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Introduction
Renal ischemia/reperfusion (I/R)-induced tubular epithelial cell
injury[1], called ischemic acute renal failure, is
associated with high mortality in
humans[2]. Protecting the kidney against I/R injury is very important during complicated renal
operations and anesthesia.
Propofol, a highly lipid-soluble anesthetic, is widely used for the induction and maintenance of general anesthesia, as well
as for intubated post-operation sedation for mechanically-ventilated adults in the intensive care unit. Propofol has been
proven to ameliorate I/R injury in several organs,
including the heart[3],
lungs[4], brain[5],
liver[6], and testicles[7]. Propofol can
also limit oxidative injury in various tissues, including the
kidneys[8]. However, the effects of propofol on renal I/R injury
have been rarely reported.
Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin, carbon monoxide, and free ferrous iron; the latter
is rapidly converted to bilirubin by biliverdin
reductase[9]. Among the 3 isoforms HO-1 is a ubiquitous and redox-sensitive
inducible stress protein that is strongly induced by various
stimuli, including heme, heavy metal, cytokines, hormones,
endotoxins, heat shock, and I/R injury, while HO-2 is the
constitutive form[10]. HO-3 has been identified, but its
function is still unknown[11]. Furthermore, HO-1 has been shown
to have protective effects against I/R injury. HO-1 induction
in donor organs of rats has been shown to ease I/R injury,
prolong graft survival, and improve the long-term function
of the grafted kidney[12]. It has also been reported that the
clipping of the renal artery in HO-1-deficient mice leads to
the exacerbation of renal damage and
death[13]. Recently, Acquaviva et al
indicated that propofol attenuated peroxynitrite-mediated DNA damage and apoptosis by the
upregulation of HO-1 expression in cultured
astrocytes[14].
Based on these data, it is reasonable for us to
hypothesize that propofol can inhibit renal I/R injury, and this effect
is partly mediated by HO-1.
Materials and methods
Materials Propofol was obtained from Astrazeneca
Pharmaceuticals (Milan, Italy). Polyclonal rabbit anti-rat HO-1
antibody and polyclonal rabbit anti-rat actin antibody were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA,
USA). Secondary horseradish peroxidase-conjugated goat
anti-rabbit IgG was purchased from Shanghai Sangon
Biotechnology (Shanghai, China). Peroxidase-conjugated goat
anti-rabbit secondary antibody was from Dako (Carpinteria,
CA, USA). The One-step RNA PCR kit (Avian
Myeloblastosis Virus, AMV) was obtained from TaKaRa Biotechnology
(Otsu, Japan). Male Sprague_Dawley rats (200_250 g, Grade
II) were from Zhejiang Academy of Medical Sciences
(Hangzhou, China).
Experimental grouping This study was approved by
our institutional animal research committee and conformed
to the Guide for the Care and Use of Laboratory Animals
published by the United States National Institutes of Health
(NIH publication No 85-23, revised 1996). Male
Sprague-Dawley rats were randomly divided into 3 groups as follows:
(i) sham-operated group, which underwent isolation of both
renal pedicles without occlusions and received saline (2 mL/h,
iv; n=5); (ii) I/R group, which was subjected to bilateral
renal ischemia followed by reperfusion and received saline
(2 mL/h, iv; n=15); and (iii) propofol group, which was
subjected to bilateral renal I/R and was treated with 10 mg/kg
propofol followed by infusion at 20
mg·kg-1·h-1, iv
(n=15). The treatments were given from 30 min before renal ischemia
to 30 min after reperfusion. The dose of propofol was
selected based on published
reports[15,16].
Induction of renal I/R and tissue preparation
The rats were anesthetized with ketamine (50 mg/kg, ip). Body
temperature was maintained at 37 °C by a heating lamp until the
rats recovered from the anesthesia. The tail vein was
cannulated for fluid or drug administration. Bilateral renal ischemia
was induced by occlusion of both renal pedicles using
non-traumatic microvascular clips. After 45 min ischemia, the
clips were removed allowing the kidneys to reperfuse.
Occlusion was verified visually by a change in the color of the
kidneys to a paler shade, and reperfusion by a
blush[17]. After 2, 6, and 24 h reperfusion, the rats of the I/R group and the
propofol group were anesthetized with ketamine (50 mg/kg,
ip). The chest and peritoneal cavities were opened carefully
and blood was drawn from the heart for the measurement of
blood urea nitrogen (BUN) and serum creatinine (SCr) levels.
The kidneys were removed and cut into 3 parts; 1 part was
fixed with 12% formaldehyde for histological and
immunohistochemical analysis and the others were frozen at -80 °C
for RNA and protein isolation. The samples of the
sham-operated rats were collected only 2 h after the sham operation.
