Preservation Injury Research Articles

Chronic renoprotective effect of pulsatile perfusion machine RM3 and IGL-1 solution in a preclinical kidney transplantation model

BackgroundMachine perfusion (MP) of kidney graft provides benefits against preservation injury, however decreased graft quality requires optimization of the method. We examined the chronic benefits of MP on kidney grafts and the potential improvements provided by IGL-1 solution.MethodWe used an established autotransplantation pig kidney model to study the effects of MP against the deleterious effects of warm ischemia (WI: 60 minutes) followed by 22 hours of cold ischemia in MP or static cold storage (CS) followed by autotransplantation. MPS and IGL-1 solutions were compared.ResultsAnimal survival was higher in MPS-MP and both IGL groups. Creatinine measurement did not discriminate between the groups, however MPS-MP and both IGL groups showed decreased proteinuria. Chronic fibrosis level was equivalent between the groups. RTqPCR and immunohistofluorescent evaluation showed that MP and IGL-1 provided some protection against epithelial to mesenchymal transition and chronic lesions. IGL-1 was protective with both MP and CS, particularly against chronic inflammation, with only small differences between the groups.ConclusionIGL-1 used in either machine or static preservation offers similar levels of protection than standard MP. The compatibility of IGL-1 with both machine perfusion and static storage could represent an advantage for clinical teams when choosing the correct solution to use for multi-organ collection. The path towards improving machine perfusion, and organ quality, may involve the optimization of the solution and the correct use of colloids.

56. Evaluation of a new solution for hypothermic machine perfusion (HMP) of the liver. II – A study in the perfused rat liver in vitro

The present study was undertaken to develop and investigate the possible benefit of a new solution for hypothermic machine perfusion (HMP) of livers for transplants. The portal vein and bile duct of Wistar rats were cannulated, the liver excised and connected on a recirculating perfusion system with 250 mL of BES-Gluconate-Polyethylene Glycol based solution (BGP-HMP) or with Bretschneider solution (HTK) at 5 °C. HMP was performed up to 24 h at a constant pressure of 40 mmH 2 O (equivalent to 25% of the normothermic portal pressure). Both perfusion solutions were air equilibrated during the HMP. After 24 h of HMP, the livers were reperfused at 37 °C with Krebs–Henseleit Dextran solution (Balaban, C.L., 2011. Artif Organs 35(5), 508–515). In both procedures, portal pressure and flow rate were measured and the intrahepatic resistance (IR) was calculated. Perfusate pH oscillations and enzymes (LDH, AST, and ALT) activities were evaluated during HMP and normothermic reperfusion. Also, the O 2 consumption, glucose, urea production and bile flow were measured during the normothermic reperfusion period. Liver perfusion variables and injury parameters during HMP Statistical differences in enzymes release ( p n = 5) during HMP. Enzyme activities in perfusate was (U/L.gliver): LDH, 10.01 ± 1.22 (HTK), 6.20 ± 1.03 (BGP–HMP); AST, 3.63 ± 0.62 (HTK), 1.25 ± 0.13 (BGP–HMP). In contrast, for ALT release no statistical differences were found. Liver function and viability during normothermic reperfusion The portal flow was 1.63 ± 0.41 vs. 2.85 ± 0.59 (mL/min.gliver) with HTK and BGP–HMP respectively. The IR and enzyme release after 90 min of reperfusion showed statistical differences between both groups. HTK solution showed higher values than BGP–HMP: IR (mmHg.min.gliver/mL) 4.96 ± 1.15 vs. 2.79 ± 0.66; LDH 43.04 ± 13.2 vs. 15.1 ± 5.43; AST 8.5 ± 2.7 vs. 3.46 ± 1.02; ALT 3.72 ± 1 vs. 1.04 ± 0.5. These results indicate that BGP–HMP is able to maintain better membrane integrity of the hepatocytes and reduce the hypothermic preservation and reperfusion injuries of the livers. Also, hematoxylin/eosin stain analysis showed better integrity in livers treated with BGP–HMP than livers perfused with HTK solution. With regard to bile flow and O 2 consumption, preserved livers with either solutions were able to produce bile, but the livers stored with BGP–HMP were able to uptake more O 2 (μmol/min.gliver) than livers preserved with HTK: 1.08 ± 0.08 vs. 0.65 ± 0.1. So, in the setted conditions, the livers are able to keep an active metabolism with both preservation solutions, but the BGP–HMP solution enhances the graft O 2 consumption capacity and reduces IR.

