Renal injury by companion kidney during hypothermic pulsatile perfusion

To study possible deleterious effects of traumatized kidneys on well harvested ones during hypothermic pulsatile perfusion, paired dog kidneys were monitored on two separate MOX-100 machines connected to pool a common perfusate. First kidneys were optimally harvested and baseline renal vascular resistance (RVR) calculated from pressure divided by flow (mm Hg/ml minute). After 1 hour second kidneys were added and observation was continued for an additional 3 hours. RVR in first kidneys increased immediately and doubled by 3 hours when ischemic traumatized second kidneys were added, 0.70 +/- 0.07 to 1.37 +/- 0.15 (p less than 0.001). No change in RVR was noted when second kidneys were optimally retrieved or if the dogs were heparinized prior to nephrectomy. Histological examination showed no evidence of vascular obstruction in any kidney, but only tubular necrosis in nonheparinized, traumatized second kidneys. Weight gain in optimally removed kidneys was 41%, but only 8% in ischemic kidneys. RVR decreased unexpectedly in ischemic kidneys during 3 hours of perfusion (p less than 0.01). Release of vasoactive substances by damaged kidneys and arteriovenous shunting may explain these findings. Separate perfusion systems seem to be justified when one of two cadaver kidneys is of marginal quality.     

Extended cadaver renal preservation with combined simple cold storage and hypothermic pulsatile perfusion

Although simple cold storage and hypothermic pulsatile perfusion are each accepted methods of cadaver kidney preservation the efficacy of combining these techniques for extended renal preservation is unclear. In 18 patients cadaver allografts were used that had been preserved with combined simple cold storage (range 6 to 20 hours) and hypothermic pulsatile perfusion (range 9 to 38 hours). The total interval of preservation for these kidneys ranged from 25 to 48 hours. Excellent post-transplant graft function was achieved in all cases, with a mean serum creatinine nadir of 1.4 mg./dl. In 14 patients (78 per cent) grafts continued to function at intervals of 3 to 38 months after transplantation. Of the remaining 4 patients 3 lost the grafts to rejection, while 1 died 7 months after transplantation with a well functioning graft. These data suggest that the combination of simple cold storage and hypothermic pulsatile perfusion provides a safe and effective method for extended renal preservation.     

Loss of myocardial viability following hypothermic perfusion storage from contaminating trace elements in the perfusate

Two groups (A and B) of isolated baboon hearts were preserved by continuous hypothermic perfusion storage for 48 hours using perfusates that, according to the manufacturers, differed only in the concentrations of the contaminating trace elements iron, lead, and arsenic. Storage with the perfusate containing the higher concentration of these elements (perfusate B) led to significantly less gain in heart mass, a greater reduction in coronary flow, coronary sinus effluent lactate, and myocardial arteriovenous oxygen difference and a greater increase in coronary sinus effluent lactate dehydrogenase, when compared with perfusate A. Group B hearts totally failed to support the circulation following orthotopic transplantation, whereas group A hearts showed excellent function. Group B hearts had undergone the typical changes of enhanced resting myocardial tension during the storage period (before warm blood reperfusion); we proposed that these changes were brought about by the production of superoxide anions and radicals by the higher relative concentration of iron, or a combination of contaminating trace elements, in perfusate B. To confirm that these perfusates did differ significantly in the concentration of these trace elements, in particular with regard to iron, the superoxide anion activity in both solutions was measured and was found to be significantly higher in perfusate B. The addition of superoxide dismutase to both solutions inhibited superoxide anion activity by more than 80%.     

Single-donor cold storage versus machine perfusion in cadaver kidney preservation

Preservation of thirty-eight consecutive renal allograft donors was studied in a prospective, randomized protocol. Procurement was in-situ cooled, en-bloc nephrectomy accomplished by a single program. Nine pairs were deleted because of nonutilization of one kidney or change in mode of preservation. The remaining twenty nine pairs were implanted by twenty two institutions through usual organ sharing policies. Results showed that posttransplantation dialysis was required in 17% of machine-perfused and 63% of cold-stored allografts, which reached statistical significance (P less than 0.01). This increased number of dialyses in patients receiving the cold-stored kidneys offsets cost savings achieved through transporting cold stored allografts. This study shows machine-perfused renal allografts to be superior to paired, cold-stored allografts when analyzed with respect to early graft function.     

