Dysregulation of renal aquaporins and Na-Cl cotransporter in CCl4-induced cirrhosis

BACKGROUND: Severe hepatic cirrhosis is associated with abnormal renal water retention. METHODS: Semiquantitative immunoblotting was employed to investigate the abundance of the major renal aquaporins (water channels) and sodium-dependent cotransporters in kidneys from control rats and rats with cirrhosis secondary to chronic CCl4 inhalation. RESULTS: The cirrhotic rats had ascites and manifested a water excretion defect detected by a standard water-loading test. The abundance of aquaporin-1 (the major aquaporin in the proximal tubule) was increased, an effect markedly accentuated in high-density membrane fractions prepared by differential centrifugation. Differential centrifugation studies demonstrated a redistribution of aquaporin-2 from high-density to low-density membranes, compatible with increased trafficking of aquaporin-2 to the plasma membrane. The abundance of aquaporin-3, but not aquaporin-2, was increased in collecting ducts of rats with CCl4-induced cirrhosis. The Na-K-2Cl cotransporter of the thick ascending limb showed no change in abundance. However, the abundance of the thiazide-sensitive Na-Cl cotransporter of the distal convoluted tubule was markedly suppressed in cirrhotic rats, possibly contributing to a defect in urinary dilution. CONCLUSIONS: In this model of cirrhosis, the development of a defect in urinary dilution may be multifactorial, with contributions from at least four abnormalities in transporter regulation: (1) an increase in the renal abundance of aquaporin-1, (2) a cellular redistribution of aquaporin-2 in the collecting duct compatible with trafficking to the plasma membrane without an increase in total cellular aquaporin-2, (3) an increase in the renal abundance of aquaporin-3, and (4) a decrease in the abundance of the thiazide-sensitive cotransporter of the distal convoluted tubule.     

Increased apical targeting of renal epithelial sodium channel subunits and decreased expression of type 2 11beta-hydroxysteroid dehydrogenase in rats with CCl4-induced decompensated liver cirrhosis

It was hypothesized that dysregulation of renal epithelial sodium channel (ENaC) subunits and/or 11beta-hydroxysteroid dehydrogenase (11betaHSD2) may play a role in the increased sodium retention in liver cirrhosis (LC). Experimental LC was induced in rats by CCl(4) (1 ml/kg, intraperitoneally, twice a week) for 12 wk (protocol 1) or for 11 wk (protocol 2). In both protocols, one group of rats with cirrhosis showed significantly decreased urinary sodium excretion and urinary Na/K ratio (group A), whereas a second group exhibited normal urinary sodium excretion (group B) compared with controls, even though extensive ascites was seen in both groups of rats with cirrhosis. In group A, protein abundance of alpha-ENaC was unchanged, whereas beta-ENaC abundance was decreased in the cortex/outer stripe of outer medulla compared with controls. The gamma-ENaC underwent a complex change associated with increased abundance of the 70-kD band with a concomitant decrease in the main 85-kD band, corresponding to an aldosterone effect. In contrast, no changes in the abundance of ENaC subunit were observed in group B. Immunoperoxidase microscopy revealed an increased apical targeting of alpha-, beta-, and gamma-ENaC subunits in distal convoluted tubule (DCT2), connecting tubule (CNT), and cortical and medullary collecting duct segments in group A but not in group B. Immunolabeling intensity of 11betaHSD2 in the DCT2, CNT, and cortical collecting duct was significantly reduced in group A but not in group B, and this was confirmed by immunoblotting. In conclusion, increased apical targeting of ENaC subunits combined with diminished abundance of 11betaHSD2 in the DCT2, CNT, and cortical collecting duct is likely to play a role in the sodium retaining stage of liver cirrhosis.     

