents a separate animal.
Figure E2. A) Receiver-operating characteristic (ROC) curve of baseline plasma soluble
receptor for advanced glycation end product levels in differentiating between the presence
and absence of non-focal loss of aeration during computed tomography scan morphology
studies on day 0 in patients with acute respiratory distress syndrome patients (N=30). The
area under the ROC curve was 0.75 (95% CI, 0.52-0.98; P=0.04) for a cut-off value of 3029
pg.mL-1, with a sensitivity of 88% (95% CI, 67-97) and a specificity of 71% (95% CI, 29-96).
B) Baseline plasma soluble receptor for advanced glycation end products (sRAGE) levels in
14

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acute respiratory distress syndrome (ARDS) patients with focal (n=7) and non-focal loss of
aeration (n=23), as assessed by computed tomography scan lung morphology on day 0.
Values are reported as means ± standard deviations and are analyzed with Kruskal-Wallis test
(nonparametric data).
Figure E3. A) Plasma soluble receptor for advanced glycation end-products (sRAGE)
baseline levels plotted against arterial oxygen tension (PaO2)/inspiratory oxygen fraction
(FiO2) show that sRAGE inversely correlated with oxygenation in patients with acute
respiratory distress syndrome (ARDS)(Spearman’s rho = -0.65 (95% CI, -0.82 to -0.37),
P<10-3). B) Plasma soluble receptor for advanced glycation end-products (sRAGE) baseline
levels plotted against lung injury score show that sRAGE positively correlated with lung
injury in patients with ARDS (Spearman’s rho = 0.79 (95% CI, 0.61 to 0.90), P<10-3). Lung
injury (or Murray) score can range from 0 to 4, with higher values indicating more severe
injury. Each data-point represents a separate patient.
Figure E4. A) Plasma and B) BAL soluble receptor for advanced glycation end-products
(sRAGE) baseline levels in patients with moderate or severe ARDS (based on Berlin
definition criteria). N = number of subjects. Values are reported as means ± standard
deviations and are analyzed with Kruskal-Wallis test (nonparametric data).
Figure E5. Baseline A) plasma, B) BAL soluble receptor for advanced glycation end-
products (sRAGE) baseline levels and C) alveolar fluid clearance (AFC) rates in 30-day
survivors (N=18) and non-survivors (N=12). Values are reported as means ± standard
deviations and are analyzed with Kruskal-Wallis test (nonparametric data).
15

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Figure E6. BAL proportions of polymorphonuclear neutrophils, mononuclear cells and
lymphocytes within the mouse model (n=4-6 for each time-point). There was no statistical
difference between groups and between time-points in injured mice.
16

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Table E1. Correlations between net alveolar fluid clearance (in % per hour) and
various clinical or biological markers of lung injury in both lung-injured mice and
patients with ARDS. Correlations have been tested with the calculation of Spearman’s rank
correlation coefficient ρ (rho). CI: confidence interval. * P<0.05.
Marker Spearman’s rank correlation coefficient
Patients with ARDS
Plasma sRAGE* ρ=-0.6 (95% CI, -0.79 to -0.29), P=0.0004
Alveolar sRAGE* ρ=-0.8 (95% CI, -0.91 to -0.63), P<0.0001
Lung injury score* ρ=-0.5 (95% CI, -0.75 to -0.19), P=0.03
PaO2/FiO2 ratio ρ=0.4 (95% CI, -0.02 to 0.64), P=0.06
Oxygenation index ρ=0.1 (95% CI, -0.25 to 0.48), P=0.49
Alveolar-to-plasma total protein ratio ρ=-0.3 (95% CI, -0.58 to 0.12), P=0.16
Static lung compliance ρ=0.2 (95% CI, -0.18 to 0.53), P=0.28
Inspiratory plateau pressure ρ=0.007 (95% CI, -0.36 to 0.38), P=0.97
Mouse model
Plasma sRAGE* ρ=-0.73 (95% CI, -0.88 to -0.45), P<0.001
Alveolar sRAGE* ρ=-0.69 (95% CI, -0.86 to -0.38), P<0.001
Histological lung injury score* ρ=-0.42 (95% CI, -0.71 to 0.009), P=0.04
PaO2/FiO2 ratio* ρ=0.46 (95% CI, 0.05 to 0.74), P=0.03
Permeability index* ρ=-0.43 (95% CI, -0.72 to -0.01), P=0.04
Plasma IL-6* ρ=-0.61 (95% CI, -0.82 to -0.26), P=0.002
Plasma TNF-α ρ=-0.11 (95% CI, -0.51 to 0.33), P=0.6
Plasma IL-17 ρ=-0.32 (95% CI, -0.65 to 0.12), P=0.1
Plasma MIP-2 ρ=-0.4 (95% CI, -0.70 to 0.03), P=0.06
Plasma KC ρ=-0.39 (95% CI, -0.70 to 0.03), P=0.06
Alveolar IL-6 ρ=-0.07 (95% CI, -0.48 to 0.37), P=0.8
Alveolar TNF-α ρ=-0.15 (95% CI, -0.54 to 0.29), P=0.5
Alveolar IL-17 ρ=0.21 (95% CI, -0.30 to 0.62), P=0.4
Alveolar MIP-2 ρ=-0.17 (95% CI, -0.55 to 0.27), P=0.4
Alveolar KC ρ=-0.40 (95% CI, -0.70 to 0.03), P=0.06
17

