I think it's just sort of. So Simon studied at Cambridge and then was at Addenbrooke's and qualified,
moved to Nottingham for his basic surgical training and came to Oxford in 2006,
taking up a position with the evidence base in transplantation and completed his thesis and then was appointed as honorary consultant,
transplant surgeon and senior clinical research fellow here in October 2017.
So and I'll introduce James at the same time. James is an academic clinical lecturer in transplantation and has a contract with University Hospital,
Coventry and the University of Oxford looking at this live donor kidney transplant and complex vascular access.
And this is the first appointment to the Coventry Oxford Transplant Network,
which is a UK transplant collaborative which is aimed at improving patient access to transplant by sharing resources to the centres.
I think we're starting with Simon. Thank you very much. Bright morning, everybody, thank you for coming.
So I wrote the title before I'd actually thought about what we're going to talk about this morning,
so actually we're going to focus on kidney preservation, having talked quite a lot in the past about the liver.
And so I'm going to talk about some of the clinical research that we've been doing and part of.
And then James is going to talk a little bit more about some of the translational in lab research.
So for those of you, the work in the chat show, this is a fairly familiar sight outside the theatres,
particularly if Sanjay's been on call for the weekend.
That's a post Sanjay weekend on that on the right hand side there, which is the mainstay of how we store kidneys in the UK at the moment.
So we put them in ice boxes, so cold fluid in ice cooled down to about four degrees.
And this is what we've been doing, probably for about 30, 40, 50 years in the UK since since we started transplanting routinely in the 1960s 1970s.
And there's a very good reason for it. It's very simple. It's very cheap. Logistically, it is straightforward.
It doesn't require power. There are no moving parts to break and nobody has to sit with it and watch and make sure that it's doing what it should do.
But it does have its limitations. So when you store an organ at four degrees, metabolism is slowed, but it's not stopped completely.
And so there's still anaerobic metabolism carrying on in the organ. You get accumulation of metabolites, you get depletion of ATP.
And so when you put the organ in and you reproduce it in your recipient, you get an ischaemia reperfusion injury,
you get inflammation, which then primes the immune system with the risk of of inflammation and damage.
There are lots of reasons why we might want to reconsider this idea of cold storage.
So over the last 10 years or so, as you can see from this graph from an SBT, our donors have been gradually getting older.
Older donors generally have more pre-existing injury from hypertension, diabetes, cardiovascular disease.
And so we're starting off with slightly more marginal kidneys than we would have been perhaps 10 20 years ago.
Equally, the type of donors that we're using has changed as well during that period of time.
So our ideal Dana Oredo deceased donor is a brainstem that donor because there's no period
of warm ischaemia prior to procurement of the organs we're seeing now and using now.
More and more donors after circulatory death, CD donors where there's a mandatory period of five minutes of warm ischaemia before you can start the
retrieval operation so these organs are more injured before we even start this preservation process.
So before we start looking at some of the tools we've got available to us,
it's worth thinking about what the ideal organ preservation method looks like.
So obviously we want to prevent damage during storage,
but we also ideally want to be able to repair some of the damage that's already occurred during that procurement process.
We'd like to be able to prolong the storage time for logistical reasons so we can
transport organs longer distances or we can accept more organs at any one time.
But also, we'd like to be able to assess the viability of organs if we could have some way of trying to work out which organs are going to behave,
work when we transplant them. That would help us to select better and possibly use some of the organs that we don't currently use.
And then moving on to the future would also like to be able to deliver therapeutic interventions to the organs before we transform them.
Second story doesn't stack up to well against all of these criteria.
Probably does help to slow organ damage during storage, although it doesn't stop it completely, but it doesn't really allow any of the others.
It doesn't repair any damage. Storage times are limited, and there's no means of viability assessment.
So even back when we started this in the 1960s, there was an interest in machine preservation of organs.
So this is Belize's hypothermic machine preservation device, so this essentially pumps cold fluid through a kidney.
And as you can see, it's pretty unwieldy and logistically challenging.
It's required a transit van to take it around a forklift truck to move it and say, perhaps unsurprisingly,
as cold storage solutions improve during the 60s and 70s, people move to cold storage for the logistical benefits.
