Tibial post fracture pain is reduced in kinin receptors deficient mice and blunted by kinin receptor antagonists.

BACKGROUND: Tibial fracture is associated with inflammatory reaction leading to severe pain syndrome. Bradykinin receptor activation is involved in inflammatory reactions, but has never been investigated in fracture pain. METHODS: This study aims at defining the role of B1 and B2-kinin receptors (B1R and B2R) in a closed tibial fracture pain model by using knockout mice for B1R (B1KO) or B2R (B2KO) and wild-type (WT) mice treated with antagonists for B1R (SSR 240612 and R954) and B2R (HOE140) or vehicle. A cyclooxygenase (COX) inhibitor (ketoprofen) and an antagonist (SB366791) of Transient Receptor Potential Vaniloid1 (TRPV1) were also investigated since these pathways are associated with BK-induced pain in other models. The impact on mechanical and thermal hyperalgesia and locomotion was assessed by behavior tests. Gene expression of B1R and B2R and spinal cord expression of c-Fos were measured by RT-PCR and immunohistochemistry, respectively. RESULTS: B1KO and B2KO mice demonstrated a reduction in post-fracture pain sensitivity compared to WT mice that was associated with decreased c-Fos expression in the ipsilateral spinal dorsal horn in B2KO. B1R and B2R mRNA and protein levels were markedly enhanced at the fracture site. B1R and B2R antagonists and inhibition of COX and TRPV1 pathways reduced pain in WT. However, the analgesic effect of the COX-1/COX-2 inhibitor disappeared in B1KO and B2KO. In contrast, the analgesic effect of the TRPV1 antagonist persisted after gene deletion of either receptor. CONCLUSIONS: It is suggested that B1R and B2R activation contributes significantly to tibial fracture pain through COX. Hence, B1R and B2R antagonists appear potential therapeutic agents to manage post fracture pain.

Cigarette Smoke Particles-Induced Airway Hyperreactivity in Vivo and in Vitro.

Cigarette smoke is a well-known strong risk factor for inducing airway hyperreactivity (AHR), but the underlying molecular mechanisms are not fully understood. In the present study, mouse in-vivo and in-vitro models were used to study effects of dimethyl sulfoxide (DMSO)-extracted cigarette smoke particles (DSP) on the airway, and to explore the underlying molecular mechanisms that are involved in DSP-induced AHR. In mouse in-vivo model, DSP (0.75, 1.5 or 3 µL/mL) was administered intranasally daily for 7 d. At the end of this period, lung functions were measured with flexiVent™. The results showed that the mice exhibited AHR in a dose-dependent manner following methacholine inhalation in vivo. In mouse in-vitro organ culture model, exposure of mouse tracheal segments to DSP (0.1 µL/mL) with or without the following pharmacological inhibitors: specific c-Jun-N-terminal kinase (JNK) inhibitor SP600125 (10 µM) or the anti-inflammatory drug dexamethasone (1 µM). DSP-induced bradykinin receptor-mediated airway contraction with increased mRNA and protein expressions for bradykinin B1 and B2 receptors could be significantly reduced by SP600125 or dexamethasone. In conclusion, the present study demonstrates that DSP could induce AHR in vivo and in vitro. In addition to this, the upregulation of bradykinin receptors in airway is most likely one of the underlying molecular mechanisms involved.

Novel insights into the critical role of bradykinin and the kinin B2 receptor for vascular recruitment of circulating endothelial repair-promoting mononuclear cell subsets: alterations in patients with coronary disease.

