original papers - Advances in Clinical and Experimental Medicine

original papers - Advances in Clinical and Experimental Medicine

original papers
Adv Clin Exp Med 2012, 21, 3, 321–329
ISSN 1899–5276
© Copyright by Wroclaw Medical University
Zehra Eren1, Jale Çoban2, Işın D. Ekinci3, Cigdem Kaspar4, Gülçin Kantarcı1
Evaluation of the Effects of a High Dose
of Erythropoietin-beta on Early Endotoxemia
Using a Rat Model
Ocena działania dużych dawek erytropoetyny beta
na wczesną endotoksemię na modelu szczurzym
1 Department
of Nephrology, Yeditepe University Hospital, İstanbul, Turkey
of Biochemistry, Yeditepe University Hospital, İstanbul, Turkey
3 Department of Pathology, Yeditepe University Hospital, İstanbul, Turkey
4 Department of Biostatistics, Yeditepe University Hospital, İstanbul, Turkey
2 Department
Abstract
Background. Endotoxins can cause serious organ damage and death by triggering the secretion of pro-inflammatory cytokines such as TNF-α, IL-6 and IL-1β in bacterial infections.
Objectives. The goal of this study was to evaluate the effects of a high dose (3000 U/kg) of erythropoietinbeta (EPO) on inflammatory cytokine levels, renal function and histological changes during the early period of
Lipopolysaccharide (LPS)-induced endotoxemia using a rat model.
Material and Methods. Male Sprague Dawley (350–400 g) rats were randomized into 3 groups: Control group
(n = 7); LPS group (received 20 mcg/kg LPS through intraperitoneal (i.p.) injection (n = 7); LPS+EPO group
(received 3000 U/kg, ip 30 minutes before LPS administration (n = 7). Four hours after the administration of
LPS, kidney tissue and serum samples were collected. Kidney function parameters, TNF-α, IL-6, IL-1β, C reactive
protein (CRP) and complete blood counts (CBC) were measured. The severity of renal tubular injury and caspase-9 immunoreactive cells was expressed as a percentage.
Results. Serum levels of urea, creatinine, TNF-α, IL-6 and IL-1β were significantly increased in the LPS group
(p < 0.0001 – p = 0.04) and were lower in LPS+EPO group (p < 0.0001, p = 0.01, p = 0.02, p = 01 and p < 0.0001,
respectively). Pretreatment with EPO significantly increased platelet counts (p = 0.00) and decreased white blood
cell counts (p = 0.02). The renal tubular injury percentage was significantly higher in the LPS group than in the
control and LPS+EPO groups (p = 0.002, p = 0.003, and p = 0.005, respectively) and caspase-9 expression was lower
in the LPS+EPO and control groups than in the LPS group.
Conclusions. EPO might have renoprotective effects against the inflammatory process and cell apoptosis during
endotoxemia (Adv Clin Exp Med 2012, 21, 3, 321–329).
Key words: erythropoietin, lipopolysaccharide, endotoxemia, acute kidney injury.
Streszczenie
Wprowadzenie. Endotoksyny mogą powodować poważne uszkodzenia narządów i zgon, wywołując wydzielanie
cytokin prozapalnych, takich jak TNF-a, IL-6 i IL-1b, w zakażeniach bakteryjnych.
Cel pracy. Ocena wpływu dużej dawki (3000 U/kg) erytropoetyny beta (EPO) na stężenie cytokin zapalnych, czynność nerek i zmiany histologiczne podczas wczesnej endotoksemii wywołanej przez lipopolisacharyd (LPS) na
modelu szczurzym.
Materiał i metody. Szczury szczepu Sprague Dawley (350–400 g) przydzielono losowo do 3 grup: grupy kontrolnej
(n = 7); grupy LPS (zwierzętom podano 20 mcg / kg LPS dootrzewnowo (ip) (n = 7)); grupę LPS + EPO (zwierzętom podano 3000 U / kg, ip 30 minut przed podaniem LPS (n = 7)). 4 godziny po podaniu LPS pobrano tkanki
nerek oraz próbki surowicy. Zmierzono wskaźniki czynności nerek, TNF-alfa, IL-6, IL-1b, białko C-reaktywne
(CRP) oraz morfologię krwi (CBC). Ciężkość uszkodzenia kanalików nerkowych i komórek immunoreaktywnej
kaspazy-9 wyrażono w procentach.
