Copyright Blackwell Munksgaard 2003
Xenotransplantation 2003: 10: 410–421 Printed in UK. All rights reserved
XENOTRANSPLANTATION ISSN 0908-665X
Ex vivo and extracorporeal perfusion with hDAF pig kidneys* Luo Y, Levy G, Garcia BM, Yang H, Phillips J, Noble L, Chakrabarti S, Grant D, Zhong R. Ex vivo and extracorporeal perfusion with hDAF pig kidneys. Xenotransplantation 2003; 10: 410–421. Blackwell Munksgaard, 2003 The present study was undertaken to determine whether human decay accelerating factor (hDAF) transgene would prevent hyperacute rejection (HAR) while perfused with human blood or extracorporeally in baboons. Four hDAF pig kidneys and three non-hDAF pig kidneys were perfused ex vivo with fresh human blood for 6 h. Additionally four hDAF pig kidneys and four non-hDAF pig kidneys were extracorporeally perfused in baboons and pigs, respectively, for 3 h. In ex vivo perfusion, the color of hDAF pig kidneys remained pink at the end of 6-h perfusion and they had normal histology, while non-hDAF kidneys developed HAR. HDAF pig kidneys had superior function over non-transgenic pig kidneys. Urine output was 17.31 ± 3.70 ml/h for hDAF pig kidneys, and only 5.81 ± 0.26 ml/h for non-hDAF kidneys (P < 0.05). Creatinine clearance was 1.16 ± 1.24 ml/min for hDAF kidneys and 0.22 ± 0.15 ml/ min for non-hDAF kidneys (P < 0.05). Other functional data including potassium, urine specific density, and osmolality were normal in the hDAF kidneys, while in non-hDAF kidneys, serum potassium was elevated to over 9 mmol/l by the end of perfusion (P < 0.01). Non-hDAF kidneys also lost more sodium through urine than hDAF kidneys (173.67 ± 14.05 mmol/l vs. 109 ± 31 mmol/l, P < 0.05). In the extracorporeal perfusion, all the baboons tolerated the procedure well with normal hemodynamic and hemotologic profiles. These baboons were well until killed 42 to 56 days after perfusion, although their antiporcine antibodies were greatly elevated. We conclude that hDAF transgene protects against HAR, allowing the pig kidney to function normally while perfused with human blood, and that extracorporeal perfusion using hDAF pig kidneys is a safe procedure in baboons.
Introduction
A shortage of human donors has prompted the research in using animal organs for transplantation in humans. In USA, kidney transplantation candidates in particular have been accumulated up to 17 883 in 1990; 31 045 in 1995, and 46 489 in 1999 [1]. Meanwhile, the maximum donors available in a year for transplantation were only 12 551 [1]. The gap between these numbers may hardly be filled without new revolutionary donor sources. In addition, many patients with renal failure have to depend on long-term dialysis with little prospect to be transplanted because of their highly sensitized status against human leukocyte antigen (HLA) of almost all the human donors [2]. 410
Yigang Luo1,2, Gary Levy3,4, Bertha M. Garcia5, Hongji Yang1,2, James Phillips6, Lee Noble7, Subrata Chakrabarti5, David Grant4,7 and Robert Zhong1,2,5,8,9 Departments of 1Surgery, 5Pathology, 8Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada, 2Multi-Organ Transplant Program, London Health Sciences Center, London, Ontario, Canada, Departments of 3Medicine, 6 Pathology, 7Surgery University of Toronto, Ontario, Canada, 4Multi-Organ Transplant Program and Toronto General Research Institute, Toronto General Hospital, University Health Network, Toronto, Ontario, Canada, 9Transplantation Group, Robarts Research Institute, London, Ontario, Canada *This work was supported by Imutran Ltd and Novartis Pharma AG Company and Transplantation Technologies Inc., Toronto, Canada.
