Slide 1

Thank you very much. I would like to thank the organising committee for this invitation to this exciting meeting and to this beautiful city.

Slide 2

I will start with this slide which shows you what we believe today about the mechanisms that participate in the progression of renal disease. In my topic today I will try to convince you that angiotensin II is a major target, if we want to regress renal fibrosis but alone is not sufficient.

Slide 3

So for people like us that were working with experimental models, we try to have models that are mimicking or that are close to renal diseases. One of those models that has been used a lot in the literature in the past 15 years is the inhibition of endogenous nitric oxide synthesis which is mainly a model of hypertension induced nephroangiosclerosis, as you can see here from this biopsy of a rat.

Slide 4

So, we used this model and we were among the first groups that reported a real regression after blockade of renin-angiotensin II. So the protocol was to allow the animals to be without nitric oxide for a period that they have developed renal lesions and in this point (note: L-NAME 4 wk in the slide) to start the treatment with angiotensin II antagonists and to compare these values (note: L-NAME+AT1 at 1wk and at 4wk) with the animals that remain untreated.

Slide 5

This is (note: Control in upper left) how it looks a control animal in the beginning, before the inhibition of nitric oxide synthesis. This is (note: L-NAME 4wk, upper second from the left) how the animals were after 4 weeks of inhibition of nitric oxide and you see (note: lower, red columns) how much the proteinuria, the plasma creatinine and the sclerotic index of these lesions were increased. So in that moment, when we started to treat the animals with angiotensin receptor antagonist losartan, this is what happened (note: L-NAME+Los 1w upper, third from the right). One week later you see (note: lower, blue columns) that the proteinuria was normalised, creatinaemia did the same thing, sclerotic index not yet normalised, but it is decreased, and it is difficult to believe that 4 weeks later this animal was like this (note: comparison of the figures up, 2nd from the left with 4th from the left L-NAME+Los 4w). You cannot dissociate the function, the structure of this animal (note: lower panel, green columns) from the control and is difficult to believe that 4 weeks ago that animals were like this (note: comparison with red columns). There was an impressive and very strong regression that happened to these animals. So, the question was that to understand if that was due to the known anti-hypertensive action of angiotensin II blockers or if it was due to another action of angiotensin II blockade.

Slide 6

And this slide says that yes in addition to reducing blood pressure with losartan, the blockade of angiotensin II reduces also the synthesis of collagen I and collagen IV. So the blockade of angiotensin II inhibited directly and clearly the synthesis of collagens.

Slide 7

Another question that we asked in this paper was to find out this regression was due just to the role of angiotensin II antagonist or if other systems such as metalloproteinases that degrade the extracellular matrix can participate. The answer was yes and in this slide you can see the activation of metalloproteinases in glomeruli. In the beginning there is no activation of this system of metalloproteinases inside the glomerulus (note: the circular form shown by the yellow arrow in left Control figure). After 4 weeks there is one (note: the white staining shown by yellow arrows in upper L-NAME 4 wk figure)  that stays with time (note: the white staining shown by yellow arrows in upper right L-NAME 8 wk figure)  but the important slide is that one (note: lower left LN+Los 1 wk figure). It is one week after the inhibition of angiotensin II. The moment that the collagen synthesis is decreased -is inhibited by the blockade of angiotensin II action - the metalloproteinase action is still active and at that moment if you remember from the slide before there is still abnormal formation of extracellular matrix. This activation disappears only when the structure is normalised again. So we believe that metalloproteinases also have contributed to the regression by degrading the excessive extracellular matrix. So, it was a combined mechanism: angiotensin II inhibition and the inhibition of angiotensin II procollagenic effect, and the degradation induced by metalloproteinases.  

