icatibant and Fibrosis

The presence of high protein levels in the glomerular filtrate plays an important role in renal fibrosis, a disorder that justifies the use of animal models of experimental proteinuria. Such models have proved useful as tools in the study of the pathogenesis of chronic, progressive renal disease. Since bradykinin and the kinin B2 receptor (B2R) belong to a renoprotective system with mechanisms still unclarified, we investigated its anti-fibrotic role in the in vivo rat model of overload proteinuria. Upon up-regulating the kinin system by a high potassium diet we observed reduction of tubulointerstitial fibrosis, decreased renal expression of α-smooth muscle actin (α-SMA) and vimentin, reduced Smad3 phosphorylation and increase of Smad7. These cellular and molecular effects were reversed by HOE-140, a specific B2R antagonist. In vitro experiments, performed on a cell line of proximal tubular epithelial cells, showed that high concentrations of albumin induced expression of mesenchymal biomarkers, in concomitance with increases in TGF-β1 mRNA and its functionally active peptide, TGF-β1. Stimulation of the tubule cells by bradykinin inhibited the albumin-induced changes, namely α-SMA and vimentin were reduced, and cytokeratin recovered together with increase in Smad7 levels and decrease in type II TGF-β1 receptor, TGF-β1 mRNA and its active fragment. The protective changes produced by bradykinin in vitro were blocked by HOE-140. The development of stable bradykinin analogues and/or up-regulation of the B2R signaling pathway may prove value in the management of chronic renal fibrosis in progressive proteinuric renal diseases.

Topics: Albumins; Animals; Bradykinin; Bradykinin B2 Receptor Antagonists; Disease Models, Animal; Female; Fibrosis; Humans; Proteinuria; Rats; Rats, Sprague-Dawley; Receptor, Bradykinin B2; Smad7 Protein; Transforming Growth Factor beta1; Up-Regulation

The albumin overload model induces proteinuria and tubulointersitial damage, followed by hypertension when rats are exposed to a hypersodic diet. To understand the effect of kinin system stimulation on salt-sensitive hypertension and to explore its potential renoprotective effects, the model was induced in Sprague-Dawley rats that had previously received a high-potassium diet to enhance activity of the kinin pathway, followed with/without administration of icatibant to block the kinin B₂ receptor (B₂R). A disease control group received albumin but not potassium or icatibant, and all groups were exposed to a hypersodic diet to induce salt-sensitive hypertension. Potassium treatment increased the synthesis and excretion of tissue kallikrein (Klk1/rKLK1) accompanied by a significant reduction in blood pressure and renal fibrosis and with downregulation of renal transforming growth factor-β (TGF-β) mRNA and protein compared with rats that did not receive potassium. Participation of the B₂R was evidenced by the fact that all beneficial effects were lost in the presence of the B₂R antagonist. In vitro experiments using the HK-2 proximal tubule cell line showed that treatment of tubular cells with 10 nM bradykinin reduced the epithelial-mesenchymal transdifferentiation and albumin-induced production of TGF-β, and the effects produced by bradykinin were prevented by pretreatment with the B₂R antagonist. These experiments support not only the pathogenic role of the kinin pathway in salt sensitivity but also sustain its role as a renoprotective, antifibrotic paracrine system that modulates renal levels of TGF-β.

Topics: Animals; Bradykinin; Bradykinin B2 Receptor Antagonists; Cell Line; Female; Fibrosis; Humans; Hypertension; Kidney Diseases; Kidney Tubules; Kinins; Metabolic Networks and Pathways; Potassium, Dietary; Proteinuria; Rats; Rats, Sprague-Dawley; Serum Albumin, Bovine; Sodium Chloride, Dietary; Tissue Kallikreins; Transforming Growth Factor beta

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.