BACKGROUND: Endothelial injury is considered critical for progression of atherosclerosis and its complications in coronary artery disease (CAD). The endothelial-supportive effects of bradykinin have mainly been attributed to activation of the resident endothelium. Here we newly investigate the role of bradykinin and its B2 receptor for the recruitment and functional activation of circulating mononuclear cell subsets with endothelial-repair promoting capacity, such as CD34(+)CXCR4(+)cells, at sites of arterial injury. METHODS AND RESULTS: Bradykinin-B2-receptor (B2R) blockade by icatibant substantially impaired recruitment of circulating CD34(+)CXCR4(+) mononuclear cells (expressing high levels of B2R) to endothelial cells in vitro and to injured arterial wall in vivo, whereas recruitment of CD14(hi) monocytes (expressing low levels of B2R) was unchanged. Moreover, the capacity of genetically B2R-deficient bone marrow cells to promote endothelial repair in vivo was markedly impaired as compared with wild-type bone marrow cells. B2R expression was reduced on CD34(+)CXCR4(+)mononuclear cells and endothelial repair-promoting early outgrowth cells, but not on CD14(hi)monocytes, from CAD patients as compared with healthy subjects. B2R stimulation induced CD18 activation in early outgrowth cells of healthy subjects, but not in early outgrowth cells of CAD patients. Adenoviral B2R overexpression enhanced in vivo vascular recruitment and rescued impaired endothelial repair capacity of early outgrowth cells from CAD patients. CONCLUSIONS: We newly report that bradykinin/B2R signaling may promote endothelial repair after arterial injury by selective recruitment and functional activation of B2R-expressing circulating mononuclear cell subsets. In CAD patients, B2R downregulation on endothelial repair-promoting circulating mononuclear cells substantially impairs the bradykinin-dependent endothelial repair, representing a novel mechanism promoting endothelial injury in CAD patients.

The kinin B1 receptor regulates muscle-specific E3 ligases expression and is involved in skeletal muscle mass control.

Regulation of muscle mass depends on the balance between synthesis and degradation of proteins, which is under the control of different signalling pathways regulated by hormonal, neural and nutritional stimuli. Such stimuli are altered in several pathologies, including COPD (chronic obstructive pulmonary disease), diabetes, AIDS and cancer (cachexia), as well as in some conditions such as immobilization and aging (sarcopenia), leading to muscle atrophy, which represents a significant contribution to patient morbidity. The KKS (kallikrein-kinin system) is composed of the enzymes kallikreins, which generate active peptides called kinins that activate two G-protein-coupled receptors, namely B1 and B2, which are expressed in a variety of tissues. The local modulation of the KKS may account for its participation in different diseases, such as those of the cardiovascular, renal and central nervous systems, cancer and many inflammatory processes, including pain. Owing to such pleiotropic actions of the KKS by local modulatory events and the probable fine-tuning of associated signalling cascades involved in skeletal muscle catabolic disorders [for example, NF-κB (nuclear factor κB) and PI3K (phosphoinositide 3-kinase)/Akt pathways], we hypothesized that KKS might contribute to the modulation of intracellular responses in atrophying skeletal muscle. Our results show that kinin B1 receptor activation induced a decrease in the diameter of C2C12 myotubes, activation of NF-κB, a decrease in Akt phosphorylation levels, and an increase in the mRNA levels of the ubiquitin E3 ligases atrogin-1 and MuRF-1 (muscle RING-finger protein-1). In vivo, we observed an increase in kinin B1 receptor mRNA levels in an androgen-sensitive model of muscle atrophy. In the same model, inhibition of the kinin B1 receptor with a selective antagonist resulted in an impairment of atrogin-1 and MuRF-1 expression and IκB (inhibitor of NF-κB) phosphorylation. Moreover, knockout of the kinin B1 receptor in mice led to an impairment in MuRF-1 mRNA expression after induction of LA (levator ani) muscle atrophy. In conclusion, using pharmacological and gene-ablation tools, we have obtained evidence that the kinin B1 receptor plays a significant role in the regulation of skeletal muscle proteolysis in the LA muscle atrophy model.

Neuronal and non-neuronal bradykinin receptors are involved in the contraction and/or relaxation to the pig bladder neck smooth muscle.

