Blockade of Kupffer cells by gadolinium chloride reduces lipid peroxidation and protects liver from ischemia/reperfusion injury

BACKGROUND/AIMS: The implication of lipid peroxidation in the inhibitory effect of GdCl3 (gadolinium chloride) on Kupffer cells activation has not been extensively investigated. The aim of this study was to examine the effect of GdCl3 inhibition of Kupffer cells activation on lipid peroxidation after severe total hepatic ischemia/reperfusion. METHODOLOGY: Male Wistar rats (n = 40) were randomly divided into a sham-operation group, a control ischemia/reperfusion group, and two ischemia/reperfusion groups pretreated with GdCl3 (10 mg and 20 mg/kg bw intravenously, 48 and 24 h prior to operation). Following 60 min of total hepatic ischemia and 120 min of reperfusion, the rats were sacrificed, and liver samples were taken for determination of malondialdehyde and light microscopy examination. Blood samples were also taken for assay of aspartate and alanine transaminase. Additional animals (n = 60) were followed up for a 7-day survival rate determination. RESULTS: Ischemia/reperfusion decreased the survival rate to 13.3%, increased (p < 0.001) the levels of aspartate and alanine transaminase in serum to 2387 +/- 75 and 2157 +/- 87 IU/L, respectively, and increased (p < 0.001) malondialdehyde levels in liver to 1.609 +/- 0.096 nmoles/g compared with 1.164 +/- 0.060 in the sham operation group. Pretreatment with GdCl3 increased the survival rate to 60%, and decreased (p < 0.001) the levels of aspartate transaminase in serum to 1549 +/- 66 and 1496 +/- 55 IU/L, the levels of alanine transaminase in serum to 1302 +/- 48 and 1305 +/- 63 IU/L, and the levels of malondialdehyde in liver to 1.132 +/- 0.034 and 1.149 +/- 0.57 nmoles/g for the lower and the higher doses of GdCl3, respectively. Histological examination showed protection of liver parenchyma in the animals treated with GdCl3. CONCLUSIONS: Experimental data suggest that GdCl3 inhibition of Kupffer cells activation protects liver from ischemia/reperfusion injury by a mechanism that reduces lipid peroxidation.     

Kupffer cell inactivation prevents lipopolysaccharide-induced structural changes in the rat liver sinusoid: An electron-microscopic study

Scanning and transmission electron-microscopic examination of the rat liver sinusoid was performed in this study after in vivo treatment of rats with gram-negative bacterial lipopolysaccharide (LPS, 1 mg/Kg(-1) body weight), with or without pretreatment with gadolinium chloride (GdCl3 10 mg(Kg(-1) body weight). Twenty-seven and 48 hours after GdCl3 administration, to inactivate/eliminate part of the Kupffer cell population, a decrease in the number of visualized Kupffer cells was observed, without evident effects on the sinusoidal endothelial cell or on the hepatocyte. Three and 24 hours after its administration, LPS produced ultrastructural changes in the sinusoid characterized by morphological evidence of Kupffer cell activation (i.e., swelling and expanded philopodia anchoring the Kupffer cell to the luminal surface of the sinusoidal wall), and a marked decrease in the population of endothelial cell fenestration. The reduction in the number of fenestrae was associated with a change in the diameter of fenestrae and can be interpreted as a component of the "capillarization" process of the hepatic sinusoid. Such ultrastructural changes were prevented by the administration of GdCl3 24 hours before LPS injection. Hence, these findings suggest that LPS-induced structural changes in the liver sinusoid are mediated by an LPS-induced Kupffer cell activation. Coupled with previous experimental data, showing similar effects of GdCl3 on one of the hepatic sinusoidal endothelial cell (SEC) functions, i.e., hyaluronan scavenging, the data presented in this study strongly support the view that Kupffer cells modulate both the hepatic SEC's functional as well as ultrastructural properties. (Hepatology 1996 Apr;23(4):788-96)     

