Normothermic Machine Perfusion of Rat Kidneys for Transplantation

Our laboratory at Sun Yat-sen University has been performing small animal normothermic machine perfusion for five years. This specific protocol outlines critical steps that are necessary for successfully performing NMP and transplantation technique of rat kidneys. To increase the probability and the reproducibility of success, our protocol including three distinctive steps:Acquire the donor kidney, ex vivo NMP, and the subsequent kidney transplantation on the recipient.

Demonstrating this procedure will be Tong Chen. After induction of anesthesia, use an electric clipper to shave the abdominal hair. Range from xiphoid process to the pubic symphysis, both side to the midaxillary line.

Use medical tape to fix the rat supine on a surgery board with a heating pad on it. Disinfect the abdominal skin with an iodine-soaked cotton ball, followed by an alcohol-soaked cotton ball for additional antisepsis. Repeat the above disinfection procedure for a total of three times.

Spread the sterile gauze on the disinfected abdominal skin. Make a laparotomy incision with a scalpel and forceps from the xiphoid process to the bladder, approximately eight centimeter in length. Retract the abdominal wall with a pull hook to expose the surgical field.

Place a ligature around the left ureter. Dissect the left ureter. Catheterize it with a 26 G tube, about 0.5 centimeter long.

Then tighten the ligature. Separate the left renal vessels respectively. Place a ligature on the renal vein.

Place a ligature on the renal artery. Tighten the ligatures on vessels sequentially. Dissect the blood vessels, ureter, and the perirenal connective tissue.

Cannulate the left renal artery with a preassembled 24 G indwelling needle. Fix the cannula with a ligature, and perfuse the kidney until it turn pale. Remove the kidney and record the appearance size and weight.

Store it in HCA solution. Expose the abdominal aorta, then collect 10 milliliter of heparinized blood. Mix 10 milliliter of donor blood with the basic perfusate in the organ chamber.

After filtration through cellular sieve to get the complete perfusate, leave about two milliliter of the mixture specimen as a baseline sample. Turn on the peristaltic pump and initiate perfusion. Fill the perfusion tube with the mixture.

Secure a sterile round yarn over the chamber with clamps. After the mixture fills the tube and flows out, connect the kidney with the perfusion system and start perfusion. Use a clamp to secure the pipeline.

Connect the ureter cannula with an extension tube. During perfusion, collect one milliliter sample every hour. Remove the ureter extension tube.

Remove the kidney from the perfusion system. Connect the indwelling to the micropump. Perfuse the kidney with pre-cooled HCA solution until the kidney turns pale.

After induction of anesthesia, use an electric clipper to shave the abdominal hair. Range from xiphoid process to the pubic symphysis, both sides to the midaxillary line. Use medical tape to fix the rat supine on a surgery board with a heating pad on it.

Disinfect the abdominal skin with an iodine-soaked cotton ball, followed by an alcohol-soaked cotton ball for additional antisepsis. Repeat the above disinfection procedure for a total of three times. Spread the sterile gauze on the disinfected abdominal skin.

Make a laparotomy incision with a scalpel and forceps from xiphoid process to the bladder, approximately eight centimeter in length. Retract the abdominal wall with a pull hook to expose the surgical field. Dissociate and ligate the right renal vessels.

Dissociate and ligate the right ureter. Excise the right kidney by cutting the vessels and the ureter. Isolate and expose the right renal vessels.

Clamp the left renal artery and the vein sequentially with vascular clamps. Excise left kidney by cutting the renal vessels and the ureter. Completely expose the vascular lumina of both artery and vein.

Respectively flush the recipient's left renal artery and vein with heparinized saline. After transferring the donor's left kidney, make an interrupted closure of the upper pole of the renal artery. Make an interrupted closure of the lower pole of the renal artery.

Make a continuous closure of the ventral aspect of the renal artery. Tighten the sutures to make sure there are no gaps in the vessel wall. Expose the dorsal aspect of the renal artery.

Make a continuous closure of the dorsal aspect of the renal artery. Make an interrupted closure of the upper pole of the renal vein. Make an interrupted closure of the lower pole of the renal vein.

Make a continuous closure of the dorsal aspect of renal vein. Fix the continuous anastomotic suture before starting making next closure. Make a continuous closure of the ventral aspect of the renal vein.

Ensure full thickness apposition of the vascular wall with each suture placement, while avoiding transmural penetration to the contralateral side. Fix the continuous anastomotic suture. Release the vascular clamps, and open the renal artery and vein.

Use a clean cotton ball to press and stop bleeding. Insert the 26 G ureteral cannula of the donor ureter into the recipient's ureter. Secure the uretal stump on both the right and the left sides.

Fix graft by suturing the connective tissue to the perirenal adipose tissue. Check out the appearance of the graft before the abdominal closure. After the procedure, suture the abdominal muscle layer and skin layer successfully, and close the abdominal cavity.

Apply lidocaine spray to the topical anastomosis. Use the heating pad to maintain the rat's body temperature during recovery. Place the rat in a metabolic cage for monitoring once ambulatory.

During NMP, vascular resistance of kidneys remained stable for three hours. The kidneys could produce urine normally during the perfusion. And the kidneys exhibit a low fractional excretion of sodium and a high fractional excretion of potassium.

Those results indicate that the kidney remained good physiological function during NMP. The postoperative seven-day survival of the NMP group is 100%showing no difference compared with the SCS group. Both recipients of NMP and SCS group exhibited comparable urine output within the first seven postoperative days.

Both the serum creatinine and the blood urea nitrogen levels on the postoperative seven day also show no significant differences between the group of NMP and SCS. Both group can see the normal glomerular architecture without inflammatory cell infiltration. The renal tubular epithelial cells are intact, without evident cellular injury, necrosis, or apoptosis, and the tubular lumen is clear of casts or deposits.

In this protocol, we demonstrate the safety and the validity of our model, which provides significant insight into pre-transplant treatment of organs, and lays way for the development of protective pretreatment of organ transplantation.