Nowadays, the machines have changed somewhat.
We cannot put it on a tabletop and one person can lift it, and this is the equivalent that we have now in our theatres.
And again, this is this is a hypothermic machine for perfusion kidneys with cold preservation fluid.
Now the benefits of this or the the reported benefits of this is you're flushing cold preservation fluid through the kidney during storage.
So you're removing some of these metabolites that are accumulating and providing substrate at the same time.
So when he was with us, John Callahan undertook a systematic review looking at hypothermic machine preservation,
and there clearly is some clinical benefit to using machine a standard static cold storage.
These are all randomised controlled trials of hypothermic machine preservation versus static cold storage as we see the risk of delayed graft.
Function is reduced by about 20 percent when you use hypothermic machine preservation.
But interestingly, there wasn't much difference in graft survival or graft function.
When you use this technology, so it does seem to prevent the reperfusion injury, but it doesn't have too much of an impact on long term outcomes.
Most of you have had good talk before about the Cape consortium,
so this was an easy funded consortium of transplant centres across Europe who were looking at organ preservation research,
and as part of that, there were three randomised controlled clinical trials and a suite of experimental research as well.
David did. The last grand round presents the results of the liver trial, the normal thymic liver preservation trial.
The next of these trials to report is the third one down there that compare randomised controlled trial,
which was looking at hypothermic kidney preservation.
And the results of this have just come out say the idea behind this trial is to see whether we can improve on hypothalamic machine preservation.
So typically, we flushed the kidneys with code preservation fluid, but there's no oxygenation.
We just literally flushing metabolites out of the kidney.
The group in Poitier, who were part of the Cape consortium, had done some experimental work, which is shown here.
Essentially, they took a DCD pig model, so 60 minutes of warm ischaemia to the kidney, removed the kidney,
placed it on a hypothermic machine preservation device for 22 hours and then transplanted back into the into the pigs again,
and they split the organs into two groups.
One group was standard hypothermic machine preservation, the type that we're used to using in clinical practise.
The second one, they actively oxygenated the profuse age.
So there's no oxygen carrier, but they're just bubbling oxygen through the the profuse that they're using in the device.
And you can see that there's a much lower peak in in creatinine after reperfusion in the transplanted animals.
And the baseline creatinine falls down much more rapidly to a lower level.
And then if you take biopsies three months after transplantation, there's much less in the way of chronic inflammation and fibrosis in the organs.
So oxidation does seem to have some benefit in preventing both short term and long term damage.
And so the purpose of the Kompass study was to test this in a clinical setting.
So this was a multicenter, randomised controlled trial across Europe, looking at oxygenated,
hypothermic machine preservation versus standard, hypothermic machine preservation.
One of the nice things about the kidney is that they come in pairs, so each donor has two.
So it's quite a nice clinical trial designed to actually just take both kidneys from one donor and treat one one way one another way.
And then you can control for that these days. The fact is, so this was a bad kidney design study.
It was blinded. So the way this worked is we had a small army of medical students and other students in the Netherlands and Belgium with vans.
She would take these devices out to the retrieval hospital, switch the oxygen on or off depending on the randomisation,
could put the kidney on the device so that they could adequately blind the surgeons in
both the retrieving and implanting centres as to whether there was oxygenation or not.
And this study was in these more marginal kidneys a DCD donors aged over 50.
The primary endpoint was graft function. OK, so one year after transplant, we looked at estimated GFR in the primary analysis.
You you see there's a slight drop in in EGFR in the standard hypothermic machine preservation group and it's improved in the oxygenated group.
It doesn't quite reach statistical significance. The problem with using graft function as an outcome is, of course,
if a kidney is not functioning one year after transplant, you can't include it in your analysis.
And so as a pre-planned sensitivity analysis,
we imputed all these groups that have failed with EGFR of 10 meals per minute and that Shane on the right hand side.
And that shows that that if you take into account the graft failures, the difference does reach statistical significance.
If you look at measured GFR, so we also measured GFR in these patients at 12 months again,
a statistically significant and probably clinically significant difference of seven men in GFR one year.
Perhaps more interesting is a significant reduction in graft failure in the oxygenated group.
