A Rat Orthotopic Renal Transplantation Model for Renal Allograft Rejection

Summary

The rat orthotopic renal transplantation model contributes to investigating the mechanism of renal allograft rejection. The current model increases the recipients’ survival without interference with blood supply and venous reflux of the lower body using an end-to-end anastomosis of kidney implantation and an end-to-side “tunnel” method of ureter-bladder anastomosis.

Abstract

Renal allograft rejection limits the long-term survival of patients after renal transplantation. Rat orthotopic renal transplantation is an essential model to investigate the mechanism of renal allograft rejection in pre-clinical studies and could aid in the development of novel approaches to improve the long-term survival of renal allografts. Donor kidney implantation in rat orthotopic renal transplantation is commonly performed by end-to-side anastomosis to recipients’ aorta and inferior vena cava. In this model, the donor’s kidney was implanted using end-to-end anastomosis to the recipients’ renal artery and renal vein. The donor’s ureter was anastomosed to the recipient’s bladder in an end-to-side ‘tunnel’ method. This model contributes to better healing of ureter-bladder anastomosis and increases the recipients’ survival by avoiding interference with blood supply and venous reflux of the lower body. This model can be used to investigate the mechanisms of acute and chronic immune and pathologic rejection of renal allografts. Here, the study describes the detailed protocols of this orthotopic renal transplantation between rats.

Introduction

Renal transplantation has become the most effective therapeutic approach for patients with end-stage renal function failure. However, T cell-mediated acute rejection and alloantibody-mediated humoral immune rejection result in the pathologic injury of renal allografts and limit the short-term and long-term survival of patients after kidney transplantation^1,2,3. Unfortunately, the effective pharmaceuticals that prevent the rejection of renal allografts are still lacking, because the exact mechanisms of immune and pathologic rejection of renal allografts are not clear. Consequently, the preclinical studies that elucidate the mechanisms of immune and pathologic rejection of renal allografts contribute to finding novel targets and developing relevant effective pharmaceuticals to prevent the rejection of renal allografts and eventually prolong the survival of patients.

Many potential immunological and pathophysiological mechanisms of renal allograft rejection have been proposed recently in rat model studies of orthotopic renal transplantation^4,5,6,7,8. These findings propose several novel targets and relevant interfering approaches as promising therapeutics to suppress renal allograft rejection, such as complement regulatory factors and anti-CD59 antibodies⁶, immunoproteasome, and epoxyketone inhibitors^7,8. Thus, rat orthotopic renal transplantation is an ideal preclinical model to investigate the mechanisms of immune rejection and pathologic injury of renal allografts after kidney transplantation.

Rat kidney transplantation has gradually shifted from heterotopic implantation of donor's kidneys⁹ to orthotopic renal implantation using end-to-side anastomosis of vessels or using end-to-end anastomosis of the ureter using a cuff method^10,11,12. The present study describes detailed protocols of the orthotopic renal transplantation between rats using end-to-end anastomosis to recipients' renal artery and renal vein, and an end-to-side "tunnel" method of ureter-bladder anastomosis, which avoids the ischemia of the lower body and the thrombosis of inferior vena cava and reduces postoperative urine leakage and the twist of the ureter.

Protocol

Inbred 8-10 weeks old male F344 and Lewis rats (200 g to 250 g) were commercially obtained. Allogeneic left kidney transplantation was performed between male F344 and Lewis rats. F344 rats were used as donors and syngeneic recipients, and Lewis rats served as allogeneic recipients. All animal handling procedures were conducted in compliance with guidelines for the Care and Use of Laboratory Animals published by NIH, and all animal experimental protocols were approved by the Animal Care and Use Committee of Chongqing University Cancer Hospital. All supplies used during surgery, including surgical instruments and solutions, are sterile. A schematic of the protocol is shown in Figure 1.

