Scientist Stories: Dianne McKay, Innate Immune Sensors in Acute Kidney Injury

So I'd like to introduce Diane McKay. Now Diane wears two hats, as a clinician and also as a researcher. And what unites her clinical practice and her research approach is something that's near and dear to us in this building. That's understanding and controlling the cascade of activities in inflammation. And what's unique about Diane is that she puts that information immediately into clinical practice. So her medical specialty is in kidney transplantation, and that's an area in which she's a nationally recognized expert. So in the process of transplant, there are a number of arrays of cellular signaling cascades that are at play, and these are ones that we are studying in vitro here and in animal models. And those occur like this. So the kidney itself is host to a large number number of immune cells. And those immune cells belong to and are made by, of course, the donor of the kidney in the transplant. And then when that kidney, when it goes into a recipient, of course, they've got their own array of immune cells, might want to reject this graft. So that's one thing that we're all familiar with. The other thing that happens is that when the kidney comes out of the donor, it's cut off from its blood supply. And so that ischemic hypoxia triggers a cytokine storm inside that kidney. So the kidney is highly activated. And then the recipient's immune system responds to that cytokine storm as well. So there's a lot of different important arrays of immune signaling and immune control that need to happen for that to be successful. The third thing that happens is that when the kidney comes out of a donor who is deceased instead of a living donor, that brain death triggers another cytokine storm. And I'm sure I have only a rudimentary understanding of this, and Diane will correct me where I'm going wrong. But Dr. McKay's intense focus, whether it's in her clinical practice or in her research laboratory, is to understand, map, and measure those innate immune responses and the receptors that trigger them and how we can harness and control that inflammation. Now, she's originally from Canada, and she did her bachelor's degree at UC Berkeley. And then got her MD from the Chicago Medical School. From there, she went to do her internship and residency at Beth Israel in New York and her fellowship at Harvard. Harvard was where her leadership was first recognized as she became the Director of Renal Pre-Transplant Medical Services at Harvard and then the Associate Director of Renal Transplantation at Brigham and Women's. While continuing her transplant medical practice, she also did postdoc research at Dana-Farber. In 2001, she moved from Boston to San Diego, and she joined the faculty of Scripps Research, and she became the director of kidney and pancreas transplant at UCSD, and the director of renal research for Scripps Clinic. She's currently a professor both in the Department of Medicine at UCSD, and also Scripps Research. She's won awards from Harvard Medical School, and also the American Society of Transplantation, She served on national advisory panels for NIDDK and other groups, and she has been the president of the American Society for Transplantation. So Diane, welcome to LJI. We're very much looking forward to hearing more about innate and adaptive recognition events in the kidney.

Thanks very much, and thanks, Erica, for the really nice introduction. And it's really quite an honor for me to be here speaking to all of you. About a little bit of work we've done in the lab looking at innate immune sensors as potential targets for acute kidney injury. But before I start and— oh, thank you. Before I tell you about a little bit of work that we've done in the lab, I want to impress upon you the importance of kidney disease. But I have to change my slides. For some reason, they don't want to change.

Is there someone else in the sound room? Oh, geez.

I guess we're stuck here.

There we go.

