Slide 1

Ok thanks very much for asking me to give the talk. Can you turn the lights down a little bit? You’ll see the colour better. Thanks. I work in an Institute of Child Health at University College London with my colleagues at Great Ormond Street Hospital in London. I’ve been asked to give you a very quick tour through some renal dysplasia syndromes associated with specific gene mutations. So, the first few minutes of the talk is just telling you about the range of renal malformations and a little bit about how the kidney and the ureter develop. Then the rest of the talk I’m going to share with you several examples of individuals that we’ve seen in our clinics who turn out to have some of these syndromes and I may have too many examples but you just cut me off when I have to stop. If I don’t get to the end, it doesn’t really matter.

Slide 2

So, these are the sort of diseases where thinking about on the left you have a normal formed kidney with a nice medulla, cortex and lots of glomeruli. The most mild renal malformation would be hypoplasia where you have too few numbers of glomeruli. Then we go into more severe entities, cystic dysplastic kidneys that maybe have a little bit of excretory function or these nasty big multicystic kidneys that have no excretory function or the most severe is one I can’t draw a picture of because it’s renal agenesis where the kidney never forms at all.
Rarely we’re able to have pathological specimens of these kidneys and the hallmark of dysplasia is seeing undifferentiated tubules surrounded by a sort of mesenchymal stromal cells.

Slide 3

Now, this symposium is called paediatric nephrology but like the last speaker I’m a great enthusiast in thinking that knowledge of these type of syndromes is not just relevant to children or paediatricians because these kids are increasingly growing up, they often even in renal failure are being saved now by advances in dialysis and transplantation and I believe that there are many individuals on adult chronic renal failure programmes and on end stage programmes that have end stage renal failure on the basis of malformations and it may not always be recognised as such.

Slide 4

So a bit about the normal anatomy of the kidney development. Here is a human embryonic kidney at about 6 weeks gestation. We see a few branches of the ureteric bud and a few little nephrons beginning. Then if we look at about half way through gestation, there’s been enormous growth, enormous amounts of nephrogenesis and collecting duct branching. When I was sort of getting interested in the area of kidney development about 18 or 19 years, there were no genes known to be associated with kidney development.

Slide 5

I think that the first one that came out was in 1991, as many of you will remember, when the Wilms’ tumour 1 gene was described and this is thought to be an important player in normal kidney development. Mice that lack WT1 never form the kidney at all.

Slide 6

It all seemed very simple then, it was just one gene but we now know that probably hundreds and thousands of genes are active in normal nephrogenesis. My colleague called Paul Winyard in London likes to take out, to grow normal embryonic kidney tissue from terminations and you can grow cells from these kidneys and start looking at gene expression in normal human embryonic kidney cells and well here are a few of them.

Slide 7

There are hundreds and thousands of genes that are active in normal kidney development in the human and perhaps mutations of any one of these would give you a kidney malformation.

Slide 8

But as nephrologists, we mustn’t forget that the urine has to get from the kidney to the outside of the body and it travels through the ureter and the bladder. Here is an embryonic bladder, again at about 7 weeks gestation there it’s just a simple epithelium and some mesenchyme around it and over the next months this forms a proper bladder. The ureter develops in a very similar way with a very highly specialised urothelia that I’ll talk about a bit more later but also a very specialised muscle layer.

Slide 9

Ureters are actually very interesting, they’re not just rigid tubes and embryonic ureters are intelligent organs and they massage the urine by peristalsis from the kidney down to the bladder. This is an embryonic ureter grown by a PhD student in our lab. Here is the bottom of the ureter, here is the top. In panel B you see a contraction starting at the top. In panel C it’s travelling down the ureter. Panel D it’s at the bottom of the ureter and presumably without these waves of normal peristalsis in development, you will end up with a functionally obstructive kidney.

