Slide 1

Well thank you very much Eric. What I’m going to do for the first 5 minutes is just give you a little, I guess a fun clinical overview of some of the entities that we’re going to be considering in the next hour and a half and then I’m going to go straight into my own talk and tell you for about 20 minutes about our efforts to try and unravel some of the genetic bases of the conditions that we’ll consider this afternoon.

Slide 2

So I think the title that the ERA-EDTA has given this session is this acronym CAKUT, which is a congenital anomalies of the kidney and urinary tract. It’s quite a nice caption phrase to describe really all of the congenital malformations that we see starting from the kidney, going down through the ureter, the bladder and even going down into the urethra. My knowledge of this is mostly from the United Kingdom prospective but I know that the story is very similar in other Western countries although we know very little about the epidemiology of these type of disorders further a field. But in the U.K., as many of you know, there are probably a little over 30.000 people with renal failure severe enough to need dialysis and transplantation and actually about 1.000 of those are children or teenagers. The commonest cause of end-stage renal failure in children and teenagers is CAKUT or congenital anomalies of the kidney and the urinary tract. Now that puts it perhaps at a couple percent of the total end-stage population in any Western country but that almost certainly underestimates the importance of these entities because you have to remember that many children grow up, they no longer die necessarily of their end-stage kidney failure and at some point they get transferred to adult care and also I think many of you will appreciate that even if you’re born with a congenital anomaly of the kidney and urinary tract, you may not, you may escape from going into end-stage renal failure in childhood only to present much later, perhaps as a young adult or even at the other end of life but the primary disease is having been born with an abnormal renal tract.
So I would guess that perhaps 10% of people with end-stage renal failure in the U.K. and other Western countries probably have renal failure on this basis.
So how do we divide up the classification of these disorders? Well, really the classification is a very histology based one and one always has to remember in clinical practice we hardly ever have access to histology. So some of the terms we use particularly to describe the kidney malformations I would say are our best guesses. We talk about hypoplasia that means kidneys with fewer glomeruli or fewer nephrons than normal, differentiation otherwise appears to have been normal but one is left with significantly fewer nephrons. Now unfortunately, thus far no one has invented a way to measure the number of glomeruli and nephrons in a living subject. But we call something renal hypoplasia, if we se a child or an individual with two small but well formed kidneys, which are functional on a renogram, for example, but they are smaller than normal. Then one has this entity called dysplasia well again another real histology diagnosis, it means if one could look inside the kidney, one would see immature structures or metaplastic structures. We very really have histology on a living subject but we use the term dysplasia when we see a kidney of abnormal shape, it can be larger or smaller than normal, sometimes containing cysts and often it has very little or no excretory function. Then the most severe in this category is agenesis, the kidney is not there at all.

Slide 3

Of course, as you know as well, renal anomalies are often associated with abnormal lower urinary tracts and this afternoon you’ll hear a talk of vescicoureteric reflux, maybe a little but about duplicated renal tracts and certainly about obstructed lower urinary tracts.

Slide 4

Just a few pictures to give you a flavour of what we’ll talk about again. This really is the most gross example of the dysplastic kidney spectrum. Let me show you here, this is a normal kidney on that side with some foetal lobulations and this very ugly beast here is what we call the multicystic dysplastic kidney. The kidney is full of abnormal undifferentiated tissues separated by massive cysts. This occurs in about 1 in 5.000 births. We can detect these things antenatally.

Slide 5

Here is a picture of a multicystic dysplastic kidney in a routine scan from a mid-gestation foetus and by isotope renography or IVP these entities classically have no function at all.

Slide 6

A very interesting thing about these cystic dysplastic kidneys that has become evident with the advent of serial routine antenatal scans is these enormous structures can involute and almost completely disappear at least they can no longer be detected by ultrasound. So there is an evolution occurring in these structures over the antenatal period and in the few years after birth. Probably many individuals who we say have got unilateral renal agenesis actually had a multicystic dysplastic kidney that has regressed.

Slide 7

We’ll certainly hear more about primary vescicoureteric reflux said to occur in about 1% of all young children and depending on which renal registry you look at said to account for between 5 or 10% of not only children but also adults with end-stage renal failure.

Slide 8

Slide 9

Finally, you’ll definitely hear more about this. Posterior urethral valves still continues now to be very, very important of end-stage renal failure in children and accounts for about a quarter of all boys with end-stage renal failure. The urethra is obstructed here by a valve. There is this characteristic key-hole appearance of the beginning of the urethra and a thick walled bladder.

