CME Slides Forum - M.C. Gubler

A joint Congress by ERA-EDTA and ISN

FSGS - PRIMARY VS. SECONDARY FORMS, CONGENITAL MUTATIONS VS NON-GENETIC TYPES

Marie Claire Gubler, Paris, France

Chair: Charles Alpers, Seattle, USA

Kerstin Amann, Erlangen, Germany

gubler

Dr. M.C. Gubler
Inserm U574, Ventre de reference MARHEA
Hopital Necker - Enfant Malades
Université Paris Descartes
Paris, France

So good evening, thank you to the organizers for this invitation to this meeting. And now we are going to speak about FSGS, primary versus secondary forms, genetic versus non-genetic forms.

As you know, FSGS was reported 50 years ago by Rich who observed this lesion in nephrotic patients dead from different complications and he observed this lesion in glomeruli located near the medulla. Since that, the lesion has been largely recognized. FSGS are characterized by their focal distribution among glomeruli and by the segmental involvement of the tuft characterized by obliteration, by deposits and fibrosis, by collapse sometimes. The lesion is surrounded by hypertrophic and vacuolised podocytes, and progressively adhesion between the sclerotic segments and the Bowman’s capsule occurs leading to this large sclerotic lesion

If we look at immunofluorescence, very often there are immune deposits or complement deposits within segmental lesions, without specific significance, and by electron microscopy we can see these sclerotic lesions with here large endocapillary foam cells and the lesion is surrounded by these abnormal podocytes which are not exactly detached but are pushed away by this material deposited between them and the basement membrane. This material is made of a collagenous component which is not normally present along the basement membrane. It is made of the ubiquitous á1-á2 collagen IV replacing the normal a3-4-5 (IV) network. So it is another element showing that there is a severe phenotypic change in these cells.

As it was beautifully described yesterday by Vivette D’Agati. several variants of the lesion have been described, either the tip variant at the tubular pole, the perihilar variant, the cellular variant or the collapsing variant and when none of these variants are present we speak about not otherwise specified variant.
Vivette D’Agati indicates that very often there is a certain degree of correlation between the type of lesion, the origin of the lesion and the prognosis.

But as you know, FSGS is a morphological lesion resulting from most often irreversible podocyte changes linked to intrinsic or extrinsic injury. But by extension and despite the fact that the lesion may develop during the course of several different disorders recognizing different causes, the term FSGS has become applied to the heterogeneous group of diseases having only in common the association of proteinuria often of the nephrotic range and FSGS.

So this is a summary of the different causes of FSGS. Usually we speak about primary FSGS or idiopathic FSGS or perhaps more precisely idiopathic proteinuria/nephrotic syndrome with FSGS when we don’t know the cause of the disease and the cause is possibly the consequence of deregulation of the immune system leading to the production of vascular permeability factors.
There are several recognized causes of FSGS. Some are genetic and they are responsible for genetic nephrotic syndrome with FSGS and most often they are the consequence of podocyte defects. But the lesion may develop when there is podocyte injury, viral, toxic for example, or in response to structural or functional glomerular abnormalities and we speak of postadaptative FSGS, for example when there is a reduced number of nephrons or when there is hemodynamic stress on a normal number of nephrons.

So what is primary FSGS or idiopathic nephrotic syndrome with FSGS? The most typical clinical feature is the rapid occurrence of florid nephrotic syndrome in children. The lesion FSGS may be observed in two different situations. One is no longer observed now because patients are very well treated. It was observed in long-lasting steroid-dependent nephrotic syndrome with numerous relapses when the biopsy was performed after several years of evolution of the disease. But most often now, it is observed in steroid-resistant nephrotic syndrome either in the initial biopsy or in a repeat biopsy when the first one has shown minimal change disease. The focal lesion is associated with diffuse alteration of the podocyte, so at the podocyte level, at the cellular level if you want, there is a diffuse disease of the podocyte. As was said yesterday, the different variants may be observed in primary FSGS with a special severity of the collapsing variant and also of the cellular variant and as you know, progression to ESRD occurs if remission of the nephrotic syndrome is not obtained. If the very aggressive treatment obtains remission, there is no progression to ESRD.

