Human Tissue Kallikrein Activity in Angiographically Documented Chronic Stable Coronary Artery Disease / Atividade da Calicreína Tecidual Humana na Doença Coronariana Crônica Estável Angiograficamente Documentada

AbstractBackground:Human tissue kallikrein (hK1) is a key enzyme in the kallikrein–kinin system (KKS). hK1-specific amidase activity is reduced in urine samples from hypertensive and heart failure (HF) patients. The pathophysiologic role of hK1 in coronary artery disease (CAD) remains unclear.Objective:To evaluate hK1-specific amidase activity in the urine of CAD patientsMethods:Sixty-five individuals (18–75 years) who underwent cardiac catheterism (CATH) were included. Random midstream urine samples were collected immediately before CATH. Patients were classified in two groups according to the presence of coronary lesions: CAD (43 patients) and non-CAD (22 patients). hK1 amidase activity was estimated using the chromogenic substrate D-Val-Leu-Arg-Nan. Creatinine was determined using Jaffé’s method. Urinary hK1-specific amidase activity was expressed as µM/(min · mg creatinine) to correct for differences in urine flow rates.Results:Urinary hK1-specific amidase activity levels were similar between CAD [0.146 µM/(min ·mg creatinine)] and non-CAD [0.189 µM/(min . mg creatinine)] patients (p = 0.803) and remained similar to values previously reported for hypertensive patients [0.210 µM/(min . mg creatinine)] and HF patients [0.104 µM/(min . mg creatinine)]. CAD severity and hypertension were not observed to significantly affect urinary hK1-specific amidase activity.Conclusion:CAD patients had low levels of urinary hK1-specific amidase activity, suggesting that renal KKS activity may be reduced in patients with this disease.

ResumoFundamento:A calicreína tecidual humana (hK1) é enzima-chave do sistema calicreína-cinina (SCC). A atividade amidásica da hK1 está reduzida na urina de pacientes com hipertensão e insuficiência cardíaca (IC); seu papel na doença arterial (DAC) coronariana ainda não está esclarecido.Objetivo:Avaliar a atividade amidásica da hK1 na urina de pacientes com DAC.Métodos:Sessenta e cinco indivíduos (18 a 75 anos) que se submeteram ao cateterismo cardíaco (CAT) coletaram amostra do jato médio de urina imediatamente antes do CAT. Baseando-se na presença de lesões coronarianas, os pacientes eram classificados em dois grupos: DAC (43 pacientes) e sem DAC (22 indivíduos). A atividade amidásica da hK1 foi estimada com o substrato cromogênico D-Val-Leu-Arg-Nan. Creatinina foi determinada pelo método de Jaffé. A atividade amidásica específica da hK1 urinária foi expressa em µM/(min . mg de creatinina) para corrigir diferenças no fluxo urinário.Resultados:A atividade amidásica da hK1 urinária foi semelhante entre os pacientes com DAC [0,146 µM/(min . mg de creatinina)] e aqueles sem DAC [0,189 µM/(min . mg de creatinina)] (p = 0,803), e permaneceu entre os baixos valores previamente publicados para pacientes com hipertensão primária [0,210 µM/(min . mg de creatinina)] e para aqueles com IC [0,104 µM/(min . mg de creatinina)], respectivamente. Nenhum efeito estatisticamente significativo da gravidade da DAC e da hipertensão sobre a atividade amidásica da hK1 urinária foi observado.Conclusão:A atividade amidásica da hK1 na urina estava reduzida nos pacientes com DAC, o que pode sugerir que a atividade do SCC renal esteja reduzida nessa doença.

Rol del sistema kallicreína kinina y su interrelación con sistemas vasoactivos durante la preñez / Role of the kallikrein kinin system and its interrelationship with vasoactive systems in pregnancy