Anti-tumor effect of fluoropyrimidines on human tumor cell lines transplanted in nude mice with CCl4-induced liver dysfunction

Tegafur (FT) is a masked compound of 5-fluorouracil (5-FU) and supposed to be activated in the liver. The present study was designed to estimate anti-tumor effect of FT on human tumors transplanted in nude mice with liver dysfunction induced by CCl4. Histologically, cirrhotic changes of liver were observed after injection with 1ml/kg 10% CCl4 twice a week for 8 weeks. Mice were transplanted with human gastric (GC-SF) or colonic cancer (CC-ZK) lines, and daily administered intragastrically with 5-FU (15mg/kg), FT (100mg/kg) or UFT (FT 20mg/kg + Uracil 44.8mg/kg) for 4 weeks. The growth of GC-SF was enhanced by liver dysfunction, but that of CC-ZK was not affected. The mean growth inhibition rates (MGIR) of CC-ZK by 5-FU, FT or UFT were 18.3, 33.1 and 54.2%, respectively, in mice without liver dysfunction, and 14.0, 50.0 and 59.5%, respectively, in mice with liver dysfunction. The MGIRs of GC-SF were 39.0, 63.8 and 48.0%, respectively, in mice without liver dysfunction, and 12.6, 53.6 and 50.0%, respectively, in mice with liver dysfunction. In both lines effect of 5-FU was reduced in liver dysfunction, but those of FT and UFT was not. These results suggest that FT and UFT can be used for cancer patients with liver dysfunction.     

Successful treatment of CCl4-induced acute liver failure with portal vein arterialization in the rat

BACKGROUND: Optimization of the conditions for regeneration is a major goal in the management of patients with acute liver failure (ALF). Previous observations suggested that hyperoxygenation of the liver may improve its regenerative capacity. Thus, this study aimed to determine whether an additional supply of oxygenated blood achieved by portal vein arterialization (PVA) is protective in rat ALF caused by toxin administration or hepatectomy. METHODS: Sprague-Dawley rats were subjected or not to PVA after CCl(4) intoxication or extended hepatectomy. PVA was performed by interposing a stent between the left renal artery and splenic vein after left nephrectomy and splenectomy. Liver injury was evaluated by the serum ALT level and necrotic cell count. Hepatocyte regeneration was assessed by calculating the mitotic index and bromodeoxyuridine (BrdU) staining. The 10-day survival was assessed in separate experimental groups. RESULTS: The pO(2) in portal blood increased significantly following PVA. In the CCl(4)-induced ALF, serum ALT levels and necrosis were significantly reduced in arterialized than non-arterialized rats. PVA greatly promotes liver regeneration in both models. Finally, PVA significantly improved survival compared to controls (CCl(4): 100 versus 40%; 90% hepatectomy: 90 versus 30%). Interestingly, in the CCl(4)-induced ALF, survival was 100% even when the shunt was closed after 48 h. CONCLUSION: These data indicate that the additional supply of arterial oxygenated blood through PVA promotes a rapid regeneration leading to the resolution of toxic-induced massive liver necrosis and a faster restoration of liver mass after partial hepatectomy in rats. Thus, PVA may represent a novel tool for optimizing hepatocyte regeneration.     

Evaluation of 2-year-old intrasplenic fetal liver tissue transplants in rats

Liver cell transplantation into host organs like the spleen may possibly provide a temporary relief after extensive liver resection or severe liver disease or may enable treatment of an enzyme deficiency. With time, however, dedifferentiation or malignant transformation of the ectopically transplanted cells may be possible. Thus, in the present study syngenic fetal liver tissue suspensions were transplanted into the spleen of adult male rats and evaluated 2 years thereafter in comparison to orthotopic livers for histopathological changes and (as markers for preneoplastic transformation) for cytochrome P450 (P450) and glutathione S-transferase (GST) isoform expression. Because inducibility of P450 and GST isoforms may be changed in preneoplastic foci, prior to sacrifice animals were additionally treated either with beta-naphthoflavone, phenobarbital, dexamethasone, or the respective solvent. In the 2-year-old grafts more than 70% of the spleen mass was occupied by the transplant. The transplanted hepatocytes were arranged in cord-like structures. Also few bile ducts were present. Morphologically, no signs of malignancy were visible. With all rats, transplant recipients as well as controls, however, discrete nodular structures were seen in the livers. Due to age, both livers and transplants displayed only a low P450 2B1 and 3A2 and GST class alpha and mu isoform expression. No immunostaining for P450 1A1 was visible. At both sites, beta-naphthoflavone, phenobarbital, or dexamethasone treatment enhanced P450 1A1, P450 2B1 and 3A2, or P450 3A2 expression, respectively. No immunostaining for GST class pi isoforms was seen in the transplants. The livers of both transplant recipients and control rats, however, displayed GST pi-positive foci, corresponding to the nodular structures seen histomorphologically. Compared to the surrounding tissue, these foci also exhibited a more pronounced staining for GST class alpha and mu isoforms and a stronger inducibility of the P450 1A1 expression due to beta-naphthoflavone. In conclusion, in contrast to the livers, no preneoplastic foci seem to appear in the intrasplenic transplants even 2 years after transplantation. This may be due either to the protection of these transplants by the orthotopic livers or to the different humoral and nerval influences at the ectopic site.     