Slide 8

That protetction was only specific and particular of our model? Because an objection can be that this has just happened to L-NAME treated rats. The answer is no and I'll pick up a paper published a few months after ours in which the authors -from the group of Doctor Ritz - did 5/6 nephrectomy and what happened is they left 8 weeks the animals after the 5/6 nephrectomy and at that moment they left some animals without treatment and some animals were treated with an angiotensin II blocker. As you can see the animals untreated progressed to renal disease but animals treated with a blocker of angiotensin II showed a clear regression because they have a better index of glomerulosclerosis compared to the beginning.

So even in the case that there is a huge reduction of renal tissue the 5/6 nephrectomy, even in the case that podocytes are very few, if you treat the animals with angiotensin II blockers, you get a real regression.

Slide 9

Here is a list of several papers now that exist in the literature and - this is not an exclusive list, it is a representative list - and you can see that now independent of the cause, the causes can be several, they can be from a physiological condition as aging to any kind of model that is actually used in the literature (note: look to the first column) and this is in rodents, mice and rats (note: look to the third column) and in this list (note: look to the second column) you can see that the main therapeutical treatment is the blockade of angiotensin II, of the renin-angiotensin system. So it is clear that when we block the renin-angiotensin system in almost any kind of model in animals, we can get a real regression.

Slide 10

Now, what about humans? The answer to this question is “yes and no” about the role of renin-angiotensin blockade. Yes, because if we look to some patients, this is 16 and this is 10, in a limited number of patients and these are data from a talk that Doctor Remuzzi gave - and it is on line in the ERA-EDTA site- and you can see that it was able to see a remission in some patients, and even better a regression in some others. But again the numbers are 16 and 10.

Slide 11

If we move to a more important number of patients this hope is somehow blunted. Of course, there is a difference between the losartan treated patients with the placebo but still these patients evolve to end-stage renal disease. So, when we move to large populations of humans, there is a break to the rate of decline of the renal failure but it has nothing to do with this spectacular regression that we see in animals.

Slide 12

The reason is to confront the experimental and the clinical data and try to understand why this is happening. So there are three major questions They are why among all the factors and systems that I have shown you in the first slide the renin-angiotensin system appears to be a major one? How can we explain the difference between animal and human studies regarding the efficiency of the renin-angiotensin system blockade? How finally can we improve the beneficial action of angiotensin II?

Slide 13

So starting from the first. It is clear and became clear when the angiotensin II blockers appeared on the market and there was an explosion in the studies of this system that the renin-angiotensin system is no longer a system that controls blood pressure and sodium. This is the major role of this system but this is not the only one. In addition to this role it appears that it is involved in several other actions.

Slide 14

In that slide I will show you what we have done concerning the ways that angiotensin II can transactivate and activate other systems, especially growth factor receptors - that are shown in this slide- and how this can go and activate collagen I synthesis.
So on the left, there is an action of angiotensin II that activates PDGF and EGF receptors, then there is the cascade of MAP kinase that is activated, and in the bottom line that gives an activation of collagen I gene. On the right, there is another kind of activation of angiotensin II. This is through TGF-beta and by activating the TGF-beta receptor and the SMAD pathway of TGF-beta there is the profibrotic effect of TGF-beta. In the middle it is an activation of angiotensin II to produce more endothelin which is a vasoconstrictor peptide displaying also profibrotic properties. So, one of the reasons that the blockade of the renin-angiotensin system is so efficient in the animals is the multitude of actions of angiotensin II.

Slide 15

Now, how can we explain this difference between animal and human studies? This spectacular recovery in animal studies, but not such good results in human studies? This is a matter of debate actually, and I have put here three reasons that I believe are the major ones.

The first one is the age of specimens. Usually, we work in experimental models with young animals and their tissues have not been exposed to hypertension, to diabetes, to obesity for years. So it is possible that it is much better to decrease the abnormal collagen in young animals, in young tissues, rather than in the old ones that is the case with humans.