54. Performance of rat liver microorgans (lmos) cold preserved in BG35 solution as the biological component of a new flat-plate model of bioartificial liver (BAL)

BAL devices constitute an innovative therapeutic approach currently accepted as a bridge to liver transplantation for patients with liver failure. Usually, the patient’s blood or plasma is circulated extra-corporeally through a bioreactor that houses a metabolically active component which performs the functions of the damaged liver. Among these, ammonia detoxification is crucial because the accumulation of this metabolite is highly toxic to the central nervous system. Our first BAL prototype consisted of a hollow fiber-based cartridge perfused with goat blood through the fiber’s lumen. This device showed an effective ammonia depuration rate using isolated hepatocytes as cellular component. But, since the “ideal” biological component should contain all liver cell types for a maximum response, we became interested in evaluating LMOs: tissue slices that retain the basic micro architecture of the liver lobe. Therefore, we built up a flat-based BAL model, suitable to use fresh or cold preserved LMOs. Accordingly, we reported the development of a novel preservation solution (BG35) that showed the same efficacy as ViaSpan ® to protect LMOs against cold preservation injury for 48 h. So, the aim of the present work was to evaluate the performance of cold preserved rat LMOs in our new BAL model. LMOs were manually cut from rat livers (338 ± 27 μm thickness, n = 25) and stored in BG35 or ViaSpan ® solutions at 0 °C up to 48 h of preservation. Freshly cut LMOs were used as controls. After the preservation period, 1.5 g of LMOs was loaded into the BAL and an ammonia overload (1.06 ± 0.12 mM, n = 9) was added to the blood before initiating the system perfusion at 9 mL/min (blood volume = 35 mL). Samples of blood and extra-fiber fluid were taken after 60 and 120 min of perfusion. We determined LDH release (%) by LMOs in the extra-fiber fluid and ammonia detoxification capacity (% of the initial dose detoxified at different times) in the perfusing blood. After 120 min of perfusion, LMOs cold preserved in BG35 or ViaSpan ® were able to detoxify 57.4 ± 12.1 % and 51.9 ± 6.0 %, respectively, of the initial ammonia overload ( n = 3). No significant differences were found with Controls (49.3 ± 8.8%, n = 3). All groups showed a good maintenance of their viability: LDH release after 120 min was 10.5 ± 5.4 % and 9.2 ± 3.4% for BG35 and ViaSpan ® preserved LMOs, respectively, similar to Controls (6.1 ± 2.2%, n = 3). In conclusion, LMOs cold preserved in both solutions showed a good ammonia detoxification capacity that enables their use as biological component of a BAL device. These results confirm that the BG35 is as efficient as ViaSpan ® to protect rat LMOs against cold preservation injury and it is considerably less expensive and easy to prepare. Also, these experiments show that the BAL prototype developed constitutes a simple and inexpensive tool to investigate the effects of preservation, cryopreservation, or culture on the LMOs functions.

52. Hypothermic preservation of rat liver microorgans (lmos) in ViaSpan® and BG35 (bes-gluconate-peg35) solutions: Study of ammonia metabolism during rewarming to evaluate their possible use as biological component of a BAL system

