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,1,1
,2,2
* INSERM, Department of Renal and Cardiac Remodeling–Team 5, Toulouse, France;
Université Toulouse III Paul Sabatier, Toulouse, France;
William Harvey Research Institute, Barts, and the London Medical School, London, UK;
Nephrology and Kidney Transplantation Department, CHU Rangueil, Toulouse University Hospital, Toulouse, France;
|| INSERM, Zootechny Department of Experimental Micro-Surgery, Toulouse, France;
¶ Sanofi-Aventis R&D, Montpellier, France;
# Department of Biophysics, Escola Paulista de Medicina, UNIFESP, São Paulo, Brazil; and
** Max-Delbrück Center for Molecular Medicine, Berlin-Buch, Germany
2 Correspondence: INSERM, Department of Renal and Cardiac Remodeling–Team 5, 1 av Jean-Poulhes, 31432 Toulouse, France. E-mail: J.L.B., jean-loup.bascands{at}inserm.fr; J.P.S., joost-peter.schanstra{at}inserm.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: kidney disease • bradykinin • unilateral ureteral obstruction • chemokine • reversion
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Regardless of the initial insult, the progression of most forms of renal disease results in tubulointerstitial fibrosis, which is the main histological hallmark of CKD (2)
. The presence of fibrosis in CKD is closely correlated to the future appearance of renal failure and has therefore been associated with poor long-term prognosis (3)
. Interstitial fibrosis is characterized by the progressive accumulation of extracellular matrix (ECM) proteins in the tubulointerstitial compartment. A multitude of events and factors (4)
were identified to be involved in the development of renal fibrosis, potentially leading to new antifibrotic strategies and compounds (5
6
7)
. However, in humans blockade of the renin-angiotensin aldosterone system remains the only effective therapy (1)
. These therapies only slow down the progression toward ESRD, and alternative molecules or therapies are still necessary.
As a general rule, the acute inflammatory response protects against infection and injury; however, the chronicity of inflammation is often deleterious (8)
. Chronic inflammation is a key event in CKD that is mainly characterized by monocyte/macrophage accumulation in the renal interstitium and is well correlated with the progression of CKD (9
, 10)
. Consequently, any strategy or agent able to limit or attenuate chronic renal inflammation should significantly slow down the rate of progression of CKD.
Lys-bradykinin and Lys-des-Arg9-bradykinin are peptides intimately linked to inflammation (11)
. These kinin peptides interact with 2 different G-protein-coupled receptors: the kinin B2 receptor (B2R) and the B1 receptor (B1R), respectively (11)
. Constitutively expressed in most tissues, the B2R has been shown to mediate most of the physiological actions of kinins. The role of the B2R has been studied in experimental CKD. We and others have observed that blockade of the B2R increased experimental renal fibrosis (12
13
14
15)
, but the ubiquitous expression of the B2R does not make it an ideal target.
In contrast, the kinin B1R, which is hardly detectable under physiological conditions, is overexpressed under inflammatory conditions in a variety of different tissues (11)
and in turn stimulates inflammation on activation. These data suggest that the B1R might be a more suitable target in chronic inflammatory renal disease.
A large number of in vitro and in vivo studies have shown that B1R induction is controlled by many proinflammatory cytokines and growth factors, including interleukin (IL) 1-β, tumor necrosis factor (TNF) -
, and interferon gamma, as well as epidermal growth factor (11)
. There is now clear evidence that induction of the B1R by many of these factors involves activation of transcription factor NF-
B, and, conversely, B1R stimulation activates NF-B (11
, 16)
. The activated B1R stimulates release of TNF- and IL-1 (17)
and is also involved in leukocyte accumulation and activation (18
, 19)
. The B1R is known to be expressed on macrophages and on fibroblasts in vitro (20)
. Furthermore we have previously shown in vivo that experimental inflammation induces functional B1R expression in renal epithelial cells (16
, 21)
. Its role in acute renal disease (ischemia-reperfusion) has been studied but yielded contradictory results most probably due to compensation in the genetically engineered B1R, B2R single and B1R/B2R double knockout mice (22
, 23)
. However, to the best of our knowledge, the role of the B1R in chronic renal inflammation and fibrosis has never been studied.
