Cold Ischemia-reperfusion Injury Research Articles

GSK3β and VDAC Involvement in ER Stress and Apoptosis Modulation during Orthotopic Liver Transplantation

We investigated the involvement of glycogen synthase kinase-3β (GSK3β) and the voltage-dependent anion channel (VDAC) in livers subjected to cold ischemia–reperfusion injury (I/R) associated with orthotopic liver transplantation (OLT). Rat livers were preserved in University of Wisconsin (UW) and Institute Georges Lopez (IGL-1) solution, the latter enriched or not with trimetazidine, and then subjected to OLT. Transaminase (ALT) and HMGB1 protein levels, glutamate dehydrogenase (GLDH), and oxidative stress (MDA) were measured. The AKT protein kinase and its direct substrates, GSK3β and VDAC, as well as caspases 3, 9, and cytochrome C and reticulum endoplasmic stress-related proteins (GRP78, pPERK, ATF4, and CHOP), were determined by Western blot. IGL-1+TMZ significantly reduced liver injury. We also observed a significant phosphorylation of AKT, which in turn induced the phosphorylation and inhibition of GSK3β. In addition, TMZ protected the mitochondria since, in comparison with IGL-1 alone, we found reductions in VDAC phosphorylation, apoptosis, and GLDH release. All these results were correlated with decreased ER stress. Addition of TMZ to IGL-1 solution increased the tolerance of the liver graft to I/R injury through inhibition of GSK3β and VDAC, contributing to ER stress reduction and cell death prevention.

Nitrite enhances liver graft protection against cold ischemia reperfusion injury through a NOS independent pathway

ABSTRACTIntroduction: Nitrite has been found to protect liver graft from cold preservation injury. However, the cell signaling pathway involved in this protection remains unclear. Here, we attempt to clarify if the NOS pathway by using the NOS inhibitor, L-NAME (L-NG-Nitroarginine methyl ester).Animals and methods: Rat livers were conserved for 24 h at 4°C in (IGL-1) solution enriched or not with nitrite at 50 nM. In a third group, rats were pretreated with 50 mg/kg of L-NAME before their liver procurement and preservation in IGL-1 supplemented with nitrite (50 nM) and L-NAME (1 mM). After 24 h of cold storage, rat livers were ex-vivo perfused at 37°C during 2 h. Control livers were perfused without cold storage.Results: Nitrite effectively protected the rat liver grafts from the onset of cold I/R injury. L-NAME treatment did not abolish the beneficial effects of nitrite. Liver damage, protein oxidation and lipid peroxidation remained at low levels in both nitrite-treated groups when compared to IGL-1 group. Antioxidant enzyme activities and functional parameters were unchanged after NOS inhibition.Conclusion: Despite NOS inhibition by L-NAME, nitrite can still provide hepatic protection during cold I/R preservation. This suggests that nitrite acts through a NOS-independent pathway.

A porcine ex vivo lung perfusion model with maximal argon exposure to attenuate ischemia-reperfusion injury.

Argon (Ar) is a noble gas with known organoprotective effects in rodents and in vitro models. In a previous study we failed to find a postconditioning effect of Ar during ex vivo lung perfusion (EVLP) on warm-ischemic injury in a porcine model. In this study, we further investigated a prolonged exposure to Ar to decrease cold ischemia-reperfusion injury after lung transplantation in a porcine model with EVLP assessment. Domestic pigs (n = 6/group) were pre-conditioned for 6 hours with 21% O2 and 79% N2 (CONTR) or 79% Ar (ARG). Subsequently, lungs were cold flushed and stored inflated on ice for 18 hours inflated with the same gas mixtures. Next, lungs were perfused for 4 hours on EVLP (acellular) while ventilated with 12% O2 and 88% N2 (CONTR group) or 88% Ar (ARG group). The perfusate was saturated with the same gas mixture but with the addition of CO2 to an end-tidal CO2 of 35-45 mmHg. The saturated perfusate was drained and lungs were perfused with whole blood for an additional 2 hours on EVLP. Evaluation at the end of EVLP did not show significant effects on physiologic parameters by prolonged exposure to Ar. Also wet-to-dry weight ratio did not improve in the ARG group. Although in other organ systems protective effects of Ar have been shown, we did not detect beneficial effects of a high concentration of Ar on cold pulmonary ischemia-reperfusion injury in a porcine lung model after prolonged exposure to Ar in this porcine model with EVLP assessment.

