#NephJC Chat
Tuesday Feb 22 20229 pm Eastern
Wednesday Feb 23 2022, 9 pm IST; 3:30 pm GMT
Am J Transplant 2022 Jan 20. doi: 10.1111/ajt.16930. Online ahead of print.
First clinical-grade porcine kidney xenotransplant using a human decedent model
Paige M Porrett, Babak J Orandi, Vineeta Kumar, Julie Houp, Douglas Anderson, A Cozette Killian, Vera Hauptfeld-Dolejsek, Dominique E Martin, Sara Macedon, Natalie Budd, Katherine L Stegner, Amy Dandro, Maria Kokkinaki, Kasinath V Kuravi, Rhiannon D Reed, Huma Fatima, John T Killian Jr, Gavin Baker, Jackson Perry, Emma D Wright, Matthew D Cheung, Elise N Erman, Karl Kraebber, Tracy Gamblin, Linda Guy, James F George, David Ayares, Jayme E Locke
PMID: 35049121
Introduction
In the quest to become immortal, humans have been experimenting since the prehistoric era. According to Hindu mythology - the Shiva Puranas (2000-3000BC), Ganesha - a god with an elephant head is a result of head and neck transplantation surgery performed by Lord Shiva. The legends and myths of transplants have inspired us to perform successful transplants today. Transplantation has revolutionized medicine by not just extending but also increasing the quality of life.
Worldwide, 5 to 10 million people require kidney replacement therapy (KRT) of which 2.6 million people received KRT. This number is an underestimate as many countries, including India and China, don't have a national end-stage kidney disease (ESKD) registry. According to the USRDS data, the overall survival of patients on maintenance hemodialysis is approximately 42% at 5 years and 40% of patients on transplant waitlists die while waiting for a kidney. This could be solved if we could use kidneys from genetically engineered pigs.
History of xenotransplantation
Attempts have been made to transplant non-human primate (NHP) kidneys in patients with ESKD but with hardly any success. In 1963, Reemtsma et al transplanted chimpanzee kidneys to 6 patients with ESKD but most of them developed rejection or infection within 8 weeks and succumbed to it. Baby Fae made the news for receiving a baboon heart transplant, but only lived for 8 weeks. Monkey and baboon kidneys were less successful as well. It was realized in the 1980s that pig kidneys were a more viable source of organs than phylogenetically similar NHPs for various reasons including the size/weight. However, when wild pig hearts or kidneys were transplanted into humans or NHPs, they were met with hyperacute rejection (HAR) within minutes which destroyed the graft within 24 hours. This was due to complement activation by antibody-mediated rejection. Humans naturally have pre-formed anti-pig antibodies that bind to the pig vascular endothelium leading to thrombosis, interstitial hemorrhage, and edema.
Pig genetic engineering
In the 1990s, genetic modification of the donor pig kidney was independently suggested by White et al (US) and Dalmasso et al (UK). The initial attempt was to introduce human complement regulatory protein to protect against recipient complement activation. This attempt extended the graft survival by only a few months.
The identification of major antigens (like α1,3 galactose or GAL) in pigs against which humans had pre-formed natural antibodies was a turning point in xenotransplantation. With the advent of CRISPR, deletion of GAL antigen was possible and this could surmount the hyperacute rejection and the life of kidney xenograft.
The Study
The major roadblocks in xenotransplants had to be addressed such as immunological and functional compatibility and transmission of porcine viruses. The only way to look into these issues was by in-vivo xenotransplant human studies. Hence, a xenotransplant program was initiated at the University of Alabama, Birmingham in 2015 (among other institutions). Pigs were reared exclusively for xenotransplant in a designated pathogen-free facility run by Revicor Inc (the same source for the pig kidney transplant reported in the news at NYU, as well as the pig heart transplant). These pigs were genetically modified to harbor 10 genetic modifications:
Insertion of
two human complement inhibitor genes,
two human anticoagulant genes,
two immunomodulatory genes, and
Deletion of
Two pig carbohydrate antigens and
Two growth hormone receptor genes.
Importantly, these pigs do not express red blood cell antigens and hence are universal donors for any blood group.
A 57-year-old human brain dead male adult with consent from the next of kin and negative flow cytometric crossmatch was included for this in-vivo human study. The human had bilateral native nephrectomies to establish anuria and then he underwent xeno-transplantion with two pig kidneys. Induction immunosuppression (ISP) consisted of methylprednisolone, anti-thymocyte globulin, and anti-CD-20. Maintenance ISP included mycophenolate mofetil, prednisone, and tacrolimus.
