Despite all recent improvements of preservation solutions, donor treatment, and harvesting protocols, renal transplantation is still afflicted by a notable degree of preservation-associated ischemia and reperfusion injury. Especially in organs that had prolonged ischemic periods or originate from extended criteria donors, early function can be seriously decreased after transplantation. Thus, delayed graft function (DGF) or even primary nonfunction of the transplanted kidney is encountered in up to 50% resulting in the requirement of hemodialysis treatment and an increased risk of acute rejection. Particularly in kidneys from brain-dead donors, DGF has been shown to independently favor the incidence of chronic nephropathy and later graft loss (1, 2).
DGF can be caused by donor and recipient factors, many of which are preexisting. Preservation, however, either through method or solution is a modality that can further diminish injury and improve results. Thus, any improvement in the preservation of grafts represents a valuable advance to enlarge the total number of viable donor organs available for transplantation.
A more and more accepted method to maintain donor organ viability during the preservation period is seen in the technique of continuous hypothermic machine perfusion (HMP).
Even though the mechanistic basis of MP is not yet fully understood, clinical studies have shown superiority of HMP over cold storage (CS) for kidney preservation (3–5).
Superiority of HMP over conventional CS was most evident in enhanced criteria grafts originating from heart-beating donors. When applied in a donated after cardiac death setting, the advantages of HMP were less evident (6–8), unless additional oxygenation of the perfusate was provided during cold perfusion (9–11).
Although the ischemic period itself predisposes the tissue to compromised resumption of cellular function on warm reperfusion, a large amount of actual preservation-induced graft injury is only manifested during the initial phase after transplantation (9, 12, 13).
These include blood endothelial interactions and the initiation of inflammatory cascades acting on vascular lining and parenchyma, still afflicted with ischemia induced dysequilibration of cellular signal homeostasis and exhausted energetic status.
To alleviate the incompetence of the energy-depleted cell to cope with the acute metabolic challenge on rearming, the concept of hypothermic reconditioning (HR) by a brief period of oxygenation immediately before transplantation has been developed by our group.
In previous experiments, we showed that short-term oxygenation of conventionally cold-stored organs before reperfusion significantly improves early functional recovery of liver and kidney grafts in vitro (14–16).
This study was now undertaken to provide preclinical in vivo evidence if and to which extent HR by 2 hr of machine perfusion subsequent to conventional CS actually improves renal outcome after transplantation.
There were no differences in cold ischemic times (mean: 21±0.2 hr) or anastomosis times (mean: 35±2 min) between the groups.
Injury Parameters During Machine Perfusion Preservation
The duration of HMP had a significant influence on tissue release of lactate dehydrogenase (LDH) and lipid peroxidation.
Activities of LDH in the circulation perfusate after 21 hr of HMP (47.8±4.9 U/L) were significantly higher than after 2 hr of HR subsequent to 19 hr of CS (24.5±1.7 U/L; P<0.05).
Similarly, concentrations of free high mobility group protein B1 (HMGB-1) (2.1±0.4 vs. 0.8±0.3 ng/mL) and lipoperoxides (17.7±1.2 vs. 7.5±0.6 nmol/mL) were found more elevated after HMP than at the end of HR (HMP vs. HR; P<0.05).
Perfusate concentrations of the endothelial injury marker von Willebrand Factor were similar after HMP and HR (HMP vs. HR: 3.6±0.3 vs. 3.3±0.4).
Renal vascular resistance averaged 0.32±0.04 after 21 hr of HMP and 0.40±0.09 at the end of HR, the difference being not significant.
Cortical Tissue Perfusion on Transplantation
Microcirculatory tissue perfusion on renal reperfusion after transplantation showed small but significant differences between the three experimental groups. Cortical erythrocyte flux tended to be higher after HMP and HR, when compared with CS (78%±9%, 78% ±11%, 59% ±10% of baseline HMP, HR, and CS, respectively, P<0.05 vs. CS, Dunnett test).
Renal Function After Transplantation
Serum creatinine rose after kidney transplantation, but levels remained significantly lower in the machine perfusion group than after CS (Fig. 1a). Interestingly, HR also significantly reduced the increase of serum creatinine after transplantation.
