Slide 1

First of all, I want to thank the organising committee for inviting me to give this talk here in Glasgow. So what I would like to show you in the next 20 minutes as Marie Claire told you is to show you how the genetic studies both in humans and in the mice have led to a better understanding of the pathogenesis of nephrotic syndrome and have pinpointed the crucial role of the podocytes in the development of certain forms of nephrotic syndromes.

Slide 2

As Marie Claire told you just before, idiopathic nephrotic syndrome as are usually classified according to their sensitivity to steroids and so there are the group of steroid-sensitive nephrotic syndromes and a subset, a large subset actually of steroid-sensitive steroid-resistant nephrotic syndrome which among those who are sensitive to cyclosporin or those to recur after transplantation who are linked to a T cell disorder.
There are a subset of other steroid-resistant nephrotic syndromes, which are actually due to structural anomalies of the glomerular filtration barrier and of course, those nephrotic syndromes are not prone to recur for transplantation and although it’s possible that the development of antibodies directed against proteins, which were not known in the native kidney could also lead to the development of proteinuria after transplantation.

Slide 3

Nevertheless, also familial cases have been reported both in steroid-sensitive and in steroid-resistant nephrotic syndromes. All the genetic studies which have been performed in the past few years, mostly involved the steroid-resistant nephrotic syndromes and this will be what I will discuss now.  In all cases also, well, in most cases also at early stages, minimal change could be found by optic microscopy. Usually patients develop focal segmental glomerulosclerosis that’s why these cases are referred either as familial nephrotic syndrome or familial FSGS in the literature.

I have listed here all these familial forms according to their mode of inheritance. So autosomal recessive forms have been described in mutations in the NPHS1 and NPHS2 genes encoding nephrin and podocin are responsible for a large number of these cases. More recently, mutations in the LAMB beta 2 gene encoding the laminin beta 2 Chain have been found in Pierson syndrome which is a syndrome associating diffused mesangial sclerosis and ocular anomalies such as mainly congenital miosis. So children are also, it’s a very severe disorder and the children also might develop neurological and neuromuscular symptoms. It’s the only gene for here which codes for protein of the glomerular basement membrane. So 2 genes also have been described in the autosomal dominant form of focal segmental glomerulosclerosis, one is the ACTN4 gene encoding alpha-actinin-4. The other one is TRPC6 encoding an ion channel. It’s also important to remember that WT1 mutations, which usually lead to Drash and Frasier’s syndromes, which are severe nephrotic syndromes associated with male pseudo-hermaphroditism can also lead to focal segmental glomerulosclerosis in very rare cases but in normal female patients and in that case these patients could transmit the disease, the mutations to their offspring. I won’t discuss here but it’s also important to remember that there could be nephrotic syndromes associated with mitochondrial cytopathy and usually the nephrotic syndrome is associated with deafness, diabetes or other extrarenal disorders, neuromuscular disorders too but proteinuria might be the first symptom.
As I told you before, besides these genetic studies in humans, knockout mouse models with the invalidation of the gene encoding protein of the podocyte also could lead to nephrotic syndrome and I’ve also given a lot of information on the structure of the slit diaphragm.

Slide 4

So to summarise, I have schematised all this data on this drawing I brought from Dontscho Kerjaschki which represents a part of the foot process and as you can see here, all these syndromes I described before due to mutations in genes are encoding proteins mostly located at the slit diaphragm except as I told you for Pierson syndrome and LAMB 2. Nevertheless, it will be important to relate the function of this protein and the function of the transcription factors WT1 and LMX1B, which is mutated in the nail patella syndrome and which has been shown recently, well, not so recently in 2002, to regulate podocin and type IV collagen at least in vitro and so it would be important to see how they could regulate also other proteins here and the same for the Smarcal gene which is mutated in Schimke disease which is a rare disorder associating nephrotic syndrome and FSGS with immunological disorder and bone dystrophy and Smarcal 1 encodes a protein which is involved in chromatin remodelling and probably also has a role as regulating the expression of some of these genes.

Slide 5

So let’s go now to the hereditary nephrotic syndrome of autosomal recessive inheritance.