Taken together, specific induction of the B1R in renal inflamed tissues, its role in inflammation, and the role of chronic inflammation in renal fibrosis led us to hypothesize that blocking the B1R could be an efficient approach to control the progression of CKD.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. For in vivo experiments, this compound was dissolved in water containing 2% dimethyl sulfoxide (DMSO) to obtain a 1 g/L solution. The SSR240612 solution was diluted with distilled water and administered by gavage at a dose of 10 mg/kg/d. Final DMSO concentration was 0.01%.
Animals
Mice invalidated for the B1R (B1–/–) were obtained as described previously (25)
. Briefly, mice were generated by gene targeting on a mixed genetic background (129/SvJxC57Bl/6J) and backcrossed 10 times to C57Bl/6J (Harlan), as previously reported (26)
. We used C57Bl/6J (Harlan) as their wild-type (B1+/+) control littermates. The mice were housed in a pathogen-free environment. All experiments reported were conducted in accordance with the NIH guide for the care and use of laboratory animals and were approved by a local animal care and use committee.
Unilateral ureteral obstruction (UUO)
B1+/+ and B1–/– male mice of 8 wk of age were used for these experiments. The unilateral ureteral ligation was performed as described previously (12)
. Briefly, under oxygen-isoflurane anesthesia and through a longitudinal, left abdominal incision, the ureter was exposed and ligated with a 6/0 nylon thread at the uretero-pelvic junction. In sham operations, the ureter was exposed but not ligated and repositioned. Mice were maintained on standard mouse chow and tap water. Treatments with the B1R antagonist were initiated either 1 d before obstruction or 3 d after and continued throughout the time of obstruction. A control group received only the vehicle (0.01% DMSO solution). At the end of the different protocols, mice were sacrificed, and the kidneys were removed and divided in different parts according to the different protocols employed.
Cell culture
HEK293T (human embryonic kidney) cells were maintained in Dulbecco’s modified Eagle medium (Gibco, Gaithersburg, MD, USA) without pyruvate and with 4.5 g/L glucose and 10% fetal calf serum at 37°C, 5% CO2. Cells were seeded in 6-well plates and were transiently transfected with either a pcDNA3 plasmid containing the GFP gene (green fluorescent protein, pGFP) or a pcDNA3 plasmid containing the gene encoding for the human B1R (phB1R) (BDKB10TN00; Missouri S&T cDNA Resource Center, Rolla, MO, USA) using JetPEI transfection reagent (Ozyme, St. Quentin Yvelines, France). Two days later, cells were pretreated or not with the peptidic antagonist des-Arg10,Leu9kallidin (10–6 M) (NeoMPS, Strasbourg, France) for 15 min and then treated with the human B1R agonist des-Arg10kallidin (10–6 M) (NeoMPS) for 4 h.
Bone marrow transplantation
Bone marrow transplantation was performed as described previously (27)
. Briefly, 8-wk-old B1+/+ male recipient mice were lethally irradiated with 10 Gy and given acidified water (pH 2) with antibiotics (100 mg/L neomycin and 60,000 U/L polymyxin B sulfate; Invitrogen SARL, Cergy Pontoise, France) until 4 wk after the transfer. Bone marrow cells were isolated from femurs and tibiae of 12-wk-old B1+/+ or B1–/– donor mice by flushing with PBS. Cells were then passed trough a 21-gauge needle, centrifuged (250 g, 5 min, 4°C), and resuspended in RPMI 1640 (Invitrogen), 2% fetal calf serum, and 5 U/mL heparin. Recipient mice received 5 x 106 bone marrow cells in 100 µl by tail-vein injection. UUO was performed 4 wk after transplantation. At the time of sacrifice, peritoneal macrophages were isolated from the abdominal cavity by peritoneal lavage, and blood was harvested. Hematologic analysis was performed by counting red blood cells, platelets, total leukocytes, neutrophils, monocytes, and lymphocytes using a Micros-60CS/18 automated counter (ABX-Diagnostics, Montpellier, France). The genomic DNA was extracted, and the genotype was determined by polymerase chain reaction (PCR) to verify the reconstitution of bone marrow after transplantation.
