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Pharmacological research in mice and human genetic analyses suggest that the kallikrein-kinin system (KKS) may regulate anxiety. We examined the role of the KKS in anxiety and stress in both species. In human genetic association analysis, variants in genes for the bradykinin precursor (KNG1) and the bradykinin receptors (BDKRB1 and BDKRB2) were associated with anxiety disorders (p < 0.05). In mice, however, neither acute nor chronic stress affected B1 receptor gene or protein expression, and B1 receptor antagonists had no effect on anxiety tests measuring approach-avoidance conflict. We thus focused on the B2 receptor and found that mice injected with the B2 antagonist WIN 64338 had lowered levels of a physiological anxiety measure, the stress-induced hyperthermia (SIH), vs controls. In the brown adipose tissue, a major thermoregulator, WIN 64338 increased expression of the mitochondrial regulator Pgc1a and the bradykinin precursor gene Kng2 was upregulated after cold stress. Our data suggests that the bradykinin system modulates a variety of stress responses through B2 receptor-mediated effects, but systemic antagonists of the B2 receptor were not anxiolytic in mice. Genetic variants in the bradykinin receptor genes may predispose to anxiety disorders in humans by affecting their function.

The kallikrein-kinin system (KKS) consists of two major cascades in mammals: "plasma KKS" consisting of high molecular-weight (HMW) kininogen (KNG), plasma kallikrein (KLKB1), and bradykinin (BK); and "tissue KKS" consisting of low molecular-weight (LMW) KNG, tissue kallikreins (KLKs), and [Lys(0)]-BK. Some components of the KKS have been identified in the fishes, but systematic analyses have not been performed, thus this study aims to define the KKS components in teleosts and pave a way for future physiological and evolutionary studies. Through a combination of genomics, molecular, and biochemical methods, we showed that the entire plasma KKS cascade is absent in teleosts. Instead of two KNGs as found in mammals, a single molecular weight KNG was found in various teleosts, which is homologous to the mammalian LMW KNG. Results of molecular phylogenetic and synteny analyses indicated that the all current teleost genomes lack KLKB1, and its unique protein structure, four apple domains and one trypsin domain, could not be identified in any genome or nucleotide databases. We identified some KLK-like proteins in teleost genomes by synteny and conserved domain analyses, which could be the orthologs of tetrapod KLKs. A radioimmunoassay system was established to measure the teleost BK and we found that [Arg(0)]-BK is the major circulating form instead of BK, which supports that the teleost KKS is similar to the mammalian tissue KKS. Coincidently, coelacanths are the earliest vertebrate that possess both HMW KNG and KLKB1, which implies that the plasma KKS could have evolved in the early lobe-finned fish and descended to the tetrapod lineage. The co-evolution of HMW KNG and KLKB1 in lobe-finned fish and early tetrapods may mark the emergence of the plasma KKS and a contact activation system in blood coagulation, while teleosts may have retained a single KKS cascade.

Radiotherapy of intrathoracic and chest wall tumors may lead to exposure of the heart to ionizing radiation, resulting in radiation-induced heart diseases (RIHD). The main manifestations of RIHD become apparent many years after treatment and include cardiomyopathy and accelerated atherosclerosis. This study examines the role of the kallikrein-kinin system (KKS) in RIHD by investigating the cardiac radiation response in a kininogen-deficient Brown Norway Katholiek (BN/Ka) rat model. BN/Ka rats and wild-type Brown Norway (BN) rats were exposed to local heart irradiation with a single dose of 18 Gy or 24 Gy and were observed for 3 to 6 months. Examinations included in vivo and ex vivo cardiac function, histopathology, gene and protein expression measurements, and mitochondrial swelling assays. Upon local heart irradiation, changes in in vivo cardiac function were significantly less in BN/Ka rats. For instance, a single dose of 24 Gy caused a 35% increase in fractional shortening in BN rats compared with a 16% increase in BN/Ka rats. BN rats, but not BN/Ka rats, showed a 56% reduction in cardiac numbers of CD2-positive cells, and a 57% increase in CD68-positive cells, together with a 52% increase in phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2). Local heart irradiation had similar effects on histopathology, mitochondrial changes, and left ventricular mRNA levels of NADPH oxidases in the two genotypes. These results suggest that the KKS plays a role in the effects of radiation on cardiac function and recruitment of inflammatory cells. The KKS may have these effects at least in part by altering Erk1/2 signaling., (©2012 AACR.)