AIMS: The current study investigates the role played by bradykinin (BK) receptors in the contractility to the pig bladder neck smooth muscle. METHODS: Bladder neck strips were mounted in myographs for isometric force recordings and BK receptors expression was also determined by immunohistochemistry. RESULTS: B2 receptor expression was observed in the muscular layer and urothelium whereas B1 expression was consistent detected in urothelium. A strong B2 immunoreactivity was also observed within nerve fibers among smooth muscle bundles. On urothelium-denuded preparations basal tone, BK induced concentration-dependent contractions which were reduced in urothelium-intact samples, by extracellular Ca(2+) removal and by blockade of B2 receptors and voltage-gated Ca(2+) (VOC) and non-VOC channels, and increased by cyclooxygenase (COX) inhibition. On phenylephrine-precontracted denuded strips, under non-adrenergic non-cholinergic (NANC) conditions, electrical field stimulation-elicited frequency-dependent relaxations which were reduced by B2 receptor blockade. In urothelium-intact samples, the B1 receptor agonist kallidin promoted concentration-dependent relaxations which were reduced by blockade of B1 receptors, COX, COX-1 and large-conductance Ca(2+) -activated K(+) (BKCa ) channels and abolished in urothelium-denuded samples and in K(+) -enriched physiological saline solution-precontracted strips. CONCLUSIONS: These results suggest that BK produces contraction of pig bladder neck via smooth muscle B2 receptors coupled to extracellular Ca(2+) entry via VOC and non-VOC channels with a minor role for intracellular Ca(2+) mobilization. Facilitatory neuronal B2 receptors modulating NANC inhibitory neurotransmission and urothelial B1 receptors producing relaxation via the COX-1 pathway and BKCa channel opening are also demonstrated. Neurourol. Urodynam. 33:558-565, 2014. © 2013 Wiley Periodicals, Inc.

Pulmonary oedema producing toxin from Mesobuthus tamulus venom augments cardio-respiratory reflexes through B2 kinin receptors.

The current study was undertaken to compare the effects of pulmonary oedema producing toxin (PO-Tx) isolated from Mesobuthus tamulus venom on cardio-respiratory reflexes with exogenously administered bradykinin (BK) and to delineate the type of BK receptors mediating these responses. Jugular venous injection of phenyldiguanide (PDG) in anaesthetized rats produced reflex bradycardia, hypotension and apnoea. The PDG-induced reflex was augmented (two folds) by PO-Tx. The pulmonary water content in PO-Tx treated group was also increased. The PO-Tx-induced reflex changes as well as pulmonary oedema were blocked by-Hoe-140 implicating the involvement of B2 kinin receptors. Exogenous BK also produced augmentation (two folds) of the PDG-induced reflexes and increased the pulmonary water content. The BK-induced augmentation was blocked by pre-treatment with des-Arg10 Hoe 140 (a B1 receptor antagonist) and Hoe 140 (B2 receptor antagonist). However, these antagonists did not prevent the development of BK-induced pulmonary oedema. Present results indicate that PO-Tx augmented the PDG-induced reflex responses similar to BK and the PO-Tx induced augmentation of reflexes is mediated through B2 receptors.

Bradykinin B2 receptor contributes to the exaggerated muscle mechanoreflex in rats with femoral artery occlusion.

Static muscle contraction activates the exercise pressor reflex, which in turn increases sympathetic nerve activity (SNA) and blood pressure (BP). Bradykinin (BK) is considered as a muscle metabolite responsible for modulation of the sympathetic and cardiovascular responses to muscle contraction. Prior studies have suggested that kinin B2 receptor mediates the effects of BK on the reflex SNA and BP responses during stimulation of skeletal muscle afferents. In patients with peripheral artery disease and a rat model with femoral artery ligation, amplified SNA and BP responses to static exercise were observed. This dysfunction of the exercise pressor reflex has previously been shown to be mediated, in part, by muscle mechanoreflex overactivity. Thus, in this report, we determined whether kinin B2 receptor contributes to the augmented mechanoreflex activity in rats with 24 h of femoral artery occlusion. First, Western blot analysis was used to examine protein expression of B2 receptors in dorsal root ganglion tissues of control limbs and ligated limbs. Our data show that B2 receptor displays significant overexpression in ligated limbs as compared with control limbs (optical density: 0.94 ± 0.02 in control and 1.87 ± 0.08 after ligation, P < 0.05 vs. control; n = 6 in each group). Second, mechanoreflex was evoked by muscle stretch and the reflex renal SNA (RSNA) and mean arterial pressure (MAP) responses to muscle stretch were examined after HOE-140, a B2 receptors blocker, was injected into the arterial blood supply of the hindlimb muscles. The results demonstrate that the stretch-evoked reflex responses were attenuated by administration of HOE-140 in control rats and ligated rats; however, the attenuating effects of HOE-140 were significantly greater in ligated rats, i.e., after 5 µg/kg of HOE-140 RSNA and MAP responses evoked by 0.5 kg of muscle tension were attenuated by 43% and 25% in control vs. 54% and 34% in ligation (P < 0.05 vs. control group; n = 11 in each group). In contrast, there was no significant difference in B1 receptor expression in both experimental groups, and arterial injection of R-715, a B1 receptors blocker, had no significant effects on RSNA and MAP responses evoked by muscle stretch. Accordingly, results obtained from this study support our hypothesis that heightened kinin B2 receptor expression in the sensory nerves contributes to the exaggerated muscle mechanoreflex in rats with femoral artery occlusion.

