Normothermic kidney preservation Sarah A. Hosgood and Michael L. Nicholson

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1 Normothermic kidney preservation Sarah A. Hosgood and Michael L. Nicholson Department of Infection, Immunity and Inflammation, Transplant Group, University of Leicester, Leicester General Hospital, Leicester, UK Correspondence to Professor Michael L Nicholson, MD, DSc, FRCS, Transplant Group, University Hospitals of Leicester, Leicester General Hospital, Leicester LE5 4PW, UK Tel: ; fax: ; mln2@le.ac.uk Current Opinion in Organ Transplantation 2011, 16: Purpose of review Kidneys from marginal or donation after cardiac death (DCD) donors are particularly susceptible to injury during hypothermic preservation and may benefit from alternative methods of preservation. Normothermic preservation can be adapted to improve the quality of kidneys for transplantation by a variety of techniques. Recent findings Extracorporeal membrane support to maintain circulation before cooling and organ retrieval has been used to improve the condition of DCD donor kidneys, with lower rates of delayed graft function (DGF) compared with standard retrieval conditions. Experimentally, normothermic perfusion has been used in conjunction with hypothermic techniques as a resuscitation technique to improve graft outcome. An ex-vivo porcine kidney model showed that energy levels could be replenished to improve tissue perfusion during reperfusion. This technique was translated into a porcine transplant model demonstrating that it was a feasible and safe method of preservation. Summary Normothermic preservation techniques have the potential to be adapted into an improved method of retaining tissue viability compared with hypothermic techniques. Furthermore, they may be used as a device to enhance and assess the condition of the kidney which would be particularly beneficial for kidneys from DCD donors. Keywords donation after cardiac death donor, kidney, normothermic preservation, oxygenation, perfusion Curr Opin Organ Transplant 16: ß 2011 Wolters Kluwer Health Lippincott Williams & Wilkins Introduction The aim of organ preservation is to maintain the organ in a viable state from the time of retrieval until transplantation. The simplest and most practiced method of preservation is to lower the temperature to reduce cellular metabolism and the requirement for oxygen [1]. However, with high rates of delayed graft function (DGF) and primary nonfunction (PNF) in extended criteria and donation after cardiac death (DCD) donor kidney transplantation, there is a need to improve preservation conditions. The alternative to hypothermic preservation is to use a normothermic temperature. Although, normothermic kidney preservation has remained an experimental technique, the potential advantages are slowly being recognized and may have a significant role in modern transplantation. This review will focus on recent attempts, development and the potential application of normothermic kidney preservation techniques. Normothermic recirculation/retrieval Traditionally, organ preservation starts with the immediate flush of the kidney with cold preservation solution to remove the blood, reduce the temperature and the lower the metabolism. In cadaveric and DCD donors this is usually performed in situ with the placement of an aortic cannula. However, some benefit has been found in maintaining circulation at normal body or room temperature using extracorporeal membrane support for a period before in-situ cooling. This technique has the advantage of perfusing the organs with oxygenated blood under controlled conditions. This may be particularly beneficial for DCD donors in maintaining tissue perfusion after the heart has stopped. In a small clinical study of DCD donors, Valero et al. [2] maintained normothermic recirculation for 60 min before total body cooling. The incidence of DGF and PNF was significantly reduced compared with standard in situ or total body cooling. Gravel et al. [3] describe a low DGF rate of 11% in controlled DCD donors using a similar technique and Lee et al. [4] found that after normothermic recirculation, 5-year graft survival of DCD donor kidneys was equivalent to heart beating and living donors. Maintaining circulation before retrieval is also thought to condition the organs with the up-regulation of adenosine receptors which may protect against preservation injury [2]. Normothermic recirculation also offers the potential to introduce therapies, again ß 2011 Wolters Kluwer Health Lippincott Williams & Wilkins DOI: /MOT.0b013e a5d