The big problem concerning primary FSGS is the possible recurrence on the grafted kidney, the frequency is variable between groups. Globally it is about one third of patients with primary FSGS who show recurrence on transplant. Recurrence of the nephrotic syndrome is associated with a poor graft survival. You can see here evolution in patients with recurrence compared with patients with primary FSGS without any recurrence. The recurrence is characterized by the occurrence of massive proteinuria within a few days following transplantation associated with diffuse effacement of foot processes.

In the transplantation department of Necker Hospital, Guillaume Canaud analyzed the evolution of 77 transplanted patients with primary FSGS and you can see that recurrence in red may occur whatever the type of FSGS.

It was exactly the same observation made by the Dutch group and you see that the distribution of the FSGS variant in the native kidney of the patient with recurrence is nearly the same. There is no perihilar variant but perhaps the group was too small to detect this variant.

In addition, in the Necker transplantation department, there is a protocol of biopsies performed when there is recurrence at 1 month, 3 months, 12 months and you can see that, at 1 month after the recurrence, only one patient has developed FSGS, and FSGS developed later on during the course of the disease.

Interesting data is shown here. You can see that when patients are treated very aggressively and for a long time and when a complete remission is obtained, no FSGS lesion develops and lesions are only observed when there is partial or no remission at all.

Perhaps more interesting, and different from the study performed by the Dutch group, you see that the type of recurrence, the type of the variant on the grafted kidney is often quite different from the type in the native kidney. It is perhaps very interesting to look at the evolution of the patients with collapsing glomerulopathy. You know this is a very severe type of FSGS progressing rapidly to ESRD and you see that after transplantation 5 of the 6 patients responded to treatment and had a normal biopsy at 1 month and later on.

So we can conclude for this experience in the group of transplantation in Necker that in primary FSGS progressing to ESRD, recurrence of proteinuria-nephrotic syndrome may occur whatever the type of the variant.
The hallmark of recurrence is the occurrence of massive proteinuria with effacement of foot processes, and FSGS develops only secondarily in patients resistant to treatment.
The type of FSGS variant in the transplanted kidney cannot be predicted from the type in the native kidney. Perhaps we can suggest that the type of FSGS variant depends on the type of aggression to the podocyte, on the genetic background of the podocyte, and on environmental factors such as treatment.

Now we switch to hereditary nephrotic syndromes. This is a large heterogenic group with diverse modes of transmission and having in common the absence of response to treatment, the absence of recurrence on the transplanted kidney. Most of them are linked to mutations in genes encoding podocyte proteins.

It is a large group. To begin with, we’ll speak about autosomal recessive nephrotic syndromes which are usually seen in children. You see that it is a long list and some of them are non-syndromic and some are syndromic. Among the non-syndromic, there are congenital and non congenital nephrotic syndromes. The first one to be discussed is the Finnish type nephrotic syndrome because the identification of NPHS1 encoding nephrin was the first gene identified in hereditary nephrotic syndrome by the group of Tryggvason in Finland and this disease you know, is characterized by its relative frequency in Finland, by the presence of massive proteinuria at birth, (the proteinuria is present before birth) and a severe course leading to death in the first years from ESRD or complications.

At the light microscopic level you see that usually glomeruli are rather large with a certain degree of mesangial proliferation and the progression leads usually to complete sclerosis of the glomeruli when the evolution to ESRD is observed.
The protein encoded by NPHS1 is nephrin. It is a large protein planted in the membrane, a transmembrane protein with a large extracellular domain. This protein is expressed specifically at the slit diaphragm and from the beginning, Tryggvason suggested that this protein by oligomerization with nephrin from adjacent podocytes could participate in the formation of the slit diaphragm. This hypothesis was confirmed. The nephrin is one of the main components of the slit diaphragm.
In Finland most mutations are truncating mutations (there is a funding effect) and they are responsible for the severe course of the disease. But in other countries, these mutations are very rare and usually they are less severe and sometimes associated with a less severe course of the disease.

