Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.
Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.
Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.
Kidney transplantation is the treatment of choice for patients with end-stage renal disease, due to associated lower rates of morbidity and mortality when compared to dialysis 1-3. Despite major improvements in patient outcomes following kidney transplantation, graft shortage still poses a severe challenge worldwide. The number of patients waiting for a kidney transplant by far exceeds the number of organs available 4-6. To increase the number of kidneys available for transplantation and to reduce patient waiting times, further sources of kidney grafts are needed.
Commonly, standard criteria donor (SCD) and extended criteria donor (ECD) kidney grafts from donation after brain death (DBD) as well as kidneys recovered from live donors (LDKT) are utilized. Since the 1990s, an increasing number of kidney grafts have been recovered in a donation after circulatory death (DCD) scenario, to further expand the donor pool 7,8. However, DCD and ECD kidney grafts demonstrate acceptable but decreased outcomes after transplantation, depending on different factors, such as donor age, warm and cold ischemia times, and the preservation technique used 9-11. Thus, additional research is required to improve the outcome of patients receiving marginal kidney grafts and to further increase the donor pool.
The porcine model of renal transplantation is well established and provides a clinical important scenario to investigate innovative approaches for the improvement of marginal kidney graft outcomes. In contrast to rodent and canine kidneys, which are unilobular, porcine and human kidneys are multilobular and are anatomically similar, particularly in regard to the arterial, venous, and urinary collecting systems 12,13. In addition, porcine and human kidneys demonstrate similarities in the pathophysiology of ischemia reperfusion injury (IRI), biochemistry, and immunological parameters 14. Thus, porcine renal transplantation is well-suited to investigate new organ preservation methods for marginal kidney grafts 15-17, model human IRI 18, study immunological pathways and allograft tolerance 19, provide surgical training 20-22, test new pharmacological therapies 23, implement new medical devices, and study new immunological mechanisms in xenotransplantation 24-26.
The renal porcine and human transplantation settings are not completely analogous. This article focuses on important technical details that will facilitate successful establishment of a renal autotransplantation model. Species-adapted pre- and postoperative housing, administration of anesthesia with close monitoring, and matched surgical techniques are described in the protocol and demonstrated in the video. Resection of the contralateral native kidney provides the opportunity to assess the function of the transplanted kidney. The placement of venous and urinary catheters and the use of metabolic cages allow more in-depth assessment. For studies aimed at investigating alternative graft preservation methods and mechanisms of IRI, autotransplantation models are superior to allotransplantation models, as they avoid the complications and confounding bias associated with rejection and use of immunosuppressive medications.
All animals received humane care and all studies we carried out in accordance with policies and guidelines of the Canadian Council on Animal Care. All procedures were carried out under Animal Use Protocols that were approved by the University Health Network Institutional Animal Care Committee.
Note: A schematic overview of the study protocol is presented in Figure 1.
Figure 1. Study protocol. Please click here to view a larger version of this figure.
1. Animals
2. Kidney Graft Retrieval
3. Kidney Graft Transplantation
4. Postsurgical Follow Up
In the following, the results of renal autotransplantation experiments (n = 4) are demonstrated. After the initial graft retrieval, the pigs recovered in their housing area. Meanwhile, the kidney grafts were stored on ice for a mean time of 7 hr 35 min (± 18 min). After reinduction of anesthesia and repeat laparotomy, the contralateral kidneys were resected and the cold-stored grafts transplanted heterotopically as described. After weaning from the ventilator, pigs were recovered from surgery and followed up for 10 days (see Figure 1). Daily (1-4 post-operative day; pod) or every second day (6-10 pod) blood samples were collected to perform blood gas analyses; to assess the renal function, serum creatinine and blood urea nitrogen (BUN) values were estimated. For comparison, the results of one allotransplanted kidney graft are presented. For immunosuppression, this pig received cyclosporine 100 mg p.o. and cortisone 250 mg i.v. b.i.d. The surgical technique used was the same as in the autotransplant protocol; no warm ischemia time was applied.