Peak serum levels of creatinine were 12.1±3.8, 7.1±1.6 (P<0.05 vs. CS), and 5.0±1.5 (P<0.05 vs. CS) after CS, HMP, and HR, respectively.
Similar results were found with respect to systemic urea concentrations (Fig. 1b).
HMP and HR resulted in significant reduction of postoperative urea concentrations compared with conventional CS. The lowest rise of serum urea levels was seen in the HR group. Peak concentrations of urea were 180±56, 110±51 (P<0.05 vs. CS), and 66±7 (P<0.05 vs. CS) after CS, HMP, and HR, respectively.
Tubular reabsorption of sodium was transiently disturbed after transplantation of cold-stored kidneys (Fig. 2). Fractional excretion of Na+ exhibited a progressive rise during the first 5 days after transplantation but normalized thereafter.
In contrast, HMP and HR resulted in a continuously unaffected reabsorption rate throughout the whole observation period.
Urine production did not differ between the groups. All animals had spontaneous urine flow immediately after revascularization and urine production remained above 500 mL/day until the end of the observation period.
No differences were observed among the groups in urinary protein content, release of LDH, or release of N-acetyl-beta-D-glucosaminidase.
Molecular Surface Activation and Signal Homeostasis
Cellular surface activation during preservation, triggering proinflammatory and innate immune response on reperfusion, was found to be influenced by the type of preservation (Fig. 3).
After conventional CS, messenger RNA (mRNA) for toll-like receptor (TLR)4 was found to be significantly reduced by HMP, as well as after HR, when compared with the cold-stored kidneys. However, no beneficial effect could be substantiated with respect to the proinflammatory adhesion molecule intercellular adhesion molecule 1 (ICAM-1), gene expression of which remained unchanged by HR and was even increased after HMP.
Additional evidence for a beneficial effect of HR on vascular signal homeostasis was seen in the preservation of the transcription factor von Kruppel-like Factor 2 (KLF-2).
Although during CS mRNA for KLF-2 declined to approximately 40% of baseline values, HR was as effective as HMP in significantly preventing the preservation-induced decrease of KLF-2 before reperfusion.
Thirty minutes after onset of reperfusion, mRNA expression of KLF-2 was still significantly lower in the CS group (0.8±02) than after HMP or HR (1.7±0.2 and 2.2±0.3 a.u., respectively), whereas differences in gene expression of ICAM-1 and TLR4 ceased to be significant among the groups. After 1 week following autotransplantation, the expression patterns did no longer differ between the three groups for either of the parameters.
Release of “Danger Associated Pattern” Molecules
Postischemic changes pertinent to the innate immune signaling were also influenced by HR. Extracellular HMGB-1 was investigated as potent stimulus to TLR receptor-mediated innate immune response (17), showing that renal release of HMGB-1 was significantly attenuated by HR, when compared with the untreated controls and was not different from the results after HMP (Fig. 4).
Light microscopy performed on tissue samples obtained after conclusion of the experiment 1 week after transplantation did not disclose significant differences among the groups. Overall, only slight alterations of normal structural appearance were observed in any group comprising limited edema and occasional swelling or vacuolization of tubular cells (Fig. 5).
To use high-risk grafts more efficiently, dynamic preservation methods should be applied, striving for reconditioning of cellular homeostasis and viability before transplantation. Ischemic storage is associated with a variety of disarrangements in cellular homeostasis, including mitochondrial redox status and energy metabolism, signal transduction, and gene up- or down-regulation (18, 19). Hypothermic pulsatile MP has often been conceived to be the optimal preservation method for kidneys (3, 5, 20) but falls back from being the general standard yet. Most renal transplants are still subjected to static CS as the easier applied technique, requiring less expenditure for logistics. One major drawback of continuous HMP lies in the necessity of being timely available at the place of organ retrieval, which may putatively complicate the retrieval procedure in peripheral hospitals. Moreover, in most cases, it is not evident at the time of kidney retrieval whether the graft is going to endure extended times of cold ischemia and might need or not need dynamic preservation instead of CS.
