Optimizing Outcomes by Preconditioning the Donor

Optimizing Outcomes by Preconditioning the Donor E. Lledó-Garcia, D. Subirá-Ríos, A. Tejedor-Jorge, J.F. del Cañizo-López, and C. Hernández-Fernández ABSTRACT The criteria that define a so-called “marginal donor” kidney have been standardized since 2002. However, every transplant center must establish its own guidelines on organ acceptability. An expanded criteria donor (ECD) kidney is age at least 60 years, or 50 to 59 years with at least two of three specified comorbidities. Cadaveric kidneys have shown worse functional and survival outcomes compared with those from living donors. Thus, all efforts should be made to minimize the effects of ischemia on standard, non– heart-beating or ECD cadaveric donor kidneys. Because of an increasing shortfall between the diminishing number of deceased donor organs available and the increasing waiting lists, an increasing number of living donor transplantations are being performed in Europe. Among deceased donor kidneys, the largest percentage corresponds to ECD—aged or comorbidity donors—and donors after cardiac death. The results of transplants with kidneys from donors over 65 years are 10% to 15% lower than those from younger donors. Older donors present more comorbidities; however, acceptable results may be obtained with careful selection and shortened cold ischemic times. If the transplant center uses these donors to expand the pool of available organs, the donor must be evaluated according to age, vascular condition, renal function, and comorbidity. If the donor is accepted, suitable questions are: Has the potential donor undergone maneuvers to improve the quality of the kidneys? Which kind of approaches should we perform? Should we only use the biopsy information for a decision? OLD PRESERVATION OF KIDNEYS continues to play a critical role in the success of deceased donor kidney transplantation (Fig 1): prolonged cold storage can lead to severe injury causing delayed graft function (DGF) and a higher rate of graft loss. Finally, ischemia-reperfusion injury (IRI) often happens following transplantation, major resection, or trauma to organs. IRI following transplantation can lead to primary nonfunction (5%), primary dysfunction (10% to 30%), or multiple organ dysfunction syndrome, resulting in morbidity and mortality in adult and pediatric cases. These three factors are not independent but interrelated, producing cumulative effects.1–3 Kidneys from deceased donors are subjected to a period of cold ischemia while awaiting tissue matching and transplantation. Unlike them, the organs from living donors do not undergo cold ischemia. As a result, renal allografts from living donors, regardless of human leukocyte antigen matching, show better survival rates. Prolonged cold ischemia of deceased donor kidneys producing apoptosis based as observed by Salahudeen,4 leads to higher incidences of graft dysfunction, of recipient mortality, and of health care

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costs. From a molecular point of view, via calcium and free radicals cold opens permeability transition pores, causing marked mitochondrial swelling, which, in turn, triggers key apoptotic events during rewarming. From a clinical point of view, reducing cold ischemia time has beneficial effects: in US reports, grafts stored for 20 to 30 hours or over 30 hours show significantly higher rates of graft loss than those cold-stored for ⬍10 hours (P ⬍ .015 for both).5 We must try to transplant cadaveric kidneys in less than 10 to 14 hours from procurement; this simple action could minimize DGF, improving renal graft outcomes and reducing costs.6 But accepting that we want to increase kidney transplant outcomes, we should use all available donors. Since extended criteria donors (ECD) and non– heart-beating doFrom the Urology Department & Experimental Urology Group (E.L.-G., D.S.-R., C.H.-F.), Hospital General Universitario Gregorio Marañón, Madrid, Spain; Transplant Group Coordinator (E.L.-G.), Spanish Urological Association; Experimental Nephrology Lab (A.T.-I.), Hospital General Universitario Gregorio Marañón, Madrid, Spain; and Artificial Circulatory Lab (J.F.d.C.-L.) Hospital General Universitario Gregorio Marañón, Madrid, Spain. 0041-1345/–see front matter doi:10.1016/j.transproceed.2010.12.020 349

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Fig 1. Deleterious effects of cold ischemia on kidney grafts.

nors (NHBD) at least theoretical offer worse short-term functional prognoses, we need to design strategies to increase organ resistance to various injuries: warm and cold ischemia as well as reperfusion. In the case of ECD, actions on the donor seem to be critical and even more so among NHBD, where strategies should also be applied to the organ itself. Preconditioning the donor starts from a basic premise: treatment before a known injury occurs can be used to minimize the severity of that injury. Solid organ transplantation, with its attendant IRI, represents an area where preconditioning of the donor or the donated organs could make a great contribution. From a practical point of view, the implementation of preconditioning uses different techniques, which rely on harnessing aspects of the innate protective mechanisms that human cells use to survive stress. Two could be the approaches: (1) physical techniques: ischemic preconditioning (IP) and (2) pharmacological techniques including administration of drugs, cytokines, and gene transfer techniques. PHYSICAL TECHNIQUES Ischemic Preconditioning

