Gadolinium chloride,a Kupffer cell inhibitor,attenuates hepatic injury in a rat model of chronic cholestasis

Abstract

The aim of the current study was to elucidate the effect of Kupffer cells inhibition on hepatic injury induced by chronic cholestasis. Sprague-Dawley rats underwent bile duct ligation (BDL) or sham operation and were treated with either saline solution or gadolinium chloride (GdCl₃, a specific Kupffer cell inhibitor, 20 mg/kg i.p. daily). Serum and liver samples were collected after 28 days. Direct and total bilirubin concentrations and serum enzyme activities of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and γ-glutamyl transpeptidase (GGT) increased following BDL (p < 0.01). On the contrary to bilirubin concentrations and AST activity, GdCl₃ partially prevented the elevation in ALP, ALT and GGT enzyme activities (p < 0.05). GdCl₃ alleviated lipid peroxidation (reflected by malondialdehyde [MDA] concentration) and increased the activities of antioxidant enzymes (i.e. catalase and glutathione peroxidase) in liver samples after BDL (p < 0.05). Fibrosis, ductular proliferation and portal inflammation were also scored in liver samples. Among morphological changes appeared following BDL (i.e. marked fibrosis, portal inflammation and ductular proliferation); only ductular proliferation was not alleviated by GdCl₃. Therefore, Kupffer cells inhibition has beneficial effects against the development of hepatic injury induced by chronic cholestasis.

Keywords

cholestasis fibrosis Kupffer cells gadolinium chloride

Introduction

Cholestasis is associated with a number of chronic liver diseases such as biliary atresia, primary biliary cirrhosis and primary sclerosing cholangitis and leads to hepatocellular injury, fibrosis, cirrhosis and ends in death from liver injury.¹ The retention of bile acids during cholestatic disorders is believed to play an important role in liver injury by inducing apoptosis or necrosis of hepatocytes, although the mechanisms involved in this toxicity are not fully understood.² Bile acids through their detergent action can disrupt cell membranes.^2,3 Additionally, by activating Kupffer cells they can also promote the generation of reactive oxygen species which, in turn, oxidatively modifies lipids, proteins and nucleic acids, and eventually cause hepatocyte apoptosis.^4,5 On the other hand, expression of death ligands in Kupffer cells may also contribute to hepatocyte apoptosis in cholestasis.^6–10

Gadolinium chloride (GdCl₃) is known to inhibit Kupffer cells functions. Number of Kupffer cells as well as tumor necrosis factor-a and interleukin-6 mRNA expression is reduced in hepatic tissue following GdCl₃ administration.¹¹ GdCl₃ also down regulates cytochrome P-450 levels.¹² Adachi et al. showed that GdCl₃ treatment during chronic exposure to alcohol ameliorates early alcohol-induced hepatic injury in rats.¹⁰ It is also demonstrated that GdCl₃ administration attenuates carbon tetrachloride- and thioacetamide-induced hepatic fibrosis.^13–15 However, the role of Kupffer cells and the beneficial effect of modulating their activity have not been fully investigated especially in a model of chronic cholestasis.

To our knowledge, there have been no reports about the effects of long-term administration of GdCl₃ on hepatic injury induced by chronic cholestasis in bile duct-ligated (BDL) rats. Our aim was to evaluate the protective role of Kupffer cells inhibition against hepatic injury and fibrosis triggered by BDL. The extent of hepatic injury was determined by measurement of hepatic enzymes activities and bilirubin concentrations in serum. Among markers of the antioxidant status of liver tissue, liver antioxidant enzymes activities as well as a lipid peroxidation marker were assessed. Moreover, in order to study the alterations in liver tissue following different experiments, the histological evaluations of fibrosis, ductular proliferation and portal inflammation were performed.

Materials and methods

Reagents

The Kupffer cell toxicant, GdCl₃ was obtained from Sigma (St Louis, MO, USA). All other materials were purchased from Merck (Darmstadt, Germany) unless otherwise stated.