Topics: Bradykinin; Bradykinin B2 Receptor Antagonists; Culture Media, Conditioned; Fibronectins; Fibrosis; Humans; Kallikrein-Kinin System; Kallikreins; Kininogens; Macrophages; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Mesangial Cells; Protease Inhibitors; Pyridines; Receptor, Bradykinin B1; Receptor, Bradykinin B2; RNA, Messenger; Thiazepines; Tissue Plasminogen Activator

Bradykinin may interfere with myocardial remodelling by promoting inflammation and mast cell activation or, alternatively, by counteracting angiotensin II-dependent collagen accumulation. The aim of the present study was to investigate the role of bradykinin B2 receptor antagonism in inflammatory and mast cell infiltration, fibroplasia and fibrosis accumulation following myocardial infarction (MI). Myocardial infarction was produced by the ligature of the left coronary artery in male Wistar rats that were 10 weeks of age. Immediately after MI, rats received the B2 receptor antagonist Hoe140 (0.5 microg/kg per min, s.c.) or saline over a period of 3 days, 1 week or 4 weeks, constituting three separate groups and their respective controls. Coronal myocardial tissue sections underwent haematoxylin and eosin, Giemsa and picrosirius red staining, as well as immunohistochemistry for alpha-smooth muscle actin (SMA). Morphometric studies were undertaken in three different myocardial regions: MI, remote non-infarcted subendocardium (non-MI SE) and remote non-infarcted interventricular septum (non-MI IVS). The MI size was comparable between Hoe140-treated groups and their respective controls (day 3: 42 +/- 4%, n = 8, vs 43 +/- 3%, n = 6; week 1: 37 +/- 5%, n = 5, vs 39 +/- 2%, n = 5; week 4: 35 +/- 3%, n = 9, vs 36 +/- 3%, n = 7). At day 3, Hoe140 treatment reduced inflammatory cell reaction within the MI (585 +/- 59 vs 995 +/- 170 cells/mm2; P = 0.02), non-MI SE (77 +/- 12 vs 214 +/- 57 cells/mm2; P = 0.02) and non-MI IVS (93 +/- 16 vs 135 +/- 14 cells/mm2; P = 0.03) regions. Mast cells were reduced within the non-MI IVS region (0.8 +/- 0.1 vs 2.5 +/- 0.4 cells/mm2; P = 0.006), but not within the MI region. In non-MI SE, mast cells were rarely found. At week 1, Hoe140 treatment reduced alpha-SMA-positive myofibroblast infiltration within the MI (2535 +/- 383 vs 5636 +/- 968 cells/mm2; P = 0.01) and non-MI SE (222 +/- 33 vs 597 +/- 162 cells/mm2; P = 0.03) regions. In the non-MI IVS region, alpha-SMA-positive myofibroblasts were rarely found. At week 4, Hoe140 treatment reduced collagen volume fraction within the MI (37 +/- 4 vs 53 +/- 4%; P = 0.03), non-MI SE (1.3 +/- 0.2 vs 2.6 +/- 0.3%; P = 0.001) and non-MI IVS (1.1 +/- 0.2 vs 1.8 +/- 0.2%; P = 0.01) regions. Bradykinin promotes inflammation, fibroplasia and fibrosis after MI. Mast cells may have a role in fibrosis deposition through a bradykinin-related mechanism.

Topics: Actins; Animals; Bradykinin; Bradykinin B2 Receptor Antagonists; Collagen; Fibrosis; Heart Ventricles; Immunohistochemistry; Inflammation; Male; Mast Cells; Myocardial Infarction; Myocardium; Rats; Rats, Wistar; Wound Healing

