Slide 1

Dear Chairman thank you very much. I would also like to thank the organisers and the EDTA for inviting me here to talk on a very hard topic; tissue protection in ischemia particularly in ischemia due to acute kidney injury.

Slide 2

Well, I will also start with a cartoon you’ve already seen and this is the pathogenesis of acute kidney injury and this was very nicely summarised by Doctor Moneteris and what we have learnt in the last years is that part of the problem with acute kidney injury is also ischemia/reperfusion injury and you already have seen all these steps starting by endothelial activation/injury, inflammation going to apoptosis also of endothelial cells and microvascular rarefaction.

Slide 3

Which is probably the problem here followed by reduced blood flow and then tissue hypoxia/ischemia.

Slide 4

There is proof for that and you already have seen some slides and I will show also some other slides by the group of Doctor Goligorsky who is actually sitting here in front of me and what they have shown is clearly if you make an ischemia/reperfusion injury, this is a model in rats and here is the clamp or the ischemic time and after that the reperfusion time starting at minute 45, you clearly see in comparison to control that there is a stop of flow in some of these capillaries which completely vanished.

Slide 5

They have shown it in this experiment. This is the normal flow in the kidney, then comes the ischemia or a clamp for 45 minutes in this model and then comes reperfusion and what you see is after a short brief increase of blood flow to the kidney there is a drop and the drop is not coming up to the normal blood flow until more than or several hours

Slide 6

and what you also see is that there are some capillaries in the kidney which are completely shut down so there is no perfusion in such a kidney which was prone to develop acute kidney injury.

Slide 7

Besides the direct effect of acute kidney injury on the endothelial cells, on the vasculature, it could also be that by triggering tissue hypoxia and ischemia and injuring also tubular cells these injured tubular cells could contribute to further damage because these tubular cells are capable of secreting angiogenic factors and it has been shown that a reduced production of such angiogenic factors may further contribute to apoptosis in this crosstalk of cells to acute kidney injury.

Slide 8

This is a very nice experiment done 6 years ago or published 6 years ago where you see here proximal tubular epithelial cells and glomerular epithelial cells and they have been co-cultured with HUVECs. This is an endothelial cell line and what you see here is hypoxic stimuli and if you apply antibodies to VEGF, which is a pro-angiogenic factor, you don’t have you see here vascular proliferation it is completely vanished, if you co-culture these HUVECs with proximal tubular epithelial cells. So this shows us that tubular epithelial cells are capable of producing also VEGF and this VEGF is important in the crosstalk with endothelial cells and maybe also in the regeneration.

Slide 9

Another experiment with similar results where the colleagues have applied VEGF antibodies or soluble VEGF receptor and what they have shown is that after applying both of these drugs there is a very rapid disintegration of the endothelial cell layer and the glomerular basement membrane showing how important it is this pro-angiogenic factor for the kidney.We know from tumour therapy with VEGF antibodies that you can induce proteinuria and you can induce renal failure in such patients and this is the experiment done in mice.

Slide 10

What you also see is that if you give these VEGF antibodies in this experiment model, there is disintegration of the glomerulus.

Slide 11

So taken together there is a concert of injury to the endothelium and the tubular cells in acute kidney injury and the question arises, can this be repaired or recovered? Here we are at this level of treatment of the tissue ischemia in acute kidney injury

Slide 12

and I have again to show a slide from the group of Goligorsky and what you see here is a silver staining of the glomerulus and the afferent arterioles.

Slide 13

After clamping and reperfusion you get loss of endothelial cells, you get a denudation of these vessels and you get a complete loss of flow in these vessels.

Slide 14

What they did after that is they transplanted endothelial cells, actually also HUVECs and could show these are labelled HUVECs that there is incorporation of HUVECs in the vessels of the kidney after the injury

Slide 15

and there is again some kind of flow in these kidneys and this also ameliorates the acute kidney injury in these animals.

Slide 16

At this point I have to say that HUVECs, we are using them also for experiments, is a quiet diverse group of cells. This is not one cell population and there are some groups from England and others and we have also shown that there are even some regenerative cells, probably such cells like endothelial progenitor cells, in these HUVECs so here we are already at a point of cell therapy of acute kidney injury. However, so far there are almost no experiments published that you can indeed regenerate destroyed endothelial or destroyed vasculature after acute kidney injury and we are also not able in several animal models to find such regeneration of vasculature. Here what I show is a very recent experiment published two months ago also by a group from the United States which made a very complicated animal model or two models. They did genetic engineering of these mice and they could label in these mice tubular cells with two kinds of dyes and what they observed after ischemia/reperfusion injury that there is a huge proliferation of tubular cells. We have known that for several years or decades but what they also have shown is that all regeneration is from cells that survive the injury and there are almost no cells coming from outside regenerating. So this is a very unusual finding for those who are working in the field of tissue regeneration but this has been now very recently published and I think we have to think over how indeed if there is regeneration of kidneys, how it happens. Some say that there are several factors secreted maybe by these cells which can do tissue regeneration.

