This transcript has been edited for clarity.
Matthew A. Sparks, MD: Welcome to Medscape InDiscussion. I'm Dr Matthew Sparks. Today, we'll be talking about xenotransplantation. Xenotransplantation has gained significant ground in recent months. How are porcine kidneys being produced? What will a potentially unlimited supply of carefully crafted pig kidneys for transplantation do for the cost and for the abundance of transplantation? And what do practitioners in adjacent sectors of the healthcare industry need to know about patients who receive a kidney xenotransplant? With me to answer some of these important questions is Dr Vineeta Kumar. She is the lead nephrologist for the living kidney donor and incompatible solid organ kidney transplant programs at the University of Alabama at Birmingham, or UAB. Her areas of expertise include kidney transplantation, living kidney donation, incompatible kidney transplant, kidney pair donation, and cardiovascular outcomes after kidney transplantation. Welcome, Vineeta.
Vineeta Kumar, MD: Delighted to be here, Dr Sparks. Thank you for having me.
Sparks: How did you get into the field of nephrology and in particular, transplantation?
Kumar: I'm a major nerd. I love detail. I love knowing things about my patients. I love the medicine of kidney dysfunction. It was a very easy choice to make to delve deeper into patients with kidney disease and restore their kidney function, and live a longitudinal journey with patients who have lost and gained their health multiple times throughout their life. Kidney patients, I think, are inspiring. They are humbling, and they refuel me and give me purpose. I wouldn't think about doing anything else.
Sparks: I don't do transplant nephrology. I chose a little bit of a different career path, but I've had so much admiration for transplant nephrology, and now it is even more exciting than ever. There are a lot of bold proclamations on where we're heading with transplantation, especially with the Advancing American Kidney Health Initiative. Can you tell us a little bit about what the goals are for the future of transplantation? Obviously, there are not enough organs. Where are we currently, how long is the waiting list, and what do we need? And then we'll talk a little bit about xenotransplantation.
Kumar: The fundamental thing we're facing with transplantation is that we know it is the gold standard for anybody with kidney failure, and for most patients with kidney failure in terms of quality of life and life giving, and yet we are not able to transplant most patients. With the Kidney Health Initiative, a big part of it's mission is to address this demand issue and increase the supply.
Sparks: One of the bold proclamations is that 80% of patients who start on kidney replacement therapy either do it at home or via transplantation. And we are far from that right now. And the other is to increase the number of kidney transplants that are done and decrease the number of discarded organs. How many transplants are done in a year in the United States?
Kumar: Excellent question. Very broadly, there are about 100,000 patients on the kidney transplant waiting list at the present time. And between all deceased and living donor kidney transplants, we do about 15,000 transplants a year. Woefully low. And more patients are added to the list every year than patients who have received a transplant, which is another truth we really need to confront and contend with.
Sparks: That brings us to this discussion on xenotransplantation. Xenotransplantation, just for our listeners, refers to transplant of an organ from another species. In this case, we're talking about a pig. What were the biggest barriers we were facing up until the publication of your paper in the American Journal of Transplantation?
Kumar: Phenomenal question. What were the barriers if kidney xenotransplantation was conceived as early as 1906 when the first recorded xenotransplantation happened? Why are we still here after over a hundred years?
Sparks: Let me get that right. 1906.
Sparks: Oh, my.
Kumar: Dr Mathieu Jaboulay was the surgeon, and the first recorded solid organ xenotransplant procedure was at that time. The one fascinating thing I love about the story is that he was the mentor and teacher to Alexis Carrel, the Nobel Prize laureate who did all the work on vascular anastomosis. The fascinating part of the story is that when he did this transplant, the source organ was from a pig. The kidney was put in the right arm of a 48-year-old woman. After the anastomosis was done, he would examine the left antecubital space every day. On day 3, the anastomosis was thrombosed, and he thought it was just his terrible surgical technique. He repeated this experiment a second time. It turns out now that we know it was actually an immunologic process — a rejection process that led to what we call thrombotic microangiopathy and the clotting of that blood vessel.
Sparks: One of the major barriers was this really robust response the human body had to this pig kidney that basically rendered it nonfunctional in a very short time after implantation, so it was nothing to do with this technique. What was it about the pig kidney that made it incompatible when placed into a human?
Kumar: In the early days, it was thought there was something about the pig itself. So in later days, they used baboons, or nonhuman primates, thinking they were closer to human physiology. Goats and other various animals were also used. And every time, it was the thought that the clotting process was the big Achilles heel. It turns out that the reason why humans react to nonhuman primate organs or other xenotransplated organs, or other animal organs, is because we have a naturally occurring antibody we make to a protein or antigen that is from an animal source.