低温机械灌注与冷藏保存在尸体肾移植中的应用

随着尸体肾移植的文泛开展,冷藏保存技术作为常规的供肾保存技术受到了研究者的挑战.近年来不断有回顾性的研究表明低温机械灌注保存能改善肾移植的近期效果,但是大规模的多中心前瞻性研究还未见报道.     

Comparison of pulsatile perfusion and cold storage for paired kidney allografts

Use of pulsatile perfusion to optimize outcomes in deceased donor kidney transplantation remains controversial. This study is a retrospective analysis of all cadaveric renal allografts procured locally by our center over a 3-year period. Kidney pairs were identified in which one kidney underwent pulsatile perfusion and transplantation at our center, whereas the contra-lateral kidney underwent cold storage and transplantation at another center. Eighty-eight percent of the exported kidneys were six-antigen matches. Study outcomes included 1-year graft and patient survival, delayed graft function, and need for posttransplant dialysis. Recipients had similar demographic and disease characteristics. Survival for pulsatile perfusion and cold storage were 95% and 88% (graft, P=0.43) and 98% and 90% (patient, P=0.36), respectively. The incidence of delayed graft function was 5% and 35% (P<0.01), whereas posttransplant dialysis was 5% and 30% (P<0.01), for pulsatile perfusion and cold storage, respectively. These data support routine use of pulsatile perfusion.     

Hepatic function in hypothermically stored porcine livers: comparison of hypothermic machine perfusion vs cold storage

Hypothermic machine perfusion (HMP) has a potential to relieve the current donor liver crisis by providing an improved and extended preservation method. This study examined the effect of HMP on hepatocellular functions, using a prototype liver transporter capable of preserving livers for 24 hours. Livers obtained from adult farm pigs (28 to 32 kg body weight) were divided into three groups: fresh control, HMP, and simple cold storage (n = 4 each). A 4-hour normothermic reperfusion of livers was conducted to assess hepato-metabolic and cellular functions. The hepatic transport function, as indicated by canalicular excretion of indocyanine green, was improved in the HMP group than in the SCS group. The overall tissue viability, as indicated by oxygen consumption levels, was notably improved in HMP and control livers as compared to the SCS group. Higher bile production in both the preserved groups as compared to the fresh control livers could be a result of biliary edema and leakage of plasma into the canaliculus. The hepato-cellular injury, measured by ALT, release was significantly greater in the SCS group as compared to the HMP and control groups. These findings suggest that HMP could be a better method to preserve hepatic function and overall tissue viability as compared to SCS. Improved hepatic functions are indirect indicators of superior microcirculation and sinusoidal endothelial cell functions. Further studies in progress will evaluate these functions to confirm the significance of these observations.     

Preservation techniques for heart transplantation: comparison of hypothermic storage and hypothermic perfusion

Preservation of the donor heart is an important and controversial subject in heart transplantation. This study compares simple hypothermic storage and hypothermic perfusion in a swine model of heart transplantation (n = 14). The donor hearts of group A (n = 7) were placed in simple hypothermic storage for 5 hours. The donor hearts of group B (n = 7) were placed onto a perfusion apparatus for 5 hours, with pressure maintained at 28 cm of H2O and a myocardial temperature of 8 to 10 degrees C. In both groups the hearts were initially protected with isosmolar potassium cardioplegic solution. The perfusate in group B contained moderate sodium, mannitol, glucose, insulin, and oxygen. The ischemic interval within both groups was 6 hours including orthotopic transplantation. Investigation was conducted at three time periods: prepreservation, postpreservation, and immediately after loading. For both groups there was nonsignificant depression of myocardial function (cardiac index, stroke index, stroke work index, ejection fraction, and wall stress) at the postpreservation period. After volume loading, for the hypothermic perfusion group there was significant improvement of myocardial function (cardiac index, p less than 0.01; stroke index, p less than 0.01) with no significant change in heart rate, systemic vascular resistance, and systolic blood pressure. There was also significant improvement in myocardial performance (p less than 0.05) for the hypothermic perfusion group after volume loading. Ultrastructural changes were minimal for both groups, and there were no major heart transplantation after 6 hours of ischemia; however, hearts retain their contractile capacity better after hypothermic perfusion than after simple hypothermic storage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of function during hypothermic renal perfusion

