Orthotopic Porcine Kidney Transplantation: A Technique to Transplant Graft Kidney in Porcine Model

Published: April 30, 2023

Abstract

Source: Liu, W. J. et al., Orthotopic Kidney Auto-Transplantation in a Porcine Model Using 24 Hours Organ Preservation and Continuous Telemetry. J. Vis. Exp. (2020).

This video describes orthotopic kidney auto-transplantation in a porcine model. This surgical procedure involves the transplantation of an animal's kidney graft at its original anatomical location. The model is used to investigate new treatments for end-stage kidney diseases in humans.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Basic techniques and common procedures

  1. Use female German landrace pigs (or comparable) for this protocol. Figure 1 depicts the summary of the described experimental protocol. Deliver the animals to the research facility 14 days before the surgery for acclimatization and house them in a temperature- and humidity-controlled barrier environment with a 12 h light and dark cycle (Figure 2). Fast the animals overnight before surgery.
  2. Premedicate by an initial intramuscular injection of azaperone (4 mg/kg) and atropine (0.1 mg/kg), followed by an injection of ketamine (15 mg/kg) 10 min later.
  3. After premedication, weigh the animal and transfer it directly from the housing facility to the central OR facility anesthesia preparation room.
  4. Cannulate one of the large ear veins using an 18 G peripheral venous catheter. Monitor the animal by a standard ECG and pulse oximetry.
  5. Initiate the anesthesia with propofol (3 mg/kg).
  6. Expose the vocal cord with a laryngoscope and insert a 7.5 mm endotracheal tube. The cuff is blocked with air according to the manufacturer's recommendations.
  7. Insert an oro-gastric drainage tube to remove fluid and air from the stomach.
  8. Insert a urinary catheter via the urethra.
  9. Subsequently, trim the skin in the area of the surgical incision.
  10. Apply eye ointment to prevent drying of the cornea during surgery.
  11. After orotracheal intubation, maintain anesthesia with isoflurane (final expiratory 1.45-2.0 Vol.%) and fentanyl (3-7.5 μg/kg/h).
  12. Ensure active intraoperative temperature control of the animal by a heating pad and using warmed air. Insert a rectal probe to monitor body temperature (target temperature 36.5 °C – 37.5 °C).
  13. Administer antibiotic prophylaxis using cefuroxime (35 mg/kg i.v.). Infuse Ringer solution at 4 mL/kg/h and increase to 8 mL/kg/h after skin incision. Administer a prophylactic dose of pantoprazole (40 mg i.v.) over the ear vein access.
  14. Perform all surgical procedures under sterile conditions according to the general principles of surgical asepsis and antisepsis. Disinfect the surgical field with povidone-iodine solution and cover with surgical drapes.

