Histidine-Tryptophan-Ketoglutarate (HTK) Is Associated with Reduced Graft Survival of Deceased Donor Kidney Transplants

Single-center studies have reported equivalent outcomes of kidney allografts recovered with histidine-tryptophan-ketoglutarate (HTK) or University of Wisconsin (UW) solution. However, these studies were likely underpowered and often unadjusted, and multicenter studies have suggested HTK preservation might increase delayed graft function (DGF) and reduce graft survival of renal allografts. To further inform clinical practice, we analyzed the United Network for Organ Sharing (UNOS) database of deceased donor kidney transplants performed from July 2004 to February 2008 to determine if HTK (n = 5728) versus UW (n = 15 898) preservation impacted DGF or death-censored graft survival. On adjusted analyses, HTK preservation had no effect on DGF (odds ratio [OR] 0.99, p = 0.7) but was associated with an increased risk of death-censored graft loss (hazard ratio [HR] 1.20, p = 0.008). The detrimental effect of HTK was a relatively late one, with a strong association between HTK and subsequent graft loss in those surviving beyond 12 months (HR 1.43, p = 0.007). Interestingly, a much stronger effect was seen in African-American recipients (HR 1.55, p = 0.024) than in Caucasian recipients (HR 1.18, p = 0.5). Given recent studies that also demonstrate that HTK preservation reduces liver and pancreas allograft survival, we suggest that the use of HTK for abdominal organ recovery should be reconsidered.     

Hypothermic storage of isolated human hepatocytes: a comparison between University of Wisconsin solution and a hypothermosol platform

Until now little is known about the functional integrity of human hepatocytes after hypothermic storage. In order to address this limitation, we evaluated several commercially available hypothermic preservation media for their abilities to protect freshly isolated hepatocytes during prolonged cold storage. Human hepatocytes were isolated from non-transplantable/rejected donor livers and resuspended in ice-cold University of Wisconsin solution (UW), HypoThermosol-Base (HTS-Base), or HypoThermosol-FRS (HTS-FRS) with or without the addition of fetal bovine serum. Cells were stored at 4°C for 24–72 h, and evaluated for hepatocyte viability (trypan blue exclusion, or labeling with fluorochromes), cell attachment, and function. The energy status of hepatocytes was evaluated by measurement of intracellular adenosine 5′-triphosphate. To determine whether the test cells expressed metabolic functions of freshly isolated cells, the activities of major phase I (cytochromes P450, FMO) and phase II (UGT, ST) drug-metabolizing enzymes were examined. Although hepatocytes are shown to be satisfactory after 24 h storage in all of the tested solutions, the cell viability, energy status, and xenobiotic metabolism following cold preservation in HTS-FRS was consistently and, in some cases, markedly higher when compared with other systems. The same metabolites for each of the tested substrates were detected in all groups of cells. Moreover, the use of HTS-FRS eliminates the need for serum in preservation solutions. HTS-FRS represents an improved solution compared to HTS-Base and UW for extending the shipping/storage time of human hepatocytes.     

Rat liver preservation

The mechanisms by which cold preservation solutions exert their protective effects are only partially understood. The consequences of mixing different solutions, with presumably different modes of action, may be additive and beneficial or may be deleterious. It is commonplace in clinical liver preservation to use Ringer's lactate (RL), Eurocollins (EC), and University of Wisconsin (UW) solution in sequence for washout of blood, precooling, and cold storage of the organ. In this study, 114 Sprague Dawley rats received orthotopic liver transplants that were flushed in various sequences with RL, EC, and UW solutions. One-week animal survival served as the criterion of preservation success. The results demonstrated that liver preservation with UW solution alone is significantly superior (P<0.01) to any combination of RL, EC, and UW solutions and may explain some of the instances of primary nonfunction in clinical liver transplantation.     

Effect of glycine on isolated,perfused rabbit livers following 48-hour preservation in University of Wisconsin solution without glutathione

Glycine has been shown to decrease membrane injury in isolated cells due to hypoxia or cold ischemia. The mechanisms of action of glycine are not known, but glycine may be useful in organ preservation solutions or in treating recipients of liver transplantation. In this study the isolated, perfused rabbit liver was used to measure how glycine affected liver performance after 48-h preservation in University of Wisconsin (UW) solution without added glutathione. UW solution is less effective for 48-h liver preservation when glutathione is omitted. Rabbit livers stored for 48 h without glutathione show a large increase in enzyme release (LDH and AST) from the liver and a reduction in bile production. The addition of 15 mM glycine to UW solution, in place of glutathione, did not improve bile production or reduce enzyme release. However, infusion of 10 mM glycine into the reperfused liver lowered LDH release significantly (from 2383±562 units/100 g to 1426±286 units/100 g) during the initial reperfusion of the 48-h preserved liver. Hepatamine, a parenteral nutrition solution containing glycine, as well as other amino acids, was also effective in lowering LDH release from the preserved liver. Although glycine reduced LDH release, it did not decrease the amount of AST released from the liver, nor did it improve bile production. Thus, we conclude that glycine, either in UW solution or given to the liver upon reperfusion, has no significantly beneficial effect as tested in this model. Further testing of glycine, however, should be conducted in an orthotopic transplant model in the rat or dog.     

