Carbon monoxide inhibits apoptosis during cold storage and protects kidney grafts donated after cardiac death

Ischemia/reperfusion (I/R) injury remains as a serious deleterious factor in kidney transplantation (KTx). We hypothesized that carbon monoxide (CO), an endogenous potent cytoprotective molecule, inhibits hypothermia-induced apoptosis of kidney grafts. Using the rat KTx model mimicking the conditions of donation after cardiac death (DCD) as well as nontransplantable human kidney grafts, this study examined effects of CO in preservation solution in improving the quality of marginal kidney grafts. After cardiac cessation, rat kidneys underwent 40 min warm ischemia (WI) and 24 h cold storage (CS) in control UW or UW containing CO (CO-UW). At the end of CS, kidney grafts in control UW markedly increased mitochondrial porin release into the cytosol and resulted in increased cleaved caspase-3 and PARP expression. In contrast, grafts in CO-UW had significantly reduced mitochondrial breakdown and caspase pathway activation. After KTx, recipient survival significantly improved with CO-UW with less TUNEL(+) cells and reduced mRNA upregulation for proinflammatory mediators (IL-6, TNF-α, iNOS). Furthermore, when nontransplantable human kidney grafts were stored in CO-UW for 24 h, graft PARP expression, TUNEL(+) cells, and proinflammatory mediators were less than those in control UW. CO in UW inhibited hypothermia-induced apoptosis and significantly improved kidney graft function and outcomes of KTx.     

Protective effect of carbon monoxide inhalation for cold-preserved small intestinal grafts

BACKGROUND: Heme oxygenase (HO)-1 system has been shown to provide protection against oxidative stress through the degradation of heme to biliverdin, free iron, and carbon monoxide (CO). This study investigated cytoprotective efficacy of CO at a low concentration on cold ischemia/reperfusion (I/R) injury of transplanted intestine. METHODS: Lewis rat recipients of syngenic orthotopic small intestinal transplantation with 6 hours UW cold preservation were either kept in room air (air-treated control) or exposed to CO (250 ppm) for 1 hour before and 24 hours after surgery. RESULTS: In air-treated grafts, mRNA levels for interleukin-6, intracellular adhesion molecule-1, cyclooxygenase-2, and inducible nitric oxide synthase promptly increased. Sequential histopathologic analysis of untreated grafts revealed initial rapid epithelial loss, subsequent recruitment of inflammatory infiltrates, and local hemorrhage in the lamina propria, which extended downward to the epithelial crypt and muscle layer with time. CO effectively blocked proinflammatory cascade during I/R injury, inhibited upregulation of inflammatory molecules and ameliorated intestinal tissue injuries. Beneficial effects of CO were associated with improved graft blood flow without inhibiting endogenous HO-1 activity. Recipient animal survival was significantly improved with CO to 100% versus 58% in air-treated controls. CONCLUSIONS: These results indicate a significant role for CO in protecting the intestine from cold I/R injury associating with small intestinal transplantation.     

Protection Against Ischemia/Reperfusion Injury in Cardiac and Renal Transplantation with Carbon Monoxide, Biliverdin and Both

Both carbon monoxide (CO) and biliverdin, products of heme degradation by heme oxygenase, have been shown to attenuate ischemia/reperfusion (I/R) injury. We hypothesized in this study that dual-treatment with CO and biliverdin would induce enhanced protective effects against cold I/R injury. Heterotopic heart and orthotopic kidney transplantation were performed in syngeneic Lewis rats after 24-h cold preservation in UW solution. While monotherapy with CO (20 ppm) or biliverdin (50 mg/kg, ip) did not alter the survival of heart grafts, dual-treatment increased survival to 80% from 0% in untreated recipients, with a significant decrease of myocardial injury and improved cardiac function. Similarly, dual-treatment significantly improved glomerular filtration rates of renal grafts and prolonged recipient survival compared to untreated controls. I/R injury-induced up-regulation of pro-inflammatory mediators (e.g. TNF-alpha, iNOS) and extravasation of inflammatory infiltrates were significantly less with dual-treatment than untreated controls. In addition, dual-treatment was effective in decreasing lipid peroxidation and improving graft blood flow through the distinctive action of biliverdin and CO, respectively. The study shows that the addition of byproducts of heme degradation with different mechanisms of action provides enhanced protection against transplant-associated cold I/R injury of heart and kidney grafts.     