2 170 Organ preservation and procurement to further protect against preservation injury or regulate molecular processes targeting ischaemia reperfusion (I/R) injury and acute rejection after transplantation. Clinically, more evidence is needed to determine the benefits of normothermic recirculation on early and longer term kidney graft function. Experimentally, techniques are being refined with methods such as using leukocyte depleted blood to improve the retrieval conditions [5 ]. Normothermic flush Achieving adequate clearance of the microcirculation is important to optimize the preservation condition. However, flushing with hypothermic solutions can cause vasoconstriction reducing the effectiveness of the perfusion. Das et al. [6] found that normothermic flushing of canine kidneys prior to a hypothermic flush eliminated the vasoconstriction effect. University of Wisconsin solution at normal body temperature has been used to flush rat kidneys, improving renal function and reducing histological change compared with flushing with Euro Collins, saline or Ringer s lactate solutions at normal body temperature [7]. University of Wisconsin has the added advantage of containing additional components to support energy production which may be particularly important at this initial stage of preservation. The most recent experimental study showed that no detrimental effect was found in porcine kidneys flushed with a novel nonphosphate buffered solution AQIX (AQIX Ltd, London, UK) at 328C [8 ]. AQIX is a newly developed normothermic preservation solution capable of supporting cellular activity at normal temperatures. A previous study showed that AQIX could sufficiently maintain viability in porcine kidneys flushed and statically stored for 2 h at 328C in the preoxygenated solution [9]. Normothermic perfusion The key element of normothermic preservation is to maintain the kidney in a normal physiological state providing oxygen and nutrients to support aerobic metabolism. This has many advantages but also a potentially important disadvantage, in that it is logistically difficult to implement into clinical practice when considering the additional equipment needed to provide metabolic support (Table 1). This situation has undoubtedly hindered both its popularity and its advancement in clinical transplantation. Table 1 Advantages and disadvantages of normothermic preservation Normothermic kidney preservation Advantages Disadvantages Aerobic metabolism Restoration of function Avoid or reduce hypothermic injury Organ assessment Resuscitation Regeneration and repair Treatment and modification Technical support/equipment Logistics of transportation Cost More practically, using a combination of hypothermic and normothermic techniques has proved to be beneficial. One of the early aims of kidney preservation was to extend the preservation period to a matter of days to facilitate cross-matching and immunological techniques. Van Der Wijk et al. [10] was able to preserve canine kidneys for a total of 144 h with autologous blood perfusion at normal body temperature for periods of 1 4 h after 96 h of hypothermic preservation before the kidneys were returned to hypothermic preservation for the remaining preservation time. Periods of 3 4 h of normothermic perfusion were deemed necessary to reverse the ischaemic damage. Similarly, Rijkmans et al. [11] was able to extend the preservation period to 6 days with a 3-h intermediate period of normothermic perfusion, again using canine kidneys. This restored energy metabolism with replenishment of adenosine levels, effectively resuscitating the organ and improving viability compared with kidneys stored under hypothermic conditions alone. Other groups followed with this intermediate normothermic perfusion again finding improved survival compared with hypothermic conditions [12,13]. With improved cross-matching techniques there is no longer the need for such prolonged preservation periods in clinical transplantation [14]. Experimentally, the concept of resuscitating the kidney has more recently been adapted for kidneys from marginal and DCD donors. Brasile et al. [15,16] found that a period of normothermic ex-vivo perfusion at the end of the preservation period could resuscitate the kidney after warm and cold ischaemic injury. This same group also showed that more prolonged periods of normothermic preservation were more beneficial than hypothermic techniques [17]. The most recent report of normothermic resuscitation by Bagul et al. [18] again found that a short period (2 h) of normothermic perfusion could enhance renal blood flow during reperfusion and reverse some of the detrimental effects of cold ischaemic injury. This technique was translated into a porcine autotransplantation model, demonstrating that it was a safe and feasible method of kidney preservation that could be easily adapted for clinical practice [19 ]. Technology The basic requirements for a normothermic preservation system are an organ chamber, perfusion pump, tubing, an oxygenator, heat exchanger and monitoring devices to measure flow, pressure and temperature. Some researchers have adapted hypothermic perfusion systems or used more elaborate custom-made devices using roller pumps