不同节段去交感神经对CCl4所致大鼠急性肝损伤的影响

目的 观察不同节段去交感神经后对CCl4所致大鼠急性肝损伤的影响.方法 健康SD大鼠分别从颈交感干、内脏大神经和肝内行去交感处理,在此基础上应用CCl4制作急性肝损伤模型.检测各组肝功能、血清肿瘤坏死因子(TNF)-α及肝组织内去甲肾上腺素(NE)浓度.结果 在急性肝损伤的情况下,颈交感神经干离断和肝内去交感组31.80±1.50、31.74±1.08和假手术组29.90±1.20比较白蛋白升高,总胆红素降低(1.80±0.14、1.70±0.23、2.05±0.18,P＜0.05、P＜0.01),肝组织内去甲肾上腺素水平下降(49.20 ±4.00、42.60±2.10、61.60±2.14,P＜0.01).血清TNF-α较假手术组降低(192±18、318±20、398 ±40,P＜0.05),而内脏大神经离断则无显著影响(476±30,P＞0.05).结论 不同节段去交感神经可显著减轻CCl4所致急性肝损伤时的肝功能损害.交感神经不同节段激活在急性肝损伤早期有促进炎症效应.     

Hepatic and mesenteric nitric oxide synthase expression in a rat model of CCl(4)-induced cirrhosis

BACKGROUND: Cirrhosis and portal hypertension are frequently linked with changes in expression of nitric oxide synthase (NOS) and/or endotoxaemia. AIMS: This study tested the following hypothesis: that inducible (i)NOS activity is increased within the visceral circulation concurrently with decreased constitutive (c)NOS activity in the hepatic sinusoids and that the concentration of NO metabolites in portal blood is consequent on endotoxin concentration. MATERIALS AND METHODS: Plasma concentrations of (nitrite + nitrate) and endotoxin, together with hepatic and mesenteric NOS activity (arginine/citrulline method) and protein expression (histochemistry) plus portal and arterial blood pressure, were determined in rats made severely cirrhotic by intragastric CCl(4) over 14 weeks (n = 6) compared with age-matched controls (n = 5). The concentrations of [nitrite + nitrate] and endotoxin in portal plasma were also directly compared in rats made cirrhotic for a period of 8-14 weeks (n = 10). RESULTS: In rats with advanced cirrhosis, arterial [nitrite + nitrate] was 93.1 (22.4) micromol/L (mean, SEM) compared with 29.1 (6.1) micromol/L in controls (P < 0.05); portal plasma [NO(2)(-) + NO3(-)] was 127.1 (27.2) compared with 24.7 (4.7) micromol/L in controls (P < 0.05). Cirrhotic rats had higher endotoxin concentration in plasma compared with controls (systemic: 85.0 (24.5) versus 1.7 (0.2) EU/ml, P < 0.05; portal: 180.3 (47.9) versus 1.7 (0.2) EU/ml, P < 0.05). The same severely cirrhotic rats possessed decreased cNOS activity in liver (2.95 [0.40] versus 5.29 [0.85] pmol/min/g; P < 0.05) and increased iNOS activity in mesentery (4.83 [1.23] versus 1.47 [0.15] pmol/min/g; P < 0.05) compared with controls. Histochemical observations confirmed these findings. Rats given CCl(4) for a period of 8-14 weeks possessed high endotoxin concentration in portal plasma, with correspondingly high [nitrite + nitrate] (r(2) = 0.954; P < 0.001). CONCLUSIONS: An endotoxin-induced increase in mesenteric iNOS activity and a decrease in hepatic cNOS activity may account for, respectively, the hyperdynamic visceral circulation and the increased intrahepatic resistance of cirrhosis.     