The technique of HR after conventional CS offers a handy alternative, allowing to be applied only if deemed necessary at the discretion of the implant team.
A major advantage of machine perfusion has often been seen in the ability to maintain higher adenosine triphosphate levels during preservation compared with simple CS (9, 21, 22), which eventually results in better equilibration of cytosolic and mitochondrial homeostasis of the graft. Thus, posttransplant renal dysfunction caused by impaired recovery of oxidative phosphorylation and failure of deenergized mitochondria to comply with the urgent need for adequate energetic support on reperfusion (15, 23) could be mitigated.
However, no comparative data are available as to whether renal tissue aerobiosis and energetic homeostasis are necessary during the whole storage period or only before warm reperfusion at the time of transplantation, which could be obtained by only a brief period of preimplantation machine perfusion, the duration of which is limited to the time available while awaiting crossmatch.
We and others could previously show in the liver that end-ischemic restoration of energy homeostasis by HMP or gaseous oxygen persufflation effectively improves graft function after transplantation (24, 25).
Herein, we could demonstrate similar effects in the kidney, but this study extends on previous reports in showing that the benefits of HR were actually comparable with the results of long-term HMP. This holds true in kidneys that are retrieved from donors with intact circulation. Whether these results might be transferable to grafts that were retrieved after cardiac arrest, where warm-ischemic tissue alterations before cold preservation add to the putative tissue injury, remains to be investigated.
In addition to parenchymal tissue, especially endothelial cells are sensitive to energy deficiency, specifically in the cold (26), and anoxic swelling of vascular endothelium during ischemia may account for microcirculatory dysfunction on reperfusion. Moreover, altered vascular or glomerular endothelium may contribute to reperfusion injury of a graft by expression of adhesion molecules, which have been shown to be a relevant denominator for postischemic tissue alteration (27, 28).
Cessation of pulsatile flow in the vasculature has been shown to result in a swift decrease of the transcription factor KLF-2 during static preservation of murine aortic grafts (29), and similar kinetics conjecturally occur in renal grafts after cessation of pulsatile perfusion (30). KLF-2 is normally operative in suppressing the production of cytokines that eventually would promote inflammatory tissue alterations on sanguineous reperfusion. Thus, one mechanistic basis for the improved renal preservation by HMP has been seen in the prevention of KLF-2 breakdown through continuous pulsatile perfusion. Similarly, pharmacologic increase of KLF-2 expression by simvastatin also prevented cellular apoptosis in cultured endothelial cells (29). In this study, we could confirm that the actual decline of KLF-2 expression on CS is prevented by HMP in renal grafts. Of note, HR was able to effectively restore normal mRNA levels of KLF-2 before transplantation, putatively resulting in an improved suppression of proinflammatory cytokine production on warm reperfusion.
HR furthermore resulted in lower systemic levels in free HMGB-1 after transplantation and in the machine perfusate. HMGB-1 is a universal sensor of nucleic acids in innate immunity signaling and one of the known ligands of TLR-mediated activation of innate immunity (17). TLRs are a family of pattern recognition receptors expressed not only on cells of the innate immune defense (31) but also on vascular endothelium (32) and renal epithelial cells (33). It is already known that ligands to TLRs are enhanced after ischemia-reperfusion (17), and there is strong evidence for a role of TLR in renal ischemia-reperfusion injury (34). TLR4-triggered signaling, for example, promoted by “DAMPs,” which interact with and activate innate TLR-bearing vascular cells, is considered to contribute to the development of atherosclerosis and chronic rejection (35).
In conclusion, HR represents an attractive alternative to long-term HMP in regard to the early functional recovery of kidney grafts from heart-beating donors.
On the other hand, obvious logistic advantages lie in the ease of simply putting the graft on CS at the time of retrieval and transportation.
After reaching the transplant clinic, the kidney can then be subjected to a short period of preimplantation machine perfusion, whereas, for example, crossmatch results are waited for, without extending cold ischemia time. We, therefore, recommend this technique for clinical testing.
MATERIALS AND METHODS
All experiments were performed in accordance with the federal law regarding the protection of animals.
