Bradykinin [corrected] B1 receptor antagonism is beneficial in renal ischemia-reperfusion injury.

Abstract

Previously we have demonstrated that bradykinin B1 receptor deficient mice (B1KO) were protected against renal ischemia and reperfusion injury (IRI). Here, we aimed to analyze the effect of B1 antagonism on renal IRI and to study whether B1R knockout or antagonism could modulate the renal expression of pro and anti-inflammatory molecules. To this end, mice were subjected to 45 minutes ischemia and reperfused at 4, 24, 48 and 120 hours. Wild-type mice were treated intra-peritoneally with antagonists of either B1 (R-954, 200 microg/kg) or B2 receptor (HOE140, 200 microg/kg) 30 minutes prior to ischemia. Blood samples were collected to ascertain serum creatinine level, and kidneys were harvested for gene transcript analyses by real-time PCR. Herein, B1R antagonism (R-954) was able to decrease serum creatinine levels, whereas B2R antagonism had no effect. The protection seen under B1R deletion or antagonism was associated with an increased expression of GATA-3, IL-4 and IL-10 and a decreased T-bet and IL-1beta transcription. Moreover, treatment with R-954 resulted in lower MCP-1, and higher HO-1 expression. Our results demonstrated that bradykinin B1R antagonism is beneficial in renal IRI.

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Brabykinin B1 Receptor Antagonism Is Beneficial in Renal
Ischemia-Reperfusion Injury
Pamella H. M. Wang1*, Gabriela Campanholle2, Marcos A. Cenedeze1, Carla Q. Feitoza1, Giselle M.
Gonc ¸alves1, Richardt G. Landgraf3, Sonia Jancar3, Joa ˜o B. Pesquero4, Alvaro Pacheco-Silva1, Niels O. S.
Ca ˆmara1,2
1Laborato ´rio de Imunologia Clı ´nica e Experimental, Division of Nephrology, Universidade Federal de Sa ˜o Paulo, Sa ˜o Paulo, Brazil, 2Laborato ´rio de Imunobiologia de
Transplantes, Department of Immunology, Universidade de Sa ˜o Paulo, Sa ˜o Paulo, Brazil, 3Laborato ´rio de Imunofarmacologia, Department of Immunology, Universidade
de Sa ˜o Paulo, Sa ˜o Paulo, Brazil, 4Department of Biophysics, Universidade Federal de Sa ˜o Paulo, Sa ˜o Paulo, Brazil
Abstract
Previously we have demonstrated that bradykinin B1 receptor deficient mice (B1KO) were protected against renal ischemia
and reperfusion injury (IRI). Here, we aimed to analyze the effect of B1 antagonism on renal IRI and to study whether B1R
knockout or antagonism could modulate the renal expression of pro and anti-inflammatory molecules. To this end, mice
were subjected to 45 minutes ischemia and reperfused at 4, 24, 48 and 120 hours. Wild-type mice were treated intra-
peritoneally with antagonists of either B1 (R-954, 200 mg/kg) or B2 receptor (HOE140, 200 mg/kg) 30 minutes prior to
ischemia. Blood samples were collected to ascertain serum creatinine level, and kidneys were harvested for gene transcript
analyses by real-time PCR. Herein, B1R antagonism (R-954) was able to decrease serum creatinine levels, whereas B2R
antagonism had no effect. The protection seen under B1R deletion or antagonism was associated with an increased
expression of GATA-3, IL-4 and IL-10 and a decreased T-bet and IL-1b transcription. Moreover, treatment with R-954 resulted
in lower MCP-1, and higher HO-1 expression. Our results demonstrated that bradykinin B1R antagonism is beneficial in renal
IRI.
Citation: Wang PHM, Campanholle G, Cenedeze MA, Feitoza CQ, Gonc ¸alves GM, et al. (2008) Brabykinin B1 Receptor Antagonism Is Beneficial in Renal Ischemia-
Reperfusion Injury. PLoS ONE 3(8): e3050. doi:10.1371/journal.pone.0003050
Editor: Patricia Bozza, Instituto Oswaldo Cruz and FIOCRUZ, Brazil
Received April 15, 2008; Accepted July 31, 2008; Published August 25, 2008
Copyright: ? 2008 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Fundac ¸a ˜o de Apoio a ` Pesquisa do Estado de Sa ˜o Paulo (FAPESP) and CNPq. Grant numbers: 04/08311-6, 05/50085-6,
06/03982-5 and 07/07139-3
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: niels@icb.usp.br
Introduction
Renal ischemia and reperfusion injury (IRI) is a leading cause of
acute renal failure in both allografts and native kidneys [1].
