Taurine as Antioxidant in a Novel Cell- and Oxygen Carrier-Free Perfusate for Normothermic Machine Perfusion of Porcine Kidneys.

Donor organ-shortage has resulted in the increased use of marginal grafts; however, normothermic machine perfusion (NMP) holds the potential for organ viability assessment and restoration of marginal grafts prior to transplantation. Additionally, cell-, oxygen carrier-free and antioxidants-supplemented solutions could potentially prevent adverse effects (transfusion reactions, inflammation, hemolysis), associated with the use of autologous packed red blood cell (pRBC)-based perfusates. This study compared 6 h NMP of porcine kidneys, using an established pRBC-based perfusate (pRBC, n = 7), with the novel cell- and oxygen carrier-free organ preservation solution Ecosol, containing taurine (Ecosol, n = 7). Despite the enhanced tissue edema and tubular injury in the Ecosol group, related to a suboptimal molecular mass of polyethylene glycol as colloid present in the solution, functional parameters (renal blood flow, intrarenal resistance, urinary flow, pH) and oxygenation (arterial pO2, absence of hypoxia-inducible factor 1-alpha) were similar to the pRBC group. Furthermore, taurine significantly improved the antioxidant capacity in the Ecosol group, reflected in decreased lactate dehydrogenase, urine protein and tubular vacuolization compared to pRBC. This study demonstrates the feasibility of 6 h NMP using a taurine containing, cell- and oxygen carrier-free perfusate, achieving a comparable organ quality to pRBC perfused porcine kidneys.

Cold Preflush of Porcine Kidney Grafts Prior to Normothermic Machine Perfusion Aggravates Ischemia Reperfusion Injury.

Normothermic machine perfusion (NMP) of kidney grafts is a promising new preservation method to improve graft quality and clinical outcome. Routinely, kidneys are washed out of blood remnants and cooled using organ preservation solutions prior to NMP. Here we assessed the effect of cold preflush compared to direct NMP. After 30 min of warm ischemia, porcine kidneys were either preflushed with cold histidine-tryptophan-ketoglutarate solution (PFNMP group) prior to NMP or directly subjected to NMP (DNMP group) using a blood/buffer solution. NMP was performed at a perfusion pressure of 75 mmHg for 6 h. Functional parameters were assessed as well as histopathological and biochemical analyses. Renal function as expressed by creatinine clearance, fractional excretion of sodium and total output of urine was inferior in PFNMP. Urine protein and neutrophil gelatinase-associated lipocalin (NGAL) concentrations as markers for kidney damage were significantly higher in the PFNMP group. Additionally, increased osmotic nephropathy was found after PFNMP. This study demonstrated that cold preflush prior to NMP aggravates ischemia reperfusion injury in comparison to direct NMP of warm ischemia-damaged kidney grafts. With increasing use of NMP systems for kidneys and other organs, further research into graft flushing during retrieval is warranted.

Influence of Vitamin C on Antioxidant Capacity of In Vitro Perfused Porcine Kidneys.

