Progrès en urologie (2014) 24, S51-S55
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Ischemia/Reperfusion during normothermic perfusion Ischémie-reperfusion en perfusion normothermique ? X. Tilloua,*, R. Thuretb, A. DoerÁerc and the members of CTAFU aUrology
and Transplantation department, University Hospital, Caen, France and Transplantation department, University Hospital, Montpellier, France cUrology and Transplantation department, University Hospital, Caen, France bUrology
KEYWORDS Ischemic normothermic preconditioning; Pharmacologic normothermic preconditioning; Ex vivo normothermic reperfusion; Remote ischemic transplantation preconditioning; Ischemic postconditioning
Summary Introduction: Cold storage of organs for preservation and transplantation is reaching its limits especially with extended criteria for heart beating donors and donation after cardiac death. We will discuss recent Àndings and perspectives in normothermic kidney preservation. Methods: A literature review was performed from original articles and syntheses selected by the search engine PubMed. Keywords used were: cold ischemia; warm ischemia, normothermic, organ preservation, preconditioning, organ perfusion. Results: We identiÀed several ways to improve kidney preservation: Ischemic normothermic preconditioning; Pharmacologic normothermic preconditioning; Ex vivo normothermic reperfusion; Remote ischemic transplantation preconditioning; Ischemic postconditioning. In clinical practice, only uses of ECMO for organ preconditioning or ex vivo normothermic organ perfusion were used. Conclusion: Promising experimental and clinical results make challenge cold preservation. The most suitable and physiological method seems to be a normothermic perfusion and conservation with autologous oxygenated blood using Extra Corporeal Membrane Oxygenation or Regional Normothermic Circulation. © 2014 Elsevier Masson SAS. All rights reserved.
*Corresponding author. E-mail adress: [email protected] (X. Tillou).
© 2014 Elsevier Masson SAS. All rights reserved.
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MOTS CLÉS Preconditionnement normothermique ischémique ; Preconditionnement normothermique pharmacologique ; Reperfusion normothermqiue ex-vivo ; Post-conditionnement ischémique
X. Tillou et al.
Résumé Introduction : la conservation hypothermique des greffons en transplantation a atteint ses limites en particulier pour les greffons à issu de donneurs à critères élargis et décédés après arrêt cardiaque. Les résultats et les perspectives récentes en conservation normothermique pour la préservation des greffons rénaux sont discutés dans cet article. Méthodes : Une revue de la littérature a été réalisée à partir des articles originaux et des synthèses sélectionnés par le moteur de recherche PubMed à l’aide des mots-clés suivants: Ischemic normothermic preconditioning; Pharmacologic normothermic preconditioning; Ex vivo normothermic reperfusion; Remote ischemic transplantation preconditioning; Ischemic postconditioning. Résultats : Plusieurs techniques d’amélioration de la conservation des greffons rénaux ont été identiÀées comme le préconditionnement ischémique normothermique, le préconditionnement normothermique pharmacologique, la reperfusion normothermique ex vivo et le postconditionnement ischémique. En pratiques cliniques, seules la circulation régionale normothermique pour le préconditionnement d’organes ou la perfusion normothermqique ex vivo des greffons ont été utilisées. Conclusion : les résultats expérimentaux et cliniques de ces techniques semblent prometteurs qui pourraient concurrencer la conservation statique hypothermique. La méthode la plus appropriée et plus physiologique semble être la perfusion normothermique et la conservation avec sang autologue oxygéné par membrane extra-corporelle d’oxygénation ou Circulation Normothermique Régionale. © 2014 Elsevier Masson SAS. Tous droits réservés.
Introduction Cold storage of organs for preservation and transplantation is reaching its limits especially with extended criteria for heart beating donors and donation after cardiac death. The objective of organ preservation is to ensure the best function after organ transplantation and reperfusion. The method developed until nowadays used cold preservation solutions to Áush and preserve organs. These solutions are designed to decrease cellular metabolism, oxygen requirement and tissue injuries [1]. To improve organ preservation, two paths are considered: normothermic conditioning of grafts before or after cold storage and normothermic oxygenated preservation. Thus, we will discuss below recent Àndings and perspectives in normothermic kidney preservation.
Methods A literature review was performed from original articles and syntheses already available and selected by the search engine PubMed of the National Library of Medicine. Keywords used for this research were: cold ischemia; warm ischemia, normothermic, organ preservation, preconditioning, organ perfusion. All items were analyzed and identiÀed. The congress abstracts were not included in our analysis.
Results Cold Ischemia/reperfusion The Ischemia/Reperfusion (I/R) phenomenon was initially considered as an event surrounding organs procurement. I/R now includes more broadly the state of the donor, with the period of brain death or that of warm ischemia in heartbeating donors. Among the many mechanisms involved in I/R injuries we would like to emphasize the following:
•
Increased production of cytokines and overexpression of MHC antigens (MHC) class I and II presenting cells, responsible for an increased immunogenicity. • The reactive oxygen species, such as free radicals, oxygen ions, and peroxide, activate the innate system of donor and recipient. • Overexpression of some ligands of Toll-Like Receptors (TLR) such as Heat Shock Proteins (HSP), interact with and activate dendritic TLR4 positive cells. Dendritic cells induce alloimmune adaptive response (acute rejection). Dendritic cells also interact with and activate vascular TLR4 positive cells, thus contributing to the development of chronic lesions. • The Toll domain of human interleukin-1 induces the activation of dependent on interferon γ (IFN-γ) pathways, which appear to be associated with dendritic cells maturation. • Mitochondrial edema induced by cold ischemia activates the apoptotic pathway, characterized, which is characterized by a translocation of cytochrome C (with others pro apoptotic proteins), and an increased Bax/Bcl-2 ratio with activation of caspase 3 at the time of reperfusion. The oxidative stress and the thermic shock being the main source of I/R organ injuries, others strategies need to be investigated to improve organ procurement and preservation.