Bioartificial livers (BAL) were designed as a bridge to maintain patients with liver failure until they recover or receive a liver transplant. In these devices, the patient’s blood or plasma is circulated extra-corporeally through a bioreactor that houses a metabolically active component, as isolated hepatic cells or cultured cells. In this work, we tested LMOs from rat liver. LMOs are tissue slices that retain the microarchitecture of the liver lobe and its physiological characteristics. The use of LMOs is also attractive because it obviates the stages of cell isolation and cultivation. So, our group has set up a technique to manually obtain LMOs and constructed a flat-based BAL model suitable to use fresh LMOs. Moreover, we have developed a novel preservation solution, BG35, with similar efficacy than ViaSpan ® to protect LMOs against cold preservation injury. This would allow having the biological component viable, functional and ready to be used when is needed for our BAL. Ammonia accumulates in the blood of patients with liver failure, causing metabolic and neurological disturbances, being crucial that any cell or tissue to be employed in a BAL can perform this task. Since the Urea Cycle is the major pathway of ammonia removal, the objective of this work was to study gene expression and activity of Carbamoyl Phosphate Synthetase I (CPSI) and Ornithine Transcarbamylase (OTC), two key enzymes, after LMOs preservation in BG35 and ViaSpan ® (0 °C, 48 h). We have assayed in vitro the LMOs ability to detoxify an overload of ammonia in order to evaluate them for their successive application into a BAL. LMOs were manually cut (338 ± 27 μm thickness, n = 25). 50 LMOs were stored at 0 °C in 60 mL of BG35 or ViaSpan ® . Freshly-cut LMOs were used as controls. After cold storage, LMOs were rewarmed (37 °C, Krebs Henseleit Rewarming solution, 120 min) in a Dubnoff metabolic shaker under 95%O 2 :5% CO 2 atmosphere. Samples were taken after 60 and 120 min. All groups showed a good maintenance of their viability. After 120 min, the amount of LDH released by LMOs cold preserved in BG35 (20.1 ± 5.6%) and ViaSpan ® (23.4 ± 5.5 %) was similar to Controls (16.0 ± 4.4 %, n = 3). The preservation conditions did not affect ammonia metabolism: after 2 h of rewarming, LMOs cold preserved in BG35 and ViaSpan ® could detoxify 29.3 ± 7.9% and 25.7 ± 4.9% of the initial overload, similarly than Controls (29.7 ± 10.7%, n = 6). Though there were differences in relative mRNA levels between Controls and the two preserved groups, the values of CPSI and OTC enzymatic activity were comparable in all studied groups. In summary, we have shown that our preservation conditions did not affect ammonia metabolism and cold preserved LMOs maintain the physiological and biochemical liver functions tested, which allows their use as biological component of a BAL system.

35. Tissue protection mediated by diallyl disulfide in rat liver hypothermic preservation

The beneficial effects of dietary garlic (Allium sativum) have been recognized for centuries. In particular, garlic consumption has been frequently associated with the reduction of multiple risk factors such as increased reactive oxygen species, high blood pressure and high cholesterol. Hydrogen sulfide (H 2 S) could be implicated in those garlic cytoprotective effects. This gas is the newest member of the gaseous transmitter family, known to promote vasodilatation, antioxidant defenses, attenuate inflammation and inhibit apoptosis. These properties make this gas ideally suited to protect tissues from ischemia–reperfusion injuries. We evaluated the cytoprotective potential of a garlic derived compound (Diallyl disulfide, DADS) during cold storage of rat livers with an isolated normothermic reperfusion experimental approach. Male adult Wistar rats (250–300 g) were hepatectomized by a widespread surgical technique. Livers were preserved in HTK solution (Custodiol®) + (1, 5, 50 and 500 μM) or without DADS (control) up to 48 h at 0–4°C, after that, they were evaluated in an ex vivo perfusion system. Fresh control livers were immediately reperfused. The following parameters were assessed: intrahepatic resistance (IR), bile production (BP), oxygen consumption (OC) and lactate dehydrogenase release (LDH). Also, 5 mg of bromosulfophtalein (BSP) was added to the perfusion media at t = 0, to evaluate depuration capacity through BSP spectrophotometric determination and thin layer chromatography (TLC) of bile samples. Hypothermic preservation injuries were manifested during reperfusion through the parameters evaluated (versus control fresh livers). Intrahepatic resistance and LDH release after 60 min of reperfusion, were lessened when DADS in a 5 μM concentration was added to the preservation solution. IR (mmHg min gliv/mL): 3.16 ± 0.60 (HTK−5 μM), 3.39 ± 0.57 (HTK); LDH activity in perfusate (U/L.gliv): 48.74 ± 2.80 (HTK−5 μM), 58.95 ± 5.50 (HTK). Also, bile production was augmented to some extent by this dose when compared to the HTK without DADS group: 0.50 ± 0.18 μL/min gliv (HTK−5 μM); 0.38 ± 0.13 μL/min gliv (HTK). Oxygen consumption constitutes a marker for active metabolism, 5 μM doses of DADS showed a 21.5% increase in O 2 uptake ( p < 0.05, vs. HTK alone). Interestingly, the other assayed doses not only did not improve protection but, in some cases, seemed to deteriorate grafts. Percentages of BSP excreted in bile at the end of ex vivo perfusion (90 min) indicated an increased depuration competence for livers preserved in HTK+5 and 50 μM of DADS: 67.17 ± 8.10% (HTK−5 μM), 60.90 ± 7.02%(HTK−50 μM) and 41.93 ± 9.86% (HTK) ( p < 0.05). TLCs revealed four spots with different R f corresponding to free BSP, BSP-GSH conjugates and BSP- γ Glu-Cys/ γ Glu species (100%). This study reflected that cold storage in HTK barely affected BSP conjugation to glutathione (GSH) since % BSP-GSH of fresh control livers did not differ from preserved ones. No statistical differences were found between preserved groups when comparing BSP-GSH proportions. In conclusion, addition of diallyl disulfide in a 5 μM concentration to preservation solution may improve static cold storage outcomes for rat livers.