Relevance of Endoplasmic Reticulum Stress Cell Signaling in Liver Cold Ischemia Reperfusion Injury.

The endoplasmic reticulum (ER) is involved in calcium homeostasis, protein folding and lipid biosynthesis. Perturbations in its normal functions lead to a condition called endoplasmic reticulum stress (ERS). This can be triggered by many physiopathological conditions such as alcoholic steatohepatitis, insulin resistance or ischemia-reperfusion injury. The cell reacts to ERS by initiating a defensive process known as the unfolded protein response (UPR), which comprises cellular mechanisms for adaptation and the safeguarding of cell survival or, in cases of excessively severe stress, for the initiation of the cell death program. Recent experimental data suggest the involvement of ERS in ischemia/reperfusion injury (IRI) of the liver graft, which has been considered as one of major problems influencing outcome after liver transplantation. The purpose of this review is to summarize updated data on the molecular mechanisms of ERS/UPR and the consequences of this pathology, focusing specifically on solid organ preservation and liver transplantation models. We will also discuss the potential role of ERS, beyond the simple adaptive response and the regulation of cell death, in the modification of cell functional properties and phenotypic changes.

Hypertonic saline solution preconditioning decreases oxidative stress and tissue lesions in warm and cold liver ischemia reperfusion injury

Hypertonic saline solution preconditioning decreases oxidative stress and tissue lesions in warm and cold liver ischemia reperfusion injury

Bioregulators of stem and progenitor cells in preservation solution reduce cold ischemia-reperfusion injury of isolated rat livers.

The effects of bioregulators of stem and progenitor cells (BSPC) of fetal tissue cytosol on rat liver during 24h hypothermic storage (HS) and following normothermic reperfusion (NR) were investigated. It was shown that BSPC presence in the preservation medium stabilized pro-oxidant-antioxidant balance impaired in liver after HS and NR, prevented the uncoupling of mitochondrial oxidative phosphorylation and ATP level decline. These consequences led to significant improvement of hepatic morphological integrity and functional state. Powerful protective effect of BSPC on liver at sub-zero temperatures can serve as basis for new approaches to extend safe time for organ preservation and foster understanding of pathways of stem and progenitor cell paracrine action. © 2016 BioFactors, 42(3):287-295, 2016.

The Protective Effects of Trypsin Inhibitor on Hepatic Ischemia-Reperfusion Injury and Liver Graft Survival.

The aim of this study was to explore the protective effects of ulinastatin (urinary trypsin inhibitor, UTI) on liver ischemia-reperfusion injury (IRI) and graft survival. We employed mouse liver cold IRI and orthotopic liver transplantation (OLTx) models. UTI was added to lactated Ringer's (LR) solution for liver perfusion and preservation in vitro or combined with UTI injection intraperitoneally to the liver graft recipient. Our results indicated that UTI supplementation protected the liver from cold IRI in a dose-dependent manner and prolonged liver graft survival from extended cold preserved liver donors significantly. The underlying mechanism of UTI on liver IRI may be mediated by inhibition of proinflammatory cytokine release, increasing the expression of the antiapoptotic gene Bcl-2 and decreasing the expression of the proapoptosis genes of Caspase-3 and Bax, and further protects hepatocytes from apoptotic death and improves liver function.

Donor pretreatment with adenosine monophosphate-activated protein kinase activator protects cardiac grafts from cold ischaemia/reperfusion injury.

Adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of energy metabolism and has been shown to be protective in ischaemia/reperfusion injury (IRI). We hypothesized that preactivation of AMPK with an activator before donor heart procurement could protect heart grafts from cold IRI. Donor Sprague-Dawley rats were injected intravenously with AMPK activator 5-amino-imidazole-4-carboxamide ribonucleotide (AICAR) or vehicle 30 min before heart procurement. Heart grafts were then preserved in histidine-tryptophan-ketoglutarate (HTK) solution at 4°C for 8 h. After preservation, grafts were immediately mounted on the Langendorff perfusion system and perfused with Krebs-Henseleit buffer at 37°C for 1 h. Adenosine triphosphate (ATP) and malondialdehyde (MDA) content in graft tissue were quantified post-preservation and post-reperfusion. After reperfusion, isolated heart function was assessed using a pressure transducer; cumulative release of creatine kinase (CK) and lactate dehydrogenase (LDH) into the perfusate was measured to assess cardiomyocyte necrosis; ultrastructural changes in the mitochondria of the grafts were examined using transmission electron microscopy (TEM). After preservation, myocardial ATP content in the pretreated hearts was significantly higher than in the control hearts (3.247 ± 0.3034 vs 1.817 ± 0.2533 µmol/g protein; P < 0.05). AICAR-pretreated heart grafts exhibited significantly higher coronary flow (9.667 ± 0.3159 vs 8.033 ± 0.2459 ml/min; P < 0.05) and left ventricular developing pressure (58.67 ± 2.894 vs 42.67 ± 3.333 mmHg; P < 0.05) than the vehicle treated after reperfusion. Cumulative release of CK (300.0 ± 25.30 vs 431.7 ± 42.39 U/l; P < 0.05) and LDH (228.0 ± 16.68 vs 366.8 ± 57.41 U/l; P < 0.05) in the perfusate was significantly lower in the AICAR-pretreated group than that in the control group. Myocardial MDA content was also reduced in the pretreated group (0.5167 ± 0.1046 vs 0.9333 ± 0.1333 nmol/mg protein; P < 0.05). TEM suggested that the mitochondrial structure of AICAR-pretreated hearts was much better preserved. Moreover, AICAR-pretreated hearts significantly diminished cytosolic cytochrome c release after reperfusion. This study demonstrates that pretreatment with AMPK activator AICAR significantly protects heart grafts from extended cold IRI. This novel protocol may be useful and feasible in clinical heart transplantation.

Acetazolamide protects steatotic liver grafts against cold ischemia reperfusion injury.

Ischemia reperfusion injury (IRI) is a primary concern in liver transplantation, especially when steatosis is present. Acetazolamide (AZ), a specific carbonic anhydrase (CA) inhibitor, has been suggested to protect against hypoxia. Here, we hypothesized that AZ administration could be efficient to protect fatty livers against cold IRI. Obese Zucker rat livers were preserved in Institut Georges Lopez-1 storage solution for 24 hours at 4°C and ex vivo perfused for 2 hours at 37°C. Alternatively, rats were also treated with intravenous injection of AZ (30 mg/kg) before liver recovery. Liver injury, hepatic function, and vascular resistance were determined. CA II protein levels and CA hydratase activity were assessed as well as other parameters involved in IRI (endothelial nitric oxide synthase, mitogen activated protein kinase family, hypoxic inducible factor 1 alpha, and erythropoietin). We demonstrated that AZ administration efficiently protects the steatotic liver against cold IRI. AZ protection was associated with better function, decreased vascular resistance, and activation of endothelial nitric oxide synthase. This was consistent with an effective mitogen activated protein kinase inactivation. Finally, no effect on the hypoxic inductible factor 1 alpha/erythropoietin pathway was observed. The present study demonstrated that AZ administration is a suitable pharmacological strategy for preserving fatty liver grafts against cold IRI.