Various methods have been suggested for assessing the viability of organs prior to transplantation but none has proved to be entirely satisfactory. The assay of renal function by normothermic perfusion has been shown to correlate with parallel transplantation studies in rabbits, and is sensitive to the damage caused by warm ischemia. In this paper we describe an attempt to simplify and adapt this assay for use as a clinically acceptable viability test, by measuring the urine/perfusate ratios of radiolabeled albumin and para-aminohippurate at a temperature of 20°C. We found that the measurements did correlate with improvements to the perfusate that result in higher adenine nucleotide levels, but failed to indicate deterioration when 24-hr preservation of rabbit kidneys was compared with 48 hr preservation. It is possible that this may be due to failure of the tests to detect vascular injury.     

Optimizing CO2 normalizes pH and enhances chondrocyte viability during cold storage

Fresh osteochondral allografts are an important treatment option for the repair of full‐thickness articular cartilage defects. Viable chondrocytes within the transplanted tissue are considered important to maintaining matrix integrity. The purpose of this study is to determine whether an increase in pH decreases chondrocyte viability during cold storage and whether equilibration of Dulbecco's modified Eagle's medium (DMEM) in 5% CO₂ normalizes pH and increases chondrocyte survival during storage at 4°C. Freshly isolated bovine articular chondrocytes cultured in alginate beads were stored for up to 5 days at 4°C or 37°C in DMEM exposed to ambient air or in DMEM equilibrated with 5% CO₂. Chondrocyte viability was determined by flow cytometry. Physiologic pH was maintained when DMEM was equilibrated with 5% CO₂, while pH increased in ambient air. After 5 days of storage at 4°C, chondrocyte necrosis was higher when stored in ambient air than if equilibrated with 5% CO₂. No decrease in chondrocyte viability was observed with storage at 37°C. In addition, chondrocyte viability in bovine cartilage osteochondral cores was examined after storage for 14 days at 4°C in DMEM with and without HEPES, and with and without 5% CO₂. Under these conditions, the superficial layer of chondrocytes was more viable when stored in DMEM with HEPES or DMEM equilibrated with 5% CO₂ than when stored in DMEM in ambient air. This data shows that an increase in pH decreased bovine chondrocyte viability when refrigerated at 4°C in DMEM, and that optimization of CO₂ normalized pH and improved chondrocyte viability during cold storage in DMEM. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:643–650, 2008     

Role of erythrocytes in short‐term rewarming kidney perfusion after cold storage

Short term normothermic reconditioning by machine perfusion after cold storage has shown beneficial effects in renal transplantation models. Systematic investigations concerning the inclusion of washed erythrocytes as oxygen carriers are lacking in this context. Porcine kidneys were subjected to 20 h of static cold storage. Prior to reperfusion, grafts were put on a machine for 2 h of oxygenated (95% O₂; 5% CO₂) rewarming perfusion. In one group (n = 6) washed erythrocytes were added to the perfusate after temperature has reached 20°C; the other group (n = 6) was run without additives. Control kidneys (n = 6) were immediately reperfused without treatment. Upon reperfusion in vitro, a more than twofold improvement of renal clearance of creatinine, urinary protein loss, fractional excretion of sodium, efficiency of oxygen utilization (TNa/VO₂) and a significant reduction of innate immune activation (HMGB1, tenascin C, expression of TLR4) was seen after machine perfusion, compared with the controls. However, no advantage could be obtained by the addition of erythrocytes and inner cortical tissue pO₂ always remained above normal values during cell‐free machine perfusion. Our data strongly argue in favor of a rewarming perfusion of cold stored donor kidneys but do not substantiate an indication for adding oxygen carriers in this particular setting.     