Inflammation plays an important role in the pathogenesis of renal
IRI, through leukocyte activation and expression of adhesion
molecules and cytokines [2–8]. Therefore, new therapeutic strategies
aiming to reduce this inflammatory response could be beneficial.
Bradykinin receptor activation may affect this inflammatory
response. Physiological effects of bradykinin are mediated by two
transmembrane G-coupled proteins, namely B1 and B2 receptors
(B1R and B2R, respectively) [9]. B2R is constitutively expressed
under physiological conditions and is responsible for most kinin
effects [9]. By contrast, B1R is normally weakly expressed, being
strongly up-regulated in the presence of inflammatory stimuli
[10,11] or its natural agonist des-Arg10-Kallidin [12]. Its role after
activation remains unclear. Several studies have shown that B1R
can influence immune responses by modulating T lymphocytes
[13,14] and leukocyte migration [14,15] and prostaglandins [16],
mast cell mediators [17], cytokines [18,19] and chemokines
production [20–22].
Regarding renal IRI, we have previously demonstrated that
B1R knockout mice were protected against IRI. In the other hand,
B2R antagonism [23] or simultaneous receptor knockouts [24] was
shown to be deleterious.
This study aimed to analyze the impact of bradykinin B1 and
B2R antagonism on renal IRI and to determine the influence of
these tratments on renal pro and anti-inflammatory molecules
expression.
Methods
Animals
Isogenic male B1R, B2R and B1B2R-deficient C57BL/6 mice
(B1KO, B2KO and B1B2KO, respectively) aged 8–12 wks (25–
28 g) were used. All mice were kindly donated by Prof. Joa ˜o Bosco
Pesquero of the Biophysics Department of the Federal University of
Sa ˜o Paulo (UNIFESP), Brazil. All animals were housed in individual
and standard cages and had free access to water and food. Wild type
C57BL/6 mice (B1B2WT), matched for age and sex, were used as
control animals. All procedures were previously reviewed and
approved by the internal Ethics Committee of the Institution.
Experimental Model of Renal IRI
Surgery was performed as previously described [3]. Mice were
briefly anesthetized with Ketamine-Xylazine (Agribrands do
Brazil, Sa ˜o Paulo, Brazil). A midline incision was made and both
renal pedicles were cross-clamped for 45 minutes. During the
procedure, animals were kept well hydrated with saline and at a
constant temperature (,37uC) by means of a heating pad device.
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Subsequently, microsurgery clamps were removed, the abdomen
closed and animals placed in single cages, warmed by indirect light
until fully recovered from anesthesia. Animals were kept under
controlled conditions until sacrifice, according to experimental
protocol at 4, 24, 48 and 120 hours after renal reperfusion. Fifteen
animals (n=15) were sacrificed at each time point. To serve as
controls,tenshamanimals(n=10)fromeachgroupweresubjectedto
the surgical procedure Groups were broken down for technical and
Table 1. Oligonucleotides sequence.
GENEFORWARDREVERSE
GATA-359-GCC TGT GCA AAA GAG ATT TCA GAT-39
59-TGA TTC ACA GAG CAT GTA GGC C-39
T-bet59-CCA GTA TCC TGT TCC CAG CC-39
59-CAT AAC TGT GTT CCC GAG GTG TC-39
MCP-159-AAG AGA ATC ACC AGC AGC AGG T-39
59-TTC TGG ACC CAT TCC TTA TTG G-39
HO-159-TCA GTC CCA AAC GTC GCG GT-39
59-GCT GTG CAG GTG TTG AGC C-39
B1R59-CCA TAG CAG AAA TCT ACC TGG CTA AC-39
59-GCC AGT TGA AAC GGT TCC-39
B2R59-ATG TTC AAC GTC ACC ACA CAA GTC-39
59TGG ATG GCA TTG AGC CAA C-39
HPRT59-CTC ATG GAC TGA TTA TGG ACA GGA C-39
59-GCA GGT CAG CAA AGA ACT TAT AGC C-39
Oligonucleotides sequence.
doi:10.1371/journal.pone.0003050.t001
Figure 1. Renal IRI and bradykinin receptors expression. Bradykinin receptors were analyzed by real-time PCR. In B1B2WT, receptors
expressions were cross-modulated (A). In B2KO, B1R expression was increased at 4 and 24 hours (B). Statistical analyses were performed by ANOVA.
