or portal hypertension

Journal of Hepatology 37 (2002) 773–780 www.elsevier.com/locate/jhep

Hemodynamic and antifibrotic effects of losartan in rats with liver fibrosis and/or portal hypertension Vincent Croquet 1,†, Fre´de´ric Moal 1,†, Nary Veal 1, Jianhua Wang 1, Fre´de´ric Oberti 1, Je´roˆme Roux 2, Eric Vuillemin 1, Yves Gallois 3, Olivier Douay 3, Daniel Chappard 4, Paul Cale`s 1,* 1

Laboratoire HIFIH, UPRES EA 2170, Universite´ d’Angers, Angers, France 2 Service d’Animalerie, Universite´ d’Angers, Angers, France 3 Laboratoire de Biochimie, Universite´ d’Angers, Angers, France 4 Laboratoire d’Histologie, Universite´ d’Angers, Angers, France

Background/Aims: To assess the effects of the early and chronic administration of losartan -a specific angiotensin II receptor antagonist- in the prevention of hepatic fibrosis and portal hypertension. Methods/Results: (1) In CCl4 rats, losartan at 5 and 10 mg/kg per day significantly decreased portal pressure (211, 218%, respectively), splenorenal shunt blood flow (260, 280%) and liver fibrosis (liver hydroxyproline and area of fibrosis) without significant changes in mortality and mean arterial pressure (MAP). (2) In bile duct ligated (BDL) rats, losartan at 5 mg/kg per day significantly decreased portal pressure (214%), splenorenal shunt blood flow (270%) and liver fibrosis. Losartan at 10 mg/kg per day significantly worsened liver and renal functions, mortality and liver fibrosis, without significant changes in portal pressure and splenorenal shunt blood flow. Losartan at 5 and 10 mg/kg per day significantly decreased MAP (224, 230%). (3) In portal vein ligated (PVL) rats, losartan significantly decreased MAP (212%) but did not change portal pressure or splenorenal shunt blood flow. Conclusions: In BDL and CCl4 rats, losartan has beneficial effects on splanchnic hemodynamics and liver fibrosis. Losartan might decrease hepatic resistances in fibrotic liver. Losartan decreased MAP except in CCl4 rats. Higher dosage of losartan had deleterious effects in BDL rats. q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. Keywords: Angiotensin II; Angiotensin II receptor antagonist; Losartan; Portal hypertension; Liver fibrosis; Hemodynamics; Rat

1. Introduction Angiotensin II is one of the most powerful vasoconstrictors. Plasma levels of angiotensin II are increased in cirrhotic patients [1]. Angiotensin II administration enhances portal pressure and hepatic resistance in humans [2,3] and collateral resistance in portal hypertensive rabbits [4]. Moreover, angiotensin II may contribute to portal hypertension (PHT) by stimulating the adrenergic vasoconstrictor Received 23 March 2001; received in revised form 16 August 2002; accepted 28 August 2002 * Corresponding author. Service d’He´pato-Gastroente´rologie, CHU, 49033 Angers Cedex 01, France. Tel.: 133-2-4135-3410; fax: 133-24135-4119. E-mail address: [email protected] (P. Cale`s). † These authors contributed equally to this work.

system (increase in central sympathetic outflow) [5] and water and sodium retention [6]. The presence of angiotensin II receptors has been recently reported on hepatic stellate cells (HSC) [7] and angiotensin II has been shown to contract these cells [7–9]. Losartan, an angiotensin II receptor antagonist, decreased the hepatic venous pressure gradient (HVPG) by 47 and 44% after 1 week of administration in cirrhotic patients with severe or moderate PHT, respectively, with a significant decrease in mean arterial pressure (MAP) [10]. Moreover, losartan significantly reduced the HVPG by 57% after 6 months administration in cirrhotic patients [11]. However, this study was not randomized or blind [12] and these beneficial effects were not confirmed in a randomized study [13]. Irbesartan, administered to cirrhotic patients for 2 months

0168-8278/02/$20.00 q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. PII: S 0168-827 8(02)00307-0

