Tissue Kallikrein-kinin System Research Articles

Ulinastatin attenuated cardiac ischaemia/reperfusion injury by suppressing activation of the tissue kallikrein-kinin system.

Ulinastatin has beneficial effects in patients undergoing coronary artery bypass grafting (CABG) surgery due to its anti-inflammatory properties, but the underlying mechanism remains unclear. We used samples from patients undergoing CABG, a model of cardiac ischaemia-reperfusion injury (IRI) in mice and murine cardiac endothelial cell cultures to investigate links between ulinastatin, the kallikrein-kinin system (KKS), endothelial dysfunction and cardiac inflammation in the response to ischaemia/reperfusion injury (IRI). These links were assessed using clinical investigations, in vitro and in vivo experiments and RNA sequencing analysis. Ulinastatin inhibited the activity of tissue kallikrein, a key enzyme of the KKS, at 24 h after CABG surgery, which was verified in our murine cardiac ischaemia-reperfusion model. Under normal conditions, ulinastatin only inhibited kallikrein activity but did not affect bradykinin (B1/B2) receptors. Ulinastatin protected against IRI, in vivo and in vitro, by suppressing activation of the kallikrein-kinin system and down-regulating B1/B2 receptor-related signalling pathways including ERK/ iNOS, which resulted in enhanced endothelial barrier function, mitigation of inflammation and oedema, decreased infarct size, improved cardiac function and decreased mortality. Inhibition of kallikrein and knockdown of B1, but not B2 receptors prevented ERK translocation into the nucleus, reducing reperfusion-induced injury in murine cardiac endothelial cells. Treatment with ulinastatin exerts a protective influence on cardiac reperfusion by suppressing activation of the kallikrein-kinin system. Our findings highlight the potential of targeting kallikrein /bradykinin receptors to alleviate endothelial dysfunction, thus improving cardiac IRI.

Interrelation among exercise training, cardiac hypertrophy, and tissue kallikrein-kinin system in athlete and non-athlete women.

Introduction: The tissue kallikrein-kinin system is an endogenous homeostatic pathway, which its stimulation is associated with cardioprotection. The present study aimed to determine the effect of exercise training on plasma tissue kallikrein (TK) and bradykinin (BK) and their association with cardiac hypertrophy. Methods: 22 non-athlete and 22 athlete women were exposed to acute (Bruce test) and chronic (12-week swimming training) exercises. 2D echocardiography was used to evaluate morphological and functional features of the heart. Plasma concentrations of TK and BK were quantified by ELISA. Results: Athletes had significantly higher values of left ventricle end-diastolic diameter index (LVEDDI) and left ventricle mass index (LVMI) than non-athletes. Exercise intervention affected echocardiographic features in neither of the study groups. Chronic exercise training notably increased plasma levels of TK and BK, which increase was more pronounced in the athletes. Plasma TK negatively correlated with LVEDDI (r=-0.64, P=0.036 and r=-0.58, P=0.027) and LVMI (r=-0.51, P=0.032 and r=-0.63, P=0.028) in the non-athlete and athlete groups. In opposition, there was a positive correlation between plasma TK and left ventricle ejection fraction in non-athletes (r=0.39, P=0.049) and athletes (r=0.53, P=0.019). Conclusion: The upregulation of the tissue kallikrein-kinin system may be a protective mechanism against excessive cardiac hypertrophy induced by chronic exercise training.

In Vitro Modeling of Bradykinin-Mediated Angioedema States.

Kinins (peptides related to bradykinin, BK) are formed from circulating substrates, the kininogens, by the action of two proteases, the kallikreins. The only clinical application of a BK receptor ligand, the B2 receptor antagonist icatibant, is the treatment of the rare hereditary angioedema (HAE) caused by the deficiency of C1-esterase inhibitor (C1-INH). Less common forms of HAE (genetic variants of factor XII, plasminogen, kininogen) are presumably mediated by increased BK formation. Acquired forms of BK-mediated angioedema, such as that associated with angiotensin-I converting enzyme (ACE) inhibition, are also known. Antibody-based analytical techniques are briefly reviewed, and support that kinins are extremely short-lived, prominently cleared by ACE. Despite evidence of continuous activation of the kallikrein–kinin system in HAE, patients are not symptomatic most of the time and their blood or plasma obtained during remission does not generate excessive immunoreactive BK (iBK), suggesting effective homeostatic mechanisms. HAE-C1-INH and HAE-FXII plasmas are both hyperresponsive to fibrinolysis activation. On another hand, we suggested a role for the alternate tissue kallikrein–kinin system in patients with a plasminogen mutation. The role of the BK B1 receptor is still uncertain in angioedema states. iBK profiles under in vitro stimulation provide fresh insight into the physiopathology of angioedema.