2. Contralateral nephrectomy and orthotopic kidney auto-transplantation

  1. During the recipient operation, adapt premedication and initial anesthesia to the restricted renal metabolism and avoid the use of ketamine. Induction is performed with propofol (3-5 mg/kg i.v.), midazolam (0,25 mg/kg i.v.), and atropine (0.1 mg/kg i.m.). Thereafter, the preoperative preparation is identical to the procedures described in section 1.
  2. Maintain anesthesia with isoflurane (final expiratory 1.45-2.0 Vol.%) and fentanyl (3 – 7,5 μg/kg/h) and propofol (2 – 4 mg/kg/h).
  3. Check and continuously monitor ECG, pulse oximetry, rectal temperature and the function of the telemetry transponder implanted in the right flank.
    NOTE: Strict anesthesia and blood pressure control is of crucial importance during the implantation procedure.
  4. In rare cases where the arterial blood pressure signal registered over the telemetry transponder is not satisfactory due to the supine position of the animal, place a further arterial catheter into the right femoral artery using percutaneous puncture and the Seldinger technique.
  5. Following sterile draping, reopen the median laparotomy and expose the surgical field using the abdominal retractor. The colon and small bowel are placed to the left side of the abdomen to expose the intact right kidney.
  6. Dissect the contra-lateral (right) kidney and its vessels from the surrounding tissue. Dissect the right renal vein and renal artery in the direction of the kidney hilum to ensure sufficient vessel length for anastomosis.
  7. Five minutes before vascular clamping, inject natrium-heparin intravenously (100 I.U./kg).
  8. Clamp the right renal artery and the right renal vein using vascular clamps. The right kidney is removed. The vessels are checked for integrity before starting the anastomoses.
  9. Place the preserved graft kidney into the abdomen and start the venous and arterial anastomoses.
  10. From this point onwards, keep the mean arterial pressure over 80-90 mm Hg to ensure good early perfusion of the kidney graft following reperfusion. Achieve this partially by adequate volume management and partially by the administration of norepinephrine (0.1 – 1.0 μg/kg/min as a continuous infusion using the mean arterial blood pressure and heart rate for monitoring the efficiency).
  11. Perform end-to-end anastomosis of the renal vein:
    1. After placing two corner stitches using 5-0 polypropylene, suture the back wall in a continuous fashion.
    2. Tie the cranial corner stitch and tie it together with the thread used for the back wall.
    3. After finishing the back wall, use the cranial corner stitch to suture the front wall in a cranio-caudal direction. Flush the vein with a heparinized saline solution (100 I.U./mL). Tie the caudal corner stitch.
      NOTE: In case of a size mismatch between the donor and recipient sides, a small growth-factor can be used to ensure a wide and sufficient anastomosis. There are many possible variations of the porcine renal vein branches. In the case of complex venous anatomy, a modified anastomosis approach is necessary (see Figure 3).
  12. Perform the end-to-end anastomosis of the renal artery:
    1. Use a 6-0 polypropylene cranial corner stitch to perform the arterial anastomosis. Placing a further caudal, supporting corner stitch which is later removed, is optional.
    2. Suture the back wall in a continuous fashion using the parachute technique. After arriving at the caudal corner remove the second corner stitch (if applicable).
    3. Suture the front wall with the other end of the double-armed 6-0 polypropylene suture. Flush the artery with a heparinized saline solution (100 I.U./mL). Tie the two threads at the caudal corner.
  13. Record the time needed for performing both anastomoses with a target warm ischemia time of <40 min.
  14. Reperfuse the kidney by opening the venous vascular clamp and subsequently the arterial clamp. Check for significant bleeding.
  15. If no significant bleeding from the anastomoses is observed, unwrap the kidney graft and pour warm normal saline solution in the abdomen covering the reperfused graft.
  16. Reposition the graft, if needed, to ensure homogeneous reperfusion and avoid congestion.
  17. Administer papaverine topically to the outside of the renal artery and the arterial anastomosis (5 mL undiluted).
  18. After reperfusion, infuse 250 mL of 20% glucose solution to induce osmotic dieresis followed by the administration of a single dose of 80 mg of furosemide.
    NOTE: Following this, initial urine production may be observed.
  19. To ensure urinary drainage, pass a 12 French pediatric urine catheter through the abdominal wall of the right flank of the animal, retroperitoneally.
  20. Secure the catheter in the ureter using ligatures (2-0 polyglactin) and block the catheter with 2 mL saline. Further sutures are used to adapt and secure the ureter to the peritoneum of the abdominal wall (2-0 polypropylene). The catheter is also secured to the skin with at least two single knot sutures (2-0 polypropylene).
  21. Close the peritoneal layer over the kidney to prevent dislocation of the kidney graft and kinking of the vascular anastomoses (3-0 polyglactin).
  22. Close the abdomen in a similar 4-layer fashion as described earlier for the graft retrieval.
  23. Following abdominal closure, maintain normothermia on the OR table.
    NOTE: Mean arterial blood pressure should be maintained over 80 mm Hg until the animal is awake and is in a prone position. Following abdominal closure, use color Doppler ultrasound to ensure adequate arterial and venous perfusion of the kidney graft (Figure 4). Monitor the animal closely until it is fully awake and can walk and drink spontaneously. The animals are given 1 L of Ringer solution during the recovery phase. Subsequently, return the animal to its box in the housing facility.

Representative Results

Figure 1
Figure 1: Study flowchart and protocol.

Abbreviations used: POD-postoperative day; ECG-electrocardiography.