Histology The kidneys were embedded in paraffin, serial
sectioned (3_4 µm thick) and stained with
HE[18]. The slides were reviewed blindly and scored with a semiquantitative
scale evaluating morphological characteristics of the tubules
by Paller's standard. Specifically, for each kidney, 100
cortical tubules from at least 10 different areas were scored. Care
was taken to avoid repeated scoring of different
convolutions of the same tubule. Higher scores represented more
severe damage (maximum score per tubule was 10), with
points given for the presence and extent of tubular epithelial
cell flattening (1 point), brush border loss (1 point), cell
membrane bleb formation (1 or 2 points), interstitial edema
(1 point), cytoplasmic vacuolization (1 point), cell
necrosis (1 or 2 points), and tubular lumen obstruction (1 or 2
points)[19,20].
Immunohistochemistry The kidney tissue sections were
hybridized with rabbit anti-rat HO-1 antibody (dilution 1:60)
at 4 °C overnight. After washing, the sections were overlaid
with peroxidase-conjugated goat anti-rabbit secondary
antibody at 37 °C for 30 min. The negative control was prepared
by PBS of the primary antibody. By counting the number of
positive-stained kidney cells in 5 high-power (×400) fields
per case, we determined the positive rate of HO-1. The
standards for the HO-1 quantification was as follows: 0%, 0 point;
<25%, 1 point; 25%_50%, 2 points; 50%_75%, 3 points; and
>75%, 4 points.
RT-PCR The total RNA was extracted by the TRIzol
reagent (Invitrogen, Carlsbad, CA, USA). Briefly, cDNA
was prepared from 1 µg total RNA using the One-step RNA
PCR kit (AMV). The primer sequences were as follows:
HO-1 sense, 5'-ACT GCT GAC AGA GGA ACA CAA A-3'; HO-1
antisense, 5'-CAA CAG GAA ACT GAG TGT GAG G-3'; GAPDH sense, 5'-AAG GTC GGA GTC AAC GGA TTT-3';
and GAPDH antisense, 5'-AGA TGA TGA CCC TTT TGG CTC-3'.
GAPDH was used as a loading control. The
amplification cycle was 95 °C for 1 min, 55 °C for 1 min, and 72 °C for
1 min, repeated for 30 cycles. RT-PCR products 8 μL were
separated by electrophoresis on 1.8% agarose gel
containing ethidium bromide 0.5 μg/mL (181 bp for HO-1 and 352 bp
for GAPDH).
Western blot analysis The kidney tissues were
homogenized in 10 mL homogenization buffer (20 mmol/L Tris, 5
mmol/L EDTA, 150 mmol/L NaCl, 1 mmol/L phenyl-methanesulfonyl fluoride) and centrifuged at
12 000×g at
4 °C for 10 min. The resulting supernatant was mixed with
loading buffer and boiled for 3 min. The
samples containing 50 µg protein were separated by 12% SDS-PAGE and
transferred to nitrocellulose membranes. The membranes were
blocked with 5% non-fat dry milk in TBST solution at room
temperature for 2 h and incubated with the specific primary
antibody against HO-1 at a dilution of 1: 200 at 4 °C overnight.
After washing with TBST, the membranes were incubated
with horseradish peroxidase-conjugated goat anti-rabbit IgG
at a dilution of 1:2000 at room temperature for 1 h. The
membranes were washed 3 times with TBST solution for 45 min
each. Immunoreactive bands were visualized and quantified
with the Quantity One Image software (Bio-Rad, CA, USA).
Statistical analysis All data were expressed as mean±
SD. SPSS 10.0 software (Chicago, USA) was used for the
data analysis. Difference was analyzed by one-way ANOVA
and H-test. P< 0.05 was considered statistically significant.
Results
Effect of propofol on serum BUN and SCr levels
The level of BUN in the I/R group (38.12±3.57 mg/dL, 43.68±9.99
mg/dL, and 99.84±8.63 mg/dL at 2, 6, and 24 h after reperfusion,
respectively) was significantly increased compared with the
sham-operated group (17.46±3.8 mg/dL,
P<0.05 at 6 h and
P<0.01 at 2 and 24 h). The level of SCr in the I/R group
(1.51±0.22 and 2.1±0.27 mg/dL at 6 and 24 h after reperfusion)
was higher than that in the sham-operated group (0.94±0.05
mg/dL, P<0.05 at 6 h and P<0.01 at 24 h). Treatment
with propofol significantly improved the renal injury
induced by I/R at 24 h (BUN, 67.48±8.28
vs 99.84±8.63
mg/dL, P<0.01; SCr, 1.39±0.15
vs 2.1±0.27 mg/dL, P<0.01).
Histopathological analysis In the I/R group, the
histopathological sections showed an obvious loss of brush
border, bleb formation, cytoplasmic vacuolization, cell
necrosis, and dilation of the renal tubules with proteinaceous
casts. The pathological changes were markedly improved
with the treatment of propofol (Figure 1). The mean
histological score by Paller's standard was obviously increased
in the I/R group (94.0±14.7, 153.0±29.7, and 251.4±33.8
at 2, 6, and 24 h after reperfusion, respectively) compared
with the sham-operated group (20.4±4.6,
n=5, P<0.01). When propofol was added, the increase of the mean histological
score caused by I/R was significantly suppressed at 6
(100.6±15.8 vs 153.0±29.7,
n=5, P<0.01) and 24 h after reper-fusion
(173.0±21.8 vs 251.4±33.8,
P<0.01) (Figure 2).
