The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to transplantation.
Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.
Kidneys are the most frequently transplanted solid organs. For patients suffering from end-stage renal disease, kidney transplantation offers better life expectancy, and improved quality of life when compared to dialysis1-4. The persisting organ shortage represents a severe problem in the field of transplant medicine (Table 1) 5.
United States* | Eurotransplant region** | |
Patients on kidney transplant waiting list | 101,563 (February, 2015) | 10,689 (December, 2014) |
Deceased donor kidneys transplanted in 2014 | 10,650 | 3,119 |
Median waiting time to deceased donor kidney transplant (in years) | Up to 5 years* | Up to 4 years** |
Table 1. Kidney Graft Shortage in US and Eurotransplant Region.
The outcome of kidney transplantation is negatively affected by the waiting time, with poorer outcome for patients subjected to prolonged dialysis6. This has triggered interest in marginal kidney grafts as an additional donor source, such as kidneys from older donors, donors with multiple comorbidities (extended criteria donors (ECD), and kidneys donated after cardiac death (DCD). Marginal donor kidneys that would have been declined in the past are now considered for transplantation7.
A major obstacle for the use of marginal kidney grafts is the preservation technique of cold anoxic storage. Currently, kidney grafts are stored statically on ice or perfused at 4 °C without oxygen. The cold anoxic preservation technique is associated with ongoing graft injury during kidney preservation and does not allow graft assessment because of the lack of metabolism and urine production. In particular, marginal kidney grafts tolerate cold storage poorly, resulting in significant kidney injury, and high rates of delayed graft function (DGF) 8,9. DGF is a prognostic factor for poor long-term graft function.
Extracorporeal kidney perfusion represents an alternative method for the preservation, assessment and repair of organs. In a porcine model, beneficial results were presented for kidneys perfused ex vivo under normothermic conditions10,11. The first clinical trial performed in 2013 demonstrated a lower rate of delayed graft function when kidneys retrieved from extended criteria donors were perfused for 1 hr immediately before transplant12.
This paper presents a model of normothermic ex vivo kidney perfusion (NEVKP). The goal of this study is to reduce the applied cold ischemia time to a minimum and extend the period of NEVKP. NEVKP is an alternative preservation method that aims to reduce the damage that can be caused by cold storage techniques.
Note: A schematic overview of the study protocol is presented in Figure 1.
Figure 1. Study Protocol. This study protocol of normothermicex vivokidney perfusion is based on a porcine model. After surgical dissection of the vessels of the kidney graft and flushing with 500 ml of histidine-tryptophan-ketoglutarate (HTK), the graft can be retrieved. After cold storage (SCS) for 3 hrs, the kidney graft is perfused normothermic ex vivo (NEVKP) for multiple hours until the designated transplantation.
All animals received humane care in compliance with the ‘‘Principles of Laboratory Animal Care’’ formulated by the National Society for Medical Research and the ‘‘Guide for the Care of Laboratory Animals’’ published by the National Institutes of Health, Ontario, Canada. The Animal Care Committee of the Toronto General Research Institute approved all studies.
1. Animals
2. Organ Retrieval
3. Normothermic Ex vivo Kidney Perfusion (NEVKP)
In the following the results of six experiments using a model of heart-beating kidney retrieval are presented. After in situ flush and kidney retrieval, the grafts were stored on ice for 3 hr (SCS) while the erythrocytes were prepared. For the clinical setting, this simulates the time required for the retrieval and the back table preparation. NEVKP was performed for 10 hr.
To maintain physiological conditions and simulate an in vivo surrounding for the kidney, the organ chamber should be heated and sealed. Perfusion and urine replacement solution should represent physiological values for blood gas analysis, oncotic pressure, and osmolarity. Normal values (baseline values) obtained from Yorkshire pigs in situ, are located in each figure description, respectively (Figures 3 - 13). The aim of NEVKP is to ensure that the graft is supplied with sufficient oxygen and nutrition. As ischemia causes vasoconstriction, thus increasing intrarenal resistance, achieving a constant flow with a stable pressure is a good indicator for adequate oxygenation. After the target graft temperature of 37 °C is reached via rewarming of the organ after SCS, flow values and intrarenal resistance remain stable with a constant physiological pressure of around 60-80 mmHg throughout the whole perfusion (Figures 3 and 4). The quantity of urine production depends mainly on the composition of the perfusion solution (Figure 5).
