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Normothermic Ex Vivo Kidney Perfusion for the Preservation of 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.


Introduction
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 dialysis [1][2][3][4] . The persisting organ shortage represents a severe problem in the field of transplant medicine ( The outcome of kidney transplantation is negatively affected by the waiting time, with poorer outcome for patients subjected to prolonged dialysis . 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 conditions 10,11 . 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 transplant 12 .
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.

Protocol
Note: A schematic overview of the study protocol is presented in Figure 1.

Organ Retrieval
1. Preoperative Procedure 1. House the male Yorkshire pigs in a research facility for at least one week to reduce their stress level. Fast the pigs for a minimum of 6 hrs before induction of the anesthesia. 2. Initiate anesthetization of the pig by an intramuscular injection of ketamine (25 mg/kg), atropine (0.04 mg/kg), and midazolam (0.15 mg/ kg). Subsequently, transport the animal from the housing facility to the operating room (OR). 3. Place the pig in a supine position on the OR table. Let it breathe 2 L of oxygen with 5% of isoflurane spontaneously. After relaxation, expose the vocal cords with a laryngoscope and spray them with 2% lidocaine to prevent a spasm caused by intubation. After intubation with a 6.5 mm tube, block the cuff with 3 -5 ml of air. Note: Capnometry reveals the correct position of the tracheal tube. 4. Lower the isoflurane gas to 2.5%. Set the ventilator to 14 -16 breaths/min and the tidal volume to 10 -15 ml/kg bodyweight. Monitor the pig closely. Heart rate and oxygen saturation are recorded by pulse oxymetry. 5. Under sterile conditions, introduce an 8.5 Fr. x 10 cm catheter into the jugular vein in Seldinger technique 13 . Therefore, use a needle to puncture the venous vessel. After introducing a wire, replace the needle with a catheter. Eliminate the wire and fix the catheter to the skin. Administer 200 ml of Ringer's lactate solution per hour throughout the surgery.
2. Surgical Procedure 1. Following disinfection and coverage of the surgical field, perform a midline incision from xyphoid to pubic symphysis. To ameliorate the exposure, extend the surgical approach with a left lateral incision. Cover the large and small bowels with a towel and position them to the left side for optimal access to the right kidney. 2. Separate the inferior vena cava (IVC) from the abdominal aorta. Ligate aortic branches from the back of the aorta. 3. After complete aortic dissection to the back, pass a ligature around the aorta cranial to the renal branches. Additionally, place two ligatures cranial of the iliac bifurcation. Place a tie around the left renal artery. 4. Free the right kidney from its adherent tissue. Dissect the renal vein, artery, and ureter. 5. Open the diaphragm and administer 1,000 IU heparin per kg donor weight into the heart. For a DCD model, inject 40 mval of KCl intracardially 3 min after systemic heparinization to induce cardiac arrest. The cardiac arrest is valued as the starting point of warm ischemia.
6. Meanwhile, for collection of blood, connect the lines of CPDA bags (citrate, phosphate, dextrose, adenosine) to the catheter introduced to the left upper jugular vein. Perform a soft spin (1,500 x g without brake). Remove plasma and buffy coat under sterile condition (biosafety cabinet class II) and store the erythrocytes for transfusion. 7. Cannulate the aorta with an organ flush line above the iliac bifurcation. Tie the ligatures at the aorta and the left renal artery. 8. Flush the kidney with histidine-tryptophan-ketoglutarate (HTK) solution with a pressure of 100 cm H 2 O. Clamp the thoracic cava and collect the blood via the jugular catheter. Cut the abdominal cava below the renal vein to secure an optimal flush of the kidney. 9. After a complete flush of the right kidney, retrieve the graft with a segment of the aorta. Cut the renal vein and leave the ureter long. Table Preparation of the Kidney Graft for the Perfusion 1. Free the kidney from adherent tissue. Close the cranial part of the aorta with a tie and cannulate the lower part with a 1/4" x 3/8" reducer. Tie off smaller arterial branches coming from the aorta. 2. Cannulate the renal vein with a 1/4" x 3/8" reducer directly. Intubate the ureter with an 8 Fr. feeding tube.

Back
3. Place the kidney on ice until the start of the NEVKP.

Preparation of the Perfusion Circuit
The perfusion circuit consists of neonatal cardiopulmonary bypass equipment (Figure 2). to the arterial line tubing, and connect the pressure lines. Connect the level sensor. 4. Connect the venous and arterial sample lines to the venous and arterial sample ports. 5. Position the organ chamber on a stand and introduce the venous and arterial tubing through the prepared holes. Fix the tubing to the table and chamber firmly. 6. Insert the suction tubing into the roller pump and position one end into the chamber to collect the fluids. 7. Connect the oxygen tubing to the gas tank containing 95% O 2 / 5% CO 2 and the oxygenator. Connect the heating unit tubing to the oxygenator and the organ chamber. 8. Use tubing clamps to close the venous and arterial outflow lines. Apply another tubing clamp to the outflow of the venous reservoir. Management System (DMS) to record the data throughout the perfusion. Activate the heating unit to warm the perfusion solution and the organ chamber to 37 °C. Open the O 2 supply. 5. Open the tubing clamp behind the venous reservoir and free the centrifugal pump head from air completely. Start the centrifugal pump at 1,000 rpm and allow the solution to be propelled throughout the circuit. Clamp the tubing bypassing the arterial filter and release air from the arterial filter. 6. Zero the pressure lines. Activate the syringe and infusion pumps.

Representative Results
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 pO 2 reveal the metabolic activity of the kidney. The oxygen consumption was calculated using the equation ((pO 2

Discussion
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, HCO 3 , 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 criteria 15 and marginal grafts 8,9 , new preservation techniques minimizing cold storage are of particular interest [16][17][18][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.

Disclosures
The authors have nothing to disclose.