Normothermic kidney conditioning Ischemic preconditioning Experimental data Torras [2], in a rat model, prepared kidney grafts for conventional cold storage by Àfteen minutes of warm ischemia and 10 minutes of reperfusion at body temperature. The beneÀcial effect of preconditioning is related to the local production of Nitric Oxide (NO). This protocol of ischemic preconditioning was
Ischemia/Reperfusion during normothermic perfusion
not conÀrmed in a pig model. Das [3] found that normothermic Áushing (with conventional preservation solution as University of Wisconsin) of canine kidneys prior to a hypothermic Áush eliminated vasoconstriction effects. Animal models highlighted molecular pathways involved in beneÀcial effects of normothermic perfusion before cold storage. Brasile [4] demonstrated that warm ischemia could enhance intragraft expression of Hem Oxygenase 1 preventing subsequent immune-mediated injury through inhibition of several immune effector functions, including lymphocyte proliferation, tumor necrosis factor and interferon synthesis, and T and natural killer cell mediated cytotoxicity. Warm ischemia could even increase nitric oxyd (NO) production preserving vascular integrity. As a consequence, elevated intragraft HO-1 and NO expression improve organ quality and long-term graft function and survival. Maintaining circulation before procurement is also thought to condition the organs with the up-regulation of adenosine receptors, which may protect against preservation injury.
Clinical practice The conventional technique of organ procurement begins with in situ placement of an aortic cannula to Áush organs with a cold preservation solution. Then the organs are stored in the same cold preservation solution at 4 °C. Some authors tried to improve this technique by maintaining circulation at normal body or room temperature using cardiopulmonary bypass by extracorporeal membrane oxygenation (ECMO) for various periods of time (45 to 90 minutes) before in-situ cooling and conventional cold storage. This technique was designed for Non Heart Beating Donors (NHBD) according to Maastricht classiÀcation. Valero opened this path [5]. The incidence of Delayed Graft Function (DGF) and Primary Non Function (PNF) was signiÀcantly reduced compared with standard in situ or total body cooling. Many others clinical studies conÀrmed these results [6–8].
Pharmacologic normothermic preconditioning Another advantage of normothermic perfusion is to prepare organs for cold storage with various treatments. Erythropoietin (EPO) preconditioning has shown interesting results [9]. EPO mediates preconditioning by limiting the destructive potential of tumor necrosis factor α and other proinÁammatory cytokines in an experimental model of rat kidney transplantation. This promising result was not conÀrmed in a porcine model. Trimetazidine demonstrated its efÀciency to prevent I/R injuries in a pig kidney warm ischemia model. Cau [10] reminded, in his study, the various effects of this molecule: decrease in intracellular acidosis, preservation of ATP production, limitation of the inÁammatory reaction, and reactive oxygen species generation, prevention of calcium overload, decrease in cell apoptosis. Gok [11] using streptokinase preÁush in a clinical model of NHBD, improved the quality of donor’s kidney. Immunosuppresive molecules have an inÁuence on I/R injury in kidneys procured from NHBD. The combination of tacrolimus and micofenolate mofetil signiÀcantly reduced TNF-alpha production at the time of reperfusion of the porcine kidney affected by ischemia. Kinsey [12] explored the lymphocyte T regulator (Treg) pathway. As demonstrated previously [13], Tregs
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suppressed inÁammation through contact mediated inhibition of immune cells and through release of soluble mediators such as TGF-beta, IL-10 (which downregulate the expression of Th1 cytokines, MHC class II antigens, and co-stimulatory molecules on macrophages, enhance B cell survival, proliferation, and antibody production), and adenosine (cytoprotection preventing tissue damage during instances of hypoxemia and ischemia). Pharmacologic stimulation of adenosine receptors (A2AR) with a speciÀc agonist increased the ability of Tregs to suppress kidney I/R injuries in a rat model of warm ischemia. Except use of streptokinase, pharmacological preconditioning during normothermic perfusion was not explored in clinical conditions.