Lesson from the GFP/CCl4 model--translational research project: the development of cell therapy using autologous bone marrow cells in patients with liver cirrhosis

The plasticity of bone marrow has been confirmed by the analysis of autopsy findings in female recipients of bone marrow cells transplanted from male donors. To establish new clinical cell therapies using autologous bone marrow cells for patients with liver failure, we developed a new in vivo model, the “green fluorescent protein (GFP)/carbon tetrachloride (CCl₄) model”. Using the GFP/CCl₄ model, we found that transplanted Liv8-negative cells efficiently repopulated into cirrhotic liver tissue and trans-differentiated into albumin-producing hepatocytes under conditions of persistent liver damage induced by CCl₄. Moreover, one marrow cell transplantation into liver cirrhosis mice improved their liver function, ameliorated liver fibrosis, and improved their survival rate. Results from the GFP/CCl₄ model showed that cell therapy using autologous bone marrow cells has the potential to become an effective treatment for patients with liver failure. Here we describe the findings from the GFP/CCl₄ model and the scope of the translational research project.     

DGAT1-deficiency affects the cellular distribution of hepatic retinoid and attenuates the progression of CCl4-induced liver fibrosis

Background

Diacylglycerol O-acyltransferase 1 (DGAT1) catalyzes the final step of triglyceride synthesis, transferring an acyl group from acyl-CoA to diacylglycerol. DGAT1 also catalyzes the acyl-CoA-dependent formation of retinyl esters in vitro and in mouse intestine and skin. Although DGAT1 is expressed in both hepatocytes and hepatic stellate cells (HSCs), we reported genetic and nutritional studies that established that DGAT1 does not contribute to retinyl ester formation in the liver.

Methods

We now have explored in more depth the role(s) of DGAT1 in hepatic retinoid metabolism and storage.

Results

Our data show that DGAT1 affects the cellular distribution between hepatocytes and HSCs of stored and newly absorbed dietary retinol. For livers of Dgat1-deficient mice, a greater percentage of stored retinyl ester is present in HSCs at the expense of hepatocytes. This is also true for newly absorbed oral [³H]retinol. These differences are associated with significantly increased expression, by 2.8-fold, of cellular retinol-binding protein, type I (RBP1) in freshly isolated HSCs from Dgat1-deficient mice, raising the possibility that RBP1, which contributes to retinol uptake into cells and retinyl ester synthesis, accounts for the differences. We further show that the retinyl ester-containing lipid droplets in HSCs are affected in Dgat1-null mice, being fewer in number but, on average, larger than in wild type (WT) HSCs. Finally, we demonstrate that DGAT1 affects experimentally induced HSC activation in vivo but that this effect is independent of altered retinoic acid availability or effects on gene expression.

Conclusions

Our studies establish that DGAT1 has a role in hepatic retinoid storage and metabolism, but this does not involve direct actions of DGAT1 in retinyl ester synthesis.     