Systemic and localized ischemia and reperfusion injury remain clinically relevant issues after organ transplantation and contribute to organ dysfunctions, among which acute kidney injury is one of the most common. An in vitro test-circuit for normothermic perfusion of porcine kidneys after warm ischemia was used to investigate the antioxidant properties of vitamin C during reperfusion. Vitamin C is known to enhance microcirculation, reduce endothelial permeability, prevent apoptosis, and reduce inflammatory reactions. Based on current evidence about the pleiotropic effects of vitamin C, we hypothesize that the antioxidant properties of vitamin C might provide organ-protection and improve the kidney graft function in this model of ischemia and reperfusion. METHODS: 10 porcine kidneys from 5 Landrace pigs were perfused in vitro for 6 h. For each experiment, both kidneys of one animal were perfused simultaneously with a 1:1 mixture of autologous blood and modified Ringer's solution at 38 °C and 75 mmHg continuous perfusion pressure. One kidney was treated with a 500 mg bolus injection of vitamin C into the perfusate, followed by continuous infusion of 60 mg/h vitamin C. In the control test circuit, an equal volume of Ringer's solution was administered as a placebo. Perfusate samples were withdrawn at distinct points in time during 6 h of perfusion for blood gas analyses as well as measurement of serum chemistry, oxidative stress and antioxidant capacity. Hemodynamic parameters and urine excretion were monitored continuously. Histological samples were analyzed to detect tubular- and glomerular-injury. RESULTS: vitamin C administration to the perfusate significantly reduced oxidative stress (49.8 ± 16.2 vs. 118.6 ± 23.1 mV; p = 0.002) after 6 h perfusion, and increased the antioxidant capacity, leading to red blood cell protection and increased hemoglobin concentrations (5.1 ± 0.2 vs. 3.9 ± 0.6 g/dL; p = 0.02) in contrast to placebo treatment. Kidney function was not different between the groups (creatinine clearance vit C: 2.5 ± 2.1 vs. placebo: 0.5 ± 0.2 mL/min/100 g; p = 0.9). Hypernatremia (187.8 ± 4.7 vs. 176.4 ± 5.7 mmol/L; p = 0.03), and a lower, but not significant decreased fractional sodium excretion (7.9 ± 2 vs. 27.7 ± 15.3%; p = 0.2) were observed in the vitamin C group. Histological analysis did not show differences in tubular- and glomerular injury between the groups. CONCLUSION: Vitamin C treatment increased the antioxidant capacity of in vitro perfused kidney grafts, reduced oxidative stress, preserved red blood cells as oxygen carrier in the perfusate, but did not improve clinically relevant parameters like kidney function or attenuate kidney damage. Nevertheless, due to its antioxidative properties vitamin C might be a beneficial supplement to clinical kidney graft perfusion protocols.

Comparison of Aerobic Preservation by Venous Systemic Oxygen Persufflation or Oxygenated Machine Perfusion of Warm-Ischemia-Damaged Porcine Kidneys.

BACKGROUND/AIM: The global shortage of donor organs for transplantation has necessitated the expansion of the organ pool through increased use of organs from less ideal donors. Venous systemic oxygen persufflation (VSOP) and oxygenated machine perfusion (OMP) have previously demonstrated beneficial results compared to cold storage (CS) in the preservation of warm-ischemia-damaged kidney grafts. The aim of this study was to compare the efficacy of VSOP and OMP for the preservation of warm-ischemia-damaged porcine kidneys using the recently introduced Ecosol preservation solution compared to CS using Ecosol or histidine-tryptophan-ketoglutarate solution (HTK). MATERIALS AND METHODS: Kidneys from German Landrace pigs (n = 5/group) were retrieved and washed out with either Ecosol or HTK after 45 min of clamping of the renal pedicle. As controls, kidneys without warm ischemia, cold stored for 24 h in HTK, were employed. Following 24 h of preservation by VSOP, OMP, CS-Ecosol, or CS-HTK, renal function and damage were assessed during 1 h using the isolated perfused porcine kidney model. RESULTS: During reperfusion, urine production was significantly higher in the VSOP and OMP groups than in the CS-HTK group; however, only VSOP could demonstrate lower urine protein concentrations and fractional excretion of sodium, which did not differ from the non-warm-ischemia-damaged control group. VSOP, CS-Ecosol, and controls showed better maintenance of the acid-base balance than CS-HTK. Reduced lipid peroxidation, as reflected in postreperfusion tissue thiobarbituric acid-reactive substance levels, was observed in the VSOP group compared to the OMP group, and the VSOP and CS-Ecosol groups had concentrations similar to the controls. The ratio of reduced to oxidized glutathione was higher in the VSOP, OMP, and CS-Ecosol groups than in the CS-HTK group and controls, with a higher ratio in the VSOP than in the OMP group. CONCLUSION: VSOP was associated with mitigation of oxidative stress in comparison to OMP and CS. Preservation of warm-ischemia-damaged porcine kidneys by VSOP was improved compared to OMP and CS, and was comparable to preservation of non-warm-ischemia-damaged cold-stored kidneys.