Slide 10

And this key-hole can be seen on the classical micturating cystogram, one can also see it on foetal scans here and sadly a few of these children continue to die with the best medical care and on an autopsy one can see the hypertrophy of the bladder with the urethral valve here.

Slide 11

So that was a little overview and now going into really for the next 20 minutes the main part of my talk. The hypothesis that many of us are working on is that many or most of these individuals will have their disease caused by mutations of genes that are active during normal renal tract development. Some evidence is emerging that this is indeed the case. It’s a very big field, we don’t have time to cover it all. I want to give you a flavour though of the breadth of the field. I want to tell you about recessive multiorgan syndrome that causes renal agenesis and dysplasia. I want to tell you about a couple of dominantly inherited renal malformation syndromes that cause renal hypoplasia and dysplasia. I then want to go into perhaps the more interesting concept to us as nephrologists and also urologists that congenital kidney disease is just confined to the urinary tract and not part of a multiorgan syndrome may also have a genetic basis. We’ll talk a bit about primary vescicoureteric reflux and renal hypoplasia. I think I won’t really have time to talk about the last two lower points, environmental influences and future perspectives about manipulating the renal tract but what I don’t want you is to go away from the talk thinking that genes are the only factors here. Almost certainly the answer to any individual disease will be an interaction of one or several important genes with the environment as well and we now know that the foetal milieu provided by the mother is very, very important in determining normal kidney development. For example, what the mother eats in terms of protein, vitamins, exposure to theratogens and so on will also be important but I won’t have time to cover those aspects.

Slide 12

Just a couple of slides here about renal developmental anatomy to show you these sort of things we’re thinking about. On the upper left here is a human normal foetal kidney about 6 weeks gestation. You can see a few branches of the ureteric buds with the little nephrons forming. A few months later in gestation you’ve had this enormous growth and differentiation but we mustn’t forget about the lower urinary tract as well because it’s developing really concurrently with the kidney.

Slide 13

And with the bladder as well at 6 weeks gestation one has a urothelium surrounded by mesenchyme and these two are going to talk to each other to become the pseudostratified urothelium and the lamina propria and the detrusor. Both in the kidney and in the ureter and in the bladder itself we have this theme of interaction between epithelium and mesenchyme and we now know that these two types of tissues talk to each other by releasing locally acting growth factors. They secrete matrices and they nurture the growth and differentiation of their partners.

Slide 14

Some cell death is very important in normal kidney development. Someone estimated that about half the cells that are born in the developing kidney are destined to die before birth. Deregulation of apoptosis is a theme that occurs in many congenital anomalies of the kidney.

Slide 15

So let’s go straight into some perhaps rather rare multiorgan syndromes that really are shedding some light on a genetic basis of normal and abnormal kidney development. If you go to the online Mendelian inheritance in man website, the McKusick website (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM), and look up recessive genetically defined multiorgan syndromes involving the kidney, you will find there are tens if not over a hundred and I just want to tell you about one over the next few slides to give you a flavour of this. This is a syndrome called Fraser syndrome not to be confused with Frasier syndrome that is something totally different, it’s to do as you know with the glomerulus. This is F.R.A.S.E.R. syndrome.

Slide 16

This is an autosomal recessive disorder and it’s characterised by the unfortunate individuals having a membrane across the eyes, fused fingers and fused toes, there’s soft tissue fusion between the digits and there is very commonly absent or dysplastic kidneys. One of the genes that is mutated in this syndrome codes for a protein that coats the surface of embryonic renal epithelia and probably mediates the cross-talk between epithelia and mesenchyme and in mice that have no FRAS1 gene that is the gene that is mutated one can see the kidneys beginning to form and then they involute by fulminant apoptosis.

Slide 17

Most of these individuals actually die soon after birth but a few of them who have not such severe kidney disease do grow up and this one of the first patients described by the clinician George Fraser. You see the membrane across the eyes. There are eyes behind it, this is a just a soft tissue membrane and the fusion between the toes and the fingers.

Slide 18

And there was a very good review recently of over 117 cases and as well as the cryptophthalmos and the syndactyly practically all of these individuals have very severe renal malformations.

Slide 19

Most of them have bilateral renal agenesis but all of the others have dysplasia or unilateral agenesis, so the kidney is a very major part of this -- recipe.