Okay, thanks. Okay. So kidney disease is a worldwide problem. It's a huge problem, which I think a lot of people don't appreciate. And we're going to— I'm going to show you a few background slides to show you that. I'm going to show you also, talk to you a little bit about the importance of kidney transplant and why we think it's an important area to target. And then the bulk of the talk will focus on our innate immune pathways that we think are probably relevant to target. So I don't know if you realize this, but kidney disease is very common in the United States. In the United States, about 1 out of 7 adults has chronic kidney disease. That comes out to be about 15% of the US population, or about 37 million people. That would be like everybody in the state of California having chronic kidney disease. It's a lot of people. Worldwide, there are reported over 800 million people with chronic kidney disease, and obviously that's And that's a big underestimate because we can't target all the countries in the world. But as shown on these little orange dots, there seems to be a geographic distribution that's becoming quite interesting. Ground zero for chronic kidney disease is in Central America, in Nicaragua and El Salvador particularly, because there's a high concentration of sugarcane farming.. And there are certain towns in Nicaragua where you'll go into the town and you'll talk to the people and everybody knows what their creatinine is. And I would venture if I reached out to all of you, there's probably very few of you who know what your creatinine is. And now there are hotspots coming popping up throughout the world. Now in Mexico, in Egypt, in southern India. And these hotspots seem to be associated with tropical countries right around the equator. And this has become such a big deal, this is called chronic kidney disease of unknown origin, that the American Society of Transplantation, but also the American Society American Society of Nephrology, which is the largest group of nephrologists in the country, are directly partnering with climate change experts to see if there's something about these tropical countries that predisposes to chronic kidney disease. And it is almost— they're wondering if this is almost a canary in the coal mine. As our planet gets warmer, are we going to start seeing more and more chronic kidney disease. The primary causes so far, as I mentioned, this is common in agricultural areas around the tropics, are thought to be pesticides, but also pathogens have been implicated, particularly leptospirosis has been thought to be a possible pathogen. In the United States, the most common cause is diabetes, but also hypertension. And other inflammatory diseases such as lupus, IgA nephropathy, which is an autoimmune disease. Many different autoimmune diseases cause kidney disease. So often we don't really know what the cause is, and thus we don't really have very effective targets. Well, as I mentioned and alluded to, kidney transplant is the best therapy for end-stage kidney disease. Because people live longer with a kidney transplant than they do on dialysis, which is the alternative therapy, and obviously have a better lifestyle. You're not hooked to a dialysis machine 3 times a week, and it's less costly. So because of this, there are a lot of people waitlisted with the United Network for Organ Sharing waitlist for transplant, as indicated in the gray line. In 2013, there were 120,000 people waitlisted for transplant. Yet only about 30 transplants performed in the United States. And that reason is because there are not enough donors available for transplant. And so put in another way, if you have an O blood type and you're here in San Diego or certainly in the Los Angeles region, you live there, and you need a kidney transplant, you're going to wait for 10 to 14 years before you get your kidney transplant. It's really that dire. So because of that, surgeons and transplant professionals are trying to push the envelope on available donors. And now more and more marginal kidneys are used. And these marginal kidneys are kidneys from a donor, a deceased donor whose brain has stopped working, but they are also older, older than 16 years old. Years of age, or they have older than 50 plus other comorbidities such as hypertension, elevated creatinine, or they die from a stroke. Another category which is being used more and more frequently is donors who, after their brain dies, their heart stops. Normally, those kidneys would have been discarded. Started, but now we're more and more we're using those kidneys. And though that seems to be an area where we can expand the potential number of donors, the problem with these marginal kidneys is they're very, very susceptible to ischemic injury. So if we have— can develop some targets to treat these donor organs, we may be able to improve the numbers of kidneys available for transplant. So this is what our— the goal is in our lab. And we work very closely with our organ bank here, which is called Lifesharing in San Diego. And we get a lot of kidneys from Lifesharing. We do a lot of experiments on discarded kidneys, but some on kidneys that are on— I'll explain that as we go