Determination of the preferred conditions for the isolated perfusion of porcine kidneys.

BACKGROUND: The isolated perfused porcine kidney (IPPK) model has been the method of choice for the early preclinical evaluation of kidney graft preservation techniques. The preferred reperfusion conditions have not yet been determined. Here, we examined the effects of pressure- or flow-controlled perfusion and oxygenation by pure oxygen or carbogen (95% O2/5% CO2) on normothermic reperfusion in the IPPK model. METHODS: Porcine kidneys were cold-stored for 24 h in histidine-tryptophan-ketoglutarate solution and reperfused for 1 h with normothermic whole blood/Krebs-Henseleit buffer medium (20/80%). Kidneys (n = 5/group) were flow-controlled reperfused with pure oxygen (1 ml/min/g; Flow-O2) or pressure-controlled reperfused (85 mm Hg mean arterial pressure) and oxygenated with either pure oxygen (Pressure-O2) or carbogen (Pressure-O2/CO2). Renal function and damage were assessed during reperfusion and NGAL and HIF-1α levels were analyzed using an ELISA. RESULTS: Pressure-O2 and Pressure-O2/CO2 were associated with significantly better renal hemodynamics and acid-base homeostasis compared to Flow-O2. Urine protein concentrations and the fractional excretion of sodium were lower with both Pressure-O2 and Pressure-O2/CO2 than with Flow-O2. NGAL and HIF-1α levels were also lower with Pressure-O2 and Pressure-O2/CO2 than with Flow-O2. Only Pressure-O2/CO2 could demonstrate a significantly increased urine production compared to Flow-O2. The structural integrity was well preserved in the Pressure-O2 and Pressure-O2/CO2 groups, whereas diffuse and global glomerular destruction was observed in the Flow-O2 group. CONCLUSION: In the IPPK model, the application of pressure-controlled reperfusion with carbogen oxygenation, and to a lesser extent with pure oxygen, maintained physiological renal function for 1 h, thus providing a reliable and reproducible ex vivo evaluation of kidney preservation quality.

Evaluation of a novel system for hypothermic oxygenated pulsatile perfusion preservation.

BACKGROUND: Recently, a novel innovative machine perfusion (MP) system for hypothermic oxygenated pulsatile perfusion called the Airdrive (AD) has been developed. The aim of the study was to evaluate the biological safety of the AD system for perfusion preservation of kidney grafts in a porcine autotransplantation model using the low-viscosity perfusion solution Polysol (PS) in comparison with cold storage (CS) using PS or the University of Wisconsin solution (UW). In addition, we evaluated real-time microcirculation parameters. At sacrifice, grafts were retrieved for histological analysis and immunohistochemistry. METHODS: After assessment of the microcirculation, left kidneys were retrieved. Following the washout, kidneys were preserved for 20 hr using AD-PS, CS-PS or CS-UW. Thereafter, contralateral kidneys were removed followed by heterotopic autotransplantation of the preserved graft. Seven days after transplantation animals were sacrificed with retrieval of the grafts for histological analysis. Renal function, renal microcirculation and tissue injury including the proliferative response of tubular epithelial cells (TECs) were compared. RESULTS: Preservation using AD-PS or CS-PS resulted in higher microcirculatory flow compared with CS-UW. Improved recovery of renal function was seen in the AD-PS and CS-PS groups compared with CS-UW. Structural integrity was better preserved using AD-PS compared with both CS groups. Proliferative response of TECs was higher in CS-UW preserved grafts compared to grafts preserved using AD-PS. CONCLUSION: This study demonstrates the biological safety of the AD system in a porcine autotransplantation model. Also, the microcirculation was better preserved and less morphological injury was observed after 20 hr MP compared with CS.

Improved preservation and microcirculation with POLYSOL after transplantation in a porcine kidney autotransplantation model.