HTK-N, a modified HTK solution, decreases preservation injury in a model of microsteatotic rat liver transplantation

Ischemia/reperfusion injury is an obstacle especially in steatotic livers, including those with steatosis induced by acute toxic stress. Recently, a modified histidine-tryptophan-ketoglutarate (HTK) solution, HTK-N, has been developed. This solution contains N-acetylhistidine, amino acids, and iron chelators. This study was designed to test the effects of HTK-N on preservation injury to rat livers after acute toxic injury. Microvesicular steatosis was induced by a single dose of ethanol (8 g/kg BW). Livers were harvested and stored at 4 °C for 8 h with HTK or HTK-N before transplantation. Tissue and blood samples were taken at 1, 8, and 24 h after reperfusion to compare serum liver enzymes (aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase), standard histology, and immunohistochemistry for myeloperoxidase (MPO), caspase-3, and inducible nitric oxide synthase. Survival was compared after 1 week. For statistics, Analysis of Variance and t test were used. HTK-N improved survival from 12.5% in HTK to 87.5% (p < 0.05). Furthermore, liver enzymes were decreased to 2-75% of HTK values (p < 0.05). Necrosis and leukocyte infiltration and MPO, caspase-3, and iNOS expression after transplantation were decreased (p < 0.05). This study demonstrates that HTK-N protects liver grafts with microvesicular steatosis caused by acute toxic injury from cold ischemic injury better than standard HTK most likely via inhibition of hypoxic injury and oxidative stress and amelioration of the inflammatory reaction occurring upon reperfusion.

Donor Management with N-Octanoyl-Dopamine Improves Renal Function and Reduces Inflammation in An Experimental Brain Death Transplantation Model

We have previously demonstrated protective effects of dopamine (DA) in renal transplantation. Recently, we have developed a non-hemodynamic dopamine derivative, i.e. N-Octanoyl-Dopamine (NOD) that is more effective than DA to protect Human-Umbilical-Vein-Endothelial-Cells (HUVEC) against cold preservation injury. In the present study we tested if NOD donor treatment has anti-inflammatory effects in a brain death animal model. Brain death (BD) was induced in Fisher rats. The kidneys were harvested after 6h and processed for further analysis. Allogeneic renal transplantations were performed to address the influence of NOD donor treatment on renal function and renal inflammation. The kidneys were harvested after 7 days. NOD treatment in brain dead donors neither influenced the hemodynamic status of the animals nor induced tachycardia. Expression of adhesion molecules was significantly reduced in donor kidneys treated with NOD (NOD vs. Control; ICAM-1, 3.4±1 vs. 5.6±2; VCAM-1, 1.6±0.9 vs. 2.9±0.8; P< 0.05). Early renal function in recipients revealed a significantly better renal function in recipients from NOD treated donors (NOD vs. Control: 0.85±0.43 vs. 1.53±0.87, day1; P< 0.05). Banff-classification revealed significantly less vasculitis (P< 0.0001) and tubulitis (P< 0.05) in NOD treated grafts 1 week after transplantation. Our data demonstrate that NOD has potent reno-protective effects. NOD donor treatment not only mitigates deterioration in renal function but also reduces renal inflammation in recipients. Hence, NOD donor treatment might be a new approach in donor management to improve graft outcome.

