Chondroitin Sulfate Protects the Liver in an Experimental Model of Extra-Hepatic Cholestasis Induced by Common Bile Duct Ligation.

During liver fibrogenesis, there is an imbalance between regeneration and wound healing. The current treatment is the withdrawal of the causing agent; thus, investigation of new and effective treatments is important. Studies have highlighted the action of chondroitin sulfate (CS) in different cells; thus, our aim was to analyze its effect on an experimental model of bile duct ligation (BDL). Adult Wistar rats were subjected to BDL and treated with CS for 7, 14, 21, or 28 days intraperitoneally. We performed histomorphometric analyses on Picrosirius-stained liver sections. Cell death was analyzed according to caspase-3 and cathepsin B activity and using a TUNEL assay. Regeneration was evaluated using PCNA immunohistochemistry. BDL led to increased collagen content with corresponding decreased liver parenchyma. CS treatment reduced total collagen and increased parenchyma content after 21 and 28 days. The treatment also promoted changes in the hepatic collagen type III/I ratio. Furthermore, it was observed that CS treatment reduced caspase-3 activity and the percentage of TUNEL-positive cells after 14 days and cathepsin B activity only after 28 days. The regeneration increased after 14, 21, and 28 days of CS treatment. In conclusion, our study showed a promising hepatoprotective action of CS in fibrogenesis induced by BDL.

Differential bradykinin B1 and B2 receptor regulation in cell death induced by hepatic ischaemia/reperfusion injury.

The biological and pharmacological effects of BK (bradykinin) are mediated by two receptors: the constitutive B2R (B2 receptor) and the inducible B1R (B1 receptor). BK plays a role in the hepatic microcirculation by inducing the PHR (portal hypertensive response) via B2R, whereas DABK (des-Arg9-BK), a B1R agonist, does not elicit the response. During IRI (ischaemia/reperfusion injury), important changes occur in the microcirculation, and cell death by necrosis and apoptosis is involved in poor graft function. The aim of the present study was to analyse the role of B1R and B2R in liver cell death induced by IRI. Livers from Wistar rats were submitted to ischaemia (4°C) for 4 or 24 h. After this period, livers were reperfused ex vivo with Krebs-Henseleit solution (37°C). BK or DABK was then injected as a bolus during reperfusion in the absence or presence of HOE-140 (a B2R antagonist) or DALBK (des-Arg(9)-Leu(8)-BK) (a B1R antagonist) respectively. Liver viability was analysed by glucose release and bile secretion. The PHR to kinins did not change. Cell death was higher in the DABK group and its antagonist significantly decreased cell death. Interestingly, the B1R antagonist did not alter the number of necrotic cells, but it decreased the number of apoptotic cells. On the other hand, the B2R antagonist decreased the number of necrotic cells, but did not alter the number of apoptotic cells. Therefore B1R may participate in apoptotic cell death signalling, and B2R may be involved in necrotic cell death.

A plant Kunitz-type inhibitor mimics bradykinin-induced cytosolic calcium increase and intestinal smooth muscle contraction.

BbKI is a kallikrein inhibitor with a reactive site sequence similar to that of kinins, the vasoactive peptides inserted in kininogen moieties. This structural similarity probably contributes to the strong interaction with plasma kallikrein, the enzyme that releases, from high-molecular weight kininogen (HMWK), the proinflammatory peptide bradykinin, which acts on B(2) receptors (B(2)R). BbKI was examined on smooth muscle contraction and Ca(2+) mobilization, in which the kallikrein-kinin system is involved. Contrary to expectations, BbKI (1.8 µm) increased [Ca(2+)](c) and contraction, as observed with BK (2.0 µm). Not blocked by B(1) receptors (B(1)R), the BbKI agonistic effect was blocked by the B(2)R antagonist, HOE-140 (6 µm), and the involvement of B(2)R was confirmed in B(2)R-knockout mice intestine. The same tissue response was obtained using a synthetic peptide derived from the BbKI reactive site structure, more resistant than BK to angiotensin I-converting enzyme (ACE) hydrolysis. Depending on the concentration, BbKI has a dual effect. At a low concentration, BbKI acts as a potent kallikrein inhibitor; however, due to the similarity to BK, in high concentrations, BbKI greatly increases Ca(2+) release from internal storages, as a consequence of its interaction with B(2)R. Therefore, the antagonistic and agonistic effects of BbKI may be considered in conditions of B(2)R involvement.

