CME Slides Forum - G.M. Ghiggeri

A joint Congress by ERA-EDTA and ISN

AN INTEGRATED PROTEOMIC APPROACH TO PLASMA AND URINARY PROTEINS IN NEPHROTIC SYNDROME

Gian Marco Ghiggeri, Genoa, Italy

Chair: Gerhard Muller, Goettingen, Germany

Robert Unwin, London, UK

gaslini

Dr. G.M. Ghiggeri
Department of Nephrology
Dialysis and Transplantation
Giannina Gaslini Children Hospital
Genova, Italy

Good morning. Chairman, colleagues thanks to the organisers for this kind invitation on this hot day in Milan. I’m Doctor Ghiggeri and I will review in this morning session a few aspects related to plasma and urinary proteins in nephrotic syndrome. The term is not referred to the broad range of nephrotic syndrome but just to the type that characterises children and is based on the pathological aspects related to minimal change, IgM proliferation and focal sclerosis. I call them hidden messages because we still do not understand what they actually mean but hopefully in the near future we will translate these messages and open probably new pathogenetic possibilities.

The answer to the former question is very easy because it is suspected a plasma factor in the genesis of the disease

and also the answer to the second question is easy because the plasma factor is probably a protein or a peptide.

This is an old question that arose almost 20 years ago in the late 80s but unfortunately in spite a lot of research we still do not know many things about the identity of the plasma factors and probably the strongest and perhaps the unique evidence supporting the concept of plasma factors is the high rate of recurrence of focal sclerosis after a renal graft.

I will focus on three points of interest. The first is the qualitative difference in serum/urine in respect to protein composition. The second topic will be the oxido-redox status of proteins mainly of albumin and finally, just a few considerations about serum peptides that is in my opinion the new frontier.

This slide shows a two-dimensional electrophoresis of urinary proteins in three different conditions; focal glomerular sclerosis, membranous nephropathies and drug-induced proteinuria that we know follows treatment with mTOR after transplants. There are not marked differences, in fact in all cases proteinuria is composed mainly of albumin and by this part of proteins that are present in all conditions and that correspond to a panel of metal glycoproteins. In general this is just a summary of what we can detect in human pathology but in general albumin and all acidic proteins are present in nephrotic syndrome and in almost every type of glomerulonephritis. Probably the peculiarity in focal sclerosis urine is the lack or at least the decrease in low molecular weight proteins compared for example, to proteinuria induced by mTOR.

Another interesting peculiarity of low molecular weight proteins in focal sclerosis is that they are not proteins but just fragments of albumin as shown by this slide that is an (immuno) Western Blot with specific anti-human albumin antibodies. As you can see here, a lot of these low molecular weight proteins are in fact the fragments of albumin.

Considering all the low molecular weight panel we could show that at least 50 proteins are in fact, fragments of albumin and also we tried to characterise by mass spec technologies all these fragments and this slide shows you just a few of the studies on characterisation. Most fragments contain only a part of albumin with a tail and a head but lack inside fragments suggesting that they derive from a strong fragmentation.

How specific are urinary fragments? I don’t know because we have to check not only nephrotic syndrome but also glomerulonephritis and as far as I know, I can say that the very low molecular weight fragments are specific for minimal change glomerulosclerosis. While they are not present in the urine for example of membranous nephropathy.

How are fragments generated? Also in this case I don’t know, maybe a part of them are produced in the urine by proteases that we know are present in all the segments of the tubular length but it is also clear that a part of fragments are produced in the serum and this is an (immuno) Western Blot of blood. So also in this case you can observe a lot of fragmentation in the plasma.

Therefore, my first conclusion is that urine and plasma in focal sclerosis are characterised by a lot of fragmentation panels.

Now, I will go to consider another aspect that probably coincides also with the first. I’ll focus on the oxido-redox status of serum albumin in FSGS. These are quite peculiar aspects and in my opinion also specific for FSGS.