Arteriogenesis is modulated by bradykinin receptor signaling.

RATIONALE: Positive outward remodeling of pre-existing collateral arteries into functional conductance arteries, arteriogenesis, is a major endogenous rescue mechanism to prevent cardiovascular ischemia. Collateral arterial growth is accompanied by expression of kinin precursor. However, the role of kinin signaling via the kinin receptors (B1R and B2R) in arteriogenesis is unclear. OBJECTIVE: The purpose of this study was to elucidate the functional role and mechanism of bradykinin receptor signaling in arteriogenesis. METHODS AND RESULTS: Bradykinin receptors positively affected arteriogenesis, with the contribution of B1R being more pronounced than B2R. In mice, arteriogenesis upon femoral artery occlusion was significantly reduced in B1R mutant mice as evidenced by reduced microspheres and laser Doppler flow perfusion measurements. Transplantation of wild-type bone marrow cells into irradiated B1R mutant mice restored arteriogenesis, whereas bone marrow chimeric mice generated by reconstituting wild-type mice with B1R mutant bone marrow showed reduced arteriogenesis after femoral artery occlusion. In the rat brain 3-vessel occlusion arteriogenesis model, pharmacological blockade of B1R inhibited arteriogenesis and stimulation of B1R enhanced arteriogenesis. In the rat, femoral artery ligation combined with arterial venous shunt model resulted in flow-driven arteriogenesis, and treatment with B1R antagonist R715 decreased vascular remodeling and leukocyte invasion (monocytes) into the perivascular tissue. In monocyte migration assays, in vitro B1R agonists enhanced migration of monocytes. CONCLUSIONS: Kinin receptors act as positive modulators of arteriogenesis in mice and rats. B1R can be blocked or therapeutically stimulated by B1R antagonists or agonists, respectively, involving a contribution of peripheral immune cells (monocytes) linking hemodynamic conditions with inflammatory pathways.

Decreased nerve growth factor upregulation is a mechanism for reduced mechanical hyperalgesia after the second bout of exercise in rats.

Delayed onset muscle soreness (DOMS) is reduced when the same exercise is repeated after a certain interval. However, the mechanism for this adaptation, called a repeated bout effect, is still not well understood. Recently, we showed that upregulated nerve growth factor (NGF) triggered by B2 bradykinin receptor (B2R) activation in exercised muscle was responsible for DOMS. In this study, we investigated whether NGF upregulation was reduced after repeated bouts of exercise in rats, and if so, whether this change occurred upstream of B2R. A bout of 500 lengthening contractions (LC) was applied on day 0 and again 5 days later. DOMS was evaluated by the mechanical withdrawal threshold of the exercised extensor digitorum longus (EDL) muscle. Mechanical hyperalgesia and NGF mRNA upregulation in EDL were observed after the first LC, but not after the second LC. We then injected HOE140, a B2R antagonist with effects lasting only several hours, once before the first LC. This blocked the development of mechanical hyperalgesia and NGF mRNA upregulation not only after the first LC but also after the second LC. This suggests that adaptation occurred upstream of B2R, as the influence of the first LC was limited to that area by HOE140.

Bradykinin promotes the chemotactic invasion of primary brain tumors.

Primary brain tumors, gliomas, diffusely invade the brain by active cell migration either intraparenchymal, along white matter tracts or along blood vessels. The close relationship of glioma with the vasculature assures a continuous supply of oxygen and nutrients essential for cell growth, and exposes cells to a variety growth factors, chemokines, cytokines, and kinins. Signals that attract glioma cells to blood vessels are poorly understood. It has been shown that vascular endothelial cells can initiate the bradykinin (BK) signaling cascade and two bradykinin receptors, B1 and B2, have been identified and cloned. In this study we show that glioma cells isolated from patient biopsies express bradykinin 2 receptors (B2R) whose activation causes intracellular Ca(2+) oscillations. Through time-lapse video-microscopy experiments we show that BK significantly enhances glioma cell migration/invasion. We further show that BK acts as a chemoattractant guiding glioma cells toward blood vessels in acute rat brain slices. The number of cells associated with blood vessels is decreased when B2R are either pharmacologically inhibited or B2R eliminated through short-hairpin RNA knockdown. These data strongly suggest that bradykinin, acting via B2R, acts as an important signal directing the invasion of glioma cells toward blood vessels. A clinically approved B2R antagonist is available that could be used as anti-invasive drug in glioma patients in the future.