Kidney resuscitation Ex vivo normothermic reperfusion Normothermic preservation is based on the idea that restoring metabolism ex vivo may allow the reversal of the detrimental effects of ischemia prior to transplantation. It allows a period of normothermic perfusion at the end of the ischemic period to « resuscitate » the organ, before transplantation. Various strategies were explored. In pigs, Hosgood [14] and Harper [15] used a period of normothermic perfusion with oxygenated autologous blood to improve post transplantation results. In these studies, the authors showed a lower level of lipid peroxidation suggesting a decreased oxidative stress. With the same study design, Bagul [16] showed normothermic perfusion with oxygenated blood was able to restore depleted ATP levels, to restore acid-base balance and reverse some of the deleterious effects of cold storage such as renal tubular injuries. The same team demonstrated later with the same porcine model, that leucocyte depletion improves renal function during reperfusion. It conÀrmed that white cells play a key role in organ I/R injuries. Mann, in pig kidneys, added to normothermic continuous perfusion (after 2 hours of cold storage) on a pulsatile machine, a selective endothelin receptor antagonist in a perfusion solution. Results on urine production of the kidneys showed restored renal function after one hour. Hosgood [17] described in 2011 the Àrst ex-vivo normothermic reperfusion after cold storage. With a plasma free red cell-based solution, the transplantation that followed gave good results.
Remote ischemic transplantation preconditioning Body and organ conditioning before normothermic reperfusion is based on remote ischemic preconditioning after elective abdominal aortic aneurysm repair, which reduces myocardial and renal injury. After study on dogs, Soendergaard [18] demonstrated on pigs that four cycles of 5 min clamping (during the venous anastomosis) of the exposed distal abdominal aorta before Ànal renal graft reperfusion in the recipient can improve initial renal plasma perfusion and glomerular function and decrease DGF. The factors transmitting the protective effect remain unclear. Three possible mechanisms have been proposed involving a neuronal signal transmission, a release of humoral mediators, or the induction of a systemic anti-inÁammatory response.
S54
Ischemic postconditioning In the Àeld of heart transplantation, Zhao [19] demonstrated that short, repeated sequences of ischemia and reperfusion after a prolonged ischemic episode, called ischemic postconditioning, reduce infarct size by about 40% after an ischemic myocardial injury. Szwarc in a mouse model [20], tried to replicate these results. Three cycles of 30 seconds of reperfusion and 30 seconds of ischemia showed renoprotective effects. Molecular pathways were not well understood but the proposed hypotheses were a decreased generation of reactive oxygen species and accumulation of intracellular Ca2+ , an activation of the reperfusion injury signaling kinase (RISK) pathway and an anti-apoptotic action by inhibiting the opening of the mitochondria permeability transition pore. Once again, it need to be assessed in preclinical model. In experimental kidney transplantation, Serviddio [21] and Jiang [22] demonstrated normothermic postconditioning is an effective strategy to reduce I/R injury. Liu [23] partially highlighted molecular pathways of ischemic postconditioning beneÀts with the major role of NO. Several other studies since the years 2000 demonstrated beneÀts of trimetazidine associated with ischemic postconditioning. In addition to repeated normothermic ischemic stimulation, trimetazidine was experimented in mouse kidney transplantation model. Association of ischemic postconditioning and trimetazidine protects the kidney by suppressing the endoplasmic reticulum and mitochondrial injury. Then this model was transposed in a pig kidney transplantation model with the same results.
Normothermic perfusion and storage Experimental data Taking into account that cold storage and oxidative stress are the main mechanisms of renal injuries, it was clearly needed to Ànd new strategies to preserve organs at body temperature with oxygenated solutions. In a rabbit kidney model, Arnaud [24] described normothermic blood perfusion added to cryoprotectants. She concluded that normothermic perfusion can produce informations in real time and has potential utility for the further understanding and treatment of I/R injuries. Impaired oxygen consumption, excessive weight gain and virtual anuria were identiÀed as potentially simple in vitro tests for kidney viability prior to transplantation. Kay [25] found no detrimental effect in porcine kidneys Áushed with a novel non-phosphate buffered solution at 32 °C. The study showed that this oxygenated solution could sufficiently maintain viability in flushed porcine kidneys and then statically stored for 2 h at 32 °C. Metcalf [26] compared hypothermic and normothermic pulsatile preservation of paired porcine kidneys during 16 hours. For normothermic perfusion, the solution used was oxygenated autologous blood supplemented by infusions of nutrients and colloid. The ability for kidneys to concentrate creatinine and withold sodium was signiÀcantly better for those preserved at body temperature. These promising results have to be conÀrmed in a transplantation model. More recently Iwai [27] studied normothermic storage with an extracellular-type solution containing trehalose. Once
X. Tillou et al.
again, interesting results were observed and the author concluded that normothermic continuous perfusion is able to prevent reperfusion injury due to temperature-dependent tissue edema. Nevertheless, these results were found in a rat model and need to be more relevant to clinical practice. Brasile [28], in the same study reported above, described a totally new concept of normothermic preservation. They called this new approach the acellular near-normothermic exsanguineous metabolic support (EMS). Their purpose was to preserve blood vessel wall function. Cellular metabolism could be adequately supported for up to 48 h at near-normothermic temperatures with stable oxidative metabolism and perfusion characteristics. Human kidneys used in this study could not be transplanted so their function were compared in parallel to canine kidney transplants which were transplanted 24 or 48 hours after procurement. Results were very promising and demonstrated that it is feasible to maintain intact human kidneys during an acellular perfusion with a cell culture-like medium administered for 48 h at 32 °C.