Angiotensin-converting enzyme inhibitors (ACE-I) protect against the development of glomerulosclerosis using mechanisms partly dissociated from their systemic antihypertensive action. The aim of the current study was to delineate the mechanism of action underlying the antifibrotic effects of the ACE-I perindoprilat in the context of macrophage-mediated scarring in human mesangial cells.. Mesangial cells were treated with macrophage-conditioned medium (MPCM) in the presence or absence of the ACE-I perindoprilat.. Forty micromol/L perindoprilat reduced MPCM-induced mesangial cell fibronectin levels by 19.4 +/- 0.6% (P < 0.001). Immunoprecipitation of 35S-methionine biosynthetically labeled fibronectin and Northern analysis suggested that the decrease in fibronectin levels was not caused by reduced synthesis. MPCM stimulated the production of matrix metalloproteinases (MMP) 2, 3, and 9 in mesangial cells; however, these were not significantly altered by ACE-I treatment, and neither was production of their tissue inhibitor of metalloproteinases (TIMP-1). Addition of exogenous bradykinin to MPCM-treated mesangial cells resulted in a 22.5 +/- 1.4% (P < 0.02) reduction in secreted fibronectin levels, while semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Southern blotting demonstrated that bradykinin B2 receptor expression was up regulated by 71 +/- 30% in MPCM-stimulated mesangial cells in response to ACE-I treatment (P= 0.032). Moreover, the bradykinin B2 receptor antagonist HOE 140 attenuated the beneficial effects of perindoprilat. MPCM-stimulated mesangial cell protein expression levels of plasminogen activator system components tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) were altered after treatment with ACE-I.. These results suggest that ACE-I-induced renoprotection, in the context of macrophage-stimulated mesangial cell scarring, is mediated, at least in part, via the actions of bradykinin.

Topics: Angiotensin-Converting Enzyme Inhibitors; Bradykinin; Bradykinin B2 Receptor Antagonists; Cells, Cultured; Fibronectins; Fibrosis; Glomerular Mesangium; Humans; Indoles; Kallikrein-Kinin System; Plasminogen Activators; Receptor, Bradykinin B2; Renin-Angiotensin System; Tissue Plasminogen Activator

Angiotensin converting enzyme (ACE) inhibitors inhibit both the formation of angiotensin II and the catabolism of bradykinin (BK). They prevent not only hypertension but also cardiac hypertrophy and fibrosis. An increase in BK level stimulates the expression of nitric oxide (NO) synthase (NOS) and induces prostaglandins, both of which are powerful vasodilator factors. The direct effect of BK against cardiac hypertrophy is still unclear. This study was performed to examine the cardioprotective effects of BK in hypertrophic models. Renovascular hypertensive (RHT) rats were treated with BK (1,000 ng/kg/day), BK+D-arginyl-[Hyp(3), Thi(5), D-Tic(7), Oic(8)]-bradykinin (HOE140) (a BK B(2) receptor antagonist), and BK+N(omega)-nitro-L-arginine methyl ester (L-NAME) (a NOS inhibitor) for 3 weeks. Blood pressure was measured and echocardiographic analysis performed during the treatment. Histological data were analyzed to confirm the hypotrophic effect of BK. Treatment with BK improved cardiac remodeling, reducing both the heart weight/body weight ratio and the left ventricular wall thickness. However, co-treatment with HOE140 or L-NAME reversed the anti-hypertrophic action of BK. In particular, cardiac fibrosis or perivascular fibrosis, along with collagen accumulation, were inhibited by treatment with BK, while HOE140 and L-NAME counteracted these changes. In addition, expressions of atrial natriuretic peptides (ANP) and brain natriuretic peptides (BNP), which are markers of cardiac abnormalities, were down-regulated by treatment with BK. These effects were reversed by co-treatment with HOE140 and L-NAME. Together, these results indicate that BK directly inhibits the progression of cardiac hypertrophy and cardiac fibrosis due to NO release via the BK B(2) receptor. The BK-NO pathway may play an important role in the progression of cardiac remodeling.

