ORIGINAL ARTICLE

Kinin B1 receptor deficiency attenuates cisplatin-inducedacute kidney injury by modulating immune cell migration

Gabriel R. Estrela & Frederick Wasinski & Danilo C. Almeida & Mariane T. Amano &

Angela Castoldi & Carolina C. Dias & Denise M. A. C. Malheiros & Sandro S. Almeida &

Edgar J. Paredes-Gamero & João B. Pesquero & Carlos C. Barros & Niels O. S. Câmara &

Ronaldo C. Araújo

Received: 10 June 2013 /Revised: 29 November 2013 /Accepted: 2 December 2013# Springer-Verlag Berlin Heidelberg 2013

AbstractCisplatin is a chemotherapeutic agent that causes severe renaldysfunction. The kinin B1 receptor has been associated with themigration of immune cells to injured tissue as well as with renalinflammation. To examine the role of the kinin B1 receptor incisplatin-induced acute kidney injury, we used kinin B1 recep-tor knockout mice and treatment with a receptor antagonistbefore and after cisplatin administration. Cisplatin injectioncaused exacerbation of renal macrophage and neutrophil mi-gration, higher levels of serum creatinine and blood urea,

upregulation of B1 receptor mRNA and an increase in pro-inflammatory cytokines expression. B1 receptor knockout miceexhibited a reduction in serum creatinine and blood urea levels,diminished apoptosis, and decreased cisplatin-induced upregu-lation of inflammatory components. Moreover, treatment withthe B1 receptor antagonist prior to cisplatin administrationnormalized serum creatinine, blood urea levels, protected fromacute tubular necrosis, apoptosis-related genes, and preventedupregulation of pro-inflammatory cytokines. Thus, we proposethat kinins have an important role in cisplatin-induced acutekidney injury by impairing immune cells migration to renaltissue during cisplatin nephrotoxicity.

Key message& Kinin B1 receptor is upregulated after cisplatin exposure.& Kinin B1 receptor deficiency diminishes the nephrotoxi-

city caused by cisplatin.& Kinin B1 receptor deficiency ameliorates the inflammato-

ry response.& Kinin B1 receptor deficiency diminishes apoptosis caused

by cisplatin.& Kinin B1 receptor antagonism ameliorates renal function

after cisplatin injection.

Keywords Kinins .Nephrotoxicity . Cisplatin .Acute kidneyinjury . Inflammation

Introduction

Cisplatin is an antitumor agent frequently used in chemother-apy; however, nephrotoxicity is recurrently associated withcisplatin treatment and is a limiting factor for its use. Renalinjury is the result of the apoptosis and/or necrosis of renal

Electronic supplementary material The online version of this article(doi:10.1007/s00109-013-1116-z) contains supplementary material, whichis available to authorized users.

G. R. Estrela : F. Wasinski : C. C. Dias : S. S. Almeida :E. J. Paredes-Gamero : J. B. Pesquero : R. C. AraújoDepartamento de Biofísica, Universidade Federal de São Paulo,São Paulo, Brazil

G. R. Estrela : F. Wasinski :D. C. AlmeidaDepartamento de Medicina, Disciplina de Nefrologia, UniversidadeFederal de São Paulo, São Paulo, Brazil

M. T. Amano :A. Castoldi :N. O. S. CâmaraInstituto de Ciências Biomédicas, Departamento de Imunologia,Universidade de São Paulo, São Paulo, Brazil

D. M. A. C. MalheirosDepartamento de Clinica Médica, Universidade de São Paulo,São Paulo, Brazil

C. C. BarrosDepartamento deNutrição, Escola deNutrição, Universidade Federalde Pelotas, Pelotas, Rio Grande do Sul, Brazil

R. C. Araújo (*)Department of Biophysics, Federal University of São Paulo, RuaBotucatu, 862, 7° andar, 04023-062 São Paulo, São Paulo, Brazile-mail: araujo.ronaldo@unifesp.br

J Mol MedDOI 10.1007/s00109-013-1116-z

cells [1–3]. Neutrophil, macrophage and T cell infiltration inthe kidney are increased after cisplatin exposure in parallelwith the expression of several cytokines and chemokines,suggesting that the immune response plays a detrimental rolein cisplatin pathology [1–4].