Genetically‑modified stem cells in treatment of human diseases: Tissue kallikrein (KLK1)‑based targeted therapy (Review).

The tissue kallikrein-kinin system (KKS) is an endogenous multiprotein metabolic cascade which is implicated in the homeostasis of the cardiovascular, renal and central nervous system. Human tissue kallikrein (KLK1) is a serine protease, component of the KKS that has been demonstrated to exert pleiotropic beneficial effects in protection from tissue injury through its anti-inflammatory, anti-apoptotic, anti-fibrotic and anti-oxidative actions. Mesenchymal stem cells (MSCs) or endothelial progenitor cells (EPCs) constitute populations of well-characterized, readily obtainable multipotent cells with special immunomodulatory, migratory and paracrine properties rendering them appealing potential therapeutics in experimental animal models of various diseases. Genetic modification enhances their inherent properties. MSCs or EPCs are competent cellular vehicles for drug and/or gene delivery in the targeted treatment of diseases. KLK1 gene delivery using adenoviral vectors or KLK1 protein infusion into injured tissues of animal models has provided particularly encouraging results in attenuating or reversing myocardial, renal and cerebrovascular ischemic phenotype and tissue damage, thus paving the way for the administration of genetically modified MSCs or EPCs with the human tissue KLK1 gene. Engraftment of KLK1-modified MSCs and/or KLK1-modified EPCs resulted in advanced beneficial outcome regarding heart and kidney protection and recovery from ischemic insults. Collectively, findings from pre-clinical studies raise the possibility that tissue KLK1 may be a novel future therapeutic target in the treatment of a wide range of cardiovascular, cerebrovascular and renal disorders.

Protective Role of Kallistatin in Vascular and Organ Injury.

Kallistatin is an endogenous protein that exerts pleiotropic effects, including vasodilation and inhibition of angiogenesis, inflammation, oxidative stress, apoptosis, fibrosis, and tumor progression. Through its 2 functional domains—an active site and a heparin-binding site—kallistatin regulates differential signaling pathways and a wide spectrum of biological functions. Kallistatin’s active site is key for inhibiting tissue kallikrein activity and stimulating the expression of endothelial nitric oxide (NO) synthase (eNOS), sirtuin 1 (SIRT1), and suppressor of cytokine signaling 3 (SOCS3). Kallistatin via its heparin-binding site blocks signaling pathways mediated by growth factors and cytokines, such as vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α), high-mobility group box-1 (HMGB1), Wnt, transforming growth factor-β (TGF-β), and epidermal growth factor. Kallistatin gene or protein delivery protects against the pathogenesis of hypertension, heart and kidney damage, arthritis, sepsis, influenza virus infection, tumor growth, and metastasis in animal models. Conversely, depletion of endogenous kallistatin by neutralizing antibody injection exacerbates cardiovascular and renal injury in hypertensive rats. Kallistatin levels are markedly reduced in rodents with hypertension, sepsis, streptozotocin-induced diabetes mellitus, and cardiac and renal injury. Kallistatin levels are also diminished in patients with liver disease, septic syndrome, diabetic retinopathy, severe pneumonia, inflammatory bowel disease, obesity, and prostate, and colon cancer. Therefore, circulating kallistatin levels may serve as a new biomarker for human diseases. This review summarizes kallistatin’s protective roles and mechanisms in vascular and organ injury and highlights the therapeutic potential of kallistatin for multiple disease states. Kallistatin was discovered in human plasma as a tissue kallikrein–binding protein.1 Sequence analysis indicated that tissue kallikrein–binding protein is a novel serine proteinase inhibitor (serpin) and was later designated kallistatin because of its ability to inhibit tissue kallikrein activity.2–9 Tissue kallikrein is a serine proteinase that cleaves low–molecular weight kininogen substrate to release vasoactive kinin peptides.10 The …

Tissue kallikrein-kinin therapy in hypertension and organ damage.