1. Donor procedure

1. Induce general anesthesia in the rat by 5% isoflurane inhalation using an induction chamber. Then, subcutaneously inject buprenorphine at 0.1 mg/kg to perform concurrent preemptive analgesia.
2. Place the rat on its back and maintain anesthesia with 2% isoflurane inhalation using a facemask over its nose and mouth. Apply eye lubricant onto the eyes to avoid drying of the cornea. Slow respiratory rate and rhythm, the disappearance of corneal reflex, and lack of response to the toe pinch indicate the effectiveness of anesthesia.
3. Shave the abdominal hair with an electric razor and sterilize the skin using 0.5% iodine and 70% alcohol.
4. Subcutaneously inject 0.5% lidocaine along the midline from the symphysis pubis to subxiphoid for local analgesics, and then incise the abdomen and pull open the incision using a retractor.
5. Take out the intestines from the right side of the incision and wrap them up with moistened gauze to prevent them from drying out. Then, expose the left kidney.
6. Segregate fat tissues from the left kidney and ureter using cotton swabs, and then dissociate the left renal artery and vein using the micro-forceps under an operating microscope with 20x magnification. Coagulate any bleeding using electrocoagulation, if necessary.
7. Perform ligation of the aorta approximately 5 mm above the left renal artery with a 4-0 polyamide monofilament suture. Then, transect the left renal vein distal to the conjunction of the left genital vein and adrenal vein.
8. Flush the kidney with ice-cold UW solution supplemented with heparin (100 U/mL) using a 24 G scalp needle from the aorta below the left renal artery until blood fades in color. Warm ischemia time is 5 min on average.
9. After transecting the left renal artery approximately 2 mm next to the aorta, dissociate the kidney and ureter with the help of micro-forceps (preserve the peripheral connective tissues to ensure blood supply to the ureter). Then, transect the ureter next to the bladder and preserve the donor left kidney in ice-cold UW solution.
10. Sacrifice the donor rat with loss of blood by transecting the aorta and subsequently placing it into a CO₂ box to ensure death.

2. Recipient procedure

1. Repeat the procedure described in steps 1.1-1.5 for the recipient rat.
2. Dissociate the left kidney and ureter as well as the left renal artery and renal vein of the recipient rat using cotton swabs and micro-forceps under an operating microscope with 20x magnification.
3. Clip the left renal artery and renal vein at the root by noninvasive micro-vascular clamps. Ligate the left ureter approximately 2-3 cm below the kidney with an 8-0 polyamide monofilament suture and transect it at ligation.
4. Resect the recipient's native left kidney by transecting the left renal artery 2 mm away from the micro-vascular clamp, and transecting the renal vein proximal to the conjunction of the left genital vein and adrenal vein. Coagulate the adrenal vein using electrocoagulation, if necessary.
5. Implant the donor kidney into the left renal fossa of the recipient rat and put ice around the implanted donor kidney. Anastomose the donor's renal artery and renal vein to the recipient's renal artery and renal vein in an end-to-end pattern using 10-0 polyamide monofilament sutures under an operating microscope with 45x magnification, as follows.
6. Anastomose the renal artery with interrupted sutures.
   1. Place the stay sutures at 12 and 6 o'clock positions of anastomosis, respectively. Equidistantly suture one side of the anastomosis between the two stay sutures with 2-3 stitches using a 10-0 Polyamide Monofilament suture.
   2. Turn over the stay sutures and similarly suture the other side of the anastomosis between the two stay sutures with 2-3 stitches.
7. Anastomose the renal vein with continuous sutures.
   1. Place the stay sutures at 6 and 12 o'clock positions of anastomosis, respectively. Suture one side of the anastomosis from the 12 o'clock position with 4-5 stitches using the running sutures, and then tie the running suture to the stay suture at the 6 o'clock position with a 10-0 polyamide monofilament suture.
   2. Turn over the stay sutures and similarly suture the other side of the anastomosis from the 6 o'clock position, and finally tie the running suture to the stay suture at the 12 o'clock position.
8. Re-perfuse the donor kidney by releasing the noninvasive micro-vascular clamp of the renal vein first; then, identify the bleeding sites and make additional stitches.
9. Release the noninvasive micro-vascular clamp of the renal artery, identify the bleeding sites and make additional stitches. Cold ischemia time is 45 min on average.
10. 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 recipient's bladder under an operating microscope with 20x magnification. Afterward, transect the end of the donor ureter with the suture outside the recipient's bladder. Let the donor ureter shrink back into the recipient's bladder.
11. Fix the donor ureter with the recipient's bladder by stitching the adventitia of the donor ureter with the muscle layer of the recipient's bladder outside at four equidistant positions using an 8-0 polyamide monofilament suture under an operating microscope with 45x magnification.
12. Place the intestines back into the abdominal cavity and close the abdominal incision with continuous sutures in the muscle layer first and then the skin layer with a 4-0 polyamide monofilament suture.
13. Place the recipient rat on a 37°C heating pad in a dry and clean cage. Wait until the rat recovers from anesthesia.
14. Subcutaneously inject buprenorphine (0.05 mg/kg) into the recipient rat every 6 h for 48 h for post-operative analgesia. Intramuscularly inject penicillin (50,000 U/kg) into the recipient rat once a day for 3 days for prevention of infection. The recipient rat is sacrificed by placing it into a CO₂ box to observe the chronic rejection of renal allograft after 10 weeks of transplantation.

Representative Results

In this rat orthotopic renal transplantation model, the recipient rats move normally after operation. To observe the chronic rejection of renal allograft, recipient rats are raised for 10 weeks after transplantation, and the total survival rate of recipient rats at this time point is approximately 90%. The major causes of death are bleeding and leakage of urine post-operation. The other major complications include bleeding during the operation, thrombosis in renal vessels, and hydronephrosis, whose respective incidence is approximately 15%, 25%, and 20%. Most of the bleeding during operation can be stopped and does not influence the survival of recipient rats. Renal grafts with embolization of vessels and hydronephrosis should be excluded from the subsequent studies.