The primary outcome was kidney function for three days post-transplant.
They modeled the protocol so that it mirrored human transplantation as much as possible.
There was a (1) Pre-transplant phase where the donors (10GE pigs) and the recipient (decedent) were carefully evaluated and prepared; (2) Transplant phase where the actual transplant was carried out with standard induction immunosuppression and (3) Post-transplant phase with maintenance immunosuppression and graft monitoring.
Figure 1: Study timeline and event summary.
Goals of the study
The basic premise of this study was to determine whether xenotransplantation could even be considered a viable option.
What are some of the questions that come to mind when considering a transplant from pigs to humans?
From an immunological stand point, the first question would be how do you even do a cross match? We are talking about two completely different species here and how do we even begin to find a compatible ‘donor’?
Even if we find a donor, isn’t there a super high risk of rejection? What if there is an on-table rejection or a hyperacute rejection?
Is this even safe?
As described in the methods section, considerable thought was put into answering these questions and each step was meticulously planned. See this news article by Arthur Caplan and Brendan Parent which discusses some of the ethical foundations for this kind of research.
Pre-transplant phase
1. Family Authorization
The decedent was a 57 year old white male (blood group AB+) with brain death secondary to blunt head trauma. After exhausting the solid organ transplant lists, next-of-kin was approached and they gave their consent for enrolling the decedent in the study.
At the time of enrolment, the decedent was 5 days post-declaration of brain death and had some degree of acute kidney injury (Serum creatinine 2.5 mg/dL). The researchers accepted that in the presence of established brain death, the decedent milieu was not ideal for assessment of kidney function. However, at the same time, they also proposed that this model would allow for addressing some of the primary goals of the study, namely, validation of the crossmatch technique, determination of risk of hyperacute rejection and life-threatening surgical complications.
2. Prospective crossmatch
This was carried out by a novel crossmatch technique using positive and negative controls (see Methods).
The selected porcine donor was a 13-month-old, 350 lb, male 10-GE pig with normal renal function (BUN 19, creatinine 1.3, assessed <60 days prior to donation) and who was negative for porcine endogenous retrovirus C and other pathogens.
Figure 2: Detection of swine leucocyte antigen and decedent flow crossmatch results. PBMCs from a 10GE pig were isolated and incubated with pretransplant sera from the decedent. Porcine PBMCs were also incubated with negative and positive control sera that were identified from screening of sera banked in the histocompatibility laboratory at the University of Alabama at Birmingham. Anti-FITC secondary antibody (goat) was used to detect antibodies in the serum that were bound to the porcine lymphocytes. Histograms are shown for all cells or for lymphocytes gated based on FSC and SSC characteristics. Prospective crossmatches were performed using frozen porcine PBMCs. Retrospective crossmatches were performed using freshly isolated porcine PBMCs
3. Xenograft Procurement
Under general anaesthesia, porcine kidneys were obtained en bloc using aseptic technique at the xenotransplant facility and transported in sterile conditions to the institution.
4. Pre-transplant xenograft histology
Prior to placement in the decedent, the pig kidneys were biopsied, which showed normal kidney histology, similar to normal human kidney histology.
Fig. S4 Pretransplant assessment of porcine kidneys. Preimplantation biopsies. PASH staining.
5. Removal of bilateral decedent native kidneys:
Anuria was established by carrying out bilateral native nephrectomies.
Transplant Phase
1. Induction immunosuppression
To mimic ‘real world’ conditions, standard induction immunosuppression using methylprednisolone and anti-thymocyte globulin was administered immediately prior to xenotransplantation.
Maintenance immunosuppression with tacrolimus was started and maintained throughout the remainder of the study
2. Transplantation of right and left kidney xenografts
Right and left 10-GE pig kidneys were transplanted separately using conventional heterotopic allotransplantation techniques.
The right ureter was anastomosed to the decedent's bladder, and the left ureter was brought through the skin as an end urostomy.
3. Visual assessment for hyperacute rejection
The kidneys were observed under direct vision for at least 60 min prior to commencement of the ureteral anastomoses.
No hyperacute rejection was observed in either xenograft
4. Visual assessment of xenograft perfusion
The xenografts were independently assessed by four different kidney transplant surgeons and all of them agreed that there was excellent color and turgor, suggestive of good reperfusion.