Histological analysis and immunohistochemistry
Four-micrometer paraffin-embedded sections were cut and used for routine staining (hematoxylin-eosin and periodic acid-Schiff staining) and immunohistochemistry. Sections were first dewaxed in toluene and rehydrated through a series of graded ethanol washes before endogenous peroxidase blockage. Specific primary antibodies were incubated (1 h at room temperature) for the detection of collagen type III (1/500) (Interchim, Montluçon, France), of F4/80 positive inflammatory cells (macrophages) (anti-mouse F4/80,1/250) (RM2900; Caltag Laboratories Inc., Burlingame, CA, USA) and -smooth muscle actin (-SMA, Dako Epos method, U7033; Dako S.A., Trappes, France). For visualization, we used the Dako Envision system. Sections were finally counterstained with hematoxylin. Negative controls for the immunohistochemical procedures included substitution of the primary antibody with nonimmune sera.
Histomorphometric analyses were performed as described previously (12)
using a commercially available image-analysis software, which allows rebuilding of a kidney section from adjacent individual captures (Explora Nova Mosaïc software, La Rochelle, France).
Isolation of RNA
Total RNA was isolated from mouse tissue or cells using Qiagen RNeasy Mini kit (Qiagen, Valencia, CA, USA), eluted in 20 µl RNase-free water and treated by DNase (Turbo DNA-free kit; Ambion, Austin, TX, USA) according to the manufacturer’s protocol. We used 1.5 µl of this solution for quantitation by a NanoDrop instrument (ND-1000 spectrophometer; Thermo Fisher Scientific, Waltham, MA, USA).
Quantification of gene expression by real-time quantitative PCR
Real-time PCR was performed using the ABI Prism7900 HT (Applied Biosystems, Foster City, CA, USA). PCR amplification was performed in a total volume of 25 µl containing 25 ng of cDNA sample, 300 nM of forward and reverse primer, and 12.5 µl of Power SYBR Green PCR Master Mix (Applied Biosystems). The reaction mixture was preheated at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. In our experimental models, the most stable mouse housekeeping gene was the 18S ribosomal RNA (18S). The primers used in this study are listed in Table 1
.
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Statistical analysis
Data are expressed as means ± SD. ANOVA with post hoc Tukey alpha test was performed for comparison between the different groups. Values of P < 0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Mice were subjected to UUO, and kidneys were harvested at different time points after obstruction (Fig. 1
). Macrophage infiltration was quantified by immunohistological staining for F4/80, and B1R mRNA expression was analyzed by real-time PCR. F4/80-positive cell infiltration started 24 h after UUO, and their number clearly increased all along the progression of the pathology (Fig. 1A
). Twenty-four hours of UUO induced a slight increase in B1R mRNA expression, which became significant at days 3 and 8 (Fig. 1B
). This induction was restricted to the obstructed kidney because UUO did not induce B1R mRNA expression in the controlateral kidney or in other tissue, including aorta, brain, mesenteric tissue, or lung (Fig. 1C
).
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Genetic and pharmacological blockade of the B1R reduces significantly the development of UUO-induced renal fibrosis
We next assessed the effect of B1R blockade on the development of renal fibrosis. Interstitial collagen accumulation was studied by histomorphometric analysis in Sirius red-stained renal sections as an index of the fibrotic response to UUO of B1–/–, B1+/+, and mice pretreated with a B1R antagonist (B1Ra, 10 mg/kg/d) (Fig. 2A, B
). After 8 d of UUO, interstitial fibrosis was significantly reduced in B1–/– (Fig. 2A
) and mice pretreated with the B1Ra (Fig. 2B
) compared to the B1+/+ group or wild-type mice treated with the vehicle only. In the different control groups (sham) no significant difference in interstitial fibrosis was observed.