The kallikrein-kinin system in diabetic nephropathy.

Diabetic nephropathy is the major cause of end-stage renal disease worldwide. Although the renin-angiotensin system has been implicated in the pathogenesis of diabetic nephropathy, angiotensin I-converting enzyme inhibitors have a beneficial effect on diabetic nephropathy independently of their effects on blood pressure and plasma angiotensin II levels. This suggests that the kallikrein-kinin system (KKS) is also involved in the disease. To study the role of the KKS in diabetic nephropathy, mice lacking either the bradykinin B1 receptor (B1R) or the bradykinin B2 receptor (B2R) have been commonly used. However, because absence of either receptor causes enhanced expression of the other, it is difficult to determine the precise functions of each receptor. This difficulty has recently been overcome by comparing mice lacking both receptors with mice lacking each receptor. Deletion of both B1R and B2R reduces nitric oxide (NO) production and aggravates renal diabetic phenotypes, relevant to either lack of B1R or B2R, demonstrating that both B1R and B2R exert protective effects on diabetic nephropathy presumably via NO. Here, we review previous epidemiological and experimental studies, and discuss novel insights regarding the therapeutic implications of the importance of the KKS in averting diabetic nephropathy.

Downregulation of Bradykinin type 2 receptor expression in cardiac endothelial cells during senescence.

OBJECTIVES: Bradykinin type 2 receptor (BK-2R) knockout mice develop microvascular dysfunction and cardiac hypertrophy. In aged human cardiac microvascular endothelium, dysfunction develops before heart failure symptoms. Since endothelial aging is an independent risk factor for cardiovascular disease, we aimed to clarify the role of kinin receptors in age-related endothelial senescence. METHODS AND RESULTS: Using qRT-PCR, a downregulation of BK-2Rs during senescence of cultured human coronary artery endothelial cells (HCAECs) and rat cardiac microvascular endothelial cells (RCMECs) was observed. BK-2R downregulation was associated with a decreased cell proliferation rate, with a growth arrest phenotype and reduced angiogenic potential. By staining senescence-associated ß-galactosidase, RCMECs from old spontaneously hypertensive rats (SHRs) were found to be significantly more senescent than those derived from age-matched WKY rats, albeit their telomere lengths were similar. Despite downregulation of BK-2Rs and BK-1Rs, a novel family member GPR-100 was highly expressed in HCAECs throughout the culture period. CONCLUSIONS: Aging cardiac endothelial cells gradually lose their capacity to express BK-2Rs, and this loss appears to be parallel with a loss of the angiogenic potential of the aging cells. Since RCMECs from hypertensive rats showed premature senescence, hypertension may predispose to cardiac dysfunction by accelerating endothelial aging.

Novel Pharmacology Following Heteromerization of the Angiotensin II Type 2 Receptor and the Bradykinin Type 2 Receptor.

The angiotensin type 2 (AT2) receptor and the bradykinin type 2 (B2) receptor are G protein-coupled receptors (GPCRs) that have major roles in the cardiovascular system. The two receptors are known to functionally interact at various levels, and there is some evidence that the observed crosstalk may occur as a result of heteromerization. We investigated evidence for heteromerization of the AT2 receptor and the B2 receptor in HEK293FT cells using various bioluminescence resonance energy transfer (BRET)-proximity based assays, including the Receptor Heteromer Investigation Technology (Receptor-HIT) and the NanoBRET ligand-binding assay. The Receptor-HIT assay showed that Gαq, GRK2 and ß-arrestin2 recruitment proximal to AT2 receptors only occurred upon B2 receptor coexpression and activation, all of which is indicative of AT2-B2 receptor heteromerization. Additionally, we also observed specific coupling of the B2 receptor with the Gαz protein, and this was found only in cells coexpressing both receptors and stimulated with bradykinin. The recruitment of Gαz, Gαq, GRK2 and ß-arrestin2 was inhibited by B2 receptor but not AT2 receptor antagonism, indicating the importance of B2 receptor activation within AT2-B2 heteromers. The close proximity between the AT2 receptor and B2 receptor at the cell surface was also demonstrated with the NanoBRET ligand-binding assay. Together, our data demonstrate functional interaction between the AT2 receptor and B2 receptor in HEK293FT cells, resulting in novel pharmacology for both receptors with regard to Gαq/GRK2/ß-arrestin2 recruitment (AT2 receptor) and Gαz protein coupling (B2 receptor). Our study has revealed a new mechanism for the enigmatic and poorly characterized AT2 receptor to be functionally active within cells, further illustrating the role of heteromerization in the diversity of GPCR pharmacology and signaling.