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 experimental models 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.

Is the expression of kinin B(1) receptor related to intrahepatic vascular response?

UNLABELLED: Bradykinin elicits an intrahepatic vascular response (IHVR) mediated by the constitutive B(2) receptor (B(2)R). The biological effects of kinins may also be mediated by the inducible B(1) receptor (B(1)R). AIM: To verify if the hepatic B(1)R expression modulates IHVR to kinins. METHOD: We evaluated the ability of bradykinin and B(1)R agonists to elicit an IHVR in normal rats and in those submitted to acute or chronic inflammatory stimuli, fibrosis, cirrhosis, or hepatic regeneration. RESULTS: Bradykinin-induced IHVR was similar in all groups. B(1)R agonists did not elicit in any of them either a hypertensive or a hypotensive response. B(1) receptor induction was observed in all experimental groups (Western blot), except for the acute inflammatory group. CONCLUSION: B(1)R hepatic expression did not modulate IHVR to kinins.

Angiotensin II or epinephrine hemodynamic and metabolic responses in the liver of L-NAME induced hypertension and spontaneous hypertensive rats.

AIM: To study hepatic vasoconstriction and glucose release induced by angiotensin (Ang)II or Epi in rats with pharmacological hypertension and spontaneously hypertensive rat (SHR). METHODS: Isolated liver perfusion was performed following portal vein and vena cava cannulation; AngII or epinephrine (Epi) was injected in bolus and portal pressure monitored; glucose release was measured in perfusate aliquots. RESULTS: The portal hypertensive response (PHR) and the glucose release induced by AngII of L-NAME were similar to normal rats (WIS). On the other hand, the PHR induced by Epi in L-NAME was higher whereas the glucose release was lower compared to WIS. Despite the similar glycogen content, glucose release induced by AngII was lower in SHR compared to Wistar-Kyoto rats although both PHR and glucose release induced by Epi in were similar. CONCLUSION: AngII and Epi responses are altered in different ways in these hypertension models. Our results suggest that inhibition of NO production seems to be involved in the hepatic effects induced by Epi but not by AngII; the diminished glucose release induced by AngII in SHR is not related to glycogen content.

Kallikrein-kinin system in hepatic experimental models.

The purpose of this brief review is to describe some characteristics of the kallikrein-kinin system (KKS) in the liver. The liver synthesizes kininogens and prekallikrein and the synthesis of both proteins is increased in rats during the acute phase reaction. It is also the main organ to clear tissue as well as plasma kallikrein from the circulation in normal and pathological conditions. Bradykinin (BK), yielded by the kallikrein-kinin system, is a potent arterial hypotensive peptide, but in the liver it induces a portal hypertensive response. The portal hypertensive action of bradykinin is mediated by B2 receptors located on sinusoidal cells of the periportal region and is followed by its hydrolysis by angiotensin-converting enzyme, which is primarily present in the perivenous (centrolobular) region.

Hemodynamic and metabolic effects of angiotensin II on the liver.

To ascertain the mechanism of interaction between angiotensins (AI and AII) and the liver, an angiotensin-converting enzyme inhibitor (captopril) and a receptor antagonist (losartan) were used. Monovascular or bivascular liver perfusion was used to assess both hemodynamic (portal and arterial hypertensive responses) and metabolic (glucose production and oxygen consumption) effects. Microphysiometry was used for isolated liver cell assays to assess AII or losartan membrane receptor-mediated interaction. Captopril abolishes portal hypertensive response (PHR) to AI but not the AII effect. AII infused via the portal pathway promotes calcium-dependent PHR but not a hypertensive response in the arterial pathway (AHR); when infused into the arterial pathway AII promotes calcium-dependent PHR and AHR. Losartan infused into the portal vein abolishes PHR to AII but not the metabolic response; when infused via both pathways it abolishes the hypertensive responses and inhibits the metabolic effects. Isolated liver cells specifically respond to AII. Sinusoidal cells, but not hepatocytes, respond to 10 nM losartan. We conclude that AI has to be converted to AII to produce PHR. Quiescent stellate cells interacts in vitro with AII and losartan. Hemodynamic responses to AII are losartan-dependent but metabolic responses are partially losartan-independent. AII hemodynamic actions are mainly presinusoidal.