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significantly reduced the HVPG by 21% in responders and MAP by 16% [14]. Angiotensin II receptor antagonists might also have a beneficial effect on liver fibrogenesis. Angiotensin II receptor antagonists significantly decrease extracellular matrix synthesis in kidney and heart fibrosis [15,16]. These effects are due to the negative control of TGF-b1, a major hepatic profibrotic cytokine [17], by angiotensin II receptor antagonists [15,16,18]. Moreover, angiotensin II might also be involved in hepatic fibrogenesis by activation of its receptors on HSC [9]. The aims of this study were to determine the effects of losartan on hemodynamics in three models of PHT, with and without liver fibrosis, and on liver fibrogenesis in rats with liver fibrosis. 2. Material and methods 2.1. Observers

losartan potassium (DuP-753, Merck and Dohme-Chibret, Paris, France) chosen in this study was 10 mg/kg per day based on the range of dosages used in spontaneously hypertensive rats: 3–30 mg/kg per day [22,23]. However, due to the very high death rate in BDL rats treated with this dosage, a 5 mg/kg per day dosage was also used. To maintain blind administration, a new placebo group was used and pooled with the previous one. Both dosages were then used concomitantly in the CCl4 model. In the CCl4 model, rats were divided into three groups: 17, 17 and 16 rats were treated with losartan 10 mg/kg per day or 5 mg/kg per day, and placebo (water), respectively. In the BDL model, rats were divided into three groups: 30, 17, and 24 rats were treated with losartan 10 mg/kg per day or 5 mg/kg per day, and placebo, respectively. PVL rats were divided into two groups: 14 and 13 rats were treated with losartan 10 mg/kg per day and placebo, respectively. SO rats were divided into two groups: 12 and ten rats were treated with losartan 10 mg/kg per day and placebo, respectively.

2.4. Biochemical dosages All rats, except those in the PVL model, underwent blood liver and renal function tests including total bilirubin, alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) activities, as well as urea and creatinine. From days 21 to 27, sham and BDL conscious rats were placed in individual metabolic cages (Iffa-Credo, L’Arbresle, France) for 24 h to measure 24-h water consumption and urinary concentrations of creatinine, sodium and potassium.

Hemodynamic measurements, biochemical dosages and morphometric studies of the liver were performed blindly by two observers who were not aware of the treatment given.

2.5. Hemodynamics

2.2. Animal models

2.5.1. Rat preparation

2.2.1. CCl4-treated rats CCl4 was given to 50 Sprague–Dawley rats (Faculty of Medicine, Angers, France) weighing around 220 g. The rats received increasing dosages of 50% CCl4 intraperitoneally diluted in liquid paraffin every 5 days for 8 weeks. Phenobarbitone induction was started 10 days before the first dose of CCl4, at a concentration of 350 mg/l in drinking water [19].

2.2.2. Bile duct ligated rats Seventy-one male Sprague–Dawley rats with an initial body weight of 180–220 g underwent bile duct ligation (BDL). The surgical procedure was performed while rats were under ether anesthesia, as previously described [20]. Briefly, the bile duct was exposed after median laparotomy and a 3-0 silk was used to ligate it in two places, then the bile duct was cut between both ligations. Rats received weekly subcutaneous injections of vitamin K1 (50 mg) [21].

2.2.3. Portal vein ligated rats Twenty-seven male Sprague–Dawley rats, with an initial body weight of 180–220 g, underwent portal vein ligation (PVL) under ether anesthesia, as previously described [19]. Briefly, a polyethylene catheter was placed along the length of the portal vein, and a 3-0 silk was used to ligate the catheter and the portal vein. The catheter was then removed.

2.2.4. Sham-operated rats Twenty-two male Sprague–Dawley rats, with an initial body weight of 180–220 g, served as sham-operated rats (SO). Rats underwent laparotomy under ether anesthesia with manipulations of the bile duct or portal vein but without ligation.