through. This is the model that we use in the lab. We use bilateral renal artery clamps. Clamping. And we clamp the renal arteries for various times depending on what we're looking at. And that's our ischemic phase. Release the clamps and then you have the reperfusion phase. And there's gross changes in the kidney that occur with each of the changes. The area— there's an area in the kidney where ischemia is most evident when you look at a sagittal section, which is shown in the red bracket, and that's the cortex and the outer medulla of the kidney. And if you look at a kidney— sham IR-injured kidney versus an injured kidney— you can see right away that there's sort of a pale region in that outer stripe of the medulla. So you can clearly see that that's the region that's really injured. And when we look histologically, we see what's called patchy necrosis. And so in the red, those are dead tubule cells, and right next to them are ones that are alive. And this histologic phenomenon is pathognomonic and has been noted for many, many years by histologists. And so the idea is that necrotic cells release DAMPs and whatever other things are inside the cells and engage pattern recognition receptors on healthy cells and extend the damage. And so our goal here is to see if we can block that cascade of injury that occurs in all kidneys that are procured for transplant. So we start first looking at cell surface. Pattern recognition receptors to see if maybe they would be a reasonable target to block. And we started looking at TLR2, and we found that there was sort of an interesting distribution of TLR2. You can see it right here, and it was localized to the cortex and the outer stripe of the medulla, which is really the very areas of the kidney that are most susceptible susceptible to ischemia. And working with Bill, wherever Bill is up there, Bill did these really pretty pictures for us. We found another interesting thing was that it was localized to the basolateral membranes of renal tubular epithelium that was in the cortex, but not deep in the medulla. So in the cortex and the outer stripe of the medulla, We definitely see it, and this was supposed to be a movie, but it isn't working, but you see it nicely lining the lateral membranes of these cells. We also found it on podocytes and arterioles in glomeruli, glomerular podocytes. So we asked whether TLR2 knockout mice could be protected from ischemic injury, and we blocked, you know, TRIF, we blocked MYD88, we blocked TRIF and MYD88, we blocked TLR4, and finally with blocking TLR2 we found really profound protection, which was a little surprising because we should have had protection with blocking MYD88 and TRIF as well, which we do see some protection, but more profound protection from TLR2, suggesting that there might be an independent dependent effect. And whether this is due to the localization of TLR2 in that region or alternative signaling pathways, we're not sure. We see less cell death and granulocyte infiltration, which is no surprise. And so then we went and asked whether it's present in human kidneys. And the way we get our human these human kidneys was through nephrectomy specimens. And we'd go to the operating room, wait for the urologist to hand the kidney to us. And these would be kidneys removed with renal cell cancer. And you would— the area distal to the renal cell cancer could be used because that's really normal tissue. And then we'd trot back to the lab, stain it, do everything. So there was a lot of time elapsed, at least 45 minutes, maybe longer of time elapsed between when we get the kidney and when we get it back. And as you can see, it looks like the receptor is sort of not nicely delineated anymore on the basolateral sides. And— but still in the humans, we saw the same distribution. It's present in the cortex, not in the deep medulla. And then we went and said, well, may it be— and in the— mouse models, the way we would do the staining— it took us a while to figure this out, but we— at the time of euthanizing the animal, you would inject formalin into the kidney so you would sort of snap-freeze everything. And if you didn't, if you did renal clamping first for about 25 minutes and then stain for the— for TLR2, It seems as though it's internalized, or it's now more cytoplasmically located. And this happens in renal tubular epithelial cells with a lot of other cell surface molecules. And we— so that was an interesting phenomenon. We don't know if that's a protective effect or whether it's internalized and shed, and we have some data suggesting that that's so. We also looked for its expression in biopsies of transplanted kidneys, just to ask whether patients that get a kidney transplant need to take immunosuppressive medications. These patients were taking tacrolimus, mycophenolate, and prednisone. And that did not have an effect on the expression of TLR2. So since this time, there have been a lot of diseases that are associated with TLR2 signaling, and a lot of people are quite excited and have been excited about trying to figure out how could we target this molecule. And this seemed like a really reasonable target for donor kidneys. And in fact, a company, Opsana