Kininogen-deficient Brown Norway Katholiek Rats Research Articles

Cleaved high molecular weight kininogen, a novel factor in the regulation of matrix metalloproteinases in vascular smooth muscle cells

We previously reported that Brown Norway Katholiek rats, which feature a deficiency of plasma kininogens, develop severe abdominal aortic aneurysm. Increased activity of matrix metalloproteinases (MMPs) in the aortic wall, leading to degradation of extracellular matrix components, is considered to play a crucial role in aneurysm formation. Using an in vitro model of vascular smooth muscle cells (VSMCs), cultured from the rat aorta, we investigated whether the cleaved form of high molecular weight kininogen, designated HKa, affects the expression of MMP-9 and MMP-2 and their tissue inhibitors (TIMPs). Treatment of VSMCs with HKa reduced in a concentration-dependent manner IL-1α-induced release of MMP-9 and MMP-2, associated with decreased MMP enzymatic activity levels in conditioned media, as demonstrated by gelatin zymography and fluorescein-labeled gelatin substrate assay, respectively. Real-time PCR revealed that HKa reduced corresponding MMP-9 mRNA levels. Further investigations showed that this effect did not result from a modified rate of MMP-9 mRNA degradation. TIMP-1 mRNA levels, already increased as a result of cytokine-stimulation, were significantly enhanced by HKa. Furthermore, we found elevated basal mRNA expression levels of MMP-2 and TIMP-2 in VSMCs derived from kininogen-deficient Brown Norway Katholiek rats. These results demonstrate for the first time that HKa affects the regulation of MMPs in VSMCs.

Tissue Kallikrein Elicits Cardioprotection by Direct Kinin B2 Receptor Activation Independent of Kinin Formation

Tissue kallikrein exerts various biological functions through kinin formation with subsequent kinin B2 receptor activation. Recent studies showed that tissue kallikrein directly activates kinin B2 receptor in cultured cells expressing human kinin B2 receptor. In the present study, we investigated the role of tissue kallikrein in protection against cardiac injury through direct kinin B2 receptor activation using kininogen-deficient Brown Norway Katholiek rats after acute myocardial infarction. Tissue kallikrein was injected locally into the myocardium of Brown Norway Katholiek rats after coronary artery ligation with and without coinjection of icatibant (a kinin B2 receptor antagonist) and N(omega)-nitro-L-arginine methylester (an NO synthase inhibitor). One day after myocardial infarction, tissue kallikrein treatment significantly improved cardiac contractility and reduced myocardial infarct size and left ventricle end diastolic pressure in Brown Norway Katholiek rats. Kallikrein attenuated ischemia-induced apoptosis and monocyte/macrophage accumulation in the ischemic myocardium in conjunction with increased NO levels and reduced myeloperoxidase activity. Icatibant and N(omega)-nitro-L-arginine methylester abolished kallikrein's effects, indicating a kinin B2 receptor NO-mediated event. Moreover, inactive kallikrein had no beneficial effects in cardiac function, myocardial infarction, apoptosis, or inflammatory cell infiltration after myocardial infarction. In primary cardiomyocytes derived from Brown Norway Katholiek rats under serum-free conditions, active, but not inactive, kallikrein reduced hypoxia/reoxygenation-induced apoptosis and caspase-3 activity, and the effects were mediated by kinin B2 receptor/nitric oxide formation. This is the first study to demonstrate that tissue kallikrein directly activates kinin B2 receptor in the absence of kininogen to reduce infarct size, apoptosis, and inflammation and improve cardiac performance of infarcted hearts.