Ex vivo Application of Carbon Monoxide in UW Solution Prevents Transplant‐Induced Renal Ischemia/Reperfusion Injury in Pigs

I/R injury is a major deleterious factor of successful kidney transplantation (KTx). Carbon monoxide (CO) is an endogenous gaseous regulatory molecule, and exogenously delivered CO in low concentrations provides potent cytoprotection. This study evaluated efficacies of CO exposure to excised kidney grafts to inhibit I/R injury in the pig KTx model. Porcine kidneys were stored for 48 h in control UW or UW supplemented with CO (CO‐UW) and autotransplanted in a 14‐day follow‐up study. In the control UW group, animal survival was 80% (4/5) with peak serum creatinine levels of 12.0 ± 5.1 mg/dL. CO‐UW showed potent protection, and peak creatinine levels were reduced to 6.9 ± 1.4 mg/dL with 100% (5/5) survival without any noticeable adverse event or abnormal COHb value. Control grafts at 14 days showed significant tubular damages, focal fibrotic changes and numerous infiltrates. The CO‐UW group showed significantly less severe histopathological changes with less TGF‐β and p‐Smad3 expression. Grafts in CO‐UW also showed significantly lower early mRNA levels for proinflammatory cytokines and less lipid peroxidation. CO in UW provides significant protection against renal I/R injury in the porcine KTx model. Ex vivo exposure of kidney grafts to CO during cold storage may therefore be a safe strategy to reduce I/R injury.     

Ex vivo carbon monoxide prevents cytochrome P450 degradation and ischemia/reperfusion injury of kidney grafts

Renal ischemia/reperfusion injury is a major complication of kidney transplantation. We tested if ex vivo delivery of carbon monoxide (CO) to the kidney would ameliorate the renal injury of cold storage that can complicate renal transplantation. Orthotopic syngeneic kidney transplantation was performed in Lewis rats following 24 h of cold preservation in University of Wisconsin solution equilibrated without or with CO (soluble CO levels about 40 microM). Ischemia/reperfusion injury in control grafts resulted in an early upregulation of inflammatory mediator mRNAs and progressive deterioration of graft function. In contrast, the grafts preserved with CO had significantly less oxidative injury and this was associated with improved recipient survival compared to the control group. Renal injury in the control group showed considerable degradation of cytochrome P450 heme proteins, active heme metabolism and increased detrimental intracellular free heme levels. Kidney grafts preserved in CO-equilibrated solution maintained their cytochrome P450 protein levels, had normal intracellular heme levels and had less lipid peroxidation. Our results show that CO-mediated suppression of injurious heme-derived redox reactions offers protection of kidney grafts from cold ischemia/reperfusion injury.     

Exogenous IL-6 Inhibits Acute Inflammatory Responses and Prevents Ischemia/Reperfusion Injury after Intestinal Transplantation

Interleukin-6 (IL-6) is a pleiotropic acute reactant cytokine involved in inflammatory responses. To explore the role of IL-6 in inflammation, this study examined the efficacy of exogenous IL-6 in preventing intestinal ischemia/reperfusion (I/R) injury associated with small bowel transplantation (SBTx). Syngenic orthotopic SBTx was performed in Lewis rats after 6-h graft preservation in University of Wisconsin (UW) at 4 degrees C. IL-6 mutein (IL-6m, 500 microg/kg), a recombinant molecular variant of human IL-6, was subcutaneously given to donors 2 h before harvesting (IL-6mD) or to excised grafts by luminal infusion (IL-6mG). Animal survival was 100% and 75% in IL-6mD (p<0.05 vs. control) and IL-6mG groups, respectively, compared with 64.3% in untreated controls. The severity of I/R injury (e.g. epithelial denudation, villous congestion) was reduced with IL-6m, in addition to a striking increase in re-epithelization. With IL-6m, neutrophil extravasation was markedly reduced in intestinal grafts and in remote organs (e.g. lung). IL-6m mediated anti-inflammatory effects through the inhibition of I/R-induced up-regulation of intragraft and circulating IL-1-beta, tumor necrosis factor-alpha (TNF-alpha) and IL-6. IL-6m also increased intestinal graft tissue blood flow. These results show that IL-6 is effective in protecting the intestine from cold I/R injury by maintaining graft blood flow and reducing pro-inflammatory cytokine up-regulation and neutrophil infiltration.     