Normothermic Acellular Ex Vivo Liver Perfusion (NEVLP): Development of a New Method for the Storage, Assessment, and Repair of Marginal Liver Grafts

Livers retrieved after cardiac death (DCD) are often declined for transplantation because of the increased risk for graft failure and bile duct injury. The current cold static preservation technique is associated with a high risk of biliary complications and does not offer the opportunity to assess graft injury and function. We compared the novel normothermic acellular ex vivo liver perfusion (NEVLP) with conventional cold static organ preservation for livers retrieved after cardiac death. We implemented spectrophotometric imaging as tool for online monitoring of blood flow and oxygen transport to tissue during NEVLP to predict organ function post reperfusion. Methods: First, pig livers were perfused (n=5) for 12 hours in a cell free, oxygenated perfusion solution (STEEN), followed by 12hr perfusion with whole blood as a model of transplantation. ALT and histology were evaluated as parameters of the liver injury, urea synthesis, bile production and oxygen consumption were determined as marker of the liver function. In a second approach, pig livers either subjected to 1hr warm ischemia plus 12hrs cold storage in UW (n=5) or 1hr warm ischemia plus 4hr cold storage plus 8hr NEVLP. After 12hr organ preservation the livers were perfused for 8hr in a pig liver transplant model. Spectrophotometric imaging was used to monitor blood flow and oxygen supply to liver tissue and bile duct tissue. Results: 12hr normothermic acellular ex vivo liver perfusion followed by 12hr whole blood reperfusion was not associated with liver injury. Serum ALT (mean 27U/L) remained normal, and histology did not show any evidence of necrosis (< 1%). Bile production (mean 3cc/hr), oxygen consumption (400mmHg), and BUN synthesis (1.8mmol/l) were within normal limits. In a second approach, NEVLP was compared to cold static storage in a DCD model using 1hr warm ischemia and 12hr preservation. NEVLP was associated with significantly decreased serum AST (26U/L vs 285U/L), decreased necrosis (10% vs 35%, p< 0.05), and increased oxygen consumption (400mmHg vs 230mmHg, p< 0.05). Cold static storage after DCD retrieval was associated with loss of peripheral arterial blood supply, while arterial blood flow was maintained in NEVLP preserved grafts. Accordingly, UW preserved grafts had massive biliary necrosis (90%), while bile ducts were normal in NEVLP preserved livers. Spectrophotometric imaging showed decreased oxygen supply especially in peripheral parts of liver and bile duct tissue. Conclusion: NEVLP allows prolonged organ storage with normal liver metabolism without inducing preservation injury. Bile duct injury after DCD liver retrieval develops in cold static, but not NEVLP preserved organs. NEVLP is a novel preservation technique for the assessment of marginal grafts. The implemented spectrophotometric imaging can monitor oxygen transport to tissue and seems to be a promising tool to predict organ function post reperfusion.

Assist devices: Machine preservation of extended criteria donors

1. Hypothermic machine perfusion (HMP) is in its infancy in clinical liver transplantation. Potential benefits include diminished preservation injury (PI) and improved graft function. 2. We have employed HMP in 21 extended criteria donor (ECD livers) at our center with excellent clinical outcomes. 3. Our experience with liver HMP is the only reported clinical experience worldwide representing a total of 41 successful liver transplant cases showing improved outcomes and diminished markers of ischemia/reperfusion injury. 4. Further multicenter and large scale trials are now warranted to further explore the benefits and applicability of liver HMP into the clinical mainstream.

Hypothermic Machine Preservation Reduces Molecular Markers of Ischemia/Reperfusion Injury in Human Liver Transplantation

Hypothermic machine perfusion (HMP) is in its infancy in clinical liver transplantation. Potential benefits include diminished preservation injury (PI) and improved graft function. Molecular data to date has been limited to extrapolation of animal studies. We analyzed liver tissue and serum collected during our Phase 1 trial of liver HMP. Grafts preserved with HMP were compared to static cold stored (SCS) transplant controls. Reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry and transmission electron microscopy (TEM) were performed on liver biopsies. Expression of inflammatory cytokines, adhesion molecules and chemokines, oxidation markers, apoptosis and acute phase proteins and the levels of CD68 positive macrophages in tissue sections were evaluated. RT-PCR of reperfusion biopsy samples in the SCS group showed high expression of inflammatory cytokines, adhesion molecules and chemokines, oxidative markers and acute phase proteins. This upregulation was significantly attenuated in livers that were preserved by HMP. Immunofluorescence showed larger numbers of CD68 positive macrophages in the SCS group when compared to the HMP group. TEM samples also revealed ultrastructural damage in the SCS group that was not seen in the HMP group. HMP significantly reduced proinflammatory cytokine expression, relieving the downstream activation of adhesion molecules and migration of leukocytes, including neutrophils and macrophages when compared to SCS controls.