Figure 3A: Reperfusion of porcine renal xenotransplants in the human decedent. Intraoperative photographs demonstrate viable kidney transplants bilaterally. (A) Reperfusion of the right kidney as shown over the course of approximately 1 min. (i) Appearance of the right kidney immediately prior to reperfusion after completion of the vascular anastomosis. Vascular clamps are present in the operative field. (ii) Appearance of the right kidney immediately after removal of vascular clamps. Note darker pink color of the kidney and the appearance of blood on the kidney surface under surgeon's hand. (iii) Appearance of the right kidney 5–10 s after removal of clamps. Reperfusion is progressing from superior to inferior pole. (iv) Appearance of the right kidney 1 min after removal of clamps. Entirety of kidney is now re-perfused.
Figure 3 C: Comparable kinetics of reperfusion and absence of hyperacute rejection for the left porcine renal xenograft.
The right kidney made urine within 23 min of reperfusion. However, urine output from the left kidney was more sluggish and scant.
5. Post-implantation xenograft biopsies
The left porcine xenograft was biopsied post-implantation and showed mild to moderate acute tubular injury with normal glomeruli. There was no evidence of endothelial injury, fibrin thrombi, or staining for IgG, IgM, or C4d, or in other words there was no evidence of rejection as was feared.
Post-implantation biopsy of the right porcine xenograft was deferred owing to the friable nature of the parenchyma.
Figure 3D: Reperfusion biopsy results of the left kidney. The investigators’ elected to not perform a reperfusion biopsy of the right kidney given the extremely delicate nature of the porcine xenografts. There was no difference in gross appearance of the kidneys at the time of biopsy
Post-Transplant Phase
1. Induction and maintenance immunosuppression
The researchers tried their best to follow routine (human) protocols of kidney transplantation. As described above, induction immunosuppression consisted of daily methylprednisolone taper with anti-thymocyte globulin for a total of 6 mg/kg, and Rituximab in a dose of 1800 mg.
Maintenance immunosuppression included mycophenolate mofetil (1000 mg twice a day), tacrolimus (1mg twice a day), and prednisone.
2. Assessment of xenograft function
Post-operatively, the decedent was maintained in the operating room, where he received intensive nursing care, monitoring, and laboratory investigations.
Urine output from each xenograft was monitored separately. Interestingly, the right kidney made 700 cc of urine within the first 24 hours but urine production from the left xenograft remained scanty.
Figure 3B: Sequential urine output after reperfusion of the right kidney is shown. Right kidney is depicted by black arrowheads. (i) and (ii) showcase urine output prior to ureteral anastomosis. Right ureter is being held in the surgeon's hand alongside collection cup. Note increased volume of urine in the cup between (i) and (ii). (iii) Urine output from the right kidney after anastomosis to the decedent bladder. Total volume in the collecting Foley bag is shown.
So, we have good perfusion, no hyperacute rejection (biopsy confirmed!) and even urine production. Does this mean the model was a success?
Well..not really!
In spite of the fact that at least one xenograft was making urine, serum creatinine did not show any decrease and nor were the kidneys able to excrete creatinine in the urine produced. The researchers were not able to explain the reason behind this.
Figure 6: Porcine renal xenotransplant function in the human decedent. (A) Cumulative posttransplant urine output from transplantation to study end from right and left xenografts. (B) BUN and creatinine in the decedent's serum. Results prior to POD 0 reflect function of decedent's native kidneys prior to native nephrectomies
3. Visual inspection of xenografts, including vascular and ureteral anastomoses
Xenografts showed good perfusion throughout the study duration.
Good blood flow in the renal arteries was confirmed with visual inspection, manual palpation and doppler survey.
Although, human mean arterial blood pressure tends to be higher than that of pigs, there was no significant bleeding or disruption of the renal parenchyma.
Then why did the left kidney not make much urine? The researchers were not able to explain the reason.
Figure 5: Longitudinal assessment of the porcine renal xenografts. Photographs from post-operative days 1 and 3 (POD 1, POD 3) were taken intraoperatively while the kidneys were in vivo. Minor blood accumulation underneath the right kidney capsule on POD 1 occurred after biopsy was taken. Yellow tinge of left kidney on POD 3 likely reflects bilirubin staining given hyperbilirubinemia in the decedent
4. Assessment of sensitization and transmission of porcine endogenous retroviruses
Daily analysis of decedent blood samples was negative for both porcine endogenous retroviruses and for chimerism.
5. Serial xenograft biopsies:
Histologic findings on post-operative day 1 were suggestive of thrombotic microangiopathy, but there was no other evidence of rejection.
Figure 10: Longitudinal analysis of porcine endogenous retrovirus transmission and microchimerism in the decedent. No PERV or microchimerism (pig-specific RPL4) was detected by RT-PCR using mRNA from different time intervals posttransplant. Pig(+) is a PERVC-positive pig control. GAPDH is an endogenous control showing presence of mRNA in all samples. Water is shown as a negative control
On post-operative day 3 there was evidence of progressive tubular injury with extensive acute tubular necrosis, but additional features of TMA including mesangiolysis were not observed.