库普弗细胞功能状态对肠缺血再灌注小鼠肝细胞凋亡和血清肿瘤坏死因子的影响

目的 研究库普弗细胞功能状态对肠缺血再灌注小鼠肝细胞凋亡和血清肿瘤坏死因子的影响。方法 封闭和不封闭BALB／c小鼠库普弗细胞(从尾静脉注射GdCl3或生理盐水27ml／kg体重)48h后，夹闭肠系膜上动脉1h后松夹，复制肠缺血再灌注模型。运用流式细胞术和酶联免疫法，分别检测缺血前、缺血60min、再灌注30min、60min肝细胞凋亡情况和血清肿瘤坏死因子的变化。结果 结果表明，肠缺血60min、再灌注30min、60min时，肝细胞凋亡数增多，血清肿瘤坏死因子水平逐渐升高；封闭库普弗细胞后，肝细胞凋亡数增多更显著，血清肿瘤坏死因子水平在同时间点上无显著差异。结论 库普弗细胞功能状态的变化对肠缺血再灌注时肝损伤有重要影响。     

Effect of matrine on Kupffer cell activation in cold ischemia reperfusion injury of rat liver

AIM: To study the effect of matrine on activation of Kupffer cell during cold ischemia and reperfusion injury in rat orthotopic liver transplantation (OLT). METHODS: 168 syngeneic SD rats were randomly divided into four groups: untreated group, small-dose treated group, large-dose treated group and sham operation group. After 5 hours of preservation in Ringer's (LR) solution, orthotopic implantation of the donor liver was performed. At 1 h, 2 h, 4 h and 24 h after reperfusion of the portal vein, 6 rats were killed in each group to collect the serum and the liver for assay and pathology. RESULTS: Matrine markedly inhibited the activation of Kupffer cells and their release of tumor necrosis factor (TNF). TNF cytotoxicity level at 2 h decreased significantly by matrine treatment (7.94+/-0.42, 2.39+/-0.19 and 2.01+/-0.13 U/ml, respectively; P<0.01), so did the other three time points. The level of hylluronic acid (HA) and alanine transaminase (ALT) decreased significantly in both treated groups, and matrine treatment markedly ameliorated focal necrosis of hepatocytes, inflammatory cells aggregating, rounding and detachment of sinusoidal endothelial cells (SEC). And no significant difference was observed between the treated groups. CONCLUSION: Matrine can inhibit the activation of Kupffer cell and prevent the donor liver from cold preservation and reperfusion injury in rat orthotopic liver transplantation.     

Kupffer cells modulate splenic interleukin-10 production in endotoxin-induced liver injury after partial hepatectomy

BACKGROUND/AIMS: This study was conducted to investigate the implication of Kupffer cells and the spleen in interleukin (IL)-10 production in endotoxin-induced liver injury after hepatectomy. METHODS: Rats were divided into five groups: the S group, sham-operation; the SG group, sham-operation followed by intravenous gadolinium chloride (GdCl(3): 7 mg/kg) administration to inhibit Kupffer cell function; the H group, two-thirds hepatectomy; the HG group, hepatectomy and subsequent GdCl(3) administration; the HGS group, hepatectomy and splenectomy with GdCl(3) administration. Lipopolysaccharide (1.5 mg/kg) was intravenously administered for each group 48 h after surgery. RESULTS: GdCl(3) treatment significantly suppressed the elevation of plasma tumor necrosis factor (TNF)-alpha levels by lipopolysaccharide administration with completely inhibited induction of hepatic TNF-alpha and IL-10 mRNAs. In the HG group, marked increase in plasma IL-10 levels associated with enhanced splenic IL-10 mRNA was observed 1 h after lipopolysaccharide administration when compared to those in the H and HGS groups. Plasma TNF-alpha/IL-10 ratio 1 h after lipopolysaccharide administration was higher in the order of H, HGS and HG groups. Hepatic parenchymal damage and the 24-h mortality were lowest in group HG, followed by groups HGS and H. CONCLUSIONS: Kupffer cells after hepatectomy may aggravate endotoxin-induced liver injury via down-regulation of IL-10 production in the spleen.     