Keywords: kidney – perfusion – pig – xenotransplantation Address reprint requests to Robert Zhong, Department of Surgery, London Health Sciences Center – University Campus, PO Box 5339, London, Ontario, Canada, N6A 5A5 (E-mail: address:
[email protected])
Received 20 March 2002; Accepted 5 August 2002
Many species have been considered for xenotransplantation. Porcine organs have emerged as the most likely candidates for transplantation into humans [3]. However, there remain many barriers to successful transplantation such as immunologic barriers, the possibility of physiologic incompatibility, the threat of zoonosis, and ethical concerns. While each of these areas is currently being investigated, it is difficult to state which will present the greatest challenge. The major limitation in using pigs is the fact that humans have preformed natural antibodies (antiaGal) directed against a specific oligosaccharide (Gala1–3Galb1–4GlcNAc; Gal) on the vascular endothelium of all of the pig’s blood vessels [4,5]. A porcine organ transplanted into an untreated,
hDAF pig kidney perfusion with human blood and on baboons non-human primate, and presumably into a human, would be subject to hyperacute rejection (HAR). HAR is caused by three components: the Gal antigen, natural anti-aGal antibodies, and complement; modification or removal of any of these components could prevent HAR. The emergence of transgenic human decay accelerating factor (hDAF/ CD55) pig in the 1990s ushered a series of experimental proofs that HAR could be overcome [6,7]. The hDAF pig kidneys can support a recipient’s life for up to 78 days in monkeys and up to 75 days in baboons after transplantation (personal communication with Dr David White and our own unpublished results). When HAR is averted, acute humoral xenograft rejection (AHXR), or termed as acute vascular rejection (AVR) or delayed xenograft rejection (DXR) occurs. This type of rejection is also believed to be associated with antiaGal antibodies. Despite intensive immunosuppressive protocols, most xenografts are rejected within a few weeks. Researchers are currently investigating the pathogenesis of AHXR and searching for novel methods to solve this problem. An alternative approach to using xenogenic organs is to use them as a bridge for clinical solid organ transplantation before a human donor is available. Extensive laboratory studies have been carried out and showed that transgenic pig liver prevents HAR and provides superior liver function when perfused with human blood [8]. Successful clinical extracorporeal perfusion with hDAF pig livers for liver failure patients has recently been reported [9]. It would be very interesting to study whether it is possible to use pig kidney extracorporeal perfusion to treat terminal renal failure patients. However, there are only a few reports of extracorporeal perfusion of pig kidneys [10–14]. When normal non-transgenic pig kidneys were used for clinical extracorporeal perfusion in two cases, the patients became unstable hemodynamically and metabolically and the perfusions did not last longer than 15 and 65 min [10]. In contrast, a study by Stork and his colleagues showed that when pig hDAF pig kidneys were perfused with human fresh blood for 1 to 3 h: (1) complement activation beyond C3 was inhibited, despite xenoantibody deposition and (2) more urine was produced [13]. However, perfusion time is relatively short in their studies. Furthermore, there has been no report of using hDAF pig kidneys for extracorporeal perfusion in both human and non-human primates. Therefore, the present studies were undertaken to test hDAF pig kidneys in both ex vivo perfusion with human blood and extracorporeal perfusion with baboons. The aims of these studies were to determine: (1) if HAR would be prevented with
hDAF pig kidneys; (2) if an hDAF pig kidney would provide a better function than a non-hDAF pig kidney; (3) if baboons would maintain a stable hemodynamic and metabolic status during extracorporeal pig kidney perfusion; and (4) if there would be any long-term complications following extracorporeal pig kidney perfusion.
Materials and methods Animals
Large white/Landrace cross strain hDAF pigs, weighing 20 to 55 kg, were supplied by a Novartis pig colony at the University of Guelph (Guelph, Ont., Canada). Age-matched, wild-type nonhDAF pigs with the same genetic background were obtained from Pig Improvement Company (Edmonton, Alta, Canada) and Premier Quality Genetics (London, Ont., Canada). Olive baboons (papio anubis), weighing 10 to 20 kg were provided by Buckshire Co. (TX, USA). All animals were treated according to guidelines published by the Canadian Council of Animal Care [22]. Experimental groups
Four groups were studied: group I, non-hDAF pig kidney perfused ex vivo with human blood (n ¼ 3); group II, hDAF pig kidney perfused ex vivo with human blood (n ¼ 4); group III, extracorporeal non-hDAF pig kidney perfusion on non-hDAF pigs (n ¼ 4); and Group IV, extracorporeal hDAF pig kidney perfusion on baboons (n ¼ 4). Blood products
Fresh frozen plasma and human blood (groups A and O) were obtained from volunteers through Canadian Blood Services. Whole blood was drawn the day before surgery and stored in citrated bags at 4C. The blood and plasma used for perfusion in any single kidney graft were the same blood type. Pig kidney procurement
The procedure was performed in accordance with the standard method in clinical kidney procurement. Briefly, after routine preparation and general anesthesia with endotracheal intubation on the donor pig, a large cross incision was performed on the abdomen. Flolan 5 ng/kg/min, i.v. was used during the procedure. The donor’s left kidney was mobilized while the infra-renal vena cava and abdominal aorta were both isolated. Heparin 300 lm/kg and Regitine 10 mg were given iv before 411
Luo et al.