Therapeutics, at the time had developed an antibody, humanized anti-TLR2 antibody that had some receptor occupancy. In Phase 1 trials, but then the company went bust and in 2019 closed its doors, and now it's working more on cancer immunotherapy. So the targeting kidney disease seemed to stop as far as any pharma interest in that. So then our focus changed to whether interest cellular pattern recognition receptors could be targeted to block ischemic injury. And we started with looking at the NLRs, which as you all know, there's lots of them and they're associated with pro-inflammatory signals, cell death signaling. They're well associated with chronic inflammatory diseases, CAPS, gout, sarcoid, NOD2 with Crohn's disease, many other diseases. In fact, there's a whole slew of them, and there's an intense interest in many diseases in looking at the NLRP family, and certainly NLRP3 is the best studied in kidney ischemia-reperfusion injury. And so we started asking whether indeed this might be a reasonable target. And the first question we asked is whether MLRP3 and ASC, which is necessary for forming the inflammasome complex, whether those were present even in human renal tubular epithelial cells. And so indeed they are in mouse, and they are indeed in human. So we had another target that we could potentially look at. And we went back to our mouse models. And found that interestingly, NLRP3 but not caspase-1 or ASC knockout kidneys were highly protected from ischemic injury. And so it suggested that NLRP was potentially uncoupled from the inflammasome. And as we now know that there's non-canonical NLRP3 signaling pathways that might be playing a role. There is less renal tubular injury in mice. All of our samples, we have— we work closely with the renal pathologist who scores all of our stuff blindly, and this was really— it was really quite profound protection from renal tubular injury. There's less IL-6, IL-18, IL-1 beta.. All of this was decreased in the NLRP3 knockout mice. And we also found— this is work again from Bill— that after ischemia-reperfusion injury, there's a ton of secreted IL-1 beta from the renal tubular epithelial cells in mice. So the question was, is Is this protection just an IL-1 beta effect? So blocking IL-1 beta, that would be a simple way because we have target— we have antagonists to the IL-1 receptor. And so we did the experiment and asked if treating mice with anakinra or IL-1 receptor knockout mice could protect the mice from injury. And in fact, the answer was no, cannot. So this is an effect that's independent of the inflammasome and is dependent on NLRP3. So the other question was, could the protection— was the protection due to inhibition of inflammatory effector cells? And so we did bone marrow transplant in chimeric mice of either NLRP3 ASC or wild-type chimeras, and we found no, that did not protect. There is some, you know, there's less inflammation, but that did not protect from the ischemic injury. And so we're coming back to the renal tubular cell as being extremely important, extremely important cell type to target in this arena. Renal tubular cells, though, when you isolate them from mice and you treat them— we've done this many different ways. We either treat it with hypoxia, reactive oxygen species. We have a big chamber that we do hypoxia reoxygenation experiments in, in various different ways. The NLRP3, AST, and caspase-1 mice are protected from necrosis. So there's something different happening in vivo, obviously, and more complex than we're seeing. There have been a lot of different inflammasome components linked to different kidney diseases. And NLRP3, while it's quite protective from ischemia reperfusion model in acute kidney in that model, when you use another model, cisplatin model, to induce acute kidney injury, you get a lot more injury with NLRP3 knockouts. ASC knockouts, you get a lot more protection when you use a different model, when you use our cisplatin model. So there's a lot we have to learn about how these components are being regulated. In vivo. That said, there's a huge interest in this area for kidney disease. And we also thought, as you know, we require priming and triggering signals going upstream. We know there's a lot of crosstalk with other innate immune receptors. So we decided to say, well, maybe we should be going upstream to look for more effective targets. And we looked at NOD2. And I also will show you a little bit of data. We looked at CD14 as potential targets. So for NOD2, we found also NOD2 was present in both human and mouse renal tubular epithelial cells. And interestingly, deleting NOD1 did not protect from ischemia-reperfusion injury, but deleting NOD2 did. But NOD1— and I didn't show the data, but it protects from neutrophil infiltration. So in most of our experiments, we use NOD1 by NOD2 double knockout mice because there's potential crosstalk. In these, there's less renal tubular epithelial cell death, for sure. And also, mice— wild-type mice transplanted with the NOD1 by NOD2 bone marrow are not protected from ischemia-reperfusion injury. Again, bringing us back to the renal tubular epithelial cell is a very important cell to target with