Limitations of normothermic preservation Logistics Normothermic perfusion and preservation of all organs seem to be the future, being the most physiological way to protect organ function. However, use of ECMO for in vivo normothermic preservation, or use of autologous oxygenated blood for ex vivo preservation require a large logistic organization. When adding oxygenation and circulation devices, this procedure requires a specialized technician to monitor pressure and perfusate Áow resistance, almost as for cardiac surgery.
Infections Bacteriological samples (blood, urine, bronchial samples) are always performed during the resuscitation of the potential donor, but the results will not be known most often after the removal of organs. Severe bacterial or mycotic sepsis (septicemia, peritonitis, meningitis infections) contraindicates organ removal. A culture of preservation Áuid of the organ removed is systematic. Despite cold storage, which decreases bacterial proliferation, graft and recipient infection had already been described. We can suppose that with normothermic perfusion or conservation, the infectious risk could increase. In the Àeld of heart and lung surgery, infection of ECMO circuits was described. In a case control study, Pieri [29] found 48% of patients treated with ECMO had infections. Gram-negative bacteria were the predominant pathogens, and Candida albicans was the most frequent isolated microorganism. These data were conÀrmed by Pluim [30] who retrospectively studied fungal ECMO infections, which reach 5%, increasing patient mortality.
Conclusion Promising experimental and clinical results in the Àeld of normothermic perfusion make challenge cold preservation. The most suitable and physiological method seems to be a normothermic perfusion and conservation with autologous oxygenated blood using Extra Corporeal Membrane
Ischemia/Reperfusion during normothermic perfusion
Oxygenation or Regional Normothermic Circulation. The major limitations are an increased logistics means and until nowadays the impossibility for normothermic organ transportation. With normothermic conditions, the risk for infection of perfusion circuits and organs remains unknown and could be a serious concern.
Disclosure of interest The authors have no conÁicts of interest to declare in relation to this review.
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Billault C, Rolland E, Vaessen C, Van Glabeke E, Gouezo R, Richard F, et al. Should temperature be monitorized during kidney allograft preservation? . Prog Urol J 2010;20:430–4. Torras J, Herrero-Fresneda I, Lloberas N, Riera M, Ma Cruzado J, Ma Grinyo J. Promising effects of ischemic preconditioning in renal transplantation. Kidney Int 2002;61:2218–27. Das S, Maggio AJ, Sacks SA, Smith RB, Kaufman JJ. Effects of preliminary normothermic Áushing on hypothermic renal preservation. Urology 1979;14:505–8. Brasile L, Buelow R, Stubenitsky BM, Kootstra G. Induction of heme oxygenase-1 in kidneys during ex vivo warm perfusion. Transplantation 2003;76:1145–9. Valero R, Cabrer C, Oppenheimer F, Trias E, Sanchez-Ibanez J, De Cabo FM, et al. Normothermic recirculation reduces primary graft dysfunction of kidneys obtained from non-heart-beating donors. Transpl Int 2000;13:303–10. Farney AC, Hines MH, al-Geizawi S, Rogers J, Stratta RJ. Lessons learned from a single center’s experience with 134 donation after cardiac death donor kidney transplants. J Am Coll Surg 2011;212:440–51;discussion 451–3. Billault C, Godfroy F, Thibaut F, Bart S, Arzouk N, Van Glabeke E, et al. Organ procurement from donors deceased from cardiac death: a single-center efÀciency assessment. Transpl Proc 2011;43:3396–7. Reznik O, Bagnenko S, Scvortsov A, Loginov I, Ananyev A, Senchik K, et al. The use of in-situ normothermic extracorporeal perfusion and leukocyte depletion for resuscitation of human donor kidneys. Perfusion 2010;25:343–8. Yang CW, Li C, Jung JY, Shin SJ, Choi BS, Lim SW, et al. Preconditioning with erythropoietin protects against subsequent ischemia-reperfusion injury in rat kidney. FASEB J 2003;17:1754–5. Cau J, Favreau F, Tillement JP, Lerman LO, Hauet T, Goujon JM. Trimetazidine reduces early and long-term effects of experimental renal warm ischemia: a dose effect study. J Vasc Surg 2008;47:852–60. Gok MA, Shenton BK, Buckley PE, Peaston R, Cornell C, Soomro N, et al. How to improve the quality of kidneys from non-heartbeating donors: a randomised controlled trial of thrombolysis in non-heart-beating donors. Transplantation 2003;76:1714–9. Kinsey GR, Huang L, Jaworska K, Khutsishvili K, Becker DA, Ye H, et al. Autocrine adenosine signaling promotes regulatory T cellmediated renal protection. J Am Soc Nephrol 2012;23:1528–37. Deaglio S, Dwyer KM, Gao W, Friedman D, Usheva A, Erat A, et al. Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J Exp Med 2007;204:1257–65.
