Orthotopic Porcine Kidney Transplantation: A Technique to Transplant Graft Kidney in Porcine Model

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Basic techniques and common procedures

1. Use female German landrace pigs (or comparable) for this protocol. Figure 1 depicts the summary of the described experimental protocol. Deliver the animals to the research facility 14 days before the surgery for acclimatization and house them in a temperature- and humidity-controlled barrier environment with a 12 h light and dark cycle (Figure 2). Fast the animals overnight before surgery.
2. Premedicate by an initial intramuscular injection of azaperone (4 mg/kg) and atropine (0.1 mg/kg), followed by an injection of ketamine (15 mg/kg) 10 min later.
3. After premedication, weigh the animal and transfer it directly from the housing facility to the central OR facility anesthesia preparation room.
4. Cannulate one of the large ear veins using an 18 G peripheral venous catheter. Monitor the animal by a standard ECG and pulse oximetry.
5. Initiate the anesthesia with propofol (3 mg/kg).
6. Expose the vocal cord with a laryngoscope and insert a 7.5 mm endotracheal tube. The cuff is blocked with air according to the manufacturer's recommendations.
7. Insert an oro-gastric drainage tube to remove fluid and air from the stomach.
8. Insert a urinary catheter via the urethra.
9. Subsequently, trim the skin in the area of the surgical incision.
10. Apply eye ointment to prevent drying of the cornea during surgery.
11. After orotracheal intubation, maintain anesthesia with isoflurane (final expiratory 1.45-2.0 Vol.%) and fentanyl (3-7.5 μg/kg/h).
12. Ensure active intraoperative temperature control of the animal by a heating pad and using warmed air. Insert a rectal probe to monitor body temperature (target temperature 36.5 °C – 37.5 °C).
13. Administer antibiotic prophylaxis using cefuroxime (35 mg/kg i.v.). Infuse Ringer solution at 4 mL/kg/h and increase to 8 mL/kg/h after skin incision. Administer a prophylactic dose of pantoprazole (40 mg i.v.) over the ear vein access.
14. Perform all surgical procedures under sterile conditions according to the general principles of surgical asepsis and antisepsis. Disinfect the surgical field with povidone-iodine solution and cover with surgical drapes.

2. Contralateral nephrectomy and orthotopic kidney auto-transplantation

1. During the recipient operation, adapt premedication and initial anesthesia to the restricted renal metabolism and avoid the use of ketamine. Induction is performed with propofol (3-5 mg/kg i.v.), midazolam (0,25 mg/kg i.v.), and atropine (0.1 mg/kg i.m.). Thereafter, the preoperative preparation is identical to the procedures described in section 1.
2. Maintain anesthesia with isoflurane (final expiratory 1.45-2.0 Vol.%) and fentanyl (3 – 7,5 μg/kg/h) and propofol (2 – 4 mg/kg/h).
3. Check and continuously monitor ECG, pulse oximetry, rectal temperature and the function of the telemetry transponder implanted in the right flank.
   NOTE: Strict anesthesia and blood pressure control is of crucial importance during the implantation procedure.
4. In rare cases where the arterial blood pressure signal registered over the telemetry transponder is not satisfactory due to the supine position of the animal, place a further arterial catheter into the right femoral artery using percutaneous puncture and the Seldinger technique.
5. Following sterile draping, reopen the median laparotomy and expose the surgical field using the abdominal retractor. The colon and small bowel are placed to the left side of the abdomen to expose the intact right kidney.
6. Dissect the contra-lateral (right) kidney and its vessels from the surrounding tissue. Dissect the right renal vein and renal artery in the direction of the kidney hilum to ensure sufficient vessel length for anastomosis.
7. Five minutes before vascular clamping, inject natrium-heparin intravenously (100 I.U./kg).
8. Clamp the right renal artery and the right renal vein using vascular clamps. The right kidney is removed. The vessels are checked for integrity before starting the anastomoses.
9. Place the preserved graft kidney into the abdomen and start the venous and arterial anastomoses.
10. From this point onwards, keep the mean arterial pressure over 80-90 mm Hg to ensure good early perfusion of the kidney graft following reperfusion. Achieve this partially by adequate volume management and partially by the administration of norepinephrine (0.1 – 1.0 μg/kg/min as a continuous infusion using the mean arterial blood pressure and heart rate for monitoring the efficiency).
11. Perform end-to-end anastomosis of the renal vein:
    1. After placing two corner stitches using 5-0 polypropylene, suture the back wall in a continuous fashion.
    2. Tie the cranial corner stitch and tie it together with the thread used for the back wall.
    3. After finishing the back wall, use the cranial corner stitch to suture the front wall in a cranio-caudal direction. Flush the vein with a heparinized saline solution (100 I.U./mL). Tie the caudal corner stitch.
       NOTE: In case of a size mismatch between the donor and recipient sides, a small growth-factor can be used to ensure a wide and sufficient anastomosis. There are many possible variations of the porcine renal vein branches. In the case of complex venous anatomy, a modified anastomosis approach is necessary (see Figure 3).
12. Perform the end-to-end anastomosis of the renal artery:
    1. Use a 6-0 polypropylene cranial corner stitch to perform the arterial anastomosis. Placing a further caudal, supporting corner stitch which is later removed, is optional.
    2. Suture the back wall in a continuous fashion using the parachute technique. After arriving at the caudal corner remove the second corner stitch (if applicable).
    3. Suture the front wall with the other end of the double-armed 6-0 polypropylene suture. Flush the artery with a heparinized saline solution (100 I.U./mL). Tie the two threads at the caudal corner.
13. Record the time needed for performing both anastomoses with a target warm ischemia time of <40 min.
14. Reperfuse the kidney by opening the venous vascular clamp and subsequently the arterial clamp. Check for significant bleeding.
15. If no significant bleeding from the anastomoses is observed, unwrap the kidney graft and pour warm normal saline solution in the abdomen covering the reperfused graft.
16. Reposition the graft, if needed, to ensure homogeneous reperfusion and avoid congestion.
17. Administer papaverine topically to the outside of the renal artery and the arterial anastomosis (5 mL undiluted).
18. After reperfusion, infuse 250 mL of 20% glucose solution to induce osmotic dieresis followed by the administration of a single dose of 80 mg of furosemide.
    NOTE: Following this, initial urine production may be observed.
19. To ensure urinary drainage, pass a 12 French pediatric urine catheter through the abdominal wall of the right flank of the animal, retroperitoneally.
20. Secure the catheter in the ureter using ligatures (2-0 polyglactin) and block the catheter with 2 mL saline. Further sutures are used to adapt and secure the ureter to the peritoneum of the abdominal wall (2-0 polypropylene). The catheter is also secured to the skin with at least two single knot sutures (2-0 polypropylene).
21. Close the peritoneal layer over the kidney to prevent dislocation of the kidney graft and kinking of the vascular anastomoses (3-0 polyglactin).
22. Close the abdomen in a similar 4-layer fashion as described earlier for the graft retrieval.
23. Following abdominal closure, maintain normothermia on the OR table.
    NOTE: Mean arterial blood pressure should be maintained over 80 mm Hg until the animal is awake and is in a prone position. Following abdominal closure, use color Doppler ultrasound to ensure adequate arterial and venous perfusion of the kidney graft (Figure 4). Monitor the animal closely until it is fully awake and can walk and drink spontaneously. The animals are given 1 L of Ringer solution during the recovery phase. Subsequently, return the animal to its box in the housing facility.