2.3. Therapeutic regimen Treatment (losartan and placebo) was given by gavage 6 days per week. Gavage was begun 24 h after surgery or the day of the first dose of CCl4 and continued until measurements were performed, i.e. 21 days in PVL rats, 28 days in BDL and SO rats, and 9 weeks in CCl4 rats. The initial dosage of

Rats were anesthetized with an intraperitoneal injection of pentobarbital (Nesdonal 0.5 g, Rhoˆ ne-Poulenc, Paris, France): 5 mg/100 g of body weight. Body temperature was maintained at 37 8C with a homeothermic blanket system (Homeothermic Blanket Control Unit, Harvard Apparatus Inc., Natick, MA, USA). All rats were given free access to food and water until 14–16 h before the measurements, when food was withdrawn. Ascites was graded with the following score: 0, no ascites; 1, ascites soaking less than half a 7:5 £ 7:5-cm compress; 2, ascites soaking less than a compress; 3, ascites soaking more than one compress.

2.5.2. Pressures MAP and heart rate were measured by a catheter (PE-10, Clay Adams, NJ, USA) inserted into the femoral artery. Another catheter (PE-10, Clay Adams) was also inserted into the femoral vein to measure cardiac output. Portal pressure was measured as previously described [24]. Briefly, a polyethylene catheter (0.7 mm diameter, Clay Adams) was inserted into a small ileal vein and gently advanced up to the bifurcation of the superior mesenteric and splenic veins. All catheters were connected to a computer via a sensor (Baxter, Uden, Germany) and a monitor (Biopac MP100A, Biopac Systems Inc., Santa Barbara, CA, USA) using the software Acknowledge 3.0 (Biopac Systems Inc., Goleta, CA, USA).

2.5.3. Blood flow Splenorenal shunt (SRS) blood flow is an index of collateral circulation blood flow. SRS blood flow was measured in anesthetized rats as previously described [25]. Briefly, the TTU probe (1R, J reflector, 1 mm diameter, Transonic System Inc., NY, USA) was placed around the SRS and connected to a dual-channel small animal flowmeter (T206, Transonic System). The TTU method was also used to evaluate cardiac output (CO) by Fick’s dilution technique as recently described in our laboratory [26]. Briefly, a 0.2-ml bolus of NaCl, at constant body temperature, was injected by a femoral vein catheter and underwent cardiac pulmonary mixing. This generated an ultrasound velocity dilution curve recorded by the TTU probe placed around the carotid artery. The area under the curve, proportional to CO, was measured off-line using software (Matlab 4.2c, Mathworks, Natick, MA, USA). CO and cardiac index (CI) were then calculated.

V. Croquet et al. / Journal of Hepatology 37 (2002) 773–780

775

Table 1 Mortality rate

CCl4

BDL

PVL Sham Total a b

Treatment

Initial number of rats

Mortality

Final number of rats

Placebo Losartan 5 mg/kg/day Losartan 10 mg/kg/day Placebo Losartan 5 mg/kg/day Losartan 10 mg/kg/day Placebo Losartan 10 mg/kg/day Placebo Losartan 10 mg/kg/day –

16 17 17 24 17 30 13 14 10 12 170

1 (6%) 2 (11%) 2 (11%) 8 (33%) a 6 (35%) 22 (73%) b 1 (7%) 0 (0%) 0 (0%) 0 (0%) 42 (25%)

15 15 15 16 11 8 12 14 10 12 128

P , 0:05 vs. sham. P , 0:05 vs. BDL placebo.

Systemic vascular resistances (SVR, dyn s cm 25 100 g 21 £ 10 3) were calculated as MAP ðmmHgÞ £ 80/CI (ml min 21 100 g 21).

3. Results 3.1. General characteristics of rats

2.6. Liver fibrosis 2.6.1. Serum hyaluronate Serum hyaluronate concentrations were determined using a radiometric technique (Amersham-Pharmacia Biotech, Uppsala, Sweden), based on the use of specific hyaluronate-binding proteins isolated from bovine-cartilage.

A total of 170 rats were used in the study. The death rate was not statistically different between the placebo and 5 mg/ kg per day losartan groups in CCl4, PVL, SO or BDL models but losartan at 10 mg/kg per day significantly increased mortality in the BDL model (Table 1). 3.2. Hydroelectrolytic data (Table 2)

2.6.2. Area of fibrosis Three liver fragments (.1 cm2 each) were randomly taken from the right, median and left liver lobes of each rat. Liver fragments were fixed in Bouin’s solution and embedded in paraffin. Four-mm-thick sections were then stained in 0.1% picrosirius red solution. The histomorphometric analysis was done on a Leica Quantimet Q570 image processor as described previously [27]. Total area of liver fibrosis was expressed as the mean of the percentage of fibrosis in the three liver fragments. In each of them, 30 fields were evaluated. Thus, the total liver area evaluated was 3 £ 73:5 mm 2.