It is well established that kinins affect the inflammatoryresponse. The physiological effects of kinins are mediated by2G-protein-coupled transmembrane receptors [5]. The B2kinin receptor (B2R) is constitutively expressed under physi-ological conditions and is responsible for the majority of kinineffects [5], including glucose homeostasis [6, 7]. On the otherhand, the B1 kinin receptor (B1R) normally has a low expres-sion level and is highly regulated by the presence of inflam-matory stimuli [8, 9]. Several studies showed that B1R mightinfluence the immune response by affecting leukocyte[10, 11], neutrophil [11], and T lymphocyte [11, 12] migra-tion, as well as modulating the release of prostaglandins [13],cytokines [14, 15], and chemokines [16, 17]. In this context, itis important to note that B1R is able to induce the release ofTNF-α and IL-1β mainly by NF-κB activation [18]. Recentwork with a high-glucose-inducing podocyte injury in ratsdemonstrates that B1R antagonism is capable to reduceNF-κB phophorylation. This in turn is able to reduce ERstress-mediated apoptosis, augmenting the bcl-2 protein levelsand down-regulating caspase-3 staining [19].

Some studies examining mice that are genetically deficientfor B1R or mice treated with a B1R antagonist showed pro-tection against different types of kidney diseases, such as renalischemia and reperfusion [20, 21], glomerulonephritis [22],obstructive nephropathy [23], and experimental focal andsegmental glomerulosclerosis [24].

This study aimed to analyze the effects of kinin B1Rdeletion and antagonism of the receptor on the nephrotoxicityinduced by cisplatin and to determine the influence of thisreceptor on renal tissue.

Materials and methods

Experimental design Male C57BL/6 and B1 receptor knock-out (B1KO; B1−/−) mice (C57BL/6 background) weighing25–30 g and 8–12 weeks of age were used for these experi-ments. Single doses of Cisplatin (20mg/kg—Bergamo, Taboãoda Serra, Brazil) were injected intraperitoneally, and the ani-mals were sacrificed 24, 48, 72, and 96 h after the injection.The animals were obtained from the Animal Care Facility at theFederal University of São Paulo (UNIFESP). All animals werehoused in individual, standard cages and had free access towater and food. All procedures were previously reviewed andapproved by the internal ethical committee of the institution.

Renal function Serum creatinine and urea levels were used todetermined renal function. Blood was collected by heart

puncture. All samples were analyzed using colorimetric as-says, using commercial kits to detect creatinine and urea CE(both from Labtest, Lagoa Santa, Brazil).

Quantification of gene expression Kidney samples were fro-zen at −80 °C. Total RNAwas isolated using TRIzol Reagent(Invitrogen, Carlsbad, CA). First-strand cDNAwas synthesizedusing the High Capacity cDNA Reverse Transcription kit(Applied Biosystems). Real-time PCR was performed usingtwo systems (Table 1): the TaqMan system (AppliedBiosystems, Carlsbad, CA) using probes for B1R(mm00432059-s1), TNF-α (mm00443258-m1), and GAPDH(mm99999915-g1); and the SYBR Green assay (ThermoScientific, Waltham, MA) using the primers described inTable 1. The cycling conditions for both the TaqMan andSYBR Green primers were as follows: 10 min at 95 °C,followed by 45 cycles of 30 s at 95 °C, 30 s at 60 °C, and30 s at 72 °C. Standard curves were performed for each primerpair to check the efficiency of amplification. Target mRNAexpression was normalized to β-actin for SYBR and toGAPDH for TaqMan and expressed as a relative value usingthe comparative threshold cycle (Ct) method (2−ΔΔCt) accord-ing to the manufacturer's instructions. The expression levels ofthe genes of interest were normalized to the control group.

