Normothermic kidney perfusion: An overview of protocols and strategies

Normothermic machine perfusion (NMP) technologies are emerging as an important adjunct in organ preservation and transplantation. NMP can enable the reduction or avoidance of cold ischemia and allows for pretransplant measurement of function and metabolic status to assess the suitability of the organ for transplantation. The key requirement of NMP is to provide an environment that is protective to the organ, ensures optimal oxygen delivery and supports metabolic function. Red blood cell‐based solutions, artificial hemoglobin solutions, and acellular solutions have all been utilized in NMP. However, there is no clear consensus on perfusion protocols. A period of NMP after hypothermic preservation is the most commonly used strategy. As an alternative, several groups have developed and tested the feasibility of more prolonged periods of NMP. There are only a few reports of the application of NMP in clinical kidney transplantation and each uses different approach and conditions. This review details the rationale for NMP protocols considering duration of NMP and different perfusate compositions in experimental and clinical models. We also include a discussion on the mechanistic action of NMP, comparison of subnormothermic and hypothermic conditions, the different logistical approaches and future requirements.


| INTRODUC TI ON
One initiative to reduce the number of patients waiting for a kidney transplant has been the use of kidneys from extended criteria donors (ECD) and donation after circulatory death (DCD) donors.
Increasing age, co-morbidities, and ischemic injury in these kidneys result in higher rates of early graft dysfunction compared to kidneys from younger, standard criteria donors (SCD). 1 Delayed graft function (DGF) complicates recovery, increases the likelihood of acute rejection and can reduce graft survival. Hypothermic preservation techniques have been pivotal in the success of organ transplantation. 2 Kidneys can be stored on ice or perfused with cold preservation solution and safely transported between donor and recipient centers. Nonetheless, in an anaerobic environment the condition of the kidney gradually deteriorates. 2 This has brought about a change in thinking in recent years. Preserving an organ at a normothermic temperature can restore cellular metabolism and replenish adenosine triphosphate (ATP) synthesis. If the optimal conditions are applied, this may prevent deterioration of the organ and promote recovery. Furthermore, by restoring function normothermic machine perfusion (NMP) also provides an opportunity to assess organ quality pre-transplantation and to serve as a platform for the delivery of pretransplant therapies to promote recovery. 3 However, there is no clear consensus on the best NMP strategy for the kidney. Figure 1 details the different published components used for NMP.
There are only a handful of clinical reports but there is an increasing volume of experimental work exploring a variety of different approaches that may lead to more widespread application. This review will examine the different protocols and practices used for kidney NMP (Table 1) and provide some insight into the mechanistic effects and future consideration of NMP. This will aid the development and translation of NMP technologies for the kidney into clinical transplantation.

| NORMOTHERMIC MACHINE PERFUS ION SYS TEMS
At present the Kidney Assist ™ Device made by Organ Assist based in the Netherlands is the only commercially available, CE marked, NMP system for the kidney. Other reported systems are in-house adaptations that use current cardiopulmonary bypass technology or custom-made perfusion devices using centrifugal or roller pumps. The UK-based company OrganOx has designed a prototype portable system for maintaining a kidney under NMP conditions for prolonged periods but at the time of writing this has not been marketed. 4  RBCs is known to increase the amount of nontransferrin bound iron. 5 This produces acute tissue iron deposition and initiates an inflammatory response. Hemolysis occurs due to an increase in unconjugated bilirubin levels and contact with the artificial surfaces of the perfusion circuit. 6 There are also time-dependent metabolic and biochemical alterations including diminished intracellular ATP levels, diminished 2,3-Diphosphoglycerate (2,3-DPG) levels, and a loss of bioactive nitric oxide (NO) derivatives. Functionally, longer-banked RBCs are less deformable and adhere excessively to the vascular endothelium. 7 Furthermore, the high doses of heparin that are added to prevent thrombosis can, paradoxically, cause increased RBC aggregation.

| Oxygen carriers
Whole blood has been used in NMP systems as a surrogate for revascularization to assess function and viability. However, its use is not considered ideal for NMP. The presence of leukocytes and platelets heightens the ischemia reperfusion injury response, with increased neutrophil infiltration, the release of reactive oxygen species (ROS) and endothelial and tubular damage. 8 Autologous whole blood with the leukocytes and platelets removed but retaining the plasma component has also been used in the development of NMP techniques. 9 Plasma contains albumin and globulins to maintain osmotic pressure, electrolytes to help maintain blood pH and immunoglobulins to fight infection and therefore beneficial for NMP conditions. 10 However, it also F I G U R E 1 Components used for normothermic machine perfusion of the kidney. HBOC, hemoglobin-based oxygen carrier; RBC, red blood cells [Correction added on November 20, 2020, after first online publication: Figure 1