A randomized prospective trial of cold storage versus pulsatile perfusion for cadaver kidney preservation

In a randomized trial conducted in 9 Ontario transplant centers, 107 cadaver kidney donors were randomized so that both kidneys were preserved either by simple cold storage (CS) or by pulsatile perfusion (PP). After exclusions and losses to follow-up, there were 90 patients in the CS group and 91 in the PP group. Total preservation times were similar in the two groups (27.7 +/- 12 and 30.5 +/- 10 hr), as was the proportion of kidneys with long (greater than 36 hr) storage times (21% and 26%). Twelve-month graft survival was 70% for CS and 75% for PP (not significant, NS); patient survival was 89% for CS and 95% for PP (NS). The incidence of kidneys that never functioned was also similar in the two groups (14% vs. 15%, respectively). However, the risk of early, reversible graft dysfunction was significantly higher for CS (44%) than for PP (31%). The same effect was observed with two independent methods of assessment of graft function. The chief effect of PP on renal function was in reduction of the mean serum creatinine at 1 week (632 mumol/L for CS vs. 403 mumol/L for PP, P less than .001). There was no significant influence of storage mode on the rapidity of urine output or number of dialyses required. No apparent effect of cyclosporine on the results with either preservation method was seen. We conclude that the case for retention of PP for kidney preservation now rests on its ability to achieve slightly better initial function than CS. However, the mild dysfunction observed with CS did not appear to translate into long-term detriment. When these considerations are weighed against the increased cost of PP, the advantage may lie with cold storage.     

Seventy-two-hour preservation of porcine liver by continuous hypothermic perfusion with UW solution in comparison with simple cold storage

Porcine livers preserved for 72 hr using continuous hypothermic perfusion (CHP) were studied in order to compare the effects of CHP on energy metabolism with those of simple cold storage (SCS). The livers of the CHP group were perfused in situ for 72 hr at 7 degrees C with University of Wisconsin (UW) solution and FC-43 as an oxygen carrier, while those of the S1 group were stored ex situ for 48 hr at 4 degrees C in UW solution, and those of the S2 group for 72 hr. They were then recirculated with human blood at 37 degrees C for 2 hr for the evaluation of their viability. At the end of preservation, significantly higher levels of total adenine nucleotides (TAN) and energy charge (EC) were observed in the CHP group compared with the S1 and S2 groups. At 2 hr recirculation, the level of TAN was significantly higher in the CHP group than in the S1 and S2 groups. The EC level was also higher in the CHP group than in the S2 group. During recirculation, the ketone body ratio (acetoacetate/beta-hydroxybutyrate) was higher in the CHP group than in the S2 group. The values in the CHP group were above 1.0 after 45 min recirculation. There were no significant differences in the pyruvate/lactate ratio and lactate level between the CHP and S1 groups. However, these values were significantly different from those in the S2 group. The present findings demonstrate that CHP using UW solution and the oxygen carrier was better able to preserve the energy metabolism of the porcine liver for 72 hr than 48-hr SCS in UW solution.     

Maintenance of lung viability for transplantation after long periods of hypothermic perfusion

A hamster-to-rat vascularized cardiac xenograft model has proven useful for xenogeneic transplantation experiments. This xenograft preparation was consistently reproducible and had a technical failure rate of less than 1%. Survival times ranged from 70 to 79 hr with a median survival time of 74.5 hr. Rejection time was well documented using electrocardiograms. Hemagglutinin and hemolysin activity of rat sera against hamster red cells were undetectable until 48 hr after transplantation. Prior to this time, electrocardiographic and histologic changes were minimal. Main histological findings of the rejected cardiac xenograft was thrombosis, congestion, massive and diffuse hemorrhage with necrosis and polymorphonuclear infiltration with a few lymphocytes.