The Renal Kallikrein-Kinin System: Its Role as a Safety Valve for Excess Sodium Intake, and Its Attenuation as a Possible Etiologic Factor in Salt-Sensitive Hypertension

The distal tubules of the kidney express the full set of the components of the kallikreinkinin system, which works independently from the plasma kallikrein-kinin system. Studies on the role of the renal kallikrein-kinin system, using congenitally kininogen-deficient Brown-Norway Katholiek rats and also bradykinin B2 receptor knockout mice, revealed that this system starts to function and to induce natriuresis and diuresis when sodium accumulates in the body as a result of excess sodium intake or aldosterone release, for example, by angiotensin II. Thus, it can be hypothesized that the system works as a safety valve for sodium accumulation. The large numbers of studies on hypertensive animal models and on essential hypertensive patients, particularly those with salt sensitivity, indicate a tendency toward the reduced excretion of urinary kallikrein, although this reduction is modified by potassium intake and impaired renal function. We hypothesize that the reduced excretion of the renal kallikrein may be attributable to a genetic defect of factor(s) in renal kallikrein secretion process and may cause salt-sensitive hypertension after salt intake.Referee: Dr. A.D. Baines, Dept. of Laboratory Medicine & Pathobiology, University of Toronto, 100 College Street, Toronto, ON, Canada, M5G 1L5

Suppressed Angiogenesis in Kininogen-Deficiencies

We investigated whether the kinin-generating system enhanced angiogenesis in chronic and proliferative granuloma and in tumor-surrounding stroma. In rat sponge implants, angiogenesis was gradually developed in normal Brown Norway Kitasato rats (BN-Ki). The development of angiogenesis was significantly suppressed in kininogen-deficient Brown Norway Katholiek rats (BN-Ka). The angiogenesis enhanced by basic fibroblast growth factor was also significantly less marked in BN-Ka than in BN-Ki. Naturally occurring angiogenesis was significantly suppressed by B1 or B2 antagonist. mRNA of vascular endothelial growth factor was more highly expressed in the granulation tissues in BN-Ki than in BN-Ka. Daily topical injections of aprotinin, but not of soy bean trypsin inhibitor, suppressed angiogenesis. Daily topical injections of low-molecular weight kininogen enhanced angiogenesis in BN-Ka. Topical injections of serum from BN-Ki, but not from BN-Ka, also facilitated angiogenesis in BN-Ka. FR190997, a nonpeptide mimic of bradykinin, promoted angiogenesis markedly, with concomitant increases in vascular endothelial growth factor mRNA. Angiogenesis in the granulation tissues around the implanted Millipore chambers containing Walker-256 cells was markedly more suppressed in BN-Ka than in BN-Ki. Our results suggest that endogenous kinin generated from the tissue kallikrein-kinin system enhances angiogenesis in chronic and proliferative granuloma and in the stroma surrounding a tumor. Thus, the agents for the kinin-generating system and/or kinin receptor signaling may become useful tools for controlling angiogenesis.

AT(2) receptor-dependent vasodilation is mediated by activation of vascular kinin generation under flow conditions.

Physiological roles of angiotensin II type 2 receptor (AT(2)) are not well defined. This study was designed to investigate the mechanisms of AT(2)-dependent vascular relaxation by studying vasodilation in pressurized and perfused rat mesenteric arterial segments. Perfusion of angiotensin II in the presence of AT(1) antagonist elicited vascular relaxation, which was completely dependent on AT(2) receptors on endothelium. FR173657 (>1 microM), a bradykinin (BK) B(2)-specific antagonist, significantly suppressed AT(2)-dependent vasodilation (maximum inhibition: 68.5% at 10 microM). Kininogen-deficient Brown Norway Katholiek rats showed a significant reduction in AT(2)-mediated vasodilatory response compared with normal wild-type Brown Norway rats. Indomethacin (>1 microM), aprotinin (10 microM) and soybean trypsin inhibitor (10 microM) also reduced AT(2)-dependent vasodilation. Our results demonstrated that stimulation of AT(2) receptors caused a significant vasodilation through local production of BK in resistant arteries of rat mesentery in a flow-dependent manner. Such vasodilation counterbalances AT(1)-dependent vasoconstriction to regulate the vascular tone.