Hydrogen Sulfide Treatment Mitigates Renal Allograft Ischemia-Reperfusion Injury during Cold Storage and Improves Early Transplant Kidney Function and Survival Following Allogeneic Renal Transplantation

Ischemia-reperfusion injury is unavoidable during organ transplantation. Prolonged ischemia-reperfusion injury is detrimental to short-term and long-term graft function and survival. H2S is a recently characterized, endogenously produced gaseous molecule with important physiological roles that has been shown to be cytoprotective during tissue ischemia-reperfusion injury. The current study aimed to determine whether H2S could mitigate cold renal ischemia-reperfusion injury in the clinically relevant context of allogeneic renal transplantation. Following bilateral native nephrectomy Lewis rats underwent renal transplantation with kidneys from Brown Norway donor rats that were flushed with cold (4C) standard University of Wisconsin preservation solution (University of Wisconsin preservation solution group) or cold University of Wisconsin preservation solution plus 150 μM NaHS (H2S group) solution. Kidneys were stored for 6 hours at 4C in the same solution. Recipient animals were monitored for 14 days or until sacrifice using metabolic cages to assess various parameters of renal graft function. H2S treatment improved early allograft survival and function, and decreased early levels of necrosis, apoptosis and Kim-1 compared to University of Wisconsin preservation solution alone. H2S treatment did not affect allograft rejection. Rather, it modulated the early allograft transcriptome to decrease the expression of renal injury, coagulation and cellular stress response genes, and increase the expression of cellular proliferation and Ifn-γ induced genes compared to University of Wisconsin preservation solution alone. To our knowledge our findings are the first to show that H2S protects donor kidneys against cold ischemia-reperfusion injury in the context of allogeneic renal transplantation. This potentially represents a novel cost-effective therapeutic solution to mitigate ischemia-reperfusion injury and improve the clinical outcomes of renal transplantation.

Carbonic Anhydrase Protects Fatty Liver Grafts against Ischemic Reperfusion Damage.

Carbonic anhydrases (CAs) are ubiquitous metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and a proton. CAs are involved in numerous physiological and pathological processes, including acid-base homeostasis, electrolyte balance, oxygen delivery to tissues and nitric oxide generation. Given that these processes are found to be dysregulated during ischemia reperfusion injury (IRI), and taking into account the high vulnerability of steatotic livers to preservation injury, we hypothesized a new role for CA as a pharmacological agent able to protect against ischemic damage. Two different aspects of the role of CA II in fatty liver grafts preservation were evaluated: 1) the effect of its addition to Institut Georges Lopez (IGL-1) storage solution after cold ischemia; 2) and after 24h of cold storage followed by two hours of normothermic ex-vivo perfusion. In all cases, liver injury, CA II protein concentration, CA II mRNA levels and CA II activity were determined. In case of the ex-vivo perfusion, we further assessed liver function (bile production, bromosulfophthalein clearance) and Western blot analysis of phosphorylated adenosine monophosphate activated protein kinase (AMPK), mitogen activated protein kinases family (MAPKs) and endoplasmic reticulum stress (ERS) parameters (GRP78, PERK, IRE, eIF2α and ATF6). We found that CA II was downregulated after cold ischemia. The addition of bovine CA II to IGL-1 preservation solution efficiently protected steatotic liver against cold IRI. In the case of reperfusion, CA II protection was associated with better function, AMPK activation and the prevention of ERS and MAPKs activation. Interestingly, CA II supplementation was not associated with enhanced CO2 hydration. The results suggest that CA II modulation may be a promising target for fatty liver graft preservation.