some of these sensors. And then we also have been looking at CD14 as a potential target. We're working with a company that has an antibody to CD14, both humanized and mouse antibody. And so we decided to do some experiments to see if this would be a reasonable target. So we do this. There have been several studies suggesting that CD14 might be a reasonable target for kidney injury in both mouse and human models. And I won't go into that. And we've been finding that we can upregulate membrane-bound CD14 as well as soluble CD14. In renal tubular epithelial cells from humans. And we have an antibody that decreases injury in a mouse model of CD14 as well, suggesting that this is a potential target as well. So now we've got several different angles to attack this at. And why this is important is that there's our current preservation times for kidneys and hearts, lungs, livers, pancreas, et cetera, are very low. Beyond these time points, there's questionable organ viability. And so we don't have much time. As you imagine, this would be really relevant for hearts, lungs. You only have a few hours to get the organ out of the recipient and into the next patient. And the current preservation methods that we use for kidneys most commonly is static cold storage because it's cheap and easy. Kidney is just washed with sterile perfusion, stored in an ice bucket basically. And then another way kidneys are preserved is with dynamic pump preservation, either hypothermic or normalthermic. And normalthermic is a a more experimental way to do this. But this pump is a really nice way to isolate the kidney and study it outside of the potential donor and downregulate things at that time. So we know that As Erica mentioned, there's multiple physiologic events that are occurring in a deceased donor, even way before that organ's removed. There's a lot of systemic inflammation, microvascular changes, and kidneys and all organs experience some ischemia-reperfusion injury prior to even removing the organ, which worsens the ischemic injury. And so, One thought is, you know, it certainly makes sense that you might be able to treat the whole donor with TLR, NLR blockers, or whatever you're going to use. But currently, it's required that you obtain consent from all the potential recipients of organs. And that's an impossible task. You cannot get— because when you're on call and you're getting a donor call, and you're having to go through the list, you'll call somebody for a kidney transplant and you'll say, we got a kidney for you, come on in. And they'll have a cold or they've got COVID or they've got something that interferes with their ability to be transplanted at that time. So you go down the list and it's quite a huge task. So it's impossible to get the heart consent, lung consent, et cetera. And the other thing is that I, I didn't show you with, but there's different— these receptors are differentially expressed in different tissue. So in the liver, TLR4 plays a bigger role than TLR2 does. In the heart, there's a lot more endothelial cells that we have to be considerate. So it becomes quite complex, and so this is doing— giving something to the whole donor is not possible. So the only way that I think we can use therapeutics is in either these kidney pumps, and now experimentally, where a lot of centers are using liver pumps. And so there's a great opportunity because there you can, while they're on the pump, you can measure a lot of different cytokines, chemokines released. You can get biopsies. All these, all the kidneys that are put on a pump get a biopsy initially, so you have initial tissue. And you can follow the outcomes of any intervention you want to use. And we have one of these pumps in the lab, and we get discarded kidneys from the organ bank and do those. And this is, we just did some work with the organ bank looking, these are kidneys that were transplanted into patients. And there you're seeing two different categories, donation after brain death, that's the standard type, and donation after cardiac death. Those are the ones that had more ischemic injury. And maybe not surprisingly, there's a lot more injury, pro-inflammatory cells or molecules released after injury in after cardiac death. So we, in conclusion, we think that TLR, NLRP3, and NOD2 are potential targets to improve donor kidney injury. CD14 might be an important upstream regulator. We need additional studies in human kidneys that are procured for transplant. And as I said, we work really closely with the organ bank and get a lot of their organs and a lot of their kidneys. And I have someone in the lab who's actually getting a lot of their lungs now and trying to understand what's happening in the organs that are actually going to be used for transplant. And then the opportunity to intervene is especially high for these marginal kidneys, which if we could downregulate some of these inflammatory responses in these kidneys that would greatly improve the number of organs available for transplant. And so that's it, short and sweet. And these are my collaborators from my lab and from UCSD, Scripps, and from here. Bill and his group has been really instrumental in helping us a lot.