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Parameter 0 1 2
i. Neutrophils in the alveolar space None 1-5 >5
ii. Neutrophils in the interstitial space None 1-5 >5
iii. Hyaline membranes None 1 >1
iv. Proteinous debris filling the airspaces None 1 >1
v. Alveolar septal thickening None 2x-4x >4x
Table E2 – Lung injury scoring system (adapted from Matute-bello et al(E2)).
18

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188x236mm (300 x 300 DPI)

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263x360mm (300 x 300 DPI)

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246x482mm (300 x 300 DPI)

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244x431mm (300 x 300 DPI)

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130x63mm (300 x 300 DPI)

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AJRCCM Articles in Press. Published on 01-May-2015 as 10.1164/rccm.

Soluble RAGE Predicts Impaired Alveolar Fluid Clearance in Acute Respiratory

Matthieu Jabaudon1,2, Raiko Blondonnet1,2, Laurence Roszyk2,3, Damien Bouvier2,3, Jules

Audard1, Gael Clairefond2, Mathilde Fournier4, Geoffroy Marceau2,3, Pierre Déchelotte5,

Bruno Pereira6, Vincent Sapin2,3 and Jean-Michel Constantin1,2

and Perioperative Medicine, Estaing University Hospital, Clermont-Ferrand, France

Corresponding author and address for reprints:

Matthieu Jabaudon, M.D., M.Sc.

Department of Anesthesiology, Critical Care and Perioperative Medicine, Intensive Care

Unit, Estaing University Hospital

Clermont Université, Université d'Auvergne, EA 7281, R2D2, Clermont-Ferrand, France

Copyright © 2015 by the American Thoracic Society

CHU Clermont-Ferrand, 1 Place Lucie Aubrac, 63003 Clermont-Ferrand Cedex 1, France

AUTHORS CONTRIBUTIONS TO THE STUDY

the article and in its revision prior to submission.

revision of this article prior to submission.

revision of this article prior to submission.

revision of this article prior to submission.

Copyright © 2015 by the American Thoracic Society

writing the article and in its revision prior to submission.

(“Programme Nouveau Chercheur de la Région Auvergne” 2013), the french Agence

Nationale de la Recherche and the Direction Générale de l’Offre de Soins (“Programme de

Recherche Translationnelle en Santé” ANR-13-PRTS-0010) and from Clermont-Ferrand

Short Running Head: sRAGE predicts alveolar fluid clearance in ARDS.

Subject Code: 4.2 ALI/ARDS: Diagnosis & Clinical Issues

Body of Manuscript Word Count: 3053

Scientific Knowledge on the Subject

Copyright © 2015 by the American Thoracic Society

What This Study Adds to the Field

content online at www.atsjournals.org

Copyright © 2015 by the American Thoracic Society

has been investigated to date.

Objectives: To verify whether sRAGE could predict AFC during ARDS.

Methods: Anesthetized CD-1 mice underwent orotracheal instillation of hydrochloric acid. At

permeability, lung histology, AFC and plasma/bronchoalveolar fluid measurements of pro-

assessed in thirty patients with ARDS.

and lung injury resolved.

of RAGE axis in the mechanisms leading to edema resolution.

(Abstract word count: 228)

Copyright © 2015 by the American Thoracic Society

ClinicalTrials.gov Identifier: NCT00811629.

Copyright © 2015 by the American Thoracic Society

Acute respiratory distress syndrome (ARDS) is a major cause of acute respiratory

develop innovative diagnostic and therapeutic approaches in ARDS patients(9).

animal and clinical studies on ARDS.

We specifically designed an experimental and a clinical study to prospectively

Copyright © 2015 by the American Thoracic Society

communication during the conference “Congrès National de la Société Française

of Intensive Care Medicine (2014)(16, 17).

Additional details are provided in the online supplement.

to orotracheal instillation of hydrochloric acid(13). After a recovery period under humidified

oxygen, mice were transferred to stabulation.

end-expiratory pressure=6 cmH2O, respiratory rate=160 breaths.min-1 and FiO2=1), before

Copyright © 2015 by the American Thoracic Society

standardized histology score was calculated(18).

groups of maximal (≥ 14%/30min) or submaximal (≥ 3%/30min, < 14%/30min) AFC(3, 22).

Our institutional review board approved protocols.

30 patients enrolled within 24 h of ARDS onset(23) in a prospective observational study of

soluble forms and ligands of RAGE in ARDS.

Baseline lung computed tomography (CT)-scan was performed; nonfocal morphology

Copyright © 2015 by the American Thoracic Society

the correlations between sRAGE and continuous variables. Receiver-operating characteristic

sided) was considered significant.