C4d as well as IgM, IgG, IgA, C1q, and C3 were negative.
Wedge biopsies from study termination demonstrated no evidence of cortical necrosis or interstitial hemorrhage.
Post-termination analysis of renal tissue confirmed expression of the human transgenes within the porcine kidney parenchyma
Figure 7: Serial histologic examination of the porcine kidney xenografts. All biopsies represent core biopsies. (A, B, G, and H) Were obtained ex vivo. (C, D, E and F) Were obtained in vivo. Sections are stained with PASH and are 10X, except for (C and D) (40X) and (F) (silver stain). C4d negative throughout. (A and B) Mild to moderate acute tubular injury from cold ischemia. Normal appearance of the capillary network, the mesangium, and the podocytes. (C and D) Glomerulus with multiple fibrin thrombi (blue circle). There is diffuse glomerular capillary congestion with swollen endothelial cells and near complete obliteration of the peripheral capillary lumina. There is presence of fibrin thrombi and fragmented red blood cells consistent with thrombotic microangiopathy (TMA). There is evidence of progressive tubular injury with extensive acute tubular necrosis (ATN). No mesangiolysis is appreciated. (E and F) Glomerular congestion and acute tubular necrosis. Endothelial cells remain segmentally swollen with partially obliterated lumina and rare fibrin thrombi with improvement of glomerular injury. (G and H) Acute tubular injury persists. Glomeruli with segmental endothelial swelling. No fibrin thrombi
6. Bilateral xenograft nephrectomies
Following termination of the study, the pig kidneys were removed and examined.
Discussion
Chronic Kidney Disease is a significant cause of morbidity and mortality worldwide. In the US alone, currently more than 800,000 people require kidney replacement therapy. Dialysis is associated with reduced quality of life and increased mortality. According to the USRDS, about 12 to 20 percent of dialysis patients die yearly, and the eight-year survival rate is only about 35 percent.
Indeed, the best method of kidney replacement therapy for these patients is kidney transplantation which offers the best possible shot at a longer and more meaningful life. Unfortunately, fewer than 25,000 kidney transplants are performed each year, and more than 90,000 people are on the transplant waiting list. It has been estimated that the number of people on the transplant waiting list at the end of the year, may actually be more than that at the beginning of the year! The reason for this is fairly simple – there simply aren’t enough donors.
Organ transplantation is still a fairly young science with the first successful kidney transplantation carried out in 1954, between identical twin brothers. With the availability of more efficacious immunosuppressants, physicians were able to break the immune barrier and transplants became possible even between completely unrelated donors and recipients. Time and again, efforts have been made to expand the donor pool with protocols being developed for ABO incompatible donation, cadaveric organ donation (neurologically determined death as well as donation after cardiac death) and even donation from sero-positive donors (HBV, HCV, HIV, etc). In spite of all these efforts, a good number of kidney disease patients die while waiting for a kidney.
In order to meet this huge demand, it seems natural that the next barrier to break would be the barrier between two different species. As early as 1963, a Tulane University surgeon transplanted chimpanzee kidneys into 13 end-stage kidney disease patients. Note that this was before the advent of modern day genetic engineering and unfortunately, this study ended with 100% mortality.
It has been proposed that non human primates have smaller kidneys than their human counterparts and in fact, pigs could be a better source of xenografts because their organs are closer in size to human organs. This concept has been studied since the 1980s and several attempts have been made to utilize pigs as potential organ donors.
Transplant surgeon Jayme Locke, M.D., who is also one of the principal investigators of this study mentioned in an interview that “The concept of being able to have an organ waiting on the shelf, waiting for the person who needs it, is just remarkable to think about, and exciting for that person,”
Pigs are easily bred and if they could be used as potential kidney donors – wouldn’t it completely solve the problem of organ availability and waiting lists?
Of course, the first step for this would be to ensure that the genetic material of these donor pigs is modified so that their organs are not rejected by human recipients. To do that, some pig antigens have to be knocked-out so that there is no immune response against pig organs. At the same time, human genes have to be knocked-in to increase the probability of successful grafting.
Researchers at the UAB and the scientists at Revivicor Inc. worked on this model and came up with the 10 gene edited pig or the 10GE pig. These pigs were raised in controlled facilities where every effort was made to keep them free from pathogens. Pigs were kept free of specified infectious agents like pig cytomegalovirus. Moreover, the porcine endogenous retrovirus C (PERV-C) was bred out.
