Endoplasmic reticulum stress inhibition protects steatotic and non-steatotic livers in partial hepatectomy under ischemia�Creperfusion

During partial hepatectomy, ischemia–reperfusion (I/R) is commonly applied in clinical practice to reduce blood flow. Steatotic livers show impaired regenerative response and reduced tolerance to hepatic injury. We examined the effects of tauroursodeoxycholic acid (TUDCA) and 4-phenyl butyric acid (PBA) in steatotic and non-steatotic livers during partial hepatectomy under I/R (PH+I/R). Their effects on the induction of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress were also evaluated. We report that PBA, and especially TUDCA, reduced inflammation, apoptosis and necrosis, and improved liver regeneration in both liver types. Both compounds, especially TUDCA, protected both liver types against ER damage, as they reduced the activation of two of the three pathways of UPR (namely inositol-requiring enzyme and PKR-like ER kinase) and their target molecules caspase 12, c-Jun N-terminal kinase and C/EBP homologous protein-10. Only TUDCA, possibly mediated by extracellular signal-regulated kinase upregulation, inactivated glycogen synthase kinase-3β. This is turn, inactivated mitochondrial voltage-dependent anion channel, reduced cytochrome c release from the mitochondria and caspase 9 activation and protected both liver types against mitochondrial damage. These findings indicate that chemical chaperones, especially TUDCA, could protect steatotic and non-steatotic livers against injury and regeneration failure after PH+I/R.     

Chemical Imaging on Liver Steatosis Using Synchrotron Infrared and ToF-SIMS Microspectroscopies

Fatty liver or steatosis is a frequent histopathological change. It is a precursor for steatohepatitis that may progress to cirrhosis and in some cases to hepatocellular carcinoma. In this study we addressed the in situ composition and distribution of biochemical compounds on tissue sections of steatotic liver using both synchrotron FTIR (Fourier transform infrared) and ToF-SIMS (time of flight secondary ion mass spectrometry) microspectroscopies. FTIR is a vibrational spectroscopy that allows investigating the global biochemical composition and ToF-SIMS lead to identify molecular species in particular lipids. Synchrotron FTIR microspectroscopy demonstrated that bands linked to lipid contribution such as -CH₃ and -CH₂ as well as esters were highly intense in steatotic vesicles. Moreover, a careful analysis of the -CH₂ symmetric and anti-symmetric stretching modes revealed a slight downward shift in spectra recorded inside steatotic vesicles when compared to spectra recorded outside, suggesting a different lipid environment inside the steatotic vesicles. ToF-SIMS analysis of such steatotic vesicles disclosed a selective enrichment in cholesterol as well as in diacylglycerol (DAG) species carrying long alkyl chains. Indeed, DAG C36 species were selectively localized inside the steatotic vesicles whereas DAG C30 species were detected mostly outside. Furthermore, FTIR detected a signal corresponding to olefin (C = C, 3000-3060 cm⁻¹) and revealed a selective localization of unsaturated lipids inside the steatotic vesicles. ToF-SIMS analysis definitely demonstrated that DAG species C30, C32, C34 and C36 carrying at least one unsaturated alkyl chain were selectively concentrated into the steatotic vesicles. On the other hand, investigations performed on the non-steatotic part of the fatty livers have revealed important changes when compared to the normal liver. Although the non-steatotic regions of fatty livers exhibited normal histological aspect, IR spectra demonstrated an increase in the lipid content and ToF-SIMS detected small lipid droplets corresponding most likely to the first steps of lipid accretion.     