201, Haemoglobin Oxygen Therapeutics LLC) is a polymerized bovine
hemoglobin-based oxygen carrier (HBOC) of low immunogenicity. It has an oxygen carrying capacity similar to that of human hemoglobin at normothermic temperatures and releases oxygen to tissues more readily than corpuscular hemoglobin. 11 Hemopure has been used for NMP in a series of experimental porcine and human kidneys that demonstrated equivalence in comparison to RBCs. 12 Nonetheless, Hemopure has not been widely tested and difficulties in supply limit its use. Furthermore, the hemoglobin contained in Hemopure has a tendency to become oxidized to the ferric (Fe 3+ ) form thus generating methemoglobin, which is toxic and increases oxidative stress.
Pyridoxylated bovine hemoglobin is another alternative oxygen carrier reported by Brasile et al. 13 Their Exsanguinous Metabolic Support (EMS) solution (Breonics) is made up of a highly enriched tissue culture-like medium containing essential and nonessential amino acids, lipids and carbohydrates supplemented with bovine hemoglobin.

| Acellular solutions
A number of experimental studies have used acellular-based mediums that have the capacity to carry oxygen. Lifor is an artificial preservation medium containing a nonprotein oxygen carrier that can be used at room temperature. 14 Aqix RS-I is a preservation medium formulated for use in tissue, organ, and cellular preservation. It is designed to maintain all aspects of cellular function across a range of temperatures (4-37.4°C) by providing natural colloidal buffering to prevent edema. 15,16 STEEN solution ™ is a preservation solution that contains a high concentration of albumin and dextran to create a high osmotic pressure. 14,17 STEEN solution is not naturally formulated for the kidney and dilution is required to prevent cellular damage and diffuse vacuolation of the tubular cells.

| Supplements
A number of essential ingredients are needed to provide volume to the perfusate and metabolic support during NMP (Table 1). In brief, the protocols list the following ingredients: crystalloid-or colloidbased solution with or without supplementation of albumin to maintain volume and prevent cellular edema, mannitol to increase osmolarity and enhance renal blood flow, vasodilators such as epoptrestenol sodium, a synthetic prostacyclin 18 , verapamil, a calcium channel blocking agent, 19 or sodium nitroprusside, 18 and corticosteroids to reduce inflammation. 12,17,20 Basic nutrient preparations such as glucose and amino acids can be administered to support metabolism. [20][21][22][23] Insulin is normally added to the parenteral solution to promote the absorption of glucose ( Table 1). The administration of multivitamins or vitamin C may also have some beneficial effects to reduce oxidative stress during NMP. 24 These preparations are based on the experiences of cardiopulmonary bypass technology without being thoroughly tested in kidney NMP models. Therefore, the true metabolic requirements of kidney NMP are unknown. The nutritional and metabolic demand may be different for kidneys from different donor types and for more prolonged periods of perfusion. Colloids such as albumin may be required to prevent cellular edema and substituting glucose with a lipid may be a more efficient source of energy.

| Antibiotics
Bacterial or fungal contamination of the preservation solution used to store deceased donor organs is common due to circumstances arising in the donor or during the retrieval process. 25

| NORMOTHERMIC MACHINE PERFUS I ON S TR ATEG IE S
Most NMP protocols combine hypothermic and normothermic techniques to provide a more practical logistical approach. Our group established a protocol of a 1 h end period of NMP using a RBC-based solution (end-ischemic approach). The protocol was based on experimental work in porcine kidneys, which demonstrated that 1-2 h of NMP was sufficient to replenish ATP and upregulate protective mechanisms after a period of hypothermic preservation. 28 The results were encouraging as 1 h NMP was associated with a significant reduction in the rate of DGF (5.6%) compared to a historical control group of kidneys undergoing static cold storage alone (36.2%). 29 We also demonstrated that after NMP, kidneys could be safely In a series of studies using a porcine autotransplant model the

