High-Na + low-K + UW cold storage solution reduces reperfusion injuries of the rat liver graft

The isolated perfused rat liver model was used to assess graft viability after 24 h of cold preservation. Two solutions were compared for liver preservation: Belzer's original UW solution (high-K ⁺ UW) and a solution containing the same components but with inverted concentrations of sodium and potassium (high-Na ⁺ UW). During the 120 min of normothermic reperfusion, livers preserved in the high-Na ⁺ UW solution released lower levels of creatine kinase-BB isoenzyme, transaminases (ALT and AST), and potassium than those preserved in the high-K ⁺ UW solution. Bile flow and biliary excretion of indocyanine green increased when livers were preserved in the high-Na ⁺ UW solution. We found no statistical differences for oxygen consumption and tissue ATP concentration. The results of this study support the concept that a high-Na ⁺ UW solution is a more effective means of preserving rat livers, at least after 24 h of cold-storage and 120 min of reperfusion in the isolated perfused model, than the original high-K ⁺ UW solution. Liver preservation in the high-Na ⁺ UW solution reduces damage to sinusoidal endothelial and hepatocellular cells. The use of an extracellular-like Belzer cold storage solution eliminates potassium-related problems in cold preservation and subsequent normothermic reperfusion while keeping all the qualities of the original UW solution. Received: 26 August 1997 Received after revision: 12 November 1997 Accepted: 28 November 1997     

The role of preservation solution on acid-base regulation during machine perfusion of kidneys

To meet the current clinical organ demand, efficient preservation methods and solutions are needed to increase the number of viable kidneys for transplantation. In the present study, the influence of perfusion solution buffering strength on renal pH dynamics and regulation mechanisms during kidney ex vivo preservation was determined. Porcine kidneys were hypothermically machine perfused for 72 h with either Unisol-UHK or Belzer-Machine Perfusion solution, Belzer-MP solution. Renal perfusate samples were periodically collected and biochemically analyzed. The UHK solution, a Hepes-based solution (35 mM), provided a more efficient control of renal pH that, in turn, resulted in minor changes in the perfusate pH relative to baseline, in response to tissue CO2 and HCO3- production. In the perfusate of Belzer-MP kidney group a wider range of pH values were recorded and a pronounced pH reduction was seen in response to significant rises in pCO2 and HCO3- concentrations. The Belzer-MP solution, containing phosphate (25 mM) as its main buffer, and only 10 mM Hepes, had a greater buffering requirement to attenuate larger pH changes.     

Static cold storage preservation of ischemically damaged kidneys. a comparison between IGL-1 and UW solution.

Especially in damaged organs, adequate organ preservation is critically important to maintain viability. Institut Georges Lopez-1 (IGL-1) is a new preservation solution, with an extracellular sodium/potassium ratio and polyethylene glycol as a colloid. The influence of warm and cold ischemia was evaluated in a rat Lewis-Lewis transplant model with a follow up of 14 days. Eight groups of donation after cardiac death donor kidneys were studied with warm ischemia of 0 and 15 min followed by 0- or 24-h cold storage (CS) preservation in IGL-1 or UW-CSS. Blood was collected daily during the first week and at day 14. Recipients were placed in metabolic cages at day 4 and 14 after transplantation allowing urine collection and adequate measurement of glomerular filtration rate. Focussing on inflammation, reactive oxygen species production, proximal tubule damage, proteinuria, histology, and renal function after transplantation we could not show any relevant difference between IGL-1 and UW-CSS. Furthermore, the combination of 15-min warm ischemia and by 24-h cold ischemia did not result in life sustaining kidney function after transplantation, irrespective of the used solution. In the present experiment, static CS preservation of ischemically damaged rat kidneys in either IGL-1 or UW-CSS rendered equal results after transplantation.     

In situ perfusion and UW solution used for storage did not decrease the incidence of ATN in kidneys harvested from hemodynamically unstable donors