Histological analyses Formaldehyde-fixed paraffin sectionsof kidneys were stained with hematoxylin and eosin (H&E).Optic light microscopy was employed to analyze the samples.Images were acquired at ×40 magnification. Epithelial des-quamation, cellular debris, flattening of the epithelium, thepresence of cylinders, and dilation of the tubular lumen wereused as criteria for tubular injury. The injuries were gradedusing a scoring procedure, in which I=0–10 % of the total

Table 1 Base pair sequences of primers used in real-time PCR assays

Gene Primers

β-actin 5′-CTGGCCTCACTGTCCACCTT-3′5′-CGGACTCATCGTACTCCTGCTT-3′

IFN-γ 5′-TCAAGTGGCATAGATGTGGA-3′5′-TGGCTCTGCAGGATTTTCAT-3′

IL-1β 5′-AGGAGAACCAAGCAACGACA-3′5′-CGTTTTTCCATCTTCTTCTTTG-3′

BAX 5′-CGGCGAATTGGAGATGAACTG-3′5′-GCAAAGTAGAAGAGGGCAACC-3′

BCL-2 5′-ACCGTCGTGACTTCGCAGAG-3′5′-GGTGTGCAGATGCCGGTTCA-3′

TNFR-2 5′-GTCGCGCTGGTCTTCGAACTG-3′5′-GGTATACATGCTTGCCTCACAGTC-3′

IFN-γ interferon gamma, IL-1β interleukin 1β, BAXBcl-2-associated X,BCL-2B-cell lymphoma 2, TNFR-2 tumor necrosis factor receptor 2

J Mol Med

kidney area was compromised; II=11–25 %; III=26–50 %;and IV≥50 %.

Flow cytometry analysis Animals were sacrificed and thekidneys were collected and digested in 4 ml collagenase typeIV (2 mg/ml) at 37 °C for 1 h. After incubation, the cells werepassed through a 70-μm strainer and then separated usingtwo-phase Percoll (35 and 70 %) centrifugation for 30 minat 1,000×g at room temperature. Leukocytes were collectedand washed in PBS-2 % fetal bovine serum (FBS). The cellswere stained for 30 min at 4 °C with the following antibodies:anti-CD11b-APC (Life Technologies, Carlsbad, CA), anti-Ly6G-PE (Biolegend, San Diego, CA), anti-CD11c-PERCP(Biolegend, San Diego, CA), anti-NK1.1-PE (eBiosciences,San Diego, CA), and anti-CD3-FITC (Life Technologies,Carlsbad, CA). We analyzed the renal immune cell popula-tions using multicolor flow cytometry. The samples wereexamined with a FACSCanto using FACSDIVA software(BD Biosciences, Franklin Lakes, NJ) and then were analyzedwith FLOWJO software (Tree Star, San Carlo, CA).Fluorescence voltages were determined using matched un-stained cells. Compensation was performed using singlecolor-stained cells. Samples were acquired up to at least200,000 events with a live mononuclear gate.

Immunohistochemistry Localization of PCNA (diluted 1:300;DAKO, Glostrup, Denmark) and Caspase-3 (diluted 1:1,000,Cell Signaling, Beverly, MA) were assessed in paraffin-embedded tissue sections. As described previously, the slideswere deparaffinized, rehydrated and antigens were retrieved ina citrate buffer solution (pH 6) at 95 °C. The endogenousperoxidase activity was blocked with 3 % hydrogen peroxidesolution, and the sections were additionally blocked withProtein Block Solution (DAKO, Glostrup, Denmark). Theslides were incubated with the primary antibody or isotypenon-specific IgG as a negative control followed by incubationwith the labeled EnVision polymer (DAKO, Glostrup,Denmark) using two sequential 30-min incubations at roomtemperature. The staining was developed by incubating for1–3 min with 3,39-diaminobenzidine plus substrate-chromogen, which stains the specific antigen brown.Hematoxylin counterstaining was also performed.

B1R antagonist treatment R-715 (Sigma Aldrich, St. Louis,MO) was used as a B1R antagonist. R-715 was injecteddifferently in different groups, one group started the antago-nist treatment 24 h prior to cisplatin injection, the secondgroup started the treatment together with cisplatin, thethird group started the treament 3 h after the cisplatininjection and the fourth group started the treatment 6 hafter the cisplatin (Fig. 1). After, all the groups weretreated with B1R antagonist once a day at a dose of800 μg/kg.

Statistical analysis All data are presented as the mean±s.e.m.Different results between groups were compared using analy-sis of variance. The value for statistical significance wasestablished at P<0.05. All statistical analyses were performedusing GraphPad Prism (GraphPad, La Jolla, CA).