TiProtec preserves endothelial function in a rat model

BackgroundCoronary artery bypass surgery provides excellent patency rates; however, the early and/or late graft failure reduces the long-term benefit of myocardial revascularization. We investigated the effectiveness of generally used saline, Custodiol solutions and a new solution (TiProtec) at preserving endothelium after cold ischemia and warm reperfusion injury. Materials and methodsAortic transplantations were performed in Lewis rats. Aortic arches were stored in saline, Custodiol, and TiProtec solutions for 2 h then were transplanted into the abdominal aorta. Two, 24 hours and 1 week after transplantation, the implanted grafts were harvested. Endothelium-dependent and -independent vasorelaxations were investigated in organ bath. DNA strand breaks were assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-method, messenger RNA expressions by quantitative real-time polymerase chain reaction, and the expression of CD-31 and alpha smooth muscle actin by immunochemistry. ResultsSeverely impaired endothelial function and integrity of implanted aortic grafts were shown after 2 h in the saline, Custodiol group (maximal vasorelaxation to acetylcholine: control: 91 ± 2%, saline: 26 ± 5%, Custodiol: 24 ± 5%, CD-31-positive area control: 96 ± 2%, saline: 35 ± 13% Custodiol: 54 ± 5%, P < 0.05, respectively); however, a preserved endothelial function was observed in the TiProtec group when compared with the saline and Custodiol group (maximal vasorelaxation: 46 ± 7%, CD-31-positive area: 54 ± 10%, P < 0.05). After 1 wk, endothelial function was partially recovered in all groups; however, it was significantly better in the TiProtec group (maximal vasorelaxation to acetylcholine: saline: 42 ± 3%, Custodiol: 48 ± 3%, TiProtec: 56 ± 3%, CD-31-positive area: saline: 56 ± 5%, Custodiol: 54 ± 4%; TiProtec: 83 ± 6%, P < 0.05, respectively). In addition, messenger RNA levels of Bax, B-cell lymphoma-2, endothelial NOS, vascular endothelial growth factor 2, and caspase-3 were significantly altered in both groups. ConclusionsTiProtec appears to be superior for the preservation of endothelial- and smooth muscle cells of bypass graft after cold storage and warm reperfusion in our murine model.

ATF3-Mediated NRF2/HO-1 Signaling Regulates TLR4 Innate Immune Responses in Mouse Liver Ischemia/Reperfusion Injury

Activating transcription factor 3 (ATF3) is a stress-induced transcription factor that has been shown to repress inflammatory gene expression in multiple cell types and diseases. However, little is known about the roles and mechanisms of ATF3 in liver ischemia/reperfusion injury (IRI). In warm and cold liver IRI models, we showed that ATF3 deficiency significantly increased ischemia/reperfusion (IR)-stressed liver injury, as evidenced by increased serum alanine aminotransferase levels, histological liver damage, and hepatocellular apoptosis. These may correlate with inhibition of the intrahepatic nuclear factor erythroid-derived 2-related factor 2/heme oxygenase-1 (NRF2/HO-1) signaling pathway leading to enhancing Toll-like receptor 4/nuclear factor kappa beta (TLR4/NF-κB) activation, pro-inflammatory programs and macrophage/neutrophil trafficking, while simultaneously repressing anti-apoptotic molecules in ischemic liver. Interestingly, activation of NRF2/HO-1 signaling using an NRF2 activator, oltipraz (M2), during hepatic IRI-rescued ATF3 anti-inflammatory functions in ATF3-deficient mice. For in vitro studies, ATF3 ablation in lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages (BMMs) depressed levels of NRF2/HO-1 and PI3K/AKT, resulting in enhanced TLR4/NF-κB activation. Pretreatment of LPS-stimulated BMMs with M2 increased NRF2/HO-1 expression, promoted PI3K/AKT, which in turn suppressed TLR4/NF-κB-mediated proinflammatory mediators. Thus, our results first demonstrate ATF3-mediated NRF2/HO-1 signaling in the regulation of TLR4-driven inflammatory responses in IR-stressed livers. Our findings provide a rationale for a novel therapeutic strategy for managing IR-induced liver injury.

Protective Effect of Intravenous High Molecular Weight Polyethylene Glycol on Fatty Liver Preservation.