Abstract The incidence of acute tubular necrosis ATN after cadaveric kidney transplantation in our centre has been in the range of 50%. A prospective study was carried out in 1991 and 1992 to assess the effect of in situ perfusion and hypothermic storage of kidneys harvested from brain-dead haemodynamically stable and unstable donors. Three litres of Ringer's solution were used for in situ perfusion. In 40 cases, the kidneys were stored in Euro-Collins (EC) solution and in the other 78 cases, in University of Wisconsin (UW) solution. Among the factors that could contribute to ATN, we analysed warm ischaemia time, anastomosis time and cold storage time. Function was considered to be delayed if the patient required posttransplantation dialysis. The donors were considered haemodynamically unstable when hypotension before harvesting was present (BP < 70 mm Hg over 2 h) despite high doses (> 15 μg/kg per minute) of dopamine or when cardiac arrest occurred at the time of harvesting and oliguria had been present for at least 2 h. Haemodynamically stable donors with a BP greater than 80 mm Hg had a normal diuresis. In all donors in this group the dose of dopamine was lower than 10 μg/kg per minute. The study showed that storage in UW solution did not influence the incidence of ATN in kidneys harvested from haemodynamically unstable donors. Differences observed in our study were due to haemodynamic status preceding donor nephrectomy and length of cold storage time.     

Retrograde oxygen persufflation in combination with UW solution enhances adenine nucleotide contents in ischemically damaged rat kidney during cold storage

Retrograde oxygen persufflation (ROP) has been reported to be beneficial to kidney preservation. The purpose of this study was to investigate whether use of ROP during cold storage (CS) with Universita of Wisconsin (UW) solution could ameliorate energy metabolism and functional recovery of ischemically injured rat kidneys and, moreover, to study the particular role of adenosine (ADO) in CS with ROP. Kidneys subjected to 30 min of warm ischemia (WI) were preserved for 24 h in 4°C UW solution with or without ROP and with or without ADO. Measurements of tissue highenergy phosphate levels showed that reduced total adenine nucleotides (TAN) after 30 min of WI further declined during the subsequent CS. In ROP kidneys, however, TAN were less reduced, suggesting that even during CS, TAN can still be regenerated in the injured kidneys when ROP is combined with UW solution. When UW did not contain ADO, regeneration of TAN by ROP was slightly less than in the case of UW with ADO. This indicates that the supply of molecular oxygen is a significant factor in TAN resynthesis during CS. There was no statistically significant difference in survival rate between the ROP and CS group, indicating that an improved energy status is not the sole determinant of functional recovery. We conclude that the gaseous oxygen supply provided by ROP during CS in UW solution ameliorates the energy state of ischemically injured rat kidneys and that exogenous ADO from the UW solution contributes to the improvement of energy metabolism to a limited extent.     

Kidney transplantation after oxygenated machine perfusion preservation with Custodiol‐N solution

Custodiol‐N, a new preservation solution, has been shown particularly suitable for hypothermic machine perfusion preservation (HMP) in isolated porcine kidneys. These preliminary results should be confirmed in an actual transplant model in vivo. Kidney function after 21 h of HMP was studied in an autotransplant model using Landrace pigs (25–30 kg; n = 6 per group). Perfusion was performed with oxygenated perfusate, using either Custodiol‐N solution including 50 g/l dextran 40 (CND) or kidney perfusion solution 1 (KPS‐1) as gold standard. Viability of the grafts was followed for 1 week after bilateral nephrectomy in the recipient pigs. HMP with CND resulted in less acute tubular injury, evaluated by levels of fatty acid‐binding protein and better clearance function during the first 24 h after Tx than with KPS‐1 (P < 0.05, resp.). Serum creatinine tended to be lower in the CND group during the whole observation period. Histological tissue scores one week after Tx were similar in both groups. Expression of endothelin‐1 as well as of Toll‐like receptor 4 15 min after reperfusion was lower in the CND group (P < 0.05), suggesting less endothelial stress response. The data provide first in vivo evidence for the suitability of Custodiol‐N as an effective perfusate for renal machine perfusion.     

Machine perfusion or cold storage in organ transplantation: indication,mechanisms, and future perspectives

Most organs are currently preserved by cold storage (CS) prior to transplantation. However, as more so called marginal donor organs are utilized, machine perfusion has regained clinical interest. Recent studies have demonstrated advantages of pulsatile perfusion over CS preservation for kidney transplantation. However, it remains unclear whether there is a significant benefit of one preservation method over the other in general, or, whether the utilization of particular preservation approaches needs to be linked to organ characteristics. Proposed protective mechanisms of pulsatile perfusion remain largely obscure. It can be speculated that pulsatile perfusion may not only provide nutrition and facilitate the elimination of toxins but also trigger protective mechanisms leading to the amelioration of innate immune responses. Those aspects may be of particular relevance when utilizing grafts with suboptimal quality which may have an increased vulnerability to ischemia/reperfusion injury and compromised repair mechanisms. This review aims to enunciate the principles of organ perfusion and preservation as they relate to indication, aspects of organ protection and to highlight future developments.     