Interleukin-10 induction after combined resection of liver and pancreas

BACKGROUND/AIMS: Recently in Japan, combined resection of liver and pancreas is being performed in cases of advanced biliary neoplasms. As we previously reported, in the rat model of combined resection of the liver and pancreas, the potential for liver regeneration after this operation was decreased compared to that after hepatectomy only. Moreover, non-parenchymal cells play an important role in the production of inhibitory factors for liver regeneration. The anti-inflammatory cytokine IL-10, downregulates the release of TNF-alpha, and affects the progressive regeneration of the hepatic parenchyma. To investigate the role of IL-10 and TNF-alpha in hepatic regeneration in the rat, we measured the levels of IL-10 and TNF-alpha in the conditioned medium of non-parenchymal cells stimulated with portal plasma. We also investigated the concentration of IL-10 and TNF-alpha in the portal plasma after combined resection of the liver and pancreas. METHODOLOGY: Adult male Sprague-Dawley rats were used. Rats were divided into 3 groups: group I underwent 70% partial hepatectomy only (Hx), group II underwent 70% partial pancreatectomy only (Px) and in group III both procedures were used, Hx plus Px (HPx). Portal plasma was harvested at 1, 3, 6, 12 and 24 hours after surgery and was used to stimulate the culture medium of non-parenchymal cells. Cytokine concentrations in the plasma and in the conditioned medium were measured by ELISA. Northern blot analysis for IL-10 mRNA was performed on liver, pancreas, kidney, lung and spleen at 1, 3 and 6 hours after surgery. RESULTS: The level of IL-10 released by non-parenchymal cells stimulated with HPx portal plasma was 154.1 +/- 20.3 pg/mL and significantly higher than when stimulated with Hx portal plasma, which was 100.1 +/- 6.4 pg/mL (P < 0.05) during the first hour. Also, the level of TNF-alpha released by Kupffer cells stimulated with HPx portal plasma was 86.6 +/- 13.4 pg/mL, significantly less than when stimulated with Hx portal plasma, which was 138.7 +/- 15.1 pg/mL (P < 0.005) during the first hour. Furthermore, the plasma levels of IL-10 in the HPx group remained significantly higher than those of the other groups from 6 hours up to 12 hours. In northern blot analyses, higher IL-10 mRNA expression were detected in the spleen and moderately high levels in the liver at 1 and 3 hours after HPx, in contrast to those after Hx. CONCLUSIONS: IL-10 expression is induced in the spleen and liver remnant just after HPx. IL-10 released by the spleen and liver might downregulate TNF-alpha production, thereby inhibiting the liver regeneration.     

Streaming liver. VI: Streaming intra-hepatic bile ducts

Thirty male adult rats, weighing 250-300 g, were injected with 0.5 muCi [3H]-thymidine. The rats were killed in groups of five, at the following times: 1 h, 14, 30, 60, 90 and 120 days. The livers were fixed in formalin, embedded in paraffin and cut into 5-microns-thick sections, which were then dipped into liquid emulsion for autoradiography. Bile ducts were evaluated in all portal tracts of a section. Obliquely cut bile ducts were excluded. The number of cells paving the duct lumen was defined as bile duct class. Class is roughly proportional to bile duct size. One hour after labelling, labelling index was 3.39 +/- 0.06% (the second number is one standard error of the mean). With time it declined, reaching 0.22 +/- 0.15 on day 60. Initially labelled cells were found in narrow bile ducts and the mean labelled cell was located at class 7.48 +/- 0.63. With time, labelled cells proceeded into wider bile ducts advancing daily 0.122 +/- 0.025 classes. Intra-hepatic bile duct epithelium renews its cells continuously in the same way as epidermis or gastro-intestinal mucosa do. It consists of two kinetic compartments, a progenitor (P) and a functional (Q) compartment. P feeds Q with cells.     

Peritoneoscopic study on rat liver cell necrosis induced by carbon tetrachloride and allyl formate

The surface changes of acute centrolobular and perilobular necrosis were studied in rat liver using peritoneoscopy. Centrolobular necrosis as induced by carbon tetrachloride and perilobular necrosis by allyl formate. Vessels observed on the control rat liver surface were identified as terminal hepatic veins by irrigation of the hepatic vein with a barium solution. Polygonal whitish markings were observed on the liver surface 48 hours after carbon tetrachloride administration. Terminal hepatic veins were regularly distributed throughout the central portions of the whitish areas. Reddish spots about 0.1 mm in diameter were scattered about the terminal hepatic veins. The reddish spots and whitish areas corresponded to the histological findings of central liver cell necrosis and infiltration by Kupffer and white blood cells. Six hours after allyl formate administration, round reddish spots about 0.3 mm in diameter were regularly distributed on the liver surface among the terminal hepatic veins. These spots corresponded to the histological findings of liver cell necrosis around the portal area. In summary, acute centrolobular and perilobular liver cell necrosis were newly observed as reddish spots by peritoneoscopy.     