Slide 20

There are strains of mice amazingly that had exactly the same mutations as humans do with Fraser syndrome, so on the top left here is a human with Fraser syndrome with the membrane across the eye. Here is a mutant mouse, human up here in B, mouse in D.

Slide 21

Mutant mice, if one looks at a neonatal mouse, a newborn mouse should have 2 kidneys like this but a mouse which has no active FRAS1 gene usually has bilateral absence of the kidneys. What’s quite interesting it’s not because the kidney was never there at the beginning, it was there at the beginning in mid-gestation it tries to begin to form and then it falls apart.

Slide 22

If one looks at the embryonic kidneys of mice with this syndrome, one finds a little embryonic kidney with many picnotic nuclei that are undergoing fulminant apoptosis. This is a phenomenon of a disappearing kidney.

Slide 23

The Fraser syndrome gene is quite interesting it’s a little bit like polycystin, it’s got a transmembrane domain and then a very long part of the protein that sticks out into the extracellular matrix and probably interacts with growth factors and other matrix components.

Slide 24

So that was one recessive syndrome now two very quick examples of dominantly inherited kidney anomalies in association with abnormalities of other organ systems. Renal cysts and diabetes syndrome and the renal coloboma syndrome. You may have some of these patients in your clinics. They certainly are there in the adult kidney dialysis clinics.

Slide 25

Renal cysts and diabetes syndrome was not recognised beyond I think maybe 5 or 6 years ago. But we think actually it’s quite common. In our 200 patients with chronic renal failure in our paediatric hospital we know of about ten families with this syndrome. It’s actually rather common. The renal phenotype is fantastically variable, renal agenesis, cysts, dysplasia, hypoplasia but the clue is that there maybe other family members with diabetes or uterus malformations and these individuals have mutations of the transcription factor called hepatocyte Nuclear Factor 1 β.

Slide 26

There are many reports in the literature now of these families but I wanted to highlight the work of Coralie Bingham in Exeter U.K. who I think did a lot of the early work to put the syndrome on the map. Here is a multi-generation family with dominant inheritance. This individual here had 2 small kidneys. This individual in generation 3 had a solitary functioning kidney. Then in generation 3 you have a cystic renal dysplasia phenotype. The same mutation all the way down the generations very different phenotype, quite common, of course, in many dominant disease including this one.

Slide 27

The HNF1β gene is expressed in the branching ureteric bud and in the embryonic collecting ducts. Probably this explains the importance in renal development.

Slide 28

Now another one you may have seen in your clinics and has been highlighted again I think for about 10 years now after its discovery was the renal coloboma syndrome. Here there’s a mutation of a transcription factor called PAX2. PAX2 like HNF1β is expressed in the branching ureteric bud and collecting duct system. These individuals have renal hypoplasia and sometimes have vescicoureteric reflux. They’re often blind because they have a malformed optic disc but many of them have very subtle eye disease and you probably have seen some of them.

Slide 29

O.k. now going on to what I think is actually more interesting for us as nephrologists and urologists. Most of our patients I think you’ll agree do not have overt multiorgan malformation syndromes. We’re dealing with individuals with congenital disease localised to the kidney, ureter, bladder or urethra. Could these two, could these people who are rather common have a genetic cause for their disease? I think the answer is probably yes. I highlight 2:2 reflux and renal displays here and renal hypoplasia.

Slide 30

Just starting with the second one. This is really an extraordinary family that one of our registrars Larissa Kerecuk has put together and you can see here over 3 generations we have 8 affected individuals, very, very variable phenotype again, again characteristic of dominantly inherited diseases. The index case here was a baby born with dysplastic kidneys with chronic renal failure. She had had two sibs with Potter’s sequence born died soon after birth with very small malformed kidneys but several others in the kindred here not so severely affected. In this single family you see individuals so badly affected they could never survive, others that went into chronic renal failure when they were in childhood and yet others who only presented in their 20s and 30s and 40s with renal impairment and proteinuria. A very nice example of CAKUT that can present to the adult clinics and its escape problems in childhood. 

Slide 31

What about primary vescicoureteric reflux? As I said very common, 1% of all children have it. When I went to medical school we were never told this could be a familial disease but it certainly is. There was an excellent review by Hollowell a few years ago and they worked out, out of about 1768 individuals who were siblings of index cases, the siblings were screened with cystogram, about 1/3 of them had reflux as well. And we think of primary VUR now as a familial disease probably with a dominant inheritance pattern but a very variable severity and expression pattern.