Sparks: One of the big advances in the field of genetics is CRISPR-Cas9. This is an interesting technique that allows the deletion or insertion of genes in a manner that is a lot easier than what was previously available. What else was done to make these organs less of a problem for the human immune system?
Kumar: The brilliant idea in all of this was that instead of enhancing and doing way too much immunosuppression in the recipient to accept these organs, how do you make the organ itself more human like? And so, in the process of doing that, along with the Gal knockout, which is the primary antigen insertion of protective genes that interfere with the coagulation cascade, another big modification was made. Complements are a huge part of this innate response humans have to anything foreign in them, and controlling the complement system became extremely important. Complement regulatory genes were something else that were introduced. The one fascinating thing is that pigs grow fast, so their organs grow really fast. A pig at birth could be a kilogram or 1300 milligrams, and then they are 130 kilograms after 6 months. Those organs are also growing at an exponential rate. One of the other things that was done was to introduce this human knockout gene to regulate the growth so the organs were still usable, normal kidneys about 10-12 centimeters in size. And if you take a pig that's 6 months old or a year old, those kidneys could be upward of 15-17 centimeters; there simply wouldn't be space to put them into humans.
Sparks: So, they made the pigs not grow as big as they naturally would.
Kumar: That's right.
Sparks: A lot of things had to happen to get to this point. We started almost 100 years ago. And now we're to the point where we want to give this a shot. Tell us about what happened and how you even get to the point where you can do this in a human. This is a brain-dead human. They're not alive. Can you tell us a bit more about how this occurred?
Kumar: Absolutely. One big roadblock we reached, which sort of hit with the nonhuman primates, was that, essentially, the nonhuman primate model for xenotransplantation is a model of incompatibility. It's a model of incompatible kidney transplant overall. This is because the frequency of a positive cross-match between the pig and nonhuman primate is 100%. And so, the survival of these knockout kidney xenografts into nonhuman primates was directly related to the titer level and the amount of immunosuppression you needed to use in the nonhuman primates. The 10 gene knockouts were all further developed to make this more acceptable to a human model; you couldn't continue to study it in a nonhuman primate. You had to go into live human trials. But how do you go into a human trial when there's still a critical knowledge gap with the genetically engineered pig made to look less foreign to the human immune system? What has been talked about is how to test this hypothesis in a human without harming them. That's where the human decedent model, the brain-dead decedent model, came into being. This came together with incredible effort and collaboration with Legacy of Hope, our institution, and the company we work with that helps this genetically engineered organ and supports the institution.
Sparks: Okay. So that's really exciting. But there is suspense. You set up the story nicely. And now you've got everyone together and what happened?
Kumar: There are a couple of things we needed to absolutely prove. The first thing was that we needed to have a clinical-grade pathogen-free environment to make sure we could get the knockout genetically engineered pigs bred in that facility. Organs were taken from that in that facility, transported to our institution again in that pathogen-free state, kept behind the barrier, and then transplanted into the decedent. There was no introduction of foreign viruses. Pigs and other animals have natural viruses — porcine viruses, cytomegalovirus (CMV). And some of them, while in humans, are not known to be pathogenic; we haven't actually transplanted pig tissue into a human to know if transferability can happen. So not only were the pigs tested and it was ensured they didn't have these viruses, but PCR tests were done. Then, from the time the organs were harvested, they had to be transported in the same safe way until they made it into the decedent body. So that was the first absolute vital portion of the xenotransplant experiment we had to contend with.
Sparks: I was going to bring this up as another barrier. They called these retroviruses. Some of them are in the pigs. And sometimes when you immunosuppress a patient, they can come out. That was also a barrier. And so in 3 days, you did not see any evidence of that occurring. Is that correct?
Kumar: Absolutely. In 3 days, we did not see evidence.
Sparks: The other thing I want to be clear of is that you gave the same cocktail of medications you typically give a human whom you might give an incompatible kidney to.
Kumar: Yes, absolutely. That brings us to our second hypotheses. The one thing that doesn't get talked about in xenotransplantation is that there's some evidence antibodies directed to human leukocyte antigen, or HLA, can cross-react with the swine leukocyte antigen, which is SLA. And two, because we know that in the nonhuman primate, it was 100% of the time a model of incompatibility, we needed to prospectively have an SLA flow cytometry cross-match for human use. So, we were able to develop this test in our histocompatibility immunogenetics lab, and we used it to prospectively test the donor pig cells. Initially, this was the circulating blood cells to the human lymphocytes, and then we retrospectively went back and took lymph nodes and did the same test; we were able to a priori show a negative cross-match that then correlated in the decedent with no occurrence of hyperacute rejection. It was important to do that because our premise was that the genetic edits were enough to where we would not have hyperacute rejection and we would not need anything more than what Dr Allan Kirk in one of his editorials called vanilla-grade immunosuppression. The immunosuppression was truly vanilla grade, which means this is what we use in our human transplants. It consisted of anti-thymocyte globulin for induction along with a high dose of corticosteroids. We did use a dose of rituximab, which is sometimes used in human-incompatible HLA transplants. And then the maintenance regimen was what is used in over 90% of the transplants in the United States, which is a combination of prednisone, tacrolimus, and mycophenolate mofetil. So yes, very vanilla grade.