BACKGROUND: The most widely used preservation method for kidney grafts is cold static storage (CS) using the University of Wisconsin (UW) solution. To date, new preservation solutions have not been able to significantly improve preservation quality of grafts. The aim of this study was to compare POLYSOL, a recently developed low viscosity preservation solution, and the UW solution for CS of porcine kidney grafts. METHODS: In a porcine autotransplantation model, real-time parameters of the renal microcirculation were evaluated using the novel oxygen-to-see (O2C) combined laser Doppler and flowmetry system. Thereafter, kidneys were retrieved and washed out with POLYSOL or UW followed by 20-h CS. After the preservation period, the contralateral kidneys were removed and the preserved kidneys autotransplanted. The microcirculation was re-assessed at 10 min after reperfusion and at 7 days posttransplant, prior to removal of the grafts for histological evaluation. RESULTS: POLYSOL was able to better preserve the microcirculation compared to UW as expressed by higher values of capillary blood flow, blood flow velocity and tissue oxygen saturation values. In addition, CS using POLYSOL resulted in improved functional recovery demonstrated by lower posttransplant serum creatinine and blood urea values in comparison to the UW group. Also, structural integrity was better preserved in the POLYSOL group, compared to UW. CONCLUSIONS: This study in a clinically relevant large animal model showed that a new preservation solution, POLYSOL, resulted in improved preservation quality of kidney grafts compared to the UW solution.

Preservation of steatotic livers: a comparison between cold storage and machine perfusion preservation.

Liver grafts are frequently discarded due to steatosis. Steatotic livers can be classified as suboptimal and deteriorate rapidly during hypothermic static preservation, often resulting in graft nonfunction. Hypothermic machine perfusion (MP) has been introduced for preservation of donor livers instead of cold storage (CS), resulting in superior preservation outcomes. The aim of this study was to compare CS and MP for preservation of the steatotic donor rat liver. Liver steatosis was induced in male Wistar rats by a choline-methionine-deficient diet. After 24 hours hypothermic CS using the University of Wisconsin solution (UW) or MP using UW-Gluconate (UW-G), liver damage (liver enzymes, perfusate flow, and hyaluronic acid clearance) and liver function (bile production, ammonia clearance, urea production, oxygen consumption, adenosine triphosphate [ATP] levels) were assessed in an isolated perfused rat liver model. Furthermore, liver biopsies were visualized by hematoxylin and eosin staining. Animals developed 30 to 60% steatosis. Livers preserved by CS sustained significantly more damage as compared to MP. Bile production, ammonia clearance, urea production, oxygen consumption, and ATP levels were significantly higher after MP as compared to CS. These results were confirmed by histology. In conclusion, MP improves preservation results of the steatotic rat liver, as compared to CS.

Machine perfusion preservation of the liver: a worthwhile clinical activity?

PURPOSE OF REVIEW: Although successful machine perfusion procedures of the liver were first performed almost four decades ago, technical and logistical constraints have prevented general acceptance. Interest in the procedure has recently been renewed due to its potential to resuscitate marginal organs. This review describes experimental and clinical liver hypothermic machine perfusion studies and current developments. RECENT FINDINGS: Experimental studies have shown that oxygenated hypothermic machine perfusion provides a complete washout and can restore parenchymal energy status, a phenomenon of particular importance in preservation of livers from compromised donors. Additionally, perfusion of the hepatic artery can prevent ischemic-type biliary lesions. Short-term and continuous hypothermic machine perfusion prior to or after cold storage preservation have proven more effective than cold storage alone. SUMMARY: The benefits of hypothermic machine perfusion for both heart-beating and nonheart-beating liver grafts seem promising in terms of expanding the donor pool. As liver hypothermic machine perfusion systems are not yet commercially available, the process is currently only clinically used for kidney grafts. Clinical application appears feasible in the near future as new, easy-to-use systems and solutions are currently under development.

Wash-out of the non-heart-beating donor liver: a matter of flush solution and temperature?