The principles of laboratory animal care (NIH Publication No. 85-23, Revised 1985) were followed.
German landrace pigs weighing 25 to 30 kg were used for the study. All animals had free access to tap water and standard pellet food. Solid food was withdrawn 24 hr before beginning of the experiment.
A porcine autotransplantation model was used as previously established in our laboratory and described by Maathuis et al. (36).
Under general anesthesia, the right internal jugular vein was cannulated with polyethylene tubing for infusion and daily collection of blood samples.
After left nephrectomy, the kidneys were flushed on the back table with histidine-tryptophan-ketoglurate (HTK) solution from a height of 100 cm until the effluent was clear.
The grafts were randomly assigned to one of the three experimental groups:
- Kidneys were simply cold stored at 4°C in HTK solution (CS; n=5).
- Kidneys were preserved by HMP with kidney perfusion solution-1 (KPS-1) solution in a Lifeport Kidney Transporter (HMP; n=5).
- Kidneys were initially preserved by conventional CS in HTK for 19 hr and then subjected to HR by approximately 2 hr of subsequent machine perfusion with KPS-1 in the Lifeport system immediately before implantation (HR; n=5).
Machine perfusion in groups 2 and 3 was performed by pressure controlled (30/20 mm Hg) perfusion with 1 L of recirculating KPS-1 solution in a pulsatile manner at 60 bpm.
A modification of the machine allowed for oxygenation of the perfusate (PO2>500 mm Hg) during preservation.
At the end of the preservation period, the kidneys were autotransplanted subsequent to removal of the native contralateral kidney. Vascular anastomoses were performed end to side (renal vein–vena cava) and end to end (left renal artery–right renal artery). At the time of reperfusion, 50 mL of 50% glucose was infused to induce osmotic diuresis.
No other diuretics were given.
The ureter was cannulated with polyethylene tubing, which was tunneled through the abdominal wall, allowing continuous visual inspection of urine production.
Renal tissue perfusion was assessed noninvasively 10 min after reperfusion as mean cortical erythrocyte flux, determined by Laser Doppler flowmetry as detailed previously (9, 37).
To account for temporal variations in blood flow, we calculated the mean flux value over 10 sec of recording, and to eliminate the influence of spatial heterogeneity, we performed measurements on four distinct places of the renal surface. All flux measurements were taken as percent variation from the baseline values obtained from the nonischemic native kidneys.
Using a jugular catheter left in place after the operation, venous blood samples were taken daily from the beginning of the intervention until the animals were killed on postoperative day 7. At the same time, urine samples were collected for biochemical analyses.
These were used for measuring the levels of creatinine, urea, sodium, and LDH, which were determined with methods of the clinical routine.
Real-time quantitative reverse transcriptase polymerase chain reaction (PCR) analysis of inflammation-related genes was performed on renal cortical biopsies taken at the end of preservation, 30 min after reperfusion, and at the conclusion of the experiment, 1 week after transplantation.
Total RNA was isolated from snap-frozen samples using TRIreagent (Applied Biosystems, Darmstadt, Germany).
Equal amounts of RNA were quantified by Nano Drop (Thermo Fisher) and complementary DNA by incubation with High Capacity cDNA RT Kit (Applied Biosystems). The PCR reaction mix was prepared by using TaqMan GenEx Master Mix (Applied Biosystems). The amount of specific mRNA in the tissue was expressed in arbitrary units after normalization for the respective individual quantities of transcripts of glyceraldehyde phosphate dehydrogenase, which was analyzed as house keeping gene. Primers for glyceraldehyde phosphate dehydrogenase (TaqMan Gene Expression Assay No. Ss03375435_u1), TLR4 (No. Ss03389780_m1), and ICAM-1 (No. Ss03392385_m1) were purchased from Applied Biosystems.
Sequences of the PCR primers for KLF-2, customized by Applied Biosystems (custom TaqMan Gene Expression Assay, Part Number 4331348), were as follows: sense GCGCTGGGCTTGGC and antisense GCGGCGTGAGGAGACC.