For example, in this study made recently by Aurelie Philippe, you see that mutations in NPHS1 gene have been found in non-congenital steroid resistant nephrotic syndrome and in this group of ten patients, the mean age at onset of nephrotic syndrome was 26 months and the mean age at ESRD 12 years. At least one of the mutations observed in these patients was classified mild allowing a normal targeting of the protein to the plasma membrane.
Globally 11% of patients with steroid resistant nephrotic syndrome before the age of 5 years have NPHS1 mutations.

So the second gene to be identified in the non-syndromic autosomal recessive nephrotic syndrome was NPHS2 encoding podocin. This group of patients with podocin mutations is characterized by early onset nephrotic syndrome in childhood, rapid progression to ESRD and no recurrence after transplantation. Histologically there are normal glomeruli when the biopsy is performed early on in course of the disease, and evolution to FSGS. Podocin is a protein expressed also at the slit diaphragm, it is an integral membrane protein with a hairpin structure and it has been shown that it interacts with nephrin and other slit diaphragm proteins.

You see that mutations in NPHS2 are frequent in SRNS: they have been detected in 42% of familial cases, in 10% of apparently sporadic cases and globally in 37% patients of children with onset of the disease before the age of 1 year. The mean age at onset of the disease is 4 years, always before 18 years, and at ESRD of 8 years. All types of mutation have been described but one is very frequent especially in Germany and in East Europe. It is responsible for 1/3 of mutated alleles and is characterized by a severe nephrotic syndrome with early onset.

It is possible to look, at the morphological level, at the consequences of NPHS2 mutations. For example, looking with antibodies against the N- terminal or the C terminal of the protein, you see in normal controls, normal expression of the protein; in patients with truncated mutations sometimes there is no expression at all, sometimes you can see a partial expression of the N-terminal domain of the protein; and in patients with missense mutations usually there is no presence of the protein along the GBM but the protein is retained within the cytoplasm.

This retention of the podocin protein within the cytoplasm of the podocytes results in the similar retention of other slit diaphragm proteins, nephrin, CD2AP and α-actinin-4.

One interesting fact was described some years ago by Tsukagushi. He observed the fact that one variant of podocin, the R229Q which is present in 2-4% of healthy controls was associated with one pathogenic mutation in patients presenting nephrotic syndrome in early adulthood and he demonstrated that this variant may be pathogenic because its binding to nephrin was decreased.
Recently in the lab, Doctor Machuca looking at a large series of patients could observe the association of this mutant, of this variant to pathogenic mutations in 27 families with, as you can see,a later onset of the disease and a later onset of progression to ESRD. In addition he could demonstrate that no patients with nephrotic syndrome after the age of 18 years had two pathogenic mutations of the gene NPHS2.

There is a third gene which may be responsible for autosomal recessive non-syndromic nephrotic syndrome, it is the gene NPHS3 or PLCE1. It has been recently identified in consanguineous families with one or two affected children and is characterized by early onset nephrotic syndrome, frequent steroid resistance and rapid progression to ESRD. The lesion is different from the classic FSGS lesion because it is what is named diffuse mesangial sclerosis characterized in early specimens by this diffuse increase in the mesangial matrix progressing to this complete sclerosis of the tuft which continues to be surrounded by large podocytes resulting in this glomerulus in collapsing glomerulopathy appearance.