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Z. Eren et al.
Wyniki. Stężenie mocznika, kreatyniny, TNF-a, IL-6 i IL-1b było znacznie zwiększone w grupie LPS (p < 0,0001 –
p = 0,04) i było mniejsze w grupie LPS + EPO (p < 0,0001, p = 0,01; p = 0,02; p = 01 i p < 0,0001). Wstępne podanie
EPO znacznie zwiększyło liczbę płytek krwi (p = 0,00) i zmniejszyło liczbę białych krwinek (p = 0,02). Odsetek
uszkodzenia kanalików nerkowych był znacznie większy w grupie LPS niż w grupie kontrolnej i grupie LPS + EPO
(p = 0,002; p = 0,003 oraz p = 0,005), a ekspresja kaspazy-9 była mniejsza w grupie LPS + EPO i kontrolnej niż
w grupie LPS.
Wnioski. EPO może wywierać działanie ochronne przed procesem zapalnym i apoptozą komórek podczas endotoksemii (Adv Clin Exp Med 2012, 21, 3, 321–329).
Słowa kluczowe: erytropoetyna, lipopolisacharyd, endotoksemia, ostra niewydolność nerek.
Acute kidney injury (AKI) is a common and
often serious clinical problem caused by several
factors such as hypovolemia, nephrotoxins, drugs,
infections. Endotoxins are a leading cause of AKI
and can trigger devastating organ damage and
death [1]. Lipopolysaccharides (LPS) are major cell
wall constituents of Gram-negative bacteria and
are responsible for well known systemic responses
in cases of sepsis. Several inflammatory mediators
cause these responses, but their mechanism of action is not fully understood [2]. Although cytokines such as TNFα, IL-1 and IL-6 are key mediators
of sepsis, little is known about their acute effects on
kidneys in the context of endotoxemia [3].
Erythropoiesis stimulating agents (ESA) are
widely used in the treatment of anemia due to
chronic renal failure. Recent studies have suggested that erythropoietin (EPO), a multifunctional molecule produced by many tissues, has
anti-inflammatory and cell protective effects that
counter the tissue damaging process playing a considerable role in the regulation of inflammatory
processes triggered by injury [4, 5]. Several studies
have evaluated the tissue-protective role of EPO
analogs, such as erythropoietin-beta (EPO-beta),
in experimental models of kidney, brain, lung, and
liver injury [6–9] ESAs have shown renoprotective effects in nephrotoxicity induced by ischemiareperfusion injury, cisplatin, cyclosporine and radiocontrast models of acute kidney injury [10–15].
ESAs have also been studied in experimental models of LPS-induced endotoxemia. The pathogenesis
of endotoxemic AKI is not fully understood [16,
17]. During endotoxemia, a broad array of humoral mediators, including cytokines, are released into
the circulation [18]. Systemic hypotension and intrarenal vasoconstriction due to an imbalance of
vasodilatory and vasoconstrictory substances results in renal ischemia and contributes to AKI [19,
20]. Furthermore, inflammatory cells infiltrate the
kidney [21] and cause tubular damage through the
release of oxygen radicals, proteases, and additional inflammatory cytokines. Most evidence suggests
that, when produced locally in response to injury,
EPO has a negative effect on this process. However, pro-inflammatory cytokines suppress the local
production of EPO in a concentration-dependent
manner. The amount of local EPO necessary for
tissue-protective effects is much higher than that
needed for hormonal effects. In addition, EPO
expression can be delayed, reaching a peak 12 or
more hours after injury [22]. Therefore, the use
of recombinant human EPO may help to heal and
restore function after an injury by producing antiapoptotic proteins and suppressing pro-inflammatory cytokines.