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[14] Hosgood SA, Barlow AD, Yates PJ, Snoeijs MG, van Heurn EL, Nicholson ML. A pilot study assessing the feasibility of a short period of normothermic preservation in an experimental model of non heart beating donor kidneys. J Surg Res 2011;171:283–90. [15] Harper SJ, Hosgood SA, Waller HL, Yang B, Kay MD, Goncalves I, et al. The effect of warm ischemic time on renal function and injury in the isolated hemoperfused kidney. Transplantation 2008;86:445–51. [16] Bagul A, Hosgood SA, Kaushik M, Kay MD, Waller HL, Nicholson ML. Experimental renal preservation by normothermic resuscitation perfusion with autologous blood. Br J Surg 2008;95:111–8. [17] Hosgood SA, Nicholson ML. First in man renal transplantation after ex vivo normothermic perfusion. Transplantation 2011;92:735–8. [18] Soendergaard P, Krogstrup NV, Secher NG, Ravlo K, Keller AK, Toennesen E, et al. Improved GFR and renal plasma perfusion following remote ischaemic conditioning in a porcine kidney transplantation model. Transpl Int 2012;25:1002–12. [19] Zhao ZQ, Corvera JS, Halkos ME, Kerendi F, Wang NP, Guyton RA, et al. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol 2003;285:H579–88. [20] Szwarc I, Soullier S, Gayrard N, Mejean C, Mourad G, Argiles A. Ischemic postconditioning prevents ischemic acute renal failure. Transpl Proc 2007;39:2554–6. [21] Serviddio G, Romano AD, Gesualdo L, Tamborra R, Di Palma AM, Rollo T, et al. Postconditioning is an effective strategy to reduce renal ischaemia/reperfusion injury. Nephrol Dial Transpl 2008;23:1504–12. [22] Jiang B, Chen Q, Liu X, Kong D, Kuang Y, Weng X, et al. Ischemic postconditioning protects renal function after 24 hours of cold preservation in a canine autotransplantation model. Transpl Proc 2012;44:1776–81. [23] Liu X, Chen H, Zhan B, Xing B, Zhou J, Zhu H, et al. Attenuation of reperfusion injury by renal ischemic postconditioning: the role of NO. Biochem Biophys Res Commun 2007;359:628–34. [24] Arnaud FG, Khirabadi BS, Fahy GM. Normothermic blood perfusion of isolated rabbit kidneys. III. In vitro physiology of kidneys after perfusion with Euro-Collins solution or 7.5 M cryoprotectant (VS4). Transpl Int 2002;15:278–89. [25] Kay MD, Hosgood SA, Harper SJ, Bagul A, Waller HL, Rees D, et al. Static normothermic preservation of renal allografts using a novel nonphosphate buffered preservation solution. Transpl Int 2007;20:88–92. [26] Metcalfe MS, Waller JR, Hosgood SA, Shaw M, Hassanein W, Nicholson ML. A paired study comparing the efÀcacy of renal preservation by normothermic autologous blood perfusion and hypothermic pulsatile perfusion. Transpl Proc 2002;34:1473–4. [27] Iwai S, Kikuchi T, Kasahara N, Teratani T, Yokoo T, Sakonju I, et al. Impact of normothermic preservation with extracellular type solution containing trehalose on rat kidney grafting from a cardiac death donor. PLoS One 2012;7:e33157. [28] Brasile L, Stubenitsky BM, Booster MH, Haisch C, Kootstra G. NOS: the underlying mechanism preserving vascular integrity and during ex vivo warm kidney perfusion. Am J Transpl 2003;3:674–9. [29] Pieri M, Greco T, De Bonis M, Maj G, Fumagalli L, Zangrillo A, et al. Diagnosis of infection in patients undergoing extracorporeal membrane oxygenation: a case-control study. J Thorac Cardiovasc Surg 2012;143:1411–6. [30] Pluim T, Halasa N, Phillips SE, Fleming G. The morbidity and mortality of patients with fungal infections before and during extracorporeal membrane oxygenation support*. Pediatr Crit Care Med 2012;13:e288–93.
Disponible en ligne sur
www.sciencedirect.com
Ischemia/Reperfusion during normothermic perfusion Ischémie-reperfusion en perfusion normothermique ? X. Tilloua,*, R. Thuretb, A. DoerÁerc and the members of CTAFU aUrology
and Transplantation department, University Hospital, Caen, France and Transplantation department, University Hospital, Montpellier, France cUrology and Transplantation department, University Hospital, Caen, France bUrology
KEYWORDS Ischemic normothermic preconditioning; Pharmacologic normothermic preconditioning; Ex vivo normothermic reperfusion; Remote ischemic transplantation preconditioning; Ischemic postconditioning
Summary Introduction: Cold storage of organs for preservation and transplantation is reaching its limits especially with extended criteria for heart beating donors and donation after cardiac death. We will discuss recent Àndings and perspectives in normothermic kidney preservation. Methods: A literature review was performed from original articles and syntheses selected by the search engine PubMed. Keywords used were: cold ischemia; warm ischemia, normothermic, organ preservation, preconditioning, organ perfusion. Results: We identiÀed several ways to improve kidney preservation: Ischemic normothermic preconditioning; Pharmacologic normothermic preconditioning; Ex vivo normothermic reperfusion; Remote ischemic transplantation preconditioning; Ischemic postconditioning. In clinical practice, only uses of ECMO for organ preconditioning or ex vivo normothermic organ perfusion were used. Conclusion: Promising experimental and clinical results make challenge cold preservation. The most suitable and physiological method seems to be a normothermic perfusion and conservation with autologous oxygenated blood using Extra Corporeal Membrane Oxygenation or Regional Normothermic Circulation. © 2014 Elsevier Masson SAS. All rights reserved.