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B1R blockade decreases inflammatory cell infiltration and myofibroblast appearance
In the tubulointerstitium of the control groups (sham) of both B1+/+ and B1–/– we found a very low level of F4/80 positive macrophages (Fig. 2C, D
) and -SMA positive myofibroblasts (Fig. 2E, F
). As expected, UUO induced a significant increase in these renal fibrosis markers. Very interestingly, this increase was prevented by the genetic and pharmacological blockade of the B1R (Fig. 2C, F
).
Delayed B1R antagonist treatment blunts the development of UUO-induced renal fibrosis
To more closely mimic the clinical situation, we investigated whether B1R blockade would result in improvement of established renal fibrosis and inflammation. We thus studied the effect of delayed B1R blockade on UUO-induced fibrosis. Three days of UUO resulted in significant B1R expression (Fig. 1B
), and kidney sections were also already strongly positive for macrophages, myofibroblasts, and collagen accumulation (Fig. 3A
, black line). We therefore decided to start B1Ra treatment 3 d after UUO (Fig. 3A
, arrow). B1Ra halted F4/80 positive macrophage infiltration, and this was associated with a significant decrease in interstitial myofibroblasts and fibrosis (Fig. 3A
, blue line). This effect was confirmed at the mRNA level because we found a significant decrease in collagen type I, III, and IV mRNA expression (Fig. 3B
). Treatment with B1Ra did not alter B2R mRNA expression (not shown).
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Delayed B1R antagonist treatment blunts connective tissue growth factor (CTGF), chemokine (CC motif) ligand (CCL) 2, and CCL7 mRNA expression
To better understand the mechanism by which B1R blockade is reducing renal fibrosis, we analyzed mRNA expression of the profibrotic cytokines transforming growth factor (TGF) -β and CTGF, and 2 chemokines shown to be critical for monocyte/macrophage recruitment, CCL2 [monocyte chemoattractant protein (MCP) -1] and CCL7 (MCP-3) (29
, 30)
. As shown in Fig. 4
, the UUO-induced expression of CTGF, CCL2, and CCL7 was blunted by delayed oral B1Ra administration. However, the treatment did not significantly affect TGF-β.
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Activation of the B1R directly induces CTGF, CCL2, and CCL7 mRNA expression in vitro
To investigate whether the B1R is able to directly stimulate cytokine and chemokine expression, we used a B1R-transfected HEK293 cell line, which is a classical cellular model commonly used to evaluate the effects of the B1R (24
, 31)
. We analyzed expression of CCL2, CCL7, and CTGF in these cells transfected with a GFP-containing plasmid, pGFP, as a control, or a human B1R-containing plasmid, phB1R (Fig. 5
). Cells were treated either with the human B1R agonist, des-Arg10 kallidin (1 µM), or the peptidic B1R antagonist, des-Arg10,Leu9 kallidin (1 µM). Treatments with either B1R-agonist or -antagonist alone were without effect on chemokine expression in control GFP-transfected cells. In B1R-overexpressing cells, the B1R agonist induced rapid expression (4 h) of CCL2, CCL7, and CTGF, and this effect was blunted by B1Ra treatment. No effect was observed on TGF-β expression (not shown).
