Marcia R. Nagaoka

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

Abstract: 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.

Participation of a galectin-dependent mechanism in the hepatic clearance of tissue-type plasminogen activator and plasma kallikrein

Tissue-type plasminogen activator (tPA) is a serine protease that plays a central role in the fibrinolytic system, activating plasminogen to plasmin, which degrades the fibrin that is present in blood clots. tPA has proven to be a potent drug in thrombolytic therapy, however, its use is limited due to the rapid clearance from circulation by active hepatic uptake [1]. The uptake mechanism for tPA in the liver involves endothelial and parenchymal cells [2]. A carbohydrate recognition system for tPA in sinusoidal endothelial cells and tPA binding to asialoglycoprotein receptor in parenchymal cells were described [3]. Uptake by sinusoidal endothelial cells involves an interaction between the carbohydrate group in the kringle1 domain and the mannose receptor. A minor involvement of fucose residue has also been suggested in the recognition of tPA by these cells [4,5]. On the other hand, uptake by parenchymal liver cells is mediated mainly by LDL receptor-related protein (LRP) [6,7], a well-known multiligand receptor that also mediates the clearance of a2Macroglobulin(a2M)-proteinases complexes [8]. We have shown that the hepatic clearance rate of complexes such as a2M-kallikrein [9] and a2M-trypsin [10] is decreased compared to the free enzyme. The 39-kDa receptor-associated protein (RAP) is a receptor antagonist that inhibits ligand interactions with the receptors that belong to the low-density lipoprotein receptor gene family. RAP can also function intracellularly as a molecular chaperone for LRP and can regulate its ligand binding activity along the secretory pathway. In addition, RAP also plays an important role in receptor folding [11]. Recently, Camani et al. [12] showed that in some cells the presence of functional LRP is not sufficient for efficient tPA degradation, suggesting that tPA degradation requires a co-receptor. Secretion of tPA is potently stimulated by bradykinin, a nonapeptide released from hydrolysis of high-molecularweight kininogen by kallikrein [13], linking the fibrinolytic and kallikrein–kinin systems. We have already shown that tPA and RPK compete for a common, but not unique, pathway for hepatic clearance [14]. In addition, a plasma kallikrein-dependent plasminogen cascade is required for adipocyte differentiation [15]. Plasma kallikrein circulates in the plasma as its zymogen, prekallikrein. After activation, plasma kallikrein is involved in different biological processes including the pathogenesis of inflammatory reaction, blood flow control, blood pressure control and intrinsic coagulation and fibrinolytic systems [16]. Recently, Akita et al. [17] showed that after partial hepatectomy an excessive amount of TNF-a, the major initiator of hepatic regeneration, may trigger the generation of TGF-h via enhancement of surface plasma kallikrein activity on stellate cells. The proteolytic activity of plasma kallikrein is modulated by plasma inhibitors and its concentration by liver clearance. Rat plasma prekallikrein and the light chain of rat plasma kallikrein (RPK) are not cleared by the isolated and exsanguinated rat liver. The binding site of RPK to hepatic cells is

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.

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.

Plasma kallikrein and tissue-type plasminogen activator compete for a common pathway into the liver.

Abstract The liver is the main organ used to clear both tissue and plasma kallikreins from the circulation. Rat plasma kallikrein (rPK) is internalized by hepatocytes by a receptor-mediated mechanism. Since plasma kallikrein and tissue-type plasminogen activator (tPA) are both important for fibrinolysis, we now compare characteristics of the clearance of both by the isolated, exsanguinated and perfused rat liver. In preliminary experiments we observed, in vitro, that rPK and tPA neither form a complex with, nor are they substrates for, each other. The characteristics of rPK and tPA clearance are similar, and we observed that 20 and 200 molar excess of tPA in the perfusion medium decreased and abolished the rPK liver clearance, respectively. These results suggest that rPK and tPA share a common pathway for liver clearance.

The hepatic clearance of recombinant tissue-type plasminogen activator decreases after an inflammatory stimulus

We have shown that tissue-type plasminogen activator (tPA) and plasma kallikrein share a common pathway for liver clearance and that the hepatic clearance rate of plasma kallikrein increases during the acute-phase (AP) response. We now report the clearance of tPA from the circulation and by the isolated, exsanguinated and in situ perfused rat liver during the AP response (48-h ex-turpentine treatment). For the sake of comparison, the hepatic clearance of a tissue kallikrein and thrombin was also studied. We verified that, in vivo, the clearance of 125I-tPA from the circulation of turpentine-treated rats (2.2 +/- 0.2 ml/min, N = 7) decreases significantly (P = 0.016) when compared to normal rats (3.2 +/- 0.3 ml/min, N = 6). The AP response does not modify the tissue distribution of administered 125I-tPA and the liver accounts for most of the 125I-tPA (>80%) cleared from the circulation. The clearance rate of tPA by the isolated and perfused liver of turpentine-treated rats (15.5 +/- 1.3 microg/min, N = 4) was slower (P = 0.003) than the clearance rate by the liver of normal rats (22. 5 +/- 0.7 microg/min, N = 10). After the inflammatory stimulus and additional Kupffer cell ablation (GdCl3 treatment), tPA was cleared by the perfused liver at 16.2 +/- 2.4 microg/min (N = 5), suggesting that Kupffer cells have a minor influence on the hepatic tPA clearance during the AP response. In contrast, hepatic clearance rates of thrombin and pancreatic kallikrein were not altered during the AP response. These results contribute to explaining why the thrombolytic efficacy of tPA does not correlate with the dose administered.

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 O2 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.

Behavioral alterations and Fos protein immunoreactivity in brain regions of bile duct-ligated cirrhotic rats

Hepatic encephalopathy (HE) encompasses a variety of neuropsychiatric symptoms, including anxiety and psychomotor dysfunction. Although HE is a frequent complication of liver cirrhosis, the neurobiological substrates responsible for its clinical manifestations are largely unclear. In the present study, male Wistar rats were bile duct-ligated (BDL), a procedure which induces liver cirrhosis, and on the 21st day after surgery tested in the elevated plus-maze (EPM) and in an open field for anxiety and locomotor activity measurements. Analysis of Fos protein immunoreactivity (Fos-ir) was used to better understand the neurobiological alterations present in BDL animals. Plasma levels of ammonia were quantified and histopathological analysis of the livers was performed. BDL rats showed a significant decrease in the percentage of entries and time spent in the open arms of the EPM, an anxiogenic effect. These animals also presented significant decreases in Fos-ir in the lateral septal nucleus and medial amygdalar nucleus. Their ammonia plasma levels were significantly higher when compared to the sham group and the diagnosis of cirrhosis was confirmed by histopathological analysis. These results indicate that the BDL model induces anxiogenic results, possibly related to changes in the activation of anxiety-mediating circuitries and to increases in ammonia plasma levels.

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.

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.