内皮祖细胞移植对大鼠肝硬化作用的研究

目的 探讨内皮祖细胞(endothelial progenitor cells,EPCs)移植对四氯化碳(carbon tetrachloride,CCl4)诱导的大鼠肝硬化的作用.方法 本组38只SD大鼠中8只大鼠为正常对照,其余30只采用25%的CCl4/橄榄油灌胃制备肝硬化模型.再将肝硬化大鼠分为3组,每组10只.12周后直接处死的为肝硬化模型组,门静脉输入大鼠EPCs为EPCs移植组,经门脉输入生理盐水为移植对照组.移植4周后检测移植组和移植对照组大鼠肝组织胶原Ⅲ(collagen Ⅲ,COL Ⅲ)、平滑肌动蛋白(smooth muscle actin α,α-SMA)和Ki67的表达,检测外周血肝功能和血凝分析.结果 肝硬化模型组大鼠肝脏体积增至正常时的2倍.EPCs移植组大鼠与肝硬化模型组比较,肝组织学活动指数(histological activity index,HAI)(F=75.062,P<0.01),丙氨酸氨基转移酶(alanine aminotransferase,ALT)(F=29.942,P<0.05),门冬氨酸氨基转移酶(aspartate aminotransferase,AST)(F=16.618,P<0.05)和总胆红素(total bilirubin,TBIL)(F=9.911,P<0.05)水平降低,白蛋白(albumin,Alb)(F=4.944,P<0.05)和Ki67(F=45.966,P<0.01)水平升高,纤维化面积(F=25.025,P<0.05)减少,α-SMA(F=7.86,P<0.05)和COL Ⅲ(F=135.787,P<0.01)表达降低;与正常肝脏相比,移植对照组HAI,ALT,AST和TBIL水平升高,Alb和Ki67水平降低,纤维化面积增加,α-SMA和COL Ⅲ表达升高(P<0.05).移植对照组大鼠凝血酶原时间延长,差异有统计学意义(P<0.05). 结论移植EPCs可以促进大鼠肝硬化的肝细胞增生,减轻肝纤维化程度.     

Induction of transplantation tolerance in humans using fetal cell transplants

When engrafted with donor stem cells and lymphoid cells, patients develop transplantation tolerance to donor antigens. We analyzed the mechanism of tolerance induction in immunoincompetent recipients whose immunity has been reconstituted by transplantation of mismatched stem cells. Seven infants or human fetuses received fetal liver transplants as a treatment for severe combined immunodeficiency disease. After reconstitution of immunity by lymphocytes developed from donor stem cells, T-cell clones were produced and analyzed. Because donors and recipients were HLA mismatched, it was easy to demonstrate the donor origin of the T-cell clones. These clones were shown to have developed tolerance to histocompatibility antigens of the stem cell donor via a process of clonal deletion (probably as a result of contact with donor-derived macrophages and dendritic cells). They were also tolerant to histocompatibility antigens of the host but through a different mechanism: many clones recognized these antigens but had no detrimental effect on the target cells exhibiting host antigens, either in vitro or in vivo. Clonal anergy was therefore the cause of this tolerance to host determinants, resulting in a lack of graft-versus-host disease and of autoimmunity. The contact between developing T cells of donor origin and host epithelial cells within the host thymus may explain this colonal anergy. It should be noted that all patients had high serum levels of interleukin-10, which might have contributed to the persistent engraftment and tolerance.     

Magnesium depletion in chronic terminal liver cirrhosis

Serum ionized magnesium represents less than 1% of the total body magnesium. The most reliable method to evaluate magnesium status is the magnesium loading test: In magnesium depletion its uptake is increased (20-50%) and is about 6% in normal magnesium status. There are no studies on magnesium status in chronic cirrhotics who may be in depletion. We performed magnesium loading test in 10 chronic cirrhotics listed for liver transplantation and in six healthy control patients. Magnesium sulphate 30 mmol was infused and urine magnesium was determined over 24 h. Serum ionized magnesium increased similarly in all patients. The uptake of magnesium was 8 +/- 8% in control patients and 34 +/- 26% in cirrhotics (p < 0.01). Chronic terminal cirrhotics are magnesium depleted which should be taken into account in case of liver transplantation and also in other interventions. Spot sampled serum ionized magnesium revealed magnesium depletion poorly.     