In this model, F344 rats are of the rat MHC (RT1) haplotype RT1^lv1 and Lewis rats are of the haplotype RT1^l. These two strains differ in the MHC class Ib locus C/E/M¹³, which does not cause acute T-cell-mediated rejection but leads to a subsequent chronic antibody-mediated rejection^4,5. Chronic allograft nephropathy is characterized by glomerular sclerosis, interstitial fibrosis, tubular atrophy, and interstitial arteriosclerosis¹⁴. At 10 weeks after transplantation, hematoxylin and eosin (HE) staining and periodic acid-Schiff (PAS) staining reveal glomerular sclerosis, interstitial fibrosis, and tubular atrophy (Figure 2A and 2B), and interstitial arteriosclerosis (Figure 2C) in renal allograft in contrast to the isograft kidney. As another characteristic of chronic glomerulopathy in the renal allograft, silver staining shows the thickening of the glomerular basement membrane in allograft kidney when compared with isograft kidney (Figure 2D), indicating the success of the present rat orthotopic renal transplantation model.

Figure 1: Schematic of the rat orthotopic renal transplantation model. (A) Resection of the donor's kidney. Ligate the aorta above the left renal artery and transect the left renal vein distal to the conjunction of the genital vein and adrenal vein. After perfusion with ice-cold UW solution, the donor's left kidney is then resected by transecting the left renal artery approximately 2 mm next to the aorta and transecting the ureter next to the bladder. (B) Resection of recipient's kidney. Following clamping of the left renal artery and renal vein at the root, the ureter is transected after ligation. The recipient's left kidney is then resected by transecting the left renal artery 2 mm from the micro-vascular clamp and transecting the renal vein proximal to the conjunction of the left genital vein and adrenal vein. (C) Implantation of the donor's kidney. The donor renal artery and renal vein are anastomosed to the recipient's renal artery and renal vein, respectively, by interrupted suture and continuous suture in an end-to-end pattern. The donor ureter is then anastomosed to the recipient's bladder using an end-to-side "tunnel" method. Please click here to view a larger version of this figure.

Figure 2: Pathological staining of renal grafts. Chronic graft nephropathy characterized by glomerular sclerosis, interstitial fibrosis, tubular atrophy, and interstitial arteriosclerosis as shown in renal allografts after 10 weeks of kidney transplantation from F344 donors to Lewis rat recipients. (A,B) Glomerular sclerosis, interstitial fibrosis, and tubular atrophy as well as (C) interstitial arteriosclerosis in renal allograft are shown by hematoxylin and eosin staining and periodic acid-Schiff staining. Scale bar: 50 µm. (D) Thickening of glomerular basement membranes (GBM) in renal allograft compared to renal isograft is shown by silver staining. The dashed squares outline the higher magnification image of GBM. Scale bar: 50 µm. Please click here to view a larger version of this figure.

Discussion

Renal transplantation in the rat is a challenging work requiring a high level of micro-surgery techniques and the operation techniques have been optimized several times. From the beginning, Gonzalez et al. implanted the donor kidney into the neck of the recipient and anastomosed the donor ureter to the skin⁹. However, because of the high incidence of urinary infection and stenosis of the donor ureter, the operation was abandoned in a short time. Subsequently, the implant operation of the donor's right kidney was improved by anastomosis of donor renal artery and renal vein to the recipient aorta and inferior vena cava¹⁵, or by anastomosis of the donor renal artery and renal vein to the recipient renal artery and inferior vena cava¹¹. Nevertheless, the right renal artery and renal vein are thin and short, increasing the difficulties and failure rate of the operation. Furthermore, this operation needed blocking of the aorta and inferior vena cava of the recipient. Therefore, ischemia of the lower body and the thrombosis of inferior vena cava resulted in high incidences of disabling and death of recipient rats. The other improved implant operations include anastomosis of donor aorta and renal vein to the recipient renal artery and renal vein using a cuff method¹⁶. Still others anastomose the ureter using the cuff method¹². However, the thinness of the renal artery and ureter increases the difficulty in producing the cuff and anastomosis thereby limiting the application of these implant operations.