Slide 17

Now I’m coming to factors which we can use or to substances which we could use in prevention of ischemia/reperfusion injury also in humans. I have to apologise, this is a very busy slide there are 3 of them and I have to point out 3 things. First these are all studies published in the last 2 years 2006, 2007 on the topic of acute kidney injury and several substances were here used.

Slide 18

Second almost exclusively the model of ischemia/reperfusion injury was used which is not quite what we see in our patients in the intensive care unit and third almost all these substances were positive.

Slide 19

I will go on now to the next slide.

Slide 20

A lot of substances, more than 50 in the last two years used and if I talked of each of these substances, I probably would be here until tomorrow morning. So I take the liberty and I will talk only on two, your very well known substances. One is erythropoietin and the second one is statins which have something to do with tissue protection and protection from ischemia.

Slide 21

I will review in brief some of these studies and we could use statins because we know they modulate the activity of the endothelium and the inflammation at the endothelium and Doctor Gueler from Hanover from my former affiliation showed in a very nice experiment that if you use here, it was cerivastatin then you can reduce ischemia/reperfusion injury in mice. What we have observed is that if you add an HO-1 inhibitor, a hemoxinase inhibitor, you can reverse all the positive effects of statins.

Slide 22

You can see it also here in this slide. This is with statin therapy and this is with the co-treatment, with the HO-1 inhibitor and interestingly, we could localise the cells expressing more HO-1 in the ischemic kidney.

Slide 23

You see it here, there was a co-localisation with the vessels in the kidney but we were surprised because all these cells were in fact, invading monocytes.

Slide 24

You can see here an experiment we have done in vitro and also in vivo showing that this HO-1 upregulation is mostly invading monocytes and it is reproducible with cerivastatin, atorvastatin and rosuvastatin showing you how complicated is this matter of cell-cell contact because it seems that even invading cells are not negative, they can also be positive.

Slide 25

Going to the second substance that is erythropoietin which is nearby VEGF also an angiogenic factor, as we have recently learnt, which could be involved in some of these steps of protection of the kidney in acute kidney injury.

Slide 26

You all know erythropoietin and you know in this cartoon it’s shown on the left hand side, it’s used for anaemia correction in our patients, for erythrocyte differentiation but what is known from recent studies that it also can be used for tissue protection because it is a very strong vasoactive or pro-angiogenic hormone. It works through its receptors on endothelial cells, also renal tissue by inhibiting apoptosis, stimulating eNOS and stimulating angiogenesis.

Slide 27

So the protection with erythropoietin could be on both sides not only on the vascular system but also on other renal tissues and I will briefly review some of the studies.

Slide 28

Again, a very busy slide with all the studies using erythropoietin for tissue protection in models of acute kidney injury like ischemia/reperfusion injury but also haemorrhagic shock or cisplatin toxicity.

Slide 29

One of the first studies was this year using 300 units of erythropoietin/kg/body weight in rats

Slide 30

and what the authors have shown is this study from the UK that you can reduce the injury to the kidney to prevent the injury to the kidney and also the tissue injury score was reduced by administrating erythropoietin

Slide 31

and they also could clearly show in the tissue that the AKT pathway was activated by erythropoietin.

Slide 32

This was known from previous studies with erythrocytes but here it was shown in kidney tissue.

Slide 33

A model which is nearer to the human situation is this one hemorrhagic shock and here too the authors used rats induced ischemia reperfusion injury and injected erythropoietin

Slide 34

and they could also show that erythropoietin was preventive in this experimental setting.

Slide 35

The last thing I want to show you is two of our studies. In mice we have used here a transplantation model of ischemia/reperfusion and injury and also administered erythropoietin and could also observe reduced apoptosis and increased HO-1 expression.

Slide 36

This was the basis for a human study which is quite advanced already and what we have done here is the study design it was designed as a prospective randomised double blinded placebo controlled study in CKD5 patients undergoing kidney transplantation, 90 patients and they received 3.000 units before erythropoietin within the first hour of transplantation after 3 and after 7 days and the primary study end point was serum creatinine and cystatin C or the need of dialysis after 42 days after transplantation.

Slide 37

These are the first safety data. We were forced by the Ethics Committee to present First Safety Data after 30 patients and what we have observed there was no adverse effect of erythropoietin. Also we applied 120.000 units and what you can see here is the first data on serum creatinine and serum cystatin C and what we see here is clearly that administering erythropoietin we have some benefit. This is not statistically significant we have to wait for the next 60 patients and we will have in about 1 month the second safety data in 60 patients and also the data on creatinine and cystatin C.

Slide 38

Taken together you see there are many substances that could be used also in the human situation for prevention of acute kidney injury we have now used erythropoietin and I hope I can present in about 1 year time more data on preventing acute kidney injury at least in the setting of kidney transplantation.

Slide 39

Thank you very much for your attention.