Results

Kinin B1 receptor is upregulated after cisplatin injection

Acute kidney injury was induced in mice by intraperitonealinjection of cisplatin. The animals were sacrificed after 24, 48,72, or 96 h. Serum creatinine levels peaked 96 h after theinjection (Fig. 2a). Blood urea levels were significantly higher72 h after the injection (Fig. 2b).

To examine the participation of B1R during cisplatin-induced acute kidney injury, we first evaluated the expressionof B1R mRNA using real-time PCR. B1R mRNAwas upreg-ulated at 24, 48, 72, and 96 h after cisplatin injection (Fig. 2c).

B1KO mice are partially protected against cisplatin-inducedacute kidney injury

WT (wild-type) and B1KO (B1 receptor knockout) animalswere treated with cisplatin and sacrificed after 96 h. B1KOmice had lower serum creatinine levels (Fig. 3a) and lowerblood urea levels (Fig. 3b) compared to control mice treatedwith cisplatin.

Because it was previously shown that cisplatin causes acutetubular necrosis, we quantified the area of necrotic tubuli.Control mice treated with cisplatin had a higher score foracute tubular necrosis. B1KO mice were protected from thiseffect (Fig. 3c).

Treatment with cisplatin causes loss of kidney function anddeath in mice; therefore, we evaluated the survival of WTandB1KO mice treated with cisplatin. The knockout miceshowed a higher survival percentage compared withthe WT mice (Fig. 3d).

Fig. 1 B1R antagonist treatment scheme. R-715 were given at fourdifferent time for different groups. The −24 group started the treatment24 h prior to cisplatin injection, the 0 group started the treatment togetherwith cisplatin administration and the +3 and +6 groups started theantagonist treatment 3 and 6 h after cisplatin, respectively

J Mol Med

B1KO mice are partially protected against cisplatin-inducedcell proliferation and apoptosis

The main effect of cisplatin treatment in the kidney isnecrosis and apoptosis. On the other hand, the remain-ing cells have the ability to dedifferentiate and

repopulate the damaged tubule. We evaluated the levelsof cell proliferation and apoptosis in the kidney usingimmunohistochemistry.

Treatment with cisplatin induced tubular proliferationin control mice, whereas B1KO mice expressed lowerlevels of PCNA, a marker for proliferation (Fig. 4a).

Fig. 2 Renal function and kininB1 receptor mRNA expressionkinetics during cisplatinnephrotoxicity. Serum creatinineis only upregulated 96 h aftercisplatin administration (a). Bloodurea levels start to rise 72 h aftercisplatin injection (b). B1RmRNA expression is rapidlyupregulated after cisplatinadministration and remains until96 h after injection (c). *p<0.05.Bars=mean and s.e.m.

Fig. 3 B1R deletion partially protects mice from cisplatin-induced acutekidney injury. Ninety-six hours after cisplatin injection, the B1KO mice(BIKO CIS) showed lower serum creatinine (a) and blood urea (b) levelscompared to the cisplatin-treatedWT (CIS). Acute tubular necrosis is also

decreased in B1KO mice after cisplatin administration (c), and B1KOmice have a higher survival percentage (d) after cisplatin administration.*p<0.05 compared to the control, #p<0.05 compared to CIS. Bars=meanand s.e.m.

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Caspase-3 expression was analyzed to evaluate apopto-sis levels. Cisplatin-treated control mice showed exten-sive expression of caspase-3, whereas B1KO miceshowed staining similar to the untreated control mice(Fig. 4b).

Both intrinsic and extrinsic apoptotic pathways were alteredin B1KO mice

Because apoptosis occurs primarily through intrinsic andextrinsic pathways, we analyzed the expression of keymolecules in these pathways using real-time PCR. Wild-type animals treated with cisplatin expressed high levels ofTNFR-2. However, this high expression level was de-creased in B1KO mice, indicating that these animals havea lower apoptotic stimulus from the extrinsic pathway(Fig. 5a). To evaluate the intrinsic pathway, we analyzedtwo genes involved in apoptosis through the mitochondrialpathway: Bcl-2-associated X protein (Bax), a pro-apoptoticprotein; and B-cell lymphoma 2 protein (Bcl-2), an anti-apoptotic protein. B1KO mice expressed higher levels ofBax and Bcl-2 compared to cisplatin-treated mice. However,we found that the Bax/Bcl-2 ratio was increased in treatedWTmice, whereas B1KO-treated mice showed a Bax:Bcl-2 ratiosimilar to untreated control mice (Fig. 5b–d).