Comparison of intermittent injection of nondepolarizing solution with a single flush of UW solution for donor heart preservation

Isolated canine hearts were preserved for 6 h at 5°C followed by normothermic reperfusion for 2 h. The dogs were divided into two groups of nine hearts each; group 1 received a nondepolarizing preservation solution in multidose, and group 2 received a single flush of University of Wisconsin (UW) solution. Serum MB-CK and mitochondrial aspartate aminotransferase (m-AAT) concentrations and calcium overload during reperfusion were lower in group 1 than in group 2. At the end of reperfusion, myocardial ATP and total adenine nucleotide concentrations were higher and mitochondrial morphology appeared more intact in group 1 than in group 2. Left ventricular diatolic function was preserved better in group 1 than in group 2. These results suggest that in 6-h heart preservation, a nondepolarizing solution applied in multidose fashion protects the myocardium from the deleterious effects of hypothermia and cardioplegia better than a single flush of UW solution.     

Warm flush at 37 °C following cold storage attenuates reperfusion injury in preserved rat livers

Abstract Pretransplant rinse solutions have been shown to reduce reperfusion injury in cold-stored liver grafts, especially at the nonparenchymal level in sinusoidal endothelial cells (SEC). In this study, different rinse temperatures were tested in a rat liver preservation model. Livers were washed out in situ via the portal vein with cold (4°C) University of Wisconsin (UW) solution, and after hepatectomy (t0), were stored for 8,16, or 24 h of cold ischemia time (CIT). After storage, livers were flushed with UW solution at either 4 °C, 20 °C, or 37 °C and reperfused for 90 min (37 °C). Control livers were reperfused at t0 without preflush. Levels of hyaluronic acid (HA), purine nucleoside phosphorylase (PNP), AST, and LDH were measured in the reperfusion medium. Bile production was monitored during reperfusion. At the end of reperfusion, liver biopsies were taken for enzyme histochemistry (5′-nucleotidase and LDH). After 8-h CIT and a flush at 4°C, a release of endogenous HA (-7 %) was observed, whereas uptake of exogenous HA occurred after the 20°C flush (2 %, P = NS) and after the 37°C flush (24 %, p < 0.001). HA release occurred at all three preflush temperatures after the 16-h CIT but was significantly lower when flushed at 37 °C (-10 %) that at 4 °C and 20 °C (-64 % and -17 %, respectively, p =0.05). After the 24-h CIT, the release of endogenous HA increased in the 4 °C and 20 °C preflush groups, but not in the 37 °C group. Levels of PNP and AST increased until the 24-h CIT in all groups but were significantly lower after preflush at 37 °C. Release of LDH did not increase with increasing periods of cold storage in any of the flush series. Compared to control livers, mean bile production during reperfusion was significantly decreased following preflush at 4°C or 37 °C after all periods of CIT. No differences in mean bile production could be demonstrated in the three preflush groups after any period of CIT. LDH activity in liver tissue was best preserved after the 8 and 16-h CIT in combination with the 37 °C preflush, indicating less hepatocellular damage. In conclusion, in cold stored rat livers flushed at 37 °C before reperfusion, SEC and hepatocellular damage is attenuated.     

Risk stratification of kidneys from donation after cardiac death donors and the utility of machine perfusion

Cantafio AW, Dick AAS, Halldorson JB, Bakthavatsalam R, Reyes JD, Perkins JD. Risk stratification of kidneys from donation after cardiac death donors and the utility of machine perfusion.
Clin Transplant 2011: 25: E530–E540. © 2011 John Wiley & Sons A/S. Abstract: There has been a dramatic increase in the utilization of kidneys from donors after cardiac death (DCD). While these organs represent an opportunity to expand the donor pool, the assessment of risk and optimal perioperative management remains unclear. Our primary aim was to identify risk factors for objective outcomes, and secondarily, we sought to determine what impact pulsatile machine perfusion (PMP) had on these outcomes. From 1993 to November 2008, 6057 DCD kidney transplants were reported to the Organ Procurement and Transplantation Network database, with complete endpoints for delayed graft function (DGF) and graft survival (GS). Risk factors were identified using a multivariable regression analysis adjusted for recipient factors. Age (50 yr) [OR 1.81, p < 0.0001] and cold ischemia time (CIT) (>30 h) [OR 3.22, p < 0.0001] were the strongest predictors of DGF. The use of PMP decreased the incidence of DGF only when donor age was >60 yr and improved long‐term graft survival when donor age was >50 yr. Donor warm ischemia time >20 min was also found to correlate with increased DGF. While the incidence of DGF in DCD kidneys is significantly higher, the only factors the transplant surgeon can control are CIT and the use of PMP. The data suggest that the use of PMP in DCD kidneys <50 yr old provides little clinical benefit and may increase CIT.     