*Corresponding author. E-mail adress: [email protected] (X. Tillou).
© 2014 Elsevier Masson SAS. All rights reserved.
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MOTS CLÉS Preconditionnement normothermique ischémique ; Preconditionnement normothermique pharmacologique ; Reperfusion normothermqiue ex-vivo ; Post-conditionnement ischémique
X. Tillou et al.
Résumé Introduction : la conservation hypothermique des greffons en transplantation a atteint ses limites en particulier pour les greffons à issu de donneurs à critères élargis et décédés après arrêt cardiaque. Les résultats et les perspectives récentes en conservation normothermique pour la préservation des greffons rénaux sont discutés dans cet article. Méthodes : Une revue de la littérature a été réalisée à partir des articles originaux et des synthèses sélectionnés par le moteur de recherche PubMed à l’aide des mots-clés suivants: Ischemic normothermic preconditioning; Pharmacologic normothermic preconditioning; Ex vivo normothermic reperfusion; Remote ischemic transplantation preconditioning; Ischemic postconditioning. Résultats : Plusieurs techniques d’amélioration de la conservation des greffons rénaux ont été identiÀées comme le préconditionnement ischémique normothermique, le préconditionnement normothermique pharmacologique, la reperfusion normothermique ex vivo et le postconditionnement ischémique. En pratiques cliniques, seules la circulation régionale normothermique pour le préconditionnement d’organes ou la perfusion normothermqique ex vivo des greffons ont été utilisées. Conclusion : les résultats expérimentaux et cliniques de ces techniques semblent prometteurs qui pourraient concurrencer la conservation statique hypothermique. La méthode la plus appropriée et plus physiologique semble être la perfusion normothermique et la conservation avec sang autologue oxygéné par membrane extra-corporelle d’oxygénation ou Circulation Normothermique Régionale. © 2014 Elsevier Masson SAS. Tous droits réservés.
Introduction Cold storage of organs for preservation and transplantation is reaching its limits especially with extended criteria for heart beating donors and donation after cardiac death. The objective of organ preservation is to ensure the best function after organ transplantation and reperfusion. The method developed until nowadays used cold preservation solutions to Áush and preserve organs. These solutions are designed to decrease cellular metabolism, oxygen requirement and tissue injuries [1]. To improve organ preservation, two paths are considered: normothermic conditioning of grafts before or after cold storage and normothermic oxygenated preservation. Thus, we will discuss below recent Àndings and perspectives in normothermic kidney preservation.
Methods A literature review was performed from original articles and syntheses already available and selected by the search engine PubMed of the National Library of Medicine. Keywords used for this research were: cold ischemia; warm ischemia, normothermic, organ preservation, preconditioning, organ perfusion. All items were analyzed and identiÀed. The congress abstracts were not included in our analysis.
Results Cold Ischemia/reperfusion The Ischemia/Reperfusion (I/R) phenomenon was initially considered as an event surrounding organs procurement. I/R now includes more broadly the state of the donor, with the period of brain death or that of warm ischemia in heartbeating donors. Among the many mechanisms involved in I/R injuries we would like to emphasize the following:
•
Increased production of cytokines and overexpression of MHC antigens (MHC) class I and II presenting cells, responsible for an increased immunogenicity. • The reactive oxygen species, such as free radicals, oxygen ions, and peroxide, activate the innate system of donor and recipient. • Overexpression of some ligands of Toll-Like Receptors (TLR) such as Heat Shock Proteins (HSP), interact with and activate dendritic TLR4 positive cells. Dendritic cells induce alloimmune adaptive response (acute rejection). Dendritic cells also interact with and activate vascular TLR4 positive cells, thus contributing to the development of chronic lesions. • The Toll domain of human interleukin-1 induces the activation of dependent on interferon γ (IFN-γ) pathways, which appear to be associated with dendritic cells maturation. • Mitochondrial edema induced by cold ischemia activates the apoptotic pathway, characterized, which is characterized by a translocation of cytochrome C (with others pro apoptotic proteins), and an increased Bax/Bcl-2 ratio with activation of caspase 3 at the time of reperfusion. The oxidative stress and the thermic shock being the main source of I/R organ injuries, others strategies need to be investigated to improve organ procurement and preservation.
Normothermic kidney conditioning Ischemic preconditioning Experimental data Torras [2], in a rat model, prepared kidney grafts for conventional cold storage by Àfteen minutes of warm ischemia and 10 minutes of reperfusion at body temperature. The beneÀcial effect of preconditioning is related to the local production of Nitric Oxide (NO). This protocol of ischemic preconditioning was
Ischemia/Reperfusion during normothermic perfusion
not conÀrmed in a pig model. Das [3] found that normothermic Áushing (with conventional preservation solution as University of Wisconsin) of canine kidneys prior to a hypothermic Áush eliminated vasoconstriction effects. Animal models highlighted molecular pathways involved in beneÀcial effects of normothermic perfusion before cold storage. Brasile [4] demonstrated that warm ischemia could enhance intragraft expression of Hem Oxygenase 1 preventing subsequent immune-mediated injury through inhibition of several immune effector functions, including lymphocyte proliferation, tumor necrosis factor and interferon synthesis, and T and natural killer cell mediated cytotoxicity. Warm ischemia could even increase nitric oxyd (NO) production preserving vascular integrity. As a consequence, elevated intragraft HO-1 and NO expression improve organ quality and long-term graft function and survival. Maintaining circulation before procurement is also thought to condition the organs with the up-regulation of adenosine receptors, which may protect against preservation injury.