In recent years, the effects of EPO in LPS-induced endotoxemia have been investigated through
studies that administered EPO at varying time
points and doses. Low doses of EPO (300 U/kg,
intravenously (i.v.)) before resuscitation were able
to eliminate renal dysfunction in hemorrhagic
shock but not in endotoxic shock induced by LPS
(6 mg/kg) in rats [15]. However, higher doses of
EPO (4000 U/kg, intraperitoneally (i.p.)) 30 minutes before the administration of a low dose of LPS
(2.5 mg/kg, i.p.) ameliorated renal dysfunction
during endotoxemia in mice [23]. In this murine
model of endotoxemia, the benefits of EPO were
due to both its antioxidant and anti-inflammatory
properties, rather than by effects on renal blood
flow or apoptosis. The subcutaneous administration of 1000 U/kg of EPO to mice improved survival when it was initiated at 30 min, 1 hour, or
2 hours after a lethal dose (10 mg/kg) of LPS. However, it had no rescuing effect if given 1 hour before
or 3, 4, or 16 hours after LPS administration [24].
Survival with EPO was associated with attenuated
apoptosis, reduced nitric oxide production and tissue hypoxia, but not with inflammatory changes.
Moreover, intravenous EPO administration at low
doses (300 U/kg) immediately before the injection of LPS (20 mg/kg, i.v.) increased the release of
TNF-α, IL-6 and IL-1 1 hour later [25].
This study aims to show the effect of a high
dose (3000 U/kg) of EPO-beta on inflammatory
cytokine levels, renal function and histological
changes during the early stage of LPS-induced endotoxemia in rats.
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Erythropoietin-beta in Early Endotoxemia
Material and Methods
Animals
The Animal Ethics Review Committee of
Yeditepe University (Istanbul, Turkey) approved
the experimental protocol. Male Sprague Dawley
rats (n = 21) reared at the Yeditepe University
Animal Research Center were used in the study.
The rats were housed at the Animal Center in
a controlled environment at a temperature of 22
± 1 °C with a 12-hour light/dark cycle. Food and
water were provided ad libitum. Animal use was in
accordance with the guidelines of European Council for animal care.
Endotoxemic Model
Endotoxemia was induced through the intraperitoneal injection 20 mg/kg of LPS (Echerichia coli
0128:B12, Sigma-Aldrich Chemical, St. Louis, MO,
USA) dissolved in 0.5 ml normal saline. The selection of a 20 mg/kg dose was based on preliminary
experiments using 0.6 mg/kg, 10 mg/kg, 15 mg/kg
and 20 mg/kg of LPS, renal and liver functions
were examined as indicators of organ damage.
The most significant change in serum urea and
alanine aminotransferase (ALT) values ​​occurred
at a dose of 20 mg/kg, and this dose was used to
induce endotoxemia. EPO-beta (3000 U/kg) was
administered through intraperitoneal injection
30 min before LPS administration. The EPO dose
was determined on the basis of previous publications [15, 23–25]. After the administration of the
endotoxin, the animals were continuously observed for 4 h and were then sacrificed.
The rats were divided into three groups: the
control group, was treated with saline (n = 7); the
LPS group was treated with 20 mg/kg intraperitoneal LPS and the LPS + EPO group was treated
with 3000 IU/kg EPO-beta (Roche, Turkey) 30 min
before LPS administration.
Measurement of Biochemical
Parameters
At the end of the experimental period, blood
samples were collected via decapitation. The
samples were centrifuged (4032 × g for 3 min) to
separate the serum. All samples were analyzed for
urea, creatinine (Cr), C-reactive protein (CRP)
and complete blood count (CBC) within 6 hours
following collection. The samples were analyzed
using commercially available clinical assay kits
with an autoanalyzer (COBAS Integra 400, Roche
Diagnostic) according to the manufacturers’ in-
structions. Urea and Cr concentrations were used
as indicators of renal function.
TNF-α, IL-6, and IL-1β
Measurement
Serum levels of TNF-α, IL-6 and IL-1β were
determined using enzyme-linked immunosorbent
assay (ELISA) kits (Rat Interleukin1-Beta ELISA
Kit, BioSource Europe, Belgium; Rat Interleukin
6 ELISA Kit, BenderMed Systems, UK; Tumor Necrosis Factor Alpha (TNF-α) ELISA Kit, Assaypro,
UK) with a DTX 880 miltimode detector (Becman
Counter, Nyon I, Switzerland) reader (Roche) and
according to the manufacturers’ instructions.