Pharmacological Activities and Hydrolysis by Peptidases of [Phospho-Ser(6)]-Bradykinin (pS(6)-BK).

Phosphorylated kininogen and some of its fragments containing serine phosphorylated bradykinin ([pS(6)]-Bk) were identified in human serum and plasma by a phosphoproteomic approach. We report the kininogenase ability of human tissue and plasma kallikreins and tryptase to generate [pS(6)]-Bk or Lys-[pS(6)]-Bk having as substrate the synthetic human kininogen fluorescent fragment Abz-MISLMKRPPGF[pS(386)]PFRSSRI-NH2. The pharmacological assays of [pS(6)]-Bk showed it as a full B2 bradykinin receptor agonist in smooth muscle, it produces a portal liver hypertensive response in rat and mouse paw edema that lasts longer than Bk. The rat hypotensive response to infusions of Bk is greater than that of [pS(6)]Bk, both if injected through femoral vein or aorta. [pS(6)]-Bk was more resistant than Bk to kininase digestion performed with angiotensin converting enzyme, neprilysin, thimet oligopeptidase, aminopeptidase P and carboxypeptidase M. (1)H-NMR experiments indicated that [pS(6)]-Bk has lower flexibility, with the pS(6)-P(7) bond restricted to the trans conformation, and can explain [pS(6)]-Bk resistance to hydrolysis. In conclusion, [pS(6)]-Bk presenting lower activity than Bk, with longer lasting effects and being slowly released by kininogenases from synthetic Abz-MISLMKRPPGF[pS(386)]PFRSSRI-NH2, suggests that phosphorylation of the kininogens can be an efficient kallikrein-kinin system regulator.

Enzyme release from injured, preserved, and ex vivo reperfused liver does not indicate malfunction.

OBJECTIVE: We compared the enzyme release from preserved and ex vivo reperfused livers after acute injury or inflammatory stimulus with organ function. METHODS: Acute injury was induced by carbon tetrachloride and inflammation was induced by turpentine oil treatments. Livers were exsanguinated and preserved for 8 or 24 hr. Enzymes were measured in preservation and reperfusion solutions, and reperfused liver function was evaluated by O(2) consumption and bromsulphalein clearance. RESULTS: The release of lysosomal enzymes was negligible in the preservation solution, and that of alanine aminotransferase and lactate dehydrogenase was similar in all groups. Release of aspartate aminotransferase and of EC 3.4.24.15 was more than that of the controls. During reperfusion liver function was normal in the injured group. CONCLUSION: Release of enzymes, mainly aspartate aminotransferase and EC 3.4.24.15, into the preservation solution is a sensitive and early indicator of either inflammatory or acute injury alterations of the preserved liver, but does not reflect organ malfunction.

Localization of EP24.15, a major liver kininase.

The liver is important for the kallikrein-kinin system modulation. This system plays a role in the inflammatory cascade with anticoagulant, profibrinolytic, and anti-adhesive attributes. The metalloendopeptidase EP24.15 is a major hepatic kininase. We studied the tissue distribution and subcellular localization of this enzyme in rat liver by cell fractionation and immunohistochemistry. Our results showed that EP24.15 is predominant in the soluble fraction of the liver homogenate and is present in the cytoplasm of hepatocytes, particularly in the perivenous zone (Z3). This localization is relevant because most hepatotoxin-induced necrosis, as well as ischemic hepatocellular injury, is predominant in Z3.

Anicteric cholangiopathy in schistosomiasis patients.

