Hassen Ben Abdennebi

Preservation of Steatotic Livers in IGL-1 Solution

A new Institut Georges Lopez (IGL‐1) solution was used to preserve steatotic livers. Steatotic (obese [Ob]) and nonsteatotic (lean [Ln]) livers from Zücker rats (n = 16, 8 Ln and 8 Ob) were preserved for 24 hours at 4°C in University of Wisconsin (UW) or IGL‐1 solution, respectively, and then perfused ex vivo for 2 hours at 37°C. Additionally, Ob and Ln livers (n = 16, 8 Ln and 8 Ob) were preserved in IGL‐1 plus Nω‐nitro‐L‐arginine methyl ester hydrochloride (L‐NAME). Hepatic injury and function (aminotransferases, bile production, bromosulfophthalein clearance), and factors potentially involved in the susceptibility of steatotic livers to ischemia‐reperfusion injury, such as oxidative stress, mitochondrial damage, and vascular resistance, were studied. Nitric oxide (NO) production and constitutive and inducible NO synthase were also measured. Steatotic and nonsteatotic livers preserved in IGL‐1 solution showed lower transaminases, malondialdehyde, glutamate dehydrogenase levels, and higher bile production than UW‐solution‐preserved livers. IGL‐1 solution protected against oxidative stress, mitochondrial damage and the alterations in vascular resistance associated with cold ischemia‐reperfusion. Thus, at the end of reperfusion period, aspartate aminotransferase levels in steatotic livers were 281 ± 6 U/L in UW vs. 202 ± 10 U/L in IGL‐1 solution. Glutamate dehydrogenase was 463 ± 75 U/L in UW vs. 111 ± 4 U/L in IGL‐1 solution, and oxidative stress was 3.0 ± 0.1 nmol/mg prot in UW vs. 2.0 ± 0.1 nmol/mg prot in IGL‐1 solution. These beneficial effects of IGL‐1 solution were abolished by the addition of L‐NAME, which implicates NO in the benefits of IGL‐1. In conclusion, IGL‐1 solution provided steatotic livers with better protection against the deleterious effects of cold ischemia‐reperfusion injury than did UW solution. Liver Transpl 12:1215‐1223, 2006.

AMPK involvement in endoplasmic reticulum stress and autophagy modulation after fatty liver graft preservation: a role for melatonin and trimetazidine cocktail

Ischemia/reperfusion injury (IRI) associated with liver transplantation plays an important role in the induction of graft injury. Prolonged cold storage remains a risk factor for liver graft outcome, especially when steatosis is present. Steatotic livers exhibit exacerbated endoplasmic reticulum (ER) stress that occurs in response to cold IRI. In addition, a defective liver autophagy correlates well with liver damage. Here, we evaluated the combined effect of melatonin and trimetazidine as additives to IGL‐1 solution in the modulation of ER stress and autophagy in steatotic liver grafts through activation of AMPK. Steatotic livers were preserved for 24 hr (4°C) in UW or IGL‐1 solutions with or without MEL + TMZ and subjected to 2‐hr reperfusion (37°C). We assessed hepatic injury (ALT and AST) and function (bile production). We evaluated ER stress (GRP78, PERK, and CHOP) and autophagy (beclin‐1, ATG7, LC3B, and P62). Steatotic livers preserved in IGL‐1 + MEL + TMZ showed lower injury and better function as compared to those preserved in IGL‐1 alone. IGL‐1 + MEL + TMZ induced a significant decrease in GRP78, pPERK, and CHOP activation after reperfusion. This was consistent with a major activation of autophagic parameters (beclin‐1, ATG7, and LC3B) and AMPK phosphorylation. The inhibition of AMPK induced an increase in ER stress and a significant reduction in autophagy. These data confirm the close relationship between AMPK activation and ER stress and autophagy after cold IRI. The addition of melatonin and TMZ to IGL‐1 solution improved steatotic liver graft preservation through AMPK activation, which reduces ER stress and increases autophagy.