Influence of Pre-, Intra- and Post-Operative Parameters of Donor Liver on the Outcome of Isolated Human Hepatocytes

The aim of the present study was to analyse, retrospectively on a large panel of patients (149), the influence of the donor liver characteristics on the outcome of human hepatocyte isolation obtained from resected liver biopsies from surgical waste after hepatectomy. Among the pre-operative parameters, the type of disease, age and sex of the patient, previous chemotherapy, alcohol or tobacco consumption did not affect the yield, viability, attachment rate and function of the isolated human hepatocytes. Pre-operative biological and anatomopathological data indicated that, while mild steatosis (≤10% steatotic hepatocytes) did also not affect the outcome of hepatocyte isolation, stronger steatosis (>10% steatotic hepatocytes) tended to decrease hepatocyte yield. Cholestasis, as assessed by γ-glutamyl transferase serum values, significantly negatively correlated with the percentage of digested liver and the yield of viable cells. Intra-operative clamping time, that is, warm ischaemia, longer than 30 min was found to decrease both the percentage of digested liver and cell yield. Among the post-operative parameters, the percentage of digested liver decreased when biopsy weights were higher than 100 g, the use of glue tended to increase both the percentage of digested tissue and the yield of viable cells. In conclusion, human diseased livers appear to be a valuable source of isolated functional human hepatocytes. We recommend, for an optimal isolation, to use liver biopsies weighing less than 100 g, to glue the section surfaces of the biopsies and to avoid the use of moderate steatotic livers (>10% steatotic hepatocytes) and cholestatic livers, as well as livers undergoing warm ischaemia or clamping during resection due to the decrease in cell yield. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Protective Effects of Different-Time-Ischemic Preconditioning on the Reperfusion Injury in Fatty Livers in Rats

Background

The present study was aimed to investigate the protective effects of different-time-ischemic preconditioning on the reperfusion injury in fatty livers in rats, and to elucidate the mechanisms underlying the protective effects and the optimal safe ischemic preconditioning time on the hepatic IR injury in steatotic livers.

Methodology/Principal Findings

A rat fatty liver model was established by high-fat diet feeding. We investigated the changes in the concentration of AST, ALT, LDH and NO in the serum, and of MDA, SOD, and MPO in the liver samples in response to different ischemic preconditioning times and ischemia-reperfusion injury. Histological analysis was performed to evaluate the results of the hepatic fatty infiltration. 1) At 24 h after 15 min ischemic preconditioning with 10 min reperfusion (15 min +10 min IP), the extent and area of the necrosis was markedly higher in the fatty liver samples with respect to IR, compared to the normal liver samples. 2) In response to the treatment of 5/8 min +10 min IP, the fatty liver group showed lower levels of serological indicators and liver MDA and MPO compared to the other groups, while the SOD activity of the fatty liver group was significantly higher than the other groups (p<0.05). Compared to the corresponding IR group, all IP groups showed a significantly higher serum NO concentration (p<0.05). Among the fatty liver groups, the 5/8 min+10 min IP group showed the highest NO concentration (p<0.05).

Conclusions/Significance

Fat infiltration could aggravate the ischemia-reperfusion injury in the rat liver. Furthermore, ischemic preconditioning could increase the tolerance of the fatty liver, which was induced by the high-fat diet, to hepatic ischemia-reperfusion injury in rats. The protocol of 5/8 min +10 min IP was the optimal regimen for the treatment of moderate and severe fatty livers.     

The role of MLKL in Hepatic Ischemia-Reperfusion Injury of Alcoholic Steatotic Livers

Alcohol-related liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) are the primary causes of chronic liver disease in western countries. Liver transplantation is currently one of the most efficient approaches to save patients with liver failure, which is often associated with hepatic ischemia-reperfusion (IR) injury. IR injury is exacerbated by hepatic steatosis, yet the mechanism remains elusive. Necroptosis is a form of regulated cell death mediated by receptor-interacting protein kinase 1 (RIP1), RIP3 and mixed lineage kinase domain-like (MLKL) protein, which has been implicated in the pathogenesis of ALD and NAFLD. Though necroptosis plays an important role in IR injury of high fat diet - induced steatotic livers, the role of necroptosis in IR injury of ethanol - induced steototic livers has not been investigated. In the present study, we used chronic plus binge alcohol (Gao-binge) feeding followed by IR surgery to investigate IR liver injury with ethanol-associated steatosis. We found that the levels of key necroptotic proteins MLKL and RIP3 increased in alcohol-fed mouse livers. Moreover, we observed increased liver injury after IR in control diet-fed mice, which was further exacerbated by alcohol feeding based on serum alanine aminotransferase (ALT) levels and TUNEL staining of necrotic cells. Hepatic neutrophil infiltration also increased in alcohol-fed mice after IR surgery. However, deletion of Mlkl did not protect against IR liver injury in alcohol-fed mice compared with matched wild-type mice. In conclusion, alcoholic steatosis promotes IR injury, which seems to be independent of MLKL-mediated necroptosis.     