2.6.3. Hydroxyproline Hepatic hydroxyproline content was measured using a modified version of the method of Jamall et al. [28] that is detailed elsewhere [29]. Hepatic protein content was measured in three liver fragments and the mean final result was expressed as micrograms of hydroxyproline per gram of protein in the liver.

2.7. Statistical analysis Qualitative variables were compared using the x 2 test with Yates’ correction or the Fischer test when necessary. Quantitative variables were expressed as mean ^ SD. Means were compared with a Student’s test or a analysis of variance using a one-way analysis of variance when necessary with Bonferroni post hoc comparisons or by non-parametric tests in nonGaussian variables. However, comparisons were only tested in each rat model, otherwise this would have introduced too many unnecessary comparisons due to the use of four rat models. In addition, differences between models were only tested in placebo groups. An a risk of less than 5% was considered to be statistically significant.

There was no significant difference in the volume of water drunk per 24 h but 24-h diuresis was significantly higher, whereas urinary Na and creatinine were significantly decreased in BDL rats compared to SO rats in placebo groups. Losartan at 10 mg/kg per day significantly decreased urinary output of Na, K, and creatinine in the BDL model. The ascites score was significantly increased in the BDL rats and losartan tended to decrease this score. The ascites score was not significantly influenced by losartan in the CCl4 model (data not shown). In the PVL model, no ascites occurred. 3.3. Liver and renal tests (Table 3) In placebo rats AST, ALP and bilirubin were significantly increased in BDL rats compared to SO rats. Losartan had no effect on liver or renal function tests in SO or CCl4 rats. In BDL rats, losartan at 5 mg/kg per day decreased ALP and bilirubin whereas at 10 mg/kg per day it increased urea, creatinine and AST activity compared to placebo rats. 3.4. Hemodynamics (Table 4) Losartan induced no significant systemic effect in the CCl4 model whereas it significantly decreased MAP in the BDL and PVL models and significantly decreased SVR in

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Table 2 Hydroelectrolytic results Sham

Water consumption (ml/24 h) Diuresis (ml/24 h) Urinary Na (mmol/24 h) Urinary K (mmol/24 h) Urinary creatinine (mmol/24 h) Ascites score a b c

BDL

Placebo (n ¼ 10)

Losartan 10 mg/kg/day (n ¼ 12)

Placebo (n ¼ 16)

Losartan 5 mg/kg/day (n ¼ 11)

Losartan 10 mg/kg/day (n ¼ 8)

24 ^ 5 13 ^ 5 1.9 ^ 0.44 2.14 ^ 0.49 0.08 ^ 0.02 0

19 ^ 3 12 ^ 4 2.37 ^ 0.44 2.44 ^ 0.43 0.09 ^ 0.01 0

32 ^ 15 26 ^ 13 a 1.32 ^ 0.65 a 1.92 ^ 0.63 0.06 ^ 0.02 a 0.9 ^ 0.6 a

28 ^ 14 23 ^ 8 1.25 ^ 0.74 1.93 ^ 0.56 0.06 ^ 0.01 0.4 ^ 0.9

30 ^ 18 20 ^ 15 0.79 ^ 0.90 b,c 1.12 ^ 0.82 b,c 0.04 ^ 0.02 b 0.3 ^ 1.0

P , 0:05 vs. sham placebo. P , 0:05 vs. BDL placebo. P , 0:05 vs. BDL losartan 5 mg/kg/day.

the BDL model with a significant increase in CI at 10 mg/kg per day compared to placebo rats. In the CCl4 model, losartan at 5 or 10 mg/kg per day induced a significant decrease in portal pressure and SRS blood flow. In BDL rats, losartan at 5 mg/kg per day significantly decreased portal pressure and SRS blood flow compared to placebo. Losartan at 10 mg/kg per day did not produce a significant splanchnic effect in the BDL or the PVL model.