Enter Mr Jim Parsons from Alabama, whose family consented to his being the model for a human recipient. This landmark study would have been impossible without the support and willingness of his family. Dr Locke has said that “Mr. Parsons and his family allowed us to replicate precisely how we would perform this transplant in a living human. Their powerful contribution will save thousands of lives, and that could begin in the very near future. Parsons' gift honors his legacy and firmly establishes the viability, safety and feasibility of this preclinical model. Because of his gift, we have proposed this to be known as ‘The Parsons Model.’”
The purpose of the “Parson’s Model” was in fact multi-fold.
Q.1 Is genetic engineering sufficient to prevent hyperacute rejection?
As mentioned in results above, there was excellent graft perfusion and no hyperacute rejection was observed either grossly or on histology.
Conclusion: Yes, genetic engineering can prevent hyperacute rejection.
Q.2 Is a negative prospective crossmatch sufficient to prevent hyperacute rejection?
Humans are not expected to possess anti-SLA (swine leukocyte antigen) antibody due to prior sensitization events. However, pre-existing anti-HLA antibody may cross-react with SLA alleles, given the sequence homology between pig and human DR, DP, and DQ antigens.
A novel crossmatch technique was developed, as described in methods above and the results were internally consistent and easily interpretable, as described in results. Additional reagent development (i.e., SLA single antigen beads) is a prospective area of research in this direction.
Conclusion: Yes, a negative prospective crossmatch may potentially reduce the chances of rejection, similar to crossmatch in human to human transplants.
Q 3 Would there be life-threatening intraoperative complications?
Pig blood pressures are significantly less than human blood pressures. However, in this model, xenograft vascular integrity was maintained at human mean arterial pressures. Moreover, the decedent remained hemodynamically stable upon reperfusion, indicating that washout of inflammatory mediators from the xenograft during reperfusion did not provoke cardiovascular collapse.
Conclusion: No life-threatening intra-operative complications
Q.4 Would porcine derived products be detectable in human blood?
There was no evidence of porcine endogenous retrovirus transmission or peripheral chimerism in the decedent based on assays developed in the UAB research laboratories. Further refinement of research laboratory assays will be necessary because veterinary diagnostic laboratories with clinical-grade porcine viral testing capabilities may not accept human specimens, and hospital clinical laboratories may not be equipped to test for porcine pathogens.
Conclusion: No pig derived products were not detectable by available assays.
Q.5 Can a pig xenotransplantation be safely carried out under the conditions necessary for the clinical trial?
The answer, as obvious from the discussion above, is yes. Thus, it would seem that the study had fulfilled all the goals it had set out to achieve.
But like all research, even this seemingly landmark study has its shortcomings:
A brain dead recipient has a hostile environment for a transplant. While it does provide a suitable model for assessment of kidney function, it does not favor survival of the graft. Further validation would be required using living recipients.
“Trying to ascertain function in the face of brain death is always going to be challenging,” Locke said. “Ultimately, we will need to move into a Phase I clinical trial in which we transplant these kidneys into a living human being where the environment is more favorable for kidney recovery.”
Moreover, as noted above, the right kidney made good urine while this was not the case in the left kidney. The cause of this is not fully understood. Additional warm ischemic time during clamping in the donor may have been contributory.
While urine was being produced, there was no clearance of creatinine from the blood and no excretion in urine. Again, the cause of this is not known.
UAB’s xenotransplantation experiment was concluded after three days. We know that rejection may develop days and even months after transplant. So, the UAB model may not be good for slower types of rejection. In other words, there was no hyperacute rejection, but there could very well be rejection at later stages.
But wait, are we missing an important step? What about consent?
Consent is a very important part of organ donation and documentation of the donor’s consent is an essential part of any transplant program. While UAB celebrates its achievement as a one of its kind ‘first’ in the world of transplantation, People for the Ethical Treatment of Animals (PETA) is already at arms and has called this ‘junk science’ and ‘Frankenscience’. PETA has declared that ‘animals aren’t tool sheds to be raided—they’re complex, intelligent individuals.’ Their stand is clear – animals are not ours to experiment with.
Conclusion
The Parson’s model aimed to answer certain basic questions on safety and feasibility of xenotransplantation and did so with some degree of satisfaction.
The researchers are confident that this model will help to breach some of the barriers in this field and pave the way for future research.
Summary by
Mythri Shankar
Assistant Professor
Department of Nephrology
Institute of Nephrourology, Bengaluru
India
Namrata Parikh
Transplant Nephrology Fellow,
University of Ottawa, Canada