La hiperfiltración glomerular y el aumento de la reabsorción de sodio son factores fundamentales para el desarrollo de la unidad feto placentaria. Dichos factores resultan de adaptaciones hemodinámicas y renales en las que participan sistemas vasoactivos. Se pudo demostrar en ratas que la activación del sistema kallicreína kinina (SKK) precede a la instalación de la hiperfiltración glomerular, dado que su inhibición por aprotinina previene el aumento del filtrado glomerular. Además, la inhibición individual o asociada de los efectores específicos del SKK, las prostaglandinas (PGs) y el óxido nítrico (ON), confirman la dependencia del filtrado glomerular del SKK durante la preñez. Encontramos también que el sistema renina angiotensina (SRA) participa en la generación de la hiperfiltración glomerular dado que ésta es afectada por la administración de bloqueantes del SRA. La inhibición máxima sobre el pico de hiperfiltración se obtuvo con el bloqueo de ambos sistemas (SKK y SRA). Además, estrategias para alterar la hiperfiltración glomerular y la reabsorción de sodio de la preñez evidenciaron alteraciones en el desarrollo de la unidad feto placentaria, menor número de crías, mayor cantidad de reabsorciones intrauterinas y retardo en el crecimiento. El tratamiento combinado de inhibidores del SKK asociados a bloqueantes del SRA o de óxido nítrico mostraron los mayores efectos. En consecuencia, demostramos que el SKK juega un rol central en los fenómenos de adaptación que acompañan la preñez normal. La interrelación del SKK con varios sistemas vasoactivos parecería formar una red que participa en las adaptaciones hemodinámicas para un adecuado desarrollo de la gestación y de la unidad feto-placentaria.

Glomerular hyperfiltration and increased sodium reabsorption are key factors for the development of the fetus and placenta in pregnancy. These adjustments result from hemodynamic and renal factors involving vasoactive systems. It was demonstrated in rats that activation of KKS precedes the installation of glomerular hyperfiltration as aprotinin prevents the increase in glomerular filtration. In addition, individual or associated inhibition of specific kallikrein kinin system effectors, prostaglandins (PGs) and nitric oxide (NO), confirm the glomerular filtration rate dependence of KKS during pregnancy. It was also found that the renin-angiotensin system (RAS) contributes to glomerular hyperfiltration as this is affected by the administration of RAS blockers. The peak of hyperfiltration maximum inhibition was obtained by the blockade of both systems (KKS and RAS). In addition, strategies used to alter the glomerular hyperfiltration and increased sodium reabsorption during pregnancy, showed abnormalities in the development of the fetus and placenta, fewer offspring, more fetus resorptions and intrauterine growth retardation. KKS inhibitors associated with RAS or nitric oxide blockers showed the greatest impact. As a consequence, it was demonstrated that KKS plays a central role in the adaptation phenomenom that accompanies normal pregnancy. The interplay of KKS with several vasoactive systems, seem to arrange a network involved in the hemodynamic adaptations to allow the proper development of pregnancy and the fetus and placenta.

Portal hypertensive response to kinin

Portal hypertension is the most common complication of chronic liver diseases, such as cirrhosis. The increased intrahepatic vascular resistance seen in hepatic disease is due to changes in cellular architecture and active contraction of stellate cells. In this article, we review the historical aspects of the kallikrein-kinin system, the role of bradykinin in the development of disease, and our main findings regarding the role of this nonapeptide in normal and experimentalmodels of hepatic injury using the isolated rat liver perfusion model (mono and bivascular) and isolated liver cells. We demonstrated that: 1) the increase in intrahepatic vascular resistance induced by bradykinin is mediated by B2 receptors, involving sinusoidal endothelial and stellate cells, and is preserved in the presence of inflammation, fibrosis, and cirrhosis; 2) the hepatic arterial hypertensive response to bradykinin is calcium-independent and mediated by eicosanoids; 3) bradykinin does not have vasodilating effect on the pre-constricted perfused rat liver; and, 4) after exertion of its hypertensive effect, bradykinin is degraded by angiotensin converting enzyme. In conclusion, the hypertensive response to BK is mediated by the B2 receptor in normal and pathological situations. The B1 receptor is expressed more strongly in regenerating and cirrhotic livers, and its role is currently under investigation.