A second issue is the kinetics. Renal progression and fibrosis is a long-term pathology and animals, especially rodents, live a much shorter life, and all these experiments that investigated progression and regression were performed in 4 weeks, 8 weeks. It is possible that the kinetics of the collagen and the stability of the collagen depend a lot on the time.

The third reason, in addition to these two, is that the fibrosis and the renal failure in humans can be a much more complex pathology than in animals and I will take here the paradigm of cancer. You know very well -all of you I believe- that several therapies are very spectacular in mice. We can take out easily the tumour for a mouse, but all of these treatments did not prove the same efficiency when they have been moved to humans. So it is possible that this also can be a reason.

All these reasons together make clear that in addition to what is known now about therapy with renin-angiotensin blockers we need additional targets.

Slide 16

What can these targets for therapy be? Among the several proposed, I have put here four that I believe are the most promising. I will not talk about the first one concerning endothelial function because that topic was brilliantly covered by the previous speaker.

Slide 17

I will give you an example or two examples of growth factor activation.

Slide 18

Concerning the PDGF receptor, it was proven by the group of Doctor Floege in Aachen that by blocking this receptor you can prevent renal disease. To remind you that this is the left part of the previous this is a transactivation by angiotensin II of EGF and PDGF receptor.

Slide 19

The group of Fabiola Terzi in Necker hospital in Paris used an animal, a mouse that was mutant to the EGF receptor specifically in the renal tissue and that receptor was not working so well. They induced nephropathy, the model of angiotensin II - induced hypertension and nephropathy in this model and the result of wild type mice is in the middle one (note: middle histology figures from left to right). They have developed -it is classically seen with angiotensin II- nephroangiosclerosis, inflammation and renal fibrosis. When they performed the same protocol and this is the third line (note: lower histology figures from left to right) in these mutant mice, there was clearly a protection in all the indexes of renal disease and renal structure that they measured.

Slide 20

So, the lack of activation of these receptors in the kidney it was a protective effect in the disease induced by angiotensin II. This was a genetic inhibition and we in the lab we used the pharmacological inhibition. We treated the animals with L-NAME and in a preventive way we treated the animals with an inhibitor of EGFR activation that is used also in humans to treat cancer. As you can see in the bottom left side, here this is the structure of the animals treated with this inhibitor, it was a clear protection/prevention of the disease. Now, in these two protocols the EGFR inactivation was in a preventive way and the objection that someone can have in the protocol of therapy is how long can we use a blocker of a growth factor receptor in chronic disease like renal disease?

This is a matter of debate. We have to test the efficiency of these drugs in a therapeutical approach not in a preventive way and maybe, this is my personal opinion, maybe we have to limit their use to a specific time cost and not to have a long treatment with these inhibitors.

Slide 21

Regarding antagonism of TGFbeta,

Slide 22

We move to the right part of that slide. What about TGF-beta and the agents that can block the action of TGF-beta?

Slide 23

It was published by the group of Doctor Calluri in Boston that in a model of nephrotoxic serum nephritis the exogenous administration of BMP-7 after the development of the disease - it was in this period (note: red and green lines in graph b)-was able to decrease the mortality rate, to improve the renal function and clearly to reverse, as you can see here (note: right middle and lower histology figures), the renal fibrosis. Again this is a very promising way, it is a very promising approach. The objection that someone can have in this approach, is how long again can we treat with BMP-7? We do not have to forget that it is a bone morphogenic protein, so we do not know what side effects this drug can have in the long-term.

Slide 24

Finally, I will finish my talk with what I believe is the most promising maybe one that can be used almost immediately it is the inhibition of the inflammatory process.

Slide 25

These are data, recent data from our lab. We were interested in the receptor of collagens that is called discoidin domain receptor 1. This receptor is a particular one, because it is like a growth factor receptor once it is activated. It is dimerized and then, there is a phosphorylation and an activation of MAP kinase pathway as was shown in vitro studies.