Sparks: I would give that vanilla with a little bit of sprinkles.
Kumar: Yes, but absolutely not Simulect [basiliximab]. We would not use Simulect for any kind of incompatible transplant recipient.
Sparks: Okay. You had the decedent body, and you had the lymph nodes and other things, and you did sort of cross-match to see how well you know what's happening. You hadn't put the kidney in yet.
Sparks: And now you say, okay, let's go ahead and do it. Yes. And then you gave all this immunosuppression, just like we said — the vanilla with sprinkles. And we'll see what happens.
Kumar: Yes. So, if I had to break this whole brain-dead model down, the pretransplant phase was about 18 or 19 hours where we took the decedent, did the bilateral medial nephrectomy, and showed they did not have any urine output. Then in the transplant phase, we did the bilateral nephrectomy or renal removal of the pig's kidneys and put them in the human. We implanted them one in each iliac fossa — right and left. This is what we would do with allotransplantation. It was important to reproduce that entire surgical experiment.
What was vitally important was that once you unclamp after the kidney was hooked up, the xenokidney was hooked up into the human. Once you unclamp, there is going to be a huge systemic response in the human, either of hypotension or hypertension. Typically, it's hypertension, but we did not know what we were going to face with the xenokidney, so we were waiting with bated breath. The clamps were then taken off and the first perfusion was established. The kidneys immediately pinked up, and they had great turgor. They started to pulse, and there was definitely a big sigh of relief everybody had. And then the decedent blood pressure shot up to the two hundreds — 220s, 230s — we were sweating bullets, and ICU management was needed. I'm trying to titrate all kinds of drips to get that blood pressure down, and we were not sure if the anastomosis would hold up. These are, after all, genetically modified kidneys. And some of these genetic modifications are happening at the endothelial level. To know there was vascular integrity was vitally important, and those kidneys held up.
Normally, the first day with transplantation, we would do postperfusion biopsies, which means we would check right away with a biopsy to confirm there was no hyperacute rejection. Obviously, hyperacute [rejection] is a clinical thing where the kidneys turn black. Because of the hypertension and risk for bleeding, we did not do postperfusion biopsies in the OR immediately after. We chose to do it the next day once we knew there was hemodynamically not going to be that type of blood pressure rise. That was another fascinating thing I wanted to share. And then afterward — immediately after perfusion within the first hour — there was urine in the Foley bag. We knew the anastomosis worked, with the artery going into the kidney and the vein taking the blood back. But whatever it was, the difference between the two ending up through the kidney, into the ureter, into the bladder, and for us to collect it — we had about 700 mL of urine output in the first 24 hours. Now, there's huge brain-dead physiology here that we had to contend with. What I mean by this is that there was a lot of physiologic derangement in the decedent already. They had blunt head trauma, and their duration of being brain dead prior to transplantation was 5 days.
Sparks: That's very fascinating. The way I looked at this as well was that the host who received this pig kidney was not ideal because of the brain-death physiology activation of different pathways you typically wouldn't see in a normal kidney transplant process. They kidney did make urine; however, the creatinine continued to rise. And you explain the differential diagnosis at that point. We just cut to the chase and say, “What did you find in the kidney and the biopsies?”
Kumar: What we found in the kidney and through biopsies was essentially thrombotic microangiopathy, which is the microvasculature in the kidney clot — significant acute tubular necrosis, acute tubular injury, and eventually acute tubular necrosis. There was some early cortical necrosis. That's day two. And by day three, the thrombotic microangiopathy had gone and early cortical necrosis that we were appreciating. And this is on explanted kidneys and not just with the biopsy we did upon termination, but the cortex actually looked healthier than it did at 48 hours.
Sparks: So mainly, is some of the kidney healing at that point?
Sparks: But no rejection.
Kumar: No rejection.
Sparks: Which is a really important point.
Kumar: Yes. Thank you for pointing that out. No rejection and no C4d stain, which is what we look for to see rejection of this kind when it involves antibodies. And we did not see that.
Sparks: Overall, this would be a success in my mind because a lot of the issues we discussed earlier were overcome. However, there were still some roadblocks as far as that this was a brain-dead human. It's not the perfect ideal host to put this kidney into. Now, juxtapose this with the publication from NYU [Langone Health] in The New England Journal of Medicine. They did see a decrease in creatinine, is that correct?