Bone morphogenetic protein-2 is a negative regulator of hepatocyte proliferation downregulated in the regenerating liver

INTRODUCTIONBone morphogenetic proteins (BMPs) were first identified in the 1960s[1]. BMPs are multi-functional growth factors that belong to the transforming growth factor beta (TGF-β) superfamily[2]. Mature BMPs are 30-38 kDa proteins that utilize BMP …     

Reversible Kupffer cell suppression in biliary obstruction is caused by hydrophobic bile acids

BACKGROUND/AIM: Biliary obstruction is associated with suppressed Kupffer cell clearance of bacteria and intracellular bactericidal activity of the phagocytes. We studied the superoxide generation of Kupffer cell in biliary obstruction and after incubation with bile acids to elucidate the mechanism of impaired intracellular killing of these phagocytes. METHODS: Kupffer cells were extracted from rats with common bile duct ligation or sham-operation. The extracted cells were tested for superoxide production immediately after extraction, 2 h and 24 h post-extraction. Superoxide generation from Kupffer cells after incubation with five bile acids (cholic acid, taurocholic acid, deoxycholic acid, chenodeoxycholic acid and ursodeoxycholic acid) at two different concentrations (0.1 mM and 1.0 mM) were also studied. Cell viability was monitored by trypan blue exclusion. RESULTS: Kupffer cells extracted from common duct-ligated animals had significantly lower superoxide production (-1.37+/-0.24 nmol O2(-)/10(6) cells) compared to that from sham-operated rats (2.54+/-0.58 nmol O2(-)/10(6) cells) and non-operated rats (2.15+/-0.76 nmol O2(-)/10(6) cells) (p<0.05). After 2 h of resting in culture medium, these cells recovered significantly in superoxide production to 2.72+/-0.63 nmol O2(-)/10(6) cells (p<0.01). A dose-related reduction in superoxide production was demonstrated when Kupffer cells were incubated with bile acids. Hydrophobic bile acids (deoxycholic acid and chenodeoxycholic acid) caused more significant suppression than with hydrophilic bile acids (cholic acid, taurocholic acid, ursodeoxycholic acid). The drop in superoxide production after bile acid treatment was not due to cell death. Washing in Hank's balanced salt solution resulted in partial recovery of Kupffer cell superoxide production. CONCLUSION: High blood levels of hydrophobic bile acids are likely to be the cause of impaired intracellular bactericidal activity of Kupffer cells in biliary obstruction.     

Neutrophil-mediated liver injury during hepatic ischemia-reperfusion in rats

BACKGROUND: Neutrophil plays an important role in hepatic ischemia-reperfusion injury. We investigated neutrophil infiltration in liver tissue, Kupffer cells' role in neutrophil accumulation, and apoptosis and regeneration of hepatocytes in liver ischemia-reperfusion injury. METHODS: Vascular microclamps were placed across the pedicles of the median and left lateral lobes for 90 minutes after 30% hepatectomy with the resection of caudate, right lateral and quadrate lobes and papillary process. Gadolinium chloride (GdCl3) was used to destroy Kupffer cells. Neutrophil activity was inhibited with Urge-8, a monoclonal antibody against neutrophil produced in our laboratory. GdCl3 (10 mg/kg) and Urge-8 (50 mg/kg) were given intravenously in respective groups. Ischemia control, GdCl3 and Urge-8 groups were compared. RESULTS: Following hepatic reperfusion, serum interleukin-8 (IL-8) levels and hepatic neutrophil counts peaked at 3 hours, and peak concentrations of alanine aminotransferase (ALT) occurred at 6 hours. Animals of the control group showed increases in neutrophil infiltration in liver tissue, liver enzyme levels, and apoptosis index of hepatocytes and decreases in overall survival rate and proliferating cell nuclear antigen (PCNA) expression of hepatocytes. The survival rates and PCNA proportion of hepatocytes were higher and the levels of hepatic neutrophil infiltration, liver enzymes, and hepatocyte apoptosis after reperfusion were lower in the GdCl3 and Urge-8 groups than those in the ischemia control group. CONCLUSIONS: Blockades of Kupffer cells' activity and neutrophil infiltration by GdCl3 and Urge-8 eliminate neutrophil-mediated hepatic injury and enhance subsequent hepatic regeneration during liver ischemiareperfusion.     