Histological Evaluation
The kidneys were removed immediately after
the animals were sacrificed (4 h after LPS administration) and submitted on Pathology Laboratory,
Yeditepe University. The tissues were fixed in 10%
buffered formalin for 24 h, and after routine tissue
processing, the tissues were embedded in paraffin. 3 serial sections of each kidney were stained
with hematoxylin and eosin for histopathological
evaluation. The severity of renal tubular injury was
scored by estimating the percentage of renal cortical or outer medulla tubules-including both distal
and proximal tubules – that showed epithelial necrosis, luminal necrotic debris, tubular dilatation,
and hemorrhage. Neutrophil infiltration was evaluated by counting the number of interstitial and
tubular neutrophils per each 10 high power field
(number of neutrophil/10 hpf; 1 hpf = the area that
is observed through 40 × 10 lens aperture of Olympus BX51 light microscope, GmbH-Germany).
Caspase-9 Immunohistochemical
Staining and Evaluation
For immunohistochemical evaluation, sections
were deparaffinized in xylene and dehydrated in
grading concentrations of ethyl alcohol.
Following deparaffinization, the slides were
boiled for 20 minutes in a 10 mM citrate buffer
(pH 6.0), cooled at room temperature for 20 minutes, and rinsed with distilled water. The slides
were immersed for 30 minutes in 0.3% hydrogen
peroxide diluted in methanol for endogenous peroxide inactivation and were then washed three
times in phosphate buffer saline (PBS, pH 7.4) at
room temperature. Subsequently, PBS containing
1% goat serum and 1% bovine serum albumin was
applied for 30 min to block non-specific binding.
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Z. Eren et al.
Next, caspase-9 (10 µg/ml, rabbit monoclonal antibody, Thermo Fisher Scientific, Fremont, CA)
was applied for 30 min at room temperature. After washing in PBS, peroxidase activity was localized with 3,3’-diaminobenzidine (DAB; DAKO
Liquid DAB-Substrate-chromogen K-3466, CA,
USA) and 0.03% hydrogen peroxide. Sections
were counter-stained with hematoxylin, cleaned
and mounted. Negative control studies were performed concurrently in the absence of the primary
antibody. Positive control studies were also performed simultaneously in sections of human tonsil tissue. Brown staining in cytoplasm, regardless
of intensity or extension was considered “positive”
and no staining was considered “negative” for caspase-9. The number of both negative and positive
cells was counted on the slides. For each slide, the
“percent caspase-9 expression” was calculated by
dividing the number of positive cells by the total
number of cells and multiplying by 100.
Statistical Analysis
Data is presented as mean ± SD. ANOVA and
Bonferroni’s tests were used to compare the means
of continuous variables that were normally distributed and had equal variances. Welch and GamesHowel tests were used to compare the means of
continuous variables that were normally distributed and did not have equal variances. The KruskalWallis and Mann-Whitney U tests were used to
compare the medians of non-normally distributed
continuous variables. Two-tailed p-values less
than 0.05 were considered statistically significant.
Statistical analyses were performed using SPSS
version 19.0 (SPSS Inc., Chicago, Ill., USA) Results
Effect of EPO-beta on Renal
Functions After Endotoxemia
Endotoxemia increased plasma urea and creatinine at 4 h. In LPS group plasma levels of urea
and creatinine were significantly higher when compared to control group (p < 0.0001 and p = 0.01,
respectively). Pretreatment with 3000 U/kg of
EPO-beta may prevent a rise in plasma urea and
creatinine levels. When compared with LPS goup,
LPS+EPO group had significantly lower levels
of urea and creatinine (95%Confidence Interval
(CI): 28.51÷63.15, p < 0.0001; 95%CI: 0.04÷0.43,
p = 0.01, respectively) (Table 1).
Effect of EPO-beta on CRP
After Endotoxemia
There was no difference in serum CRP levels
during the first 4 h of endotoxemia. Compared
with the control group, no significant changes in
serum CRP levels were observed in the LPS group
(p = 0.78) (Table 1).