3 Normothermic kidney preservation Hosgood and Nicholson 171 and dialysis circuits to obtain optimal renal function using physiological pressures and temperatures [20 23]. The normothermic system report by Brasile et al. [24,25] the exsanguinous metabolic support (EMS) system used a pressure-controlled perfusion system including an oxygenator and pulsatile pump with controllers to maintain PaO 2, PaCO 2, ph and temperature. Kidneys were perfused at a subnormal temperature of 328C and mean arterial pressure of approximately 35 mmhg. Bagul et al. [18] and Hosgood et al. [19 ] preserved porcine kidneys at a more physiological temperature (37 388C) and mean arterial pressure of mmhg using adapted paediatric cardiopulmonary bypass technology. This isolated organ preservation system (IOPS) is perhaps the most modern and simple system described, using a clinical grade pediatric centrifugal pump, membrane oxygenator, venous reservoir, heat exchanger and PVC tubing (Fig. 1). Normothermic solutions The composition of the perfusate is a vital to ensure adequate delivery of nutrients and oxygen to maintain cellular integrity and vascular processes. A blood-based solution is the most natural choice of perfusate with the red blood cells providing an adequate oxygen carrying Figure 1 Schematic diagram of the isolated organ perfusion system indicating the direction of blood flow capacity [26]. However, blood-based perfusates do have their limitations. Early studies found that haemolysis, platelet activation and degradation of products during perfusion caused an increase in resistance and tissue oedema during prolonged periods of preservation [27]. Nonetheless, with the use of centrifugal pumps which reduce the risk of stress and haemolysis and membrane oxygenators enabling filtration and improved oxygenation, the use of a blood-base perfusates has remained a feasible option for shorter preservation periods. Bagul et al. [18] and Hosgood et al. [19,28] successfully used diluted leukocyte depleted autologous blood supplemented with a nutrient and electrolyte solution to preserve porcine kidneys for a short duration on their IOPS. An alternative to blood is to use an artificial blood substitute. Perfluorochemical and haemoglobin solutions can be used to deliver oxygen at normothermic temperatures. Perfluorocarbons (PFC) are hydrocarbons in which all or most of the hydrogen atoms are replaced with fluorine. They have twice the density of water and a high capacity for dissolving respiratory gases. The solubility of dissolved oxygen in PFC is approximately 25 times greater than blood or water, making them a very effective oxygen carrier [29,30]. PFCs are lipophilic therefore for intravascular use they must be formulated as an emulsion. This has proved to be problematic with the surfactants used to emulsify them being linked to side effects such as anaphylaxis, hypotension, reduced platelet counts and complement activation when administered by intravascular infusion [31]. Other problems include the variable half lives of different PFCs, instability due to particle size and inability to be sterilized [31]. The early EMS system described by Brasile et al. [25] used an acellular normothermic solution based on a modified cell culture medium containing essential and nonessential amino acids with a range of other nutrients and PFC emulsion (Perflubron). Haemoglobin-based solutions such as stroma-free haemoglobin have also been found to be problematic causing toxic effects on the kidney [26]. However, pyridoxalated haemoglobin-polyoxyethylene (PHP) solution was deemed to be a more stable solution. Brasile et al. [16,32] has since replaced the PFC with pyridoxylated haemoglobin in their perfusion medium. New more stable second generation PFCs are being developed and several are undergoing clinical trials to assess their safety. Humphreys recently used a commercially made PFC, Oxygent (Alliance Pharmaceutical Corp, San Diego, CA, USA) to provide oxygenation and reduce ischaemic injury to the kidney during warm ischaemia by retrograde infusion through the urinary collecting system in rabbit kidneys [33]. These new PFC solutions may hold more promise for future development of normothermic preservation perfusates.

4 172 Organ preservation and procurement Table 2 Parameters to assess viability during normothermic kidney perfusion Viability assessment; kidney function Perfusion parameters Renal function Tubular function Intracellular enzymes molecular markers Haemolysis Urine output Filtration fraction AST, GGT Renal blood flow Creatinine levels Fractional excretion Na þ Free iron Intrarenal resistance Creatinine clearance Total protein LDH Oxygen consumption Acid base balance Oxidative damage AST, aspartate aminotransferase; GGT, gamma-glutamyltransferase; LDH, lactate dehydrogenase. Viability testing With the restoration of metabolism, normothermic preservation techniques offer the potential to assess viability before transplantation. This may be particularly advantageous for extended criteria or DCD donor kidneys to prevent the transplantation of nonfunctioning kidneys. Specific tests such as the pressure/flow index and measurement of intracellular enzymes are used in hypothermic machine perfusion to predict viability [34,35]. However, the efficacy in detecting nonviable organs has not been fully determined. Normothermic perfusion could offer a more accurate and immediate assessment of viability compared with hypothermic techniques. Various tests including renal and tubular cell function, perfusion parameters and change in kidney weight could be used in addition to the analysis of the perfusate measuring intracellular enzymes or biomarkers of injury for a more comprehensive assessment of the condition of the kidney. Many of these parameters have been used experimentally