Suppression of Dextran Sulfate Sodium-Induced Colitis in Kininogen-Deficient Rats and Non-peptide B2 Receptor Antagonist-Treated Rats

Various proinflammatory mediators are believed to be involved in the processes and symptoms of ulcerative colitis (UC). To determine whether endogenous kinin enhances the severity of UC, we induced experimental colitis (EC) in kininogen-deficient mutant rats and tested the effect of a non-peptide B2 receptor antagonist. EC was induced in male kininogen-deficient Brown Norway-Katholiek rats (BN-Ka) and normal Brown Norway-Kitasato rats (BN-Ki) with 5% dextran sulfate sodium (DSS). Sprague-Dawley rats (SD) were also used. Colon length, body weight and hematocrit were determined for 7 days. Effects of FR173657, an orally active B2 antagonist, were tested. The colon length was shortened in BN-Ki with DSS treatment, but not in BN-Ka, and the difference between their lengths was significant. The hematocrit value was also reduced in BN-Ki, and the difference in hematocrit between BN-Ki and BN-Ka was significant. In SD, shortening of the colon and reduction in hematocrit were also observable, and both were blunted by FR173657. The survival rate in SD given DSS for 7 days was 68%, but FR173657 treatment restored it significantly to 100%. These results suggest that the endogenous kinins generated from the kallikrein-kinin system have a significant role in the development of EC.

Pivotal Role of Renal Kallikrein-Kinin System in the Development of Hypertension and Approaches to New Drugs Based on This Relationship

Renal kallikrein is one of the tissue kallikreins, and the distal nephron is fully equipped as an element of the kallikrein-kinin system. Although a low excretion of urinary kallikrein has been reported in essential hypertension, the results from studies on patients with hypertension are not consistent. Congenitally hypertensive animals also excrete lowered levels of urinary kallikrein, but the effects of this are yet unknown. Extensive genetic and environmental studies on large Utah pedigrees suggest that the causes of hypertension are closely related to the combination of low kallikrein excretion and the potassium intake. Mutant kininogen-deficient Brown Norway-Katholiek rats, which cannot generate kinin in the urine, are very sensitive to salt loading and to sodium retention by aldosterone released by a non-pressor dose of angiotensin II, which results in hypertension. The major function of renal kallikrein-kinin system is to excrete sodium and water when excess sodium is present in the body. Failure of this function causes accumulation of sodium in the cerebrospinal fluid and erythrocytes, and probably in the vascular smooth muscle, which become sensitive to vasoconstrictors. We hypothesize that impaired function of the renal kallikrein-kinin system may play a pivotal role in the early development of hypertension. Inhibitors of kinin degradation in renal tubules and agents, which accelerate the secretion of urinary kallikrein from the connecting tubules and increase the generation of urinary kinin, may be novel drugs against hypertension.

Ebelactone B, an inhibitor of urinary carboxypeptidase Y-like kininase, prevents the development of deoxycorticosterone acetate-salt hypertension in rats

Kininogen-deficient Brown Norway Katholiek rats (BN-Ka) excrete little urinary kinin, compared with normal rats of the same strain (BN Kitasato rats (BN-Ki)). Deoxycorticosterone acetate-salt treatment increased systolic blood pressure in both rats, but much faster in BN-Ka than in BN-Ki. Daily subcutaneous administration of ebelactone B (15 and 5 mg/kg/day), a rat urinary carboxypeptidase Y-like kininase inhibitor, significantly reduced systolic blood pressure in BN-Ki, but not in BN-Ka. This treatment significantly increased urinary Na + excretion and reduced Na + concentration in the erythrocytes in BN-Ki, but not in BN-Ka. An angiotensin-converting enzyme inhibitor, lisinopril (5 mg/kg/day s.c.), did not reduce the systolic blood pressure in either BN-Ki or BN-Ka. These results suggested that ebelactone B has promise as a preventive agent for the development of hypertension acting through the inhibition of urinary kinin degradation.