Slide 10

Just as in the kidney many genes are active in nephrogenesis in the ureter, probably many hundreds of genes are also active and this is a microarray study actually in mice from a lab in Australia that shows over 200 genes are upregulated as the normal ureter develops.

Slide 11

So we have many candidates to think about. So now let me go through the cases that I wanted to share with you to give you a flavour of some of the genes that can be mutated. In each case there’s a clinical history and then, I’ll tell you what the gene mutated is.

Well this girl is now 11 years old and she had an antenatal diagnosis of a unilateral multicystic dysplastic kidney. After she was born, in the first few years of life her GFR was about 60 ml/min.

Slide 12

The multicystic kidney involuted, as they do often and the kidney opposite it obviously wasn’t normal. It was in the normal range of size but if you or I have a normal solitary kidney, it should be bigger than normal. So she had bilateral kidney disease. This is what her multicystic kidney would have looked like before birth. This is not hers but just to show you what these things are like.

Slide 13

Then as she went through childhood, she put on a bit of weight and then she became overtly diabetic. She walked into the clinic one day when she was 11 or 12 and said ‘oh I’ve lost 3 kg since the last visit. I’m very thirsty, I’m passing lots of urine.’ She had developed severe diabetes mellitus. Now she turns out to have a heterozygous mutation of a gene called hepatocyte Nuclear Factor-1β and if people tell you having a single kidney doesn’t affect your life, this is not true because she then went on and had an episode of severe diarrhoea and vomiting and went into acute renal failure and then she has been on dialysis.

Slide 14

Now what are these HNF1β genes? There are two of them 1β and the other 1α. 1α I’m not going to talk about but 1β is the thing we’re interested in as nephrologists. HNF1β is a transcription factor, it’s expressed in the normal developing kidney but also in other organs such as the pancreas. When the gene goes wrong, you get kidney malformations and you can develop diabetes mellitus as well.

Slide 15

This is a knockout model where the gene was specifically knocked out in the collecting ducts in the medulla of mice and you get a type of cystic disease in mice. The HNF1β transcription factor usually upregulates other genes.

Slide 16

One of the genes it upregulates interestingly is Pkhd1 that is the gene mutated in humans with autosomal recessive polycystic kidney disease. So in the kidney HNF1β is trying to tell tubules to remain differentiated and don’t form cysts.

Slide 17

HNF1β is expressed in a normal human foetal collecting duct system.

Slide 18

Now, in the last 7 years we’ve been beginning to test for this gene in our patients we see in London in our Chronic renal failure clinic and we believe that this probably must be the commonest gene mutation we get in children with kidney malformations. We’ve found so far 22 index cases positive out of 91 cases we have tested.

Slide 19

The phenotype in the renal disease is very variable. Initially when this was described 7 years ago, it was called the renal cyst and diabetes syndrome but we find the renal phenotype doesn’t only include cysts, you can have dysplastic kidneys, bright echogenic kidneys and we also have a few children with hydronephrosis and megaureter with HNF1β mutations.

Slide 20

Ok let’s go onto another gene, another child. This was a girl, now 2 years old she actually had a normal antenatal renal scan but soon after birth she was unwell. She was found to have a raised blood creatinine and an ultrasound showed two small kidneys, we presume hypoplastic kidneys. She had visual impairment and abnormal visual evoked potentials

Slide 21

 I don’t have a picture of her own fundus but it would have looked like this.

Slide 22

At the back of both of her eyes she had flat pale optic disks, she had bilateral optic nerve colobomas and she turns out to have a mutation of another transcription factor, Paired box 2 gene and mutations of this gene were described about 12 years ago as causing the renal coloboma syndrome. The renal disease is again quite variable. Kidneys are usually hypoplastic and often there is associated vescicoureteric reflux. Her father has abnormal optic disks and he’s awaiting a renal ultrasound.

Slide 23

The PAX2 gene is expressed in the normal human foetal ureter and collecting duct system in a pattern not dissimilar from HNF1β.