Acute cholecystitis in combination with chronic hepatitis and cirrhosis of the liver

An analysis of surgical treatment of cholelithiasis, acute cholecystitis was made which had appeared in 107 patients against the background of chronic diseases of the liver. Current diagnostic methods were used for differential diagnostics of jaundice: USI, CT, retrograde cholecystopancreatography, duodenoscopy. All the patients were operated upon against the background of hepatotropic and anti-ulcer therapy being performed. The main component of anesthesia during operation in most patients was epidural blockade which unlike multicomponent anesthesia had no negative effects on the indices of bilirubin in blood and aminotranspherase activity. Cholecystectomy, sanitation and decompression of bile ducts in patients with acute cholecystitis which appeared against the background of chronic lesion of the liver allowed to eliminate the source of portal toxemia, liquidate the mechanical source of cholestasis that, as a rule, results in liquidation of acute hepatic failure. The optimal method of differential diagnostics of jaundices was intraoperative cholangiography and choledochotomy.     

术后应用肠外营养对肝细胞癌合并肝硬化病人的影响

目的探讨肝细胞癌合并肝硬化病人术后的营养支持。方法24例行根治性切除的肝癌病人随机分为PN组（n=12）和rhGH＋PN组（n=12）。术前、术后1d和术后6d测肝功能、血糖、AFP、血清前白蛋白、转铁蛋白及血清GH、IGF-1、IGFBP-3，用RT—PCR法检测肝组织（包括癌组织，癌旁组织和术后6d肝穿刺组织）ALBmRNA、IGF-1mRNA、IGFBP-3mRNA的表达，肝组织行Ki67免疫组织化染色。同时选12例因胆石症或肝血管瘤行手术的病人作为正常对照。结果肝细胞癌合并肝硬化病人血清前白蛋白，转铁蛋白低于正常对照组，血清GH水平高于正常对照组，而血清IGF-1、IGFBP-3水平低于正常对照组；术后6d，rhGH＋PN组血糖、血清前白蛋白、转铁蛋白、血清GH、IGF-1、IGFBP-3水平、肝ALBmRNA、IGF—1mRNA、IGFBP-3mRNA表达水平、肝Ki67指数均高于PN组。血清GH水平与血糖呈正相关，血清IGF1、IGFBP-3水平与血清AST、ALT、TBIL呈负相关，与血清ALB、前白蛋白，转铁蛋白呈正相关。结论rhGH＋PN有利于肝细胞癌合并肝硬化病人术后生长激素／胰岛素样生长因子-1轴的恢复，检测血清IGF-1、IGFBP-3水平有助于了解短期营养支持的效果和选择营养素。     

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吴肇汉 ,男 ,出生于1938年 1月。现任上海医科大学附属中山医院外科教研室副主任、外科实验室主任、临床营养研究中心主任。外科教授。博士研究生导师。中华医学会外科学会副主任委员 ,上海医学会普外科学会主任委员 ,中华医学会外科学会肠内、肠外营养学组副组长。兼任《中国实用外科杂志》等 12本杂志副主编或编委。195 7～ 196 2年于上海第一医学院医疗系学习。 1986～1987年于美国Ｓｔａｎｆｏｒｄ大学医学院作访问学者 ,1993年 7月至 10月在美国Ｐｉｔｔｓｂｕｒｇｈ大学医院作访问学者。主要从事胃肠外科和外科营养方面的科研和临床工…     

Long-term fate of heterotopic liver transplants in rats with portal vein inflow only

Previous studies have suggested that hepatic arterial flow in heterotopic partial liver transplants is necessary to ensure graft survival and regenerative capacity. This report presents findings in a syngeneic rat strain (Lewis) that partial liver transplants can be successfully heterotopically transplanted in the long term with the only inflow coming from the portal vein. When the host liver undergoes a nearly complete resection at 3-4 weeks, the transplanted liver regenerates to maintain the health of the host. Moderate to massive hepatocellular necrosis occurs in the first 3 months postoperatively, with recovery by 4-5 months. Liver transplants 8-10 months postoperatively appear architecturally normal. No host liver tissues were found to be regenerating after subtotal host liver resection. We conclude that portal vein reconstruction without hepatic arterial inflow can sustain a partial liver transplant in the long term, replacing the function of the host liver.