Slide 1

Thank you very much. Thank you also all for coming. As you know this session is organised by the EUTox group which is involved in research on uremic toxins and which is a European group. Without further a due we go ahead with the presentation and I will talk about new data on uremic toxicity apart from the topics which will be dealt with by Ziad and Alessandra which are mostly related to homocysteine, to calcium-phosphorous metabolism.

Slide 2

So for convenience and as usual I will subdivide these molecules into the 3 major physical-chemical groups which are the water soluble compounds, the protein bound compounds and the larger so-called ‘middle molecules’. But water soluble compounds of course we’ve been concentrating on urea as an index of dialysis adequacy and also of solute retention but urea as such was not such an impressive uremic toxin which nevertheless doesn’t mean that all the molecules in this group are inert.

Slide 3

One example of this is asymmetric dimethylarginine or ADMA which repeatedly has been shown to be linked to vascular damage like in this study by Zoccali et al but there are more studies like that around. As you can see here, there is a real significant correlation between ADMA concentration and intima media thickness as an index of vascular damage.

Slide 4

It has long been thought that it’s a structural counterpart which is symmetric dimethylarginine or SDMA was an inert molecule but this has proved not to be true lately, especially in this study from the Kilstein group in Hanover where it was shown that SDMA like ADMA but using a different pathway was also inhibiting the function of nitric oxide synthase or the generation of nitric oxide as a protector of the endothelium and in this way could also be linked to the potential of damaging the cardiovascular system.

Slide 5

Our own group has been studying this own molecule for its impact on leukocyte function and especially on generation of oxygen free radicals and could demonstrate that this SDMA also was indeed increasing monocyte SDMA production. Studies done by Eva Schepers in our lab which will also be presented at this congress and we also could show that part of this effect was related to calcium influx since inhibiting calcium influx by administration of ACE inhibitors was neutralising entirely this effect. Another part we used was the NFKB way which was also enhanced and if we neutralised the latter by administering N-acetylcysteine, again this increase of leukocyte activity was totally neutralised. It should be noted that ADMA has no significant effect at all.

Slide 6

Other studies from our group which had been performed earlier had demonstrated also other guanidines because ADMA and SDMA are guanidines, that also other guanidines were interfering with leukocyte function and there was leukocyte activation by many of those and indicated by the generation of TNF-α in this way this study also demonstrated that the different groups of uremic toxins are not separate but that they are influencing each other and that in this case the small guanidines are affecting the production of a ‘middle molecule’. But one could say so what, since these are small water soluble compounds and urea is also what we do with urea will be representative enough for these guanidines so that we know what we are doing if we are determining Kt/V. So for that reason after that we embarked on a study evaluating the kinetics of the guanidines and to our big surprise could see that most of these had a distribution of volume which was significantly larger, up to three times even larger than that of urea which was resulting in a smaller effective removal which can be solved only by lengthening dialysis or increasing its frequency.

Slide 7

Of course, these are mathematical kinetic modelling studies, so for that reason we tried to confirm this by direct measurements and came to the same conclusion. Here is the evolution within the red blood cell of guanidine concentration. These are the results for urea here in purple and you can see that for most of the guanidines the removal from inside the red blood cell is lagging behind to the one of urea. So conclusion here some water soluble compounds are active and they obviously are not behaving kinetically like urea. So, we should look further than urea kinetic modelling and Kt/V urea in interpreting uremic retention removal.

Slide 8

Let’s move to the protein bound molecules which by definition do not behave like urea because their protein binding is hampering their removal. There are some clinical data linking them to clinical outcome. There is more infectious disease with higher p-cresol for instance, as a typical protein bound molecule. There is a correlation between p-cresol and clinical uremic symptoms. There is a correlation with negative outcome and in a recent study from the Louvain group also with cardiovascular mortality.

Slide 9

Now p-cresol in reality is not present in the body of uremic patients it’s conjugated it’s p-cresylsulphate and there was an error in the interpretation of the results due to the fact that most estimations of concentration were done after acidification for the protein binding of the compound and this acidification is hydrolysing the p-cresylsulphate conjugate in to p-cresol and sulphate and as a consequence we were erroneously measuring p-cresol but nevertheless, there are good reasons to accept that there is a correlation between what we measured before as p-cresol and what we presently see as p-cresylsulphate. In any case if we are estimating p-cresylsulphate with a method where there is no acidification involved, we see here this p-cresylsulphate peak here in the hands of – et al who made the detection in 2005 and here in our own hands with a very – and high p-cresylsulphate peak which is very well distinguished of other uremic retention solutes.