Intestinal damage mediated by Kupffer cells in rats with endotoxemia

AIM：To determine the in vivo effects of phagocytic blockade of Kupffer cell(KC)on the release of proinflammatory cytokines in small intestinal lesion and on the integrity of intestinal tract by using gadolinium chloride(GdCl3)during early endotoxemia.METHODS:Wistar rats were divided into three groups:GropA,rats were injected with endotoxin(E.coliO111:B4,adose of 12mg&#183;kg^-1)only;GroupB,rats were pretreated intravenously with 25mg of GdCl3per kg24hare given endotoxin;and Group C,sham operation only.All animals were sacrificed 4h after endotoxin injecton.In portion of the rats of three groups.bile duct was cannulated,which the bile was collected externally.Morphological changes of ileum were observed under light microscopy and electronic microscopy.The KC were isolated rfrom rats by collagenase perfusion and inKC,expression of TNF-αand IL-6mRNA were determined by RT-PCRanalysis.Plasma and bile TNF-αandIL-6Levels were determined by enzyme-linked immunosorbent assay(EISA)&gt;RESULTS:In group A,there were neutrophil infiltration and superficial epitelial necrosis of the ilealvvilli,sloughing of mucosal epithelium.and disappear ance of somevilli.In groupB.the ileal mucosal damage was much reduced.which in groupC,no significant morphological changes were seen,GdCl3pretreatment decreased significantly the expression of TNF-αand IL-6mRNA ingroupB(4.32&#177;0.47and4.05&#177;0.43)when compared to groupA(9.46&#177;1.21and9.04&#177;1.09)(P&lt;0.05).There was no significant expression of TNF-αand IL-6mRNA in groupC(1.03&#177;0.14and10.4&#177;0.13).In rats of groupA,the levels of TNF-αand IL-6in bile and plasma were 207&#177;29ng&#183;L^-1,1032&#177;107ng&#183;L^-1,213&#177;33ng&#183;L^-1,and 1185&#177;127ng&#183;L^-1,respectively.In groupB,they were113&#177;18ng&#183;L^-1,repectively.In groupC,they were 67&#177;10ng&#183;L^-1,72&#177;13ng&#183;L^-1,109&#177;18ng&#183;L^-1,and 118&#177;22ng&#183;L^-1respectively.There were significant difference between the three group(P&lt;0.05).CONCLUSION:KC release cytokinesTNF-αand IL-6causing damage to the integrity of intestinal epithelium and play a crucial role in the initiation and progression of intestinal mucosal damage during early endotoxemia.     

Cell proliferation and oncogene expression after bile duct ligation in the rat: Evidence of a specific growth effect on bile duct cells

The proliferative response of the rat liver was measured after temporary or permanent total biliary obstruction (BDO) and in different regions after selective ligation of the lobar ducts draining the right 60% of the hepatic mass. The results were compared with those after 70% partial hepatectomy (PH). Cell proliferation was assessed globally by measuring DNA synthesis and stratified to the separate cell populations with cytostaining techniques that allowed distinction of hepatocytes, duct cells, and nonparenchymal cells (NPCs). In selected experimental groups, gene expression was determined of transforming growth factor-β1 (TGFβ-1), prothrombin, c-erb-B2, transforming growth factor alpha (TGFα), human Cyclophilin (CyP), and 28S ribosomal RNA. The stimulation of a proliferative response to total BDO required obstruction for longer than 24 hours, but after this deligation did not switch off regeneration. In the first week after permanent BDO, there was progressive infiltration of NPCs, fibrous linkage of some portal areas, and a crescendo of DNA synthesis that was obvious at 24 hours, maximal at 48 hours, and back nearly to baseline at 6 days. At the 2-day mark, the bile duct cells had a 17-fold increase in proliferation, accompanied by a threefold to fourfold increase in hepatocyte renewal. Little or no increase in expression of TGFα or the hepatocyte-specific prothrombin gene was detectable in the first 48 hours, whereas levels of the oncogene c-erb-B2 that is associated with cholangiocarcinoma were expressed from 48 to 96 hours. Livers subjected to regional BDO with or without immunosuppressive treatment with FK 506 and cyclosporine had an inflammatory reaction only on the side with ligated ducts. DNA synthesis increased in both the obstructed and freely draining lobes to approximately half the level that occurred after total BDO. The proliferation of the obstructed side was similar to the mixed duct cell/hepatocyte response after total BDO, but this almost exclusively involved duct cells on the freely draining side. In contrast to the findings after BDO, livers after PH regenerated maximally at 24 hours rather than 48 hours, had a predominantly noninflammatory hepatocyte as opposed to duct cell response, and had marked expression of the prothrombin and TGFα genes but only weakly and late of c-erb-B2 messenger RNA. The results show that the liver responds as a whole and in a biologically intelligent way to the nature of the injury inflicted on any part of it. It further implies the presence of humoral communications and control networks that assure organ homeostasis and relate this to total body homeostasis.     