Hydrogen Inhalation Ameliorates Oxidative Stress in Transplantation Induced Intestinal Graft Injury

Ischemia/reperfusion (I/R) injury during small intestinal transplantation (SITx) frequently causes complications including dysmotility, inflammation and organ failure. Recent evidence indicates hydrogen inhalation eliminates toxic hydroxyl radicals. Syngeneic, orthotopic SITx was performed in Lewis rats with 3 h of cold ischemic time. Both donor and recipient received perioperative air or 2% hydrogen inhalation. SITx caused a delay in gastrointestinal transit and decreased jejunal circular muscle contractile activity 24 h after surgery. Hydrogen treatment resulted in significantly improved gastrointestinal transit, as well as jejunal smooth muscle contractility in response to bethanechol. The transplant induced upregulation in the inflammatory mediators CCL2, IL‐1β, IL‐6 and TNF‐α were mitigated by hydrogen. Hydrogen significantly diminished lipid peroxidation compared to elevated tissue malondialdehyde levels in air‐treated grafts demonstrating an antioxidant effect. Histopathological mucosal erosion and increased gut permeability indicated a breakdown in posttransplant mucosal barrier function which was significantly attenuated by hydrogen treatment. In recipient lung, hydrogen treatment also resulted in a significant abatement in inflammatory mRNA induction and reduced neutrophil recruitment. Hydrogen inhalation significantly ameliorates intestinal transplant injury and prevents remote organ inflammation via its antioxidant effects. Administration of perioperative hydrogen gas may be a potent and clinically applicable therapeutic strategy for intestinal I/R injury.     

Protection against ischemia/reperfusion injury by the cavitary two-layer method in canine small intestinal transplantation with reduction of reactive oxygen species

BACKGROUND: Ischemia and reperfusion (I/R) injury is a major determinant of early graft dysfunction and long-term graft survival in small intestinal transplantation. The cavitary two-layer method (TLM) has been reported to be superior to the University of Wisconsin cold storage method (UWM) in long-term preservation of canine small intestine. This study was designed to evaluate the protective effect of the cavitary TLM against I/R injury in canine small intestinal transplantation. METHODS: Intestinal grafts harvested from beagles were allotransplanted after 24-hour preservation by UWM (group 1) or the cavitary TLM (group 2). The graft in the controls (group 3) was immediately allotransplanted without preservation. I/R injury was assessed by functional success rates, biochemical assay, graft adenosine triphosphate (ATP) and lipid peroxidation (LPO) concentrations, and histopathologic examination including TUNEL staining for apoptosis. RESULTS: In group 1, ATP recovery was delayed after reperfusion, and most recipients died with hemorrhage of the grafts and lungs. In group 2, graft ATP concentrations recovered rapidly, and I/R injury was prevented with reduced LPO production, resulting in good outcome. CONCLUSIONS: The cavitary TLM protected intestinal grafts against I/R injury evidenced by maintenance of graft ATP levels and reduction of LPO production compared with UWM in canine small intestinal transplantation.     

Hydrodynamic plasmid DNA gene therapy model in liver transplantation

BACKGROUND: There is great interest in the field of transplantation to genetically modify grafts to decrease preservation injury or allograft rejection. Although adenoviral gene transfer has been effective in experimental liver transplantation, viral toxicity and safety concerns limit potential use in clinical trials. Therefore, the purpose of this study was to develop a model of nonviral gene transfer in the liver transplant setting, allowing for efficient transgene expression. MATERIALS AND METHODS: Orthotopic syngeneic rat liver transplantation was performed with 3 h cold ischemia using University of Wisconsin (UW) preservation. A hydrodynamic gene transfer technique was developed where plasmid DNA was delivered to the liver graft by ex vivo rapid infusion of DNA in UW via the IVC with other vessels clamped. Expression plasmids for the marker genes luciferase and secreted human alpha1-antitrypsin (alpha1-AT) were used. Hepatic injury was assessed by graft histology and liver transaminases. Transgene expression was determined by hepatic luciferase relative light units activity (RLU) and serum alpha1-AT protein levels. Variables examined included the effect of (a) volume injected on the intravenous pressure in the liver graft; (b) injury to the liver, as measured by hepatic enzymes and histopathology; (c) variable expression between lobes; (d) volume of UW that the plasmid is administered in; (e) amount of DNA plasmid; (f) type of the promoter used; (g) clamp time; as well as (h) the time course of the marker gene expression. RESULTS: Control rats underwent standard orthotopic syngeneic rat liver transplantation and had no detectable hepatic luciferase activity or serum human alpha1-AT. The optimal DNA plasmid dose was found to be 400 mug/liver graft, as there was no increase in the luciferase expression by increasing the dose. Furthermore, cytomegalovirus promoter yielded greater expression than Rous sarcoma virus. A high injection pressure gradient allowed for more efficient transgene expression, but produced greater liver injury shown by elevated transaminases and centrilobular necrosis. Lowering injection volume from 75 to 50% of graft weight decreased liver injury by 4.5-fold. Although higher UW injection volumes were associated with increased expression, volumes of only 50% led to luciferase expression up to 10,000,000 RLU/mg; this expression was homogeneous between the different liver lobes. Human alpha1-AT was detected in recipient blood as early as 6 h, peaked at 24 h, and remained high for 5 days. CONCLUSIONS: We have developed a nonviral gene transfer technique where hydrostatic pressure across the cold-preserved liver vascular bed allows for efficient plasmid DNA delivery. This simple strategy should prove useful to genetically modify liver grafts in the transplantation setting.     