So thank you.

Are there mouse-sized kidney pumps? Yeah, yeah, we spent a huge amount of time trying to get a mouse pump, but the problem is the perfusion pressure destroys the endothelium, and so it doesn't work, although we spent way too much money and time trying to do that. So that's not a surmountable problem? I mean, at least it hasn't been. So far, not. I mean, so a lot of what people do is move to rat models, which are a little bit bigger, but that mouse kidney and the vessels are so small and so delicate that we haven't been able to do it. And I don't want to even tell you how much money we spent and time on that, but it's— but I'm happy to— if somebody has an idea of how to do that, I'm happy to revisit that. Yes. I was going to ask you, so, two questions. The first, the receptors that involve the TLR and the NLR, do those change at the transcriptional level during ischemia, or do any, like, innate immune signatures increase?

Yes.

Yeah, in the kidney. Oh yeah. What's the timing of that? Well, it's, it's sort of variable, and, and so, yeah, and, and the different ones change at different times. So we see big upregulation of TLR2, we see big upregulation of NLRP3 with the early inflammation. And that happens, I want to say, I mean, we look at like 24 hours, but probably. And do you think that there is a DAMP response involved there? Yeah. Yeah. And so we've— What DAMPs do you think are involved? That's a good question. We tried to figure that out. So we also spent a lot of money on this, on doing mass spec, and we started analyzing under all sorts of different circumstances, and it's a big mess. That's all I have to say. And I'm not going to spend any more money on that. It's just there's so much stuff that's released in the half— I can't talk. I mean, some people talk HMGb1, you know, and then do ex vivo, but I think realistically. Yeah, no, yes. And, and, and I— but the problem is, and, and I think we want to go back to the real situation in a patient, right? And the problem is you clamp the renal arteries, or in a— in any of these things, you're going to have bowel leakage. So you've got a lot of PAMs. So nothing's sterile. And so I think the targets should be really the receptors, which are— you know, there's fewer of them than really targeting any particular DAMPs. I mean, while it's a very interesting question, it's hard to really precisely, I think, get at it. Yeah, protein mass spec would definitely give you some of the cytokines, but you may want to think about also— I know you just said you didn't want to— but small molecules. Yeah. Is that right? Absolutely, absolutely. But we—

no.

You're 100% right, and I agree with you, but it's an undertaking that's huge and one that I don't know if that is translatable therapeutically.

So—

Thank you.

Hi.

I had a quick question about your chimera experiment. I may have missed it, but you showed that when the immune system is deleted for TLR2 and LRP3, okay, you don't protect. Have you shown shown that with the wild-type immune system, that NLP3 knockout recipient or TLR2 recipient, that you do have the prediction?

Yes, yes, you do. I, I do. I'm sorry I didn't show that. Yes, absolutely. Yes, it's an important control.

Yes, you mentioned that in most of the cases the patients have severe autoimmune disease, right?

Well, that, that can be a cause for kidney disease, absolutely.

Yeah.

Yeah.

So that means that the patients have a lot of pathogenic autoreactive T cells.

Yes.

Do you think that it might be possible that some of these autoreactive T cells are targeting TLR2, for instance, and that that's actually—

Yeah, I think that's a really interesting point, and kind of what I was I was sort of implying by my first slides with all of the chronic kidney disease of unknown origin, I think there's probably mechanisms like that that are leading to kidney injury, either through innate immune receptors, obviously other mechanisms. But we see so much in the clinic of people coming in, you know, like lupus, Lupus is a good example, you know, IgA nephropathy is another great example. We don't know what the inciting cause is, but—

But it's probably going to be different targets.

Yeah, I know.