4. Discussion 4.1. Hemodynamics 4.1.1. CCl4 model Losartan had no systemic hemodynamic effect whereas it significantly prevented the increase in portal pressure (211 and 218%, respectively at 5 and 10 mg/kg per day) and in SRS blood flow (260% and 280%). Losartan was well tolerated in this model. The decrease in SRS blood flow might be partly due to the reduction in portal pressure since it has already been demonstrated that the baseline values of collateral blood flow and portal pressure were well correlated in rats [30] and humans [31]. However, the discrepancy between the marked decrease in SRS blood flow and the slight reduction in portal pressure suggests that losartan reduces SRS blood flow through other mechanisms. Indeed, angiotensin II induces contraction and proliferation of human activated HSC unlike in quiescent HSC [7]. Thus, losartan could lower intrahepatic vascular resistance, as previously suggested [10,12], but

3.5. Fibrosis measurements (Table 5) Losartan caused a significant decrease in serum hyaluronate level at both dosages in CCl4 rats. Losartan at 5 mg/kg per day significantly reduced the area of fibrosis and hepatic hydroxyproline content in the BDL and CCl4 models whereas, at 10 mg/kg per day, the area of fibrosis and hepatic hydroxyproline content were also significantly decreased in the CCl4 model but significantly increased in the BDL model.

Table 3 Liver and renal tests Sham Placebo (n ¼ 10)

Urea (mmol/l) Creatinine (mmol/l) AST (UI/l) d ALT (UI/l) d Alkaline phosphatases (UI/l) d Bilirubin (mmol/l) d b c d a

BDL Losartan 10 mg/kg/day (n ¼ 12)

0.08 ^ 0.01 0.07 ^ 0.01 64 ^ 22 58 ^ 9 144 ^ 103 138 ^ 60 51 ^ 40 45 ^ 19 146 ^ 26 154 ^ 41 1.3 ^ 0.5 1.4 ^ 0.5

CCl4

Placebo (n ¼ 16)

Losartan 5 mg/kg/day (n ¼ 11)

Losartan 10 mg/kg/day (n ¼ 8)

0.07 ^ 0.03 48 ^ 14 519 ^ 309 a 56 ^ 28 339 ^ 199 a 75 ^ 35 a

0.11 ^ 0.07 49 ^ 26 721 ^ 689 66 ^ 56 186 ^ 107 b 47 ^ 46

0.32 ^ 0.29 b,c 0.09 ^ 0.01 89 ^ 41 b,c 83 ^ 54 1163 ^ 1097 211 ^ 86 49 ^ 23 66 ^ 24 235 ^ 56 75 ^ 55 70 ^ 47 6.1 ^ 6.2

P , 0:05 vs. BDL placebo. P , 0:05 vs. BDL losartan 5 mg/kg/day. Comparison with Kruskal–Wallis test but results expressed as mean ^ SD. P , 0:05 vs. sham placebo.

Placebo (n ¼ 9)

Losartan 5 mg/kg/day (n ¼ 15)

Losartan 10 mg/kg/day (n ¼ 10)

0.10 ^ 0.02 84 ^ 21 374 ^ 342 219 ^ 275 154 ^ 73 2.9 ^ 3.4

0.13 ^ 0.05 85 ^ 15 225 ^ 76 71 ^ 28 149 ^ 85 3.0 ^ 3.1

Table 4 Hemodynamic data PVL

BDL

Placebo (n ¼ 10) Losartan 10 mg/kg/day (n ¼ 12)

Placebo (n ¼ 12) Losartan 10 mg/kg/day (n ¼ 14)

Placebo (n ¼ 16) Losartan 5 mg/kg/day (n ¼ 11)

Mean arterial pressure (mmHg) 114 ^ 13

97 ^ 11 a

114 ^ 12

399 ^ 39

a

395 ^ 29

Heart rate (beats/min) 21

448 ^ 37

240 ^ 26 a

211 ^ 14

7.6 ^ 1.0

14.7 ^ 2.2 a

0.3 ^ 0.1

3.7 ^ 2.0 a

41 ^ 10

Systemic vascular resistance (dyn s cm 25 100 g 21)

411 ^ 37

314 ^ 35

Portal pressure (mmHg)

7.8 ^ 1.2

Splenorenal shunt blood flow (ml/min)

0.3 ^ 0.1

a d b e f c

P , 0:05 vs. P , 0:05 vs. P , 0:05 vs. P , 0:01 vs. P , 0:05 vs. P , 0:05 vs.

sham placebo. PVL placebo. BDL placebo. CCl4 placebo. CCl4 placebo. BDL losartan 5 mg/kg/day.