Slide 6

Before going on further, what I want to stress here is genetic heterogeneity of this disorder since at least already 3 genes have been already characterised but there is another locus, which is already known which has already been identified also the gene hasn’t been yet identified. In our experience in Necker we have collected 80 families with autosomal recessive steroid-resistant NS and only 35 of them have podocin mutations and in 18 of the families unlinked to the podocin locus we have performed a genome wide scan and haven’t been able to find one locus but on the contrary we have found 3 different loci each of them quite large and containing a large number of genes which impair the identification of the new genes but what I mean here is that it means that at least 4 new genes are to be found involved in nephrotic syndrome.

Slide 7

So the congenital nephrotic syndrome of the Finnish type is probably the most well known familial form of nephrotic syndrome. It is the most severe form with a massive proteinuria outburst and large placenta and a very severe outcome. It’s as you all know due to mutations in the NPHS1 gene encoding nephrin and nephrin is a large transmembrane protein with a large extracellular domain with immunoglobulin-like domains which are able to homophylic interactions and its nephrin is a major component of the slit diaphragm as you can see here.

Slide 8

Mutations of the NPHS1 gene are found, too frequent mutations are found in Finland but nevertheless, mutations have been also found in patients from other origin and over 60 disease causing mutations have been detected until now, more than half of them being missense mutations.  The group of culturing has tested 25 of them in a functional assay and have shown that the mutant protein, 16 of the 21 missense mutations they tested are not transported, are not trafficking correctly in the cell and are retained in the endoplasmic reticulum probably due to a misfolding of the protein. They have also shown that it’s possible to rescue this defective trafficking by a drug which is a 4-phenylbutyrate in 6 of them. This is really a very interesting finding because it proposes or at least, it gives some ideas of potential treatments in some kind of mutations. It’s important to notice that NPHS1 mutations were not always found in all patients with congenital nephrotic syndrome. On the contrary, that some mutations and especially the R1160X mutation, it’s a stop codon mutation could be found in some less severe forms of congenital nephrotic syndrome. It means that in these cases the children have a very severe nephrotic syndrome at birth but then improve their condition and so it has a lot of implications for the treatment of the patients and also for genetic counselling.

Slide 9

The second disease I want to speak of today is the familial steroid-resistant nephrotic syndrome so almost more than 10 years ago in the department of nephrology in Necker we characterised patients with an autosomal recessive form of NS with an early onset in childhood, a rapid progression to end-stage renal disease, no recurrence after transplantation and FSGS in optic microscopy. A few years later, we found that the NPHS, a new gene NPHS2 encoding a new protein called podocin which was responsible for the disease and podocin appeared to be a podocyte protein, as you can see here and it has a strange, a curious hairpin-like structure which means it’s a cytoplasmic protein with a 1 membrane domain, which anchors the protein in the plasma membrane. We also have been able to show by electron microscopy, by immunoelectron microscopy that podocin is located at the cytoplasmic face of the slit diaphragm as expected from the potential structure of podocin.

Slide 10

So initially podocin mutations were found in familial cases but very fast it has been also found in sporadic cases and until now there have been three large scale mutation studies, which have been published accounting for more than 400 patients and with sporadic cases of steroid NS, usually in children and you see that a mean of more than 10% of the patients have mutations, two pathogenic mutations in the podocin gene. Interestingly, it shows that as we knew from the start that the patients have a very early onset of nephrotic syndrome and in the cohort of Friedhelm Hildebrandt 40 patients have even started their NS before, almost at birth which means that when you do not find mutations in the NPHS1 gene in congenital nephrotic syndrome it is very important to look for mutations in the podocin gene. On the contrary no patients out of 18 which we have tested in our cohort who start their NS after 15 years of age were found with pathogenic mutations.

Slide 11

So it’s really a severe disorder mostly when you have 2 pathogenic mutations and so there were either misframe shift or non-missense mutations but we also found some missense mutations which are described here. You see the same kind of functional test as a group of – indeed we tested what was the cellular localisation of the mutant protein and as you can see here, most of them are in yellow here are retained in the endoplasmic reticulum, whereas a few of them only are correctly targeted to the plasma membrane and what is clear is that there is a clear phenotype genotype correlation since all the mutant proteins targeted to the plasma membrane have a less severe phenotype than those which are retained in the endoplasmic reticulum.
Once again, this finding is really important because as you know that there might be some drugs able to retarget the mutant protein to the plasma membrane that could be a target for future therapies.