Slide 26

When we took animals that were lacking the expression of this receptor, and we applied to these animals the model of angiotensin II-induced nephroangiosclerosis, this is the picture of the wild type animals (note: middle histology panel), and these again are the classical lesions that you can see with angiotensin II characterised by a huge inflammation, perivascular, periglomerular, by fibrosis, by tubular atrophy, and this is the picture that we got with DDR1 in the null mice (note: right histology panel). You can see that this animal is clearly protected against inflammation and against fibrosis.

Slide 27

This is when we quantify these indexes of structure. You see that the sclerotic glomeruli were much less in this animals that the perivascular infiltrate was almost normal and while the wild type progressed with time, the knockout animals remained a little bit proteinuric, but stable.

Slide 28

In this case we believe that the angiotensin II activates this receptor. By activating this receptor, there is the MAP kinase pathway activation which leads to inflammatory cytokine excretion and inflammation which in turn activates collagen which binds to this receptor and we have a circle here that is a positive feedback and keeps the fibrosis going. Once this activation happens, this system does not need angiotensin anymore. It is a self-sustained system.

Slide 29

So, to conclude and to answer the question that is the title of this topic: “Can we reverse renal fibrosis?” The answer is definitely yes when we use angiotensin II inhibitors, but we have to fulfil one condition: to be a rodent.

If we move to humans then the story is a little bit different. The angiotensin II, yes certainly is a central player of the development of renal disease and this is due to multiple actions that angiotensin II has in addition to classical prohypertensive and sodium handling. It participates almost to everything that has to do with vascular disease, and I believe that everyday we are going to discover more and more actions of angiotensin.

It is certainly the central player, certainly the blockade of the action in humans is necessary. But, also it is clear from the accumulation of the clinical data that it is not sufficient. For this reason it is necessary to continue to identify additional targets, additional treatments, and we can do this by moving in different directions. One is for instance, to do models that are even closer to human disease, to do a better molecular refined analysis, and of course, to use urinary proteomics.

Slide 30

In this slide is the summary of what we know today about the progression and the study of profibrotic systems (note: shown in red) and this is also a list that is always increasing. In the blue part here, you see what has been proven to be efficient in animal studies, I insist in animal studies. This (note: shown in yellow) is to my knowledge what the different labs are investigating to find out whether we can regress (not prevent). In the prevention all these systems are very efficient. The question now is how can we do regression?

Slide 31

I will finish by thanking all of my collaborators of all these years. Jean Jacques Boffa who was the initiator of the regression study, Sandrine Placier who was important for her very strong technical support and especially Jean Claude Dussaule and Pierre Ronco that have been my friends and close collaborators for all these last 15 years. Thank you for your attention.

Slide 32

Chairman: Thank you very much for this very interesting talk which is open for discussion, comments from the floor. So I have a question for you. Just in the last slide you presented the possibility that angiotensin II may over stimulate some genes and it is necessary to use macroarrays, so I would like to know if you have preliminary data concerning this situation of down or up regulation of genes after angiotensin II stimulation?

Dr Chatziantoniou: Yes, we have some data that are ongoing and we have compared animals that progress with animals that regress and we have found some interesting differences, mainly the pathway of TGF-beta and BMPs not the usual suspects -that was also a surprise- it was some members of this family that are not supposed to be in the kidney. We are looking to confirm these data and it is preliminary to advanced more, but it looks like that pathway -the TGF-beta/BMP pathway- is important in the regulation of progression and regression.

Chairman: Thank you Christos.

Question: I have got one very short comment. The difference between humans and animals and positive experiments in the regression of animals and the negative in the everyday clinical practice in humans is in the dose of use of ACE inhibitors or angiotensin receptor blockers. For example, in our experiment in a 5, 6 nephrectomy model we used the dose of enalapril corresponding to 3 g/day in humans. So the rationale for such a high dose is that we believe that such a high dose is necessary for blockade of the renin-angiotensin system inside the kidney in the renin-angiotensin system. So in the future maybe new drugs which can be new potent than these currently available is the solution of the regression.