Slide 32

If you don’t believe it, well of course, we only show, always show the most extraordinary families. This kindred here we have 7 out of 13 individuals from our clinic in London affected by VUR and/or reflux nephropathy.

Slide 33

So one candidate of gene for this came up a couple of years ago and these are the uroplakin genes. Uroplakin genes code for proteins that coat the surface of the urinary tract, the bladder, the ureter and the renal pelvis. They form an asymmetric unit membrane that stops urine back-leak from the bladder and ureter into the rest of the body. Here is the protein on the surface of the foetal bladder.

Slide 34

What caught our interest is that mutant mice have very, very severe VUR and congenital hydronephrosis. Here on the right we’re looking up into the ureter from the bladder of a newborn mouse and you see this golf-hole ureter orifice rather like some humans have with severe primary reflux.

Slide 35

Well, it turns out that the uroplakins are probably not going to be the answer for the everyday primary reflux we see in the clinic but occasionally one can find de novo mutations of the uroplakin genes that cause severe reflux and severe renal failure. This is one such case here that was written up in the JASN last year.

Slide 36

So what we and I think other people have decided to do with the primary reflux condition, we think that this is going to have rather complicated genetics, there maybe several loci perhaps interacting in any one individual. So in the U.K. we’ve made a very large collection of affected families over the last few years. We now have over 250 sibling pairs and we’ll be going on to a whole-genome search using this facility. We’ll see what loci or genes fall out.

Slide 37

Now really sort of coming to the end of my bit. But I said I’d mention duplex kidneys and duplicated urinary tracts. Again one can find families where the disorder seems to track in an autosomal dominant manner although sometimes it seems to be able to miss a generation, so the severity is very variable. There are lots of mouse mutants out there which get duplicated urinary tracts and we’ve been diligently sequencing these genes in human families such as this but not really come up with anything as yet.

Slide 38

So I’m going to finish there because I won’t have time to talk about environmental influences but just to remind you what I talked about. I told you a little bit about recessively inherited multiorgan syndromes. I talked about 2 dominantly-inherited multiorgan syndromes. I introduced the concept that some more common renal anomalies like primary reflux may have a genetic cause all be it the genetics maybe rather complex but don’t forget that environmental influences are going to be important as well and perhaps will modulate the severity of disease in some of these genetic disorders.

Slide 39

This is my last slide I think. These are I think the important areas to be working on over the next 10 years. Defining genes in non-syndromic human renal tract malformations, making large connections of patients is going to have to be on an international scale followed by genome searches for loci, perhaps we’ll be looking for things like very small microdeletions. I think, I’m a real believer that we should be establishing clinics where you have a nephrologist, urologist, clinical geneticist and maybe a foetal medicine expert all together because you need a lot of different expertise to be able to diagnose some of these syndromes but I think that’s no excuse for not trying to do it. I think in the future we’re going to be isolating and manipulating human kidney and bladder progenitor cells and trying to understand interactions between the genome and environment.

Slide 40

Well, I’m going to stop there. This is a lot of work from a lot of people in our college, University College London and elsewhere in the U.K., elsewhere in the world. I’ll stop there. Eric thanks.

Slide 41

Chairman: Thank you for this elegant overview. Are there any questions in the audience? Yes please.

Question: It was very nicely presented. I think I would like to come to one point. What the real reason is? If you look, let’s say for the ESCAPE study, where 400 patients are included, 70% of these patients have renal hypoplasia in one or the other forms. If you look for the genetics, then 10% have the monogenetic disorders you presented. Other 15% have point mutations from which you can’t say at the beginning are they causing the disease or not but most likely they do because in the control group there are no such anomalies. However then we must develop a concept of how this is working. Also the diversity of clinical manifestations and so it seems not to be in the majority that one gene is only working.

Prof Woolf: Yes. I’m glad you brought your brilliant ESCAPE study and this is exactly the kind of study that should be done on very large numbers of clinically well-documented patients. I think you know I totally agree with you that the genetics may turn out to be rather complex. I mean, there are many genes acting in nephrogenesis that are yet to be discovered. They could explain some of the other patients but again we could also be looking at sometimes the interaction of more than one gene giving the disease in a patient and environmental factors as well. I think it’s going to be a really big challenge unravelling this and we’re going to need a lot of help from genetic departments not just to find the genes but in terms of genetic counselling as well. Because when we find these mutations or genetic variations we need to think very carefully about what advice we give to the individuals about what is the risk when they start to have their own family. We may overestimate the risk. Maybe it’s less than we think.