A Rat Orthotopic Renal Transplantation Model for Renal Allograft Rejection

Rat orthotopic renal transplantation is an important model to investigate the mechanism of renal allograft rejection in preclinical studies and could aid in developing novel approach to improving the long-term survival of renal allografts. End-to-end anastomosis of renal vessels in this surgery avoids ischemia of the lower body and the thrombosis of inferior vena cava, while anastomosis of ureter in an end-to-side tunnel method reduces postoperative urine leakage and the twist of the ureter. This method provides insight into investigating the mechanisms of immune rejection and injury of renal allografts after kidney transplantation.

Begin by incising the abdomen using a surgical scissor along the midline from the symphysis pubis to the subxiphoid. Then pull open the incision using a retractor. Take out the intestines from the right side of the incision and wrap them with moistened gauze to prevent them from drying out.

Then expose the left kidney. Dissociate the left renal artery and vein using micro-forceps and cotton swabs under 20 times magnification. Ligate and transect the adrenal vein and genital vein if necessary.

Dissociate the ureter with the help of micro-forceps, preserving the peripheral connective tissues to ensure blood supply to the ureter. Dissociate and clip or ligate the aorta approximately five millimeters above the left renal artery with a vascular clamp or 4-0 polyamide monofilament suture. Then transect the left renal vein distal to the conjunction of the left genital vein and adrenal vein.

Using a 24 gauge scalpel needle, flush the kidney from the aorta below the left renal artery with ice cold UW solution supplemented with 100 units per milliliter Heparin until blood fades in color. Transect the left renal artery next to the aorta, then transect the ureter next to the bladder and dissociate the kidney with the help of micro-forceps and scissors. Preserve the donor left kidney in ice cold UW solution.

Under 20 times magnification, dissociate the left renal artery and renal vein of the recipient rat using cotton swabs and micro-forceps. Ligate the adrenal vein and genital vein if necessary. Clip the left renal artery and renal vein at the root using microvascular clamps, then transect the renal artery and renal vein three millimeters away from the microvascular clamp.

Dissociate and ligate the left ureter approximately two to three centimeters below the kidney with an 8-0 polyamide monofilament suture and transect it at the ligation. Resect the recipients native left kidney by dissociating the kidney with the help of micro-forceps and scissors. Implant the donor kidney into the left renal fossa of the recipient rat and put ice around the implanted donor kidney.

Switch to 45 times magnification, then anastomose the donors renal artery and renal vein to the recipients renal artery and renal vein in an end-to-end pattern using 10-0 polyamide monofilament sutures. To anastomose the renal artery with interrupted sutures, place the stay sutures at 12 and six o'clock positions of anastomosis. Equidistantly suture one side of the anastomosis between the two stay sutures with two to three stitches.

Turn over the stay sutures and similarly suture the other side of the anastomosis between the two stay sutures with two to three stitches. To anastomose the renal vein with continuous sutures, place the stay sutures at the six and 12 o'clock positions of the anastomosis. Suture one side of the anastomosis from the 12 o'clock position with four to five stitches using the running sutures.

Turn over the stay sutures and similarly, suture the other side of the anastomosis from the six o'clock position. Reperfuse the donor kidney by releasing the non-invasive microvascular clamp of the renal vein, identify the bleeding sites and make additional stitches if necessary. Next, release the non-invasive microvascular clamp of the renal artery, identify the bleeding sites and make additional stitches if necessary.

Under 20 times magnification, ligate or stitch the end of the donor ureter with a 4-0 polyamide monofilament suture as a tow to drag the end through the tunnel of the recipients bladder. Fix the donor ureter with the recipients bladder by stitching the adventitia of the donor ureter with the muscle layer of the recipients bladder outside at four equidistant positions using an 8-0 polyamide monofilament suture. Afterward, transect the end of the donor ureter with the suture outside the recipients bladder and allow the donor ureter to shrink back into the recipients bladder.

Place the intestines back into the abdominal cavity and close the abdominal incision with continuous sutures in the muscle layer and then the skin layer with a 4-0 polyamide monofilament suture. At ten weeks after transplantation, hematoxylin and eosin staining and periodic acid shift staining revealed glomerular sclerosis, interstitial fibrosis, tubular atrophy and interstitial arterial sclerosis in the renal allograft in contrast to the isograft kidney. Silver staining showed the thickening of the glomerular basement membrane in the allograft kidney when compared with the isograft kidney, indicating the success of the rat orthotopic renal transplantation model.

Because the vascular hole of the renal is very thin, to avoid the wrong suturing, remember to perform the anastomosis on the high magnification field.