Hipertensão portal é a complicação mais comum das doenças crônicas do fígado, tais como cirrose. A resistência intravascular aumentada observada na doença hepática é devida a alterações na arquitetura celular e contração ativa das células estreladas. Neste trabalho revisamos aspectos históricos do estudo do sistema calicreína-cinina e os resultados de nossos estudos do papel deste nonapeptídeo no controle do tono vascular intra-hepático em condições normais e modelos experimentais de agressão hepática usando a perfusão de fígado isolado de rato (mono e bivascular) e células hepáticas isoladas. Nós demonstramos que: 1) o aumento da resistência vascular intrahepática induzido pela bradicinina é mediado por receptores B2, envolve a participação de células endoteliais sinusoidais e células estreladas e não é alterada pela presença de inflamação, fibrose ou cirrose; 2) a resposta hipertensiva induzida pela bradicinina no sistema arterial hepático é cálcio-independente emediada por eicosanóides; 3) bradicinina não tem efeito dilatador na circulação intra-hepática; 4) após exercer efeito vasoconstritor intra-hepático, a bradicinina é degradada pela enzima conversora de angiotensina. Em conclusão, a resposta hipertensiva à bradicinina é mediada pelo receptor B2 em condições normais e patológicas. Receptor B1 é expresso mais fortemente nos fígados em regeneração e cirróticos e seu papel está sob investigação.

El kininógeno de alto peso molecular: su participación en la respuesta inflamatoria y en la angiogénesis. Propiedades y posible aplicación terapéutica / High molecular weight kininogen in inflammation and angiogenesis: a review of its properties and therapeutic applications

The plasma kallikrein-kinin system (KKS) participates in the pathogenesis of inflammatory reactions involved in cellular injury, coagulation, fibrinolysis, kinin formation, complement activation, cytokine secretion and release of proteases. It has been shown that KKS activation in the systemic inflammatory response syndrome results in decrease of its component plasma proteins. Similar changes have been documented in diabetes, sepsis, children with vasculitis, allograft rejection, disseminated intravascular coagulation, patients with recurrent pregnancy losses, hereditary angioedema, adult respiratory distress syndrome and coronary artery disease. Direct involvement of the KKS in the pathogenesis of experimental acute arthritis and acute and chronic enterocolitis has been documented by previous studies from our laboratory using experimental animal models. It has been found that in HK deficient Lewis rats, experimental IBD was much less severe. We showed a genetic difference in kininogen structure between resistant Buffalo and susceptible Lewis rats, which results in accelerated cleavage of HK and it is responsible for the susceptibility to the inflammatory process in the Lewis rats. It has been demostrated that therapy with a specific plasma kallikrein inhibitor (P8720) modulated the experimental enterocolitis, arthritis and systemic inflammation. Furthermore, it has been shown that a bradykinin 2 receptor (B2R) antagonist attenuates the inflammatory changes in the same animal model. We have showed that a monoclonal antibody targeting HK decreases angiogénesis and arrests tumor growth in a syngeneic animal model. In summary, these results indicate that the plasma KKS plays a central role in the pathogenesis of chronic intestinal inflammation, arthritis and angiogenesis.

Se ha demostrado la participación del sistema plasmático de kalikreína-kininas (KKS) en el proceso inflamatorio, el cual incluye reacciones de daño celular, coagulación y fibrinólisis, formación de kininas, activación del complemento, secreción de citoquinas y liberación de proteasas. El KKS se encuentra activado en el síndrome de respuesta inflamatoria sistémica con una disminución en la concentración plasmática de las proteínas que lo constituyen. También se ha demostrado una activación similar en la diabetes, choque séptico, vasculitis en infantes, enfermedad injerto-huésped, coagulación intravascular diseminada, pacientes con abortos de repetición, angioedema hereditario, el síndrome de estrés respiratorio del adulto y enfermedad coronaria arterial. Mediante el uso de modelos animales experimentales, nuestro laboratorio ha demostrado una participación directa del KKS en la patogénesis de la artritis experimental aguda y la enterocolitis aguda y crónica. Se ha demostrado que en la rata tipo Lewis, cuando es deficiente de kininógeno de alto peso molecular (HK), la enfermedad inflamatoria intestinal es menos severa comparada con la presentada en ratas con niveles normales de HK como la Buffalo. Nosotros mostramos una diferencia entre el gene que codifica la molécula del kininógeno de la rata tipo Buffalo (resistentes) y Lewis (susceptibles), que resulta en un incremento de la actividad proteolítica de kalikreína sobre su substrato HK, lo cual predispone a las ratas Lewis al desarrollo de la enfermedad inflamatoria crónica. Se ha demostrado una disminución en las manifestaciones inflamatorias sistémicas de la enterocolitis y artritis experimental mediante el uso de un inhibidor específico de la kalikreína (P8720). Además, el antagonista del receptor 2 de la bradikinina (BR2) atenuó los cambios inflamatorios en el mismo modelo animal. Asimismo, se ha demostrado que las ratas Lewis deficientes de kininógeno desarrollaron inflamación intestinal sistémica menos severa. Mediante el uso del anticuerpo monoclonal C11C1 contra HK se logró una disminución de la angiogenesis y, consecuentemente, el crecimiento tumoral. En conclusión, los resultados demuestran que el sistema plasmático de KKS desempeña un papel preponderante en la patogénesis de la artritis reumatoide, la enfermedad intestinal crónica y en el proceso angiogénico.