Tissue kallikrein is a serine proteinase that cleaves low molecular weight kininogen to produce kinin peptides, which in turn activate kinin receptors to trigger multiple biological functions. In addition to its kinin-releasing activity, tissue kallikrein directly interacts with the kinin B2 receptor, protease-activated receptor-1, and gamma-epithelial Na channel. The tissue kallikrein-kinin system (KKS) elicits a wide spectrum of biological activities, including reducing hypertension, cardiac and renal damage, restenosis, ischemic stroke, and skin wound injury. Both loss-of-function and gain-of-function studies have shown that the KKS plays an important endogenous role in the protection against health pathologies. Tissue kallikrein/kinin treatment attenuates cardiovascular, renal, and brain injury by inhibiting oxidative stress, apoptosis, inflammation, hypertrophy, and fibrosis and promoting angiogenesis and neurogenesis. Approaches that augment tissue kallikrein-kinin activity might provide an effective strategy for the treatment of hypertension and associated organ damage.

Kallikrein-kinin in stem cell therapy.

The tissue kallikrein-kinin system exerts a wide spectrum of biological activities in the cardiovascular, renal and central nervous systems. Tissue kallikrein-kinin modulates the proliferation, viability, mobility and functional activity of certain stem cell populations, namely mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), mononuclear cell subsets and neural stem cells. Stimulation of these stem cells by tissue kallikrein-kinin may lead to protection against renal, cardiovascular and neural damage by inhibiting apoptosis, inflammation, fibrosis and oxidative stress and promoting neovascularization. Moreover, MSCs and EPCs genetically modified with tissue kallikrein are resistant to hypoxia- and oxidative stress-induced apoptosis, and offer enhanced protective actions in animal models of heart and kidney injury and hindlimb ischemia. In addition, activation of the plasma kallikrein-kinin system promotes EPC recruitment to the inflamed synovium of arthritic rats. Conversely, cleaved high molecular weight kininogen, a product of plasma kallikrein, reduces the viability and vasculogenic activity of EPCs. Therefore, kallikrein-kinin provides a new approach in enhancing the efficacy of stem cell therapy for human diseases.

Lack of Plasma Kallikrein-Kinin System Cascade in Teleosts

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 [Lys0]-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 [Arg0]-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.

Intestinal tissue kallikrein-kinin system in inflammatory bowel disease

Tissue kallikrein cleaves kininogens to release kinins. Kinins mediate inflammation by activating constitutive bradykinin receptor-2 (BR2), which are rapidly desensitized, and induced by inflammatory cytokines bradykinin receptor-1 (BR1), resistant to desensitization. Intestinal tissue kallikrein (ITK) may hydrolyze growth factors and peptides, whereas kinins are responsible for capillary permeability, pain, synthesis of cytokines, and adhesion molecule-neutrophil cascade. Our and others results have demonstrated ITK in intestinal goblet cells and its release into interstitial space during inflammation. Kallistatin, an inhibitor of ITK, has been shown in epithelial and goblet cells, and was decreased in inflamed intestine as well as in plasma compared with noninflammatory controls. BR1 was upregulated in patients with inflammatory bowel disease (IBD), and it has expressed in an apical part of enterocytes in inflamed intestine, but in the basal part in normal intestine. ITK and BR1 were visualized in macrophages forming granuloma in Crohn's disease. In animal studies BR2 blockade decreased intestinal contraction, but had limited effect on inflammatory lesions. BR1 was found to be upregulated in animal inflamed intestine, in part dependent on tumor necrosis factor alpha (TNF-α). A selective BR1 receptor antagonist decreased morphological and biochemical features of experimental intestinal inflammation. Both BR1 and BR2 mediate epithelial ion transport that leads to secretory diarrhea. The upregulation of BR1 in inflamed intestine provides a structural basis for the kinins function, suggesting that a selective BR1 antagonist may have potential in therapeutic trial of IBD patients.