Intraluminal Polyethylene Glycol Stabilizes Tight Junctions and Improves Intestinal Preservation in the Rat

Rapidly progressing mucosal breakdown limits the intestinal preservation time below 10 h. Recent studies indicate that intraluminal solutions containing polyethylene glycol (PEG) alleviate preservation injury of intestines stored in UW-Viaspan. We investigated whether a low-sodium PEG solution is beneficial for intestines stored in histidine-tryptophane-ketoglutarate (HTK) preservation solution. Rat intestines used as control tissue (group 1) were perfused with HTK, groups 2 and 3 received either a customized PEG-3350 (group 2) or an electrolyte solution (group 3) intraluminally before cold storage. Tissue injury, brush-border maltase activity, zonula occludens-1 (ZO-1) and claudin-3 expression in the tight junctions (TJ) were analyzed after 8, 14 and 20 h. We measured epithelial resistance and permeability (Ussing chamber) after 8 and 14 h. Group 2 had superior morphology while maltase activity was similar in all groups. TJ proteins rapidly decreased and decolocalized in groups 1 3; these negative events were delayed in group 2, where colocalization persisted for about 14 h. Intestines in group 2 had higher epithelial resistance and lower permeability than the other groups. These results suggest that a customized PEG solution intraluminally reduces the intestinal preservation injury by improving several major epithelial characteristics without negatively affecting the brush-border enzymes or promoting edema.

Role of Translocator Protein in Renal Ischemia Reperfusion, Renal Preservation and Acute Kidney Injury

Role of Translocator Protein in Renal Ischemia Reperfusion, Renal Preservation and Acute Kidney Injury

Ezrin functionality and hypothermic preservation injury in LLC-PK1 cells

Renal epithelial cells from donor kidneys are susceptible to hypothermic preservation injury, which is attenuated when they over express the cytoskeletal linker protein ezrin [34]. This study was designed to characterize the mechanisms of this protection. Renal epithelial cell lines were created from LLC-PK1 cells, which expressed mutant forms of ezrin with site directed alterations in membrane binding functionality. The study used cells expressing wild type ezrin, T567A, and T567D ezrin point mutants. The A and D mutants have constitutively inactive and active membrane binding conformations, respectively. Cells were cold stored (4°C) for 6–24h and reperfused for 1h to simulate transplant preservation injury. Preservation injury was assessed by mitochondrial activity (WST-1) and LDH release. Cells expressing the active ezrin mutant (T567D) showed significantly less preservation injury compared to wild type or the inactive mutant (T567A), while ezrin-specific siRNA knockdown and the inactive mutant potentiated preservation injury. Ezrin was extracted and identified from purified mitochondria. Furthermore, isolated mitochondria specifically bound anti-ezrin antibodies, which were reversed with the addition of exogenous recombinant ezrin. Recombinant wild type ezrin significantly reduced the sensitivity of the mitochondrial permeability transition pore (mPTP) to calcium, suggesting ezrin may modify mitochondrial function. In conclusion, the cytoskeletal linker protein ezrin plays a significant role in hypothermic preservation injury in renal epithelia. The mechanisms appear dependent on the molecule’s open configuration (traditional linker functionality) and possibly a novel mitochondrial specific role, which may include modulation of mPTP function or calcium sensitivity.

Impact of a new refrigerator on the preservation of hepatic grafts

Current medical transplantation methods focus on solutions for major problems such as the shortage of donors. To overcome these issues, expanding organ preservation time has become a major concern. A new refrigerating chamber has been recently developed, which can cool the inside of a material to the required temperature by frequently sensing the temperature of both inside and surface of the materials. The purpose of this study is to evaluate the usefulness of a new refrigerating system in hepatic preservation. The liver grafts were harvested from rats and divided into two groups. Group A consisted of grafts preserved in chilled University of Wisconsin solution (UW) solution (on ice) for 24, 72 and 168 h. Group B consisted of grafts preserved in the UW solution in a new refrigerator at 4°C. In group B, aspartate aminotransferase released into effluent after cold storage for 72 h showed a marked decrease compared to group A (P < 0.05). The levels of ammonia and lactate decreased significantly in group B (P < 0.05). In group B, the levels of adenosine triphosphate were significantly preserved after cold storage for 24 h and 72 h compared to group A (P < 0.05). Immunohistochemistry showed positive cells for heme oxygenase-1 were significantly increased in group B after cold storage. This new refrigerator can improve preservation injury of hepatic grafts and may provide an innovative technique for liver transplantation.