But it would make sense because you see an effect with deletion of TLR2 but not of the downstream molecules.

Yes. So that would be consistent with that TLR2 TLR2 is recognized as a target for this autoreactive T cell. Yeah, I think it's really—

Or for— Yeah.

I mean, yeah.

I'm not excluding one or the other.

I'm just saying—

Yes.

So it's not really clear to me where the damage is initiated. If you're doing a transplant, is the damage initiated within the donor itself when you're taking out that kidney, or is this a process that occurs once you do the transplantation?

Both. Both. When you take the kidney out, you've got ischemic injury and all sorts of bad humors.

Is it too late then?— to think about a treatment if you're driving that epithelial necrosis at the time you're actually just taking out the kidney.

Yeah, no, that's the very point, is that that's an area in transplantation that we haven't targeted that we really ought to target. That, I think, is a new frontier of therapies in transplant. Normally what happens, you come in, you're going to get your transplant. You go to the OR. On the way to the OR, you're getting IV steroids. You're getting, you know, thymoglobulin. Then you're getting your immunosuppression, heavy immunosuppression early on, and tapers down. So the cat's out of the bag, I think, by the time you're putting that kidney in. You know, we have good outcomes. Mind you. But what are our good outcomes measured by? What's the measure? But I think the real opportunity that we have in the field of transplant to make some headway, to increase the number of marginal organs, to probably make those kidneys last longer, because, you know, we use a lot of therapies on a kidney that has already preexisting damage. And the The amount of preexisting damage certainly is quite clearly associated with the length of ischemia. So that's— I just think it's a really important therapeutic. I'm not the only one that thinks that, obviously.

Yeah, I'm sorry.

So you feel that those preexisting damages are very much organ-specific? Because obviously in heart, that's very well known, there are many pathways in reoxygenation. Including ion channels, there are sensors that have been shown to promote— and there was work on TRPM2, for example, in the kidney. Do you feel that there's a general kind of approach that could be taken for all transplantation, or is it very much specific for each organ?

Yeah, I think it's going to be organ-specific. I think that the data so far looking at the Liver ischemia is different than kidney ischemia, that's different than heart and lung. Lung is really even more complex, as I mentioned, someone in my lab is working on lung right now. And one problem with lung is that there's a lot more pathogens in the lung at the onset. So there are complexities to these things that I think, you know, they're nuances that lead to differences, essentially. Off-the-wall question. Absolutely. So they were breeding these pigs that lacked histocompatibility antigens precisely for the purpose of obtaining organs for transplants. What's the status on that? One of my favorite people in the field of xenotransplantation has said to me, xenotransplantation is an amazing, great idea and always will be. I don't do zeno, so I don't really get it. But, you know, I don't mean to be glib about it, but you're right. If we can, you know— One keeps reading about it. No, no, I know. And a lot of— there's a lot of interest and there's a lot of work in that area. And it would be great if you could go to your pig farm and pick out your organ of interest, and for sure that would be great. And I'd invest heavily in that. Company, if there was such a thing. But so far we're not there. I think we may get there.

I don't know. Why aren't we there yet?

What are we missing? There's a lot of problems that I think need to be surmounted. Not only just looking at, you know, what are the important targets, but pigs' organs have retroviruses. That after you transplant— I read an article about it. Yeah, they're trying to— they're breeding those out. But still, I think in the pig transplant that was done in New York into the human, yeah, that patient was eventually— had evidence of pig retroviruses. I think there's a lot more to go, and I really did forget to somebody who does that a lot. I don't do that. That's David Sacks who said it was always a good idea and always will be. I know, I think he was just probably joking at the time.

Um, two questions. One is, um, what about adaptive immunity? I've read papers that said that gamma interferon was critical for ischemia reperfusion injury, and what about targeting, you know, adaptive immunity in some way?