Slide 24

Ok, family 3. This is a boy now 5 years old. He presented with scaly dry skin, icthyiosis and undescended testicles. He was found to have a hypoplastic left kidney and two of his mother’s brothers also had icthyiosis. One of his uncles had a solitary functioning kidney and went into end stage renal failure in his 20s and died of a heart attack quite recently.

Slide 25

Now, the index case and his two uncles have what we call Kallman syndrome. Theirs is x-linked, it’s x-linked recessive and the mutated gene is expressed in the ureteric bud system but also in the front of the brain explaining the fact that these patients have no sense of smell and they also have hypogonadotrophic hypogonadism and poor fertility. In this case the icthyiosis, this dry skin was caused by a contiguous gene deletion of the steroid sulphatase gene.

Slide 26

This is a work from Paris several years ago that immunostained normal human embryos with an antibody to the protein coded by the Kallman gene protein called anosmin. You can see it beautifully coating the surface of the ureteric bud but very interestingly, it’s also expressed in a basement membrane like pattern in glomeruli and perhaps one can conceive two hits here, either if you have a mutation maybe the kidney doesn’t form at all or if it does form, you later go on to get glomerular disease and progressive renal failure because the gene is also needed to maintain glomeruli.

Slide 27

Ok family 4. A girl presented to us at 2 years of age. She had hidden eyes. She had membranes across her eyes technically called cryptophthalmos. She had a web across her larynx, fused fingers and toes, abnormal genitalia and a malformed hind gut. She had a solitary pelvic kidney and a previous sibling never made it to birth. This sibling was terminated with bilateral renal agenesis. She has the so-called the Fraser syndrome not to be confused with the Frasier syndrome.

Slide 28

Fraser syndrome is rather rare, about 1/20,000 live births. It’s autosomal recessive, you have cryptophthalmos, syndactyly, abnormal genitalia but all the patients essentially have renal malformations.

Slide 29

A quarter of them have bilateral agenesis and nearly all of the others have dysplasia or unilateral agenesis.

Slide 30

This is one of the longer surviving Fraser syndrome patients. Actually one of the first cases described by Doctor Fraser in England. Very rare for them to survive and you can see she still has cryptophthalmos, fused fingers and toes. A few years ago Peter Scambler in our institute found the gene that was mutated. It’s a very large gene that sits in the cell membrane that coats the surface of renal epithelia and a bit like polycystin it has an enormous extracellular portion that presumably sticks out into the extracellular part of the tissues and may interact with growth factors there or matrix molecules.

Slide 31

There are mice that have mutations of the Fraser gene and instead of their kidneys normally starting to develop midway through gestation the kidney rudiment just begins to form and then it involutes, it commits suicide via massive apoptosis.

Slide 32

Family 5, can I go on for a bit maybe? A white female. By day 3 of life her plasma creatinine was very raised; 378 µM. Imaging failed to detect the right kidney. The left kidney had abnormal cortical echogenicity and its malformed pelvis was connected to a tortuous ureter with severe reflux. The bladder interestingly had two diverticula. Aged 12 years of age she had renal failure. Still she’s quite rather stable actually treated with vitamin D and Epo etc. She has no other syndromic features but she has a de novo mutation of a gene called UPIIIa.

Slide 33

What is UPIIIa you ask? Well, it’s one of a member of uroplakin proteins that coat the surface of the renal pelvis, the ureter and the bladder. Here is an embryonic ureter grown in culture by one of our PhD students Clare Gannon, she’s stained it for uroplakin in green its beautiful sinuous structure, smooth muscle actin in red.

Slide 34

If you are sitting in the middle of the ureter or bladder looking at the surface of the urothelium with an electron microscope, uroplakin proteins would appear here in plaques and they coat the surface of the urothelium and prevent the ingress of urine back into the rest of the body.