Successful 72-hour cold storage of dog kidneys with UW solution

Effects of three cold-storage solutions on kidney function in dogs were examined with the isolated perfused (IPK) kidney model and the autotransplant model. EuroCollins' (EC) solution, phosphate-buffered sucrose solution, and a new solution developed at the University of Wisconsin (UW) were studied. Kidneys were cold-stored for 48 hr or 72 hr. With the IPK model, cold storage for 48 hr or 72 hr in each of the three solutions caused creatinine clearance to decrease by 80%-90%. More protein was excreted by kidneys stored for 48 hr in PBS solution than by kidneys stored in EC or UW solution; protein excretion after 72 hr of storage was similar for kidneys stored in EC or UW solution. Sodium reabsorption decreased after 48 hr or 72 hr of storage, but was higher in kidneys stored in UW solution (83% and 56%, respectively) than in EC solution (52% and 22%, respectively). With the autotransplant model, 40% of the kidneys were viable after 48-hr storage in PBS solution, but 80% viable when stored in EC solution and 100% were viable when stored in UW solution. All kidneys were viable when stored for 72 hr in UW solution; none were viable when stored for 72 hr in EC solution. These results suggest that UW solution effectively preserves kidneys for 72 hr. We previously reported successful 72-hr pancreas preservation. Recently UW solution was able to preserve canine livers for 30 hr. Thus, this single solution appears to be effective for preserving all intraabdominal organs and may simplify cold storage of organs for transplantation.     

Treatment of transplant kidneys during machine perfusion

The increasing use of donation after circulatory death (DCD) and extended criteria donor (ECD) organs has raised awareness of the need to improve the quality of kidneys for transplantation. Treating kidneys during the preservation interval could improve early and long-term graft function and survival. Dynamic modes of preservation including hypothermic machine perfusion (HMP) and normothermic machine perfusion (NMP) may provide the functional platforms to treat these kidneys. Therapies in the field of regenerative medicine including cellular therapies and genetic modification and the application of biological agents targeting ischaemia reperfusion injury (IRI) and acute rejection are a growing area of research. This review reports on the application of cellular and gene manipulating therapies, nanoparticles, anti-inflammatory agents, anti-thrombolytic agents and monoclonal antibodies administered during HMP and NMP in experimental models. The review also reports on the clinical effectiveness of several biological agents administered during HMP. All of the experimental studies provide proof of principle that therapies can be successfully delivered during HMP and NMP. However, few have examined the effects after transplantation. Evidence for clinical application during HMP is sparse and only one study has demonstrated a beneficial effect on graft function. More investigation is needed to develop perfusion strategies and investigate the different experimental approaches.     

Electrolyte equilibration of human kidneys during perfusion with HTK-solution according to Bretschneider

Twelve surgically removed human kidneys (mainly tumor kidneys) were investigated. The investigations comprised perfusion criteria (perfusion flow, perfusion pressure, perfusion resistance, electrolyte equilibration). During perfusion of the kidneys with HTK solution, the perfusion resistance was nearly three times as high in human kidneys as in canine kidneys perfused under the same conditions in previous studies. Beside possible species differences the raised perfusion resistance may be explained by the greater trauma to the human kidneys due to the surgery, the primary ischemic stress which cannot be avoided clinically and the often nonoptimal initial diuresis. Nevertheless definitive perfusion is possible under clinical conditions although pronounced increases of perfusion resistance may occur. As indicated by the raised perfusion resistance of human kidneys under clinical conditions as compared with canine kidneys in an experimental model, electrolyte equilibration of human kidneys was protracted. For this reason, a duration of perfusion of at least 10 min is necessary in clinical application of HTK solution, i.e., longer than in animal experiments.     