BACKGROUND AND AIMS: Ischemically damaged donor livers are prone to graft non-function. This can in part be explained by a suboptimal wash-out during procurement. An enriched machine perfusion (MP) preservation solution for livers, named Polysol, was developed. The aim of this study was to investigate the type of flush solution, temperature and anticoagulant content on the wash-out of the non-heart-beating donor (NHBD) rat liver. METHODS: Rat livers were flushed after 30 min warm ischemia. After excision, livers were reperfused at 37 degrees C, with analysis of damage and function, concerning (1) solutions (University of Wisconsin (UW), histidine-tryptophan-ketoglutarate (HTK) and Polysol); (2) temperature (4 degrees C, 18 degrees C and 37 degrees C); (3) addition of heparin and (4) wash-out followed by 24 h MP. RESULTS: (1) Reperfusion results were inferior in the UW group; (2) less damage and improved function were seen after wash-out using Polysol at 37 degrees C; (3) No effects were seen of the addition of heparin to Polysol; (4) MP after wash-out using HTK resulted in more liver damage and decreased liver function as compared with wash-out using Polysol. CONCLUSIONS: Polysol is applicable as a flush solution for the NHBD liver, resulting in equal to better wash-out as compared with UW and HTK. The best temperature for this NHBD wash-out is 37 degrees C.

Improved machine perfusion preservation of the non-heart-beating donor rat liver using Polysol: a new machine perfusion preservation solution.

Waiting lists for transplantation have stimulated interest in the use of non-heart-beating donor (NHBD) organs. Recent studies on organ preservation have shown advantages of machine perfusion (MP) over cold storage (CS). To supply the liver with specific nutrients during MP, the preservation solution Polysol was developed. The aim of our study was to compare CS in University of Wisconsin solution (UW) with MP using UW-gluconate (UW-G) or Polysol in an NHBD model. After 30 minutes of warm ischemia, livers were harvested from rats for preservation by either CS, MP-UW-G, or MP-Polysol. After 24 hours of preservation, livers were reperfused with Krebs-Henseleit buffer (KHB). Perfusate samples were analyzed for liver damage and function. Biopsies were examined by hematoxylin and eosin staining and transmission electron microscopy. Liver damage was highest after CS compared with the MP groups. MP using Polysol compared with UW-G resulted in less aspartate aminotransferase (AST) and alanine aminotransferase (ALT) release. Perfusate flow, bile production, and ammonia clearance were highest after MP-Polysol compared with CS and MP-UW-G. Tissue edema was least after MP-Polysol compared with CS and MP-UW-G. In conclusion, preservation of the NHBD rat liver by hypothermic MP is superior to CS. Furthermore, MP using Polysol results in better-quality liver preservation compared with using UW-G.

Improved rat liver preservation by hypothermic continuous machine perfusion using polysol, a new, enriched preservation solution.

For experimental machine perfusion (MP) of the liver, the modified University of Wisconsin solution (UW-G) is most often used. In our search for an enriched MP preservation solution, Polysol was developed. Polysol is enriched with various amino acids, vitamins, and other nutrients for the liver metabolism. The aim of this study was to compare Polysol with UW-G for MP preservation of the liver. Rat livers were preserved during 24 hours with hypothermic MP using UW-G (n = 5) or Polysol (n = 5). Hepatocellular damage (aspartate aminotransferase [AST], alanine aminotransferase [ALT], lactate dehydrogenase [LDH], alpha-glutathione-S-transferase [alpha-GST]) and bile production were measured during 60 minutes of reperfusion (37 degrees C) with Krebs-Henseleit buffer. Control livers were reperfused after 24 hours of cold storage in UW (n = 5). MP using UW-G or Polysol showed less liver damage when compared with controls. Livers machine perfused with Polysol showed less enzyme release when compared to UW-G. Bile production was higher after MP using either UW-G or Polysol compared with controls. In conclusion, machine perfusion using Polysol results in better quality liver preservation than cold storage with UW and machine perfusion using UW-G.