*B1R versus B2R, p,0.05. # B2KO versus B1B2WT, p,0.05.
doi:10.1371/journal.pone.0003050.g001
Bradykinin B1R and Renal IRI
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scientific purposes to allow 15 surgeries per day, 5 from each group,
namely, 15 IRI surgeries or 15 sham-operated animals per day.
Analysis of Renal Function
Serum creatinine was used for renal function assessment. Blood
samples were collected at 4, 24, 48 and 120 hours post reperfusion
from the abdominal inferior cava vein immediately before induced
death. Serum samples were analyzed on a Cobas Mira Plus
(Roche, Mannhein, Germany), using the modified Jaffe ´ technique.
Caspase-3 Activity Assay
Kidneys were collected at 24 hours and homogenized in 1 mL of
ice-cold 40 mmol/L Tris-HCl pH 7.6 with 1% triton X-100.
Extracted kidneys were centrifuged and supernatants collected.
Protein extracts (50 mg) were diluted in 1 mL of protease assay
buffer (20 mmol/L Pipes, 100 mmol/L NaCl, 10 mmol/L DTT,
1 mmol/L EDTA, 0.1% CHAPS, 10% sucrose, pH 7.2) contain-
ing 10 mL of Ac-DEVD-AFC substrate (PharMingen, BD
Biosciences, San Jose, USA) and incubated for 1h at 37uC.
Samples were analyzed using a fluorimeter (F-2000, Hitachi,
Japan) at 505 nm.
Antagonist and agonist treatments
All treatments were administrated i.p. 30 minutes prior to
ischemia. For B1R and B2R antagonism, wild-type animals were
treated with R-954 or HOE-140 (200 mg/kg), while for B1R
agonism, animals were treated with des-Arg9-BK (DABK) at a
concentration of 600 mg/kg. Agonist and antagonists were kindly
donated by Dr. Pierre Sirois from the Universite ´ de Sherbrooke,
Que ´bec, Canada. All doses were based on two previously published
studies that had used these same compounds for in vivo treatment
[25,26].
Gene Profiles
Kidney samples were quickly frozen in liquid nitrogen. Total
RNA was isolated by TRIzol Reagent (Invitrogen, California, USA)
methodology. First-strand cDNAs were synthesized using MML-V
reverse transcriptase (Promega,Madison,USA). Real-time PCR was
performed using TaqMan PCR assays as followed: IL-1b
(Mm00434228_m1), IL-4
(Mm00439616_m1),Bcl-2 (Mm00477631_m1)
(Mm00432042_m1) plus the housekeeper gene hypoxanthine
guanine phosphoribosyltransferase (HPRT) (Mm00446968_m1)
(Applied Biosystem, California, USA). Real-time PCR was per-
formed for GATA-3, T-bet, MCP-1, HO-1, B1R and B2R
expression using SYBR Green assay (Applied Biosystem). In this
case, another specific SYBR Green HPRT was used. Sequences of
oligonucleotides are depicted in Table 1. Cycling conditions were:
10 minutes at 95uC followed by 45 cycles of 30 seconds at 95uC,
30 seconds at 58uC and 30 seconds at 72uC. Relative quantification
of mRNA levels was performed using the comparative threshold
cycle method (described in detail in User Bulletin 2; PerkinElmer,
Applied Biosystems, Branchburg, NJ, 1997). Briefly, the target gene
amount was normalized to the endogenous reference (HPRT) and
then compared against a calibrator (sample with the lowest
expression, namely, sham-operated animals), using the formula
22DDCT. Hence, all data were expressed as an n-fold difference in
relation to the expression of matched controls (sham). Analyses were
performed with the Sequence Detection Software 1.9 (SDS).
(Mm00445259_m1),IL-10
Badand
Statistical Analysis
All data were described as mean6S.E.M. Different results
among groups were compared using Kruskal-Wallis One Way
Analysis of Variance on Ranks (ANOVA) or by the T-test. Results
were considered significant when p,0.05. Survival curves were
estimated by the Kaplan-Meier test. All statistical analyses were
performed with the aid of SigmaStat Statistical Software 2.0
(Jandel Corporation, TX, USA).