Kidneys from B1KO mice showed decreased infiltrationby immune cells

We evaluated cell infiltration in the kidney after cisplatininjection. First, we observed that B1KO mice treatedwith cisplatin had a lower percentage of cells expressingthe lymphocyte antigen 6 complex, locus G (Ly6G)compared to treated wild-type mice, maintaining a nearlybasal state (Fig. 6a). A larger number of cells isolatedfrom the kidneys of cisplatin-treated mice expressedCD11b (Fig. 6b), a macrophage marker. Moreover, inB1KO mice, fewer CD11c+ cells infiltrated the kidneycompared to WT mice (Fig. 6c). We also analyzedNK1.1+ cells (Fig. 6d) and observed increased kidneyinfiltration in WT cisplatin-treated mice. The deletion ofB1R was able to diminish NK1.1+ cell infiltration, cor-roborating the observed protection conferred by B1Rdeficiency.

Cytokines are expressed at different levels in B1KO mice

We analyzed the mRNA expression of various cytokinesusing real-time PCR on kidney tissues. Cisplatin-treatedB1KO mice showed lower levels of IL-1β, similar to controlmice (Fig. 7a). However, the expression of IFN-γ was

Fig. 4 Both proliferating cellnuclear antigen (PCNA) andcaspase-3 expression are inhibitedin B1KO mice. Representativepictures of kidney slicesincubated with anti-PCNA andanti-caspase-3. B1KO mouserenal tissue shows protection aftercisplatin injection from PCNAexpression (a) and caspase-3 (b)expression. The pictures weretaken with an originalmagnification of ×40. *p<0.05compared to the control, #p<0.05compared to CIS.Bars=mean ands.e.m.

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downregulated in WT cisplatin-treated mice, whereas thelevels of this cytokine were higher in B1KO treated mice(Fig. 7b). It is known that cisplatin can augment TNF-α levels.The B1R deficiency reduced the cisplatin-induced TNF-αincrease (Fig. 7c).

Early treatment with a B1R antagonist improves renalfunction and kidney structure after cisplatin exposure

Next, we evaluated whether early or delayed treatmentwith an antagonist of B1R, R-715, could have a protec-tive effect on cisplatin-induced acute kidney injury.Treatment starting together with cisplatin administration,3 and 6 h after cisplatin injection, prevents partiallyrenal injury (Fig. 8a–c). In contrast, early treatment withR-715 (24 h prior to cisplatin administration) was ef-fective at reducing kidney pathology. The animals treat-ed with R-715 showed lower levels of serum creatinine(Fig. 8a), lower levels of blood urea (Fig. 8b) and astrong protection against acute tubular necrosis showingthe same histological score of control group (Fig. 8c).

B1R antagonist attenuates apoptosis-related genes and renalinflammation induced by cisplatin

We analyzed the mRNA expression of key molecules ofapoptosis and three important cytokines using real-timePCR on kidney tissues. Cisplatin treatment augments theTNFR-2 mRNA levels and B1R antagonism was capable

to maintain these levels at basal state (Fig. 9a). The Bax/Bcl-2 ratio evaluation showed that only early treatmentwith B1R antagonist (−24 h) is able to attenuates theupregulation of Bax/Bcl-2 ratio (Fig. 9b). TNF-α and IL-1β are two important cytokines mediating the inflamma-tion in cisplatin-induced acute kidney injury. The groupstreated with B1R antagonist showed no upregulation ofthese cytokines after cisplatin administration (Fig. 9c, d).IFN-γ is downregulated after cisplatin injection and onlythe early treatment with the R-715 was able to upregulatethis cytokine when compared with the WT cisplatin-treated mice (Fig. 9e).

Discussion

B1R is involved in many inflammatory disorders [21,23–25]; therefore, we hypothesized that deletion or antag-onism of B1R could have a protective role in cisplatin-induced acute kidney injury. Several studies show thatdeletion or antagonism of this receptor is associated withdecreased inflammation and kidney fibrosis [26, 27], butno data have been reported concerning cisplatin-inducedacute kidney injury.