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Specific deletion of the B1R on blood circulating cells does not affect the development of UUO-induced fibrosis
As the B1R is expressed on infiltrating macrophages as well as on resident renal cells in the inflamed kidney, we investigated whether specific B1R knockout on blood circulating cells (including inflammatory cells) modifies the development of UUO-induced fibrosis in B1+/+ mice. To verify that the mice were successfully reconstituted with bone marrow, peritoneal macrophages and blood of the recipient mice were harvested before the sacrifice, counted, and genotyped. As shown in Fig. 6A
, no difference was observed in the number of circulating hematopoietic cells in wild-type mice with B1+/+ or B1–/– marrow. Moreover, in wild-type mice reconstituted with B1–/– marrow, the B1R mRNA corresponding band was not detected in the blood as well as in peritoneal macrophages (Fig. 6B
). These results demonstrated that the mice were successfully reconstituted and that blood circulating cells were primarily from the donor. B1–/– bone marrow (B1–/–Bm) -transplanted wild-type mice subjected to 8 d UUO did not display significant differences in chemokine mRNA expression (CCL2, CCL7), macrophage infiltration (F4/80 immunohistochemistry), and fibrosis development (Sirius red) compared to obstructed B1+/+Bm-transplanted mice (Fig. 6C
). These results strongly support the hypothesis that the effect of B1R blockade on interstitial fibrosis is mainly mediated by resident renal cells, most likely by reducing chemokine expression and subsequently reduced inflammatory cell recruitment.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 3
, 32)
. As recently reviewed (33)
, chronic inflammation is one of the novel risk factors contributing to the increased mortality seen in CKD patients. Therefore, it is hypothesized that reduction of renal interstitial inflammation has the potential to reduce the progression of interstitial fibrosis and thus prevent ESRD (7
, 34)
. To the best of our knowledge, even if a number of promising strategies are emerging (7
, 34)
, we still do not have effective therapeutic strategies or molecules able to specifically target and reduce chronic renal inflammation without blocking other important pathophysiological functions. We present here data that support the hypothesis that specific blockade of the kinin B1R might be a promising strategy to reduce renal chronic inflammation and subsequently blunt the development of renal fibrosis. Several lines of evidence support this hypothesis. We first demonstrated that the B1R contributed to the progression of UUO-induced renal inflammation and fibrosis. We next showed that delayed treatment with an orally active nonpeptide B1R antagonist in the UUO model significantly decreased the progression of established renal fibrosis. We further showed, using the UUO model associated with bone marrow transplantation experiments and in vitro studies, that this effect was partly mediated via a mechanism involving the inhibition of CTGF and chemokine expression by resident renal cells. To our knowledge this is the first in vivo study reporting that B1R blockade could become an efficient antifibrotic strategy.
We first demonstrated that during UUO, B1R mRNA expression was significantly increased in the obstructed kidney. Very interestingly, this induction seemed to be restricted to the site of the pathology, because there was no marked difference of B1R expression in other tissues. The effects of genetic ablation of the B1R (B1–/–) showed reduced macrophage recruitment associated with a reduction in interstitial fibrosis. This suggests that B1R blockade is modifying the inflammatory response. This was consistent with the attenuated inflammatory response observed in B1–/– mice in different inflammatory models, including pleurisy and paw edema (25)
, as well as in a model of intestinal ischemia and reperfusion injury (35)
.
It is well known that compensatory mechanisms occur in genetically engineered animals. We have previously reported increased renal B1R expression in B2–/– mice (36)
. Similarly, increased renal B2R mRNA expression in B1–/– mice has been reported (37)
. We have also shown that in vivo B2R activation reduces UUO-induced renal fibrosis (12)
. Therefore, it is possible that the B2R is involved in the effects observed in B1–/– mice. However, B1R antagonist treatment, mimicking the effects observed in B1–/– mice, did not induce renal B2R mRNA expression. Thus, the reduction in UUO-induced renal fibrosis observed in B1–/– mice is most likely not due to the modification of kinin B2R expression.