Fate of hepatocyte and sinusoidal lining cell function and kinetics after extended cold preservation and transplantation of the rat liver

We investigated the chronological profile of graft damage and recovery after liver cold ischemia-reperfusion (I/R) injury, with particular attention to the role of apoptosis on hepatocyte and sinusoidal endothelial cell (SEC) damage. Male Lewis rats underwent rearterialized orthotopic liver transplantation using grafts subjected to a short (University of Wisconsin [UW] solution for 1 hour [UW1h]) and prolonged period (UW16h) of cold preservation. Experiments were performed immediately after preservation and 4 hours, 24 hours, 3 days, and 7 days after reperfusion. At each time, graft function, incidence of apoptotic cells, expression of the epitope recognized by a monoclonal antibody specific to rat SECs (SE-1), and incidence of proliferating cells were estimated. In the UW16h group, the proportion of apoptotic SECs was markedly elevated at 4 hours. The incidence of hepatocyte apoptosis was very low, although massive hepatocyte necrosis was evident at 24 hours. The incidence of proliferating hepatocytes and SECs peaked at 3 days, then returned to normal by 7 days. SE-1 expression was reduced immediately after preservation, followed by a marked reduction at 4 and 24 hours after reperfusion, and expression returned to normal by 7 days. Although SEC apoptosis was induced in the early phase of cold I/R injury, hepatocyte damage developed without the occurrence of apoptosis. Regeneration of both hepatocytes and SECs after cold I/R injury peaked at 3 days and was complete by 7 days, whereas functional recovery of these cell populations was complete 3 days after reperfusion. (Liver Transpl 2002;8:370-381.)     

Addition of adenosine monophosphate-activated protein kinase activators to University of Wisconsin solution: a way of protecting rat steatotic livers.

This study investigates how the addition of trimetazidine (TMZ) and aminoimidazole-4-carboxamide ribonucleoside (AICAR) to University of Wisconsin (UW) solution protects steatotic livers. Steatotic and nonsteatotic livers were preserved for 24 hours at 4 degrees C in UW and UW with TMZ and AICAR (separately or in combination) and then perfused ex vivo for 2 hours at 37 degrees C. Adenosine monophosphate-activated protein kinase (AMPK) or nitric oxide (NO) synthesis inhibition in livers preserved in UW with TMZ was also investigated. Hepatic injury and function (transaminases, bile production, and sulfobromophthalein clearance) and factors potentially involved in the susceptibility of steatotic livers to ischemia-reperfusion (I/R), including vascular resistance, mitochondrial damage, adenosine triphosphate depletion, and oxidative stress were evaluated. AMPK, NO synthase (NOS), nitrate, and nitrite levels were also determined. The addition of TMZ and AICAR (separately or in combination) to UW reduced hepatic injury, improved functionality, and protected against the mechanisms responsible for the vulnerability of steatotic livers to I/R. Like AICAR, TMZ increased AMPK, constitutive NOS, and nitrates and nitrites, and conversely, AMPK or NO synthesis inhibition abolished the benefits of TMZ. In conclusion, TMZ, by means of AMPK, increased NO, thus protecting steatotic livers against their vulnerability to I/R injury. TMZ and AICAR may constitute new additives to UW solution in steatotic liver preservation, whereas a combination of both seems unnecessary.     