Ezrin functionality and ischemia‐reperfusion (I/R) injury in transplantation

Organ transplantation is limited by organ donation and the inevitable I/R injury associated with organ preservation. To date, little clinical advancements in I/R injury have been made because the basic molecular mechanisms remain poorly understood. The objective was to characterize the role of ezrin in preservation injury since it is hypothesized that the sub‐lamellar cytoskeletal system is an important mediator of ischemic injury. Ezrin dissociates from the cytoskeletal structures during cold ischemia in renal tubules. In LLC‐PK1 cells, preservation injury is exacerbated with ezrin knock‐down (siRNA) and attenuated with ezrin over expression. Point mutations of ezrin at T567A potentiated whereas T567D attenuated preservation injury when expressed in LLC‐PK1 cells, implicating the binding functionality of ezrin as important for protection. Specific ezrin binding sites were also identified on purified mitochondria from renal tubules and exogenous recombinant ezrin attenuated the sensitivity of the mitochondrial permeability transition pore to calcium. Finally, dissociation of the ezrin analog moesin in liver and sinusoidal endothelial cells was also observed with preservation. In conclusion, ezrin plays an important causal role in I/R injury via its linker functionality and possibly by a novel mitochondrial binding role. Supported by NIH R01 DK087737.

Role of Translocator Protein in Renal Ischemia Reperfusion, Renal Preservation and Acute Kidney Injury

Translocator protein (TSPO), formerly known as peripheral-type benzodiazepine receptor (PBR), has been described in several tissues and characterized as one of the main elements of steroidogenesis. However, TSPO is also involved in other pathways and cell functions, such as apoptosis regulation, protein import, membrane biogenesis, cell cycle regulation, oxygen homeostasis and mitochondrial membrane fluidity regulation. In the kidney, TSPO is normally located in the distal parts of the nephron from the thick ascending limb of the loop of Henle to the medullary collecting ducts. However when the kidney is submitted to a stress such as ischemia reperfusion injury there is a defined change in TSPO expression towards more proximal areas of the nephron, and the protein can be detected as high as proximal tubular cells and the Bowman Capsule. As the injury persists, TSPO is also located in invading mononucleated cells, in a pattern reproducing invasion by CD4+ helper T cells, and in the damaged vessels where TSPO is expressed both in endothelial and smooth muscle cells. Herein we review the potential use of TSPO-directed treatment for ischemia reperfusion injury, particularly regarding pre-conditioning of the organ. We also detail the relationship of proximal TSPO staining with the intensity of the injury, particularly the implication of monomeric (18 kDa) TSPO and its role in hypoxia-reoxygenation and apoptosis prevention. The potential implications of the protein with regeneration processes activated in response to injury and their relation with embryogenesis pathways are discussed.

Implications of a positive crossmatch in liver transplantation: A 20-year review

Whether a positive crossmatch result has any relevance to liver transplantation (LT) outcomes remains controversial. We assessed the impact of a positive crossmatch result on patient and graft survival and posttransplant complications. During a 20-year period, 2723 LT procedures with crossmatch results were identified: 2479 primary transplants and 244 retransplants. The rates of positive B cell and T cell crossmatches were 10.1% and 7.4%, respectively, for primary transplants and 14.6% and 6.4%, respectively, for retransplants (P = 0.049 for a B cell crossmatch). Across all primary transplants, females (P < 0.001) and patients with autoimmune hepatitis (P < 0.001) had greater frequencies of positive crossmatches. There was no effect from race or age. For both primary transplants and retransplants, patient survival and graft survival were not affected by the presence of a positive crossmatch. With respect to posttransplant complications, there were no differences in rejection episodes (hyperacute, acute, or chronic) or technical complications (biliary and vascular) between negative and positive crossmatch groups. However, there were significant differences in the pathological findings of preservation injury (PI) on liver biopsy samples taken at the time of transplantation and within the first week of transplantation (P = 0.003 for B cells and P = 0.03 for T cells). In summary, a positive crossmatch had no significant impact on patient survival or graft outcomes. However, there was a significantly higher incidence of PI in primary LT recipients with a positive crossmatch. This finding is important for a broader understanding of PI, which may include a significant immunological component.