Effect of EPO-beta on
Hemoglobin (Hb), White Blood
Cells (WBC) and Platelets (PLT)
After Endotoxemia
Pretreatment with 3000 IU/kg EPO-beta significantly increased platelet counts and decreased
Table 1. Laboratory findings of study groups
Tabela 1. Badania laboratoryjne w grupach
Control group
(Grupa kontrolna)
(n = 7)
LPS group
(Grupa LPS)
(n = 7)
LPS + EPO group
(Grupa LPS + EPO)
(n = 7)
P value
(Istotność statystyczna)
P*
P**
Urea (mmol/L)
103.21 ± 12.04
255.66 ± 3.46
126.42 ± 3.72
< 0.0001
< 0.0001
Creatinine (µmol/L)
29.04 ± 2.64
51.04 ± 15.84
29.92 ± 7.04
0.01
0.01
CRP (mg/dl)
0.23 ± 0.07
0.26 ± 0.07
0.29 ± 0.04
0.78
0.67
WBC (× 104/µL)
9.78 ± 2.03
12.83 ± 3.63
7.94 ± 2.43
0.21
0.02
Hb (g/dl)
13.24 ± 1.68
13.75 ± 1.57
14.78 ± 0.44
0.80
0.39
PLT (× 104/µL)
3.16 ± 1.12
5.16 ± 2.02
9.56 ± 2.43
0.25
0.005
Data is presented as mean ± SD. P*: between control and LPS group, p**: between LPS and LPS+EPO group, CRP: C reactive protein, WBC: white blood cells, Hb: Hemoglobin, PLT: Platelet.
Dane przedstawiono jako średnie ± SD. P*: między grupą kontrolną i LPS, p**: między LPS i LPS + EPO, CRP – białko
C-reaktywne, WBC – krwinki białe, HB – hemoglobina, PLT – płytki krwi.
325
Erythropoietin-beta in Early Endotoxemia
Effect of EPO-beta on Tubular
Injury After Endotoxemia
white blood cell counts. Compared with the LPS
group, the LPS+EPO group had significantly decreased white blood cell counts (95%CI: 0.44÷9.33,
p = 0.02). Platelet counts were significantly higher
in the EPO+LPS group than in the control or LPS
groups (p < 0.0001 and p = 0.005, respectively).
LPS and EPO application did not cause significant changes in hemoglobin levels during the first
4 h of endotoxemia (Table 1).
Compared with the LPS group, the LPS+EPO
group had significantly decreased tubular epithelial necrosis, tubular dilatation and hemorrhage
in the kidney cortex or outer medulla during the
early stages of LPS-induced endotoxemia (95%CI:
16.01÷55.66, p = 0.01) (Fig. 2).
Effect of EPO-beta on TNF-α,
IL-6, and IL-1β Levels After
Endotoxemia
Effect of EPO-beta on
Neutrophil Infiltration of the
Kidney After Endotoxemia
Plasma concentrations of TNF-α, IL-6, and
IL-1β increased significantly 4 h after LPS administration (p = 0.001, p < 0.0001, and p < 0.0001,
respectively). Pretreatment with 3000 U/kg of
EPO-beta may prevent a rise in inflammatory
cytokines. Compared with the LPS group, the
LPS+EPO group had significantly decreased levels of TNF-α, IL-6, and IL-1β (95%CI: 0.46÷8.12,
p = 0.02; 95%CI: 8.39÷19.50, p < 0.0001; 95%CI:
2.98÷9.35, p = 0.0001, respectively) (Fig. 1).
In LPS administered group the number of
neutrophils detected in the kidney was increase
when compared with control group, but this increase was not statistically significant (p = 0.42).
Although the number of neutrophils was lower
in LPS+EPO group than in the LPS group but the
difference did not reach statistical significance
(p = 0.11).
Fig. 1. Changes in serum TNF-α, IL-6, IL-1β at 4 h after LPS administration
*LPS group vs. Control group: 95%CI: 3.02÷11.05, p = 0.001; 95%CI: 18.93÷30.58, p < 0.0001; 95%CI: 4.54÷11.21,
p < 0.0001, respectively
#LPS group vs. LPS+EPO group: 95%CI: 0.46÷8.12, p = 0.02; 95%CI: 8.39÷19.50, p = 0.001; 95%CI: 2.98÷9.35,
p < 0.0001, respectively
Ryc. 1. Zmiany TNF-alfa, IL-6, IL-1b w surowicy 4 godziny po podaniu LPS
* grupa LPS vs grupa kontrolna: 95% CI: 3,02 ÷ 11,05, p = 0,001; 95% CI: 18,93 ÷ 30,58, p < 0,0001; 95% CI: 4,54 ÷
11,21, p < 0,0001;
# grupa LPS vs grupa LPS + EPO: 95% CI: 0,46 ÷ 8,12, p = 0,02; 95% CI: 8,39 ÷ 19,50; p = 0,001; 95% CI: 2,98 ÷ 9,35;
p < 0,0001
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Z. Eren et al.