Fig. 1. A diagram of ex vivo perfusion.
in situ flushing with 4C Euro-Collin solution, about 700 ml, through aorta cannula, was carried out. The flushing drainage was obtained by a vena cava cannula. The graft was taken out, weighed and placed into icy Euro-Collin’s solution before being put on perfusion circuit. The cold ischemia time of all ex vivo perfusion grafts was within 30 min, while that of extracorporeal perfusion graft, within 2 h. Ex vivo perfusion
The ex vivo perfusion circuit was based on modifications of our liver perfusion circuit (8). Briefly, the graft kidney was immersed in the perfusion chamber and connected to the perfusion system by cannula via the renal artery and renal vein. The outflow from a Integrated Handshell Venous Reservoir (dideco D902 Lilliput 2) was pumped by a Medtronic bio-pump (Bio-medicus 550 Bioconsule) through an infant hollow fiber oxygenator and heat exchanger into the renal artery of the graft. The outflow from the renal vein of the graft was returned to the reservoir by gravity (Fig. 1). A total of 1000 to 1500 ml of human blood and fresh frozen plasma in same blood type was heparinized [10 units of heparin in 1 ml of perfusate, keeping activated clotting time (ACT) > 1000 s and hematocrit (Hct) between 20 and 30%] and used for each perfusion experiment with the temperature adjusted between 37 and 39C. Perfusion pressure was kept between 70 and 90 mmHg. Creatinine, serum potassium, blood gases, count of blood cells (CBC), and xeno-antibody titre were measured pre-perfusion (0 h), then hourly until the end of perfusion. The ureter was cannulated with a No. F8 tube for collection of urine during perfusion. Urine samples were collected hourly as well until the end of perfusion and analyzed for creatinine, sodium, potassium, cell count, glucose, protein, osmolality, and specific density. Urine output was also recorded hourly. The kidneys were ex vivo perfused for 6 h unless the perfusion flow was spontaneously stopped. Tissue samples were taken 412
pre-perfusion, at 1 h and at the end of perfusion, for hematoxylin and eosin (H&E), Maritus scarlet blue (MSB) and immunostaining of C3, fibrin, IgG, IgM and IgA. It should be noted that there was no significant blood loss during the perfusion even with heavily heparinized perfusate. Instead, perfusate was gradually concentrated with increase of hematocrit because of the urine produced by the graft. Therefore, normal saline was added to perfusate periodically to keep hematocrit within 20 to 30%. Extracorporeal perfusion
Baboon or pig was prepped under general anesthesia with endotracheal intubation and central venous pressure line via either right femoral vein branch or left jugular vein. The perfusion system, including a kidney perfusion chamber and a single Medtronic bio-pump (Bio-medicus 550 Bio-consule), was primed with citrated blood (from an allogeneic donor with a same blood type of recipient) and normal saline of a total volume around 400 to 500 ml (Fig. 2). No reservoir or oxygenator was used. The kidney graft was immersed in the perfusion chamber and joined to the circuit through the renal vein and renal artery. Inflow to the graft was from the femoral artery of the recipient animal via the Medtronic bio-pump to the renal artery. Outflow of the graft was from the renal vein to the femoral vein of the recipient animal. The ureter was cannulated with a No. F8 tube for collection of urine during perfusion. Recipient baboon was given heparin 1000 unit per hour i.v., but no blood transfusion was given. ACT was kept >250 to 350 s. Hematocrit was maintained as 20 to 30%. Blood gases and electrolytes were kept within a clinically acceptable range. The graft color, blood flow, perfusion pressure and hourly urine output were recorded. The perfusion arbitrarily ended at the end of 3 h. The kidney graft was biopsied at pre-perfusion, 1 h and at the end of perfusion (3 h) for H&E, MSB, immunostaining of C3, fibrin, IgG, IgM, IgA. In order to obtain postperfusion follow-up data, the baboon native kidneys were kept intact. Post-perfusion, the pig recipients were extubated and kept alive for at least 2 days before euthanasia, while the baboon recipients were kept for 42 to 56 days, with no immunosuppression. Weekly physical examination and blood work including blood gas, liver function and renal function, CBC and antiporcine hemolytic antibody titer were performed on all recipient baboons. After killing, an autopsy was performed and lung, liver, heart, and kidney tissues were collected for pathologic examinations.
hDAF pig kidney perfusion with human blood and on baboons
Fig. 2. Surgical model of extracorporeal perfusion. (1) Cannulation of femoral vein and artery for inflow and outflow; (2) jugular vein cannulation for central venous pressure; (3) kidney perfusion chamber; (4) bio-pump; and (5) collection of urine produced by the perfusion kidney.
Pathology scoring
Two independent pathologists scored the morphology of biopsy specimens from a range of 0 to 4, with 0 being normal and four representing severe change. Statistics
The data was analyzed using anova. A P-value of