There is this question then is this p-cresylsulphate also toxic compared to p-cresol the mother compound? In reality we found a totally different result but nevertheless toxicity namely this p-cresylsulphate is increasing the activity of red blood cells and in this way may contribute to vascular damage. This is seen for monocytes and lymphocytes and not significantly but still the same then also for granulocytes.

Slide 10

So it’s thus exactly the inverse of the mother compound p-cresol which is an inhibitor of the white blood cell. This has more to do with vascular damage. It also says that the conjugate not necessarily behaves like the mother compound and that also conjugates may exert toxicity. The same can be learned also from indoxyl sulphate. Here data from the Marseille group. Another sulphate, so another conjugate from the indole group where also the repair capacity and the functional capacity of endothelial cells is inhibited by this indoxyl sulphate. So taking together indeed protein bound compounds are also playing a role.

Slide 11

The question is since we know that standard dialysis isn’t very well in this, are there other means to give or to enhance their removal? Therefore, we embarked upon this comparative study which contained more than protein bound solutes but I’ll show you only the data for the protein bound solutes and where we evaluated different convective strategies being pre-dilution, hemodiafiltration versus hemofiltration first where you can see that the hemodiafiltration, in other words adding diffusion to convection is significantly better at removing most of the protein bound compounds apart from Cmpf, carboxymethyl-propyl-furan-proprionic acid whereby Natalie Meert in our lab which is also in progress and shown at this meeting.

Slide 12

On the other hand, if we then compared post dilution with pre-predilution hemodiafiltration there was not much of a difference for most of the protein bound compounds but here the post dilution was superior for Cmpf and more importantly in the other analysis also for β2 microglobulin and for the water soluble compounds. So all in all we think that for removing protein bound compounds you’re better off with convection and if you use convection that the preferred one is post dilution and for that reason we also went for this other study but now I’m pushing on the wrong button, there is this longitudinal study going over 9 weeks also by Natalie Meert where you can see all these protein bound compounds and where you can see that the strongest protein bound ones p-cresylsulphate and Cmpf are going down progressively and significantly after this 9 week course. This again, seems to confirm in comparison in this study with high flux hemodialysis that this convective strategy is better at removal. There must be other solutions like absorption but we are still far away to come to decent conclusions in this context.

Slide 13

We finish with the ‘middle molecules’ β2 microglobulin our classical marker. In a subanalysis of the HEMOSTUDY related to outcome the lower the β2M, the better the outcome of these patients, the global outcome also the outcome related to infectious disease. Although we could consider β2M here there is this recent paper in Circulation which was shown to me by Tillman Drueke and which is very interesting because it’s proteomic analysis in people without any kidney dysfunction and looking for a biomarker of vascular disease of atherosclerosis and where this β2M came out as the best biomarker for this problem.

Slide 14

There are also Japanese data showing β2M in the vessel wall of atheromatosis, so in this way this could be not only a marker but also a culprit. There are more molecules involved with cardiovascular things anything which is in red are middle molecules and they all have a cardiovascular impact somehow demonstrated so in this way it makes some logic to open up the membranes, to go for high flux and/or convection.

Slide 15

But are there then clinical data confirming this? Yes first of all, secondary analysis or retrospective analysis. For instance this subanalysis of the HEMOSTUDY superiority for the high flux. The same for the 4D study originally designed for fat for the statin administration but also better for high flux at subanalysis than for different brands and types of low flux membranes.

Slide 16

Finally, in an RCT the MPO, the membrane permeability outcome in the subpopulation with serum albumin below 4g/l a significantly better outcome for the high flux as compared to the low flux at subanalysis also for the diabetic population.

Slide 17

Is there more than increasing pore size? Therefore, we went for this study the GENIUS, it’s a batch dialysis system that allows to bring the fresh dialysate and dispend dialysates into the same bowl but they are nicely separated here illustrated by a computer model where the blue is the fresh and the red to yellow is the dirty dialysate. But the interesting thing of this system is that you have only one pump for blood and dialysate and so whatever you do if you do 4 hours or 6 or 8 hours you do everything else the same, the dialysate flow, the total dialysate flux is the same, blood flux is also the same. So this study went for the impact of time on uremic toxins without anything else. As you can see quite impressively, there is an increase with the hours from 4 in red up to blue 8 hours even for the small water soluble compounds but very impressively also and more significantly for phosphate and for β2M. If you express this as a percentage it’s even more impressive it’s versus 4 hours and especially for β2M there is an almost doubling of the removal.

Slide 18

So vascular lesions seem to a large extent related to molecules which are difficult to remove by standard dialysis. New strategies are available to enhance their removal and outcome studies confirm the clinical benefit.

Slide 19

This is the composition of the EUTox group. I thank you for your attention.