Currently, the commonly used rat orthotopic renal transplantation model is performed in the left kidney by anastomosis of the left donor renal artery and renal vein to the recipient aorta and inferior vena cava, and anastomosis of the donor bladder patch to the recipient bladder¹⁰. The left renal artery and renal vein are long enough to facilitate anastomosis. Nevertheless, the anastomosis to the recipient aorta and inferior vena cava still cannot avoid the ischemia of the lower body and the thrombosis of inferior vena cava, thus requiring a high level of micro-surgery experience to shorten the operation time. The current rat orthotopic renal transplantation model is modified from Reuter S. et al.¹⁷ via end-to-end anastomosis of the left donor renal artery and renal vein to the recipient renal artery and renal vein, and anastomosis of the donor ureter to the recipient's bladder using a "tunnel" method. In this model, there is no need to clip the recipient aorta and inferior vena cava so that the physiological systemic circulation of the recipient rat is not interrupted, and the survival rate is improved without crippling (over 90%). Moreover, the "tunnel" method of anastomosis of the donor ureter to the recipient's bladder is easy to operate and reduces the incidence of urine leakage after operation by stitching the donor bladder patch.

However, some details in this model still need to be noticed. First, the intestines should be kept moist outside by covering them with moistened gauze. Otherwise, the recipient would die from intestinal necrosis after the operation. Second, the connective tissues of the donor ureter should not be completely removed to ensure the blood supply to it. Third, similar weights of donor and recipient rats should be granted to ensure that the donor and recipient vessels are of the same caliber. A significantly different caliber of donor and recipient vessels would increase the bleeding or thrombosis after anastomosis. Fourth, a suitable length of the anastomosed vessels should be kept, especially for the veins by transecting the donor renal vein distal from the conjunction of the genital vein and adrenal vein and transecting the recipient renal vein proximal to the conjunction of the genital vein and adrenal vein. Anastomosed vessels that are overlong or over-short may result in twisting of vessels, poor blood flow, and blood leakage. Fifth, the anastomosis process should be performed under a high magnification field, such as x45 fold, owing to thin walls of the renal artery and renal vein. A poorly resolved field would cause wrong sutures of vessel walls. Sixth, to reduce the thrombosis after the operation, heparin solution (100 U/mL) must be dropped on the anastomosed vessels during the anastomosis process. Seventh, the whole layer of the donor ureter should not be stitched when fixing it to the recipient's bladder because stenosis of the ureter and hydronephrosis would increase.

The limitations of this model are a high requirement of micro-surgery skills and a certain probability of complications, including bleeding, urine leakage, thrombosis, and hydronephrosis. The major postoperative complications are similar among different orthotopic renal transplantation techniques in rats, which include bleeding, urine leakage, thrombosis, and hydronephrosis resulting from ureter anastomosis stenosis. However, in contrast to the anastomosis of the donor renal artery and renal vein to the recipient renal artery and inferior vena cava¹¹, the current technique mildly increases the thrombosis in renal vessels because of the thinness of anastomosis but avoids the interference with lower limb circulation. In contrast to the anastomosis of the renal artery and renal vein using a cuff method¹⁶, the current technique decreases the twist of vessels. In comparison with end-to-end anastomosis of the ureter using the cuff method¹², the current technique mildly increases the stenosis of ureter anastomosis but reduces the difficulty in operation. The improvement of the micro-surgery techniques by continuous practice can reduce the complications, increase the survival rate of recipient rats and increase the utilization rate of the models in the subsequent experiments. The current model provides a reference to scientists who study renal transplantation.

Materials

10-0 Polyamide Monofilament suture	B.Braun Medical Inc.	G0090781
4-0 Polyamide Monofilament suture	B.Braun Medical Inc.	C1048451
8-0 Polyamide Monofilament suture	B.Braun Medical Inc.	C2090880
Buprenorphine	US Biological life Sciences	352004
Electrocoagulator	Electrocoagulator	ZJ1099
F344 and Lewis rats	Center of Experimental Animals (Tongji Medical College, Huazhong University of Science and Technology, China)	NA
Gauze	Henan piaoan group Co., LTD	10210402
Heating pad	Guangzhou Dewei Biological Technology Co., LTD	DK0032
Heparin	North China Pharmaceutical Co., LTD	2101131-2
Injection syringe (1 ml and 10 ml)	Shandong weigao group medical polymer Co., LTD	20211001
Isoflurane	RWD Life Science Co., LTD	21070201
Penicillin G Sodium	Wuhan HongDe Yuexin pharmatech co.,Ltd	69-57-8
Scalp needle (24 G)	Hongyu Medical Group	20183150210
Shaver	Beyotime	FS600
Small animal anesthesia machine	RWD Life Science	R500
Small Animal Surgery Kit	Beyotime	FS500
Sodium chloride injection	Southwest pharmaceutical Co., LTD	H50021610
Surgical operation microscope	Tiannuoxiang Scientific Instrument Co. , Ltd, Beijing, China	SZX-6745
Swab	Yubei Medical Materials Co., LTD	21080274
Tape	Minnesota Mining Manufacturing Medical Equipment (Shanghai) Co., LTD	1911N68
UW solution	Bristol-Myers Squibb Company	17HB0002