Why do we consider the oxido-redox status of albumin? This is a new research that started very recently when biochemical studies showed that albumin is the major antioxidant in plasma. This is also reasonable because in plasma we know that antioxidants such as glutathione have a very, very reduced level. For example, the level of glutathione that is the major antioxidant substance is one hundred times lower than the level, for example, in the red blood cell but in general in all kinds of cells suggesting that glutathione is the major antioxidant inside the cell but not outside the cell. This is the importance of albumin because albumin introduced in plasma with a unique, I’ll show you later the structure of albumin, but with a unique free SH of the sequence of albumin introduces the same level of glutathione of red blood cells. So it constitutes by any fact the most important source of free glutathione in plasma.

This is a tri-dimensional structure. Albumin is made up by 35 SHs that are linked in 14 disulfide bonds but one free sulfidric group in position 34 here in this pocket remains free to react with plasma. The antioxidant function is probably the function of this free SH group.

Just a few biochemical lines to put the basis for the analysis of serum albumin nephrotic syndrome. When a thiol group is oxidated it is introduced first one oxygen here, then a second oxygen here and these are unstable derivates but after the third oxygen is put, the sulfonic acid is formed that is a stable derivate. The sulfonic acid contains three more oxygens that means 48 daltons more than the normal SH group.

So if we look to plasma albumin nephrotic patients, it appears that the molecular weight, this is serum albumin in patients with FSGS is higher just by 48 Daltons that is a very, very small change but enough to suggest that the free SH group, as here shown, was modified to a sulfonic derivate.

We also developed technologies to apply to plasma for detecting oxidated albumin

and the basic procedure is very simple because it is based on the assumption that if a free SH is oxidated, the content of the free SH decreases and since free SH groups are easily detected by alkylation for example, with maleimide that may be linked to biotin, the assay is very simple because with a streptavidin you can detect the free SH group and in case of oxidation you do not detect any kind of reaction. This is what happens with plasma from children with FSGS. Here is albumin that has been alkylated by maleimide and as shown here, you can detect a lot of albumin here and here is the deep decrease of albumin since the unique SH group had been oxidated.

Another possibility to show oxidated derivate of albumin is to measure their charge because the sulfonic group introduces one acidic charge and therefore oxidated albumins are more acidic than the normal ones.

Here in orange is an oxidated isoform of albumin as compared with a green from that is normal. This is a two-dimensional analysis that is called titration electrophoresis that carefully detects every ionised group in a broad range of pH.

Finally, we recently have developed specific cyanines that can be utilised in two-dimensional gels, this is an example, this is FSGS plasma and this is normal plasma and this is a very interesting technique because it may also be utilised also in – that is a direct comparison of two different plasma samples utilising two different cyanines. Also in this case here you can appreciate that the amount of the SH is decreased in patients with FSGS.

Finally, I have just given you a few hits on the new frontiers because now we are testing the possibility that also other serum peptides are present in plasma and serum of patients with FSGS.

The study is now in due course, so we cannot give you any data about the significance and the preponderance but I’d just like to show you that this is a linear mass spec analysis of the plasma from controls. Sorry for the Italian but these are nephrotic patients, these are children and these are adults.

Just to summarise the results on plasma peptides I can tell you that there are at least four peptides that have an increased level in the patients with FSGS. One of them that is fragments of C3 that is called C3f may have interest because may play some functional effects in vitro studies and also in animals.

So utilising an ‘a priori’ statistical approach and utilising the panel of peptides we could differentiate normal patients from nephrotic children and adult children with ESRD but I told you this is an ongoing study, so we need much more careful studies on many patients to propose peptides as markers of the disease.

I have just a few conclusions because we are faced with hidden messages so I am in difficulty to grasp any concrete message. However in my opinion proteolysis of major plasma proteins and oxidation of albumin are two main features in FSGS and this aspect in my opinion also should support unexplored mechanism also for the pathogenesis of the disease because in some way oxidation and proteolysis may be also directed not only towards proteins but also towards membrane proteins and in some way may produce some negative effect on the podocyte.