The kallikrein-kinin system (KKS) has been described as an important mediator of physiologic processes. Kallikreins use kininogen (KNG) as substrate to generate bradykinin, the main active peptide of the KKS that acts through two types of receptors, the B(1)R and the B(2)R. The goal of this study was to characterize some components of the KKS in different compartments of the ovary during the bovine ovulation process. The KNG, B(1)R and B(2)R mRNA expression patterns were assessed in theca and granulosa cells, as well as the bradykinin concentration and kallikrein-like activity in follicular fluid of bovine periovulatory follicles. To obtain a periovulatory follicle (≥12 mm), twenty-seven cows were submitted to estrus synchronization protocol and ovariectomized by colpotomy at 0, 3, 6, 12 or 24h after a GnRH-analog injection (gonadorelin; 100 μg, IM). Follicular fluid was aspirated for enzymatic assays while granulosa and theca cells were harvested for mRNA analysis. The mRNA expressions in follicular cells were evaluated by real-time RT-PCR and data representation related to the cyclophilin housekeeping gene. The bradykinin concentration and kallikrein-like activity were measured in follicular fluid by enzymatic immunoassay and selective substrate cleavage, respectively. The B(2)R expression in theca cells and B(1)R expression in theca and granulosa cells showed different profiles during the periovulatory period (P<0.05). The bradykinin concentration and kallikrein-like activity in the follicular fluid were different (P<0.05) due to the time during the ovulation process. KNG mRNA expression was similar for both follicular cell types (P>0.05). Taken together, these results provide an important characterization of the presence and possible KKS regulation during the bovine ovulation., (Copyright © 2011. Published by Elsevier Inc.)

The aim of the present study was to investigate whether pharmacological enhancement of the renal kallikrein-kinin system using the vasopeptidase inhibitor omapatrilat plays a direct role in modulating the fibrotic responses of human mesangial cells to injury. Treatment with 40 micromol/L omapatrilat was able to reduce macrophage-conditioned medium (MPCM)-induced fibronectin levels without affecting mRNA expression. MPCM injury also suppressed kallikrein and low molecular weight kininogen mRNA. Omapatrilat was able to attenuate this suppression. Bradykinin levels in contrast were increased by MPCM and treatment with omapatrilat further augmented levels. Co-incubation with the bradykinin B2 receptor antagonist HOE 140 attenuated the omapatrilat-induced lowering of fibronectin. Moreover, inhibition of cGMP release had a similar effect. Paradoxically, RT-PCR and Southern blotting demonstrated that bradykinin B2 receptor mRNA levels were down regulated in response to omapatrilat. Western blotting supported this data. Supernatant levels of tissue plasminogen activator (tPA), a product of bradykinin stimulation, were decreased by omapatrilat while cell associated tPA levels were increased. Matrix metalloproteinase-9 (MMP-9) mRNA expression was up regulated by omapatrilat treatment, although no difference in active zymogen levels was observed. In conclusion enhancement of kallikrein-kinin system appears to play a direct role in promoting anti-fibrotic responses in MPCM-injured human mesangial cells., (Copyright 2006 S. Karger AG, Basel.)

Inflammation is accompanied by activation of the plasma kallikrein-kinin system (KKS). KKS activation has been demonstrated in a variety of inflammatory human diseases. To further explore the participation of KKS in arthritis and inflammatory bowel disease, we used two experimental animal models in arthritis and enterocolitis. We found that activation of KKS is associated with arthritis induced by intraperitoneal injection of peptidoglycan-polysaccharide polymers (PG-PS) as well as the enterocolitis and systemic inflammation induced also by PG-PS when injected into the intestinal wall of genetically susceptible Lewis rats. We postulated that KKS participates in the pathogenesis of inflammatory reactions involved in cellular injury, coagulation, fibrinolysis, kinin formation, complement activation, cytokine secretion, and release of proteases. We demonstrated that therapy with a specific plasma kallikrein inhibitor modulated the experimental enterocolitis, arthritis, and systemic inflammation. The fact that deficiency of plasma high molecular weight kininogen in the genetically susceptible Lewis rat results in decreased chronic enterocolitis and systemic inflammation also supports our hypothesis. We suggest that KKS plays a similar role in idiopathic human intestinal inflammatory disease and arthritis, making kallikrein-kinin system proteins appealing targets for drug therapy in chronic inflammatory diseases such as rheumatoid arthritis and Crohn's disease.