Ischemia reperfusion injury (IRI) leads to significant tissue damage in liver surgery. Polyethylene glycols (PEGs) are water soluble nontoxic polymers that have proved their effectiveness against IRI. The objective of our study was to investigate the potential protective effects of intravenous administration of a high molecular weight PEG of 35 kDa (PEG 35) in steatotic livers subjected to cold ischemia reperfusion. In this study, we used isolated perfused rat liver model to assess the effects of PEG 35 intravenous administration after prolonged cold ischemia (24 h, 4°C) and after reperfusion (2 h, 37°C). Liver injury was measured by transaminases levels and mitochondrial damage was determined by confocal microscopy assessing mitochondrial polarization (after cold storage) and by measuring glutamate dehydrogenase activity (after reperfusion). Also, cell signaling pathways involved in the physiopathology of IRI were assessed by western blot technique. Our results show that intravenous administration of PEG 35 at 10 mg/kg ameliorated liver injury and protected the mitochondria. Moreover, PEG 35 administration induced a significant phosphorylation of prosurvival protein kinase B (Akt) and activation of cytoprotective factors e-NOS and AMPK. In conclusion, intravenous PEG 35 efficiently protects steatotic livers exposed to cold IRI.

Isoflurane does not protect from brain death-associated aggravation of cold hepatic ischemia-reperfusion injury.

Previous studies have shown that brain death aggravates cold ischemia-reperfusion injury in liver transplantation. Isoflurane, a volatile anesthetic, has been indicated to reduce warm hepatic ischemia-reperfusion injury. Herein, we studied in Sprague-Dawley rats whether isoflurane is capable of ameliorating brain death-associated aggravation of cold hepatic ischemia-reperfusion injury. Brain-dead animals were treated for 30 min with isoflurane (MAC 1.5%; n=8). Animals without isoflurane treatment served as controls (n=8). Another 13 animals without induction of brain death served as sham controls. After a 4-h period portal venous blood perfusion, hepatic microcirculation and bile flow were determined. Livers were recovered and stored for 24 h in 4°C cold HTK solution, followed by reperfusion with 37°C Krebs-Henseleit-buffer for 60 min. Liver enzymes in the effluent and bile flow were analyzed. Hepatocellular morphology was determined by histology and immunohistochemistry. Brain death reduced portal venous blood perfusion and bile flow, induced heme oxygenase-1 (HO-1), and resulted in hepatocellular damage. Isoflurane treatment did not prevent the reduction of portal venous blood perfusion or bile flow or the induction of HO-1. Accordingly, isoflurane was not capable of reducing the hepatocellular injury. Isoflurane does not protect from brain death-associated aggravation of cold hepatic ischemia-reperfusion injury.

Infusion of mesenchymal stem cells protects lung transplants from cold ischemia-reperfusion injury in mice.

Cold ischemia-reperfusion injury (IRI) is a major cause of graft failure in lung transplantation. Despite therapeutic benefits of mesenchymal stem cells (MSCs) in attenuating acute lung injury, their protection of lung transplants from cold IRI remains elusive. The present study was to test the efficacy of MSCs in the prevention of cold IRI using a novel murine model of orthotopic lung transplantation. Donor lungs from C57BL/6 mice were exposed to 6 h of cold ischemia before transplanted to syngeneic recipients. MSCs were isolated from the bone marrows of C57BL/6 mice for recipient treatment. Gas exchange was determined by the measurement of blood oxygenation, and lung injury and inflammation were assessed by histological analyses. Intravenously delivered MSC migration/trafficking to the lung grafts occurred within 4-hours post-transplantation. As compared to untreated controls, the graft arterial blood oxygenation (PaO2/FiO2) capacity was significantly improved in MSC-treated recipients as early as 4 h post-reperfusion and such improvement continued over time. By 72 h, oxygenation reached normal level that was not seen in controls. MSCs treatment conferred significant protection of the grafts from cold IRI and cell apoptosis, which is correlated with less cellular infiltration, a decrease in proinflammatory cytokines (TNF-α, IL-6) and toll-like receptor 4, and an increase in anti-inflammatory TSG-6 generation. MSCs provide significant protection against cold IRI in lung transplants, and thus may be a promising strategy to improve outcomes after lung transplantation.

CD39 Is Critical for Inducing Tolerance in Murine Allogeneic Liver Transplantation.