How does B1R blockade reduce the inflammatory response in this model of renal fibrosis? As shown by a number of studies, interstitial inflammation mediated by macrophages constitutes an early and major event in response to UUO (38
, 39)
. The precise molecular mechanism of the development of renal fibrosis is not yet fully elucidated, but key mediators, including the major profibrotic cytokines TGF-β and CTGF, were clearly identified (4
, 28
, 40
, 41)
. As expected, we observed an increased TGF-β and CTGF mRNA expression in UUO-induced fibrosis. Interestingly, the blockade of the B1R was not associated with a decrease in TGF-β mRNA expression but reduced its downstream mediator CTGF. This observation confirms in vitro experiments reporting that activation of the B1R stabilized CTGF mRNA, without modifying TGF-β expression (20)
. In addition, this is the first demonstration that B1R antagonism reduces CTGF mRNA expression in vivo. Furthermore a number of chemokines are involved in renal monocyte/macrophage infiltration (9)
. The role of CCL2 (MCP-1) (30)
and even more recently the role of CCL7 (MCP-3) (29)
was clearly established in this process. Our data showed reduced renal expression of these 2 chemokines in response to B1Ra treatment.
The next issue was to determine whether the B1R is directly involved in this attenuated chemokine response observed in vivo. To verify this hypothesis we used HEK cells transfected with the human B1R to study the effect of B1R stimulation on CTGF and chemokine expression. We observed that B1R stimulation is able to rapidly (4 h) induce a significant increase in CTGF, CCL2, and CCL7 mRNA expression, which was abolished by a specific B1R antagonist. Although the mechanism by which the B1R stimulates CTGF mRNA overexpression has been previously shown to be mediated by mRNA stabilization (20)
, the effects of B1R activation on CCL2 and CCL7 mRNA expression most probably involves NF-B, as it was shown that the B1R activates this transcription factor (42)
. In addition, down-regulation of CCL2 and CCL7 expression was observed by inhibition of NF-B (43
, 44)
.
These results thus suggest that direct stimulation of CTGF and chemokine expression by the B1R could be involved in the proinflammatory and profibrotic actions of the B1R, although the identity of the cell type involved in this effect remains to be determined. Almost any renal cell type can express functional chemokines in vivo (30)
, and the B1R is potentially expressed on infiltrating macrophages and on resident renal cells in the inflamed kidney. We were not able to perform colocalization experiments, as antibodies against the mouse B1R are not commercially available. Therefore, we carried out bone marrow transplantation experiments to clarify whether decreased expression of CTGF and chemokines induced by B1Ra treatment could be mediated by infiltrating inflammatory or resident renal cells, or both. We performed UUO in wild-type mice reconstituted with either B1+/+ bone marrow or bone marrow lacking the B1R gene (B1–/–). Eight days after UUO, there was no difference between mice with B1+/+ or B1–/– bone marrow, showing that the absence of the B1R on monocytes/macrophages was without effect on inflammatory cell infiltration, chemokine expression, and interstitial fibrosis. Thus, although the B1R is also expressed on monocytes/macrophages (11)
, these apparently did not participate in the anti-inflammatory and antifibrotic effects of B1R antagonism. These data strongly suggested that the beneficial effect of the B1R blockade was mainly mediated by resident renal cells.
As recently reviewed, a number of experimental data showed that the progression of renal fibrosis is a reversible process (45)
. Our data showed that, in UUO-induced fibrosis, delayed B1Ra administration leads to reversion of fibrosis. However, we must remain cautious in our interpretation, because the UUO model is an accelerated model of interstitial fibrosis. We are currently validating these data on chronic kidney disease models where interstitial fibrosis appears more progressively.
Because of its inducible character, mainly localized at the site of inflammation, it has been suggested that blocking the B1R will be potentially without significant side effects. This has drawn attention to the B1R as a new therapeutic target for the treatment of pathologies related to chronic inflammation, such as airway inflammation, diabetic neuropathy, arthritis, and chronic and neuropathic pain (46)
. An additional advantage over other potential antifibrotic agents is that the B1R antagonist is already orally available (24)
.
Because failure of many organs [lung (47)
, liver (48)
, and heart (49)
] is often associated with the development of fibrosis, our study should be considered as a "proof of concept" of the therapeutic potential associated with B1R blockade.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication July 21, 2008. Accepted for publication August 21, 2008.
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REFERENCES |
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