Volume 133, Issue 6, Pages 2019-2028 (December 2007) Bradykinin Attenuates Hepatocellular Damage and Fibrosis in Rats With Chronic Liver Injury  Pau Sancho–Bru, Ramón Bataller, Guillermo Fernandez–Varo, Montserrat Moreno, Leandra N. Ramalho, Jordi Colmenero, Montserrat Marí, Joan Clària, Wladimiro Jiménez, Vicente Arroyo, David A. Brenner, Pere Ginès  Gastroenterology  Volume 133, Issue 6, Pages 2019-2028 (December 2007) DOI: 10.1053/j.gastro.2007.09.023 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Expression of B1, B2, kininogen (kng), and kallikrein-1 (klk1). (A) Western blot analysis of B1 and B2 receptors in normal and cirrhotic human livers and in activated HSCs. Red ponceau staining was performed to ensure equal protein loading. Graphs show the densitometry quantification of Western blots. *P < .05 compared with normal tissue. ☐, Normal liver; ■, cirrhotic liver. (B) Gene expression analysis by quantitative reverse-transcription PCR of components of the KKS in human normal and cirrhotic livers as assessed by quantitative PCR. Gene expression is the mean of 6 different tissues. *P < .05 compared with normal tissue. ☐, Normal liver; ■, cirrhotic liver. (C) Gene expression analysis of components of the KKS in human quiescent and activated HSCs as assessed by quantitative PCR. *P < .05 compared with quiescent cells. ☐, Quiescent HSCs; ■, activated HSCs. (D) Gene expression analysis of components of the KKS in human hepatocytes as assessed by quantitative PCR. (E) Gene expression analysis of components of the KKS in human liver macrophages as assessed by quantitative PCR. Gastroenterology 2007 133, 2019-2028DOI: (10.1053/j.gastro.2007.09.023) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Effects of BK infusion on hepatocellular damage in CCl4-treated rats. (A) Representative photomicrograph of saline-infused control and CCl4-treated rat livers stained with B1- and B2-receptor antibodies. Magnification, 400×. (B) Effects of BK on ALT and AST serum levels. ANOVA, P < .01; ##P < .01 compared with control rats infused with saline. *P < .05 and **P < .01 compared with CCl4-treated rats infused with saline. BK was infused at 1 ng and 100 ng/kg/min. (C) Representative photomicrographs of liver specimens stained with H&E. Rats treated with CCl4 showed areas of intense hepatocellular necrosis, inflammation, hepatocyte ballooning, and apoptosis (arrows). Both 1 ng and 100 ng/kg/min of BK markedly attenuated hepatocellular damage. Magnification, 200×. (D) TUNEL quantification of liver specimens. BK infusion did not significantly reduce the number of apoptotic cells per field. Gastroenterology 2007 133, 2019-2028DOI: (10.1053/j.gastro.2007.09.023) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Effect of BK infusion on collagen deposition, myofibroblast accumulation, inflammatory cells, and gene expression. (A) Representative photomicrograph of Sirius red staining. Saline-infused CCl4-treated rats showed a marked bridging fibrosis throughout the parenchyma. BK infusion markedly attenuated fibrosis development. Magnification, 40×. (B) Morphometric quantification of positive area for Sirius red staining. (C) Representative photomicrograph of α-SMA staining. CCl4-treated rats receiving saline showed a marked accumulation of α-SMA–positive cells that colocalized with areas of fibrosis. BK infusion reduced the accumulation of positive cells. Magnification, 40×. (D) Morphometric quantification of positive area for α-SMA–positive cells. (E) Representative photomicrograph of CD43 staining. CCl4-treated rats receiving saline showed an increase of infiltration of CD43-positive inflammatory cells scattered in the liver parenchyma (arrows). BK infusion