Treatment and sample collection

Male Sprague-Dawley rats (230–260 g) were used in this study. The rats were housed in an animal room monitored at 24 ± 2°C and with a 12-h light-dark cycle, given unlimited access to standard laboratory chow and water. All animal procedures were in accordance with ‘Guide for the Care and Use of Laboratory animals’ (NIH US publication no. 85-23 revised 1985). BDL was performed under general anesthesia. In brief, through a midline abdominal incision the common bile duct was exposed and ligated in two places with a silk thread and then sectioned between the ligatures. In the sham-operated rats, used as controls, the bile duct was exposed but no ligation was performed.

The rats were randomly divided into four groups each containing 10 animals. In the control- and BDL-saline groups, rats were treated with sterile saline solution (1 ml/kg/day). In the control- and BDL-GdCl₃ groups, rats received GdCl₃ (20 mg/kg/day i.p.) dissolved in normal saline, following sham and BDL operations, respectively. All the animals were sacrificed after 28 days with an overdose of sodium pentobarbital. Liver samples were collected rapidly and snap frozen in liquid nitrogen for biochemical measurements, or were immersed in 10% buffered formalin and then embedded in paraffin for histological studies. Serum samples separated from blood were obtained via a cardiac puncture.

Biochemical measurements

Among biochemical markers of liver injury, total and direct bilirubin concentrations and enzyme activities of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ-glutamyl transpeptidase (GGT) were measured in serum samples using commercially available kits (Zistshimi, Tehran, Iran).

The extent of lipid peroxidation was estimated in liver homogenates by measurement of malondialdehyde (MDA) formation using the thiobarbituric acid method.¹⁶ Among antioxidant enzymes, activities of catalase (CAT) and glutathione peroxidase (GPx) were determined in liver tissue. Measurement of CAT activity was done according to Aebi, in which the decomposition of H₂O₂ is estimated by measuring the decrease in absorbance at 240 nm.¹⁷ GPx activity was measured in accordance with method of Paglia and Valentine.¹⁸ In brief, GPx catalyses the oxidation of glutathione by H₂O₂ in a reaction coupled with the conversion of nicotinamide adenine dinucleotide phosphate (NADPH) to NADP⁺. The change in absorbance at 340 nm is the basis for measuring GPx activity in this method. Total protein was assessed using the method of Lowry et al.¹⁹

Histological study

Formalin-fixed paraffin-embedded sections of the right liver lobe were cut at 3 μm and stained with hematoxilin and eosin, and Masson’s Trichrome. Fibrosis, ductular proliferation and portal inflammation were scored by a single pathologist who was blinded to the treatment groups. Fibrosis was staged 0–4 using Scheuer’s scoring system (0: no fibrosis, 1: expansion of portal tract without linkage, 2: portal expansion with portal to portal linkage, 3: extensive portal to portal and focal portal to central linkage and 4: cirrhosis).²⁰ Ductular proliferation was measured in 50 portal areas and scored using the following grading system: 0, <10% of portal areas involved; 1, 10%–50% of portal areas involved; 2, >50% of portal areas involved; 3, circumferential involvement of at least 50% of the portal area without significant expansion of portal tract; 4, circumferential involvement of at least 50% of the portal area with significant expansion of portal tract; 5, same as 4 plus bridging of the portal tracts. Based on Kamal Ishak’s scoring system, portal inflammation was staged 0–4 as follows: 0: none, 1: mild, some or all portal areas, 2: moderate, some or all portal areas, 3: moderate/marked, all portal areas, 4: marked, all portal areas.²¹

Statistical analysis

Data are presented as mean ± standard error of the mean (SEM). All data analyses were performed with SPSS software (version 16.0; SPSS Inc., Chicago, USA), using one-way analysis of variance (ANOVA) followed by Tukey’s test. p < 0.05 was considered significant.

Results

Three animals died in BDL-saline group, whereas treatment with GdCl₃ decreased the mortality to 1 out of 10. Sham-operated animals treated with GdCl₃ or saline showed no mortality.

