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.
Modellen af svin nyretransplantation giver en unik mulighed for at fremme inden for human transplantation på grund af ligheder i kirurgiske aspekter, fysiologi, biokemi og immunologi 14.
Afhængigt af formålet med den eksperimentelle undersøgelse, modellen af renal autotransplantation har flere fordele sammenlignet med den allotransplantation model. Selv om flere grupper rapporterer god nyretransplantatfunktion efter allotransplantation 28, immunosuppression hos grise er udfordrende, især i nyretransplantation. Præoperativ blodprøve analyser for at sikre kompatibilitet for svin leukocyt antigen (SLA) er mulige, men dyrt og upraktisk 14. Postoperativt, foreslås immunosuppressive midler såsom tacrolimus og ciclosporin (calcineurinhæmmere, CNI) administreres oralt eller iv 28. Oral administration er upraktisk, da grisene normalt nægter at sluge oral medication. Desuden kan intestinale forhindringer undgå tilstrækkelig absorption af immunosuppressive medikamenter og vedligeholdelse af terapeutiske lægemiddelkoncentrationer. Den kontinuerlige infusion af CNI s iv i aktive dyr er teknisk krævende. Iv bolus administration medfører høje spidsværdier, som forårsager toksicitet. Således til undersøgelse af nye konserveringsteknikker, modellen af renal autotransplantation har flere fordele. I de repræsentative resultater af allotransplantated nyregraft påvist ovenfor, en forsinket og øget top af kreatinin og BUN angiver afvisning, der blev påvist ved histologisk vurdering.
Den porcine model af autotransplantation er tidligere blevet anvendt til at undersøge nye konserveringsteknikker 14,18,29. Imidlertid varierer de rapporterede postoperativ serumkreatinin og BUN værdier autotransplanted svin i en hjerte-bankende scenarie betydeligt afhængig af det eksperimentelle system 22,30 </sup>. Hjerte-bankende donor protokol præsenterer vi her resulterer i en lav postoperativ serumkreatinin højdepunkt på 2,8 mg / dl (± 0,7) og BUN toppunkt på 25,3 mg / dl (± 7,4). Disse resultater er sammenlignelige med de lave spidsværdier præsenteret af Hanto og kolleger 28 og Snoeijs og kolleger 31.
For at sikre et vellykket resultat efter nyretransplantation i et svin autotransplantation model, har vi identificeret flere vigtige tekniske faktorer, der minimerer antallet af visse komplikationer. Anvendelsen af histidin-tryptophan-ketoglutarat-opløsning (HTK) reducerer risikoen for vasospasme på grund af dets lavere indhold af kalium i forhold til University of Wisconsin (UW) opløsning. For yderligere at mindske risikoen for vasospasme ved punktet for reperfusion, kan verapamil injiceres i den renale arterie, og papaverin kan administreres topisk under hentning og efter reperfusion. Desuden er en kontinuerlig drop af norepinephrin titreret til at opretholdedet systoliske blodtryk over 100 mmHg sikrer en homogen reperfusion. Det er nyttigt at opretholde denne blodtrykket i det mindste indtil grisen er placeret udsat. Endvidere positioneringen af transplanterede transplantat er vigtigt at forhindre kinkning af de nyligt anastomoseres blodkar. Derfor er det nyttigt at resecere den kontralaterale venstre nyre før sy anastomoser af implantatet for at undgå omfattende mekanisk manipulation. Efter endt ureterale anastomose, indpakning tyndtarmen omkring det transplanterede transplantat sikrer sin position efter lukning af bugvæggen. Komplikationer såsom tarm hindringer, der skyldes kinkning af tarmen ses sjældent, men kan føre til alvorlige komplikationer, herunder ileus, perforering af tarm og død. Samlet set nøjagtig kirurgisk teknik, opmærksom anæstesi og nøje overvågning under opfølgning sikre god klinisk resultat og transplantatfunktion.
Arterielle og venøse anastomoser kan PERFORMED hjælp af forskellige teknikker. Ortotopisk placering af implantatet tillader end-to-end anastomoser af den renale arterie og vene. I tilfælde af heterotopisk transplantation, kan implantatet anbringes i den kontralaterale nyre fossa til end-to-end anastomoser, onto iliaca fartøjer, eller den distale aorta direkte. Heterotopisk transplantation med anastomoser til aorta og cava direkte i ende-til-side-teknik foretrækkes i denne model, da det kan reducere risikoen for trombose og vasospasme 32. Anatomiske variationer med meget tidlige venøse bifurkationer kan føre til behovet for at sy to separate venøse anastomoser. Hvis arterien eller venen er forholdsvis korte, kan implantatet drejes 180 ° for at få længden af skibene. Ureter side-til-side anastomose kan opnå gode eksperimentelle resultater uden at komplicere forsnævringer eller urin lækage.
Generelt er den porcine model af nyretransplantation giver fordele i forhold til andre dyremodeller. Som described ovenfor, er der visse ligheder mellem porcin og human indstilling, der tillader relativt hurtig translation af nye teknikker til klinisk praksis. Teknikken med transplantation er teknisk lettere i forhold til gnavermodeller. Desuden ved placering af venøse katetre, perifere blodprøver kan opsamles let og behandles til yderligere undersøgelse. Indsamlingen af urin tillader yderligere vurdering af nyreskade og funktion. At indsamle urinprøver, kan en perkutan kateter indføres i urinblæren. At undgå manipulation af grisen, bør den distale ende blive tunnel subkutant på bagsiden af dyret. En anden mulighed for urinopsamling er anvendelsen af metaboliske bure, som tillader lange indsamlingsperioder at estimere kreatinin clearance og koncentration af yderligere biomarkører i urinen. Ultralydsundersøgelser, CT scanninger, og MRI-billeder er mulige. Donation efter kredsløbssygdomme død protokoller kan efterlignes ved at anvende varmiskæmi før hentning. Endvidere svin er relativt nemme at håndtere, hvis kastreret at begrænse deres aggressive adfærd.
Ulemper omfatter de høje omkostninger af animalsk køb, bolig, kirurgisk og andet medicinsk udstyr, og arbejdskraft. Disse faktorer betyder, at det er ikke muligt at medtage et stort antal dyr i hver undersøgelsesgruppe. Endvidere sammenlignet med gnavermodeller, et begrænset antal referencer er tilgængelige i litteraturen til svin normative biologiske data. Som et alternativ til vurdering af nye udviklede teknikker, såsom hidtil ukendte konserveringsmetoder, har andre grupper beskrevet normotermiske ex vivo reperfusion som et alternativ til nyretransplantation 33,34. Denne teknik er lettere at udføre og billigere. Men standardiseret nyre graft transplantation giver en model mere ligner den kliniske praksis og tillader længere følge op perioder. Derfor tjener det til en mere realistisk transplantat vurdereling.
Som konklusion porcine model af heterotopisk renal autotransplantation tilvejebringer en klinisk vigtig scenario for undersøgelse af nye hidtil ukendte fremgangsmåder til forbedring af nyregraft resultater. Især denne protokol har vigtige tekniske detaljer, der vil lette en vellykket etablering af en nyre autotransplantation model og giver mulighed for hurtig oversættelse af ny viden til kliniske forsøg.
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 |