Fig. 2. Histopathologic changes in kidney after LPS. A–C: Corresponding hematoxylin and eosin stained kidney sections of control (A), LPS (B) and LPS+EPO (C) groups. Control (A) and LPS+EPO (C) groups showed minimal or no
histopathologic alteration related to tubular injury. In LPS (B) group mild tubular vacuolization could be seen (H&E,
×200). D–F: Representative kidney sections of control (D), LPS (E) and LPS+EPO (F) groups showing cytoplasmic
immunohistochemical expression of Caspase-9 in renal tubulary epithelial cells of cortical and outer medullary
tubules (×200)
Ryc. 2. Zmiany histopatologiczne w tkankach nerek po podaniu LPS. Wycinki tkanki nerek zabarwione hematoksyliną i eozyną A) grupy kontrolnej, B) grupy LPS i C) grupy LPS + EPO. W grupie kontrolnej (A) i LPS + EPO (C) zaobserwowano minimalne histopatologiczne zmiany związane z uszkodzeniem kanalików lub ich brak. W grupie LPS (B)
widać łagodną wakuolizację kanalików (H & E, × 200). Reprezentatywne wycinki tkanki nerek D) grupy kontrolnej, E)
grupy LPS i F) grupy LPS + EPO pokazują cytoplazmatyczną immunohistochemiczną ekspresję kaspazy-9 w komórkach nabłonkowych kanalików części zewnętrznej i rdzennej nerek
Effect of EPO-beta on
Immunohistochemical Expression of
Caspase-9 as a Marker of Apoptosis
After Endotoxemia
The expression of caspase-9 was increased
in the kidney of rat with LPS-induced endotoxemia and was reduced the kidney of rat pretreated
with EPO. Compared with the control group, the
kidneys of rats in the LPS group showed a significantly higher caspase-9 positive cell percentage (95%CI: 3.29÷40.38, p = 0.01). Pretreatment
with 3000 U/kg EPO-beta significantly decreased
the proportion of caspase-9 positive cells (95%CI:
0.65÷36.02, p = 0.04) during the first 4 h of endotoxemia (Fig. 2).
Discussion
This study shows that a high dose of EPO-beta
(3000 U/kg) may have protective effects on both
the function and histological appearance of kidneys during the early stages of LPS-induced endotoxemia in rats. The beneficial effects of EPO-beta
administration on renal function were measured
by serum urea; the effects on tubular histological
changes were assessed with hematoxylin and eosin
staining. These protective effects were associated
with a reduction in inflammatory cytokines such
as TNF-α, IL-1β and IL-6 as well as prevention of
activation of apoptotic pathways secondary to the
reduction of caspase-9 activation.
There are several known properties of EPO
that may be involved in the renoprotective effects
observed in this study. The molecular mechanisms
underlying the tissue protective effect of exogenous EPO are still not completely understood,
and only a few components have been identified
or common pathways proposed [22, 26]. The best
known property of EPO is its ability to bind to the
erythropoietin receptor (EPOR) that is expressed
in the kidney by endothelial cells, mesangial cells,
and the epithelial cells of the tubule and collecting
duct [27]. When bound to the EPOR, EPO activates the receptors associated with Janus kinase
2 (JAK 2), leading to tyrosine phosphorylation of
the EPOR [28]. Erythropoietin signals through the
phosphatidylinositol 3-kinase (PI3K)/Akt signal
transducer and activates transcription 5 (STAT5),
mitogen-activated protein kinase (MAPK) and
protein kinase C pathways; these pathways serve
as antiapoptotic mediators of EPO-induced cytoprotection.
The most probable renoprotective effect of
EPO is its ability to reduce or prevent cell death.