Hourly measurements of venous and arterial pO2 reveal the metabolic activity of the kidney. The oxygen consumption was calculated using the equation ((pO2art – pO2ven) x flow / weight) (Figure 6) 14. During the perfusion pH, HCO3, and electrolytes are stable without requiring interventions (Figures 7 – 10). Real-time AST and lactate measurements serve to monitor cellular damage. No increase of parameters of cell injury is detected during the NEVKP period (Figures 11 and 12). The osmolarity of the perfusion solution is stable (Figure 13). Histological assessment reveals minor changes (Figure 14 – 16).
Figure 3. Mean arterial flow with standard deviation (ml/min). Throughout the perfusion the flow remains in a physiological range. Porcine physiological values, measured in situ: mean art. flow: 170 ± 57 ml/min (range 83 – 325 ml/min).
Figure 4. Intrarenal resistance (IRR), mean and standard deviation (mmHg/ml/min). The mean arterial pressure (MAP) remains constant between 60 and 80 mmHg. The intrarenal resistance is below 0.5 mmHg/ml/min constantly.
Figure 5. Total urine volume, mean and standard deviation (ml). The total urine volume mainly depends on the composition of the perfusion solution. The higher the oncotic pressure and the osmolarity, the lower the urine production.
Figure 6. Oxygen consumption, mean and standard deviation (ml/min/g).
Figure 7. pH venous, mean and standard deviation. The pH remains constant in a physiological range without administration of bicarbonates. Porcine physiological values, measured in situ: pH 7.46 ± 0.06 (range 7.34 – 7.63).
Figure 8. HCO3 venous, mean and standard deviation (mmol/l). The HCO3 remains in a physiological range without administration of bicarbonates. Porcine physiological values, measured in situ: HCO3 30.3 ± 2.4 mmol/L (range 21.6 – 35.8 mmol/L).
Figure 9. Venous sodium concentration, mean and standard deviation (mmol/L). The sodium remains in a physiological range. Porcine physiological values, measured in situ: 137.1 ± 3.8 mmol/L (range 118.7 – 140.9 mmol/L).
Figure 10. Venous potassium concentration, mean and standard deviation (mmol/L). The potassium remains constant in a physiological range. Porcine physiological values, measured in situ: 3.85 ± 0.46 mmol/L (range 3.5 – 5.36 mmol/L).
Figure 11. Venous aspartate aminotransferase, mean and standard deviation (AST; U/L). In the ex vivo normothermic kidney perfusion, AST demonstrates a cell injury marker. AST values are low throughout perfusion.
Figure 12: Lactate, mean and standard deviation (mmol/L). In the ex vivo normothermic kidney perfusion, lactate represents a cell injury marker. The values are stable throughout the perfusion.
Figure 13: Osmolarity of the serum, mean and standard deviation (mosm/L). A constant osmolarity in the perfusion solution secures low but constant urine production. Porcine physiological values, measured in situ: 282 ± 1.7 mosm/L (range 279 – 283 mosm/L).
Figure 14: Histology (H&E). 50X / 200X magnification of corticomedullary junction showing mild tubular vacuolization. No signs of necrosis. Please click here to view a larger version of this figure.
Figure 15: Histology (PAS). 50X / 200X magnification of corticomedullary junction showing mild tubular vacuolization. No signs of necrosis. Please click here to view a larger version of this figure.
Figure 16: Histology (TUNEL staining). 25X / 200X magnification. Very occasionally nuclei are stained demonstrating very low rates of apoptosis. Please click here to view a larger version of this figure.
This study demonstrates that NEVKP with an erythrocyte-based solution can be performed with excellent results for a prolonged period of time in a porcine model. During the 10 hrs ex vivo perfusion the kidneys demonstrated stable perfusion parameters, active renal metabolism, homeostasis, and minimal renal injury.