Background: ATP is abundant in the cell cytosol and important for cell metabolism. Once cells die or are injured, ATP is released into the extracellular space and activates immune cells as a DAMP (damage-associated molecule pattern). CD39 regulates immune activation by hydrolysis of extracellular (e)ATP. We have found that conventional mouse liver myeloid(m) DC express comparatively high levels of surface CD39 and that this contributes to protection of the liver from cold ischemia reperfusion injury after syngeneic liver transplantation (LTx). We hypothesized that CD39 might play an important role in “spontaneous” transplant tolerance after allogeneic mouse LTx. METHODS: 10-12 week-old male B6 WT or CD39 KO (B6 background) livers were transplanted orthotopically into normal 10-12 week-old male C3H mice. Graft rejection was assessed by host survival and histology and also by serum ALT on day 4 after LTx. Incidences of regulatory T cells (Treg) and IFN-γ+ CD8+ T cells in the graft were quantified by flow cytometry at day 4 post LTx. Anti-donor T cell proliferative responses were evaluated by CFSE-MLR. RESULTS: Whereas all the WT B6 liver transplanted C3H mice survived more than 100 days, animals that received CD39KO livers all died within 40 days (MST: WT: >100d vs CD39 KO 8d; p<0.01). Serum ALT at day 4 post LTx was significantly higher in mice given CD39KO livers than in those with WT livers (WT: 480±226 IU/l vs CD39KO 860±484IU/l; p<0.05). The frequency of Treg in liver graft non-parenchymal cells after LTx was significantly lower in CD39KO livers than in WT livers (WT: 3.4±0.5% vs CD39KO 2.6±0.2%; p<0.05). Moreover IFN-γ production by CD8+ liver T cells was significantly higher in CD39KO allografts than in WT allografts (WT: 38.8±1.7% vs CD39KO 46.6±2.0%; p<0.05). Splenic T cells from CD39KO liver transplant recipients exhibited greater proliferation against donor splenocytes than those from WT liver allografted mice (WT: 27.2±5.8% vs CD39KO 36.3±5.7%; p<0.05). CONCLUSION: CD39 deficiency in liver grafts induces stronger anti-donor T cell responses and breaks “spontaneous” tolerance after mouse liver Tx. CD39 may be an important molecule for promoting liver allograft tolerance.

New Insights in Fatty Liver Preservation: A Role for Carbonic Anhydrase II.

Purpose: During liver graft preservation, the switch to anaerobic metabolism provokes accumulation of lactic acid and leads to lower pH. Studies on the relevance of acidic pH are controversial. It can either activate proteases and induce cell death or protect cell against apoptosis. Carbonic anhydrases (CA) are ubiquitous metalloenzymes involved in many physiological and pathological processes, including pH and CO[/sub]2[/sub] homeostasis. However, the role of CA in liver graft preservation has not been investigated. The aim of this work was to evaluate the effects of CAII by its addition to the IGL-1 preservation solution or when it was inhibited by acetazolamide (AZ). A close CAII relationship with well known protective factors (AKT, MAP kinases) and apoptosis prevention was established Experimental: Steatotic livers from obese male Zucker rats (9 weeks aged) were classified as follows: Group1: Livers preserved for 24h (4°C) in IGL-1 solution and then subjected to “ex vivo” perfusion (2h at 37°C); Group2: same as Group1 but IGL-1 supplemented with CAII (10 μg/ml) and Group 3: Same as Group 1 but livers were pretreated with AZ (i.v. 30mg/Kg) before liver preservation. Liver injury (transaminases) and function (bile production) were measured. Akt, GSK3β, Caspase 3, Caspase 9 and MAPKs (p38, ERK and JNK) were also determined by western blot techniques. Results and Discussion: Fatty livers preserved in IGL-1+CAII showed lower injury and better function when compared to IGL-1 alone. IGL-1+CAII solution induced a significant phosphorylation of Akt and significant decreases in MAPKs (ERK, p38 and JNK) after 2h-reperfusion. This was consistent with a major decrease of liver apoptosis parameters (caspase 3, caspase 9, and GSK3β). Surprisingly, the AZ pretreatment protected efficiently steatotic liver grafts, as revealed by lower transaminases, higher bile production and decreases in MAPK levels. AZ showed differential protective mechanism of liver as it fails to activate Akt. Also, it reduced vascular resistance which was not observed with CAII addition. Conclusion: Data reported here, demonstrated that CAII is a potential pharmacological target for preserving liver grafts against cold ischemia reperfusion injury.