Results
Bradykinin receptor expression in renal IRI
B1KO, B2KO, B1B2KO and wild-type mice were subjected to
renal IRI and kidney expression of bradykinin receptors were
analyzed at 4, 24, 48 and 120 h of reperfusion. In wild-type mice,
Figure 2. Animal survival and renal function after bradykinin
receptor deletion or antagonism. Animal survival was assessed by
Kaplan-Meier curve for 5 days after reperfusion. B2KO had the lowest
survival at 120 hours of reperfusion (A). Renal function was estimated
by serum creatinine levels measured by modified Jaffe ´ method. B1KO
protection was observed at all times, while B2KO protection was only
seen at 4 and 24 hours (B). Thirty minutes prior to the ischemic insult,
wild-type animals were treated with B1R or B2R antagonists (R-954 and
HOE-140, respectively, both at 200 mg/kg) via i.p and plasmatic
creatinine levels were assayed at 24 hours of reperfusion. Only the R-
954 treatment resulted in lower SCr levels (C). Statistical analyses were
performed using ANOVA. *B1KO versus B1B2WT, p,0.05 and #B2KO
versus B1B2WT, p,0.05. ***IR+R-954 versus IR and IR+HOE-140, p,0.05.
doi:10.1371/journal.pone.0003050.g002
Bradykinin B1R and Renal IRI
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receptors expressions were alternated (Figure 1A), suggesting a
cross-modulation between them. In B2KO, B1R expression was
increased at 4 and 24 hours (Figure 1B). In B1KO mice, B2R
expression was not modified (data not shown).
Bradykinin receptor importance on survival was also analyzed
and mortality of B2KO mice was increased at 120h post ischemia
(Figure 2A).
Modulation of renal function by Bradykinin receptors
deletion or antagonism
All animals subjected to renal ischemia showed a significant rise
in serum creatinine levels (sCr) after 4, 24 and 48 hours of
reperfusion, in comparison to sham-operated (data not shown). All
values returned to normal at 120 hours (Figure 2B).
B1KO had a significant reduction in sCr at all times analyzed,
meanwhile B2KO mice presented lower sCr values only at 4 and
24 h. These results showed that B1R deletion was beneficial
throughout different reperfusion times; meanwhile protection
under B2R absence was only seen at the beginning of this process.
Moreover, B1R antagonism (R-954) significantly reduced sCr
levels at 24 hours (0.9360.40 in treated versus 2.2260.14 mg/dL
in untreated animals, p=0.005), which was not observed by B2R
antagonism (HOE-140) (Figure 2C). Since a significant protection
was only achieved by B1R antagonism, we decided to analyze
whether its agonist (DABK) would have an impact on renal IRI. In
this case, sCr levels were unaffected (2.2260.14 in untreated versus
1.9560.20 mg/dL in treated animals).
Bradykinin receptors and apoptosis after renal IRI
Cell death was estimated by the anti-apoptotic Bcl-2 and the
pro-apoptotic Bad mRNA expression and caspase-3 activity, a
central protease in apoptosis that is induced by renal ischemia. We
observed that B1KO had increased Bcl-2 (Figure 3A) and
decreased Bad (Figure B) expressions compared to wild-type and
B2KO animals. Furthermore, caspase-3 activity of B1KO was
significantly lower than wild type and B2KO (Figure 3C). These
results thereby indicate that B1KO protection was associated with
lower apoptosis.
Pro and anti-inflammatory molecule expression in B1R
knockout or antagonism after renal IRI
Pro and anti-inflammatory molecules were measured at
24 hours of reperfusion in B1R knockout or antagonist-treated
mice. We observed that the pro-inflammatory transcriptional
factor T-bet and the cytokines IL-1b were significantly reduced by
B1R knockout (Figure 4a and b) and antagonism (Figure 5a and
b). In the other hand, the anti-inflammatory components GATA-
3, IL-4 and IL-10 were increased in knockout mice (Figure 4c, d
and e) and R-954 treated group (Figure 5d, e and f). We previously
have observed that B1R deletion increased HO-1 and decreased
MCP-1 expression [27]. Herein, similar results were achieved by
B1R antagonism (Figure 5c and g). B1R agonist was not to modify
the expression of any molecule analyzed (Figure 5).
Renal function and the expression of some cytokines were also
studied in B1B2KO mice. At 24 hours after IRI, these animals
presented high levels of sCr (2.4760.17 mg/dL in B1B2KO versus
2.2260.14 mg/dL in B1B2WT) and thus, they were not protected
from injury. They also presented a pro-inflammatory profile
similar to wild-type animals (Figure 6b, c, d and e).