All pigs were in good clinical condition during the follow up period. The serum creatinine and BUN values revealed the highest increase at day one after surgery (Crea 2.8 ± 0.7 mg/dl, BUN 25.3 ± 7 mg/dl) and decreased until pod 10 (Crea 1.7 ± 0.4 mg/dl, BUN 10.7 ± 4 mg/dl) close to the initial baseline values. The allotransplanted kidney graft demonstrated higher creatinine and BUN values after good initial graft function, when compared to the autografts, most likely due to rejection (Figure 2 and 3). Acid-base hemostasis (Figure 4) and electrolyte levels (Figure 5) were stable without intervention. Histological examination showed preserved tubulointerstitium in the autotransplanted kidney (Figure 6), and diffuse interstitial inflammation, tubulitis, and glomerulitis in the allotransplanted kidney (Figure 7).
Figure 2. Serum creatinine values. Serum creatinine values (mean and standard deviation) for baseline and 10 after surgery. Please click here to view a larger version of this figure.
Figure 3. Serum BUN values. Serum BUN values (mean and standard deviation) for baseline and 10 days after surgery. Please click here to view a larger version of this figure.
Figure 4. Acid-base hemostasis. Acid-base hemostasis (mean and standard deviation) for baseline and 10 days after surgery. Please click here to view a larger version of this figure.
Figure 5. Electrolyte levels. Electrolyte levels (mean and standard deviation) for baseline and 10 days after surgery. Please click here to view a larger version of this figure.
Figure 6. Histology (H&E), 100X magnification. Normal tubulointerstitium in the autotransplanted kidney 10 days after surgery. Please click here to view a larger version of this figure.
Figure 7. Histology (H&E), 100X magnification. Extensive interstitial inflammation, tubulitis, and glomerulitis, consistent with rejection, in the allotransplanted kidney 10 days after surgery. Please click here to view a larger version of this figure.
The model of porcine kidney transplantation provides a unique opportunity to further the field of human transplantation due to similarities in surgical aspects, physiology, biochemistry, and immunology 14.
Depending on the purpose of the experimental study, the model of renal autotransplantation has several advantages compared to the allotransplantation model. Although several groups report good renal graft function after allotransplantation 28, immunosuppression in pigs is challenging, especially in renal transplantation. Preoperative blood sample analyses to ensure compatibility for swine leukocyte antigen (SLA) are feasible, but expensive and impractical 14. Postoperatively, proposed immunosuppressive agents such as tacrolimus and cyclosporine (calcineurin inhibitors, CNI) are administered orally or i.v. 28. Oral administration is impractical, as pigs usually refuse to swallow oral medication. Furthermore, intestinal obstructions might obviate sufficient absorption of immunosuppressive medications and maintenance of therapeutic drug levels. The continuous infusion of CNI's i.v. in active animals is technically demanding. I.v. bolus administration leads to high peak values, which cause toxicity. Thus, for the investigation of new preservation techniques, the model of renal autotransplantation has several advantages. In the representative results of the allotransplantated kidney graft demonstrated above, a delayed and increased peak of creatinine and BUN indicate rejection, which was demonstrated by histological assessment.
The porcine model of autotransplantation has previously been used to investigate new preservation techniques 14,18,29. However, the reported postoperative serum creatinine and BUN values of autotransplanted pigs in a heart-beating scenario vary considerably depending on the experimental system 22,30. The heart-beating donor protocol we present here results in a low postoperative serum creatinine peak of 2.8 mg/dl (± 0.7) and BUN peak of 25.3 mg/dl (± 7.4). These results are comparable with the low peak values presented by Hanto and colleagues 28 and Snoeijs and colleagues 31.
To ensure a successful outcome after renal transplantation in a porcine autotransplantation model, we have identified several key technical factors that minimize the rate of certain complications. The use of histidine-tryptophan-ketoglutarate solution (HTK) reduces the risk of vasospasm due to its lower content of potassium when compared to University of Wisconsin (UW) solution. To further decrease the risk of vasospasm at the point of reperfusion, verapamil can be injected into the renal artery, and papaverine can be administered topically during retrieval and after reperfusion. In addition, a continuous drip of norepinephrine titrated to maintain the systolic blood pressure above 100 mmHg ensures a homogeneous reperfusion. It is useful to maintain this blood pressure at least until the pig is positioned prone. Furthermore, the positioning of the transplanted graft is important to prevent kinking of the newly anastomosed blood vessels. Therefore, it is helpful to resect the contralateral left kidney prior to sewing the anastomoses of the graft to avoid extensive mechanical manipulation. After finishing the ureteral anastomosis, wrapping small intestine around the transplanted graft secures its position after closure of the abdominal wall. Complications such as bowel obstructions due to kinking of the intestine are rarely observed but can lead to severe complications, including ileus, bowel perforation, and death. Overall, accurate surgical technique, attentive anesthesia and close monitoring during follow up ensure good clinical outcome and graft function.
