CME Slides Forum - R. Schiffmann

A joint Congress by ERA-EDTA and ISN

PROGRESSION OF FABRY NEPHROPATHY

Raphael Schiffmann, Dallas, USA

Chair: Joao Paulo Oliveira, Porto, Portugal

David Warnock, Birmingham, USA

baylor

Dr R. Schiffmann
Institute of Metabolic Disease
Baylor Research Institute
Dallas, TX, USA

Good morning. I would like to thank Professor Warnock and the organisers for inviting me. If I say anything stupid, that’s because I’m not a nephrologist, I’m a neurologist but there was confusion in the term, so I ended up here.

Fabry disease is a deficiency of a lysosomal enzyme called α-galactosidase A which breaks down the terminal galactose of a number of glycosphingolipids but in particular globotriasylceramide which I’ll abbreviate as Gb3. As a result there is a so-called storage disorder that can be seen classically in walls of vessels in other organs. Basically it’s an enzyme that’s expressed in every cell in the body and classically on electron microscopy you can see lysosomal inclusions in endothelial cells and in a number of other cells. One can divide the disease into specific signs and non-specific signs which are the vast majority of the disease. So the patients are often identified because they have cornea verticillata or changes in skin called angiokeratoma. However, in childhood they may develop anhydrosis or hypohydrosis, neuropathic pain and febrile crises, as well as abdominal pain and diarrhoea but the importance of this disorder lies in the fact that usually in adulthood they develop or they tend to develop progressive renal disease, a variety of cardiac manifestations and an increased risk for ischemic stroke. I’d like to emphasise that this is an -- disorder where the males are predominantly affected but the females are also affected and this is because in the organs there is no cross-correction between the deficient cells and the replete cells. So the replete cells don’t correct even though there is an -- inactivation - those cells with a normal enzyme don’t correct the cells in the females that are deficient. The incidence is being constantly revised but it’s at least 1:40,000. So in terms of the kidney involvement this is really a central issue in this disease and this is why we are here.

The classical and the only specific abnormalities to my knowledge in the kidney pathology are those lysosomal inclusions which are indicated, the majority of them are in podocytes but they also are in epithelial cells as well as in endothelial cells.

This is a toluidine blue section and it’s easy to see them on electron microscopy and here is a podocyte and a vascular endothelial cell with some inclusions. There is some debate as to what is the importance of these inclusions in terms of the pathogenesis of the disease.

So the progression of the disease can be looked at easily at the level of the glomerulus.

So initially they have the specific abnormality in the light microscopy that can be appreciated that I described and at that time the glomerulus will still look ok here in PAS staining.

The next phase as I understand has mesangial widening and then progressive focal and general glomerulosclerosis and then of course, there is also additional interstitial disease and a global demise of the kidney.

That includes the tubular system and a distal tubule seems to be predominantly involved and as a result they have among other things difficulty to concentrate the urine or hypostenuria.

There’s another way to look at the storage material, the Gb3 material and this is with a very specific antibody that was developed a number of years ago by a Japanese group and what’s nice about it is that it’s specific, so in fact it stains only or it identifies only Gb3 in affected individuals and it’s completely negative in a healthy individual. So you can imagine the goal of a treatment for example ERT should be to make the antibody staining completely negative.

So I’ll describe briefly some of the results of two retrospective studies. The first one was published a number of years ago when I was still at NIH and the second one was an international cohort that was really led both by Genzyme which helps a lot and by Professor Warnock.

The NIH renal natural history cohort concentrated on the males. So these are the most affected individuals at the time we looked at 105 males and we looked at the various characteristics, the clinical symptoms, the age of diagnosis as well as the age of onset of chronic renal insufficiency, proteinuria, hypertension, ESRD and death.

The international cohort on the other hand was a lot larger and it included males and females, so that was really critical to obtain.

Overall the renal abnormalities in Fabry are of course, progressive renal failure, proteinuria, nephrotic syndrome. I mentioned the hypostenuria and of course hypertension and anaemia that you are very familiar with as well.