UNLABELLED: We previously reported that in anicteric patients with the isolated form of schistosomiasis (without co-morbidities) an ursodeoxycholic acid-sensitive increase in serum gamma-glutamyltransferase activity (gammaGT) occurs. We now describe the presence of cholangiopathy in these patients. METHODS: Sixteen adult anicteric patients with the isolated form of schistosomiasis mansoni were carefully selected: nine with increased gammaGT and seven with normal gammaGT. High sensitive C-reactive protein (CRP), to exclude inflammatory status, hyaluronic acid (HA), and other laboratory parameters were determined. The ultrasonographic study measured spleen length, portal vein and splenic vein diameters, and the portal flow. Magnetic resonance cholangiopancreatography (MRCP) images were interpreted by a blind observer. MRCP was deemed abnormal when focal narrowing and/or paucity of second and third order biliary branches and/or irregularities in the contours of biliary pathways were identified. RESULTS: Both groups (normal and elevated gammaGT) have preserved hepatic function tests (HA, serum albumin, prothrombin time) and clinical significant portal hypertension (low platelet count and ultrasonographic parameters). MRCP was abnormal in all patients with elevated gammaGT but in only 3 of the 7 patients with normal gammaGT (p=0.003). CONCLUSION: Magnetic resonance cholangiopancreatography characterized a cholangiopatic disorder in anicteric patients with the isolated form of schistosomiasis, even preceding laboratory test alterations.

Hepatic conversion of angiotensin I and the portal hypertensive response to angiotensin II in normal and regenerating liver.

BACKGROUND AND AIM: Angiotensin I (AI) and angiotensin II (AII) induce a portal hypertensive response (PHR) and the liver is able to convert AI into AII to trough the action of the angiotensin-converting enzyme (ACE). Our purpose was to characterize angiotensin I liver conversion. METHODS: AI, AII or angiotensin (1-7) were used in monovascular or bivascular perfusions. RESULTS: The maximum gain in portal pressure induced by AII took place significantly earlier (P = 0.031) than that occurring after an equimolar AI infusion. The AI-induced PHR was abolished both by captopril or losartan, whereas the AII-induced PHR was not affected by captopril, but was abolished by losartan. Angiotensin (1-7) has no hemodynamic effect in the perfused liver. After partial hepatectomy, the AII-PHR pattern changes from a rapid return to baseline values to a pattern where there was no return to baseline values (3-7 days ex-surgery). In the bivascular perfusion system when AII was infused in the arterial branch in the retrograde mode of perfusion (peptide available only to the periportal zone), the PHR was at least 50% of that obtained when the prograde mode was used (peptide available to the periportal and perivenous zones). CONCLUSION: AI does not induce PHR; this effect is a result of its mandatory conversion into AII by the ACE and the sequential action of AII on the AII receptor type 1 located in the hepatic periportal zone. AII induced PHR pattern changes during liver regeneration.

Fate of bradykinin on the rat liver when administered by the venous or arterial route.

BACKGROUND AND AIM: Bradykinin (BK) infused into the portal vein elicits a hypertensive response via the B2 receptor (B2R) and is efficiently hydrolyzed by the liver. Our purpose was to characterize the mechanism of interaction between BK and the liver. METHOD: BK, HOE-140 (a B2R antagonist), des-R(9)-BK (a B1R agonist) and enzyme inhibitors were used in monovascular or bivascular perfusions and in isolated liver cell assays. RESULTS: Des-R(9)-BK did not elicit a portal hypertensive response (PHR); BK infused into the hepatic artery elicited a calcium-dependent PHR and a calcium-independent arterial hypertensive response (HAHR), with the latter being almost abolished by naproxen. BK has a predominant distribution in the extracellular space and an average hepatic extraction of 8% in the steady state. Hydrolysis products of infused BK (R(1)-F(5) and R(1)-P(7)) did not elicit PHR. Angiotensin converting enzyme (ACE) is concentrated in the perivenous region and B2R in the periportal region. Microphysiometry showed that BK (and not a B1 agonist) interacts with stellate cells and the endothelial sinusoidal/Kupffer cell fraction. This effect was inhibited by the B2R antagonist. CONCLUSIONS: Events can be summarized as: the hypertensive action of BK on sinusoidal cells of the periportal region is followed by its hydrolysis by ACE which is primarily present in the perivenous region; there is no functional B1R in the normal liver; BK induces HAHR via eicosanoid release and PHR by a distinct pathway on the B2R. Our data suggest that BK may participate in the modulation of sinusoidal microvasculature tonus both in the portal and the arterial routes.