Figure 2
Figure 2: Animal housing facility with real-time and continuous telemetry monitoring of up to 6 animals.

(A) Schematic blueprint of our facility suitable for the housing and telemetry monitoring of up to 6 animals. The size of the single holding boxes was determined based on the guidelines of the EU Directive 2010/63 and ETS 123 Appendix A. Panels A-E show representative images of the organization of our facility. (B) Animal room for the housing of 6 animals. (C) Observation room with a PC used for the continuous registration of telemetry data. (D) Real-time video and thermal footage of the animals. (E) Individual holding ensuring acoustic and olfactory contact of the animals with their companions to avoid social isolation.

Figure 3
Figure 3: Orthotopic kidney auto-transplantation and anatomical variations and reconstruction possibilities.

(A, B) The steps of the orthotopic kidney auto-transplantation model in case of a "standard" vascular anatomy. (C) Variation 1: while one larger vein comes with the donor kidney, there are two veins on the recipient side. Management: the smaller vein is closed by a ligature and the anastomosis is performed end to end between the renal veins. (D) Variation 2: while one larger vein comes with the donor kidney, there is no suitable recipient vessel on the contralateral side (e.g., size mismatch). Management: end to side anastomosis of the renal vein to the inferior vena cava. (E) Variation 3: two similar-sized veins on both sides. Management: reconstruction by two venous anastomoses. (F) Variation 4: while two similar-sized veins come with the donor kidney, there is no suitable recipient vessel on the contralateral side. Management: end to side anastomosis of the renal vein to the inferior vena cava in the case of two renal veins. (G) Variation 5: a donor kidney comes with a vein showing an early bifurcation, while there is one large vein on the contralateral side. Management: end-to-end anastomosis of the short common channel of the donor renal vein with one large vein on the recipient side. (H) Variation 6: while the donor kidney comes with a single renal vein with an early bifurcation, there is no suitable recipient vessel on the contralateral side. Management: end to side anastomosis of the short common channel of the donor renal vein to the inferior vena cava. This figure depicts a handful of the more frequent variations and is not statistically comprehensive in terms of all variations possible in German landrace pigs. Abbreviations used: KG-kidney graft; RK-right kidney; IVC-inferior vena cava; AO-aorta.

Figure 4
Figure 4: Representative color Doppler ultrasound images, directly after orthotopic kidney auto-transplantation and abdominal closure.

(A) Color Doppler ultrasound is performed directly following the implantation of the kidney and abdominal closure, to ensure good arterial and venous perfusion of the kidney graft and to screen for potential iatrogenic vascular kinking. Ultrasound was also used daily and on-demand, based on the clinical performance of the animal to screen for various problems. (B-E) Representative ultrasound images of a kidney graft following implantation. The image of the kidney graft with and without color Doppler (B, C) shows an excellent arterial (D) and venous perfusion (E). This figure shows representative images from the same animal.

Divulgaciones

The authors have nothing to disclose.