Activation of the plasma kallikrein-kinin system on human lung epithelial cells

Activation of the tissue kallikrein-kinin system (KKS) plays a major inflammatory role in the lung, but the contribution of the plasma KKS remains unclear. Plasma KKS involves assembly and activation of high molecular weight kininogen (HK) and plasma prekallikrein (PPK) on cell surfaces, resulting in the liberation of the inflammatory peptide, bradykinin (BK), from HK by plasma kallikrein (PK). To this end, we determined the possible contribution of plasma KKS in BK formation using airway epithelium. The HK binding proteins, urokinase plasminogen activator receptor, cytokeratin 1 and gC1qR, were expressed on transformed A549 and BEAS-2B cell lines, as well as on normal lung tissue, but Mac-1 was absent. A549 cells bound FITC-labelled HK, which was only partially inhibited by a combination of antibodies to the HK binding proteins. HK-PPK complex activation on the transformed epithelial cell lines, as well as primary epithelial cells, resulted in PK formation and liberation of BK. HK-PPK activation was inhibited by cysteine, BK and protamine, and by novobiocin, a heat shock protein 90 (HSP90) inhibitor. In summary, lung epithelial cells support the assembly and activation of the plasma KKS by a mechanism dependent on HSP90, and could contribute to KKS-mediated inflammation in lung disease.

Delivery of Recombinant Adeno-Associated Virus-Mediated Human Tissue Kallikrein for Therapy of Chronic Renal Failure in Rats

The tissue kallikrein-kinin system is important in regulating cardiovascular and renal function, and dysregulation of the system has been implicated in heart and kidney pathologies. These findings suggest that if balance can be restored to the kallikrein-kinin axis, then associated disease progression may be attenuated. To test this hypothesis, recombinant adeno-associated virus (rAAV)-mediated human tissue kallikrein (HK) expression was induced in a rodent model of chronic renal failure involving 5/6 nephrectomy (nephrectomy plus 70% reduction of remaining kidney). rAAV-HK treatment attenuated the rise in blood pressure, glomerular sclerosis, and tubulointerstitial injury observed in this model. rAAV-HK treatment also attenuated renal function decline as measured by urinary microalbumin, osmolarity, and cGMP levels. Reverse transcriptase-polymerase chain reaction analysis showed that rAAV-HK-treated rats had higher levels of bradykinin receptor-2 (B(2)R) and dopamine receptor-1 mRNAs. In contrast, angiotensin II receptor-1, endothelin receptor-A, and vasopressin receptor-2 mRNAs were markedly downregulated in kidneys from HK-treated rats. Bradykinin induced similar changes in receptor levels and prevented transforming growth factor-beta(1)-induced tubulointerstitial fibrosis. The effects of bradykinin could be reversed with the B(2)R antagonist HOE-140. Together, these findings suggest that restoration of the kallikrein-kinin system reduces kidney injury and protects renal function in 5/6-nephrectomized rats via changes in the expression and activation of G protein-coupled receptors including B(2)R.

Review: Angiotensin-converting enzyme in skeletal muscle: sentinel of blood pressure control and glucose homeostasis

Recent evidence suggests a coordinated regulation by the local renin-angiotensin system (RAS) and tissue kallikrein-kinin system (TKKS) of blood flow and substrate supply in oxidative red myofibres of skeletal muscle tissue during endurance exercise. The performance of these myofibres is dependent on the increased oxidation of substrates facilitated by augmenting nutritive blood flow and glucose uptake. Humoral factors released by the contracting fibres, such as adenosine and kinins, are suggested to be responsible for this metabolic adjustment. The considerable drain of blood volume and the enormous consumption of glucose during endurance exercise require a control mechanism for the maintenance of blood pressure (BP) and glucose homeostasis. This is achieved by the sympathetic nervous system and its subordinate RAS, which is located in the nutritive vessels and parenchyma of the red myofibres. The angiotensin-converting enzyme (ACE) is the primary enzyme responsible for kinin degradation during exercise, underscoring the important interrelationship between the RAS and the TKKS in the critical role of kinins in the multifactorial regulation of muscle bioenergetics and glucose and BP homeostasis. Importantly, overactivity of the ACE, as occurs in individuals displaying risk factors such as overweight, causes exaggerated BP response and reduced glucose disposal. If they persist over years, compensatory responses to this ACE overactivity, such as hypersecretion of insulin and compliance of the vessel walls, will inevitably be exhausted, leading ultimately to the manifestation of type 2 diabetes and hypertension. This concept also provides a unifying explanation for the beneficial effects of ACE-inhibitors and Angiotensin II receptor antagonists in the treatment of hypertension and insulin resistance.