Roles for the kallikrein-kinin system in inflammation have been investigated extensively, and many reviews on this topic have been published during the 50 years since the discovery of bradykinin in 1949. Recent progress in the field has been remarkable with the help of experiments using gene-targetted transgenic or knockout mice, which have added further valuable information in addition to previous results obtained from pharmacological and biochemical studies using purified and isolated components of the system. Furthermore, much knowledge has been accumulated as a result of the development of various bradykinin agonists and antagonists. In this review, we focused on the data obtained from the kininogen-deficient rat, which is a natural mutant, and discuss the results in comparison with those from bradykinin receptor knockout mice. These data have clarified that endogenous bradykinin exerts a most important role in inflammatory exudation along with prostanoids, preferentially to histamine, serotonin, or neuropeptides. In inflammatory pain perception also, bradykinin produced in the local perivascular spaces stimulates polymodal pain receptors in conjunction with co-helpers such as prostanoids, vanilloids, and neuropeptides. These important roles are concluded based on consistent results obtained from experiments using several antagonists of bradykinin, kininogen-deficient rats, and bradykinin receptor knockout mice.

Bradykinin and kallidin are released during dermal injury and inflammation as a result of activation of kallikreins which cleave high- and low-molecular weight kininogen (HMW and LMW kininogen, respectively). In the skin, kinins are involved, e.g., as co-mitogens in cellular proliferation or in processes propagating pain and inflammation. The aim of our study was to investigate the specific occurrence of mRNAs for components of the kallikrein-kinin system in normal human skin and in skin biopsies of patients with selected skin diseases (psoriasis, lichenificated atopic eczema, basalioma). In normal skin, reverse transcription polymerase chain reaction (RT-PCR) with specific primer pairs followed by separation of products by polyacrylamide gel electrophoresis (PAGE) revealed the presence of mRNAs for tissue kallikrein, for the B2 and the B1 bradykinin receptors, but not for kininogen. In biopsies of lichenificated atopic eczema and basalioma, additionally, the mRNAs for HMW and LMW kininogen were detected, whereas in psoriatic skin mRNA for HMW kininogen was not expressed. These differences in mRNA expression may reflect the different contribution of kallikrein-kinin system components to the maintenance of chronic skin diseases like psoriasis. In acute dermal reactions occurring in lichenificated atopic eczema or in basalioma, tissue mRNA for HMW kininogen appears to be arisen from sources not pre-existing in normal skin.

The kallikrein-kinin system (KKS) plays an important role in the regulation of renal function. Endogenous kinins modulate renal microvascular resistance, medullary blood flow, and distal nephron sodium and water reabsorption. All the components of the KKS, including tissue kallikrein, kininogen, kininase II, and kinin receptors are expressed within the kidney, establishing a paracrine system capable of controlling local nephron functions. In this review, data will be presented demonstrating that the developing kidney expresses an endogenous, functionally active KKS. Molecular studies have shown that gene expression of the renal KKS in the rat is activated postnatally, and that the intrarenal distribution of KKS components is subject to developmental control. Furthermore, the developmental expression of KKS appears to be regulated primarily at the transcriptional level. Ontogenetic studies have also revealed that the bradykinin B-2 receptor gene is overexpressed in the developing rat kidney. As kinins are potent vasoactive and growth-promoting factors, it is proposed that endogenous kinins mediate developmental renal growth and differentiation, and modulate the maturational changes which occur in renal hemodynamics.

Tissue kallikrein is a serine proteinase which processes kininogens to release bioactive kinins. Kinins mediate a variety of biological processes through the interaction with kinin receptors. Kinins are involved in the regulation of blood pressure and local blood flow, vasodilation, smooth muscle contraction and relaxation, production of pain and inflammation, and stimulation of cell proliferation. The tissue kallikrein-kinin system has been implicated in a number of pathophysiological processes such as hypertension, allergy and diabetes mellitus. In the present study, we have identified the expression and localization of components of the kallikrein-kinin system in the human eye by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analyses, and in situ hybridization histochemistry. RT-PCR and Southern blot analyses have detected mRNAs of the key components of the system including tissue kallikrein, low molecular weight kininogen, and bradykinin B1 and B2 receptors at high levels in human retina, choroid and ciliary body, and relatively low levels in the optic nerve. In situ hybridization has identified cellular localization of these four mRNAs in ocular tissues. They are expressed in retinal neuronal cells including the outer nuclear layer, inner nuclear layer and ganglion cell layer. These mRNAs were also identified in endothelial cells of ocular blood vessels, ciliary muscle and lens epithelial cells. The sense riboprobes showed negative staining, which indicates the specificity of the antisense riboprobes. These results suggest that the tissue kallikrein-kinin system is produced endogenously in human ocular tissues. Similar expression patterns of kallikrein, kininogen and kinin receptors indicate that the kallikrein-kinin system may function in an autocrine or paracrine fashion in the eye.