And if you look at the mechanism for that,
you see there's a halving in the risk of acute rejection during the first year transplant with oxygenated machine preservation.
So there does seem to be something in this the clinical data to support that animal data
from prior to the one year after transplantation with oxygenated machine preservation,
we're seeing improved function, less graft failure and a reduction in the risk of acute rejection.
And it seems to be that reduction in the risk of acute rejection that seems to mediate the differences in function and graft failure.
In terms of the mechanisms, well, if you're oxygenating, you will reduce the risk of ischaemic damage,
build up of metabolites during preservation, and so you get less human ischaemia,
reperfusion injury and if you reduce the ischaemia reperfusion injury, there'll be less inflammation pastes reperfusion,
which will result in less priming of the innate immune response and the subsequent adaptive immune response.
And we seem to be seeing less in the way of acute rejection, but also less in the way of chronic inflammation and fibrosis later on.
So hypothermic machine preservation, then if we add oxygen to it,
does seem to do a little better than cold storage, we can prevent damage during storage.
We can repair damage to a degree that's occurring during preservation, which might allow us to store kidneys for a little bit longer.
But we still don't have a means of assessing viability of organs during preservation to see which ones we think are going to work or not.
Nor do we have to do that.
We've really got to be able to store our organs in a functioning state, and that's where normal thermic preservation comes in.
So we're now producing the kidneys with oxygenated blood at body temperature, providing nutrients so that they can metabolise as normal,
which has the potential to allow us to assess viability and deliver therapeutics on the circuit.
David Nasrallah in our last round round present the results of the liver study using normal smoke machine preservation
and certainly in the liver that does appear to be a reduction in the reperfusion injury following transplantation.
But perhaps more interestingly, there was also a difference in discard rate between the two arms in that study,
which suggests that the surgeons were able to use information from the device to make a decision as to whether they could transplant a liberal or not,
and potentially improve utilisation of livers that would otherwise be discarded.
So what about the kidney?
Well, you can't really talk about normal thermic preservation of the kidney without talking about the work of Mike Nicholson and Sarah HostGator,
now in Cambridge. He really does most of the initial clinical research in this area, so they have a a slightly less developed circuit.
You can see on the right hand side that consisting of a bunch of pumps and an oxygenated and derived from a paediatric anaesthetic machine, I think.
And they took 18 kidneys that they accepted otherwise for transplant,
and they placed them on this circuit for an hour just after cold storage prior to transplantation and then transplanted the kidneys.
And you can see that they get quite impressive delayed graft function rates when they do this.
So we would expect and kidneys like this to see a delayed graft function rate of perhaps 30 percent to 40 to 50 percent.
And they saw in their 18 kidneys a delayed graft function rate of just over five percent.
So this does seem to be having some some effect in repairing the organs prior to reperfusion.
What about viability assessment? So they took this one step further, and they took a whole bunch of discarded kidneys,
the kidneys that were not accepted for transplant anywhere in the UK.
They took these kidneys, they refused them and they developed what they call the QC school,
which is a sort of clinical score for assessing organ viability based upon macroscopic perfusion, blood flow, urine output.
They then applied this score prospectively to a further group of discarded kidneys and demonstrated that they were able
to transplant some of these kidneys that were declined by all UK centres and actually improved the transplant rate.
And so the five kidneys that they transplanted for had immediate function, and they all did OK after a year.
So. The challenge with their method is they're only producing for a very short period of time,
so they're only producing for an hour immediately prior to the transplantation.
So you're not preventing any of the damage to the cause during storage,
but they do seem to be able to reverse some of that damage prior to transplantation.
It's clearly doesn't have any logistical benefits because you're not prolonging preservation time, but it does seem to allow viability assessments.
So off the back of this in the liver research,
Emory wasn't back when she was here as a defo student along with Peter and Constantine developed a prototype.
Normally, semi kidney preservation device based upon the liver circuit that we've used previously,
and Emery used first a series of Pekinese and then discarded human kidneys,
and was able to demonstrate that she could refuse them under stable conditions
for up to 24 hours a far longer than anybody else has managed to this point.
So this shows the development of a device is constantly mammary on the right hand side there.
So it started off as a bunch of lab equipment and turned into a quite nice looking prototype, which has been developed further since then.