A Novel Preservation Solution Prolonged Cold Storage in Mouse Cardiac Grafts.

Background: We developed a novel non-potassium preservation solution contains L-cysteine, glycine, ascorbic acid/ascorbic acid-2-phosphate magnesium to prolonged mouse cardiac grafts cold storage. Methods: B6 mice underwent heterotopic heart transplantation and the animals were derived into 3 groups: recipients with non-preserved grafts (control group), recipients with grafts preserved in HTK solution for 24 and 48 hours (HTK-group), and recipients with grafts preserved in the new solution, called Solution II for 24 and 48 hours (S-II group). Recipient serum and grafts were harvest for assay. Results: In both 24hr- and 48hr-preservation series (24h- and 48h-series), time for restoration of heartbeat after reperfusion in S-II group was significantly shorter than in HTK group (HTK-24h: 306.2±39.6sec, S-II-24h: 110.5±20.3sec, p<0.01; HTK-48h: 1537.5±454.7sec, S-II-48h: 640±286sec, p<0.01). In 48h-series, Release of serum LDH, CK in S-II-group was significantly lower to compare with HTK group. The graft survival of HTK-48h group is 0 day (n=9), but S-II group is 9d, 17d, >100d×2. Graft ATP level of S-II-48h was significantly higher than HTK-48h. In both 24 and 48h-series, S-II significantly inhibited graft apoptosis. Moreover, immunohistochemistry staining revealed that the CD68-positive macrophage infiltration in S-II group was significantly lower compared with HTK group. In 24h-series, graft 8-OHdG positive cells (%) were significantly lower in S-II compared with HTK. Consistent with histologic findings, serum 8-OHdG levels were significantly inhibited in S-II group. Furthermore, the mRNA expression of the IL-6, HO-1, iNOS, CD11b in S-II-48h group, were significantly lower, wherever Nrf2, NF-κB, Arg-1, HIF-1α, TGF-β were significantly higher than HTK-48h group (p<0.01). Conclusion: Hence our data indicated that the novel preservation solution impairs graft macrophage infiltration, inhibits inflammatory cytokines and myocardial apoptosis which results in attenuating mouse cardiac grafts cold ischemia-reperfusion injury and prolonging cold storage.

S8-3 Hydrogen sulphide: The magic bullet in organ transplantation?

Ischemia reperfusion injury (IRI) is unavoidable in renal transplantation and is associated with increased rates of acute tubular necrosis, delayed graft function and impaired early and long-term graft function and survival. Gasotransmitters, particularly H2S, may be cytoprotective in models of IRI in various tissues including the kidney. We aimed to investigate the utility of H2S in mitigating renal IRI during transplantation, thereby improving subsequent renal graft function and survival compared to current standard clinical practices. Our two main objectives were to characterize the real-time protective properties of supplemental H2S against prolonged periods of warm renal IRI as well as determining its protective role in organ preservation during extremes of cold storage and transplantation in murine models. Our results demonstrated, for the first time, that supplemental H2S treatment during prolonged cold ischemia associated with transplantation dramatically improved renal graft survival and function and mitigated graft injury and inflammation compared to grafts that were preserved in standard University of Wisconsin organ preservation solution. In addition, we found that H2S treatment improved real-time renal vascular flow patterns as well as function and decreased the renal injury and inflammation associated with prolonged warm renal IRI. Overall, exogenous H2S may protect kidneys from both prolonged warm and cold IRI associated with renal transplantation. Considering that more transplant centers are utilizing grafts near the limits of cold and warm ischemic times to accommodate their expanding waiting lists, H2S treatment may represent a novel, potent and cost-effective solution to mitigate transplantation induced IRI.