This protocol describes all of the key steps to successfully establish a porcine orthotopic kidney auto-transplantation model using an organ preservation time of 24 hours and peri-operative telemetric monitoring. The major advantage of this large animal auto-transplantation model over ex-vivo studies on small animal models are a surgical anatomical and the pathophysiological similarities to the clinical setting. Our team at the university hospital RWTH Aachen will be demonstrating the procedures.
After confirming the appropriate level of sedation in an overnight fasted adult female pig, make a three to five centimeter incision in the groin and dissect the femoral artery in a 360 degree fashion. Use a number 11 scalpel to make an arteriotomy and insert the arterial sensor of the telemetry device. After making a subcutaneous pouch in the left flank, tunnel the telemetry transponder into the flank and fix the transponder to the muscle fascia with a 3-O polypropylene single knot suture.
Next, make two small incisions on the left and right side of the thorax and tunnel the red and white ECG electrodes to their designated location. When the electrodes have been secured, commence registration of the telemetry data. Upon stable signal acquisition, close the groin incision with sutures and spray film dressing and place a suitable sized porcine jacket onto the animal.
14 days after the first surgery, make a four to five centimeter incision in the jugular groove on the right side of the anesthetized animal and dissect the subcutaneous and muscle tissue to expose the juggler vein. Use a peel away sheet introducer or the Seldinger method to insert the jugular catheter. Secure the catheter with a 5-0 polypropylene suture and close the incision in two layers.
Following surgical disinfection and sterile draping, perform a medium laparotomy to open the abdomen and use a standard abdominal retractor to expose the surgical field. Open the peritoneal layer and use a monopolar cautery, bipolar forceps, and fine scissors to dissect the left kidney and ureter from any adherent tissue. Then use a 3-0 Polyglactin suture to ligate and divide the left ureter at least 10 centimeters distal to the kidney hilum.
To retrieve the graft kidney, use vascular clamps to close the renal artery and vein close to the aorta and vena cava. And cut the vessels above the clams. Then give the kidney to the back table team for additional preparation and use a 5-0 polypropylene suture to close the stump of the renal artery and the renal vein.
Immediately after retrieval, use a standard 14 gauge peripheral catheter to cannulate the renal artery and flush the kidney with at least 500 milliliters of ice cold organ preservation solution. After the flush, wrap the graft in sterile organ bags for storage in organ preservation solution with a target cold ischemic time of 24 hours at four degrees Celsius using a computer controlled cooling circuit. Two to five minutes before vascular clamping, intravenously inject 100 international units per kilogram of natrium heparin and subsequently remove the contralateral right kidney as demonstrated and reopen the median laparotomy.
Two to five minutes before vascular clamping, intravenously inject 100 international units per kilogram of natrium heparin and subsequently remove the contralateral right kidney as demonstrated. Place the preserved graft kidney into the surgical field and initiate the administration of 0.1 to 1 microgram per kilogram per minute of norepinephrine as continuous infusion using the mean arterial blood pressure and heart rate to monitor the efficiency. For end-to-end anastomosis of the renal vein, use 5-0 polypropylene to place two corner stitches and suture the back wall in a continuous fashion.
After completing the back wall, use one end of the cranial corner stitch to suture the front wall in a craniocaudal direction and flush the vein with a 100 international unit per milliliter of Heparinized saline solution. For end-to-end anastomosis of the renal artery, use a 6-0 polypropylene cranial corner stitch before using the parachute technique to suture the back wall in a continuous fashion as demonstrated. Suture the front wall with the other end of the double armed 6-0 polypropylene suture and flush the artery with 100 international units per milliliter of Heparinized saline solution.
After anastomosis completion, tie the two threads at the caudal corner and sequentially open the venous vascular and arterial clamps to allow reperfusion of the kidney. Following reperfusion, topically administer five milliliters of papaverine to the outside wall of the renal artery and intravenously administer 250 milliliters of 20%glucose solution in a single 80 milligram dose of Furosemide. Next, pass a 12 French pediatric urine catheter through the abdominal wall and skin and use ligatures to secure the catheter to the ureter.
Close the peritoneal layer over the kidney to prevent dislocation of the graft and kinking of the vascular anastomosis. And close the abdomen in four layers as demonstrated. Finally, use color Doppler ultrasound to ensure adequate arterial and venous perfusion of the kidney graft before returning the fully recumbent animal to the housing facility.
In this representative set of five porcine orthotopic kidney auto-transplantation experiments, the transponder implantation and auto-transplantation procedures were successful in each animal with sufficient telemetry signals registered over the observation period. Following abdominal closure, color Doppler ultrasound revealed a satisfactory arterial and venous perfusion of the kidney in all cases. Serum potassium and creatine values peaked on postoperative days three to four and showed gradual recovery afterwards.
The blood pH remained within normal range and the urine output recovered to normal values over the first four postoperative days. This protocol can be used to answer various questions about the effects of organ preservation techniques or ex-vivo or in-vivo therapies in a clinically significant large animal model.