One of the challenges with the kidney is that, unlike the liver, the kidney produces urine,
and so if you leave it on a circuit for too long, then it will obviously produce. You're in the second volume will decrease.
And so if somehow you have to replace that volume, see some of the early experiments that Anne-Marie did,
she added in Ringer's Lactate to try and maintain the circulating volume.
But you find that very quickly during preservation, you get electrolyte imbalances,
imbalances, and it's very difficult to maintain perfusion for long periods of time.
And so one of the things that she tested during her experiments with this idea of urinary circulation,
so you simply take the urine that the kidneys excreted made it back into the circuit to maintain homeostasis.
And that actually seems to work quite well.
So the graphs here, the black dots, the the kidneys without urinary circulation and then the red ones are the ones with urinary circulation,
and you can see that you can preserve a kidney without recirculation, just using me as lactate for perhaps up to six hours or so.
But then you start to get electrolyte imbalances and eventually the resistance increases.
The perfusion pressures decrease in the unable to maintain perfusion for longer periods of time.
With urine recirculation, you can get a nice, stable preservation for 24 hours or even longer.
So no Muslim fusion seems to take most of our boxes, we can prevent damage, we can repair damage.
We can potentially store for long periods of time up to and beyond 24 hours,
and we potentially have a means of viability assessment and enough time on the circuit to be able to deliver therapeutic interventions.
So where are we taking this next? Well, we've got funding from the NIH are free for a phase one clinical trial of this prototype device and the aims
of that to assess safety and feasibility of an MP for the first time in human kidneys for up to 24 hours.
At the same time, we'll collect samples to look for biomarkers for perfusion and predicting viability of dying of kidneys
and to look at the post-transplant outcomes of these kidneys will compare them to to historical controls.
So we're going to take 36 kidneys. We're going to perfused them in gradually increasing durations from six to 12 to 24 hours,
and there'll be a safety stop after each phase of the trial to look at the results and make sure that things are working as we expect.
And then each patient will be matched to historical controls from our from our own cohort to see whether there's any impact on clinical outcomes.
Primary outcomes, purely a safety outcome. 30 day graft survival.
And then we'll also look at graft function, long term graft survival and the incidence of delayed in primary function.
And then we'll also be taking samples looking at perfusion parameters and biomarkers as potential predictors of organ viability.
So I'm going to hand over to James now, he's going to talk about some of the more sort of lab and translational research.
Evan, thanks very much, Simon. So.
I'm going to talk a bit about the science side of what we've been doing in the department, and it seems like this might be a little bit back to front,
but they they kind of run in parallel and I'm going to go back over a little bit of background as well,
just so it fits in with with the science as well as the clinical. And so, I mean, as transplant surgeons, our objective is to transplant more organs.
We don't want to compromise patient's outcome and we want better graft survival, the EU on fewer kidneys that don't work.
And I've shown a graph on the left side which which, if you ignore the Green Line at the bottom,
shows the outcomes over five years of all deceased and the transplants.
And actually, if you look at the one year numbers, if you look here, you've got a round about 90 95 percent survival at one year.
So the survival rates are very good. They're not good enough. We want them better, but they're very good.
And in fact, they're so good that drug companies have almost stopped doing clinical trials
into immunosuppression because you can't really power a trial to go from 95,
96, 97 percent without, without many, many, many patients.
So we need to try and find better ways of improving these outcomes and say the way we've been doing this is.
So. Simon showed this graph and apologised for the repetition, but but the question is, is there a need to do this?
Well, as he explained, the donors are getting older and there's not much we can do about that.
And if you look about 280 patients last year died whilst waiting for either a kidney or a pancreas.
So this certainly is a need to do this. And where can we find the resource for this?
We're transplanting what we think is as many kidneys as we're offered, but actually we're not.
So this is consistent over 10 years, about 10 or 12 percent off of kidneys that are retrieved are not transplanted.
So of those we we think about, half of them are probably transplants.
So how do we go about assessing this and when we get this, what we think the problem here is what's in the box?
Okay, well, that's in the box. It's a kidney. And for those non transplant surgeons, it looks okay.
You see that kidney thing. That's great. It looks fine.