Insulin in UW solution exacerbates hepatic ischemia / reperfusion injury by energy depletion through the IRS-2 / SREBP-1c pathway.

Ischemia / reperfusion (I / R) injury is related to tissue graft energy status. Insulin, which is currently used in the University of Wisconsin (UW) preservation solution with insulin (UWI), is an anabolic hormone and was shown to exacerbate the hepatic I / R injury in our previous study. In this study, the energy status and regulation of metabolism genes by insulin were investigated in liver grafts preserved by UW solution. Insulin could significantly decrease adenosine triphosphate (ATP) level after 3 hours of preservation, as well as total adenine nucleotides (TANs) and energy charge (EC) levels. Energy regeneration deteriorated in the grafts preserved by insulin in terms of ATP and EC levels at 24 hours after transplantation. The insulin signal was transduced through the insulin receptor substrate-2 (IRS-2) pathway and the activity of IRS-2 was decreased gradually at the messenger ribonucleic acid (mRNA) level during cold preservation. Downstream targeting genes such as sterol regulatory element-binding protein-1c (SREBP-1c), glucokinase (GKC), and fatty acid synthase (FAS) genes, as well as phospho-glycogen synthase kinase-3beta (GSK-3beta) were activated and they showed the similar expression profiles during cold preservation. Lipoprotein metabolism was accelerated by insulin through upregulation of the activity of apolipoprotein C-III (Apo C-III) during cold preservation. The insulin-like growth factor-binding protein-1 pathway was inhibited during cold preservation. In conclusion, insulin in UW solution exacerbates hepatic I / R injury by energy depletion as the graft maintains its anabolic activity. The key enzyme activities of the energy-consuming process of glycogen and fatty acid synthesis as well as lipoprotein metabolism were accelerated by insulin through the IRS-2 / SREBP-1c pathway.     

Effect of hyperbarically oxygenated-perfluorochemical with University of Wisconsin solution on preservation of rat small intestine using an original pressure-resistant portable apparatus

BACKGROUND: Perfluorochemicals (PFC) are chemical substances that have a higher oxygen solubility under hyperbaric oxygen (HBO) pressure. This study investigated the effect of cold HBO-PFC/University of Wisconsin (UW) solution on preservation of rat small intestinal graft. METHODS: We manufactured an air-tight, pressure-resistant tank made of stainless steel with high thermal conductivity. Rat ileal grafts were placed in a custom-made silicon-gum bag with UW solution, which was immersed in 5 atm HBO-PFC solution in the tank (Group P-5). The tank was kept at 4 degrees C. We compared the ATP concentration and mucosal permeability in Group P-5 with grafts preserved in 1 atm oxygenated-PFC/UW solution (Group P-1) and simple cold storage in UW solution (Group C). Histologic study was also performed. RESULTS: PO(2) in UW solution after 48 h preservation were 1852 +/- 37, 499 +/- 13, and 173 +/- 3 mmHg (Group P-5, P-1 and C, respectively, mean +/- SD). At 48 h of preservation, graft ATP concentration was significantly greater in Group P-5 compared to that in Group P-1 and Group C. Mucosal hyperpermeability as well as mucosal morphologic changes were also ameliorated in Group P-5. CONCLUSION: HBO-PFC can supply a greater amount of oxygen to UW solution. Indirect measures of oxygen metabolism such as ATP content and lactate production suggested improvement in maintaining graft oxygen metabolism.     

Apoptosis and regeneration of sinusoidal endothelial cells after extended cold preservation and transplantation of rat liver

BACKGROUND: Sinusoidal endothelial cells (SECs) are particularly susceptible to cold ischemia-reperfusion (I/R) injury. We have examined the process of injury and recovery of graft after cold-preserved liver transplantation, with special focus on the proliferation of SECs and regulatory mechanisms involved. METHODS: Male SD rats were divided into two groups according to length of cold preservation time in University of Wisconsin [UW] solution of graft: UW1h group and UW12h group. Graft function, incidence of apoptosis, proliferation of SECs and the expression of related regulatory factors were assessed after orthotopic liver transplantation (OLT). RESULTS: SECs are more sensitive to apoptosis induced by cold I/R injury compared with hepatocytes. Using bromodeoxyuridine and rat endothelial cell antigen-1 double immunostaining assay, SECs exhibited a delayed proliferation in comparison with hepatocytes, reaching a peak at 72 hr in UW1h group and 96 hr in UW12h group, respectively. Vascular endothelial growth factor increased at 24 hr after reperfusion, and peaked at 72 hr in both groups. Flt-1 and flk-1 expression was found to be mainly limited to SECs, with a peak in expression occurring between 72 and 96 hr, which coincided with the peak in SEC proliferation in UW1h group. However, flt-1 was found to be reduced significantly at any time throughout the experiments in UW12h group compared to sham. CONCLUSION: The delayed recovery of rat liver after extended cold preservation and transplantation correlates with a retarded regeneration of SECs due to increased apoptosis and reduced expression of flt-1. These results suggest that SECs play an important role in cold-preserved liver transplantation.     