Hepatic clearance of tissue-type plasminogen activator and plasma kallikrein in experimental liver fibrosis.

UNLABELLED: We have previously shown that tissue-type plasminogen activator (tPA) and rat plasma kallikrein (RPK) share a common, but not unique, pathway for liver clearance. AIM: To evaluate the hepatic clearance of both proteases in experimental liver fibrosis. METHODS: The hepatic clearance of these proteases was studied in porcine serum-induced liver fibrosis using the isolated and perfused rat liver model. To better interpret the results, we also studied four other experimental groups: the turpentine oil-induced acute-phase response (AP group), AP group followed by GdCl3 administration (AP/Gd group), CCl4-induced cirrhosis (CCl4 group) and normal group. RESULTS: The tPA clearance decreased significantly by both fibrotic and cirrhotic rat livers whereas the RPK clearance was not altered by the fibrotic rat liver. The hepatic clearance of tPA was reduced in the AP and AP/Gd groups; on the other hand, RPK clearance was increased in the AP group and, interestingly, this effect was neutralized by concomitant GdCl3 administration. CONCLUSIONS: We observed that tPA and RPK clearances were affected differently by fibrosis as well as by different stimuli of the acute-phase response, despite the fact that they share a common hepatic clearance mechanism in normal livers, and they were equally affected in cirrhosis.

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.

Fígado e endocitose mediada por receptor (lectinas hepáticas) / Liver and receptor-mediated-endocytosis (hepatic lectins)

A concentraçåo das substâncias presentes no plasma é o resultado de um balanço entre as velocidades com que elas entram e deixam o espaço intravascular. Este artigo é atualizaçåo de importante mecanismo pelo qual o fígado remove glicoproteínas da circulaçåo: a endocitose mediada por receptor

Proteína C e correçåo cirúrgia da hipertensåo portal na esquistossomose / Protein C and surgical procedures for schistosomiasis portal hypertension

A proteína C (PC) do sistema plasmático de anticoagulaçäo pode estar diminuída em portadores da forma hepatoesplênica da esquistosomose (EHE), o que constitui fator de risco trombótico. Para avaliar o efeito de cirurgia da hipertensåo portal (HP) sobre a PC, determinamo-la (teste imuno-enzimático, Boehringer Mannheim Diagnostica) em 9 portadores da EHE antes e, em 5 deles, 6 dias após cirurgia que inclui a esplenectomia. Como controle estudamos 11 doadores de sangue sadios e 8 indivíduos eletivamente à colecistectomia. Os 28 indivíduos estudados eram HBsAg-negativos, nåo-alcólatras e nåo-transfundidos. A concentraçåo plasmática média da PC nos sadios (1,1 +- 0,1 U/mL) nåo diferiu das médias obtidas no pré (1,1 +- 0,1 U/mL) e no 6§ dia pós-operatório (1,1 +- 0,2 U/mL) dos colecistectomizados. Por outro lado, a média do grupo EHE (0,7 +- 0,1 U/mL) foi anterior (p<0,001) à grupos de controle. Em nenhum dos 19 controles a PC foi inferior a 0,6 U/mL, o que ocorreu em 33 por cento dos EHE, diminuiçåo esta acompanhada de hipoalbuminemia. Assim como a colecistectomia, a cirurgia de HP nåo alterou a PC diminuída (<0,6 U/mL) no pré-operatório näo a tiveram normalizada no pós-operatório. Os resultados evidenciam que atos cirúrgicos, per se, näo modificam a concentraçåo plasmática da PC e sugerem que a diminuiçåo desta reflete síntese hepática insuficiente e nåo consumo crônico. Na EHE, consumo crônico pode, como no caso da protrombina, ser corrigido pela esplenectomia.