Enzyme-linked immunosorbent assay kits were used according to the instructions of the manufacturers to analyze urine or serum levels of N-acetyl-beta-D-glucosaminidase (BlueGene Biotech, Shanghai, China), von Willebrand Factor (Cusabio Biotech, Wuhan China), and high-mobility group protein B1-HMGB-1 (IBL-international, Hamburg, Germany).
After the experiments were finished, renal tissue was collected from every animal, cut into small specimens (3-mm thickness), and fixed in 4% buffered formalin. The specimens were embedded in paraffin and cut into 2 μm sections using a microtome. Hematoxylin and eosin staining was used to assess morphologic integrity of the renal parenchyma.
All values were expressed as means±standard error. After proving the assumption of normality, differences between groups were tested by analysis of variance followed by multiple comparison of the means with the Student-Newman-Keuls test, unless otherwise indicated. Statistical significance was set at P value less than 0.05.
The authors are indebted to Mario Fox for the biochemical and molecular analyses and to Mario Sitzia for excellent help during the surgical operations.
1. Perico N, Cattaneo D, Sayegh MH, et al.. Delayed graft function in kidney transplantation
. Lancet 2004; 364: 1814.
2. Hauet T, Gibelin H, Richer JP, et al.. Influence of retrieval conditions on renal medulla injury: Evaluation by proton NMR spectroscopy in an isolated perfused pig kidney model. J Surg Res 2000; 93: 1.
3. Moers C, Smits JM, Maathuis MH, et al.. Machine perfusion
or cold storage in deceased-donor kidney transplantation
. N Engl J Med 2009; 360: 7.
4. Treckmann J, Moers C, Smits JM, et al.. Machine perfusion
versus cold storage for preservation
of kidneys from expanded criteria donors after brain death. Transpl Int 2011; 24: 548.
5. Yuan X, Theruvath AJ, Ge X, et al.. Machine perfusion
or cold storage in organ transplantation
: Indication, mechanisms, and future perspectives. Transpl Int 2010; 23: 561.
6. Watson CJ, Wells AC, Roberts RJ, et al.. Cold machine perfusion
versus static cold storage of kidneys donated after cardiac death: A UK multicenter randomized controlled trial. Am J Transplant 2010; 10: 1991.
7. Nicholson ML, Hosgood SA, Metcalfe MS, et al.. A comparison of renal preservation
by cold storage and machine perfusion
using a porcine autotransplant model. Transplantation
2004; 78: 333.
8. Evenson AR. Utilization of kidneys from donation after circulatory determination of death. Curr Opin Organ Transplant 2011; 16: 385.
9. Minor T, Sitzia M, Dombrowski F. Kidney transplantation
from NHBD after oxygenated low flow machine perfusion preservation
with HTK. Transpl Int 2005; 17: 707.
10. Lindell SL, Compagnon P, Mangino MJ, et al.. UW solution for hypothermic machine perfusion
of warm ischemic kidneys. Transplantation
2005; 79: 1358.
11. Weegman BP, Kirchner VA, Scott WE III, et al.. Continuous real-time viability assessment of kidneys based on oxygen
consumption. Transplant Proc 2010; 42: 2020.
12. Menger MD, Lehr HA, Messmer K. Role of oxygen
radicals in the microcirculatory manifestations of postischemic injury. Klin Wochenschr 1991; 69: 1050.
13. Lemasters JJ. V. Necrapoptosis and the mitochondrial permeability transition: Shared pathways to necrosis and apoptosis. Am J Physiol 1999; 276: G1.
14. Stegemann J, Minor T. Energy charge restoration, mitochondrial protection and reversal of preservation
induced liver injury by hypothermic oxygenation prior to reperfusion. Cryobiology 2009; 58: 331.
15. Koetting M, Frotscher C, Minor T. Hypothermic reconditioning
after cold storage improves postischemic graft function in isolated porcine kidneys. Transpl Int 2010; 23: 38.
16. Minor T, Stegemann J, Hirner A, et al.. Impaired autophagic clearance after cold preservation
of fatty livers correlates with tissue necrosis upon reperfusion and is reversed by hypothermic reconditioning
. Liver Transpl 2009; 15: 798.