The question, of course, is what is this sequence of events? So here we depicted the time to onset to a number of basic complications in these individuals. So what you can see is that the onset of renal insufficiency is usually in the third decade of life and it’s interesting that hypertension is not present in a lot of these patients until they develop chronic renal insufficiency and then of course, its incidence tremendously increases. Then in our cohort the median survival was 55 years. So it’s clearly a disorder that ends up killing the patients. But however, as you see what is completely absent here from this diagram is proteinuria which as I’ll point out is absolutely a critical manifestation of the disease.

So to look at in a different way this time using the international cohort, this is time to event analysis for the first event whether it’s renal such as ESRD, a cardiac problem, a stroke or death and the idea here is to show you that even females are affected. So even thought they develop this complication at a significantly later age compared to the males it’s still significant if you had a control group of healthy females that would have run somewhere here. So it clearly is a disorder of females as well as of males.

So the international study allowed us to define better the rate of progression of the disease in terms of the estimated GFR, so this is the GFR calculated by the MDRD formula which we fully realise has its problems.
So what you can see first of all is that the males even though those that after follow up that we had did not reach ESRD had on average a significant acceleration or decline of their GFR. So it’s about 3-4 the normal rate. The females actually clearly progress at a slower rate and this is a rate that on average is a little bit higher than normal. The best normal rate I found is 0.75 ml/min/year and this is about 1. However, the individuals who ended up developing ESRD had a much faster progression and there the difference between males and females is not large suggesting that secondary processes get into work.

So this international cohort study looked carefully at proteinuria because it turned out that it plays a major role as an indicator of progression of the disease. We didn’t have information on proteinuria in all the patients but what is remarkable is the range of proteinuria. So it ranged from 0 to a nephrotic level, although curiously I don’t know of too many patients who actually developed nephrotic syndrome even though they have tremendous proteinuria. Also interesting is to note that a very small number of individuals ended up on ACE inhibitors because this is a retrospective study at a time where people didn’t realise the full effect of these drugs. Obviously if you look at the age, the people who had GFR below 60 ml/min were clearly a lot older than those that had better glomerular function obviously because this is a progressive disorder.

So the critical thing this is a very important slide in this story is that this looks at the urinary protein and so either there is no proteinuria, mild proteinuria or significant proteinuria that’s above 1 g per 24 hours and you can see that this slope of decline in males and females tends to be larger the more proteinuria there is. So the proteinuria clearly is associated with an acceleration of the decline of the glomerular function in this disease.
You see that in females in fact if they don’t have proteinuria, they don’t have renal disease. Their decline is actually equivalent on average to the normal population but as they accelerate as they develop some of them develop significant and overt proteinuria. So this is an important thing.

Well, probably the other single largest modifier is the residual enzyme activity. So we defined 0 as an activity that is patients who were considered non mutant versus individuals who have real residual enzyme activity which is in practice over 2% but the test has to be done appropriately. But it shows that people who have a little bit of residual enzyme activity 2, 3, 4 up to 8% of normal in this group have a significant delay in the onset of chronic renal insufficiency. Now the importance of that means that in order to have an effect on the natural history of the disease it’s probably enough to correct or to increase the enzyme activity a little bit however in every cell of the organ. So you don’t need necessarily to have a marked effect on the disease to normalise or come close to normalising the enzyme level in the cell in the kidney.

So briefly, I want to conclude with the concept of threshold. As I showed you there is a clear correlation between the severity of the disease and the residual enzyme activity.

We demonstrated here 2 or 3 years ago in the lab. So what we did is we cultured skin fibroblasts from a large variety of patients of males, of course, with an increasing residual enzyme activity. So you have here the percent of normal that is progressively increasing and we looked at the amount of Gb3 in the cell. What you can see is that as long as you have residual enzyme activity I emphasise in these experimental conditions, in these cells up to about 4% of residual activity you can clearly see in light microscopy here the so-called storage material. Once you go beyond that you essentially do not see any. So this is the idea that the amount of storage obviously depends on the residual enzyme activity.

