Clinical Biochemistry 44 (2011) 601–604

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Clinical Biochemistry
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c l i n b i o c h e m

Increased levels of soluble receptor for advanced glycation end products (sRAGE) and
high mobility group box 1 (HMGB1) are associated with death in patients with acute
respiratory distress syndrome
Tsukasa Nakamura a, Eiichi Sato a, Nobuharu Fujiwara a, Yasuhiro Kawagoe a,
Sayaka Maeda b, Sho-ichi Yamagishi b,⁎
a
Division of Nephrology, Department of Internal Medicine, Shinmatsudo Central General Hospital, Matsudo 270-0034, Japan
b
Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, Kurume 830-0011, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Objectives: Receptor for advanced glycation end products (RAGE) plays a role in inflammatory reactions.
Received 3 October 2010 Soluble RAGE (sRAGE) level is elevated in patients with acute respiratory distress syndrome (ARDS).
Received in revised form 23 December 2010 However, which clinical parameters and inflammatory biomarkers including sRAGE are associated with death
Accepted 24 December 2010 in ARDS patients remain unknown.
Available online 3 January 2011
Design and methods: We examined whether sRAGE level was independently associated with death in 20
ARDS patients with severe infection.
Keywords:
ARDS
Results: Compared with age- and sex-matched control subjects, blood pressure levels were lower and KL-
sRAGE 6, high mobility group box 1 (HMGB1), interleukin-6 and sRAGE levels were higher in ARDS patients. In
HMGB1 multivariate analysis, sRAGE was associated with death in ARDS patients, but severity of illness was not.
HMGB1 was a sole independent correlate of sRAGE.
Conclusions: This study demonstrated that sRAGE was independently associated with death in ARDS
patients. Our present results suggest active involvement of HMGB1–RAGE axis in poor prognosis of ARDS.
© 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Introduction has been implicated as a putative danger signal in a variety of

Acute respiratory distress syndrome (ARDS), the most severe
form of acute lung injury, is a major cause of rapid-onset respiratory
failure in critically ill patients, which could account for the high
disability rate in these subjects [1,2]. A variety of injurious insults
could elicit diffuse alveolar damage, which is followed by alveolar
edema and interstitial fibrosis [3]. Although the etiology of ARDS is
multifactorial, uncontrolled inflammation has recently been consid-
ered to play a central role in the pathogenesis of this devastating
disorder [4,5].
There is a growing body of evidence that receptor for advanced
glycation end products (RAGE) is mainly involved in inflammatory
reactions in various disorders such as atherosclerotic cardiovascular
disease, rheumatoid arthritis, and acute lung injury [6–9]. Further,
high mobility group box-1 (HMGB1), one of the ligands for RAGE,
Abbreviations: ARDS, acute respiratory distress syndrome; RAGE, receptor for advanced
glycation end products; HMGB1, high mobility group box 1; sRAGE, soluble RAGE; BAL,
bronchoalveolar lavage; BP, blood pressure; APACHE, Acute Physiology and Chronic Health
Evaluation; ELISA, enzyme-linked immunosorbent assay; IL-6, interleukin-6; SD, standard
deviation.
⁎ Corresponding author. Fax: +81 942 31 7873.
E-mail address: shoichi@med.kurume-u.ac.jp (S. Yamagishi).
inflammatory conditions as well [10–12]. HMGB1–RAGE interaction
promotes chemotaxis and maturation of immune cells, enhances the
expression of adhesion molecules in endothelial cells, and stimulates
the production of cytokines by various types of cells, thereby
evoking local and systemic inflammation [10–12].
Recently, HMGB1 was found to be a late mediator of endotoxin-
induced acute lung injury in mice [13], and serum levels of HMGB1
were increased in septic shock patients and positively associated with
sepsis-related organ failure assessment score [14]. In addition,
blockade of the HMGB1 and RAGE axis was reported to ameliorate
acute pulmonary inflammatory responses after endotoxin instillation,
including lung edema with neutrophil accumulation and increased
production of cytokines [13–16].
Moreover, soluble RAGE (sRAGE) has been identified in human
plasma [17–19]. RAGE is expressed in alveolar type 1 cells and most
abundant in the lung [20,21]. In endotoxin-induced acute lung injury
mouse models, sRAGE levels in bronchoalveolar lavage (BAL) fluid
and serum were elevated and correlated with the severity of lung
damage [20,21]. These observations suggest that serum level of sRAGE
is a biomarker of severity and clinical outcomes of ARDS subjects.
Therefore, in this study, we examined whether sRAGE level was
independently associated with the severity of lung injury and/or
death in 20 ARDS patients with severe infection. We further examined