A nonsynonymous gene variant in the adiponutrin gene is associated with nonalcoholic fatty liver disease severity

We explored the role of the adiponutrin (PNPLA3) nonsynonymous-rs738409 single nucleotide polymorphism (SNP) in genetic susceptibility to nonalcoholic fatty liver disease (NAFLD) and whether this SNP contributes to the severity of histological disease. Two hundred sixty-six individuals were evaluated in a case-control association study, which included 172 patients with features of NAFLD and 94 control subjects. The rs738409 G allele was significantly associated with NAFLD (P < 0.001; OR 2.8 95%, CI 1.5–5.2), independent of age, sex, body mass index (BMI), and Homeostasis Model Assessment (HOMA) index. When we tested the hypothesis of a relation between the SNP and the histological spectrum of NAFLD, a significant association was observed [chi2 19.9, degree of freedom (df): 2, P < 5 × 10⁻⁵, adjusted for HOMA and BMI]. The degree of liver steatosis, as evaluated by liver biopsy, was significantly associated with the rs738409 G allele. Patients with CC genotype showed a lower steatosis score (14.9% ± 3.9) in comparison with the CG genotype (26.3% ± 3.5) and GG genotype (33.3% ± 4.0) (P < 0.005). The proportion of the total variation attributed to rs738409 genotypes was 5.3% (β 0.23 ± 0.07; P < 0.002). Our data suggest that the rs738409 G allele is associated not only with fat accumulation in the liver but also with liver injury, possibly triggered by lipotoxicity.     

Theaflavin attenuates ischemia-reperfusion injury in a mouse fatty liver model

The incidence of non-alcoholic fatty liver disease (NAFLD) has been increasing, and there is a shortage of liver donors, which has led to the acceptance of steatotic livers for transplantation. However, steatotic livers are known to experience more severe acute ischemia-reperfusion (I/R) injury than normal livers upon transplantation. In the present study, we investigated the role of theaflavin, a polyphenol substance extracted from black tea, in attenuating acute I/R injury in a fatty liver model. We induced I/R in normal and steatotic livers treated with or without theaflavin. We also separated primary hepatocytes from the normal and steatotic livers, and applied RAW264.7 cells, a mouse macrophage cell line, that was pretreated with theaflavin. We observed that liver steatosis, oxidative stress, inflammation and hepatocyte apoptosis were increased in the steatotic liver compared to the normal liver, however, these changes were significantly decreased by theaflavin treatment. In addition, theaflavin significantly diminished the ROS production of steatotic hepatocytes and TNF-α production by LPS-stimulated RAW264.7 cells. We concluded that theaflavin has protective effects against I/R injury in fatty livers by anti-oxidant, anti-inflammatory, and anti-apoptotic mechanisms.     

Chronic steatosis aggravates cold-storage induced acute ischemic injury in rat donor livers through the perturbation of lipophagy

In this study, we explored the effects of cold ischemia on chronic steatosis and lipid signaling in vivo. Sprague Dawley (SD) rat models of chronic steatosis were established. Pathological observations and liver indices were assessed through hematoxylin-eosin (HE)- and Oil Red O staining. Autophagy and metabolism in adipose tissue were analyzed under post-ischemia and hypoxic conditions via western blotting and immunofluorescent analysis.We found that cold ischemia treatment exacerbated hepatic steatosis and reduced lipid phagocytosis. This manifested as a loss of Microtubule-associated protein 1A/1B-light chain 3 (LC3) and Perilipin 2 (PLIN2), and lower levels of autophagy. Cold ischemia also inhibited lipophagy in transplanted rat livers, most notably in moderate-to-severe steatosis models. Ischemia and hypoxia inhibited lipid phagocytosis and increased lipid accumulation.Collectively, these data show that chronic steatosis aggravates cold storage induced acute ischemic injury in rat donor livers through the inhibition of lipophagy. Moderate-to-severe steatosis therefore influences the postoperative recovery of liver transplant recipients, which should be immediately transplanted to reduce the risk of cold ischemia.     