The balance between the production of tumor necrosis factor-alpha and interleukin-10 determines tissue injury and lethality during intestinal ischemia and reperfusion

A major goal in the treatment of acute ischemia of a vascular territory is to restore blood flow to normal values, i.e. to "reperfuse" the ischemic vascular bed. However, reperfusion of ischemic tissues is associated with local and systemic leukocyte activation and trafficking, endothelial barrier dysfunction in postcapillary venules, enhanced production of inflammatory mediators and great lethality. This phenomenon has been referred to as "reperfusion injury" and several studies demonstrated that injury is dependent on neutrophil recruitment. Furthermore, ischemia and reperfusion injury is associated with the coordinated activation of a series of cytokines and adhesion molecules. Among the mediators of the inflammatory cascade released, TNF-alpha appears to play an essential role for the reperfusion-associated injury. On the other hand, the release of IL-10 modulates pro-inflammatory cytokine production and reperfusion-associated tissue injury. IL-1beta, PAF and bradykinin are mediators involved in ischemia and reperfusion injury by regulating the balance between TNF-alpha and IL-10 production. Strategies that enhance IL-10 and/or prevent TNF-alpha concentration may be useful as therapeutic adjuvants in the treatment of the tissue injury that follows ischemia and reperfusion.

Regulation of the kinin receptors after induction of myocardial infarction: a mini-review

It is well known that the responses to vasoactive kinin peptides are mediated through the activation of two receptors termed bradykinin receptor B1 (B1R) and B2 (B2R). The physiologically prominent B2R subtype has certainly been the subject of more intensive efforts in structure-function studies and physiological investigations. However, the B1R activated by a class of kinin metabolites has emerged as an important subject of investigation within the study of the kallikrein-kinin system (KKS). Its inducible character under stress and tissue injury is therefore a field of major interest. Although the KKS has been associated with cardiovascular regulation since its discovery at the beginning of the last century, less is known about the B1R and B2R regulation in cardiovascular diseases like hypertension, myocardial infarction (MI) and their complications. This mini-review will summarize our findings on B1R and B2R regulation after induction of MI using a rat model. We will develop the hypothesis that differences in the expression of these receptors may be associated with a dual pathway of the KKS in the complex mechanisms of myocardial remodeling.

Vasopressin and bradykinin receptors in the kidney: implications for tubular function

Vasopressin and bradykinin are two of the most important peptides in regulating vascular tone, water, and ionic balance in the body, adn thus they play a key role in controlling blood pressure. In addition to being a potent vasoconstrictor, Vasopressin also has an antidiuretic activity in the kidney, whereas kinins regulate renal blood flow in addition to their vasodilatory and natriuretic activity. We review here the primary evidence for the localization of the vasopressin and kinin receptors and their role in ionic and water regulation in the kidney.

Molecular biology of the kallikrein-kinin system: from structure to function

The participation of the kallikrein-kinin system, comprising the serine proteases kallikreins, the protein substrates kininogens and the effective peptides kinins, in some pathological processes like hypertension and cardiovascular diseases is still a matter of controversy. The use of different experimental set-ups in concert with the development of potent and specific inhibitors and antagonists for the system has highlighted its importance but the results still lack conclusivity. Over the last few years, transgenic and gene-targeting technologies associated with molecular biology tools have provided specific information about the elusive role of the kallikrein-kinin system in the control of blood pressure and electrolyte homeostasis. cDNA and genomic sequences for kinin receptors B2 and B1 from different species were isolated and shown to encode G-protein-coupled receptors and the structure and pharmacology of the receptors were characterized. Transgenic animals expressing an overactive kallikrein-kinin system were established to study the cardiovascular effects of these alterations and the results of these investigations further corroborate the importance of this system in the maintenance of normal blood pressure. Knockout animals for B2 and B1 receptors are available and their analysis also points to the role of these receptors in cardiovascular regulation and inflammatory processes. In this paper the most recent and relevant genetic animal models developed for the study of the kallikrein-kinin system are reviewed, and the advances they brought to the understanding of the biological role of this system are discussed.