An automated and portable low-flow pulsatile perfusion system for organ preservation

While machine preservation reduces the incidence of delayed graft function in renal transplant recipients, it is only used in 10% of kidney transplantations. The performance of our portable, lowflow-pulsatile organ perfusion system was examined in a canine kidney autotransplantation model. Grafts were stored for 72 h by simple cold preservation in University of Wisconsin (UW) solution, or by high or low-flow machine preservation. After preservation, the grafts were autotransplanted and the animals were followed for 15 days. Graft function was better in machine-preserved kidneys. Tissue biochemistry indicated that machine preservation resulted in higher levels of adenine nucleotides and better histological integrity than the cold storage. While histology and biochemistry of machine-preserved groups were similar, electromicroscopy of high-flow grafts showed mild accumulation of intravenous debris and endothelial swelling. This study shows that a simplified machine perfusion technique is effective for organ preservation.     

新型器官保存液低温保存猪肾效果的研究

目的研究新型器官保存液Lifor液对猪肾脏的低温保存效果。方法 24只白色杂种猪,随机均分为Lifor液保存组和威斯康星大学保存液（UW液）组各12只,建立离体肾脏非循环灌注模型,供肾取出后分别以0~4℃的Lifor液和UW液灌注并低温保存,再根据保存时间随机分成2个亚组,分别为保存24h、48h组,然后行猪自体肾移植。比较两组肾脏低温保存结束后其病理学及肾皮质三磷腺苷（ATP）含量的改变,并观察自体肾移植后肾功能恢复情况。结果供肾离体保存24h、48h后,Lifor液组与UW液组的肾组织病理学改变基本一致,肾皮质ATP含量比较差异无统计学意义（P〉0.05）。移植后两组血清肌酐水平比较差异亦无统计学意义（P〉0.05）。结论 Lifor液低温保存肾脏的效果与UW液相当,且成本低廉,因此更具有临床应用前景。     

机器灌注在心脏死亡捐献供肾保存中的应用进展

机器灌注在保存心脏死亡捐献(DCD)供肾过程中,比单纯静态冷保存具有更多的优越性。随着我国DCD供肾的应用,机器灌注已受到各大移植中心的重视,并越来越多地应用于捐献器官的保存。本文就机器灌注的工作原理、机制及在保存DCD供肾过程中的应用研究进展作一综述。     

Postoperative recovery of mitochondrial function of the human liver graft procured and preserved with University of Wisconsin (UW) solution

Changes in arterial blood ketone body ratio (KBR) were investigated in 47 human liver transplantations. Of the 20 grafts preserved with University of Wisconsin (UW) solution, 10 had a cold preservation period of less than 10 h (UWS group) and 10 of more than 10 h (UWL group). In 27 other cases, grafts were preserved with EuroCollins (EC) solution for less than 10 h (EC group). In the EC group, KBR increased over 0.7 within 6 h after reperfusion of the graft in 17 cases (63%) and within 24 h in 7 cases (26%). In the 3 other cases, KBR failed to recover, and these patients underwent retransplantation. In the UW group, KBR recovered within 6 h in 13 cases (65%) and within 24 h in 7 cases (35%). There were no significant differences between the UWS and UWL groups. It is shown that the mitochondrial function of liver grafts preserved with UW solution can be well maintained even after extended preservation periods of more than 10 h.     

Pulsatile machine perfusion vs. cold storage of kidneys for transplantation: a rapid and systematic review

OBJECTIVE: To identify and prioritize key areas for further research in kidney preservation systems. MATERIALS AND METHODS: We conducted a systematic review and meta-analysis of the effectiveness of machine perfusion and cold storage techniques in reducing delayed graft function (DGF) and improving graft survival in recipients of kidneys from beating and non-heart-beating donors. Literature quantifying the link between DGF and graft survival was used to evaluate the potential long-term impact of machine perfusion and cold storage systems. Cox proportional hazards modelling was used to predict graft survival and graft years gained over 10 yr. Monte Carlo sensitivity analysis was conducted to evaluate stochastic uncertainties within the model. RESULTS: Machine perfusion leads to a relative risk of DGF of approximately 80% (67%, 96%) compared with cold storage, although the evidence base is limited in quality and study size. Direct evidence on graft survival at 1 yr demonstrates no statistically significant difference between machine perfusion and cold storage. Predictions based upon quantifying the link between DGF and graft survival suggest potential improvements of between 0 and 6% at 10 yr. DISCUSSION: Studies of high methodological quality and sufficient size are required to determine whether machine preservation leads to reduce rates of DGF. Predicted impact on graft survival implies that direct evidence would require a large population followed up over a long period of time. Registry database analysis supported by validation of the link between DGF and graft survival may be preferable and more feasible than randomized controlled trials.