In this study, we observed that the absence of this receptoris protective for kidney injuries induced by cisplatin in mice.We also demonstrated that early treatment with the B1Rantagonist R-715 has a protective effect on the kidney in miceadministered cisplatin by i.p. injection.

Fig. 5 mRNA expression ofapoptosis-related genes. Cisplatininjection causes apoptosis after96 h. B1KO mice express lowermRNA levels of TNFR-2 (a) andhave a decreased BAX:BCL-2ratio (b to d), showing partialprotection from apoptosis viaboth the extrinsic and intrinsicpathway. *p<0.05 compared tothe control, #p<0.05 compared toCIS. Bars=mean and s.e.m.

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Upregulation of B1R in the kidney of control mice 24 to96 h after cisplatin injection strongly suggests the participationof this receptor in kidney injuries caused by cisplatin. Weanalyzed serum creatinine and blood urea levels 24 to 96 hafter cisplatin injection to identify the timepoint associatedwith the most severe acute kidney injury. We observed thehighest levels of creatinine 96 h after the cisplatin injectionand the highest levels of blood urea 72 and 96 h after cisplatininjection. These results in the context of B1R upregulationafter cisplatin treatment directed our analysis to the effects of

cisplatin injection in B1KO mice 96 h after injection. Thelower levels of serum creatinine and blood urea observed inB1KOmice after cisplatin injection are indicative of decreasedrenal injury in the absence of B1R. Several authors have shownthat cisplatin leads to acute tubular necrosis [1, 3]. We alsoobserved that cisplatin-treated mice had a higher score of acutetubular necrosis. These results were not observed in B1KOmice treated with cisplatin, corroborating observations from astudy of renal ischemia and reperfusion-induced acute kidneyinjury [20]. The protective effect demonstrated in the survival

Fig. 6 B1R deletion decreases immune cell migration. Ninety-six hoursafter cisplatin administration, the B1KO mice showed lower levels ofneutrophils (a), macrophages (b), dendritic cells (c) and NK cells (d)

infiltrating the kidney. *p<0.05 compared to the control, #p<0.05 com-pared to CIS. Bars=mean and s.e.m. SSCA, side or orthogonal scatter:measures cell complexity or granularity

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curve indicates that B1R expression increases the risk of kidneyinjury after cisplatin treatment in mice.

It is known that cisplatin causes an increase in neutrophilmigration to renal tissue [2, 3, 28]. Some authors also showthat neutrophils express the B1R protein under inflammatory

conditions, and B1R activation induces neutrophil migration[11, 18]. In this work, we show that B1R deficiency decreasesneutrophil migration to the injured kidney tissue. Similarly,macrophages are implicated in many different models ofkidney inflammation, mainly in the pathogenesis of ischemic

Fig. 7 B1R deletion modulatescytokine expression after cisplatinexposure. Four days aftercisplatin injection, cytokineexpression is altered in the kidney.B1KO mice show decreasedexpression of IL-1β (a) andTNF-α (c) and restored IFN-γ (b)expression to normal levels.*p<0.05 compared to the control,#p<0.05 compared to CIS.Bars=mean and s.e.m.

Fig. 8 B1R antagonism protectsrenal function after cisplatininjection. After 96 h of cisplatinexposure, treatment with a B1Rantagonist diminished serumcreatinine levels (a). Blood urealevels were decreased with earlytreatment with the B1R antagonist(b). Acute tubular necrosis scoreis lower after cisplatin exposure inB1R antagonist treated mice (c).*p<0.05 compared to the control.Bars=mean and s.e.m.