Liver Grafts for Transplantation from Donors with Diabetes: An Analysis of the Scientific Registry of Transplant Recipients Database

Patients with a history of diabetes mellitus (DM) have worse survival than those without DM after liver transplantation. However, the effect of liver grafts from DM donors on the post-transplantation survival of recipients is unclear. Using the Scientific Registry of Transplant Recipients database (2004–2008), 25,413 patients were assessed. Among them, 2,469 recipients received grafts from donors with DM. The demographics and outcome of patients were assessed. Patient survival was assessed using Kaplan–Meier methodology and Cox regression analyses. Recipients from DM donors experienced worse graft survival than recipients from non-DM donors (one-year survival: 81% versus 85%, and five-year survival: 67% versus 74%, P<0.001, respectively). Graft survival was significantly lower for recipients from DM donors with DM duration >5 years (P<0.001) compared with those with DM duration <5 years. Cox regression analyses showed that DM donors were independently associated with worse graft survival (hazard ratio, 1.11; 95% confidence interval, 1.02–1.19). The effect of DM donors was more pronounced on certain underlying liver diseases of recipients. Increases in the risk of graft loss were noted among recipients from DM donors with hepatitis-C virus (HCV) infection, whereas those without HCV experienced similar outcomes compared with recipients from non-DM donors. These data suggest that recipients from DM donors experience significantly worse patient survival after liver transplantation. However, in patients without HCV infection, using DM donors was not independently associated with worse post-transplantation graft survival. Matching these DM donors to recipients without HCV may be safe.     

Human Hepatocyte Growth Factor (hHGF)-Modified Hepatic Oval Cells Improve Liver Transplant Survival

Despite progress in the field of immunosuppression, acute rejection is still a common postoperative complication following liver transplantation. This study aims to investigate the capacity of the human hepatocyte growth factor (hHGF) in modifying hepatic oval cells (HOCs) administered simultaneously with orthotopic liver transplantation as a means of improving graft survival. HOCs were activated and isolated using a modified 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH) model in male Lewis rats. A HOC line stably expressing the HGF gene was established following stable transfection of the pBLAST2-hHGF plasmid. Our results demonstrated that hHGF-modified HOCs could efficiently differentiate into hepatocytes and bile duct epithelial cells in vitro. Administration of HOCs at the time of liver transplantation induced a wider distribution of SRY-positive donor cells in liver tissues. Administration of hHGF-HOC at the time of transplantation remarkably prolonged the median survival time and improved liver function for recipients compared to these parameters in the other treatment groups (P<0.05). Moreover, hHGF-HOC administration at the time of liver transplantation significantly suppressed elevation of interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) levels while increasing the production of IL-10 and TGF-β1 (P<0.05). HOC or hHGF-HOC administration promoted cell proliferation, reduced cell apoptosis, and decreased liver allograft rejection rates. Furthermore, hHGF-modified HOCs more efficiently reduced acute allograft rejection (P<0.05 versus HOC transplantation only). Our results indicate that the combination of hHGF-modified HOCs with liver transplantation decreased host anti-graft immune responses resulting in a reduction of allograft rejection rates and prolonging graft survival in recipient rats. This suggests that HOC-based cell transplantation therapies can be developed as a means of treating severe liver injuries.     