It's well flushed. Someone's very kindly bunched it for us. But what we don't know?
Once they transplant it, is it going to be this or is it going to be this?
Because you can't tell from just looking the kidney? And so that's where the normal foamy perfusion ideally comes into play.
I think Simon talks about Mike Nicholson's work, which has got some, which is.
Fine, if you just take some very simple measures. But there's nothing else in there that tells us about what's inside.
And so our aims in the department were both clinical and scientific.
The clinical the funding for the phase one trial has been achieved thereafter funding for a larger,
randomised trial to see whether this technology can be used and then a conditioning unit.
That's the goal. We have kidneys that come from around the country or from wherever they've not be
transplanted and we assess them and then we decide with their transplants well.
And if they are, we either transplant themselves or we send them elsewhere to be transplanted.
And in parallel with that, we will have got some scientific goals.
So using discarded human kidneys for research is challenging because they're a very heterogeneous group.
There's a huge variation in what you get an Anne-Marie's data, a fantastic.
Some of these kidneys are going round the houses and had very, very long periods of cold ischaemia.
And so we needed a better model to assess the science than discarding human kidneys because we wanted to look at things like mechanisms of action.
And we wanted to add novel therapies to see why that would take us.
And so if you think Mike's machine looks a bit Heath Robinson, then you can just take a look at ours.
And I've put this up because this was very much in the early days where we we've
got a sophisticated pump and and the technology that you can't see there,
that actually does the work.
But this is a device where you pump hot pump from centrifugal pump blood into the oxygen data up into the renal artery here.
And then it drains down the renal vein and then back around the circuit.
And we've modified that because if you just buy these disposables, they're two thousand euros each.
So if you imagine we go to our abattoir, we get a pig kidney, we get some pig blood,
which is essentially free, and then we need two thousand euros to do the experiment.
So that's completely unrealistic in any walk of research. And so by doing it this way, we can reuse components.
We can buy them separately.
We can do it in a way that we can actually get some information by pumping large numbers of kidneys without spending lots of money.
And the classic is the one that we started with.
And then this is what's the the open circuit because the vein is not granulated.
So the renal vein is draining directly into the chamber Philip blood rather than having it suspended in a as you saw on the previous.
And so one of our early questions which one's better does it matter what we use because the open circuit is much,
much easier to use in the cloud, so it's much less. And so we had a medical student who came in.
It's an experiment with us and and we just compare the two.
This was right at the bottom of our learning curve three four years ago when we first started.
And so it's not perfect, perfect experiments.
But what it sort of showed initially was there wasn't much difference in terms of perfusion characteristics on she.
The closed circuit seemed to produce more urine.
Now there's a theory that if you've got a candidate in the vein, it's a bit like having it attached to the IVC.
You've got some venous pressure, whereas if you've got the renal vein draining into it into a canister, there's no venous pressure at all.
So maybe you increase the pressure across the glomerulus and and you and you help, but that's possible.
But actually, when we looked at the kind of markers of injury in the other parameters, it wasn't consistent.
So the lactate was higher in the closed circuit, the HGH was in the open.
And so in the end, for the sake of ease, we went to the open circuit.
OK. But with these pig kidneys, we could not perfusion and beyond about eight hours.
The human kidneys seem to work. OK. Kidneys are much more sensitive.
They're much more visa spastic. And so we couldn't control the CO2 levels.
And in parallel with what we were doing, there was a Canadian group who just did this beautiful pig,
also transplant model where the kidneys were pumped for 16 hours transplanted and the results were fantastic.
So we thought, Oh, goodness me, what are they doing that we're not? Why can they do it?
And we can't. And so we just it made some small changes.
We change things like the vasodilator, OK.
And so this is three groups. It's we used to you say, do not to proceed because that's what my next news.
That's what others it used and it seemed to work.
Our colleagues in Groningen, I'm glad Brooke is not here because he would tell me, I'm not pronouncing it right.
Groningen in the Netherlands, they don't use any vasodilator.
And the Katz Group, they use Verapamil, so we compared the three, and what we found is there wasn't much difference.
Interestingly, even if you didn't use a visa to later at all.
The sodium nitric oxide kidneys look like they produce more urine.