Clinical practice The conventional technique of organ procurement begins with in situ placement of an aortic cannula to Áush organs with a cold preservation solution. Then the organs are stored in the same cold preservation solution at 4 °C. Some authors tried to improve this technique by maintaining circulation at normal body or room temperature using cardiopulmonary bypass by extracorporeal membrane oxygenation (ECMO) for various periods of time (45 to 90 minutes) before in-situ cooling and conventional cold storage. This technique was designed for Non Heart Beating Donors (NHBD) according to Maastricht classiÀcation. Valero opened this path [5]. The incidence of Delayed Graft Function (DGF) and Primary Non Function (PNF) was signiÀcantly reduced compared with standard in situ or total body cooling. Many others clinical studies conÀrmed these results [6–8].
Pharmacologic normothermic preconditioning Another advantage of normothermic perfusion is to prepare organs for cold storage with various treatments. Erythropoietin (EPO) preconditioning has shown interesting results [9]. EPO mediates preconditioning by limiting the destructive potential of tumor necrosis factor α and other proinÁammatory cytokines in an experimental model of rat kidney transplantation. This promising result was not conÀrmed in a porcine model. Trimetazidine demonstrated its efÀciency to prevent I/R injuries in a pig kidney warm ischemia model. Cau [10] reminded, in his study, the various effects of this molecule: decrease in intracellular acidosis, preservation of ATP production, limitation of the inÁammatory reaction, and reactive oxygen species generation, prevention of calcium overload, decrease in cell apoptosis. Gok [11] using streptokinase preÁush in a clinical model of NHBD, improved the quality of donor’s kidney. Immunosuppresive molecules have an inÁuence on I/R injury in kidneys procured from NHBD. The combination of tacrolimus and micofenolate mofetil signiÀcantly reduced TNF-alpha production at the time of reperfusion of the porcine kidney affected by ischemia. Kinsey [12] explored the lymphocyte T regulator (Treg) pathway. As demonstrated previously [13], Tregs
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suppressed inÁammation through contact mediated inhibition of immune cells and through release of soluble mediators such as TGF-beta, IL-10 (which downregulate the expression of Th1 cytokines, MHC class II antigens, and co-stimulatory molecules on macrophages, enhance B cell survival, proliferation, and antibody production), and adenosine (cytoprotection preventing tissue damage during instances of hypoxemia and ischemia). Pharmacologic stimulation of adenosine receptors (A2AR) with a speciÀc agonist increased the ability of Tregs to suppress kidney I/R injuries in a rat model of warm ischemia. Except use of streptokinase, pharmacological preconditioning during normothermic perfusion was not explored in clinical conditions.
Kidney resuscitation Ex vivo normothermic reperfusion Normothermic preservation is based on the idea that restoring metabolism ex vivo may allow the reversal of the detrimental effects of ischemia prior to transplantation. It allows a period of normothermic perfusion at the end of the ischemic period to « resuscitate » the organ, before transplantation. Various strategies were explored. In pigs, Hosgood [14] and Harper [15] used a period of normothermic perfusion with oxygenated autologous blood to improve post transplantation results. In these studies, the authors showed a lower level of lipid peroxidation suggesting a decreased oxidative stress. With the same study design, Bagul [16] showed normothermic perfusion with oxygenated blood was able to restore depleted ATP levels, to restore acid-base balance and reverse some of the deleterious effects of cold storage such as renal tubular injuries. The same team demonstrated later with the same porcine model, that leucocyte depletion improves renal function during reperfusion. It conÀrmed that white cells play a key role in organ I/R injuries. Mann, in pig kidneys, added to normothermic continuous perfusion (after 2 hours of cold storage) on a pulsatile machine, a selective endothelin receptor antagonist in a perfusion solution. Results on urine production of the kidneys showed restored renal function after one hour. Hosgood [17] described in 2011 the Àrst ex-vivo normothermic reperfusion after cold storage. With a plasma free red cell-based solution, the transplantation that followed gave good results.
Remote ischemic transplantation preconditioning Body and organ conditioning before normothermic reperfusion is based on remote ischemic preconditioning after elective abdominal aortic aneurysm repair, which reduces myocardial and renal injury. After study on dogs, Soendergaard [18] demonstrated on pigs that four cycles of 5 min clamping (during the venous anastomosis) of the exposed distal abdominal aorta before Ànal renal graft reperfusion in the recipient can improve initial renal plasma perfusion and glomerular function and decrease DGF. The factors transmitting the protective effect remain unclear. Three possible mechanisms have been proposed involving a neuronal signal transmission, a release of humoral mediators, or the induction of a systemic anti-inÁammatory response.