So in this system we define a threshold between 4-5 and here we do the same, we can quantify this by looking at the cells in different ways, by doing FACS analysis and we even looked at skin biopsies and we saw that we didn’t see vascular endothelial inclusions in patients in our group that had a residual enzyme activity over 2% of normal. So just a little bit of residual enzyme activity is enough to modify the metabolism.

So in conclusion Fabry disease causes a global nephropathy. It is in many ways similar to other progressive proteinuric disorders but diabetes is, of course, a typical one. The importance of the statement is that all of you even if you haven’t seen a single Fabry patient all of you nephrologists know how to treat this disease because it also responds in a typical manner all the way from ACE inhibitors and ARBs as you’ll hear and this is a major contribution by Professor Warnock who realised you can use simple methods to modify the natural history. The disease progression depends obviously on the sex. In males it progresses faster than females, on the degree of protein excretion on the residual enzyme activity and of course as always, on the blood pressure and we are sure that other genetic epigenetic environmental modifiers exist and we hope to identify them in the coming years.

I want to thank, to acknowledge first of all the NIH group in particular the nephrologists at NIH that always helped us with the nephrological aspects of the disease.

The international cohort in particular Doctor Warnock as well because without him I think we would not have this extremely crisp and excellent analysis of this cohort. Thank you very much.

Chairman: Thank you Raphael. Our next speaker is here but we have time for a couple of questions. Please identify yourselves that’s helpful. Don’t be bashful come on. Raphael I’m going to ask you a question about residual activity. It’s astounding to me that by giving enzyme once every 2 weeks we can actually do something for this disease. But your cut off in the Brant paper as I remember was less than 1%. If they had more than 1% residual activity that was helpful, that protected the kidney.

Question: I’m sorry I misspoke, you’re absolutely right in the cardiac -- things are different -- reported this natural history of a cardiac variant. Those subjects have 5-6% of activity typically we don’t have kidney problems but they still progress and develop end stage restrictive cardiomyopathy and terrible things. So there’s a difference. We had a problem with the heart.

Dr. Schiffmann: Yes so the concept of threshold is of course important and is organ specific. If you reduce progressively the enzyme activity from the normal range to 0 the organ that is the most susceptible to the decline, to a decrease in α-galactosidase A activity is the heart. If you reduce it further you then included susceptibility to the kidney disease and in fact if you reduce it all the way to 0 to my experience it’s only then that you have a substantial increase in the risk of stroke. So the susceptibility of every organ is different. Inside every organ I’m sure every cell type has a different threshold as well.

Chairman: Ok, that’s very helpful another question one more. Doctor West, Michael West.

Question: Thank you I just wondered whether the interstitial compartment here is maybe more important than we think. I mean we all look at those nice electron micrographs that show a lot of effect on podocytes and people have focused on that. Very important work on proteinuria as you presented here so clearly it’s not a pure interstitial disease, that’s not what I’m arguing but I just wonder whether the interstitial component may not be more important and the reason for that is we see even in children that they may present with tubulointerstial disease and nephrogenic -- they may present with -- with Fabry disease we’ve seen that and when you look at them, you biopsy those children they do already have some glomerular involvement. But they still show a lot of tubular involvement you showed that on your slides here. So I just wonder whether that might be an important consideration because no one’s really looked at that in any way. Not to say that proteinuria is not important obviously you’ve shown that here very nicely.

Dr. Schiffmann: No I completely agree and again I’m not a nephrologist but there is no question I mean one cannot separate the glomerulus from the tubular part of the nephron and the interstitium is absolutely a critical part and the reason there is proteinuria is actually tubular disease where it loses the ability to reabsorb some of this stuff that is filtered. So yes you’re absolutely right.

Chairman: Ok thank you very much Raphael we’ll go on to the next presentation.