Ca2+ signalling of kinins in cells expressing rat, mouse and human B1/B2-receptor

With respect to functional aspects, the kallikrein–kinin-system can be divided into a plasma kallikrein–kinin-system and a tissue kallikrein–kinin-system. At least four functional kinin peptides act via two different G-protein-coupled receptors, an inducible B1-receptor and a constitutively expressed B2-receptor. B1R and B2R couple to Gq/11 and lead via phospholipase C to Ca2+ mobilization. In humans both, bradykinin and kallidin are agonists on the B2-receptor. In contrast, bradykinin is believed to be the only kinin acting on the B2R in rats and mice. However, recently we have isolated a kallidin-like-peptide from plasma and urine of rats.Until now the kinin ligand–receptor interactions were mainly characterized in binding studies. However, receptor affinity does not inevitably correspond with the intrinsic activity of an agonist.The aim of the present study was to investigate intracellular calcium mobilization to quantify mouse, rat and human B1- and B2-receptor activation by bradykinin, kallidin, des-Arg9-bradykinin, des-Arg10-kallidin, and of the two novel rat kinins, kallidin-like-peptide and des-Arg10-kallidin-like-peptide.In cells stably expressing the human, rat, and mouse B2-receptor, respectively, bradykinin, kallidin, and kallidin-like-peptide were nearly equipotent (EC50, 10−12M) at eliciting Ca2+-transients. Their des-Arg-derivatives were 103-fold less potent.In cells expressing B1-receptor the des-Arg derivatives elicited Ca2+-signals at an EC50 in the order of 10−9M. Major differences between these peptides could not be observed. Bradykinin, kallidin, and kallidin-like-peptide caused a Ca2+-signal at substantially higher concentrations in the order of 10−7M.The data show that des-Arg9-bradykinin, des-Arg10-kallidin, and des-Arg10-kallidin-like-peptide are equipotent agonists at the B1-receptor. Bradykinin, kallidin and kallidin-like-peptide are equipotent agonists at the B2-receptor.

Mechanisms of Disease: the tissue kallikrein–kinin system in hypertension and vascular remodeling

The pathogenesis of arterial hypertension often involves a rise in systemic vascular resistance (vasoconstriction and vascular remodeling) and impairment of salt excretion in the kidney (inappropriate salt retention despite elevated blood pressure). Experimental and clinical evidence implicate an imbalance between endogenous vasoconstrictor and vasodilator systems in the development and maintenance of hypertension. Kinins (bradykinin and lys-bradykinin) are endogenous vasodilators and natriuretic peptides known best for their ability to antagonize angiotensin-induced vasoconstriction and sodium retention. In humans, angiotensin-converting enzyme inhibitors, a potent class of antihypertensive agents, lower blood pressure at least partially by favoring enhanced kinin accumulation in plasma and target tissues. The beneficial actions of kinins in renal and cardiovascular disease are largely mediated by nitric oxide and prostaglandins, and extend beyond their recognized role in lowering blood pressure to include cardioprotection and nephroprotection. This article is a review of exciting, recently generated genetic, biochemical and clinical data from studies that have examined the importance of the tissue kallikrein-kinin system in protection from hypertension, vascular remodeling and renal fibrosis. Development of novel therapeutic approaches to bolster kinin activity in the vascular wall and in specific compartments in the kidney might be a highly effective strategy for the treatment of hypertension and its complications, including cardiac hypertrophy and renal failure.

Contribution of Coagulation Pathways and Fibrinolysis in Generation of Kinins.

BACKGROUND. Classically, there are two main pathways by which kinins are generated: the plasma and tissue kallikrein-kinin systems. Each of this complex multi-protein system includes tissue or plasma kallikrein, which generate kinins from high or low molecular weight kininogen. In addition to plasma and tissue kallikreins, other serine proteases may have a kinin-forming capacity.OBJECTIVES. The main purpose of this study was to characterize the kininogenase activity of plasma kallikrein, thrombin (factor IIa), factor Xa, and plasmin in a purified system. We also aimed at characterizing the kinetic profile of generation of kinins in a whole blood system where coagulation and fibrinolysis were monitored by thromboelastography.METHODS. Bradykinin (BK) released from both low and high molecular weight kininogens and quantified with a specific competitive enzyme immunoassay was used to calculate the kinetic parameters for the different kininogenase activities. Physicochemical and pharmacological characterization of immunoreactive kinins confirm their nature. The kinetic profile of kinin generation was studied during the process of coagulation which was initiated with kaolin or with tissue factor and monitored by thromboelastography.RESULTS. BK was released not only by plasma kallikrein; both plasmin and factor Xa hydrolyzed high molecular kininogen to produce BK. In the case of thrombin, the release of both BK and kallidin was detected from low and high molecular weight kininogen. When applied to whole blood, this analytical approach indicated that the kinin-forming capacity of the coagulation intrinsic pathway was higher than that of the extrinsic pathway. The triggering of fibrinolysis by tissue plasminogen activator dramatically potentiated the release of des-Arg9-BK.CONCLUSIONS. Although plasma kallikrein is considered as being the main kinin-forming enzyme, other serine proteases of the coagulation and the fibrinolysis pathways are able to generate kinins and could play a role in different pathologies where kinins are potentially involved.