Luminescence-Based Assay to Screen Preservation Solutions for Optimal Ability to Maintain Viability of Rat Intestinal Grafts

Background

Segmental intestinal transplantations from living, genetically related donors provide advantages compared with those from cadaveric subjects. However, successful preservation during ischemic cold storage is critical for living donor grafts. Thus, the development of preservation solutions that maintain graft viability is essential for success. Herein we have reported application of a cell-based viability assay in multiwell plates to assess the effectiveness of various solutions to preserve intestinal grafts.

Methods

Freshly isolated intestinal chips from luciferase transgenic rats were placed in 96-well tissue culture plates for incubation at 4°C for 24 hours in various preservation solutions: ET-Kyoto (ET-K), University of Wisconsin (UW) solution, Euro-Collins (EC) solution, histidine-tryptophan-ketoglutarate (HTK) solution, lactated Ringer’s (LR) solution, or saline.

Results

As indicated by a higher level of luminescence, intestinal chips preserved in UW, HTK, or ET-K solution contained more viable cells, than those preserved in EC, LR, or saline solution. After exposure to the preservation solutions for 1 hour, the mucosal layer chips showed lower cell viability than the muscle layer chips.

Conclusion

Our data demonstrated that ET-K and UW solutions used together with intestinal chips of Luciferase transgenic rat and in vivo imaging provided optimal viability during ischemic cold storage prior to transplantation. Further development of preservation conditions to minimize the loss of viability of intestinal grafts before clinical transplantation is essential to improve outcomes.     

ET-Kyoto solution plus dibutyryl cyclic adenosine monophosphate is superior to University of Wisconsin solution in rat liver preservation

ET-Kyoto solution (ET-K) is an extracellular-type organ preservation solution containing the cytoprotective disaccharide, trehalose. A previous study reported the supplement of dibutyryl cyclic adenosine monophosphate (db-cAMP) in conventional ET-K to attenuate lung ischemia-reperfusion injury. In this study, the efficacy of this modified ET-K for liver preservation was investigated by comparison with University of Wisconsin solution (UW). ET-K was supplemented with db-cAMP (2 mmol/L). Lewis rats were randomly assigned to two groups, and liver grafts were flushed and stored at 40C for 24 h with ET-K or UW before syngeneic liver transplantation. The graft function and histological changes at 4 h posttransplant as well as 7-day survival were evaluated. Recipient rat survival rate was significantly higher in the ET-K group than in the UW group. Preservation in ET-K resulted in a significant reduction in serum parenchymal transaminase level and promotion of bile production in comparison with UW. The serum hyaluronic acid level, an indicator of sinusoidal endothelial cell injury, was significantly lower after ET-K preservation than that in UW. Histologically, at 4 h after transplantation, the liver grafts preserved in UW solution demonstrated a greater degree of injury than those in ET-K, which appeared to be apoptosis, rather than necrosis. The continuity of the sinusoidal lining was better preserved in ET-K than in UW. In conclusion, ET-K supplemented with db-cAMP is superior to UW in rat liver preservation. This modified ET-K might therefore be a novel candidate for the procurement and preservation of multiple organs.     

Preservation of pancreas in the University of Wisconsin solution supplemented with AP39 reduces reactive oxygen species production and improves islet graft function

Ischemia-reperfusion injury (IRI) results in increased rates of delayed graft function and early graft loss. It has recently been reported that hydrogen sulfide (H₂S) protects organ grafts against prolonged IRI. Here, we investigated whether the preservation of pancreas in University of Wisconsin (UW) solution supplemented with AP39, which is a mitochondrial-targeted H₂S donor, protected pancreatic islets against IRI and improved islet function. Porcine pancreata were preserved in the UW solution with AP39 (UW + AP39) or the vehicle (UW) for 18 h, followed by islet isolation. The islet yields before and after purification were significantly higher in the UW + AP39 group than in the UW group. The islets isolated from the pancreas preserved in UW + AP39 exhibited significantly decreased levels of reactive oxygen species (ROS) production and a significantly increased mitochondrial membrane potential as compared to the islets isolated from the pancreas preserved in the vehicle. We found that the pancreas preserved in UW + AP39 improved the outcome of islet transplantation in streptozotocin-induced diabetic mice. These results suggest that the preservation of pancreas in UW + AP39 protects the islet grafts against IRI and could thus serve as a novel clinical strategy for improving islet transplantation outcomes.     