All biochemical markers of liver injury including ALT, AST, ALP and GGT enzyme activities along with direct and total bilirubin concentrations increased significantly following 28 days of BDL (p < 0.01, Table 1 ). GdCl₃ administration partially prevented the elevation in ALP, ALT and GGT serum enzyme activities but these markers did not reach normal levels (p < 0.05 and p < 0.01 compared to BDL-saline and sham-operated controls, respectively). GdCl₃ had no effect on AST enzyme activity and direct and total bilirubin concentrations.

Table 1.

Biochemical markers of liver injury determined in serum samples

	Control-saline	Control-GdCl3	BDL-saline	BDL-GdCl3
AST (U/l)	230 ± 11	243 ± 50	512 ± 34^a	540 ± 49^a
ALT (U/l)	37 ± 2.6	42 ± 6.8	167 ± 9.2^a	132 ± 9.5^a,b
ALP (U/l)	356 ± 14	367 ± 55	955 ± 40^a	761 ± 50^a,b
GGT (U/l)	4.6 ± 0.3	3.5 ± 0.5	25.8 ± 2.2^a	18.6 ± 1.2^a,b
Direct bilirubin (μmol/dl)	0.3 ± 0.1	0.3 ± 0.1	8.9 ± 2.3^a	8.3 ± 1.9^a
Total bilirubin (μmol/dl)	0.4 ± 0.2	0.5 ± 0.2	9.5 ± 2.5^a	8.9 ± 2.2^a

Data are expressed as mean ± SEM.

^a p < 0.01 compared to control-saline and control-GdCl₃ groups.

^b p < 0.05 compared to BDL-saline group.

Table 2 shows that the hepatic MDA concentration and CAT and GPx activities were worsened after BDL (p < 0.01). However, these markers were improved by GdCl₃ administration (p < 0.05).

Table 2.

Malondialdehyde (MDA) concentration and antioxidant activities of catalase (CAT) and glutathione peroxidase (GPx) in liver tissue

	Control-saline	Control-GdCl3	BDL-saline	BDL-GdCl3
MDA concentration (nmol/100 mg protein)	40.2 ± 5.8	46.8 ± 3.2	84.4 ± 5.2^a	63.1 ± 4.8^a,b
CAT activity (IU/mg protein)	525 ± 20	514 ± 18	233 ± 24^a	315 ± 17^a,b
GPx activity (IU/mg protein)	112.7 ± 8.4	94.4 ± 5.3	35.4 ± 3.5^a	61.8 ± 4.4^a,b

Data are expressed as mean ± SEM.

^a p < 0.01 compared to control-saline and control-GdCl₃ groups.

^b p < 0.05 compared to BDL-saline group.

Biliary obstruction for 28 days resulted in marked morphological changes in the liver tissue, including the formation of extensive fibrosis in periportal areas, portal tracts inflammation, and widespread ductular proliferation (Figure 1 ). On the contrary to ductular proliferation, degrees of fibrosis and inflammation were reduced by GdCl₃ treatment (p < 0.05 compared to BDL-saline group, Table 3 ).

Table 3.

Histologic assessment of fibrosis, ductular proliferation and portal inflammation

	Control-saline	Control-GdCl3	BDL-saline	BDL-GdCl3
Fibrosis	0 ± 0	0 ± 0	2.32 ± 0.24^a	1.40 ± 0.24^a,b
Ductular proliferation	0 ± 0	0 ± 0	3.38 ± 0.18^a	3.20 ± 0.20^a
Portal inflammation	0 ± 0	0 ± 0	1.50 ± 0.22^a	0.80 ± 0.20^a,b

Data are expressed as mean ± SEM.

^a p < 0.01 compared to control-saline and control-GdCl₃ groups.

^b p < 0.05 compared to BDL-saline group.