Ischemic preconditioning reduces endoplasmic reticulum stress and upregulates hypoxia inducible factor-1α in ischemic kidney: the role of nitric oxide.

BackgroundAlthough recent studies indicate that renal ischemic preconditioning (IPC) protects the kidney from ischemia-reperfusion (I/R) injury, the precise protective mechanism remains unclear. In the current study, we investigated whether early IPC could upregulate hypoxia inducible transcription factor-1α (HIF-1α) expression and could reduce endoplasmic reticulum (ER) stress after renal I/R and whether pharmacological inhibition of nitric oxide (NO) production would abolish these protective effects.MethodsKidneys of Wistar rats were subjected to 60 min of warm ischemia followed by 120 min of reperfusion (I/R group), or to 2 preceding cycles of 5 min ischemia and 5 min reperfusion (IPC group), or to intravenously injection of NG-nitro-L-arginine methylester (L-NAME, 5 mg/kg) 5 min before IPC (L-NAME+IPC group). The results of these experimental groups were compared to those of a sham-operated group. Sodium reabsorption rate, creatinine clearance, plasma lactate dehydrogenase (LDH) activity, tissues concentrations of malonedialdehyde (MDA), HIF-1α and nitrite/nitrate were determined. In addition, Western blot analyses were performed to identify the amounts of Akt, endothelial nitric oxide synthase (eNOS) and ER stress parameters.ResultsIPC decreased cytolysis, lipid peroxidation and improved renal function. Parallely, IPC enhanced Akt phosphorylation, eNOS, nitrite/nitrate and HIF-1α levels as compared to I/R group. Moreover, our results showed that IPC increased the relative amounts of glucose-regulated protein 78 (GRP78) and decreased those of RNA activated protein kinase (PKR)-like ER kinase (PERK), activating transcription factor 4 (ATF4) and TNF-receptor-associated factor 2 (TRAF2) as judged to I/R group. However, pre treatment with L-NAME abolished these beneficial effects of IPC against renal I/R insults.ConclusionThese findings suggest that early IPC protects kidney against renal I/R injury via reducing oxidative and ER stresses. These effects are associated with phosphorylation of Akt, eNOS activation and NO production contributing thus to HIF-1α stabilization. The beneficial impact of IPC was abolished when NO production is inhibited before IPC application.

Pharmacological strategies against cold ischemia reperfusion injury

Importance of the field: Good organ preservation is a determinant of graft outcome after revascularization. The necessity of increasing the quality of organ preservation, as well as of extending cold storage time, has made it necessary to consider the use of pharmacological additives. Areas covered in this review: The complex physiopathology of cold-ischemia–reperfusion (I/R) injury – and in particular cell death, mitochondrial injury and endoplasmic reticulum stress – are reviewed. Basic principles of the formulation of the different preservation solutions are discussed. What the reader will gain: Current strategies and new trends in static organ preservation using additives such as trimetazidine, polyethylene glycols, melatonin, trophic factors and endothelin antagonists in solution are presented and discussed. The benefits and mechanisms responsible for enhancing organ protection against I/R injury are also discussed. Graft preservation was substantially improved when additives were added to the preservation solutions. Take home message: Enrichment of preservation solutions by additives is clinically useful only for short periods. For longer periods of cold ischemia, the use of such additives becomes insufficient because graft function deteriorates as a result of ischemia. In such conditions, the preservation strategy should be changed by the use of machine perfusion in normothermic conditions.

Attenuation of endoplasmic reticulum stress and mitochondrial injury in kidney with ischemic postconditioning application and trimetazidine treatment