Materials

Anesthesia materials, drugs and medications
Atropine sulfate solution for injection, 100mg Dr. Franz Köhler Chemie GmbH, Bensheim, Germany 1821288 parasympatholytic agent, premedication
Bepanthen ointment for eyes and nose Bayer Vital AG, Leverkusen, Germany 1578675 eye ointment
BD Discardit II syringes, 2ml, 5ml, 10ml,20ml Becton Dickinson GmbH, Heidelberg, Germany 300928, 309050,309110, 300296  syringes
BD Micolance 3 (20G yellow) Cannula Becton Dickinson GmbH, Heidelberg, Germany 305888  venous catheter
BD Venflon Pro Safety (20G pink)  Becton Dickinson GmbH, Heidelberg, Germany 4491101 venous catheter
Buprenorphine (Buprenovet)  Bayer Vital AG, Leverkusen, Germany 794-996  analgesia
Cefuroxime 750mg, powder for preparing injection solution FRESENIUS KABI Deutschland GmbH, Bad Homburg, Germany J01DC02  antibiotics
Covidien Hi-Contour, Endotracheal Tube 7,5 with Cuffed Murphy Eye Covidien Deutschland GmbH,Neustadt/Donau, Germany COV-107-75E  endotracheal Tube
FENTANYL 0,5 mg Rotexmedica solution for injection Rotexmedica GmbH Arzneimittelwerk, Trittau, Germany 4993593 opioide analgetic agent
Furosemide-ratiopharm 250 mg/25 ml solution for injection Ratiopharm GmbH, Ulm, Germany  1479542 loop diuretics
Glucose 5% solution for infusion (500ml, 250ml) B. Braun Deutschland GmbH & Co. KG, Melsungen, Germany 3705273, 03705422 infusion fluid
Glucose 20% solution for infusion  B. Braun Deutschland GmbH & Co. KG, Melsungen, Germany 4164483 osmotic diuresis
Heparin-Sodium 5000 I.E./ml  B. Braun Deutschland GmbH & Co. KG, Melsungen, Germany 15782698 anticoagulant
Isoflurane-Piramal (Isoflurane)  Piramal Critical Care Deutschland GmbH, Hallbergmoos, Germany 9714675 volatile anaesthetic agent
Ketamine (Ketamine hydrochloride) 10% Medistar Arzneimittelvertrieb GmbH, Ascheberg, Germany 4230 general anaestetic agent
MIDAZOLAM 15mg/3ml  Rotexmedica GmbH, Arzneimittelwerk, Trittau, Germany 828093 hypnotica, sedative agent
NaCl 0,9% solution for infusion (500ml,1000ml) B. Braun Deutschland GmbH & Co. KG, Melsungen, Germany 864671.8779 infusion fluid
Norepinephrine (Arterenol)  Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany 16180  increase in blood pressure
Pantoprazole 40mg/solution for injection Laboratorios Normon,Madrid, Spain  11068  proton pump inhibitor
Paveron N 25mg/ml solution for injection (Papaverine Hydrochloride) LINDEN Arzneimittel-Vertrieb-GmbH, Heuchelheim, Germany 2748990 spasmolytic agent for vasodilatation
Propofol 1% (10mg/ml) MCT Fresenius FRESENIUS KABI Deutschland, GmbH, Bad Homburg, Germany 654210 general anaesthetic agent
Ringer solution  B. Braun Deutschland GmbH & Co. KG, Melsungen, Germany 1471411  infusion fluid
Urine catheter ruffle 12CH  Wirutec Rüsch Medical Vertriebs GmbH, Sulzbach, Germany RÜSCH-180605-12  transurethral urine catheter
Surgical materials
Appose ULC Skin Stapler  Covidien Deutschland GmbH,Neustadt/Donau, Germany 8886803712 skin stapler
Feather Disposable Scapel (11)(21)  Feather, Japan  8902305.395 scapel
Prolene 2-0, blue monofil VISIBLACK, FS needle Johnson & Johnson Medical GmbH- Ethicon Deutschland, Norderstedt, Germany EH7038H  skin
Prolene 5-0 (simply angulated, C1 needle) blue monofil VISI-BLACK Johnson & Johnson Medical GmbH- Ethicon Deutschland, Norderstedt, Germany EH7227H  vascular
Prolene 5-0 (double armed, C1 needle) 60cm Johnson & Johnson Medical GmbH- Ethicon Deutschland, Norderstedt, Germany KBB5661H  vascular
Prolene 6-0 (double armed, C1 needle) 60cm Johnson & Johnson Medical GmbH- Ethicon Deutschland, Norderstedt, Germany EH7228H vascular