Immunohistochemical expression of HO-1 in the
kidney tissues In the propofol-treated rat kidney tissues, a
pronounced increase in intensity of HO-1 immunostaining
was observed in the proximal and distal tubuli in the cortex
and prominently in the outer strip region of the outer
medulla when compared with the I/R group (0.36±0.17
vs
0.04±0.09, 1.48±0.23 vs 0.76±0.26, and 1.36±0.26
vs 0.64±0.17, n=5, P<0.05 at 2, 6, and 24 h after reperfusion, respectively),
while it was not detected in the tissues from the
sham-operated group (Figures 3, 4).
mRNA expression of HO-1 in the kidney tissues
At 6 and 24 h after reperfusion, the transcription level of HO-1
increased in the I/R group compared with the sham-operated
group (P<0.01, n=4). However, the transcription level of
HO-1 was comparable with or without I/R treatment at 2 h.
Interestingly, propofol dramatically elevated the HO-1
transcription level 2 h after reperfusion
(P<0.01, n=4), and also increased the HO-1 transcription level 6 and 24 h after
reperfusion (P<0.05, n=4) (Figure 5).
Quantitation of the HO-1 protein expression in the
kidney tissues using Western blot analysis The induction of
HO-1 was not only at the transcription level, but also
occurred at the expression level. The expression of HO-1
markedly increased in the I/R-treated kidneys 6_24 h after
reperfusion (P<0.01, n=4), whereas the HO-1 protein was
expressed at an exceedingly low level in the I/R and
sham-operated group 2 h after reperfusion. In contrast, the
expression of the HO-1 protein was observed 2 h after reperfusion,
and increased 6 and 24 h after reperfusion in the
propofol-treated kidneys than that in I/R-treated kidneys
(P<0.05, n=4) (Figure 6).
Discussion
In the current study, we demonstrated that propofol
treatment significantly reduced renal dysfunction and injury in
renal I/R rats. Furthermore, these protective effects are in
part mediated by the induction of HO-1.
Propofol is known to exert protective effects against I/R
injury on various organs[3_7,21]. Our data also demonstrated
that propofol effectively reduced the increase of BUN and
SCr levels, and the mean histological score induced by I/R.
The morphological abnormality of kidney tissues was eased
by treatment with propofol, which suggests that propofol
has protective effects against renal I/R injury. The
production of free radicals and subsequent lipid peroxidation plays
a key role in I/R injury. Propofol has an antioxidant property,
and the majority of studies attributed this capacity to the
phenolic structure of propofol. Propofol appears to inhibit
lipid peroxidation either by reacting with lipid peroxyl
radicals to form the relatively stable propofol phenoxyl
radical[22], or by scavenging peroxynitrite, which is an important molecule
in the cellular toxicity of I/R[23], or both. The other important
factors involved in the development of I/R injury are the
activation of neutrophils overloaded with cellular calcium
and the opening of the mitochondrial permeability transition
pore (MPTP). Propofol can inhibit the activity of
neutro-
phils[24] and calcium influx across plasma
membranes[25]. It has also reported that the protective effect of propofol against
I/R injury was accompanied by less MPTP opening in
isolated hearts[26]. These mechanisms may partially explain our
results. Moreover, we found that propofol ameliorated renal
I/R injury accompanied with an up-regulation of HO-1
expres-sion.
Scapagnini et al suggested that the ultimate stimulation
of the HO-1 pathway was likely to account for the
antioxidant/anti-inflammatory properties of bioactive
polyphenols[27]. The induction of HO-1 has been shown to play an
impor-tant role in the adaptive protection of tissues against I/R
injury[12,13,28]. The end products of heme degradation such as
biliverdin, bilirubin, and CO provide important physiological
roles. Both biliverdin and bilirubin could remove reactive
oxygen species generated by I/R and are thereby regarded
as potent endogenous antioxidants[29]. CO relaxes blood
vessels[30] and inhibits the proliferation of vascular smooth
muscle cells[31]. In addition, CO is capable of suppressing
platelet aggregation[32]. These features suggest that HO-1
plays a critical role in microcirculation. Our data showed
that when induced 6 h after reperfusion, propofol effectively
potentiated the transcription and expression levels of HO-1.
In the propofol-treated group, both the transcription and
expression of HO-1 could be detected, even 2 h after
reperfu-sion. Moreover, propofol treatment significantly reduced
the renal dysfunction and histological damage caused by I/R.
These data, together with previously published
reports[14,27], suggest that the induction of HO-1 might be an important
mechanism for the protective effects of propofol against
renal I/R injury.
In conclusion, propofol attenuates renal I/R injury in
rats, which might provide an effective strategy for renal
protection during anesthesia. Furthermore, HO-1 might
be a new protective pathway of propofol against renal I/R
injury.
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