Dr Chatziantoniou: This is an interesting point because there is controversy about this. Yes, I agree that maybe it is a possibility to even more efficiently block the renin-angiotensin system. For instance, Aliskeren that is going to come out by Novartis actually in the United States, maybe can provide the answer to this, but people that work on the renin-angiotensin system -and I will note Doctor Corvol who is very well known expert- they don’t believe it. They believe that blocking the renin-angiotensin system completely would be lethal for humans and they believe so, because there was a paper in Natural Medicine last year where they found a mutation in humans that if the renin-angiotensin system is not working there is a lethality perinatally (before birth or after birth). It is clear to the humans that the mutation that does not allow the activation of the renin-angiotensin system is lethal. So maybe it is a question of even better blocking the renin-angiotensin, but still I don’t think that could resolve all the problems. The reason is -this is my personal belief- that after a while when we have all these lesions in the kidney, when you have all these hemodynamic changes inside the kidney, the kidney does not need anymore angiotensin II, to keep this disease going. The matrix is there, the kidney cannot work, it is no longer a question of producing collagen. We have to help the kidney to take out the excessive matrix. That is why we have to attack the other systems that are around and that keep this disease to be self-sufficient.

Question: Thank you very much for the important lesson that man is a good model for rats. On a more serious tone, I would like to ask you what happened to BMP-7? You went very rapidly over BMP-7; there were very nice, promising results some years ago. How about the application of this protein to human beings?

Dr Chatziantoniou: The problem of BMP-7 in that part was that it was exogenous administration of BMP-7. It was huge amounts of BMP-7. It was also peritoneal injection every day. I doubt that this can ever happen in a human and because it is bone morphogenic, because it gives calcifications, I believe that with time, a molecule like this will have side effects that are going to be worse than the protection. The question is “can we find a molecule that mimics”? Because BMP-7 has two actions. One is anti-fibrotic, the other is pro or bone morphogenic. So, a drug that mimics only the anti-fibrotic effect without having the part that is the secondary bad effect would be fine. I know -but I don’t have experience of this, I don’t have the drug- that they are working to produce this drug. The other question that is interesting about this system is that all the studies that created a benefit by doing BMP-7 always were exogenous. We don’t know well the endogenous regulation of BMPs in general. We don’t know whether really this system is activated or not, and our preliminary data that I do not want to advance anymore says that yes, it is a member of this family, but it is not the BMP-7 that is involved in the kidney.

Chairman: Ok thank you before closing, please one more.

Question: Excuse me a question. In summary you just said there is the possibility to reverse sclerosis and reverse renal function. Can you speculate how these glomeruli are detected get reconstructed? Obviously you have to repopulate cells and is there any idea from where these cells are coming from?

Dr Chatziantoniou: It is a difficult, you say, to repopulate but the data that I showed you with 5/6, nephrectomy -it is clearly there that we have only one sixth of glomeruli. The podocytes are always hypertrophic, but still if you get a regression with angiotensin II. So even if they are 1/6 present of their number still, you can get improvement. Now, there are so many cells that are involved in the reconstruction of the kidney, and all of the results, as you saw from the first talk and in part of the second talk on the regeneration, they come from the acute renal failure. The chronic renal failure is a different story I think, that we are going to advance in the next years to come. But, we need time and maybe better models to better investigate how this can happen. If you want, from all the cells that we know in the kidney, they can all be regenerated except the podocytes but, this is a dogma: Who knows what we will find in a few years? Maybe it is not the failure of the condition of just one cell, it is maybe not just one cell the limited facto,r but the combination of not having endothelial cells working well with smooth muscles cells they have lesions, with tubular cells that they are apoptotic that provides the renal failure, and if we treat these different cells at the same time, maybe we will get regeneration.

Chairman: Thank you Christos.