There is a problem which has not been resolved concerning these mutations in children. You can see that in some patients with a mutation there is a normal response to steroids or cyclosporine treatment and a complete remission of the disease. In addition, in some families, individuals with the same mutations as the patients are quite well. Here you have two young patients with a severe disease progressing to ESRD during the first year of life with this homozygous mutation and the father with the same mutations is quite well without any proteinuria at 46 years. The explanation for this variable expression of the mutations may be that there is in this disease an oligogenic heredity, or that other genes need to be mutated for the expression of the disease, or perhaps there is a protective effect of other genes of the phospholipases C family. But the response is not known yet.

Globally looking at autosomal recessive non-syndromic nephrotic syndrome you see that no mutations are found in half of the families. So other loci have to be identified.

One example is given here. In collaboration with the group from Tunisia, Corinne Antignac localized another locus on chromosome 17 by studying two families, two consanguineous families with nephrotic syndrome and FSGS leading to ESRD in young adulthood.

I have no time to discuss the various syndromic autosomal recessive FSGS. You can see that they may develop with all types of diseases. Just a word about Pierson disease. It is the only one which is not directly linked to disease of the podocyte but which is linked to a laminin (a component of the GBM) defect.

So there are also autosomal dominant nephrotic syndromes with FSGS, they may be syndromic or non syndromic in the same way.

But here the difficulty is that most of the mutated genes are not known. Two genes have been identified, the first one is ACTN4 encoding the α-actinin 4 protein able to link actin filaments. This gene has been identified by the study of these three large families with an autosomal dominant heredity. The nephrotic syndrome usually appears during adulthood, the disease has incomplete penetrance and the mechanism of the disease is still being discussed but it has been now shown that the mutated protein is misfolded leading to aggregation and rapid degradation in the podocyte.

You see here a recent paper from the group showing that in patients with this type of mutation, in the podocyte, in the cytoplasm of the podocyte, there are some aggregates of material, of dense material. And by using immunofluorescence studies, this material seems to be aggregation of the mutated protein.

The other gene, FSGS2, which has been identified in the autosomal dominant nephrotic syndrome is TRPC6. The gene has been localized by Winn by looking at a large family with autosomal dominant inheritance of proteinuria, nephrotic syndrome occurring during the third decade or later, frequent progression to ESRD, FSGS. Later on, she and Reiser could identify the gene, it is a gene encoding TRPC6 expressed in the podocyte where it interacts with nephrin and podocin. It is a calcium channel regulating calcium entry into the cells and it is suggested that mutated proteins enhance TRPC6-mediated calcium signals in response to diverse stimuli suggesting a mechanism for the progressive alteration of podocyte function.

The problem concerning autosomal dominant FSGS is that usually no linkage to these different genes is observed. For example, in our group, no linkage to ACTN4 has been observed in the families studied in Necker. Moreover, Kaplan did not find any mutation in ACTN4 gene in 80 additional families studied by his group.
In our group, linkage to TRPC6 was observed in 10 families out of 12 but no mutation was observed and globally only one mutation of TRPC6 was observed in a large group studied.
So these two genes are very important but they are rarely involved. Other loci/genes have to be identified and for that it is necessary to collect large families with autosomal dominant proteinuria nephrotic syndrome.

In addition, there are some syndromic autosomal dominant FSGS and the best characterized perhaps are Epstein/Fechtner syndrome and Denys-Drash and Frasier syndrome. As you know Fechtner syndrome is a rare autosomal dominant disorder with variable penetrance it is characterized by the association of proteinuria and nephrotic syndrome and inconstant hematuria, FSGS and sensorineural hearing loss. It is associated with microthrombocytopenia and in some cases cataract and neutrophil inclusions.

It was long considered an Alport syndrome variant but mutations in MYH9, the gene encoding a cytoskeleton component, the non-muscle myosin heavy chain 9 expressed in podocytes, inner ear cells and platelets, have been identified, explaining the clinical expression of the disease. More recently it has been shown and it is very interesting to consider that polymorphisms and not true mutations but polymorphisms of the gene are associated with FSGS on the one hand and hypertensive ESRD in non-diabetic African Americans. These findings show that polymorphisms in this gene may be associated with a susceptibility to develop severe disease.