La glucosa en conjunto con péptidos derivados de cininógenos podrían actuar desde el aparato digestivo como bloqueadores de la acción diurética-natriurética del péptido natriurético auricular / Glucose in conjunction with peptides derived from kininogens might act from digestive tract as blockers of ANP mediated diuresis-natriuresis

This paper describes long term research efforts wich have lead: 1) to the identification of peptides present in pepsanurin, a peptidic fraction obtained by pepsin hydrolisis of plasma globulins that inhibits the renal excretory action of atrial natriuretic peptide (ANP) and 2) to the discovery of an unexpected role of glucose, as a requisite, for these inhibitory effects. The active peptides identified in pepsanurin are derived from plasma kininogens, substrates of the kallikrein-kinin system. Pro-kinins of 15, 16 and 18 aminoacids, and bradykinin itself, block ANP-induced diuresis and natriuresis when injected iv, ip or into, the duodenal lumen of anesthetized rats in picomol doses. Furthermore, a novel 20 aminoacids fragment derived from kininogen dominium-1, named PU-D1, is the most potent and longer lasting blocker of ANP renal effects. The anti-ANP effects of those peptides are prevented by B2- kinin receptor antagonists. The inhibition of ANP by kinins and PU-D1 was evident only in rats infused with isotonic glucose; whereas the excretory effect of ANP was not affected in fasted rats not infused, or infused with saline. These findings provide evidence that glucose facilitates liquid retention through a kinin-mediated inhibition of ANP excretory action that may be related to the prandial cycle

Efecto cardio y vasculoprotector de las kininas y los inhibidores de la enzima de conversión / Cardiovascular protective effect of kinins and converting enzyme inhibitors

ANTECEDENTES: Las kininas son oligopéptidos que generados localmente en corazón y vasos sanguíneos estimulan la producción endotelial de factores relajantes (prostaciclina, óxido nítrico y factor endotelial hiperpolarizante). El nivel de kininas depende de: a) la tasa de producción por kalikreínas y b) la tasa de destrucción por kininasas, entre las que se encuentra la enzima de conversión de angiotensina (CE). Los inhibidores de la enzima de conversión (CEI) empleados con notable éxito en el tratamiento de la hipertensión arterial y la insuficiencia cardíaca actuarían en parte prolongando la vida media del péptido. El objetivo de este trabajo es examinar: a) si los componentes del sistema kalikreína-kininógeno-kininas (SKKK) se liberan al perfusado en corazones aislados y perfundidos, b) la influencia de ramiprilat sobre la tasa de secreción de kininas y c) la acción biológica de las kininas en una preparación de vasos coronarios aislados. MATERIAL Y METODO: Los corazones de Ratas Wistar (300 ñ 30 gr) se aislaron y perfundieron en forma retrógrada con buffer Krebs-Henseleit equilibrado con 95 por ciento de oxígeno y 5 por ciento de anhídrido carbónico a un flujo constante (5 ml/min) mediante una bomba de perfusión Gilson. Kalikreína se midió por su capacidad de generar kininas; kininógeno, por una modificación de un método previamente descripto; y las kininas extraídas con un Set-Pack Cartridge, se dosaron por RIA. RESULTADOS: Los componentes del SKKK se forman y liberan continuamente en la preparación de corazón aislado (hasta 120 minutos). El pretratamiento con puromicina, un inhibidor de la síntesis proteica, descendió significativamente el release de kalikreína y kininógeno, confirmando la síntesis ex novo en el propio tejido cardíaco. El agregado del CEI, ramiprilat, incrementó significativamente el nivel de kininas en el perfusado. Kalikreína, per se, actuando sobre su sustrato en la propia pared del vaso libera kininas que relajan los vasos coronarios. Las kininas generadas permanentemente en la pared vascular en pequeñas cantidades se ponen de manifiesto en presencia de ramiprilat, relajando los vasos coronarios. El agregado de HOE 140, un inhibidor selectivo B2 de las kininas, bloquea el efecto relajante, confirmando que tanto la acción de las kalikreínas liberadas localmente como el efecto de los CEIs es mediado por kininas endógenas...