0009-9120/$ – see front matter © 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.clinbiochem.2010.12.014
602 T. Nakamura et al. / Clinical Biochemistry 44 (2011) 601–604

which clinical parameters and inflammatory variables could be Table 2

independent correlates of elevated sRAGE level in ARDS subjects. Characteristics of survivors and non-survivors of ARDS patients.

Characteristics Survivors Non-survivors p-Value

Age (years) 67.2 ± 10.5 62.1 ± 12.1 –

Patients and methods Sex (M/F) 5/5 7/3 –
SBP (mm Hg) 114.0 ± 9.3 108.8 ± 7.6 0.17
Twenty ARDS patients with severe infection (12 males and DBP (mm Hg) 69.6 ± 7.5 67.8 ± 6.9 0.38
Heart rate (bpm) 94.6 ± 5.7 96.2 ± 9.6 0.76
8 females, mean age; 64.6 years (pneumonia n = 12, pyelonephritis
APACHE II 19.9 ± 2.2 20.5 ± 1.9 0.52
n = 2, peritonitis n = 2, pancreatitis n = 1, cholangitis n = 1, colitis Lung injury score 2.1 ± 0.4 2.4 ± 0.5 0.25
n = 1, unknown origin n = 1) and 20 age-matched healthy volunteers KL-6 (U/mL) 395.0 ± 73.1 413.0 ± 130.4 0.97
(11 males and 9 females, mean age; 63.9 years) were included in this HMGB1 (ng/mL) 1.8 ± 0.9 2.7 ± 0.8 0.012
study. Research Ethics Committee of our hospital approved this study IL-6 (pg/mL) 934.0 ± 879.0 1521.0 ± 781.4 0.69
sRAGE (pg/mL) 1796.8 ± 418.5 2230.5 ± 287.3 0.002
protocol, and informed consent was obtained from all the alert
Endotoxin (pg/mL) 1.4 ± 0.3 1.4 ± 0.5 0.68
subjects or first-degree relatives of drowsy patients. The criteria of
American European Consensus Conference were used for the
diagnosis of ARDS [22]. Patients with malignancy, ischemic heart
disease, alcohol abuse, tuberculosis, collagen disease and liver disease Results
were excluded from the study.
At admission, blood pressure (BP) was measured in supine Table 1 shows the clinical and laboratory data of ARDS patients and
position and blood was drawn from the antecubital vein for age- and sex-matched healthy volunteers. Systolic BP (SBP) and
determinations of biochemical variables. Severity of ARDS was diastolic BP (DBP) were significantly lower and heart rate was higher
evaluated clinically by both Acute Physiology and Chronic Health in ARDS patients than those in healthy volunteers (p b 0.01). Serum
Evaluation (APACHE) II score and lung injury score [23,24]. Serum levels of KL-6, HMGB1, IL-6, HMGB1, sRAGE, and endotoxin were
level of KL-6, a lung epithelial cell injury marker [25], was measured significantly elevated in ARDS patients compared with healthy
by a commercially available kit (electrochemiluminescence immu- volunteers. APACHE II score and lung injury score in ARDS subjects
noassay) (Eizai, Tokyo, Japan). Serum HMGB1 level was measured were 20.2 and 2.24, respectively. As shown in Table 2, serum levels of
with an enzyme-linked immunosorbent assay (ELISA) as described HMGB1 and sRAGE were significantly higher in non-survivors of ARDS
previously [26]. Validated inter-assay and intra-assay coefficients of patients compared with survivors, although there were no significant
variation were b 10%, and detection limit of HMGB1 was 0.3 ng/mL. differences of other clinical parameters and inflammatory biomarkers
Interleukin-6 (IL-6) and sRAGE levels were determined with between the two groups. In the univariate analysis, HMGB1 (p b 0.05)
commercially available ELISA kits (R&D systems, Minneapolis, MN, and sRAGE (p b 0.01) were positively associated with death in ARDS
USA) [27,28]. Inter-assay and intra-assay coefficient of variations of patients (Table 3). Because the parameters could be closely correlated
sRAGE were 7.7% and 5.7%, respectively [28]. Serum endotoxin level with each other, to determine independent determinants of death,
was determined by a turbidimetric time assay using an ET-2000 multiple stepwise regression analysis was performed. This analysis
toxinometer (Wako, Tokyo, Japan). Each sample was stored at showed that sRAGE was independently correlated to death in ARDS
−80 °C and analyzed in a single analytical run. All patients received patients (R2 = 0.289) (Table 3). Further, multiple stepwise regression
intensive therapy for ARDS such as mechanical ventilation, direct analysis revealed that HMGB1 was a sole independent correlate of
hemoperfusion with polymyxin B-immobilized fiber, vasopressor sRAGE in ARDS patients (R2 = 0.813) (Table 4).
agents and antibiotics, and followed until death or 28 days. Data are
expressed as mean ± standard deviation (SD). Mann–Whitney's Discussion
U test was performed for comparison of the parameters between
ARDS patients and healthy volunteers and between survivors and In this study, we demonstrated for the first time that sRAGE and
non-survivors. Univariate and multivariate stepwise regression HMGB1 levels were correlated with each other and HMGB1 was a sole
analysis was performed for correlates of death and sRAGE levels. independent correlate of sRAGE in ARDS patients with severe
Statistical significance was defined as p b 0.05. All statistical analyses infection. Furthermore, we found here that baseline sRAGE level
were performed with the use of the SPSS system (SPSS Japan Inc., was elevated in non-survivors compared with survivors and
IBM company, Tokyo, Japan).