did not effect the infiltration of CD43 cells. Magnification, 100×. (F) Quantification of positive CD43 cells per field. (G) Pro-collagen α-1(I) gene expression was increased markedly in CCl4-treated rats receiving saline. BK infusion reduced collagen expression. (H) TGF-β1 gene expression was increased markedly in CCl4-treated rats receiving saline, which was reduced by BK infusion. ANOVA analysis, P < .05. Posttest analysis, ##P < .01 or #P < .05 compared with rat infused with saline. *P < .05 compared with CCl4-treated rats infused with saline. BK was infused at low (0.1 ng/kg/min) and high (100 ng/kg/min) concentrations. Gastroenterology 2007 133, 2019-2028DOI: (10.1053/j.gastro.2007.09.023) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Effects of BK infusion on hepatocellular apoptosis in a Fas-stimulated mouse model. (A) Effects of BK on ALT and AST serum levels. ANOVA, P < .001; post-test, #P < .01 compared with control mice infused with saline. *P < .01 compared with Jo2-injected mice infused with saline. BK was infused at 100 ng/kg/min. (B) Representative photomicrographs of liver specimens stained with TUNEL. Magnification, 400×. (C) TUNEL quantification of liver specimens. BK infusion markedly attenuated the number of apoptotic cells per field. Kruskal–Wallis test, P < .01; post-test, #P < .01 vs saline; *P < .05 vs Jo2. Gastroenterology 2007 133, 2019-2028DOI: (10.1053/j.gastro.2007.09.023) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Effects of BK on cultured rat hepatocytes. (A) Primary rat hepatocytes were preincubated for 15 minutes with B1-receptor antagonist des-arg10-HOE-140 or B2-receptor antagonist HOE-140 and stimulated with BK (0.1 μmol/L) for 1 hour. Nuclear translocation of the NF-κB p65 subunit was assessed by Western blot of nuclear extracts. BK (0.1 μmol/L) increased p65 translocation to the nucleus, indicating an induction of NF-κB activity. B1-receptor antagonist did not have an effect on p65 translocation induced by BK, whereas the B2-receptor antagonist blocked the effects of BK. The constitutively activated transcription factor, Oct-1, was used as a protein loading control. Graph shows the densitometry quantification of p65 with respect to Oct-1 expression. (B) Primary rat hepatocytes were stimulated with BK (0.1 μmol/L) for 30 minutes. ERK, AKT, and JNK phosphorylation was assessed by Western blotting. BK induced ERK and AKT but not JNK phosphorylation in the absence of actinomycin D (Act) and tumor necrosis factor α (tnf-α). BK reduced JNK phosphorylation induced by TNF-α. Figures are representative of 3 independent experiments. Gastroenterology 2007 133, 2019-2028DOI: (10.1053/j.gastro.2007.09.023) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Effects of BK on human HSCs. (A) BK induced a marked increase in [Ca2+]i. Incubation of cells with the B2-receptor antagonist HOE-140, but not the B1-receptor antagonist des-Arg10-HOE-140, prevented the increase of [Ca2+]i. ♦, BK 1 μmol/L; ■, BK 0.1 μmol/L; ▲, BK 0.01 μmol/L; ○, HOE-140 + BK 0.1 μmol/L; –, des-Arg10-HOE-140 + BK 0.1 μmol/L. (B) BK (0.1 μmol/L) stimulation reduced the expression of procollagen αI(I) and TGF-β1, but not TIMP-1. *P < .05 vs buffer. B2-receptor antagonist HOE-140, or B1-receptor antagonist des-Arg10-HOE-140 failed to block the effects of BK. ■, Collagen αI(I); , TGF-β1; , TIMP-1. (C) Representative zymography analysis of gelatinolytic activity in HSC supernatants. (D) BK (0.1 μmol/L) stimulated the activity of metalloproteinase 2 (mmp-2) that was quantified by a densitometry analysis. *P < .05 vs buffer. Gastroenterology 2007 133, 2019-2028DOI: (10.1053/j.gastro.2007.09.023) Copyright © 2007 AGA Institute Terms and Conditions