Urine production and kidney injury depend on the composition of the perfusion solution. It is important to keep oncotic pressure and osmolarity of the perfusate within a physiologic range. In particular, a low oncotic pressure will result in an unphysiologically high urine production with significant kidney edema and increasing markers of kidney injury. STEEN solution containing albumin is chosen in this model to regulate the oncotic pressure and to simulate physiologic conditions for the kidney. Sodium bicarbonate and calcium gluconate are added to the system to achieve physiological values of pH, HCO3, sodium, potassium, calcium, and chloride. The selection and dosage of the vasodilator is important to secure sufficient blood flow and oxygen supply.
The technique of normothermic ex vivo kidney perfusion has several limitations. Ex vivo perfusion is not associated with hormonal support of the kidney, which could negatively impact longer perfusion periods. In addition, the new technology, at this point in time, is associated with increased costs. Future improvements might simplify the technology and reduce the costs. The development of a portable kidney perfusion device might allow to completely avoid cold kidney storage in the future.
The severe and persisting organ shortage leads to an increased use of marginal organs (ECD or DCD kidney grafts) 7. Currently, organ preservation is based on static cold storage or hypothermic machine perfusion. As a prolonged cold ischemic time has a significant impact on the outcome of kidney function of standard criteria15 and marginal grafts8,9, new preservation techniques minimizing cold storage are of particular interest16-19.
A major obstacle to use marginal grafts more extensively is the inability to assess the quality and viability of organs prior to transplantation. Currently, only clinical parameters such as donor age, donor related diseases, and warm ischemia time of the grafts are used for the decision of whether an organ is accepted or declined for transplantation. By preserving the graft under normothermic conditions, graft assessment based on perfusion characteristics and data is possible. Real-time parameters such as renal vascular flow, pressure, intrarenal resistance, urine production, oxygen consumption, and kidney injury parameters (such as AST and lactate) are supposed to be useful parameters to assess the viability of the graft.
In addition, the active metabolism during NEVKP allows the application of repair strategies to improve marginal kidney grafts prior to transplantation. For example, inhibition of pro-inflammatory pathways, immunomodulation, gene transfer, as well as stem cell administration could be future techniques to modify kidney grafts during the preservation time and improve recipient outcome.
The authors have nothing to disclose.
We want to thank Sorin Group (Milano, Italy) for providing us with custom made kidney perfusion circuits. Furthermore we thank XVIVO Perfusion Inc. (Goteborg, Sweden) for providing us with Steen solution, BBraun AG (Melsungen, Germany) for the supply with syringe pumps, and Rieber GmbH & Co KG (Reutlingen, Germany).
Neonatal cardiopulmonary bypass technology | SORIN GROUP Canada Inc (Markhan, Canada) | Custom made | Neonatal venous reservoir D100 (500 mL, 1/16" in- and outflow), neonatal oxygenator D100, centrifugal pump head (Revolution), arterial bubble filter (D130) |
Heart lung machine, Stöckert S3 | SORIN GROUP Canada Inc (Markhan, Canada) | Custom made | Centrifugal pump, roller pump, control panel (sensors for pressure, flow, temperature, bubbles, and level), oxygen blender, heater unit |
Tubing | SORIN GROUP Canada Inc (Markhan, Canada) | 01906BPC SG XS | 3/16"x 1/16" |
019071PC SG XS | 1/4"x 1/16" | ||
019060PC SG XH | 3/8"x 1/16" | ||
Tubing connectors | SORIN GROUP Canada Inc (Markhan, Canada) | Various sizes | |
STEEN solution | XVIVO (Göteborg, SWE) | 19004 | 200 mL |
Ringer's lactate | Baxter (Mississauga, ON, CAN) | JB2324 | 175 mL |
Sodium bicarbonate | Hospira (Montréal, QC, CAN) | 6625050 | pH dependent |
Calcium gluconate | Pharmaceutical Partners of Canada (Richmond Hill, ON) | C31110 | Calcium dependent |
Heparin | Sandoz Canada Inc (Toronto, ON, CAN) | 10750 | 1000 IU |
Amino acid and glucose, Travasol 10% | Baxter (Mississauga, ON, CAN) | JB6760 | 1 mL/h |
Fast acting insulin, Novorapid | Novo Nordisk Canada Inc (Mississauga, ON, CAN) | DS6H748 | 5 IE/h |
Verapamil | Sandoz Canada Inc (Quebec, QC, CAN) | 52216 | 0,25 mg/h |