The tissue kallikrein-kinin system protects against cardiovascular and renal diseases and ischemic stroke independently of blood pressure reduction

Tissue kallikrein (hK1) cleaves low-molecular-weight kininogen to produce kinin peptide, which binds to kinin receptors and triggers a wide spectrum of biological effects. Tissue kallikrein levels are reduced in humans and in animal models with hypertension, cardiovascular and renal diseases. Transgenic mice or rats over-expressing human tissue kallikrein or kinin B2 receptor are permanently hypotensive, and somatic kallikrein gene delivery reduces blood pressure in several hypertensive rat models. Moreover, kallikrein gene delivery or kallikrein protein infusion can directly improve cardiac, renal and neurological function without blood pressure reduction. Kallikrein has pleiotropic effects in inhibiting apoptosis, inflammation, proliferation, hypertrophy and fibrosis, and promoting angiogenesis and neurogenesis in different experimental animal models. Kallikrein's effects can be blocked by kinin B2 receptor antagonists. Mechanistically, tissue kallikrein/kinin leads to increased nitric oxide levels and Akt activation, and reduced reactive oxygen species formation, TGF-beta1 expression, MAPK and nuclear factor-kappaB activation. Our studies indicate that tissue kallikrein, through the kinin B2 receptor and nitric oxide formation, can protect against oxidative damage in cardiovascular and renal diseases and ischemic stroke. These novel findings suggest that kallikrein/kinin may serve as new drug targets for the prevention and treatment of heart failure, renal disease and stroke in humans.

Rat tissue kallikrein releases a kallidin-like peptide from rat low-molecular-weight kininogen.

The kallikrein-kinin system is subdivided into the plasma and tissue-kallikrein-kinin system, with bradykinin (BK) and kallidin (KAL) (Lys(0)-bradykinin) as functional peptides. This occurs in both humans and other mammals. Both peptides are released by plasma and tissue-kallikrein. BK, but not KAL, has been detected in rats until now. One can explain this observation by the structural differences found in the sequence of rat high- and low-molecular kininogen containing an Arg-residue instead of a Lys-residue in front of the N-terminus of the BK sequence. Nevertheless, we were able to measure a kallidin-like peptide (KLP), in rat plasma and urine, using a specific KAL antiserum. In order to confirm our data, we isolated low-molecular-weight kininogen from rat plasma and incubated it with purified rat glandular kallikrein. The generated peptide was retained on a high-pressure liquid chromatography column and displaced by an excess of angiotensin I. The KLP-containing fraction was identified with the KLP radioimmunoassay. A specific ion signal with a mass to charge ratio (m/z) of 1216.73 was detected with matrix-assisted laser desorption/ionization mass spectrometry. As proposed earlier, the structure of this peptide is Arg(1)-KAL, instead of Lys(1)-KAL. The structural similarity between the Lys- and the Arg-residue explains the high crossreactivity (80%) of KLP with the specific KAL antibody. The incubation of KLP with angiotensin-converting enzyme yields two molecules with masses of 913.4 and 729.3 containing the sequence H-Arg-Arg-Pro-Pro-Gly-Phe-Ser-Pro-OH and H-Arg-Arg-Pro-Pro-Gly-Phe-OH. The enzymatic cleavage could be inhibited by captopril. The data suggest that in rats, as in other mammals, the tissue kallikrein-kinin system mediates its physiological effects via a kallidin-like peptide, which is Arg(1)-kallidin (Arg(0)-bradykinin).