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Ischemic postconditioning In the Àeld of heart transplantation, Zhao [19] demonstrated that short, repeated sequences of ischemia and reperfusion after a prolonged ischemic episode, called ischemic postconditioning, reduce infarct size by about 40% after an ischemic myocardial injury. Szwarc in a mouse model [20], tried to replicate these results. Three cycles of 30 seconds of reperfusion and 30 seconds of ischemia showed renoprotective effects. Molecular pathways were not well understood but the proposed hypotheses were a decreased generation of reactive oxygen species and accumulation of intracellular Ca2+ , an activation of the reperfusion injury signaling kinase (RISK) pathway and an anti-apoptotic action by inhibiting the opening of the mitochondria permeability transition pore. Once again, it need to be assessed in preclinical model. In experimental kidney transplantation, Serviddio [21] and Jiang [22] demonstrated normothermic postconditioning is an effective strategy to reduce I/R injury. Liu [23] partially highlighted molecular pathways of ischemic postconditioning beneÀts with the major role of NO. Several other studies since the years 2000 demonstrated beneÀts of trimetazidine associated with ischemic postconditioning. In addition to repeated normothermic ischemic stimulation, trimetazidine was experimented in mouse kidney transplantation model. Association of ischemic postconditioning and trimetazidine protects the kidney by suppressing the endoplasmic reticulum and mitochondrial injury. Then this model was transposed in a pig kidney transplantation model with the same results.
Normothermic perfusion and storage Experimental data Taking into account that cold storage and oxidative stress are the main mechanisms of renal injuries, it was clearly needed to Ànd new strategies to preserve organs at body temperature with oxygenated solutions. In a rabbit kidney model, Arnaud [24] described normothermic blood perfusion added to cryoprotectants. She concluded that normothermic perfusion can produce informations in real time and has potential utility for the further understanding and treatment of I/R injuries. Impaired oxygen consumption, excessive weight gain and virtual anuria were identiÀed as potentially simple in vitro tests for kidney viability prior to transplantation. Kay [25] found no detrimental effect in porcine kidneys Áushed with a novel non-phosphate buffered solution at 32 °C. The study showed that this oxygenated solution could sufficiently maintain viability in flushed porcine kidneys and then statically stored for 2 h at 32 °C. Metcalf [26] compared hypothermic and normothermic pulsatile preservation of paired porcine kidneys during 16 hours. For normothermic perfusion, the solution used was oxygenated autologous blood supplemented by infusions of nutrients and colloid. The ability for kidneys to concentrate creatinine and withold sodium was signiÀcantly better for those preserved at body temperature. These promising results have to be conÀrmed in a transplantation model. More recently Iwai [27] studied normothermic storage with an extracellular-type solution containing trehalose. Once
X. Tillou et al.
again, interesting results were observed and the author concluded that normothermic continuous perfusion is able to prevent reperfusion injury due to temperature-dependent tissue edema. Nevertheless, these results were found in a rat model and need to be more relevant to clinical practice. Brasile [28], in the same study reported above, described a totally new concept of normothermic preservation. They called this new approach the acellular near-normothermic exsanguineous metabolic support (EMS). Their purpose was to preserve blood vessel wall function. Cellular metabolism could be adequately supported for up to 48 h at near-normothermic temperatures with stable oxidative metabolism and perfusion characteristics. Human kidneys used in this study could not be transplanted so their function were compared in parallel to canine kidney transplants which were transplanted 24 or 48 hours after procurement. Results were very promising and demonstrated that it is feasible to maintain intact human kidneys during an acellular perfusion with a cell culture-like medium administered for 48 h at 32 °C.
Limitations of normothermic preservation Logistics Normothermic perfusion and preservation of all organs seem to be the future, being the most physiological way to protect organ function. However, use of ECMO for in vivo normothermic preservation, or use of autologous oxygenated blood for ex vivo preservation require a large logistic organization. When adding oxygenation and circulation devices, this procedure requires a specialized technician to monitor pressure and perfusate Áow resistance, almost as for cardiac surgery.
Infections Bacteriological samples (blood, urine, bronchial samples) are always performed during the resuscitation of the potential donor, but the results will not be known most often after the removal of organs. Severe bacterial or mycotic sepsis (septicemia, peritonitis, meningitis infections) contraindicates organ removal. A culture of preservation Áuid of the organ removed is systematic. Despite cold storage, which decreases bacterial proliferation, graft and recipient infection had already been described. We can suppose that with normothermic perfusion or conservation, the infectious risk could increase. In the Àeld of heart and lung surgery, infection of ECMO circuits was described. In a case control study, Pieri [29] found 48% of patients treated with ECMO had infections. Gram-negative bacteria were the predominant pathogens, and Candida albicans was the most frequent isolated microorganism. These data were conÀrmed by Pluim [30] who retrospectively studied fungal ECMO infections, which reach 5%, increasing patient mortality.
Conclusion Promising experimental and clinical results in the Àeld of normothermic perfusion make challenge cold preservation. The most suitable and physiological method seems to be a normothermic perfusion and conservation with autologous oxygenated blood using Extra Corporeal Membrane
Ischemia/Reperfusion during normothermic perfusion
Oxygenation or Regional Normothermic Circulation. The major limitations are an increased logistics means and until nowadays the impossibility for normothermic organ transportation. With normothermic conditions, the risk for infection of perfusion circuits and organs remains unknown and could be a serious concern.
Disclosure of interest The authors have no conÁicts of interest to declare in relation to this review.
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