Table 3
Univariate and multivariate analyses for determinants of death.
Table 1
Characteristics of the patients. Univariate Multivariate

Characteristics ARDS Control p-Value Characteristics β SE p-Value β p-Value

Age (years) 64.7 ± 11.3 63.9 ± 5.5 – Age 0.232 0.010 0.326
Sex (M/F) 12/8 11/9 – Sex 0.204 0.235 0.388
SBP (mm Hg) 111.4 ± 8.7 129.7 ± 4.2 p b 0.01 SBP 0.307 0.013 0.188
DBP (mm Hg) 68.7 ± 7.1 75.9 ± 3.1 p b 0.01 DBP -0.131 0.017 0.582
Heart rate (bpm) 95.4 ± 7.7 73.7 ± 4.0 p b 0.01 Heart rate 0.106 0.016 0.656
APACHE II 20.2 ± 2.0 – – APACHEII 0.153 0.059 0.520
Lung injury score 2.24 ± 0.45 – – Lung injury score 0.287 0.259 0.221
KL-6 (U/mL) 404.0 ± 103.0 151.5 ± 44.0 p b 0.01 KL-6 0.089 0.001 0.708
HMGB1 (ng/mL) 2.3 ± 0.94 n.d. – IL-6 0.349 0.000 0.132
IL-6 (pg/mL) 1227.5 ± 863.6 2.4 ± 0.8 p b 0.01 sRAGE 0.537 0.000 0.015 0.537 0.015
sRAGE (pg/mL) 2013.7 ± 414.2 410.1 ± 188.7 p b 0.01 HMGB1 0.506 0.111 0.023
Endotoxin (pg/mL) 1.4 ± 0.4 n.d. – Endotoxin -0.025 0.294 0.917

SBP, systolic blood pressure; DBP, diastolic blood pressure; bpm, beats/minutes, β: Regression coefficients. Female = 0, male = 1.
HMGB1, high mobility group box-1; IL-6, interleukin-6; sRAGE, soluble RAGE. SE: standard error.
n.d., Not detectable. R2 = 0.289.
 T. Nakamura et al. / Clinical Biochemistry 44 (2011) 601–604 603

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