Arterial and venous anastomoses can be performed using different techniques. Orthotopic placement of the graft allows end-to-end anastomoses of the renal artery and vein. In the case of heterotopic transplantation, the graft can be positioned in the contralateral renal fossa for end-to-end anastomoses, onto the iliac vessels, or the distal aorta directly. Heterotopic transplantation with anastomoses to aorta and cava directly in end-to-side technique are preferred in this model as it can reduce the risk of thrombosis and vasospasm 32. Anatomical variations with very early venous bifurcations might lead to the need of sewing two separate venous anastomoses. If the artery or vein are relatively short, the graft can be turned 180° to gain length of the vessels. Ureteral side-to-side anastomosis can achieve good experimental results without complicating strictures or urinary leak.
In general, the porcine model of renal transplantation offers advantages compared to other animal models. As described above, certain similarities exist between the porcine and the human setting, which allows relatively fast translation of new techniques into clinical practice. The technique of transplantation is technically easier compared to rodent models. In addition, by placement of venous catheters, peripheral blood samples can be collected easily and processed for further investigation. The collection of urine allows further assessment of kidney injury and function. To collect urine samples, a percutaneous catheter can be inserted into the urinary bladder. To avoid manipulation by the pig, the distal end should be tunneled subcutaneously to the back of the animal. Another option for urine collection is the use of metabolic cages, which allow prolonged collection periods to estimate the creatinine clearance and concentration of additional biomarkers in the urine. Sonography, CT scans, and MRI images are possible. Donation after circulatory death protocols can be mimicked by applying warm ischemia prior to retrieval. Furthermore, pigs are relatively easy to handle if castrated to limit their aggressive behavior.
Disadvantages include the high costs of animal purchase, housing, surgical and other medical equipment, and manpower. These factors mean that it is not feasible to include large numbers of animals in each study group. Furthermore, compared to rodent models, a limited number of references are available in the literature for pig normative biological data. As an alternative for the assessment of new developed techniques, such as novel preservation methods, other groups have described the normothermic ex vivo reperfusion as an alternative to renal transplantation 33,34. This technique is easier to perform and less expensive. However, standardized kidney graft transplantation provides a model more similar to the clinical practice and allows longer follow up periods. Therefore, it serves for a more realistic graft assessment.
In conclusion, the porcine model of heterotopic renal autotransplantation provides a clinical important scenario to investigate innovative novel approaches for the improvement of kidney graft outcomes. In particular, this protocol features important technical details that will facilitate successful establishment of a renal autotransplantation model and allows the rapid translation of new findings to clinical trials.
The authors have nothing to disclose.
We thank the Sorin Group (Milano, Italy), XVIVO Perfusion Inc. (Goteborg, Sweden), and Braun AG (Melsungen, Germany) for their support. We highly appreciate the support of the John David and Signy Eaton Foundation.