Kallikrein–kinin in stroke, cardiovascular and renal disease

Tissue kallikrein, a serine proteinase, produces the potent vasodilator kinin peptide from kininogen substrate. The levels of tissue kallikrein are reduced in humans and animal models with hypertension, cardiovascular and renal disease. Using transgenic and somatic gene transfer approaches, we investigated the role of the tissue kallikrein-kinin system in cardiovascular, renal and central nervous systems. A single injection of the human tissue kallikrein gene in plasmid DNA or an adenoviral vector resulted in a prolonged reduction of blood pressure and attenuation of hypertrophy and fibrosis in the heart and kidney of several hypertensive animal models. Furthermore, enhanced kallikrein-kinin levels after gene transfer exerted beneficial effects, with protection against cardiac remodelling, renal injuries, restenosis, cerebral infarction and neurological deficits in normotensive animal models without haemodynamic effects, indicating direct actions of kallikrein independent of its ability to lower blood pressure. The effects of kallikrein were mediated by the kinin B2 receptor, as the specific B2 receptor antagonist icatibant abolished the actions of kallikrein. Moreover, kallikrein-kinin exhibited pleiotropic effects by inhibiting apoptosis, inflammation, hypertrophy and fibrosis, and promoting angiogenesis and neurogenesis in the heart, kidney, brain and blood vessel. Exogenous administration of kallikrein also led to increased nitric oxide (NO)/cGMP and cAMP levels, and reduced NAD(P)H oxidase activities, superoxide formation and pro-inflammatory cytokine levels. These results indicate a novel role of kallikrein-kinin through the kinin B2 receptor as an antioxidant and anti-inflammatory agent in protection against stroke, cardiovascular and renal disease, and may uncover new drug targets for the prevention and treatment of heart failure, vascular injury, end-stage renal disease and stroke in humans.

Induction of tissue kallikrein in human carotid atheroma does not lead to kallikrein-kinins pathway activation

The impairment of the tissue kallikrein-kinin system (KKS) may result in atheroma development. To determine the involvement of KKS in pathophysiology of human atherosclerosis, we examined the expression of all components of this system as well as angiotensinogen (another tissue kallikrein (TK) substrate), at messenger ribonucleic acid (mRNA) and protein levels in the human carotid artery with and without atheroma. mRNA levels were compared with semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) between atheroma plaque and intact tissue obtained during carotid endarterectomy in 15 patients. The cellular localization of the transcripts and proteins was analyzed with in situ hybridization and immunohistochemistry. TK activity was measured using chromogenic substrate. The kininogen mRNA was not detected in carotid wall. The TK mRNA was increased four-fold and TK activity 23-fold in atheroma plaque compared with intact tissue. No difference was observed for B1, B2 receptors, kallistatin, angiotensinogen and protein-kinase G type 1alpha (PK-G) mRNAs. The TK and angiotensinogen transcripts as well as kininogen and angiotensinogen proteins were present in both intimal and medial cells. The kininogen immunoreactivity was weaker in atheroma. All KKS components were synthesized in arterial wall except kininogen probably coming from plasma. The absence of PK-G mRNA down-regulation in atheroma suggests that the kallikrein induction does not lead to KKS activation.

The Plasma and Tissue Kininogen-kallikrein-kinin System: Role in the Cardiovascular System

Bradykinin and Lys-bradykinin are potent peptide mediators implicated in several physiopathological effects in mammals. They act through activation of G-protein-coupled constitutive B(2) or inducible kinin B(1) receptors linked to signaling pathways involving increased intracellular Ca(++) concentrations and/or release of mediators including arachidonic acid metabolites, NO and EDHF. In the cardiovascular system, the kallikrein-kinin system exerts a fine control of vascular smooth muscle tone and arterial blood pressure, and plays a significant cardioprotective effect. This has been lately confirmed in experimental studies employing transgenic mice overexpressing human tissue kallikrein and animals with knockout of kinin B(1) and B(2) receptor gene. Disturbances in this system are associated with arterial hypertension, myocardial ischaemia and other clinical complications. Inhibitors of kininase II (angiotensin-converting enzyme) have been prescribed successfully to patients with cardiovascular diseases, but there is still a great interest in developing drugs or pharmacological strategies that augment the activity of kininogen-kallikrein-kinin system in pathological conditions. Delivery of adenovirus vector containing the human tissue kallikrein gene (gene kallikrein therapy) has emerged as a great potential to satisfy these conditions. This review provides a summary of plasma and tissue kallikrein-kinin system, focusing on the pharmacological properties, kinin receptors and drugs reported to interfere with their actions. The modulatory effects of the kallikrein-kinin system on cardiovascular system, particularly in regulating smooth muscle tone and arterial blood pressure and in preventing myocardium ischaemia have also been explored in the review.