409 ^ 55 64 ^ 36

25 ^ 11

100

426 ^ 39 a

95 ^ 18 a

39 ^ 6

23 ^ 5

Cardiac index (ml.min g 21)

100 ^ 17 d

CCl4

72 ^ 21 b 395 ^ 58

a

Losartan 10 mg/kg/day (n ¼ 8) 66 ^ 25 b 385 ^ 52

Placebo (n ¼ 15) Losartan 5 mg/kg/day (n ¼ 15) 100 ^ 7 386 ^ 54

b

99 ^ 12

97 ^ 23

422 ^ 30

397 ^ 45

44 ^ 20

37 ^ 16

71 ^ 24

85 ^ 33

159 ^ 20 a

92 ^ 15 b

83 ^ 20 b

190 ^ 43 a

221 ^ 118

259 ^ 158

14.1 ^ 2.6

14.8 ^ 2.8 a

12.7 ^ 2.0 b

13.0 ^ 2.8

14.3 ^ 1.9 a

11.6 ^ 3.0 e

12.7 ^ 2.2 f

4.3 ^ 2.5

3.3 ^ 3.1 a

1.0 ^ 0.9 b

2.0 ^ 1.9 a

0.4 ^ 0.3 e

0.8 ^ 1.1 f

2.5 ^ 2.3 c

44 ^ 14

a

Losartan 10 mg/kg/day (n ¼ 15)

V. Croquet et al. / Journal of Hepatology 37 (2002) 773–780

Sham

777

778

V. Croquet et al. / Journal of Hepatology 37 (2002) 773–780

Table 5 Liver fibrosis data Sham Placebo (n ¼ 10) 50 ^ 21 Serum hyaluronate (mg/l) a Area of liver fibrosis (%) 1.9 ^ 1.2 Hepatic hydroxyproline 0.05 ^ 0.02 content (mg/g of liver proteins) a b d c e

BDL

CCl4

Losartan 10 mg/kg/day (n ¼ 12)

Placebo (n ¼ 16)

Losartan 5 mg/kg/day (n ¼ 11)

Losartan 10 mg/kg/day (n ¼ 8)

41 ^ 12 1.4 ^ 0.6 0.05 ^ 0.03

574 ^ 326 b 7.6 ^ 0.7 b 0.25 ^ 0.10 b

440 ^ 373 657 ^ 393 4.8 ^ 0.6 c 9.9 ^ 2.2 c c 0.15 ^ 0.10 ND e

Placebo (n ¼ 15)

Losartan 5 mg/kg/day (n ¼ 15)

Losartan 10 mg/kg/day (n ¼ 15)

222 ^ 211 b 10.5 ^ 3.2 b 0.35 ^ 0.14 b

81 ^ 54 d 58 ^ 26 d 8.2 ^ 2.9 d 8.3 ^ 3.1 d 0.25 ^ 0.08 d 0.24 ^ 0.13 d

Comparison with Kruskal–Wallis test but results expressed as mean ^ SD. P , 0:05 vs. sham placebo. P , 0:05 vs. CCl4 placebo. P , 0:05 vs. BDL placebo. Not done due to the increase in the area of liver fibrosis and the correlation between both parameters (r ¼ 0:66, P , 0:0001 in other BDL rats).

only in PHT with liver fibrosis, i.e. when HSC are activated. Thus, in the CCl4 model the portal pressure reduction is probably mostly due to a direct effect of losartan on intrahepatic vascular resistance.

rats might be related to the slight decrease in MAP and the lack of effect on hepatic resistances. 4.2. Liver fibrosis