Enzyme-Linked Immunosorbent Spot (ELISpot) monitoring of cytokine-producing cells for the prediction of acute rejection in renal transplant patients

The purpose of this study was to evaluate T-cell immunity markers using serial post-transplantation monitoring of cytokine-producing cells during the first post-transplant months for the prediction of acute rejection and potentially chronic rejection of kidney allograft. We followed 57 kidney allograft recipients for meanly 3 years post-transplantation. Blood samples were collected pre-transplant, 2, 4 and 12 weeks post-transplant. The frequencies of IL-10-, IL-17- and IFN-γ-producing cells were determined in all time-points using ELISPOT assay. The results of ELISpot monitoring and levels of IL-23 and TGF-β were compared between recipients with acute (n = 12) or chronic rejection episodes and patients with stable graft function (n = 45). In all post-transplant time-points, significantly high frequencies of IFN-γ- and IL-17-producing cells and low frequency of IL-10-producing cells were observed in rejection group versus patients with stable graft function (P<0.0001). TheROCcurve analysis for determining the reliability of cytokine-producing cells for the prediction of acute rejection revealed that AUC was 0.046 for IL-10 (P<0.001), 0.927 for IL-17 (P<0.001) and 0.929 for INF-γ-producing cells (P<0.001). Our results indicate that analyzing the frequencies of INF-γ/IL-10/IL-17-producing cells may define a reliable panel for the prediction of acute rejection within the first post-transplant year which could also be applicable for the prediction of chronic rejection episodes.     

Impact of normothermic preservation with extracellular type solution containing trehalose on rat kidney grafting from a cardiac death donor

Background

The aim of this study was to investigate factors that may improve the condition of a marginal kidney preserved with a normothermic solution following cardiac death (CD) in a model of rat kidney transplantation (RTx).

Methods

Post-euthanasia, Lewis (LEW) donor rats were left for 1 h in a 23°C room. These critical kidney grafts were preserved in University of Wisconsin (UW), lactate Ringer''s (LR), or extracellular-trehalose-Kyoto (ETK) solution, followed by intracellular-trehalose-Kyoto (ITK) solution at 4, 23, or 37°C for another 1 h, and finally transplanted into bilaterally nephrectomized LEW recipient rats (n = 4–6). Grafts of rats surviving to day 14 after RTx were evaluated by histopathological examination. The energy activity of these marginal rat kidneys was measured by high-performance liquid chromatography (HPLC; n = 4 per group) and fluorescence intensity assay (n = 6 per group) after preservation with UW or ETK solutions at each temperature. Finally, the transplanted kidney was assessed by an in vivo luciferase imaging system (n = 2).

Results

Using the 1-h normothermic preservation of post-CD kidneys, five out of six recipients in the ETK group survived until 14 days, in contrast to zero out of six in the UW group (p<0.01). Preservation with ITK rather than ETK at 23°C tended to have an inferior effect on recipient survival (p = 0.12). Energy activities of the fresh donor kidneys decreased in a temperature-dependent manner, while those of post-CD kidneys remained at the lower level. ETK was superior to UW in protecting against edema of the post-CD kidneys at the higher temperature. Luminescence intensity of successful grafts recovered within 1 h, while the intensity of grafts of deceased recipients did not change at 1 h post-reperfusion.

Conclusions

Normothermic storage with extracellular-type solution containing trehalose might prevent reperfusion injury due to temperature-dependent tissue edema.     

Scavenger receptor BI facilitates hepatic very low density lipoprotein production in mice

Scavenger receptor BI (SR-BI) is a selective uptake receptor for HDL cholesterol but is also involved in the catabolism of apolipoprotein (apo)B-containing lipoproteins. However, plasma levels of apoB-containing lipoproteins increase following hepatic SR-BI overexpression, suggesting that SR-BI not solely mediates their catabolism. We therefore tested the hypothesis that hepatic SR-BI impacts on VLDL production. On day 7 following adenovirus (Ad)-mediated overexpression of SR-BI, VLDL-triglyceride and VLDL-apoB production rates were significantly increased (P < 0.001), whereas VLDL production was significantly lower in SR-BI knockout mice compared with controls (P < 0.05). In mice injected with AdSR-BI, hepatic cholesterol content increased (P < 0.001), microsomal triglyceride transfer protein activity was higher (P < 0.01) and expression of sterol-regulatory element binding protein (SREBP)2 and its target genes was decreased (P < 0.01). Conversely, in SR-BI knockout mice, microsomal triglyceride transfer protein activity was lower and expression of SREBP2 target genes was increased (P < 0.01). Finally, we demonstrate in vitro in isolated primary hepatocytes as well as in vivo that cholesterol derived from HDL and taken up via SR-BI into the liver can be resecreted within VLDL. These data indicate that hepatic SR-BI expression is linked to VLDL production, and within liver, a metabolic shunt might exist that delivers HDL cholesterol, at least in part, to a pool from which cholesterol is mobilized for VLDL production. These results might have implications for HDL-based therapies against atherosclerotic cardiovascular disease, especially with SR-BI as target.     