Topics: Adrenergic beta-Antagonists; Animals; Atrial Natriuretic Factor; Blood Pressure; Body Weight; Bradykinin; Coronary Circulation; Echocardiography; Enzyme Inhibitors; Fibrosis; Hypertension, Renal; Hypertrophy, Left Ventricular; Male; Myocardium; Natriuretic Peptide, Brain; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Organ Size; Rats; Rats, Wistar; RNA, Messenger; Ventricular Remodeling

Angiotensin-converting enzyme (ACE) inhibitors reduce the progression of various fibrotic renal diseases both in humans and in animal models. Unilateral ureteral obstruction (UUO) is an animal model of accelerated renal tubulointerstitial fibrosis that is attenuated by ACE inhibition. Although ACE inhibitors increase bradykinin concentrations in addition to their effect on angiotensin II formation, the role of bradykinin in renal fibrosis has not been studied. We show here that genetic ablation (B2(-/-) mice) or pharmacological blockade of the bradykinin B2 receptor increases UUO-induced interstitial fibrosis in mice, whereas transgenic rats expressing increased endogenous bradykinin show reduced UUO-induced interstitial fibrosis. The increased interstitial fibrosis in B2(-/-) mice was accompanied by a decreased activity of plasminogen activators (PAs) and metalloproteinase-2 (MMP-2), enzymes involved in ECM degradation, suggesting that the protective effects of bradykinin involve activation of a B2 receptor/PA/MMP-2 cascade. This ability of bradykinin to increase PA activity was confirmed in primary culture proximal tubular cells. Thus, in both mice and rats, bradykinin B2 receptor activation reduces renal tubulointerstitial fibrosis in vivo, most likely by increasing ECM degradation.

Topics: Animals; Bradykinin; Cell Division; Collagen; Disease Models, Animal; Extracellular Matrix; Extracellular Matrix Proteins; Female; Fibrosis; Immunoenzyme Techniques; Kidney; Male; Matrix Metalloproteinase 2; Mice; Mice, Inbred C57BL; Mice, Knockout; Nephritis, Interstitial; Plasminogen Activators; Rats; Receptor, Bradykinin B2; Receptors, Bradykinin; Tissue Kallikreins; Ureteral Obstruction

Chronic elevations in plasma angiotensin II (AngII) are associated with an efflux of plasma macromolecules into the perivascular and contiguous interstitial space. This is followed by the appearance of macrophages and type I collagen-producing, fibroblast-like cells that precede the accumulation of fibrous tissue at these sites. Whether this perivascular and interstitial fibrosis is a direct effect of AngII on collagen turnover of these cells or an indirect response mediated by nitric oxide, prostaglandins and/or bradykinin released in response to AngII, is uncertain.. We measured perivascular and interstitial fibrosis (picrosirius-stained tissue) in response to 14-day infusion of AngII (150 ng/kg/min, s.c.) in male Sprague-Dawley rats. Treated animals were compared to untreated controls and to groups receiving AngII together with either an NO-synthase inhibitor [NG-nitro-L-arginine methyl ester (L-NAME) 10 mg/kg/day in drinking water], a cyclo-oxygenase inhibitor (indomethacin, 2 mg/kg/day in drinking water), or a bradykinin B2 receptor antagonist (Hoe140, 115 ng/kg/min, s.c.).. When left and right ventricles of treated rats were compared to untreated controls, AngII led to a respective 68 and 48% increase in perivascular collagen volume fraction (PCVF) and a 54 and 22% increase in interstitial collagen volume fraction (ICVF). Co-administration of AngII + L-NAME did not attenuate either PCVF or ICVF while indomethacin significantly attenuated PCVF by 37 and 33% of left and right ventricle, respectively, but did not alter ICVF in either ventricle when compared to AngII-treated animals. Co-administration of AngII + Hoe140 completely prevented perivascular and interstitial collagen accumulation with the extent of perivascular fibrosis comparable to untreated controls.. The perivascular and interstitial fibrosis of the rat right and left ventricles seen in association with the exogenous administration of AngII is mediated by the release of bradykinin and prostaglandins, and therefore is an indirect response to elevated circulating AngII.

Topics: Angiotensin II; Animals; Arginine; Bradykinin; Bradykinin Receptor Antagonists; Collagen; Cyclooxygenase Inhibitors; Fibrosis; Indomethacin; Male; Myocardium; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Prostaglandins; Rats; Rats, Sprague-Dawley