Yeah, I think adaptive immune cells should be targeted because the minute you put the kidney in, then— but what I'm talking about is going before you even get that kidney in, while it's on the pump, before it's seen the recipient's—

I mean—

But even some of the resident cells might respond to ischemia reperfusion. Respond to the inflammatory injury by making gamma interferon.

No, absolutely, and that might be an important— I don't mean to say that this is— there's just a one-off here. Sure, for sure, absolutely.

What about TLR2?

Is the idea that it's seeing peptidoglycan from microbes, or a DAMP, some kind of an endogenous?

We think it's— I mean, it's In the real situation, it's both, obviously. But in our mouse models where we've taken renal tubular epithelium, stuck them in culture for a while, and then treated them with necrotic supernatant from other renal tubular epithelial cells and— Sterile. Sterile. Sterile as sterile can be. And we see induction of injury. Is hitting the—

Yeah, for sure. Have you looked at IL-18 as some role?

I'm sorry?

IL-18? Yeah.

Have you looked at it?

Because it's also produced by cleavage of the inflammasome.

Yeah, IL-18 is—

Sorry, induces gamma interferon. Sure. IL-18 is downregulated in I mean, in the NLRP3 knockouts, we see less IL-18. How much it plays a role, I don't know. Independently, we haven't really spent a lot of time looking. We haven't looked at individual cytokines per se, as we haven't looked—

we could, we should. [INAUDIBLE] Blocking IL-1, you still have the damage, but blocking IL-18, what happens?

I have to go back, I don't remember, but if we had an effect, it'd be right on the top of my head right now. Thank you.

Sorry.

Yes? What kind of research is being done towards addressing what causes kidney disease in people? Maybe people should be thinking about targeting that instead of transplantation. Yeah, no, I agree with you. 1000%. I think that these have not been looked at as important targets for some of the diseases that we commonly see in nephrology. In our nephrology clinics, we have patients come in all the time that, you know, you say, I know you've been exposed to something that's caused this autoimmune disease, that's caused this, and And I think there's a lot of people in nephrology that think that these might be important targets. I read a paper not too long ago where someone said that this is going to— sort of NLRP3 blockers are going to be our new statins. We're all going to be taking them someday to downregulate. But yeah, no, you're absolutely right. I think that this— if we have a better understanding about how some these things are regulated, maybe we'll have targets for other diseases that are quite common, kidney diseases. And also, like, you said pesticides and pathogens, right? What pesticides? What— yeah, there's a list of pesticides that have been implicated in some of this, and several pathogens have been implicated in them. Leptospirosis is one pathogen that comes to mind because it does bind TLR2, and it's been shown to induce kidney injury in that model. So there may be parallels, right? There may be very, very much may be parallels, and there may be crosstalk, but there's something about all that kidney disease in these around the equator that really needs to be— some attention needs to be paid to that. And that's why I mentioned this upcoming meeting for the American Society of Nephrology. There's going to be— there's a huge— we have a big meeting in November. About 13,000 to 15,000 nephrologists are going to be sitting around. And a big focus is on climate change and infections and how this leads into— how induces kidney disease. It's really, I think, very important.

What happens differently in a living donor where they have all the time in the world to prepare?

Do they suppress their immune system in some way? So a live donor, it's much easier. The donor is in the operating suite right next to the recipient. Kidney's taken out, truck, truck, truck, goes on, and it's max 45 minutes. If you have a good surgeon, you can get it in there, you know, a little faster than that. And those kidneys, there's a huge amount of epidemiologic data showing a live donor kidney lasts longer, significantly longer, than a deceased donor kidney. And if you look at the different deceased donor kidneys, the ones from marginal donors do not last as long. And even to bring it home further, if you are over the age of 60, 60 in Southern California and you want to get a kidney transplant, you're only going to get a marginal kidney. You're not going to get a standard kidney unless you have a live donor. So we spent a lot of time trying to tell everybody to get a live donor when they're in the clinics. Any other questions? If not, let's thank Pam.

Okay.

Thanks a lot.