Relationship between ischemia/reperfusion injury and the stimulus of fibrogenesis in an experimental model: comparison among different preservation solutions

Background and Aims

Orthotopic liver transplantation (OLT) has been the standard treatment for end-stage acute and chronic liver disease. Ischemia-reperfusion (I/R) injury is one of the major causes of poor graft function early after OLT, and adversely influencing graft and patient survivals. It is unknown whether I/R injury influences liver fibrogenesis.

Materials and Methods

Livers from 25 adult male Wistar rats were randomly assigned into 5 experimental groups according to the preservation solution: saline solution (SS); University of Wisconsin (UW) solution; Fructose 1, 6-biphosphate (FBP); S-Nitroso-N-Acetylcysteine (SNAC): or UW + SNAC (SNAC+UW). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactic dehydrogenase (LDH) were determined in preservation solution samples at 2, 4, and 6 hours. After 6 hours of cold ischemia, ex situ reperfusion was applied to the liver for 15 minutes. Serum AST, ALT, LDH, and renin levels were determined. Fresh liver slices were processed for histological studies, determination of thiobarbituric acid reactive substances, catalase, and glutathione, and expression of TGF-β1 and angiotensin II AT1 receptor.

Results

AST was significantly lower during cold storage with UW than with the older media (P = .001); ALT was lower in the FBP group (P = .023) and LDH was lower in the FBP and SNAC groups (P = .007). After reperfusion, serum AST, ALT, LDH, and TBARS showed no significant differences among the groups. Catalase was significantly lower in the SS and FBP groups (P = .008 and P = .006, respectively). Compared with UW, glutathione concentrations were significantly higher in SS, FBP, and SNAC 200 (P = .004). Renin levels were significantly lower in the FBP group (P = .022). No histological signs of preservation injury were observed in the hepatic sample. No expressions were detected of TGF-β1 or AT1 receptor.

Conclusion

In this experimental model of early reperfusion injury, preservation changes related to higher levels of renin, which suggest its role in fibrogenesis. FBP was associated with lower renin levels than other solutions including UW.     

Efficacy and safety of Celsior preservation fluid in liver transplantation: one-year follow up of a prospective, multicenter, non-randomized study

The purpose of this prospective, nine-center, non-randomized study was to assess the efficacy and safety of Celsior preservation fluid in liver transplantation using unselected donors. As data comparing allograft outcomes following liver transplantation using Celsior and University of Wisconsin (UW) preservation fluids are limited, we also compared our cohort with matched controls selected from the European Liver Transplant Registry (ELTR) who received total liver grafts preserved with UW solution during the same period. One hundred and forty patients who received livers preserved with Celsior were included. The primary endpoint, graft loss at one-yr post-transplantation, was observed in 24 patients (17.1%) which was not significantly different from the 20.0% pre-defined threshold rate (95% confidence interval [CI] 10.9, 23.4; p=0.398). Predictive factors for graft loss on univariate analysis were moderate-to-severe steatosis on the donor graft (5/22 patients with graft loss vs. 8/107 patients without, p=0.046) and duration of warm ischemia (1.4±1.1 h in patients with graft loss vs. 0.9±0.5 h in patients without, p=0.034). Hepatic artery thrombosis and stenosis occurred in seven (5.0%) and six (4.3%) patients, respectively. The comparison of our patients to 420 ELTR controls showed that one-yr graft survival rates (Celsior: 82.9%, 95% CI 75.8, 88.2; UW: 78.6%, 95% CI 74.4, 82.2) and Kaplan-Meier one-yr graft survival distributions (p=0.285) were similar. Within the cold ischemia time achieved in our study, liver preservation with Celsior appeared efficient and safe. Comparison with ELTR patients suggested that liver allograft survival was similar using Celsior or UW solution for preservation of unselected donor grafts.