Slide 12

It’s more important than by the fact that it’s not only found in vitro but also in vivo. Marie Claire Gubler was able to show here in this nice picture that patients with compound heterozygous mutations with 1 frame shift and one missense mutation the podocin is retained in the cell. Here it’s difficult to show where.

Slide 13

So nevertheless, is it possible to have a NPHS2 mutations in the late-onset FSGS? Actually the group of Martin  Pollack in Boston identified 6/30 patients with late-onset autosomal recessive FSGS, which were found with mutations of podocin. Actually they have 1 podocin mutation and 1 variant, the R229Q which is a polymorphism found in 3-4 % of the normal population but when associated with a mutation can lead to a late-onset of nephrotic syndrome. What they showed is that this variant leads to a decreased binding of nephrin to podocin, which might explain why it’s not a neutral polymorphism and its association with mutation could lead to a phenotype. It has also been shown that the R229Q allele is associated with almost 3-fold increased risk of presenting microalbuminuria. In our experience in Necker once again we found this association of the R229Q variant with the pathogenic mutations in 11 families and as you can see here, the mean age of onset is far more elevated than in the patients with 2 pathogenic mutations since it’s of 14 years with a mean onset, a mean age at end-stage renal disease around 20 years. So,  the R229Q variant is a common non neutral polymorphism, which can contribute to the development of FSGS.

Slide 14

Then as I told you, initially patients with podocin mutations shouldn’t recur the disease after transplantation since they have received a transplant which does not have mutations in the podocin gene. Nevertheless, it’s true that recurrence of the disease has been reported in patients with 2 mutations in the podocin gene, at least you see, it means with a frequency of 1-15 which is, of course, very, far lower than what is reported in the current cases of steroid-resistant nephrotic syndrome. There have also been cases of recurrence reported associated with variants in the heterozygous state of NPHS2 but as the role of this variant is not known even the pathogenesis of the disease it’s not so strange that the recurrence of the transplantation with this mutation.
However, even if it’s now not very clear why these patients do recur after transplantation, in these cases it might be the recurrence of antibodies, podocin antibodies after the graft. What is true is that the patients with NPHS2 mutations with homozygous or compound heterozygous mutations have a highly reduced risk of recurrence of nephrotic syndrome after transplantation.

Slide 15

Slide 16

If we go now to the autosomal forms of FSGS so the first gene to be identified is ACTN4 which encodes alpha-actinin 4 which is an actin binding protein which is highly expressed in podocytes. ACTN4 mutations have been found in 3 large families with autosomal dominant FSGS but probably ACTN4 are not a frequent cause of autosomal dominant FSGS since in 18 new families only 1 mutation was found.

Slide 17

So there have been also I’m going fast to finish, there have been also mutations in the TRPC6 gene which have been recently found in families from New Zealand, in large families from New Zealand and once again, it has been shown that there is a large genetic heterogeneity and a lot of other loci should be identified.

Slide 18

I do not have time to speak of the mouse models.

Slide 19

Nevertheless, I would like to speak of the role of CD2AP because it’s an adapter protein that acts to cluster CD2 which is a T cell receptor and anchors CD2 to the skeleton. Unexpectedly mice lacking CD2AP develop proteinuria and renal failure.

Slide 20

Some patients with primary FSGS have splicing variant in FSGS. So it could be a link between the immunological forms and the structural forms of nephrotic syndrome.

Slide 21

So just in conclusion, I would like to mention that the podocyte and especially the slit diaphragm proteins play a crucial role in the formation and maintenance of the glomerular filtration barrier. Hereditary steroid-resistant nephrotic syndromes are genetically heterogeneous. Mutations in the podocin gene can be observed both in familial and sporadic cases, mostly in patients with early onset and even in congenital cases. We think that screening for podocin, WT1 gene mutations and even also podocyte protein encoding genes should be performed in children with FSGS. Screening for the R229Q variant should be performed in adults with FSGS.

Slide 22

Thank you for your attention.