But if you look at the error bars, it's essentially one kidney will just be an outlier there and produce lots of urea.
But what we found was striking. Because. The sodium nitrate precede kidneys, which is the ventilator we've been using all along,
got a profound lactic acid process and the price drops dramatically.
OK. And so you do a little bit more digging a bit more research,
and you find that one of the metabolic by-products of sodium patricide is a cyanide type compound,
which interferes with oxidative respiration and effectively stops the kidneys respiratory.
So we stop using that and we started using verapamil. And then we had a fantastic Italian nephrologist who came over and spent a year with us.
So this is the Italian influence, and we wanted to tinker a bit with the preservation solution we were using because as you heard,
Simon said, the Nixon Group used Ringer's lactate. Anne-Marie used albumin, human albumin and in the liver trial gel fusion was used.
And so it's there's so many questions that need answering, which is the best one, which is the right one to use.
And so what we did here is we we took the pig blood.
It came from the abattoir with the kidney and we split it into its component fractions.
And then we resuspended the red cells with a known amount of plasma, took out all the noise, get all the immune cells.
And and we're a little bit more meticulous about some of the sterility.
And if we filtered the blood and it was still white cells in it, we felt it again.
What we found is the perfusion got better and say, suddenly we got abattoir pig kidneys and we could perfused them for 18 hours,
which is what we've been wanting to achieve as a sort of model of what we wanted to compare with the human setting.
They produce urine. The perfusion was reasonably stable, they acknowledge.
At the end, if you look at the lactate does start increasing, but that's one or two kidneys. And so.
If you don't believe me, I thought I show you some of those pictures. So this is a kidney on the machine.
It looks shiny because it's covered in cling film, which again is a very cost effective way of keeping the kidney warm and moist.
And so at one hour, it's reasonably pink and perfused in 18 hours the same.
So you can pretty much tell when the kidneys dying because it starts looking black and blockchain horrible.
And that kidney looks okay. So it starts to achieve this objective of a research model of kidneys that was a cost effective way of doing.
And so we wanted to look a bit more into some of the mechanisms of action.
We know that mitochondria are crucial parties,
so we know there's hundreds of factors that are important in in all of this clotting cascade, et cetera.
But we wanted to look at the mitochondrial function in kidneys undergoing Nelson Fusion.
And so this is funded by Academy Medical Sciences grants, and we used Animal House pigs, not slaughterhouse pigs for this model.
Okay. And so pigs were anaesthetised at the laparotomy, and then they had one kidney removed and flushed, completely healthy.
It's a beautiful living donor kidney. This is the gold standard kidneys and uninjured kidney, and it's just flushed and put a nice temporarily.
The contralateral kidney is then clamped for 60 Minutes to give it a 60 minute warm ischaemic.
So it's pretty injured. You can see how injured it is. You compare that picture to the one before where the kidney look nice and pink and healthy.
So this is a pretty skinny kidney. And then on top of that, we want to put it on ice or on the pump for 24 hours.
So it has a period of warm ischaemic injury where it's got the oxygenated blood in it and it's got
a bit of cold ischaemic injury where it's sitting in a box of ice and then we fuse both of them.
Excuse me, for eight hours on the machine. OK, so what we found initially?
Well, if you compare the two groups and interestingly, that doesn't appear to be much difference.
So the Nicholson score tells you fellows important and we looked at this.
And so but it doesn't seem to matter, but all of this injured group.
Two kidneys didn't make it for eight hours. They made it to four or five hours and they were essentially dying.
It was a terminal model. The kidneys looked the purple holy perfused, and so the graft doesn't really give the game away.
So. So we split the grooves and we split them into injured kidneys that that appeared to recover healthy kidneys, which is the blue line.
And then the grey line at the bottom is those kidneys that didn't, did not profuse and did not survive and were not viable.
And the line starts to split.
So perhaps flow is important, but more crucially, when we start looking at measures that aren't just perfusion parameters like oxygen consumption,
you might think it's logical and obvious, but actually we haven't seen this before.
The oxygen consumption drops dramatically. They stop consuming oxygen in these kidneys and start dying and their lactate levels go up.
So we start to find objective measures of viability during an MP other than just blood flow.