Discussion
A previous study by our group demonstrated that bradykinin
receptors may influence tissue outcome in renal IRI [27]. B2R are
constitutively expressed, while B1R are inducible. In the present
study, IRI induced B1R expression and a cross-modulation
between receptors was observed. Cross-modulation has been
previously described in different experimental models of intestinal
[28], cardiac [29–31] and renal [31,32] insults.
Our previous work has demonstrated that sCr levels, measured
24 hours after renal ischemia, were much lower in B1KO than in
wild-type mice [27]. In this study, B1KO protection was
Figure 3. Cell death modulation under B1R-knockout. Apoptosis was estimated by Bcl-2 and Bad expression and caspase-3 activity, at
24 hours of reperfusion. Bcl-2 and Bad expression were measured by real-time PCR, and caspase-3 activity by fluorimetric assay. B1KO animals
presented higher Bcl-2 expression (A) along with lower Bad expression (B) and caspase-3 activity (C), indicating a lower degree of apoptosis. Statistical
analyses were performed using ANOVA.* B1KO versus B1B2WT and B2KO, p,0.05.
doi:10.1371/journal.pone.0003050.g003
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confirmed at all stages. Moreover, we found that B1R antagonist
R-954 also able to reduce sCr levels.
Concerning B2R and renal IRI, our previous paper showed that
sCr levels were slightly lower in B2KO when compared to wild-
type mice. However, this difference did not reach statistical
significance. Here, the use of a larger number of B2KO animals
resulted in a significant decreased of sCr exclusively at the
beginning of reperfusion. In contrast, this protection was not
mimicked by B2R antagonism and was not associated with any
inflammatory molecule here analyzed. This led us to believe that
B2KO protection might rely on expression and function of other
molecules not measured here. In agreement with this, a previous
study have shown that in renal IRI, early B2R deletion was
associated with decreased serum creatine levels, ROS production,
cell death and TNF-a and MCP-1 expression [23]. However, in
the study of Chiang et al. [23] animals were subjected to a less
severe IRI, which may explain the slight difference of renal
dysfunction and tissue responses between our studies. Even more,
they only observed tissue outcome at 24 hours of reperfusion,
herein the analyses were made at 4, 24, 48 and 120 hours.
The 24-hour post ischemia period is commonly considered as
the most deleterious in renal IRI, corresponding with the highest
probability of cell death. Serum creatinine levels of all ischemic
groups analyzed peaked at this 24 hr period, indicating that
maximum renal damage occurred. We evaluated several mole-
cules associated with cell death at 24 hrs and found their
expression to be decreased in B1KO mice. Moreover, we found
that the renal protection observed in B1KO was associated with a
lower degree of apoptosis. Thus, B1R deletion may protect renal
cells from death during IRI.
Concerning the immunological aspect of renal IRI, T lympho-
cytes are considered important mediators of this injury, since T cell
depletion significantly improves renal function [4,6,7]. In this
context, CD4 T cells have been a focus of research due to their
Figure 4. Pro and anti-inflammatory molecule expression in B1KO and wild type animals. All molecule expressions were measured by
real-time PCR at 24 hours of reperfusion. B1KO group had lower pro-inflammatory molecule expression (T-bet and IL-1b) (A and B) and higher anti-
inflammatory response (GATA-3, IL-4 and IL-10) (C, D and E). Statistical analyses were performed using the t-test.* B1KO versus B1B2WT, p,0.05.
doi:10.1371/journal.pone.0003050.g004
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Figure 5. Pro and anti-inflammatory molecules expression after B1R antagonist and agonist treatment. All molecule expressions were
estimated by real-time PCR at 24 hours of reperfusion. B1R antagonism (R-954) resulted in lower pro-inflammatory molecule expression (T-bet, IL-1b
and MCP-1) (A, B and C) and higher anti-inflammatory response (GATA-3, IL-4, IL-10 and HO-1) (D, E, F and G). Molecule expression after B1R agonism
(DABK) were similar to non-treated mice. Statistical analyses were performed using ANOVA.*IR+HOE-140 versus IR, p,0.05.
doi:10.1371/journal.pone.0003050.g005
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Figure 6. B1B2KO renal function and expression of pro and anti-inflammatory molecules. Renal function was estimated by serum
creatinine levels measured using the modified Jaffe ´ method. At 24 hours of reperfusion, B1B2KO presented high levels of serum creatinine and they
were similar to wild-type mice (A). Thus, no renal protection was observed under simultaneous receptors deletion. Inflammatory molecule
expressions were measured by real-time PCR. IL-1b (B), T-bet (C), IL-4 (D) and IL-10 (E) expressions were similar between the double-knockout strain
and wild-type mice. Statistical analyses were performed using the T-test.* B1B2KO versus B1B2WT, p,0.05.