4.1.2. BDL model Losartan at 5 or 10 mg/kg per day significantly decreased MAP (224% and 231%, respectively) and SVR. A decrease in MAP requiring irbesartan cessation in 12% [14] to 22% [32] of cirrhotic patients has already been reported. Losartan at 5 mg/kg per day significantly prevented the increase in portal pressure (214%) and in SRS blood flow (270%). Another mechanism involved in the BDL model might be arterial splanchnic vasoconstriction in response to systemic vasodilation as described with nitrates [33]. The lack of effect of losartan at 10 mg/kg per day on splanchnic hemodynamics could be due to its harmful effects, especially the worsening of liver fibrosis. Indeed, losartan at 10 mg/kg per day had deleterious effects with a significantly higher mortality rate, a more marked decrease in MAP associated to a significant increase in CO, renal failure and increased AST activity. However, no harmful effect was observed after the chronic administration of 30 mg/kg per day in spontaneous hypertensive rats [23]. The metabolism of losartan is hepatic with 50–60% biliary elimination [34]. Thus, in case of biliary obstruction, plasmatic accumulation of the losartan metabolite, which has a greater pharmacological activity than losartan, may be partially responsible for deleterious systemic effect. In addition, cholestasis per se [35] could worsen the hemodynamic effects of losartan. 4.1.3. PVL model In PVL and SO rats, the decrease in MAP after losartan at 10 mg/kg per day was less (215% and 212%, respectively) than in BDL rats and did not influence CO. The lack of effect of losartan on splanchnic hemodynamics in PVL

Losartan significantly decreased parameters reflecting liver fibrosis at both doses in CCl4 rats and at low dose the BDL model. This might contribute to the decrease in hepatic resistance. In progressive fibrosis in other organs, particularly the heart and kidney, TGF-b1 production is enhanced by angiotensin II and is lowered by its antagonists [15,18]. Moreover, losartan resulted in a decrease in extracellular matrix production in these organs [36,37]. In BDL rats, where the expression of AT-1 gene was 6 times higher than in sham rats, irbesartan significantly decreased the mRNA expression of TGF-b1 [38]. Candesartan significantly decreased hepatic hydroxyproline content, plasma collagen 7-S, P-III-P and hyaluronic acid, the area of fibrosis and the mRNA expression of TGF-b1 in rats with liver fibrosis induced by administration of pig serum [39]. The inhibition of angiotensin converting enzyme by perindopril in choline-deficient rats [40] or by captopril in BDL rats [41] decreased the liver fibrosis development. Finally, there is a significant relationship between TGF-b1 or angiotensinogen genotypes and the development of hepatic fibrosis in patients with chronic hepatitis C [42]. All these results confirm that angiotensin II plays a role in liver fibrosis and that its inhibition is a therapeutic option. The anticholestatic effect of losartan at low dose in our BDL rats could be due to a non specific improvement of liver fibrosis as described with silymarine in BDL rats [43] or with octreotide in the CCl4 model [29]. However, a significant decrease in serum bilirubin concentrations has been observed in cirrhotic patients with severe portal hypertension treated with losartan for only 7 days [10]. In conclusion, the early and chronic administration of losartan had beneficial effects in two models of liver fibro-

V. Croquet et al. / Journal of Hepatology 37 (2002) 773–780

sis. Splanchnic hemodynamics improved with a reduced portal pressure and collateral blood flow in BDL and CCl4 rats. MAP was reduced in the BDL model only. In addition, the development of liver fibrosis was reduced in both liver fibrosis models. Moreover, losartan had no effect on splanchnic hemodynamics in prehepatic PHT despite a decrease in MAP. These results suggest that losartan might improve splanchnic hemodynamics via a splanchnic vasoconstriction secondary to arterial hypotension and/or via a decrease in hepatic resistances. The administration of sartans might be beneficial in the treatment of fibrosis or in early cirrhosis whereas its administration should be considered with caution in decompensated cirrhosis with regard to MAP decrease. In addition, a dual dose effect in biliary cirrhosis and species differences to angiotensin II receptors AT1 [44] should be taken into account.

[17]

Acknowledgements

[18]

V.C. received a grant from the Fondation pour la Recherche Me´ dicale. We thank Mrs. Dale Roche for her contribution.

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