And the other interesting thing is, despite the urine output of these injured kidneys, they're not functioning OK, so they're not clearing creatinine.
If you look at the orange line, no ground clearance to the healthy kidneys as you would expect pretty consistent creatinine clearance.
Same with the urine album creatinine ratio. So what that shows is is leaky glomerular due to injury, just like albumin into the urine.
So the healthy kidneys don't do that and the injured ones do. So measuring function is not very helpful because they're not functioning.
It doesn't tell you whether they're viable or not. It just tells you that they're not working. It doesn't tell you whether they'll recover.
So we looked at the mitochondria. And say the healthy kidneys, the graph on the left,
the mitochondrial respiration is pretty stable and the way you do this just out of interest is if anyone is particularly interested in this,
you take the kidney biopsies at different time points, you homogenised the tissue and then you extract the mitochondria fresh.
So you have to do this at the time of the experiment. You can't just sort of put it in the freezer and say, maybe we'll do this next month.
So it's quite time and resource intensive. And once you've got these mitochondria extracted,
you put them in a chamber clerk's electrode and you pass oxygen into it and then you measure
the current across the chamber and it tells you how much oxygen they are using at that time.
And so it's a measure of mitochondrial respiration and measure of mitochondrial function.
So it's probably the most accurate way of assessing how these mitochondria function. And so the healthy ones just sort of keep functioning.
The injured ones, although it's only small numbers,
there is a suggestion in these mitochondria that managed to get eight hours that they start recovering.
So the graphs, you have to believe me with you, I have faith starts going up,
but we acknowledge this is small numbers and we acknowledge it's a suggestion rather than any significance.
So the other thing you notice is that the levels are a bit lower.
So if you look at the the y axis, we've got two just below 20 and you've got around about 50 here, which sort of fits with an injured kidney.
And then we we split the grass again.
To see whether the injured kidneys that didn't perform as well were different to those that did, and apart from perhaps this anomalous reading here,
the injured kidneys that don't make it sort of the mitochondria appear not to function,
as well as the ones that either healthy or the skinny ones that recover.
So we had some suggestion that mitochondria function is important during this, which again, it makes sense, doesn't it?
You need mitochondria, but this this was work that hadn't been done before.
And so then I'll just touch briefly on some of the science that Anne-Marie did.
So this is one of the human kidneys that was discarded on the left.
And you can see the picture doesn't come out well, but it's quite patchy here, and this kidney would have been turned down for the characteristics.
So a transplant surgeon somewhere would have looked in Sydney,
and that's not well fused or transplanted, actually when you put it on the machine and it's OK.
Sorry about the mug thing. And so we wanted to look at whether you can demonstrate any regeneration or repair during perfusion.
Hepatocyte growth factor is a mark of this. That's produced is a protein produced by many different cells, not just the liver,
which is involved in tissue regeneration, tissue repair and so across perfusion.
You can see the levels increase. So the kidney appears to be producing levels of hepatocyte growth factor.
It pays to be able to produce proteins, which demonstrates its functioning to some degree.
And this is a clever scatterplot. This is work that others did.
It's on kidney tissue from Anne-Marie's experiments, and it essentially shows the change in protein expression across the perfusion.
So the left lt1 one is a scatterplot, and it shows the difference in upregulated proteins and downregulated proteins throughout the perfusion.
Each dots the different protein. The heat map on the left is just a different way of demonstrating this.
So it goes from zero, 14, 24 hours and each line is a protein.
And red doesn't mean good in green, so red doesn't mean bad in green means good.
It just tells you whether it's been upregulated or downregulated. And again, that doesn't mean it's good or bad.
It might be a heat shock protein. It might be a marker of inflammation.
It may be a repair protein. It just tells you that there's a change.
And so what we see during perfusion is we see a change in the protein profile of the kidney, which again, that's fascinating to know.
This helps you with determining pathway analysis, and it helps you with hypothesis generation.
You look at these proteins think, where do we look next? How can we find out what's going on?
So this is crucial. What? And then we decided to have a bit of fun and add some therapies to it.
He got a kidney in a machine. You can do what you like, you can add whatever you like to.