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acute kidney injury [3, 29]. This has also been demonstrated ina cisplatin model of acute kidney injury [30]. We observed asmaller percentage of infiltrating macrophages in the kidneytissue of B1KO mice, which is in accordance with these andother authors who show that B1KO mice have lower macro-phage infiltration in a model of obstructive nephropathy andglomerulonephritis [22, 23]. These data are also in agreementwith the decreased number of infiltrating dendritic cells inB1KO mice after cisplatin injection compared to WT-treatedmice. Recently, it has been shown that dendritic cells contrib-ute to TNF-α production during ischemic renal injury [31].Here, we suggest that the same mechanisms present in theischemic renal injury model are relevant to the cisplatin neph-rotoxicity model because both injury models lead to an in-crease in infiltrating dendritic cells and inflammation state andin our model of injury it is diminished in B1KO mice, whichshow an improved inflammatory status. In terms of inflam-matory cell infiltration, Kim et al.[32] showed that cisplatintreatment increases the number of NK and NKT cells in thekidney. Our results corroborate these data because cisplatin-treated WT mice have higher levels of NK1.1+ cells, whichare decreased in B1KO mice. Moreover, cisplatin nephrotox-icity leads to apoptosis and necrosis [1, 3, 33]. TNFR-2 hasbeen show to play a major role in TNF-α-mediated apoptosisand necrosis in cisplatin nephrotoxicity [1] through the extrin-sic apoptotic pathway. Cisplatin toxicity causes the release ofcytochrome c, which is associated with mitochondrial

dysfunction and leads to higher caspase 3 activity, subsequent-ly activating the intrinsic apoptotic pathway. Cisplatin-treatedB1KO mice express lower levels of TNFR-2 mRNA as wellas an altered ratio of Bax/Bcl-2 mRNA levels, suggesting thatB1R signaling is involved in activation of these pathways inresponse to cisplatin. B1R deficiency also decreased expres-sion of caspase-3, as assessed by immunohistochemistry.Together, these results show that both the intrinsic and extrin-sic pathways of apoptosis are increased by B1R induction andsignaling. Additionally, cytokines are involved in cisplatin-induced acute kidney injury, and IL-1β levels increase aftercisplatin administration [30, 34]. IFN-γ downregulation oc-curs in some types of renal diseases and in cisplatin nephro-toxicity, and deletion of this cytokine causes severe injuryafter cisplatin administration [35]. The lower mRNA levelsof IL-1β and TNF-α and the smaller decrease of IFN-γexpression observed in B1KO mice after cisplatin administra-tion further elucidates the inflammatory scenario in cisplatin-induced acute kidney injury in the absence of the B1R gene.

Because chemotherapy treatment is normally planned inadvance, the results observed with R-715 treatment demon-strate the possibility of using B1R antagonists to reduce unde-sired renal side effects and increase the efficiency of cisplatintreatment for cancer, more studies are necessary to show thisefficiency. B1R antagonism has been used in the treatment ofseveral different types of diseases in animals [21–24]. Here, weobserved that early treatment with R-715 is more beneficial,

Fig. 9 B1R antagonismmodulates apoptosis related genes and cytokinesexpression after cisplatin exposure. Cisplatin administration leads toapoptosis and inflammation. B1R antagonism treatment was able todiminish TNFR-2 mRNA levels (a), early treatment with R-715 dimin-ishes bax/bcl-2 ratio (b). Cytokine expression is altered in the kidney after

cisplatin injection. B1R antagonist-treated mice show decreased expres-sion of IL-1β (c) and TNF-α (d) and only early treatment restored IFN-γ(c) expression. *p<0.05 compared to the control, #p<0.05 compared toCIS. Bars=mean and s.e.m.

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reducing serum creatinine, blood urea levels, acute tubularnecrosis, apoptosis-related genes, and inflammation.

It is known that cisplatin treatment has side effects other thankidney injury. The kinin B1KO mice exhibit kidney protectionagainst cisplatin-induced nephrotoxicity; however, approxi-mately 80 % of the animals died after treatment, which canbe related to other side effects. We observed that B1KO micehave no protection against the hepatotoxicity and bone marrowsupression induced by cisplatin treatment (supplementary files)and it could be related with B1KO mortality. Nevertheless, theanimals had a significant superior lifespan compared to thewild-type animals treated with cisplatin.

Our study is the first to evaluate the role of B1R in cisplatinnephrotoxicity. In summary, the results show that B1R isinvolved mechanistically in the development of kidney injuryin a cisplatin-induced acute kidney injury model and thateither B1R deficiency or inhibition with B1R antagonists areable to reduce the severity of these injuries. Therefore, ourfindings should provide new and valuable perspectives oncisplatin nephrotoxicity management.

Acknowledgements This work was supported by FAPESP (Fundaçãode Apoio a Pesquisa do Estado de São Paulo), grant 2011/03528-0.

Conflict of interest All the authors declared no competing interests.

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