| Temperature
Hypothermic preservation is based on the principle that at 4°C the rate of metabolism is approximately 10% of the level at normal physiological temperature. 34 This slows the depletion of ATP and also inhibits the degrading processes (phospholipid hydrolysis). However, during static cold storage the gradual depletion of It is proposed that NMP at physiological temperatures (36-37°C) can replenish ATP and prevent ischemic injury. However, there is a risk that, due to the high metabolic demand, the conditions could accelerate the breakdown of ATP and cause reversion to anaerobic glycolysis. To reduce the metabolic rate but maintain aerobic metabolism and viability, NMP can be performed using subnormothermic temperatures (21-32°C). However, one consideration is that the oxygen carrying capacity of RBCs is reduced at lower temperatures and may potentially cause kidney injury. 37 Encouragingly, one study found that the outcome of using RBCs at 22°C for 4 h was similar to NMP with Hemopure at 22°C. There was no comparison to NMP at 37°C but both conditions were superior to cold storage. 38 For more prolonged periods (24 h), the acellular approach using STEEN solution at 21°C supported a lower and more stable vascular resistance compared to blood-based solutions at 37°C. 21 The work by Brasile et al also advocated the use of a subnormothermic temperature of 32°C using their EMS medium. 13 The gradual transition from cold to warm, described by Minor et al, is also a promising strategy. 16,32 The protocol involves a 90-min controlled phase of rewarming before NMP at 35°C using STEEN solution. This could protect against mitochondrial and cellular injury by reducing the levels of damage-associated molecular patterns (toll-like receptor 4 and high mobility group box 1 protein) during reperfusion. 39 Furthermore, it could more efficiently optimize ATP and oxygen consumption levels during reperfusion compared to the immediate transition from 4°C. 39

| Oxygenation
The majority of reported NMP systems use high levels of oxygen, either 100% or a 95% oxygen/5% carbon dioxide (carbogen) balance to maintain pH delivered through a membrane oxygenator. Typically, the partial pressures of oxygen are supraphysiological (70-75 kPa).
The delivery of 100% oxygen during NMP with an acellular-based perfusate is capable of maintaining the PO 2 above 500 mm Hg (66.67 kPa) across a range of temperatures. 37 The NMP conditions described by Brasile and Weissenbacher used a PO 2 that was closer to physiological levels (26 kPa). 4,13,33 There is no clear evidence to suggest the optimal oxygen concentration. However, due to the kidney's high metabolic demand and the unique regulation of renal metabolism, a range of concentrations appear to be tolerated without adverse consequences. 37

| MECHANIS MS OF AC TION
There is little information on the mechanistic effects of NMP. From experimental evidence we have shown that inflammatory cytokines (IL-6, IL-8) and heat shock proteins (HSPs) are upregulated during a 1 h period of NMP. 40 There is an assumption that these processes help to condition the kidney in preparation for reperfusion. A recent analysis of the transcriptional changes in pairs of human kidneys rejected for transplantation showed some support for this theory.
A 1 h period of NMP activated protective stress responses and promoted cell survival and proliferation. 41 This suggests that even a short period of NMP after cold ischemia can promote recovery during reperfusion. However, a short duration of NMP is unlikely to demonstrate the effect of repair.
The release of inflammatory cytokines and chemokines into the perfusate during NMP warrants further investigation. This may be related to the type of donor, previous ischemic injury, or caused by the perfusion conditions themselves. The passage of donor leukocytes from the interstitial compartment of the kidney into the circulation may also be a source of inflammation. The incorporation of specialist filters into the circuit could reduce circulating leukocytes and cytokines to reduce inflammation. 41,42 Brasile et al demonstrated that a 24 h period of NMP could facilitate recovery of a kidney and upregulate repair processes after ischemic injury. NMP conditions could be enhanced further by the addition of mesenchymal stromal cells 33 . The delivery of cellular therapies during NMP is a topical area of research but beyond the scope of this review.

| VIAB ILIT Y A SS E SS MENT
One particular benefit of NMP is that it may allow an assessment of the quality of a kidney prior to transplantation. We have formulated a basic scoring system (1 best, 5 lowest quality) using the macroscopic appearance, renal blood flow, and urine production during 1 h of NMP. It can be calculated in real time and has some association with early graft function. 43  There is one report of using NMP technology to avoid any exposure to cold ischemia. 47

| SUMMARY AND CON CLUS ION
There is an increasing amount of evidence from experimental work in support of using NMP technology in kidney transplantation. Nonetheless, the application of NMP into clinical practice has been limited.
Despite the potential benefit in the application of NMP techniques, logistical requirements are at the forefront. Prolonged periods of NMP, or indeed complete avoidance of cold ischemia, may well be the best conditions in which to preserve a kidney. However, the practicalities of applying these should not be under-estimated. Ultimately, NMP strategies require significant financial resources and will only be introduced more widely if they can be shown to have significant benefits over current hypothermic techniques.

D I SCLOS U R E
The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.