Bradykinin promotes migration and invasion of human immortalized trophoblasts.

Having demonstrated that the bradykinin B2 receptor (B2R) is expressed in cells that participate in trophoblast invasion in humans and guinea-pigs, we investigated the role of bradykinin (BK) on cell migration and invasion in the HTR-8/SVneo trophoblast cell line using wound healing and invasion assays. First, we documented that HTR-8/SVneo cells expressed kallikrein, B2R, B1R, MMP-2 and MMP-9 using immunocytochemistry. Incubation with BK (10.0 microMol/L) for 18 hours increased the migration index 3-fold in comparison to controls or to cells preincubated with the B2R antagonist HOE-140. BK (10.0 microMol/L) incubation yielded a similar number of proliferating and viable cells as controls, therefore the enhanced closure of the wound cannot be attributed to proliferating cells. Incubation with BK (10.0 microMol/L) for 18 hours increased the invasion index 2-fold in comparison to controls or to cells preincubated with the antagonist of the B2R. Neither the B1R ligand Lys-des-Arg9 BK, nor its antagonist Lys-(des-Arg9-Leu8), modified migration and invasion. Further support for the stimulatory effect of B2R activation on migration and invasion is provided by the 3-fold increase in the number of filopodia per cell versus controls or cells preincubated with the B2R antagonist. Bradykinin had no effect on the cellular protein content of the B2R, nor the MMP-9 and MMP-2 gelatinase activity in the culture media varied after incubation with BK. This study adds bradykinin-acting on the B2R-to the stimuli of trophoblast migration and invasion, an effect that should be integrated to other modifications of the kallikrein-kinin system in normal and pathological pregnancies.

Contribution of bradykinin B2 receptors to the inhibition by valsartan of systemic and renal effects of exogenous angiotensin II in salt-repleted humans.

To investigate whether bradykinin (BK) participates in the inhibition of renal effects of exogenous angiotensin II (AngII) by AngII type 1 receptor (AT1R) blockade, eight salt-repleted volunteers underwent four p-aminohippurate- and inulin-based renal studies of AngII infusion at increasing rates of 0.625, 1.25, and 2.5 ng.kg.min(-1) for 30 min. Studies 1 and 2 were preceded by 3 days of placebo, whereas studies 3 and 4 used 240 to 320 mg.day(-1) valsartan. Bradykinin B2-type receptor (BKB2R) antagonist icatibant (50 mug.kg(-1)) was coinfused in studies 2 and 4. Mean blood pressure (MBP), glomerular filtration rate (GFR), renal blood flow (RBF), and renal sodium excretion (UNaV) were measured. In study 1, MBP rose by 12.8%, UNaV decreased by 68%, and GFR and RBF also fell (p < 0.001 for all). In study 2, GFR and RBF fell as in study 1, but the rise in MBP and the fall in UNaV were accentuated [+20.0%, analysis of variance (ANOVA), p < 0.02 versus study 1 and -80.0%, p < 0.05, respectively]. In study 3, AngII had no effects, and in study 4, renal hemodynamics remained unaffected, but MBP still rose and UNaV fell (ANOVA, p < 0.02 and 0.005 versus study 3, respectively). Icatibant accentuated AngII-induced changes in MBP and UNaV. Previous AT1R blockade prevented any systemic and renal effects of AngII, but significant changes in MBP and UNaV still followed AngII plus icatibant even after AT1R blockade. BK, through BKB2Rs, participates in the inhibitory action of AT1R blockers toward actions of exogenous AngII on MBP and UNaV in healthy humans.