Slide 35

There’s a family of uroplakin proteins that I’m not going to talk about but this girl had a mutation of uroplakin III a and part of the molecule sticks out into the urinary lumen and part of the molecule is cytoplasmic and has got also phosphorylation sites that are sometimes disrupted by mutations.

Slide 36

A very brief bit of cell biology here. Our collaborator Jenny Southgate in the University of York has been transfecting wild type uroplakin into normal human urothelial cells and you can see that the red uroplakin appears on the apical surface of these cells but if you transfect the missense mutant in these cells, it appears to be retained as blobs within the urothelial cells and never makes it to the cell membrane. So this is why I say we as nephrologists shouldn’t forget the lower urinary tract and the ureter and the bladder

Slide 37

Do I have time for a bit more?
This is a mutant mouse from Henry Sun’s group in New York. The mutant uroplakin III mouse has a massive big hole where the junction of the ureter and the bladder should be and they get vescicoureteric reflux and hydronephrosis.

Slide 38

This is the last family I’ll show you before I stop. We don’t have an answer for this family but the index case is now 7 years old. She presented with oligohydramnios. After she was born it was found she had severe renal failure with scans compatible with renal hypoplasia or dysplasia.

Slide 39

One of our research fellows Larissa wrote up this family last year, this is the index case here. She has two previous siblings that died with bilateral renal agenesis. She has one brother with a small hypoplastic kidney. Her father has hypoplastic kidneys. Their grandfather now dead on dialysis had a solitary functioning kidney. He married twice. His daughter here has renal hypoplasia and her son has received a transplant, he had renal dysplasia. They have no syndromic features at all. This is an autosomal dominant gene travelling through the family that causes renal hypodysplasia.

Slide 40

We analysed PAX2, HNF1β and other genes, the sequences are all normal. This is probably a new renal malformation gene waiting to be discovered.

Slide 41

Last slide here. Sometimes when I talk to my colleagues in London about genetic testing, they are a bit cynical. They say well you can make a genetic diagnosis but what’s the point? It’s not going to help in the treatment of these children. My own feeling is the main reason to make a genetic diagnosis is to give the parents and later on the child a reason why the kid has been born with sometimes very malformed kidneys. But it does open up a Pandora’s box which I don’t have time to talk about today and that Pandora’s box is one of genetic counselling of the kid and also of the wider family.

Slide 42

That’s why I believe that if a nephrologist undertakes this sort of work, it should be in conjunction with clinical geneticists and that’s why I work with our clinical genetics team Raoul Hennekam, Maria Bitner and others at the institute because I think this is a great area where’s it’s too much for a nephrologist to do. You have to work with other specialists as well. I’ll stop there. Thanks.

Slide 43

Chairman: Yes we have time for a couple of questions. Please there’s a microphone there.

Question: Thank you. What is known about the process of involution of the multicystic dysplastic kidney? What cell biologic process? And what can be learned about renal development from the fact that it’s unilateral and not bilateral?

Prof Woolf: Yes, well about 10 years ago one of our research fellows Paul Winyard, he took samples of multicystic dysplastic kidney either antenatally or ones that were removed postnatally by the surgeons and if you try and count the incidence of apoptotic cells, it’s higher than in tie matched normal kidneys that you can get. So there’s certainly apoptosis going on and that probably contributes to the involution as well. There are some interesting features about these massive multicystic kidneys. I don’t think there is any animal model which really models them that well. These can be enormous things that fill the whole abdomen. The other question that you raise is why are they often unilateral, how can we have a genetic defect present in every cell of the body and just have one side affected? Well in answer to that I think that often the other side is affected but in a more subtle way, maybe hypoplastic or mildly dysplastic. The other is idea that you may need a second genetic hit to get the phenotype so that in the precursor of the ureteric bud on one side of the body there would be an additional somatic mutation that summates with the germ line one to give you the severe phenotype. That’s simple speculation. Maybe it’s something to do with mitochondrial genes or yes must be telomeres.

Chairman: Ok thank you I’m afraid we have to continue.