Just a word concerning the Denys-Drash syndrome and Frasier syndrome which are associated with mutations in the gene WT1 encoding a transcription factor.
Denys-Drash syndrome is characterized by the association typically of male pseudohermaphroditism, high risk for nephroblastomas, early onset nephropathy, rapid progression to ESRD. And the lesion is that of diffuse mesangial sclerosis (as in PLCE1 mutation) which is characterized by the diffuse increase in mesangial matrix associated with hypertrophy of podocytes, and progression to complete sclerosis of the tuft always surrounded by these large podocytes.

One interesting feature is that very often in the superficial cortex of the patient with Denys-Drash syndrome there are small immature glomeruli progressing to glomerulosclerosis suggesting that perhaps in this disease there is a defect in glomerular development associated with the defect in the podocyte leading to nephrotic syndrome. As you know, the incomplete phenotype of DDS is essentially seen in females who have normal genital development.
In this disease the genetic defect is mutation located within exons 8 or 9 of WT1, changing the zinc-finger structure of the protein and modifying its binding to DNA and its target genes.

Frasier’s syndrome is linked to WT1 mutation. It is also characterized by male pseudo-hermaphroditism, with complete sex reversal in this case and streak gonads, risk of gonadoblastomas and progressive glomerulopathy which is often the first symptom of the disorder because the patients look strictly normal females, proteinuria usually occurs before the age of 6 years, then nephrotic syndrome develops and ESRD is usually present between 10-35 years of age. On initial renal biopsy, glomeruli look normal then this type of glomerulosclerosis developed. And as was said by Agnes Fogo before, there is diffuse alteration of the GBM in Frasier, as in Denys-Drash syndrome.

Here, the genetic lesion is in the splice II domain of the gene and results in the absence of three aminoacids. This mutation demonstrates that the strict equilibrium between the different isoforms of WT1 with or without KTS is necessary for normal kidney and genital development.

In conclusion, FSGS is a non-specific glomerular lesion. It is observed in a large number of quite different diseases. Morphological analysis and identification of the different variants may orient towards the basic defect. However, clinical and family investigations are of the highest importance to recognize the cause of the disease, the development of FSGS being rather a marker of the severity of the disease. In primary FSGS the best prognostic marker remains the answer to steroid treatment.

In the past few years the molecular genetic basis of several steroid-resistant nephrotic syndromes have been characterized and most defects affect genes encoding podocyte proteins of the slit diaphragm or the cytoskeleton. Their identification led to a better understanding of the podocyte function and its major role in the control of the glomerular filtration barrier.

A nice model of the structure of the slit diaphragm has been proposed by Kerjaschki and was shown yesterday by Vivette D’Agati
And I would like to emphasize that most histological lesions in genetic diseases of the podocyte are unspecific.

Molecular genetic investigations are difficult because of the genetic heterogeneity of the disease, however the identification of the genetic basis and the detection of mutations in patients are useful at the clinical level because they lead to the absence of prescription of unnecessary immuno-suppressive therapy, the prediction of the absence of recurrence of the disease after transplantation and the possibility of genetic counselling and prenatal diagnosis.
This slide reminds us that mutations in the four genes are detected in approximately 85% of patients with congenital or infantile steroid resistant nephrotic syndromes, so it is necessary to look for them.

From a practical point of view in congenital nephrotic syndrome the first gene to be tested is NPHS1 then NPHS2 and LAMB2. In steroid resistant syndrome occurring before the age of 15 years, NPHS2 is the first to be tested. In diffuse mesangial sclerosis NPHS3, WT1 then LAMB2 and in steroid nephrotic syndrome occurring in adults, what we do in our lab, is to look for the podocin variant R229Q and if the variant is present, we test for a second pathogenic mutation.