Bradykinin decreases podocyte permeability through ADAM17-dependent epidermal growth factor receptor activation and zonula occludens-1 rearrangement.

Recent data show that increases in bradykinin (BK) concentration contribute to the beneficial effects of angiotensin-converting enzyme inhibitor (ACEI) treatment in chronic kidney disease. However, the possible role of BK in attenuated proteinuria, often seen in ACEI-treated patients, is not well studied. Here, we report that BK decreases mouse podocyte permeability through rearrangement of the tight junction protein zonula occludens-1 (ZO-1) and identify some of the major signaling events leading to permeability change. We show that BK2 receptor (BK2R) stimulation transactivates the epidermal growth factor receptor (EGFR). EGFR transactivation is mediated by a disintegrin and metalloenzyme (ADAM) family members, which are required for both extracellular signal-regulated kinase (ERK) and EGFR activation by BK. Using a gene-silencing approach we observed that both BK-induced ERK activation and BK-induced permeability decrease in podocytes is attenuated by ADAM17 down-regulation, and we identified epiregulin (ER) as the EGFR ligand participating in ADAM-dependent BK2R-EGFR cross-talk. EGFR inhibition attenuated both ZO-1 rearrangement and BK-induced permeability decreases in podocyte. We propose that ZO-1 redistribution is an important element of BK-induced permeability change and the signaling events involved in ZO-1 rearrangement include transactivation of the EGFR via ADAM17 activation and ER shedding. Our data indicate that ADAM17 and the EGFR may be potential novel therapeutic targets in diabetic nephropathy and other chronic kidney diseases.

Role of kinin B2 receptor signaling in the recruitment of circulating progenitor cells with neovascularization potential.

Reduced migratory function of circulating angiogenic progenitor cells (CPCs) has been associated with impaired neovascularization in patients with cardiovascular disease (CVD). Previous findings underline the role of the kallikrein-kinin system in angiogenesis. We now demonstrate the involvement of the kinin B2 receptor (B(2)R) in the recruitment of CPCs to sites of ischemia and in their proangiogenic action. In healthy subjects, B(2)R was abundantly present on CD133(+) and CD34(+) CPCs as well as cultured endothelial progenitor cells (EPCs) derived from blood mononuclear cells (MNCs), whereas kinin B1 receptor expression was barely detectable. In transwell migration assays, bradykinin (BK) exerts a potent chemoattractant activity on CD133(+) and CD34(+) CPCs and EPCs via a B(2)R/phosphoinositide 3-kinase/eNOS-mediated mechanism. Migration toward BK was able to attract an MNC subpopulation enriched in CPCs with in vitro proangiogenic activity, as assessed by Matrigel assay. CPCs from cardiovascular disease patients showed low B(2)R levels and decreased migratory capacity toward BK. When injected systemically into wild-type mice with unilateral limb ischemia, bone marrow MNCs from syngenic B(2)R-deficient mice resulted in reduced homing of sca-1(+) and cKit(+)flk1(+) progenitors to ischemic muscles, impaired reparative neovascularization, and delayed perfusion recovery as compared with wild-type MNCs. Similarly, blockade of the B(2)R by systemic administration of icatibant prevented the beneficial effect of bone marrow MNC transplantation. BK-induced migration represents a novel mechanism mediating homing of circulating angiogenic progenitors. Reduction of BK sensitivity in progenitor cells from cardiovascular disease patients might contribute to impaired neovascularization after ischemic complications.

Bradykinin and high glucose promote renal tubular inflammation.