Recurrent Hepatitis C After Liver Transplantation

Infection with hepatitis C virus is the most common indication for liver transplantation in the United States. Although recurrence of hepatitis C virus infection is universal following transplantation, the natural history of posttransplantation hepatitis C varies. In general, however, posttransplant hepatitis C virus infection progresses relatively quickly, with 10%-20% of patients developing cirrhosis within 5 years. Risk factors for severe recurrent hepatitis C include donor age, female sex, treatment of rejection, preservation injury, and high viral load pretransplant or early posttransplant. Type of allograft, infection with cytomegalovirus, or type of calcineurin inhibitor used may not play a role. Treatment with interferon + ribavirin in recurrent hepatitis C virus shows mixed results. Sustained virologic response has been observed in only 8%-30% of patients, and side effects of these medications are considerable. Protease inhibitors are not yet approved for the posttransplant population, but clinical trials are under way.

Hyperbaric oxygen therapy reduces the severity of ischaemia, preservation and reperfusion injury in a rat model of liver transplantation

BackgroundApproaches to increase organ availability for orthotopic liver transplantation (OLT) often result in the procurement of marginal livers that are more susceptible to ischaemia, preservation and reperfusion injury (IPRI). MethodsThe effects of post-OLT hyperbaric oxygen (HBO) therapy on IPRI in a syngeneic rat OLT model were examined at various time-points. The effects of IPRI and HBO on hepatocyte necrosis, apoptosis, proliferation, and sinusoidal morphology and ultrastructure were assessed. ResultsPost-OLT HBO therapy significantly reduced the severity of IPRI; both apoptosis [at 12h: 6.4±0.4% in controls vs. 1.6±0.7% in the HBO treatment group (p < 0.001); at 48h: 2.4±0.2% in controls vs. 0.4±0.1% in the HBO treatment group (p < 0.001)] and necrosis [at 12h: 18.7±1.8% in controls vs. 2.4±0.4% in the HBO treatment group (p < 0.001); at 48h: 8.5±1.3% in controls vs. 3.4±0.9% in the HBO treatment group (P= 0.019)] were decreased. Serum alanine transaminase was reduced [at 12h: 1068±920IU/l in controls vs. 370±63IU/l in the HBO treatment group (P= 0.030); at 48h: 573±261IU/l in controls vs. 160±10IU/l in the HBO treatment group (P= 0.029)]. Treatment with HBO also promoted liver regeneration [proliferation at 12h: 4.5±0.1% in controls vs. 1.0±0.3% in the HBO treatment group (p < 0.001); at 48h: 8.6±0.7% in controls vs. 2.9±0.2% in the HBO treatment group (p < 0.01)] and improved sinusoidal diameter and microvascular density index. ConclusionsHyperbaric oxygen therapy has persistent positive effects post-OLT that may potentially transfer into clinical practice.

A new preservation solution for lung transplantation: Evaluation in a porcine transplantation model

Lung preservation injury is still a major problem in lung transplantation. The aim of the current study was to evaluate the effects of a new preservation solution (Custodiol-N) for lung preservation. Using an in vivo pig model, 7 lungs each were preserved for 24 hours after perfusion with: low-potassium dextran (LPD) solution as control (Group I); base solution of Custodiol-N without iron chelators (Group II); Custodiol-N (Group III); or Custodiol-N supplemented with dextran 40 (Group IV). Four animals received a sham operation. After left lung transplantation and contralateral lung exclusion, hemodynamics and blood gases were monitored for 6 hours; tissue samples were taken at the end of the experiments. All animals survived the transplantation procedure. Base solution- and Custodiol-N-preserved lungs (Groups II and III) showed graft function similar to that of LPD-preserved lungs (Group I), showing a trend toward improved values. Custodiol-N with dextran (Group IV) led to a significant reduction of mean pulmonary arterial pressure (20 ± 2 vs 28 ± 3 mm Hg, p < 0.01) and pulmonary vascular resistance (410 ± 51 vs 588 ± 83 dyne/s/cm(5), p < 0.01), and oxygenation ratio was significantly higher (536 ± 52 vs 313 ± 107 mm Hg at 6 hours, p < 0.01) and PCO(2) values were significantly lower (51 ± 9 vs 77 ± 5 mm Hg at 6 hours, p < 0.01) at 6 hours compared with LPD (Group I). Custodiol-N (Groups II to IV) showed a trend toward a lower wet/dry ratio and reduced oxidative stress; in the presence of dextran (Group IV), the difference was again statistically significant, when compared with LPD (Group I). Custodiol-N solution is a new alternative preservation solution for lung transplantation that offers significantly superior protection compared with LPD when dextran 40 is added.