Anesthesia Equipment | |||
Anesthesia Machine, Optimax | Moduflex Anesthesia Equipment | SN5180 | |
Infusion Pump 3,000 | SIMS Graseby LTD. | SN300050447 | |
Infusion Pump Line | Smith Medical ASD Inc. | 21-0442-25 | |
Intravenous permanent catheter (9.5 Fr) | Cook Medical Company | G01865 | |
Isoflurane Vapor 19.1 | Draeger Medical Canada Inc. | N/A | |
Mallinckrodt, Tracheal Tube, 6.5 mm | Covidien Canada | 86449 | |
Temperature Therapy Pad | Gaymar Industries Inc | TP26E | |
Ventilator, AV 800 | DRE Medical Equipment | 40800AVV | |
Warm Touch, Patient Warming System | Nellcor/ Covidien Canada | 5015300A | |
Name | Company | Catalog Number | Comments |
Surgical Equipment | |||
Abdominal Retractor | Medite GmbH | 07-0001-00 | |
Aorta/vein punch 4.0 mm, round | Scanlan International Inc. | 1001-602 | |
De Bakey, Atraumatic Peripheral, Clamp | Aesculap Inc. | FB463R | |
De Bakey-Beck, Atraumatic Vena Cava, Clamp | Aesculap Inc. | FB519R | |
De Bakey, Atraumatic Mini-Bulldog, Straight | Aesculap Inc. | FB422R | |
De Bakey, Atraumatic Mini-Bulldog, Curved | Aesculap Inc. | FB423R | |
De Bakey, Atraumatic Coarctation Clamp, Angled | Aesculap Inc. | FB453R | |
Dissection Blade #11 | Feather Safety Razor Co. | 089165B | |
Connector (1/4") with male luer lock | Sorin Group Inc. | AB1452 | |
Liver Admin Set (flush line) | CardioMed Supplies Inc | 17175 | |
Maxon, 1 | Covidien Canada | 606173 | |
Med-Rx Suction Connecting Tube | Benlan Inc. | 70-8120 | |
Organ Bag | CardioMed Supplies Inc | 2990 | |
Potts – De Martel, Scissors | Aesculap Inc. | BC648R | |
Renal artery cannula, 1.6" | Sorin Group Inc. | VC-11000 | |
Sofsilk, 2-0 | Covidien Canada | S405 | |
Sofsilk, 3-0 | Covidien Canada | S404 | |
Satinsky, Suprahepatic Cava Clamp | Aesculap Inc. | FB605R | |
Suction Tip | Tyco Healthcare Group LP | 8888501023 | |
Surgipro II, 6-0 | Covidien Canada | VP733X | |
Valleylab, Cautery Pencil | Covidien Canada | E2515H | |
Valleylab, Force Tx | Valleylab Inc. | 216151480 | |
Valleylab, Patient Return Electrode | Covidien Canada | E7507 | |
Name | Company | Catalog Number | Comments |
Medication | |||
Atropine Sulfate 15 mg/30 ml | Rafter 8 Products | 238481 | |
Buprenorphine 0.3 mg/ml | RB Pharmaceuticals LDT | N/A | |
Ceftiofur 3 mg/mL | Pfizer Canada Inc. | 11103 | |
Cefazolin 1 g | Pharmaceutical Partners of Canada Inc. | 2237138 | |
Fentanyl Citrate 0.25 mg/5 ml | Sandoz Canada Inc. | 2240434 | |
Heparin 10,000 iU/10 ml | Sandoz Canada Inc. | 10750 | |
Histidine-tryptophan-ketoglutarate (HTK) solution | Methapharm | CU001LBG | |
Isoflurane 99.9%, 250 ml | Pharmaceutical Partners of Canada Inc. | 2231929 | |
Ketamine Hydrochloride 5000 mg/50 ml | Bimeda-MTC Animal Health Inc. | 612316 | |
Lactated Ringer’s + 5% Dextrose 1 L | Baxter Corporation | JB1064 | |
Lactated Ringer’s 1 L | Baxter Corporation | JB2324 | |
Metronidazole 500 mg/100 ml | Baxter Corporation | 870420 | |
Midazolam 50 mg/10 ml | Pharmaceutical Partners of Canada Inc. | 2242905 | |
Norepinephrine 16 mg/250 mL Dextrose 5% | Baxter Corporation | N/A | |
Pantoprazole 40 mg | Sandoz Canada Inc. | 2306727 | |
Papaverine 65 mg/2 mL | Sandoz Canada Inc. | 9881 | |
Propofol 1000 mg/100 ml | Pharmascience Inc. | 2244379 | |
Saline 0.9%, 1 L | Baxter Corporation | 60208 | |
Solu-Medrol 500 mg | Pfizer Canada Inc. | 2367963 | |
Verapamil | Sandoz Canada Inc. | 2166739 | |
Xylocaine Endotracheal 10 mg/50 ml | AstraZeneca | 2003767 |