Discussion

The purpose of this study was to evaluate the effect of Kupffer cell inhibition on hepatic injury in a rat model of chronic BDL. Chronic BDL leads to cirrhosis after 28 days and has been used extensively in a wide variety of experimental studies.^22–25 Our results indicate that chronic BDL elevates biochemical markers of liver injury as well as MDA concentration, and reduces antioxidant enzyme activities. BDL also leads to the development of fibrosis, portal inflammation and ductular proliferation in liver tissue. Interestingly, administration of GdCl₃ reversed most of aforementioned values but failed to normalize them.

Hepatocyte apoptosis is a key component in the formation of hepatic injury,^26,27 and is linked to a diverse group of hepatic diseases including cholestasis-induced liver damage.²⁶ Canbay et al. showed that inflammation, regeneration and fibrosis can all be stimulated by apoptosis.²⁷ Engulfment of apoptotic bodies triggers Kupffer cells to release inflammatory cytokines and increase the expression of death ligands which are associated with more apoptosis and collateral damage to the liver tissue.⁷ Kupffer cell inhibition reduces engulfment of apoptotic bodies and expression of death ligands.⁷ These findings are in concordance with our results demonstrating less hepatic inflammation and injury following GdCl₃ treatment.

We showed that GdCl₃ alleviates oxidative stress reflected by improvement in liver antioxidant enzyme activities and MDA concentration. Bile acids have been regarded as important mediators of oxidative stress.²⁸ Bile acids cause direct hepatic injury and also activate hepatocytes and leukocytes to release reactive oxygen species and toxic free radicals.^4,5,28–31 Since antioxidant agents can alleviate cholestasis-induced hepatic injury,^32,33 it is postulated that oxidative stress may play a role in the development of hepatic injury following cholestasis. Our findings suggest that Kupffer cells at least in part are responsible for deterioration of oxidative status of liver tissue observed after chronic cholestasis.

Our results demonstrate that although hepatic fibrosis was not diminished completely, it was improved following GdCl₃ administration. A number of mechanisms may be responsible for this observation. For instance, activated Kupffer cells release profibrogenic mediators like transforming growth factor-β (TGF-β) which are involved in hepatic stellate cells transformation to myofibroblasts.³⁴ Hepatic stellate cells and their activated form myofobroblasts, according to their responsibility in production of collagen fibers, play a key role in fibrosis.³⁵ Rivera et al. presented that destruction of Kupffer cells blunts the increase in TGF-β expression and fibrosis following carbon-tetrachloride treatment.¹³ In this respect, it is also demonstrated that GdCl₃ stimulates Kupffer cells to release matrix metalloproteinase-13 in two different models of hepatic fibrosis.^36,37 Matrix metalloproteinases are responsible for degrading fibrotic fibers and their increased expression precedes less fibrotic tissue formation.³⁸ On the contrary to fibrosis, ductular proliferation was not mediated by GdCl₃ treatment. Therefore, Kupffer cells may not be essential for occurrence of bile duct proliferation.

Our findings may also have clinical relevance. Cholestasis can happen following formation of bile duct strictures, which can be traumatic, postoperative, infectious or inflammatory.³⁹ The postoperative strictures are the most frequent and can occur after iatrogenic injuries during cholecystectomy or anastomotic operations.³⁹ Although the timing development of biliary cirrhosis following biliary obstruction is under debate, biliary cirrhosis can occur as early as 20 weeks following bile duct injury.⁴⁰ Thus, early repair of biliary stricture is recommended to prevent liver fibrosis and cirrhosis. Sometimes health service problems or imposed risk of morbidity and mortality following early invasive procedures for removing bile duct strictures decreases the benefits of invasive interventions. In these situations patients can get benefit from drugs that delay the development of hepatic injury until treated more efficiently.

In conclusion, destruction of Kupffer cells by long-term administration of GdCl₃ diminishes cholestasis-induced hepatic injury. Changes appeared following BDL, like elevation in the biochemical markers of liver injury and MDA concentration together with decrease in liver antioxidant enzyme activities were reversed by GdCl₃. Kupffer cell inhibition also improved the histological state of the liver tissue in cholestatic rats. Further studies are needed to elucidate the clinical and therapeutic implications of Kupffer cells inhibitors in different models of hepatic injury.