Direct mechanical preconditioning in which the target organ is exposed to a brief ischemia period prior to prolonged ischemia has the benefit of reducing IRI,7 but its main disadvantage is trauma to major vessels and stress to the target organ. IP utilizes endogenous mechanisms in skeletal muscle, liver, lung, kidney, intestine, and brain in animal models to convey varying degrees of protection from IRI. To date, there are few human studies, but recent reports suggest that human liver, lung, and skeletal muscle acquire similar protection after IP. Specifically, preconditioned tissues exhibit reduced energy requirements, altered energy metabolism, better electrolyte homeostasis, and

genetic reorganization, giving rise to the concept of “ischemia tolerance.” IP also induces “reperfusion tolerance” with fewer reactive oxygen species and releases of activated neutrophils, reduced apoptosis, and better microcirculatory perfusion compared to nonpreconditioned tissue. Systemic I/R injury is also diminished by preconditioning. IP is ubiquitous, but more research is required to fully translate these findings to the clinical arena.

Remote Interorgan Preconditioning

Remote interorgan preconditioning (RIPC) is a recent observation in which brief ischemia of one organ has been shown to confer protection on distant organs without direct stress to the actual organ of interest.8 Remote intraorgan preconditioning was first described in the heart where brief ischemia in one territory led to protection in other areas. Translation of RIPC to clinical application has been demonstrated by the use of brief forearm ischemia to precondition the heart prior to coronary bypass and to reduce endothelial dysfunction in the contralateral limb. Recently protection of the heart has been demonstrated by remote hind limb preconditioning in children who underwent surgery for congenital heart disease with cardiopulmonary bypass. The RIPC stimulus presumably induces release of biochemical messengers, which act either by the bloodstream or by neurogenic pathways, resulting in reduced oxidative stress and preservation of mitochondrial function. Studies have demonstrated endothelial nitric oxide, free radicals, kinases, opioids, catecholamines, and KATP channels as candidate mechanisms for remote preconditioning. Experiments have shown suppression of proinflammatory genes, expression of antioxidant genes, and modulation of gene expression by RIPC as a novel method of IRI injury prevention.

PRECONDITIONING TO OPTIMIZE OUTCOMES

Postconditioning

Finally, postconditioning is an innovative, promising option.9 Completely applied to the recipient, this alternative consists of brief episodes of reperfusion alternating with reocclusion applied during the first minutes of reperfusion after prolonged ischemia. This maneuver not only alters the dynamics of reperfusion, but induces a cellular protection comparable to that of preconditioning and probably acting on the same end effectors (Fig 2). In summary, regarding physical techniques, three points should be borne in mind.10 Pre- and postconditioning are attractive strategies for organ protection in transplantation, since the start of both ischemia and reperfusion are predictable. In experimental conditions, preconditioning has been shown to be effective for liver and kidney transplantation. There is not yet evidence to indicate its systematic application in the clinical setting. Remote ischemic preconditioning offers some positive evidence, essentially focused on cardiac and vascular surgery. PHARMACOLOGICAL TECHNIQUES

Four aspects define the current situation of pharmacological techniques as alternatives to preconditioning either of the organs or the recipients: administration of molecules/drugs may be directed to the donor before ischemia-reperfusion; Their mechanisms of action may be generation of protective factors (heat shock proteins) or direct endothelialprotective effects on the target organ; Only experimental publications are available on this strategy. We could not find clinical trials published in MedLine. Administration of some amino acids such as taurine has been reported to be useful to protect kidneys against the consequences of ischemia and transplantation.11 Guan et al presented an experimental study using intravenous infusion of taurine to the donor before nephrectomy.11 This donor preconditioning with taurine protected kidney grafts from injury (apoptosis, necrosis), improved graft function, and increased the regenerative potential most likely via mechanisms including antioxidant effects. Glutamine (GLN), an amino acid that is conditionally essential during critical illness and injury, has been shown to reduce cell and organ damage induced by endotoxemia or ischemia.12 GLN induces endogenous heat shock protein 70 (HSP 70) expression in animals and humans, thus conferring cytoprotection against various stressors. In a syngeneic rat kidney transplantation model with severe preservation reperfusion injury induced by 40 hours of cold storage, GLN donor pretreatment showed potential benefits. Fuller et al hypothesized that GLN donor pretreatment, initiated 24 hours before organ procurement, induces renal HSP 70