Sempermed derma PF Surgical Gloves Seril Gr. 7, 7.5, 8 Semperit investment Asia Pte Ltd, Singapore 4200782, 4200871, 4200894  surgical gloves
Sentinex® PRO Surgical Gowns Spunlace XL 150cm Lohmann & Rauscher GmbH & Co. KG, Neuwied, Germany 19302 surgical gown
Telasorp Belly wipes (green 45x45cm) PAUL HARTMANN AG, Heidenheim, Germany 4542437  abdominal towel
Pediatric urine catheter  Uromed Kurt Drews KG, Oststeinbeck, Germany PZN 03280856  used for the uretero-cutaneus stoma
VICRYL- 0 MH Plus  Johnson & Johnson Medical GmbH- Ethicon Deutschland, Norderstedt, Germany V324  fascial closure
VICRYL – 3-0, SH1 Plus needle, 75cm Johnson & Johnson Medical GmbH- Ethicon Deutschland, Norderstedt, Germany W9114  subcutaneous suture, peritoneal suture
VICRYL – 3-0, SH1 Plus needle, 75cm Johnson & Johnson Medical GmbH- Ethicon Deutschland, Norderstedt, Germany V780  subcutaneous suture, peritoneal suture
VICRYL – ligatures Sutupak purple braided, 3-0 Johnson & Johnson Medical GmbH- Ethicon Deutschland, Norderstedt, Germany V1215E  threads for ligature
3M™ Standard Surgical Mask 1810F  3M Deutschland GmbH, Neuss, Germany 3M-ID 7000039767  surgical mask
Surgical instruments
Anatomical forceps Standard  ASANUS Medizintechnik GmbH, Tuttlingen, Germany PZ0260  anatomical forceps
Atraumatic tweezers steel, De Bakey Tip 1,5mm 8" ASANUS Medizintechnik GmbH, Tuttlingen, Germany GF0840  anatomical atraumatic forceps
Bipolar forceps 16 cm straight, Branch 0,30 mm pointed, universal fit Bühler Instrumente Medizintechnik GmbH,Tuttlingen, Germany 08/0016-A  biopolar forceps
Bulldog clamp atraumatic, curved, De bakey 78 mm, 3" ASANUS Medizintechnik GmbH, Tuttlingen, Germany GF0900  bulldog clamps
DE BAKEY-SATINSKY vascular clamp 215mm ASANUS Medizintechnik GmbH, Tuttlingen, Germany GF1661  vascular clamp
Dissecting scissors Mayo, 250 mm, 10" ASANUS Medizintechnik GmbH, Tuttlingen, Germany SC2232  Scissors for dissection
Dissecting scissors Metzenbaum-Fino, 260 mm, 101/4" ASANUS Medizintechnik GmbH, Tuttlingen, Germany SC2290  Scissors for dissection
Draeger CATO Anesthetic machine with PM8050 Monitor Dräger, Drägerwerk AG & Co. KGaA, Lübeck, Germany 106782  Ventilation System
Fine Tweezers, ADSON 180 mm  ASANUS Medizintechnik GmbH, Tuttlingen, Germany ADSONPZ0571  fine forceps
Gosset abdomenal wall spreader  CHIRU-INSTRUMENTE, Kaierstuhl, Germany 09-621512  abdominal retractor
Microsurgical/watermaker tweezers LINZ 150mm 6" Ergo round handle ASANUS Medizintechnik GmbH, Tuttlingen, Germany MN0087  fine microsurgical forceps
Surgical forceps Standard 5 3/4"  ASANUS Medizintechnik GmbH, Tuttlingen, Germany PZ1260  surgical forceps
Surgical scissors standard pointed-blunt (thread/cloth scissors)175 mm, 7" ASANUS Medizintechnik GmbH, Tuttlingen, Germany SC1522  surgical scissors
Further material
Heating pad  Eickemeyer – Medizintechnik für Tierärzte KG, Tuttlingen, Germany 648050 MHP-E1220  maintain body temperature during surgery
Laryngoscope, customized  Wittex GmbH, Simbach, Germany  333222230 expose the vocal cord
Rectal temperature probe  Asmuth Medizintechnik, Minden, Germany ASD-RA4  measure body temperature
Ultrasound device, Sonosite Edge-II  FUJIFILM SonoSite GmbH, Frankfurt, Germany V21822  ultrasound and color Doppler
Urine bag 2000ml Volume  ASID BONZ GmbH, Herrenberg, Germany 2062578 disposable urine bag connected to the uretero-cutaneous fistula catheter

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Orthotopic Porcine Kidney Transplantation: A Technique to Transplant Graft Kidney in Porcine Model. J. Vis. Exp. (Pending Publication), e20914, doi: (2023).

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