Kalikreína vascular y cardíaca en hipertensión asociada a alteraciones del metabolismo lipídico / Cardiovascular kallikrein in hypertension associated to lipidic metabolic disorders

ANTECEDENTES: La hipertensión arterial y la hiperlipemia dañan el endotelio vascular y predisponen al desarrollo de lesiones ateromatosas y enfermedades coronarias. Las células endoteliales y musculares son capaces de modular la función cardiovascular mediante la producción de factores autocrino/paracrinos. Se ha descripto la presencia de un sistema generador de kininas en vasos y corazón. El propósito de este estudio es examinar si existe modificación de la actividad kininogenásica en tejido cardiovascular de ratas dislipidémicas e hipertensas por inhibición de la producción del factor relajante derivado del endotelio. MATERIAL Y METODO: Se utilizaron ratas Wistar macho (430 ñ 50 g peso), las que se sometieron a los siguientes tratamientos: control (n= 10): dieta normal; lípidos (n= 10): dieta con sobrecarga de 16 por ciento de ácidos grasos saturados y 2 por ciento de colesterol durante 8 semanas; L-NAME (n= 10): administración oral de un inhibidor de la síntesis de factor relajante endotelial, L-nitro arginina metil éster, 100 mg/kg/ día durante las 2 últimas semanas del período experimental; lípidos + L-NAME (n= 10): administración de dieta con sobrecarga grasa durante 8 semanas y de L-NAME durante las 2 últimas semanas. Se midió la presión arterial, la concentración de lípidos plasmáticos y la actividad kininogenásica en homogenados de corazón y vasos sanguíneos (pgBK/mg/h). Resultados: La manipulación dietética de los animales no produjo cambios en su presión arterial, pero fue efectiva para instaurar un perfil lipoproteico aterogénico (p< 0,05). La administración de L-NAME produjo un aumento significativo de los niveles tensionales (p< 0,05), sin modificar las concentraciones de los metabolitos plasmáticos dosados. La actividad kininogenásica presente en homogenados de corazón aumentó significativamente en los animales sometidos a sobrecarga dietética de grasa y ese incremento fue aún mayor cuando se les adicionó al agua de bebida el inhibidor de la síntesis de factor relajante endotelial (p< 0,05). Un patrón semejante se observa en homogenados de aorta, arterias caudales y venas caudales (p< 0,05). CONCLUSIONES: El incrmento local de la capacidad generadora de kininas en tejido cardiovascular podría representar un esfuerzo tisular por contraponerse a la agresión endotelial causada por el aumento de los lípidos plasmáticos y el aumento de la presión arterial. Las kininas liberadas in situ tendrían efectos hemodinámicos y metabólicos ...

Vascular-derived kinins and local control of vascular tone

The vascular itself, through a complex interplay of endocrine, neurocrine and autoparacrine mechanisms, plays an active role in vascular homeostasis. The endothelial cell senses humoral and hemodynamic changes and respondes by secreting a variety of metabolically active substances that act locally causing either vasodilatation or vasoconstriction. Kallikrein (KK) and the nRNA for KK are present in arteries and veins. Vascular KK releases Kinins from kininogen which circulate in plasma and is also present in vascular tissue. Vascular-derived kinins induce vasodilatation through the release of endothelial compounds ( prostacyclin, EDRFs and cytochrome P-450). Disturbance in the delicate balance between vasodilators and vasoconstrictiors may play a role in the development of hypertension. Vascular kallikrein (VKK) was significantly (P < 0.05) elevated after 2 weeks of development of renovascular and mineralocorticoid hypertension, and blood pressure was only slightly elevated. However, VKK decreased in both experimental models when blood pressure was incresed. It is possible that the increase in VKK in the early stages resulted in incresead local vasodilatory activity, thus counteracting the rise in blood pressure. As hypertension developed, KK was significantly decreased in arteries. The decrease in arterial KK during established hypertension is most likely secondary to high blood pressure....