The in utero programming effect of increased maternal androgens and a direct fetal intervention on liver and metabolic function in adult sheep

Epigenetic changes in response to external stimuli are fast emerging as common underlying causes for the pre-disposition to adult disease. Prenatal androgenization is one such model that results in reproductive and metabolic features that are present in conditions such as polycystic ovary syndrome (PCOS). We examined the effect of prenatal androgens on liver function and metabolism of adult sheep. As non-alcoholic fatty liver disease is increased in PCOS we hypothesized that this, and other important liver pathways including metabolic function, insulin–like growth factor (IGF) and steroid receptivity, would be affected. Pregnant ewes received vehicle control (C; n = 5) or testosterone propionate (TP; n = 9) twice weekly (100 mg; i.m) from d62–102 (gestation 147 days). In a novel treatment paradigm, a second cohort received a direct C (n = 4) or TP (20 mg; n = 7) fetal injection at d62 and d82. In adults, maternal TP exposure resulted in increased insulin secretion to glucose load (P<0.05) and the histological presence of fatty liver (P<0.05) independent of central obesity. Additionally, hepatic androgen receptor (AR; P<0.05), glucocorticoid receptor (GR; P<0.05), UDP- glucose ceramide glucosyltransferase (UGCG; P<0.05) and IGF1 (P<0.01) expression were upregulated. The direct fetal intervention (C and TP) led to early fatty liver changes in all animals without differential changes in insulin secretion. Furthermore, hepatic phosphoenolpyruvate carboxykinase (PEPCK) was up-regulated in the fetal controls (P<0.05) and this was opposed by fetal TP (P<0.05). Hepatic estrogen receptor (ERα; P<0.05) and mitogen activated protein kinase kinase 4 (MAP2K4; P<0.05) were increased following fetal TP exposure. Adult liver metabolism and signaling can be altered by early exposure to sex steroids implicating epigenetic regulation of metabolic disturbances that are common in PCOS.     

The impact of cortisol in steatotic and non‐steatotic liver surgery

The intent of this study was to examine the effects of regulating cortisol levels on damage and regeneration in livers with and without steatosis subjected to partial hepatectomy under ischaemia–reperfusion. Ultimately, we found that lean animals undergoing liver resection displayed no changes in cortisol, whereas cortisol levels in plasma, liver and adipose tissue were elevated in obese animals undergoing such surgery. Such elevations were attributed to enzymatic upregulation, ensuring cortisol production, and downregulation of enzymes controlling cortisol clearance. In the absence of steatosis, exogenous cortisol administration boosted circulating cortisol, while inducing clearance of hepatic cortisol, thus maintaining low cortisol levels and preventing related hepatocellular harm. In the presence of steatosis, cortisol administration was marked by a substantial rise in intrahepatic availability, thereby exacerbating tissue damage and regenerative failure. The injurious effects of cortisol were linked to high hepatic acethylcholine levels. Upon administering an α7 nicotinic acethylcholine receptor antagonist, no changes in terms of tissue damage or regenerative lapse were apparent in steatotic livers. However, exposure to an M3 muscarinic acetylcholine receptor antagonist protected livers against damage, enhancing parenchymal regeneration and survival rate. These outcomes for the first time provide new mechanistic insight into surgically altered steatotic livers, underscoring the compelling therapeutic potential of cortisol–acetylcholine–M3 muscarinic receptors.