doi:10.1371/journal.pone.0003050.g006
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differentiation to either a Th1 (pro inflammatory) or a counterbal-
ancing Th2 (anti inflammatory) profile. In renal IRI, animals lacking
the transcriptional factor of the Th2 profile (STAT-6) develop more
severe renal damage, while deletion of the Th1 transcriptional factor
STAT-4 results in mild protection. Furthermore, activation of the
Th2 transcriptional factor GATA-3 followed by IL-4, IL-5, IL-6, IL-
10and IL-13 production wasshown tobeprotective,whereas higher
expression of the transcriptional factor T-bet, and IL-12, IL-1b and
IFN-c was deleterious [6,8,33–35]. All of these studies point out that
tissue outcome after renal IRI may be influenced by anti and pro
inflammatory responses.
To investigate possible immunological responses that were
involved in B1KO protection, the expression of some anti and pro
inflammatory components were analyzed in the kidneys. We found
that B1R gene deletion or antagonism resulted in higher
expression of the anti-inflammatory GATA-3, IL-4 and IL-10
and lower transcription of the pro-inflammatory T-bet and IL-1b.
Other studies have associated B1R activation with enhanced
inflammation [29,36,37]. Ni and colleagues demonstrated in a rat
model that overexpression of B1 receptors exacerbated paw edema
induced by carrageenan, and rendered transgenic mice more
susceptible to LPS-induced endotoxic shock [28]. Liesmaa and
collaborators identified increased expression of B1R in endothe-
lium of failing human hearts. They demonstrated that B1R
knockout mice, in contrast to B2R knockout mice, did not
spontaneously develop heart failure and presented an altered
inflammatory response, suggesting that B1R play an essential role
in the initiation of inflammation following myocardial ischemia
[37]. Engagement of the B1R has pro-inflammatory effects,
including promotion of leukocyte traffic, edema and pain.
Moreover, it has been demonstrated that B1R activation can
enhance the release of prostaglandins [16], mast cell mediators
[15], cytokines, mainly IL-1b [18,19] and chemokines and
leukotrienes in different cell types [20–22].
Our results suggest a major influence of B1R in renal ischemia,
since treatment with its antagonist resulted in functional improve-
ment. B1R antagonist also changed the profile of pro and anti
inflammatory molecules, whose expression was induced by IRI.
Animals treated with R-954 showed an increased anti-inflammatory
cytokine profile, whereby GATA-3, IL-4 and IL-10 were up
regulated while T-bet and IL-1b expressions were down regulated.
Even more, the expression of MCP-1 was reduced while HO-1
was up regulated by B1R antagonism, confirming previous
observations from our group that showed a similar expression
profile in B1KO mice [27]. Ischemic organ outcome is determined
mainly by the tuned balance between aggression and cytoprotec-
tion. HO-1 is a protective enzyme able to generate carbon
monoxide (CO), biliverdin and free iron, through heme metab-
olism (reviewed in Camara NO and Soares MP, 2005 [38]), which
can be protective against ischemic insults [39]. Sacerdoti and
colleagues have recently demonstrated that HO-1 expression is
inversely correlated with MCP-1 [40].
Herein, we also showed that simultaneous deletion of both
receptors resulted in extremely high levels of sCr and in a pro-
inflammatory profile. In concordance with this, worse prognoses of
double knockout mice were also observed in pancreas [32] and in
another study of renal [24] IRI. Kakoki et al. linked this deleterious
effect to an enhanced pattern of oxidative stress [24]. Here, we
demonstrated that B1B2KO mice expressed a pro-inflammatory
phenotype that might explain part of their renal dysfunction.
Taken together, our results showed that the protection found
under B1R gene deletion or antagonism involves a shift towards an
anti-inflammatory profile, concomitant with down regulation of
pro-inflammatory molecules and upregulation of anti-inflamma-
tory ones. Since B1R antagonism proved to be protective, we
believe that this may represent a new therapeutic strategy against
renal IRI.
Author Contributions
Conceived and designed the experiments: PHMW NOSC. Performed the
experiments: PHMW GC MAC CF GG. Analyzed the data: PHMW GC
MAC CF GG RL SJ JBP APS NOSC. Contributed reagents/materials/
analysis tools: PHMW MAC RL SJ JBP APS. Wrote the paper: PHMW
NOSC.
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