It's a fantastic model for testing things on. So I'm not going to steal and Caitlin Rosenberg's thunder because she did a lot of this work for her,
her defence and hasn't presented that work yet, but that we've got a collaboration with her.
The MEAP collaboration, which is between a group in our house in Denmark and Rotterdam in the Netherlands,
where we produce pig derived ADP's drive pig stem cells.
And we've been doing the normal thermic perfusion side of that.
So the initial bit was a dose testing.
So you take two million, 10 million and 50 million stem cells and you give them to a kidney during machine perfusion.
And the questions were very simple. Where do they go? Can we find them?
These questions are relatively unanswered. Do they have any effect?
If so, what effect? So we weren't trying to be too clever. We just wanted to see if we could answer some simple questions.
As part of this, there's a big auto transplant model. The results are currently being analysed.
I'm not going to talk about that today, but that's a part of the sort of bigger picture with this project.
And so these graphs just show the the groups of control two million, 10 million and 50 million stem cells.
And so essentially what it showed was it didn't really matter how many stem cells you you give them.
It doesn't seem to change what happens during perfusion. So the perfusion characteristics were the same.
The injury markers were the same. The haemolysis, the likes it, they were all essentially the same.
There wasn't any difference in anything that you measured. OK?
The perfusion tended to dip a bit when you gave lots and lots of stem cells, but it seemed to recover.
And so you could. Is that positive or negative? Well, it means you can give lots of stem cells.
It doesn't appear to clog up the capillary system. It doesn't appear to have an effect on perfusion.
So that may be positive because it means you can give lots of stem cells if they work or also means you don't have to give lots of stem cell.
But we don't really know what they do. And so we have done some work looking at whether it improves cytokine release and growth factors.
Haven't got time to go into all of that because I want to show you some pretty pictures because that's more interesting.
So we've we took some biopsies from these kidneys to see if we could find the stem cells.
And these are labelled these stem cells with something called Q dots.
And the slightly perverse side of this is if you and what you do is you take this big population of stem cells,
which have been expanded in a very clever way and you add kudos to them.
And then that affects their viability and they probably don't work. But if you don't add kudos, you can't find that you've got no idea why they go.
So for this experiment, we added these because we wanted to see where they go.
So it's probably not fair assessing how well they work and trying to find where they go in the same experiment.
But this is a slide of a kidney, and this was the two million stem cells group, and you can see one can see one get.
So we found one, which was which was I mean, that was fantastic. We found stem cells could well then us.
Now, one kudo doesn't mean one stem cell because some of the stem cells take up lots and lots and lots of kudos.
And then if the stem cell dies, it releases the kudos. So actually, it tells you that there's probably a stem cell there,
but there's definitely acute, OK, they're on the stem cell has been there or something.
But then if you go on and look at the 10 million, there's more some hair.
Some air. Down there and there in the climate, realists, interestingly, that's where they all appear to be.
And then you look at the 50 million and there's more still. So it's not quantified, but there appears to be a dose response.
So the more stem cells you give, the more you can find. And again, that's interesting.
That's useful. That wasn't this has not been been done before.
Again, they appear to be in the climate. That's probably because they get stuck in the capillaries.
We don't know for sure,
but they go into the blood supply and then they probably get stuck in the end of the because they're bigger than the calibre of the capillary.
Makes sense. And so said, this work will lead on to more, there's lots more facets to this,
but this is just to give you an idea of some of the things we're doing and where we're taking the preservation.
And so, so overall, in our kind of progression towards both these clinical and scientific goals,
Simons told you the trials have been completed, the hope them coach native fusion.
The funding for the phase one trial has been complete and we started to work on and produce a rapid,
reproducible model of kidney perfusion using abattoir pig kidneys, which is cost effective and can be used reused consumables.
We've got some promising data on mitochondrial recovery.
We've got lots to do on M.S. therapy and we got plenty of work ongoing and we've we've just got a little grant to do to look at a fusion solution.
We've got some work on MRI imaging and giving those to the kidney.
So there's lots and lots of work to be done here. So thank you very much for listening.
I hope that's been interesting. We've got lots of collaborators, lots of people to thank.
I hope I haven't missed anybody. If I have, I'm sure they're not here.
Happy to take any questions.