Footnotes

The authors report no conflicts of interest.

This research received no specific grant from any funding agency in the public, commercial, or not for-profit sectors.

References

Guicciardi

Gores

. Bile acid-mediated hepatocyte apoptosis and cholestatic liver disease. Dig Liver Dis 2002; 34: 387–392.

Perez

Briz

. Bile-acid-induced cell injury and protection. World J Gastroenterol 2009; 15: 1677–1689.

Dial

Rooijakkers

Darling

Romero

Lichtenberger

. Role of phosphatidylcholine saturation in preventing bile salt toxicity to gastrointestinal epithelia and membranes. J Gastroenterol Hepatol 2008; 23: 430–436.

Sokol

Straka

Dahl

Devereaux

Yerushalmi

Gumpricht

. Role of oxidant stress in the permeability transition induced in rat hepatic mitochondria by hydrophobic bile acids. Pediatr Res 2001; 49: 519–531.

Ljubuncic

Fuhrman

Oiknine

Aviram

Bomzon

. Effect of deoxycholic acid and ursodeoxycholic acid on lipid peroxidation in cultured macrophages. Gut 1996; 39: 475–478.

Sun

Wada

Maemura

Uchikura

Hoshino

Diehl

. Hepatic allograft-derived Kupffer cells regulate T cell response in rats. Liver Transpl 2003; 9: 489–497.

Canbay

Feldstein

Higuchi

Werneburg

Grambihler

Bronk

. Kupffer cell engulfment of apoptotic bodies stimulates death ligand and cytokine expression. Hepatology 2003; 38: 1188–1198.

Kojima

Suzuki

Tsuchiya

Konno

Baba

Nakamura

. Regulation of pro-inflammatory and anti-inflammatory cytokine responses by Kupffer cells in endotoxin-enhanced reperfusion injury after total hepatic ischemia. Transpl Int 2003; 16: 231–240.

Fischer

Cariers

Reinehr

Haussinger

. Caspase 9-dependent killing of hepatic stellate cells by activated Kupffer cells. Gastroenterology 2002; 123: 845–861.

10.

Adachi

Bradford

Gao

Bojes

Thurman

. Inactivation of Kupffer cells prevents early alcohol-induced liver injury. Hepatology 1994; 20: 453–460.

11.

Kono

Fujii

Asakawa

Yamamoto

Maki

Matsuda

. Functional heterogeneity of the kupffer cell population is involved in the mechanism of gadolinium chloride in rats administered endotoxin. J Surg Res 2002; 106: 179–187.

12.

Badger

Kuester

Sauer

Sipes

. Gadolinium chloride reduces cytochrome P450: relevance to chemical-induced hepatotoxicity. Toxicology 1997; 121: 143–153.

13.

Rivera

Bradford

Hunt

Adachi

Schrum

Koop

. Attenuation of CCl(4)-induced hepatic fibrosis by GdCl(3) treatment or dietary glycine. Am J Physiol Gastrointest Liver Physiol 2001; 281: G200–207.

14.

Muriel

Escobar

. Kupffer cells are responsible for liver cirrhosis induced by carbon tetrachloride. J Appl Toxicol 2003; 23: 103–108.

15.

Ide

Kuwamura

Kotani

Sawamoto

Yamate

. Effects of gadolinium chloride (GdCl(3)) on the appearance of macrophage populations and fibrogenesis in thioacetamide-induced rat hepatic lesions. J Comp Pathol 2005; 133: 92–102.

16.

Ohkawa

Ohishi

Yagi

. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979; 95: 351–358.

17.

Aebi

. Catalase in vitro. Methods Enzymol 1984; 105: 121–126.

18.

Paglia

Valentine

. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967; 70: 158–169.

19.

Lowry

Rosebrough

Farr

Randall

. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265–275.

20.

Scheuer

. Classification of chronic viral hepatitis: a need for reassessment. J Hepatol 1991; 13: 372–374.

21.

Ishak

Baptista

Bianchi

Callea

De Groote

Gudat

. Histological grading and staging of chronic hepatitis. J Hepatol 1995; 22: 696–699.