BackgroundEndoplasmic reticulum (ER) and mitochondria have been implicated in the pathology of renal ischemia/reperfusion (I/R). In the present study, we investigated whether the use of ischemic postconditioning (IPostC) and trimetazidine (TMZ) separately or combined could reduce ER stress and mitochondria damage after renal ischemia.MethodsKidneys of Wistar rats were subjected to 60-min of warm ischemia followed by 120-min of reperfusion (I/R group, n = 6), or to 6 cycles of ischemia/reperfusion (10-s each cycle) just after 60-min of warm ischemia (IPostC group, n = 6), or to i.p. injection of TMZ (3 mg/kg) 30-min before ischemia (TMZ group, n = 6), or to the combination of both treatments (IPostC+TMZ group, n = 6). The results of these experimental groups were compared to those of a sham-operated group in which rat renal pedicles were only dissected. Sodium reabsorption rate, creatinine clearance lactate deshydrogenase (LDH) activity in plasma, and concentration of malonedialdehyde (MDA) in tissue were determined. In addition, Western blot analysis was performed to identify the amounts of cytochrome c, c-JunNH2-terminal kinase (JNK), voltage-dependent anion channel (VDAC), glycogen synthase kinase 3-beta (GSK3-β), and ER stress parameters.ResultsIPostC or/and TMZ significantly decreased cytolysis, oxidative stress and improved renal function in comparison to I/R group. IPostC but not TMZ significantly attenuated ER stress parameters versus I/R group. Indeed, it down-regulated the glucose-regulated protein 78 (GRP78), the activating transcription factor 4 (ATF4), the RNA activated protein kinase (PKR)-like ER kinas (PERK), the X box binding protein-1 (XBP-1) and the caspase12 protein levels. TMZ treatment significantly augmented GSK3-β phosphorylation and reduced levels of cytochrome c and VDAC phosphorylation in comparison to IPostC application. The combination of both treatments gave a synergetic effect. It significantly improved the survival rate, attenuated cytolysis, oxidative stress and improved renal function.ConclusionThis study revealed that IPostC protects kidney from I/R injury by suppressing ER stress while the beneficial effects of TMZ are mediated by mitochondria protection. The combination of both treatments ameliorated functional recovery.

Addition of carvedilol to University Wisconsin solution improves rat steatotic and nonsteatotic liver preservation

Here we examine the effect of adding carvedilol (CVD) to University of Wisconsin (UW) solution on the preservation of steatotic and nonsteatotic livers during cold ischemia and after normothermic reperfusion. We used an isolated perfused rat liver model. The following protocols were evaluated. Protocol 1 concerned the effect of CVD after cold ischemia. Steatotic and nonsteatotic livers were preserved for 24 hours in UW solution alone or with CVD. Livers without cold ischemia were used as controls. Transaminases were evaluated in the flushing effluent. Protocol 2 involved the effect of CVD after reperfusion. Both liver types were preserved for 24 hours in UW solution alone or with CVD and then perfused ex vivo for 2 hours at 37°C. Livers flushed and perfused without ischemia were used as controls. Hepatic injury and functionality [transaminases, bile production, and hepatic clearance of sulfobromophthalein (BSP)] were evaluated after reperfusion. In addition, factors potentially involved in hepatic ischemia‐reperfusion injury, including oxidative stress (malondialdehyde and superoxide anion levels), mitochondrial damage (glutamate dehydrogenase activity), microcirculatory disorders (flow rate and vascular resistance), and adenosine triphosphate (ATP) depletion, were evaluated after reperfusion. After cold ischemia, steatotic livers preserved in UW solution showed higher transaminase levels than nonsteatotic livers. After reperfusion, steatotic livers preserved in UW solution showed higher transaminase levels and lower bile production and BSP clearance than nonsteatotic livers. Alterations in the perfusion flow rate and vascular resistance, mitochondrial damage, and reduced ATP content were more evident in steatotic livers preserved in UW solution. The addition of CVD to UW solution reduced hepatic injury, obstructed its mechanisms, and improved hepatic functionality in both liver types. We conclude that CVD is a useful additive for UW solution that improves the preservation of steatotic and nonsteatotic livers subjected to prolonged cold ischemia. Liver Transpl 16:163–171, 2010.

New preservation strategies for preventing liver grafts against cold ischemia reperfusion injury

Background:  In spite of improvements in University of Wisconsin (UW) preservation solution, the injury from grafts during cold storage is an unresolved problem in liver transplantation. The aim of the present study was to evaluate the beneficial effect on ischemia–reperfusion injury associated with liver transplantation of the inversion of K+ and Na+ concentrations and the replacement of hydroxyethyl starch (HES) by polyethylene glycol (PEG) in UW preservation solution.