17. Li J, Gong Q, Zhong S, et al.. Neutralization of the extracellular HMGB1 released by ischaemic-damaged renal cells protects against renal ischaemia-reperfusion injury. Nephrol Dial Transplant 2011; 26: 469.
18. Goes N, Hobart M, Ramassar V, et al.. Many forms of renal injury induce a stereotyped response with increased expression of MHC, IFN-gamma, and adhesion molecules. Transplant Proc 1997; 29: 1085.
19. Kouwenhoven EA, de Bruin RW, Bajema IM, et al.. Cold ischemia augments allogeneic-mediated injury in rat kidney allografts. Kidney Int 2001; 59: 1142.
20. Polyak MM, Arrington BO, Stubenbord WT, et al.. The influence of pulsatile preservation
on renal transplantation
in the 1990s. Transplantation
2000; 69: 249.
21. Mcanulty JF, Huang XQ. The effect of simple hypothermic preservation
with Trolox and ascorbate on lipid peroxidation in dog kidneys. Cryobiology 1996; 33: 217.
22. Mangino JE, Lindell S. Hypothermic machine perfusion
of kidneys. In: Uygun K, Lee CY, eds. Organ preservation
and reengineering. Norwood, MA, Artech House 2011, pp 35.
23. Legrand M, Mik EG, Johannes T, et al.. Renal hypoxia and dysoxia after reperfusion of the ischemic kidney. Mol Med 2008; 14: 502.
24. De Rougemont O, Breitenstein S, Leskosek B, et al.. One hour hypothermic oxygenated perfusion (HOPE) protects nonviable liver allografts donated after cardiac death. Ann Surg 2009; 250: 674.
25. Minor T, Koetting M, Koetting M, et al.. Hypothermic reconditioning
by gaseous oxygen
improves survival after liver transplantation
in the pig. Am J Transplant 2011; 11: 2627.
26. Steinlechnermaran R, Eberl T, Kunc M, et al.. Respiratory defect as an early event in preservation
-reoxygenation injury of endothelial cells. Transplantation
1997; 63: 136.
27. Menger MD, Vollmar B. Adhesion molecules as determinants of disease: From molecular biology to surgical research. Br J Surg 1996; 83: 588.
28. Schwarz C, Regele H, Steininger R, et al.. The contribution of adhesion molecule expression in donor kidney biopsies to early allograft dysfunction. Transplantation
2001; 71: 1666.
29. Gracia-Sancho J, Villarreal G Jr, Zhang Y, et al.. Flow cessation triggers endothelial dysfunction during organ cold storage conditions: Strategies for pharmacologic intervention. Transplantation
2010; 90: 142.
30. Tullius SG, Garcia-Cardena G. Organ procurement and perfusion before transplantation
. N Engl J Med 2009; 360: 78.
31. Boros P, Bromberg JS. New cellular and molecular immune pathways in ischemia/reperfusion injury. Am J Transplant 2006; 6: 652.
32. Faure E, Thomas L, Xu H, et al.. Bacterial lipopolysaccharide and IFN-gamma induce Toll-like receptor 2 and Toll-like receptor 4 expression in human endothelial cells: Role of NF-kappa B activation. J Immunol 2001; 166: 2018.
33. Wolfs TG, Buurman WA, van Schadewijk A, et al.. In vivo expression of Toll-like receptor 2 and 4 by renal epithelial cells: IFN-gamma and TNF-alpha mediated up-regulation during inflammation. J Immunol 2002; 168: 1286.
34. Robson MG. Toll-like receptors and renal disease. Nephron Exp Nephrol 2009; 113: e1.
35. Land WG. The role of postischemic reperfusion injury and other nonantigen-dependent inflammatory pathways in transplantation
2005; 79: 505.
36. Maathuis MH, Manekeller S, van der PA, et al.. Improved kidney graft function after preservation
using a novel hypothermic machine perfusion
device. Ann Surg 2007; 246: 982.
37. Minor T, Isselhard W, Yamaguchi T. Involvement of platelet activating factor in microcirculatory disturbances after global hepatic ischemia. J Surg Res 1995; 58: 536.