Maybe this is quite a similar conclusion but proteases and oxidations probably play as I told you a role also in the pathogenesis of disease and in my opinion serum peptides, albumin fragments and also oxidation of albumin just reflect an aspect of their activation.

The real final message is for me still obscure so I propose you in Greek for those who studied old Greek at Lyceum may be easy to translate, so far my position is that for me there are hidden messages.

Chairman: Thank you very much Dr. Ghiggeri. This presentation is open for discussion. Could you wait for the microphone please?

Question: A very nice talk I just have two questions. One in your opinion is the urinary sample better than the plasma sample? In your opinion, to use a plasma serum sample is better or the urinary sample is more reflective of FSGS? That’s the first question. Second question is how can your result interpret the progression of the FSGS?

Dr. Ghiggeri: The second question was clear to me I have no data on progression just on acute phases. Sorry the first part I didn’t understand.

Question: The first question was comparing your plasma measurements with your urine which do you think is giving you actually more information? Is it your mass spec analysis of plasma versus urine in the same setting of FSGS? You referred in your presentation largely to plasma. The same sort of measurements you make in serum and in urine.

Dr. Ghiggeri: The analysis of urine peptides is very difficult and as far as we know urine peptides and just on plasma peptides.

Chairman: Any other questions? Perhaps I can ask you, your last but one slide the principal component analysis that you’ve used to try and separate out your groups, have you identified yet any particular proteins that are good discriminators that help you to separate your groups? Obviously you’re putting in all the data you have but are you able to sort of subtract and then eventually find which group of proteins or which particular proteins seem to be important in that clustering?

Dr. Ghiggeri: Well, probably peptides are more specific than proteins, so the focus is now on peptides that in fact, can identify a peculiar group of FSGS but as far as I know, no proteins can clearly separate the different groups of patients.

Question: We’re seeing quite a number of diseases so what is the specificity for C3 fragments in FSGS?

Dr. Ghiggeri: This is a good point probably you know the aspect of C3f. The C3f in FSGS is a bit different from other C3f I know for example cancer, in cancer you can detect high levels of C3f but also in cancer maybe proteases are activated but I cannot give you details about this but these are different C3f just a bit bigger.

Question:Actually in any disease where you have interstitial inflammation you will have production of C3 by the proximal tubular cells, by the dendritic cells, by the macrophages and all of these potentially can appear in the urine depending on the degree of inflammation.

Dr. Ghiggeri: But I refer to plasma C3f not urine C3f. So I agree with you maybe C3f is a marker of inflammation, oxidation or something like this but we detect it in plasma not in urine. Therefore, urine has no role in the tubular aspect.

Question: But if you go to the circulation then if you go for instance to patients who have SLE they have very high conversion of C3 and they also will have high increased levels of C3f, C3d etc.

Dr. Ghiggeri: Well I just told you probably C3f is different, C3f isotypes are specific for different -- because probably they are produced by endoproteases that are activated just in different diseases. I know for example, cancer, prostate cancer for example. The first papers on C3f but also in acute myocardial infarction you know a lot of studies but in all cases different C3f. So my opinion is that C3f is a good marker.

Question: Can I just end then perhaps by asking you, you mentioned proteases, as well as oxidation in proteinuric renal disease there is a lot of interest now in urinary proteases that may have direct tubular effects. Do you have any thoughts about that from your own studies certainly looking at what might be in serum and plasma?

Dr. Ghiggeri: I’ve tried to characterise proteases and in fact, we have data but the analysis of serum and urinary proteases is very difficult and I cannot actually give you any details because we have confusing data.

Chairman: Ok well thank you. Well if there are no more questions I’ll call this session to an end. Thank you to the speakers and thank you to the audience for attending.