The use of the University of Wisconsin (UW) and Euro-Collins (EC) solutions either alone or in a combined method

From June 1988 to October 1990, a total of 100 orthotopic liver transplantations (OLTs) in 91 patients were performed at the Hospital Clínic of Barcelona. Euro-Collins (EC) solution was used as the flush and storage solution in 29 livers, and the University of Wisconsin (UW) solution was used in 24. A combined method, consisting of flushing and harvesting the liver with UW solution through the portal vein and with EC solution through the aorta, was used in the remaining 47 livers. Livers harvested using such a combined method showed substantially better postoperative function in terms of AST, ALT, and prothrombin activity than those harvested in EC solution alone. Although AST and ALT values were lower in patoents whose livers were harvested using the combined method than with UW alone, differences were not significant. On the other hand, prothrombin activity was consistently better in the UW group. Bilirubin levels, platelet count, and bile output showed no difference among the three groups. We conclude that the combined use of UW and EC solutions for flushing and harvesting is not hazardous to human liver preservation and, in fact, may considerably reduce the amount of UW solution needed and, consequently, the costs.Preliminary results from this study were presented at the First International Congress of the Society for Organ Sharing in Rome in June 1991 and will also appear in Transplantation Proceedings.     

Effect of blood group and HLA matching on pancreas graft survival with the use of UW solution

Pancreas graft survival is influenced by various donor and recipient factors. Factors that have posed serious problems to pancreas transplantation have included the limited cold ischemia time, early graft thrombosis, and rejection. A limited cold ischemia time not only causes problems in terms of logistics but also implies limitations with regard to HLA matching and organ exchange. Between August 1988 and August 1989 we performed a prospective, nonrandomized European multicenter study to evaluate the effect of University of Wisconsin (UW) solution on pancreas graft survival. In addition, donor and recipient factors were collected and their influence on graft survival analyzed. Overall pancreas graft survival at 1 and 4 years was 67% and 59%, respectively (n=62). When only simultaneous pancreas and kidney transplants were included, the graft survival was 70% and 63% at 1 and 4 years, respectively. The incidence of pancreas graft thrombosis was 8%. Cold ischemia time was not found to significantly influence pancreas graft survival even when it exceeded 12h. Factors that did were HLA-DR matching, simultaneous pancreas and kidney transplantation versus pancreas transplantation alone, and ABO blood group matching. We feel that the use of UW solution for pancreas preservation has contributed to improved pancreas graft survival and has reduced early graft thrombosis despite much longer cold ischemia times of over 12 h. Given this and the significant effect of HLA and blood group matching, we conclude that more attention should be paid to preoperative matching and organ exchange in order to further improve pancreas graft survival.     

A comparison of histadine lactobionate solution with University of Wisconsin solution for rat liver and heart preservation

We developed a new solution mainly composed of Na-lactobionate and histidine (HL) and compared the effectiveness of this solution with that of University of Wisconsin (UW) solution using orthotopic liver and heterotopic heart transplantation in rats. The new solution has a higher sodium content and a lower potassium content (Na, 90 mEq/l; K, 45 mEq/l) than UW. Hydroxyethyl starch, adenosine, dexamethasone and insulin are not included. Buffering capacity is increased by adding histidine (90 mm/l) together with KH₂PO₄ (20 mm/l). Rat liver was perserved in either UW or HL solution hypothermically for 24 h and then transplanted orthotopically into the recipient rat. The heart was preserved in either solution for 18 h and transplanted heterotopically into the recipient rat. The 1-week survival rate for rats receiving livers preserved in UW for 24 h at 4°C was 29% (5/17). In contrast, the new solution (HL) gave a 78% (11/14) survival rate (P < 0.01). The 1-week heart graft survival rate, using UW solution was 50% (3/6), following 18-h cold preservation, whereas all hearts (7/7) continued to beat for over a week using new HL solution (P < 0.05). These results demonstrated that the new HL solution, with a substantial buffering capacity, was superior to UW solution in rat liver and heart preservation.     

Effect of S-adenosylmethionine on hepatic injury from sequential cold and warm ischemia

We investigated whether S-adenosylmethionine (SAM) treatment improved ischemic injury using perfused rat liver after sequential periods of 24 h cold and 20 min re-warming ischemia. SAM (100 micromol/L) was added to University of Wisconsin (UW) solution and Ringers lactate solution. After cold and sequential warm ischemia, releases of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP) markedly increased during reperfusion. The increase in PNP was significantly reduced by SAM treatment. While the concentration of reduced glutathione (GSH) in ischemic livers significantly decreased, the concentration of glutathione disulfide (GSSG) increased. This decrease in GSH and increase in GSSG were suppressed by SAM treatment. Lipid peroxidation was elevated in cold and warm ischemic and reperfused livers, but this elevation was also prevented by SAM treatment. Hepatic ATP levels were decreased in the ischemic and reperfused livers to 42% of the control levels. However, treatment with SAM resulted in significantly higher ATP levels and preserved the concentration of AMP in ischemic livers. Our findings suggest that SAM prevents oxidative stress and lipid peroxidation and helps preserve hepatic energy metabolism.