Kumar: Absolutely. In fact, they took the kidney. The kidney was a Gal knockout kidney with thymus under the capsule. So, it's a model of tolerance. The decedent had less physiologic…
Sparks: Can we stop right there? What was that under the capsule?
Kumar: The thymus.
Sparks: The thymus was under the capsule. The kidney.
Sparks: Wow. That's fascinating.
Kumar: That's the model of tolerance they were going for, meaning that the kidney itself came with some type of protection, and the kidney was placed in the thigh and not in the iliac fossa and left to the outside. So, it's a much shorter surgery, if you will. The anastomoses are not the same. The ureter was not hooked up, but the important thing was that there was less physiologic derangement in this decedent. The mechanism of injury for the decedent death was not reported, the duration of brain death was not reported, and the cause of termination was not reported in that brain-dead model. Having said that, that those kidneys made upward of 20 liters of urine. So that is significant. And worth noting that they did see clearance, but they gave a cumulative clearance for the original kidneys that were left in place and the transplanted kidney. The declining creatinine they showed was between all those organs together. There was no native kidney removal to show that all the clearance came from the transplanted organs.
Sparks How long do you think it will take to get to a point where we can offer this to humans or even see our first human trial and someone who's not brain dead?
Kumar: The important thing for us to discuss is what knowledge gaps we have filled between the two kidneys that were done in New York and the kidney we did — and we've done one since then. The knowledge gaps we can clearly say we filled are that the hyperacute rejection can be avoided in humans. We've now shown it among two centers, so two different operations. The other thing is that the critical gene deletion is definitely the alpha-Gal knockout gene. That is absolutely essential. These are the two things we can hang our hat on. The knowledge gap that persists is the function in a living human, given the limitations of the decedent model. And the reason I say that it's a limitation of the decedent model is that, for us, it was vitally important that we used every potential disease donor and maximize the chances of using their organs. Only if they were not usable, then we were going to do this experiment in the decedent. This particular decedent that we used had a creatinine going into transplantation of 2.5 mg/dL with liver dysfunction, along with all the other derangements. That was the only way we were then willing to use them for the experiment. It would be great if we got a decedent much earlier in the course, and they would have to have some process that absolutely didn't allow them to be donors, like metastatic malignancy. It's very hard to come by these type of decedents if you really want to be true to using every organ you can for transplantation.
Sparks: So, we're talking about 1 year before we can get to a living human, then maybe the clinical trials would follow thereafter. I was thinking maybe decades or 10-20 years if everything goes well. We might get to that point.
Kumar: I think that the going into the humans needs to start as a clinical trial. The one-off in emergency indications that happened with the Maryland Heart group probably is not the best way to further this field. A phase 1 clinical trial with a limited N like three or five is probably the way to go. It's very conceivable that if we can get some of these questions answered, especially PERV-related questions answered, that we move to that phase 1 clinical trial within a year. Now to make it a reality and to make it available to all our patients, that's a different story. And you're right. We're probably looking at the magnitude of a decade or two.
Sparks: PERV is porcine, endogenous retrovirus. And that was the virus there's still questions about.
Sparks: That's great. I want to wrap up and talk about some takeaways. For me as a nephrologist, I get questions about this all the time from patients and people I know. It's very exciting, and I think we did a great job of discussing the limitations, barriers, and some of the success we've had recently. Any last words you might have for our listeners?
Kumar: Sure. Being a history buff, I'll go back to a saying from one of my personal heroes in transplant, Tom Starzl. I had never met him but read a lot of his works. One of his lines was “history tells us that procedures that were inconceivable yesterday are barely achievable today and often become routine tomorrow.” That's been a guiding light. People have talked about xenotransplantation being just around the corner, but it's a very long corner. And the adage “when pigs fly,” as in that it's an impossibility. Again, I lean on Allan Kirk in one of his editorials when he said that while the pigs are not flying yet, they've hit the runway. I would love for people to remember that and keep the faith. Let's end with a beautiful quote from George Orwell from Animal Farm, where he said, “The creatures outside looked from pig to man, and from man to pig, and from pig to man again; but already it was impossible to say which was which.” So, I am hoping that one day “making a pig of yourself” can have a whole new meaning.
Sparks: Thank you so much, Dr Kumar, and for allowing us to have a peek around that corner. We really appreciate your time and your expertise.
Kumar: Thank you for having me here. It is exciting to share and discuss this journey. And I think only through sharing and discussion can we keep each other informed and honest and motivated to keep doing what we do, which is to get up every day and come to work and take care of patients.
Sparks: Thanks for listening. I'm Dr Matthew Sparks for Medscape InDiscussion.
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Cite this: Behind the Scenes: Pig-to-Human Kidney Xenotransplantation at The University of Alabama - Medscape - Sep 15, 2022.