BACKGROUND: The role of the kallikrein-kinin system in diabetic nephropathy remains controversial. METHODS AND RESULTS: High-glucose (HG) super-induced interleukin (IL)-6, CCL-2, transforming growth factor (TGF)-beta, vascular endothelial growth factor (VEGF) and B(2)K receptor (B(2)KR) mRNA in cultured proximal tubular epithelial cells (PTEC), whereas bradykinin (BK) upregulated IL-6, CCL-2 and TGF-beta mRNA. HG activated mitogen-activated protein kinase (MAPK) p42/p44 and protein kinase C (PKC) signals, whereas BK only activated MAPK. Tubular expression of these mediators and tissue kallikrein 1 (KLK1) was confirmed in human diabetic kidney biopsies. Inhibition of MAPK p42/p44 by PD98059 partially reduced HG and BK induction of IL-6, CCL-2 and TGF-beta, whereas inhibition of PKC by staurosporine partially reduced HG- but not BK-induced overexpression of these cytokines and that of VEGF. Staurosporine and PD98059 synergistically reduced the effect of HG on IL-6, CCL-2 and TGF-beta expression. The B(2)KR blocker, icatibant, downregulated BK- and HG-induced MAPK p42/p44 but not HG-induced PKC activation and partially reduced both HG- and BK-induced IL-6, CCL-2 and TGF-beta secretion. HG stimulated expression of KLK1 and low-molecular-weight kininogen (LMWK) and its downstream effects were attenuated by aprotinin (tissue kallikrein inhibitor). The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist, rosiglitazone, attenuated HG-induced PKC but not HG- or BK- induced MAPK p42/44 activation and reduced HG-stimulated VEGF, along with IL-6, CCL-2 and TGF-beta secretion. Rosiglitazone plus icatibant further reduced these effects of HG. CONCLUSIONS: In conclusion, HG stimulates tubular proinflammatory, profibrotic and angiogenic signals, which is partly mediated through BK via MAPK signalling and partly through PKC independent of BK. The potential therapeutic role of complementary B(2)KR blockade and PPAR-gamma activation deserves clinical investigation.

Atorvastatin protects against angiotensin II-induced injury and dysfunction in human umbilical vein endothelial cells through bradykinin 2 receptors.

The morphological and functional integrity of the endothelial cell (EC) is compromised in many cardiovascular diseases such as atherosclerosis, hypertension, and diabetes. Angiotensin II (Ang II) plays important roles in the initiation and progression of these diseases. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) may have cholesterol-independent pleiotropic effects on preventing the EC injury and dysfunction that occurs in these diseases, and the protective effects may relate to bradykinin 2 receptors (B2Rs). Our study was designed to test the hypothesis that atorvastatin, via B2Rs, protects the viability and function of EC exposed to Ang II independent of hemodynamics. The experimental results showed that the cytotoxic effects of Ang II on human umbilical vein endothelial cells were significantly ameliorated by atorvastatin pretreatment (LDH tests, MTT assay, and propdium iodide (PI)/Annexin V-stating analysis), and atorvastatin treatment simultaneously enhanced expression of endothelial nitric oxide synthase and yielded of nitric oxide (NO) and cyclic guanosine monophosphate, but both effects were attenuated by the B2Rs antagonist HOE-140. This study proves the hypothesis and may be pertinent to the complex mechanism of action of statins explaining their long-term beneficial effects in maintaining the morphological and functional integrity of vascular ECs.

Endogenous bradykinin contributes to increased plasminogen activator inhibitor 1 antigen following hemodialysis.

Oxidative stress and inflammation predict cardiovascular events in chronic hemodialysis patients. Hemodialysis activates the kallikrein-kinin system, increasing bradykinin. Bradykinin promotes inflammation but also stimulates endothelial release of tissue-plasminogen activator and inhibits platelet aggregation. Understanding the detrimental and beneficial effects of endogenous bradykinin during hemodialysis has implications for the treatment of cardiovascular disease in the hemodialysis population. To test the hypothesis that bradykinin contributes to the inflammatory and fibrinolytic responses to dialysis, we conducted a double-blind, randomized, placebo-controlled crossover study comparing the effect of the bradykinin B(2) receptor blocker HOE-140 with vehicle on markers of oxidative stress, inflammation, fibrinolysis, and coagulation in nine hemodialysis patients without coronary artery disease. Bradykinin receptor antagonism did not affect the mean arterial pressure or heart rate response to dialysis. Monocyte chemoattractant protein 1 (MCP-1) peaked postdialysis; HOE-140 blunted the increase in MCP-1 (5.9 +/- 5.9 versus 25.6 +/- 20.1 pg/ml, P = 0.01). HOE-140 also abolished the increase in plasminogen activator inhibitor 1 (PAI-1) antigen observed at the end of dialysis. In contrast, HOE-140 significantly accentuated the effect of dialysis on F(2)-isoprostanes and P-selectin. Taken together, these results suggest that endogenous bradykinin contributes to increases in MCP-1 and PAI-1 antigen after hemodialysis via its B(2) receptor. Factors that increase the production of bradykinin or decrease its degradation may enhance the inflammatory response to hemodialysis.