Erythropoietin-beta in Early Endotoxemia
The present study showed that the renoprotective
effects of single, high dose (3000 U/kg) of EPObeta are probably related to its ability to reduce tubular epithelial necrosis, tubular dilatation, luminal necrotic debris and hemorrhage in the kidney
cortex or outer medulla during the early stages of
LPS-induced endotoxemia. Moreover, immunohistochemical expression of caspase-9 showed that
the caspase cascade was inhibited by preventing
the mitochondrial pathway; a significant decrease
in the caspase-9 positive cell count was observed
during the first 4 h of endotoxemia.
The protective effects of EPO have been demonstrated in several experimental models [7, 9, 13,
29]. Most notably, the ability of EPO to protect
kidneys from ischemia-reperfusion injury (IRI)
has been shown in various animal studies [11, 12].
Although recent studies [13, 20–22] did not show
a beneficial effect of EPO on histological findings
and apoptosis, in the present study the authors find
a positive effect of EPO during endotoxic injury.
The different doses of EPO used in these experimental models may explain the observed discrepancies. Three earlier studies used much lower doses of
EPO (up to 100 U/kg) than the amount used in this
study [15, 24, 25]. In addition, one earlier study [15]
that used 4000 U/kg EPO only detected TUNEL
positive apoptotic cells and did not examine tubular
injury after LPS application. Thus, the authors suggest that the renoprotective effects of EPO during
early endotoxemia are dose-dependent.
The protective effects of EPO against endotoxemia may involve a reduction in pro-inflammatory cytokines such as TNF-α, IL-6, IL-1β.
Some models of renal ischemia-reperfusion injury
(IRI) [11, 12] have shown that EPO reduces various cytokines, but models of endotoxemia have
been unable to demonstrate this effect [15, 24, 25].
Earlier studies have shown that TNF-α is detectable in serum during the first 4 h of endotoxemia
and peaks at 2 h [31]; other studies measured inflammatory cytokines at 1 h [25], 16 h [13] and
an unknown time [24] after LPS administration.
This study showed that plasma concentrations of
TNF-α, IL-6, IL-1β increased significantly 4 h after
LPS administration, and pretreatment with a high
dose (3000 U/kg) of EPO-beta may reduce rise in
inflammatory cytokines.
327
No difference was detected in serum CRP levels during the first 4 h of endotoxemia in present
experiment. A recent study reported that CRP increased at 6 h of endotoxemia and that EPO administration further enhanced its rise during this
acute phase [23]. This result is intriguing because
another experiment of transgenic mice showed that
CRP has a protective effect by increasing cytokine
binding [31] during endotoxemia. Current results
suggest that this mechanism of protection may be
more effective in a later stage of endotoxemia.
In the present study, platelet counts were significantly higher in the EPO group, but there was
no difference in hemoglobin levels or red blood cell
counts among the groups. Therefore, the authors
suggest that the renoprotective effect of EPO in
early endotoxemia is not due to the mitigation of
ischemia via the correction of anemia, increase in
the red blood cell mass and subsequent increase in
the oxygen-carrying capacity of the blood. Many
clinical and experimental studies indicate that ESAs
augment thrombopoeisis and that a high dose of
EPO increases intracranial hemorrhage and mortality rates [32]. The fact that a high dose of EPObeta, even when administered early, leads to an
increased platelet count and a potential increase in
the risk of thromboembolic events may limit its use
as a renoprotective agent in cases of sepsis.
One limitation of this study is the high dose of
LPS used in the experiment. This dose was determined based on preliminary studies and titrated
to cause more significant increases in liver and
kidney function markers. The selected model may
reflect serious clinical conditions characterized by
rapid deterioration of organ functions and the role
of EPO as rescue therapy. A second limitation is
the short observation time; nevertheless, the study
was designed to examine the effects of EPO during
the early stages of LPS-induced endotoxemia.
In summary, this study suggests that a high
dose of EPO-beta may have protective effects during the early stages of LPS-induced endotoxemia.
Further studies with varying doses and observations times can further establish the renoprotective effects of ESAs and define the range of possible clinical applications.
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Erythropoietin-beta in Early Endotoxemia
Address for correspondence:
Zehra Eren
Yeditepe University Hospital
Devlet Yolu Ankara Cad.No:102/104
34752, Kozyatağı, Istanbul
Turkey
Phone: +902165784096
E-mail: [email protected]
Conflict of interest: none declared
Received: 6.12.2011
Revised: 4.01.2012
Accepted: 6.06.2012
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