during normothermic preservation and in isolated kidney perfusion systems to successfully assess viability and could be adapted for clinical use [19,23,36 42] (Table 2). Normothermic kidney manipulation/ conditioning Another clear advantage of restoring metabolism during preservation is the opportunity to manipulate and treat the kidney before transplantation. Normothermic preservation supports cellular processes with the active production of ATP [18]. Brasile et al. [43] found that the protective gene hemeoxygenase-1 could be up-regulated after treating kidneys with cobalt protoporphyrin (CoPP), a heme analog during normothermic perfusion after canine kidneys were exposed to both warm and cold ischaemic injury. The same group also demonstrated that with the addition of a fibroblast growth factor, active cellular recovery was evident after kidneys had been exposed to 2 h of warm ischaemic injury [44]. In the same study, ex-vivo gene transfection of the fibroblast growth factor also lead to transcription and protein synthesis during 24 h of ex-vivo normothermic perfusion in nontransplanted human kidneys [44]. This finding is direct evidence that normothermic preservation techniques could be adapted as a device to manipulate the kidney, regulating specific genes to target the long standing problems such as I/R injury, acute rejection and renal transplant fibrosis. Normothermic preservation also enables the treatment of kidneys with therapeutic agents to enhance the preservation condition and target the mechanisms of I/R injury after transplantation. The anti-inflammatory and oxygen scavenging properties of the gaseous molecules carbon monoxide and nitric oxide have recently been investigated in an ex-vivo porcine kidney model [28]. Kidneys treated with a novel carbon monoxide releasing molecule (CORM-3) during 2 h of normothermic perfusion had improved renal and tubular cell function and lower intrarenal resistance after reperfusion compared with kidneys treated with a nitric oxide donor. Pharmacological interventions during normothermic perfusion have the added advantage that the agent is directly targeting the kidney in isolation rather than the complexity of treating the recipient. Conclusion Kidneys from marginal and DCD donors require additional support during preservation to maintain tissue viability. Maintaining circulation before retrieval, continuously during the preservation period or for a short period to resuscitate the kidney appears to be advantageous compared with hypothermic techniques. Furthermore, normothermic preservation techniques have a greater potential than simply preserving viability. They may be used as a method to protect against preservation and I/R injury, resuscitate the kidney to instigate repair mechanisms, regulate specific genes targeting acute and chronic conditions and also allow a comprehensive assessment of the kidney before it is transplanted. Nonetheless, more experimental evidence is needed to determine the efficacy of these techniques. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest Additional references related to this topic can also be found in the Current World Literature section in this issue (p. 263). 1 Fuller BJ, Lee CY. Hypothermic perfusion preservation: the future of organ preservation revisited? Cryobiology 2007; 54:

5 Normothermic kidney preservation Hosgood and Nicholson Valero R, Cabrer C, Oppenheimer F, et al. Normothermic recirculation reduces primary graft dysfunction of kidneys obtained from nonheart-beating donors. Transpl Int 2000; 13: Gravel MT, Arenas JD, Chenault R, et al. Kidney transplantation from organ donors following cardiopulmonary death using extracorporeal membrane oxygenation support. Ann Transplant 2004; 9: Lee CY, Tsai MK, Ko WJ, et al. Expanding the donor pool: use of renal transplants from nonheart-beating donors supported with extracorporeal membrane oxygenation. Clin Transplant 2005; 19: Reznik O, Bagnenko S, Scvortsov A, et al. The use of in-situ normothermic extracorporeal perfusion and leucocyte depletion for resuscitation of human donor kidneys. Perfusion 2010; 25: This small clinical study describes the use of normothermic recirculation with leukocyte depleted blood. It highlights the potential of the technique to expand the organ donor pool. 6 Das S, Maggio AJ, Sacks SA, et al. Effects of preliminary normothermic flushing on hypothermic renal preservation. Urology 1979; 14: Hughes JD, Chen C, Mattar SG, et al. Normothermic renal artery perfusion: a comparison of perfusates. Ann Vasc Surg 1996; 10: Kay MD, Hosgood SA, Harper SJ, et al. Normothermic versus hypothermic ex vivo flush using a novel phosphate-free preservation solution (AQIX) in porcine kidneys. J Surg Res [Epub ahead of print] This study used a novel normothermic preservation solution to flush kidneys immediately after retrieval. This technique could be developed to completely negate the need for hypothermic temperatures. 9 Kay MD, Hosgood SA, Harper SJ, et al. Static normothermic preservation of renal allografts using a novel nonphosphate buffered preservation solution. Transpl Int 2007; 20: Van der Wijk J, Slooff MJ, Rijkmans BG, et al. 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