22.

Fernandez-Martinez

Perez-Alvarez

Tsutsumi

Shibayama

Muriel

. Chronic bile duct obstruction induces changes in plasma and hepatic levels of cytokines and nitric oxide in the rat. Exp Toxicol Pathol 2006; 58: 49–58.

23.

Ebrahimkhani

Kiani

Oakley

Kendall

Shariftabrizi

Tavangar

. Naltrexone, an opioid receptor antagonist, attenuates liver fibrosis in bile duct ligated rats. Gut 2006; 55: 1606–1616.

24.

Ebrahimkhani

Sadeghipour

Dehghani

Kiani

Payabvash

Riazi

. Homocysteine alterations in experimental cholestasis and its subsequent cirrhosis. Life Sci 2005; 76: 2497–2512.

25.

Nezami

Talab

Emami

Assa

Rasouli

Asadi

. Chronic upregulation of the endogenous opioid system impairs the skin flap survival in rats. Ann Plast Surg 2009; 63: 558–563.

26.

Rust

Gores

. Apoptosis and liver disease. Am J Med 2000; 108: 567–574.

27.

Canbay

Friedman

Gores

. Apoptosis: the nexus of liver injury and fibrosis. Hepatology 2004; 39: 273–278.

28.

Copple

Jaeschke

Klaassen

. Oxidative stress and the pathogenesis of cholestasis. Semin Liver Dis 2010; 30: 195–204.

29.

Becker

Reinehr

Graf

vom Dahl

Haussinger

. Hydrophobic bile salts induce hepatocyte shrinkage via NADPH oxidase activation. Cell Physiol Biochem 2007; 19: 89–98.

30.

Ljubuncic

Tanne

Bomzon

. Evidence of a systemic phenomenon for oxidative stress in cholestatic liver disease. Gut 2000; 47: 710–716.

31.

Decker

. Biologically active products of stimulated liver macrophages (Kupffer cells). Eur J Biochem 1990; 192: 245–261.

32.

Soylu

Aydogdu

Basaran

Altaner

Tarcin

Gedik

. Antioxidants vitamin E and C attenuate hepatic fibrosis in biliary-obstructed rats. World J Gastroenterol 2006; 12: 6835–6841.

33.

Yang

Lee

Huang

Wang

Hou

Lee

. Effects of N-acetylcysteine administration in hepatic microcirculation of rats with biliary cirrhosis. J Hepatol 2008; 49: 25–33.

34.

Kolios

Valatas

Kouroumalis

. Role of Kupffer cells in the pathogenesis of liver disease. World J Gastroenterol 2006; 12: 7413–7420.

35.

Moreira

. Hepatic stellate cells and liver fibrosis. Arch Pathol Lab Med 2007; 131: 1728–1734.

36.

Hironaka

Sakaida

Matsumura

Kaino

Miyamoto

Okita

. Enhanced interstitial collagenase (matrix metalloproteinase-13) production of Kupffer cell by gadolinium chloride prevents pig serum-induced rat liver fibrosis. Biochem Biophys Res Commun 2000; 267: 290–295.

37.

Sakaida

Hironaka

Terai

Okita

. Gadolinium chloride reverses dimethylnitrosamine (DMN)-induced rat liver fibrosis with increased matrix metalloproteinases (MMPs) of Kupffer cells. Life Sci 2003; 72: 943–959.

38.

Han

. Matrix metalloproteinases, the pros and cons, in liver fibrosis. J Gastroenterol Hepatol 2006; 21 Suppl 3: S88–91.

39.

Kocher

Cerna

Havlik

Kral

Gryga

Duda

. Percutaneous treatment of benign bile duct strictures. Eur J Radiol 2007; 62: 170–174.

40.

Sikora

Srikanth

Agrawal

Gupta

Kumar

Saxena

. Liver histology in benign biliary stricture: fibrosis to cirrhosis and reversal? J Gastroenterol Hepatol 2008; 23: 1879–1884.