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expression that attenuates early structural damage and functional impairment among 40 hours, cold-preserved, syngeneic rat kidney grafts. GLN donor pretreatment significantly increased intragraft HSP 70 expression.12 The GLN group showed severe tubular damage with significantly less papillary necrosis and GLN significantly reduced the number of apoptotic cells. Administration of calcineurin inhibitors has also been reported in the experimental setting to be useful to decrease the consequences of organ ischemia. Otean et al observed that donor pretreatment with FK506 reduced reperfusion injury, accelerating intestinal graft recovery in rats.13 The main conclusions of this work were: Donor pretreatment with the calcineurin inhibitor tacrolimus in the experimental setting reduced IRI by limiting graft inflammatory activation. The Mechanisms are HSP-72 upregulation to enhance cell regeneration and inhibit nuclear factor-kB, an endothelial proinflammatory factors. Phosphodiesterase type 5 inhibitors constitute a promising option to precondition and protect organs against consequences of ischemia-reperfusion. Kukreja summarized experimental and clinical information:14 Experimental data in animals have shown sildenafil to display a preconditioning-like effect against IRI in the intact heart. The mechanisms include: nitric oxide generated from eNOS/iNOS, activation of protein kinase signaling, and opening of mitochondrial ATP-sensitive potassium channels. The treatment attenuated cell apoptosis and necrosis. The clinical uses of this strategy have been to treat pulmonary arterial HT, endothelial dysfunction, and heart and lung transplant models. We also have reported experimental results demonstrating beneficial effects of both oral and intravenous sildenafil, administered to the donor before a period of warm ischemia and reperfusion. After autotransplantations both renal vascular flow and nitric oxide levels were significantly higher in all periods among the sildenafil group of animals, showing that it may constitute a useful preconditioning drug both for donors and recipients.15 GENE THERAPY

Gene therapy is a promising therapeutic option. Success is currently limited to small animal experimental models.16,17 The goal is to achieve a suitable vector with low immunogenicity and high predictable protein expression at the desired time and site. Antioxidative molecules, antiapoptotic molecules, or heat shock proteins could be induced in the course of this process. Some reported experimental achievements include: successful transfection of the superoxide dysmutase gene,

Fig 2. Ischemic postconditioning: scheme of action.

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regulation of cytokine balance, local production of blockade of T-cell factors promoting graft survival, decreased chronic inflammatory injury, and production of enzymes promoting remodeling of vessel walls. Research is imperative and essential to the future of this alternative. In conclusion, The deceased donor organ is subjected to a multitude of stresses: harvest, cold storage, and reperfusion of the organ. The key points are to reduce cold ischemia and prevent IRI. Pre- and postconditioning strategies need to be translated to solid clinical trials. Remote ischemic preconditioning is the strongest option clinically. Data on pharmacological preconditioning are predominantly experimental. Developing effective, feasible gene therapy strategies to reduce donor organ damage are important for the future. International, multicenter collaborations should be established to translate experimental strategies into feasible clinical studies. REFERENCES 1. Kälble T, Lucan M, Nicita G, et al: EAU guidelines on renal transplantation. European Urology 47:156, 2005 2. Sellers MT, Velidedeoglu E, Bloom RD, et al: NHS Economic Evaluation Database (NHS EED). Expanded-criteria donor kidneys: a single-center clinical and short-term financial analysis— cause for concern in retransplantation. NHS Economic Evaluation Database (NHS EED). Produced by the Centre for Reviews and Dissemination 3. Knoll G, et al: Trends in kidney transplantation over the past decade. Drugs 68(suppl 1):3, 2008 4. Salahudeen AK: Cold ischemic injury of transplanted kidneys: new insights from experimental studies. Am J Physiol Renal Physiol 287:F181, 2004 5. Salahudeen AK, May W: Reduction in cold ischemia time of renal allografts in the United States over the last decade. Transplant Proc 40:1285, 2008

LLEDÓ-GARCÍA, SUBIRÁ-RÍOS, TEJEDOR-JORGE ET AL 6. Goh CC, Ladouceur M, Peters L, et al: Lengthy cold ischemia time is a modifiable risk factor associated with low glomerular filtration rates in expanded criteria donor kidney transplant recipients. Transplant Proc 41:3290, 2009 7. Pasupathy S: Ischemic preconditioning protects against ischemia-reperfusion injury: emerging concepts. Eur J Vasc Endovasc Surg 29:106, 2005 8. Karbanda RK, et al: Translation of remote ischemic preconditioning into clinical practice. Lancet 374:1557, 2009 9. Zhao ZQ, Corvera JS, Halkos ME, et al: Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol 285:H579, 2003 10. Huang Y: Can ischemic preconditioning alone really protect organs from ischemia reperfusion injury in transplantation. Transplant Immunol 20:127, 2009 11. Guan X, et al: Donor preconditioning with taurine protects kidney grafts from injury after experimental transplantation. J Surg Res 146:127, 2008 12. Fuller TF, et al: Glutamine donor pretreatment in rat kidney transplants with severe preservation reperfusion injury. J Surg Res 140:77, 2007 13. Otean M, et al: Donor pretreatment with FK506 reduces reperfusion injury and accelerates intestinal graft recovery in rats. Surgery 141:667, 2007 14. Kukreja RC: Pharmacological preconditioning with sildenafil: basic mechanisms and clinical implications. Vascul Pharmacol 42:219, 2005 15. Lledó-García E, Subirá-Ríos D, Rodríguez D, et al: Sildenafil as a protecting drug for warm ischemic kidney transplants: experimental results. J Urol 182:1222, 2009 16. Moore DJ, Markmann JF, Deng S: Avenues for immunomodulation and graft protection by gene therapy in transplantation. Transpl Int 19:435, 2006 17. Ritter, Kupiec-Weglinski JW: Gene therapy for the prevention of ischemia/reperfusion injury in organ transplantation. Curr Gene Ther 5:101, 2005