Role of the liver in regulating numbers of circulating neutrophils

Neutrophils (polymorphonuclear leukocytes [PMNs]) carry potent destructive enzymes that can destroy invasive bacteria or damage normal tissue. PMNs have a half-life of only 6 hours in the blood, but the details of this homeostasis are unknown. In a rat model of endotoxemia, P-selectin was selectively up-regulated in hepatic sinusoids and veins where it was necessary for phagocytosis of PMNs by Kupffer cells in the liver, as opposed to the spleen or the lungs. Apoptotic PMNs appeared in the lungs and spleen only after inactivation of Kupffer cells by gadolinium chloride (GdCl(3)). Blocking of Fas protein reduced the number of apoptotic cells in the liver; binding of annexin V to phosphatidylserine (PS) reduced the number of PMNs phagocytosed by Kupffer cells. The results support a clearance pathway in which apoptosis and phagocytosis are effected by Kupffer cells after P-selectin-mediated sequestration. (Blood. 2001;98:1226-1230)     

GdCl_3 abates hepatic ischemia-reperfusion injury by inhibiting apoptosis in rats

BACKGROUND:Gadolinium chloride(GdCl3)is a specific inhibitor of Kupffer cells(KCs),which are important promoters of various liver injuries.It is therefore of interest to explore the role of KCs in liver ischemia-reperfusion injury and their relations with apoptosis caused by ischemia-reperfusion injury. METHODS:One hundred male Wistar rats(190-210 g, 6-7 weeks old)were divided into two groups at random, GdCl3 group and control group.Samples were collected at 0.5,1,6,12,and 24 hours from each group after rep...     

Glycine and uridine prevent D-galactosamine hepatotoxicity in the rat: role of Kupffer cells

Extrahepatic factors, such as increased gut permeability and bacteria from the gut, have been shown to play a role in D-galactosamine toxicity in rats. Because bacterial endotoxin activates Kupffer cells, the purpose of this study was to clarify the role of Kupffer cells in the mechanism of D-galactosamine hepatotoxicity in rats and determine whether uridine, a compound that rescues animals from D-galactosamine toxicity, affects Kupffer cells. Rats were fed control or glycine (5%) containing diets to prevent Kupffer cell activation or treated with gadolinium chloride (GdCl3, 20 mg/kg) to destroy Kupffer cells selectively before injection of D-galactosamine (500 mg/kg, intraperitoneally). D-galactosamine caused panlobular focal hepatocellular necrosis, polymorphonuclear cell infiltration, and increased serum transaminases significantly at 24 hours. Dietary glycine or pretreatment with GdCl3 prevented these effects. D-galactosamine caused a transient increase in circulating endotoxin that was maximal at 1 hour and was blunted significantly by dietary glycine. Additionally, antisera to tumor necrosis factor-alpha (TNF-alpha) prevented hepatotoxicity caused by D-galactosamine. Moreover, apoptosis in hepatocytes caused by D-galactosamine occurred before necrosis (6 hours) and was prevented by glycine, GdCl3, TNF-alpha antiserum, and uridine. Thus, it was hypothesized that TNF-alpha from Kupffer cells causes apoptosis after D-galactosamine administration in the rat. Indeed, increases in TNF-alpha messenger RNA (mRNA) were detected as early as 2.5 hours after D-galactosamine treatment. Previous work proposed that uridine blocks D-galactosamine toxicity by preventing inhibition of mRNA synthesis. In view of these results, the possibility that uridine might affect Kupffer cells was investigated. Uridine significantly blunted the increase in [Ca2+]i and release of TNF-alpha caused by endotoxin in isolated Kupffer cells and prevented apoptosis caused by D-galactosamine treatment in vivo. These data support the hypothesis that uridine prevents D-galactosamine hepatotoxicity not only by rescuing the hepatocyte in the late phases of the injury but also preventing TNF-alpha release from Kupffer cells thereby blocking apoptosis that occurs early after D-galactosamine treatment. Taken together, these data strongly support the role of Kupffer cell activation by endotoxin early after D-galactosamine treatment as an important event in the mechanism of hepatotoxicity in the rat.