Proteasome inhibitors protect the steatotic and non-steatotic liver graft against cold ischemia reperfusion injury.

BACKGROUND The dramatic shortage of organs leads to consider the steatotic livers for transplantation although their poor tolerance against ischemia reperfusion injury (IRI). Ubiquitin proteasome system (UPS) inhibition during hypothermia prolongs myocardial graft preservation. The role of UPS in the liver IRI is not fully understood. Bortezomib (BRZ) treatment at non-toxic doses of rats fed alcohol chronically has shown protective effects by increasing liver antioxidant enzymes. We evaluated and compared both proteasome inhibitors BRZ and MG132 in addition to University of Wisconsin preservation solution (UW) at low and non-toxic dose for fatty liver graft protection against cold IRI. EXPERIMENTAL Steatotic and non-steatotic livers have been stored in UW enriched with BRZ (100 nM) or MG132 (25 μM), for 24h at 4°C and then subjected to 2-h normothermic reperfusion (37 °C). Liver injury (AST/ALT), hepatic function (bile output; vascular resistance), mitochondrial damage (GLDH), oxidative stress (MDA), nitric oxide (NO) (e-NOS activity; nitrates/nitrites), proteasome chymotrypsin-like activity (ChT), and UPS (19S and 20S5 beta) protein levels have been measured. RESULTS ChT was inhibited when BRZ and MG132 were added to UW. Both inhibitors prevented liver injury (AST/ALT), when compared to UW alone. BRZ increased bile production more efficiently than MG132. Only BRZ decreased vascular resistance in fatty livers, which correlated with an increase in NO generation (through e-NOS activation) and AMPK phosphorylation. GLDH and MDA were also prevented by BRZ. In addition, BRZ inhibited adiponectin, IL-1, and TNF alpha, only in steatotic livers. CONCLUSION MG132 and BRZ, administrated at low and non toxic doses, are very efficient to protect fatty liver grafts against cold IRI. The benefits of BRZ are more effective than those of MG132. This evidenced for the first time the potential use of UPS inhibitors for the preservation of marginal liver grafts and for future applications in the prevention of IRI.

Bortezomib enhances fatty liver preservation in Institut George Lopez-1 solution through adenosine monophosphate activated protein kinase and Akt/mTOR pathways

The aim of this study is to investigate the protective mechanisms induced by bortezomib added to Institut George Lopez (IGL)‐1 preservation solution to protect steatotic livers against cold ischaemia reperfusion injury and to examine whether these mechanisms occur through the activation of adenosine monophosphate activated protein kinase (AMPK), Akt/mTOR pathways.

Role of sirtuins in ischemia-reperfusion injury.

Ischemia-reperfusion injury (IRI) remains an unresolved and complicated situation in clinical practice, especially in the case of organ transplantation. Several factors contribute to its complexity; the depletion of energy during ischemia and the induction of oxidative stress during reperfusion initiate a cascade of pathways that lead to cell death and finally to severe organ injury. Recently, the sirtuin family of nicotinamide adenine dinucleotide-dependent deacetylases has gained increasing attention from researchers, due to their involvement in the modulation of a wide variety of cellular functions. There are seven mammalian sirtuins and, among them, the nuclear/cytoplasmic sirtuin 1 (SIRT1) and the mitochondrial sirtuin 3 (SIRT3) are ubiquitously expressed in many tissue types. Sirtuins are known to play major roles in protecting against cellular stress and in controlling metabolic pathways, which are key processes during IRI. In this review, we mainly focus on SIRT1 and SIRT3 and examine their role in modulating pathways against energy depletion during ischemia and their involvement in oxidative stress, apoptosis, microcirculatory stress and inflammation during reperfusion. We present evidence of the beneficial effects of sirtuins against IRI and emphasize the importance of developing new strategies by enhancing their action.