CME Slides Forum - X. Jeunemaitre

A joint Congress by ERA-EDTA and ISN

WNK KINASES AND HYPERTENSION

Xavier Jeunemaitre, Paris, France

Chair: Xavier Jeunemaitre, Paris, France

Gerjan Navis, Groningen, Netherlands

jeunemaitre

Prof Xavier Jeunemaitre
INSERM U772
Collège de France
Paris, France

Thank you very much. I would like also to thank the organisers for selecting me for presentation of WNK and hypertension. I would like to try to convince you in a few minutes that these kinases are of interest in the field of hypertension.

The human WNK family was described after the cloning of the WNK1 by the group of Melanie Höök in 2000 and since then there have been 4 members of this family and you can see that this --- kinase pathway is a form of first small aminopeptide domain and then a large kinase domain which is relatively homologous from one to the other one and then an auto inhibitory domain and two coiled-coil domain generally except for the WNK2 and some proline-rich region which might be important to the linkage or to the interference with other proteins. The WNK motif as you can see at the bottom of the slide is corresponding to the fact that this lysine which is usually present in the MAP kinase pathway is present in subdomain I instead of subdomain II.

So as you may know, of course, genes and hypertension is a complicated story and if you want to summarise the thing there are 3 major types of hypotheses that we can make. Either there are some very rare and monogenic forms of Mendelian hypertension and we have just an example with Liddle’s syndrome. Some rare variants that might be responsible for the protection-susceptibility to environment especially salt if we are looking at nephrology. Some common polymorphisms and we have seen especially with my co-chair the fact that trying to find some loci in essential hypertension is relatively complex. Two of these hypotheses look at the WNK pathway itself.

So first the Mendelian form and you probably know the Gordon syndrome or pseudohypoaldosteronism type 2 or familial hyperkalemic hypertension which is a rare Mendelian form of hypertension with autosomal dominant transmission. We have gathered about 20-25 families in France for the moment in the last 5 years. There is a tendency for hyperkalemia despite the fact that you have normal creatinine levels, metabolic acidosis with hypercloremia, low renin and aldosterone has to be interpreted of course depending on the level of potassium. Variability of phenotype not a such high blood pressure compared to Liddle’s syndrome for example but as you can see from this family that I have just selected from ours you have in the adult some sort of light to mild to moderate form of hypertension together with hyperkalemia with a strong tendency to metabolic acidosis and hypercloremia but for example in these two children you have still normal blood pressure despite the fact that you have a complete phenotype in terms of the biological phenotype.

For all there is a very strong tendency to be sensitive to hydrochlorothiazide. This condition has been looked at in terms of genetics for several years and there have been at least 3 loci that have been determined but as you see they don’t explain all the cases and familial cases of this condition. The first one is on chromosome 1. No gene has been identified yet and the two genes on chromosome 17 and chromosome 12 are corresponding to the two types of kinase that have been described before, WNK1 for chromosome 12 and WNK 4 for chromosome 17.

Actually the WNK 1 locus was first identified in a French pedigree coming from the North of France where we had the chance to have a lot of patients with this condition with hyperkalemia and hypertension. We have linkage on chromosome 12 and working with Rick Lifton he was able to identify another family where he had a marker which was a new allele corresponding to a large deletion of intron 1 which is there and actually we found the same type of deletion in the French family. In these families we had the notion that together with this intron 1 deletion we had an over expression of the gene at least in leukocytes.

The chromosome 12 is in part corresponding to the chromosome 17 at least for this locus on chromosome 12 p and 17 q and there was indeed a gene on 17q which is WNK 4 and the mutations that have been found on WNK 4 are completely different. It’s not intron deletion that we had before but a change of residue in this acidic domain close to the coiled-coil domain 1 and you see this missense mutation found in the pedigrees from Lifton’s laboratory and also this one on the basic residue very close to the coiled-coil domain II and the hypothesis was that probably these mutations are changing the interaction with other proteins.

Actually these two kinases WNK1 and 4 are expressed not only at least for WNK1 in different tissues but mainly in the kidney in the renal tubule and in the distal convoluted tubule and in the collecting --- duct, this is CCD with the kinase domain for both of them. You see the homology between the 2, the 2 transcripts. WNK 1 is a little more complicated with two transcripts that will go further on it.

An interesting thing for this WNK1 and WNK 4 is that they are expressed in the distal tubule where you have a lot of transporters already involved in monogenic traits or in the transport of sodium or potassium or in the aldosterone machinery as has been reviewed elegantly by David -- group in Hypertension in 2007. So the hypothesis was and it has been demonstrated in vitro and in vivo that changing the expression of WNK 1 or mutating WNK 4 will then imply a change in regulation of different genes which are mainly NCC for the sodium chloride transport or ENaC or ROMK.

As you see these things are a little bit complicated these kinases are regulating factors and they can for WNK 4 interact with NNC directly or probably also with WNK 3 but not for WNK 1 which might itself interact with WNK4 and the two isoforms which are kidney specific isoforms, long isoforms possibly interacting with each other. Depending on the situation that you are looking at the distal convoluted tubule or the CNT things might be different in terms of relationship so it is a relatively complex manner and I encourage you to look at our poster or to hear Mrs Hadchouel’s talk tomorrow about the knockout of the kidney specific isoform and see its consequences.

The WNK 1 promoter has actually two promoters and then depending on these two promoters we end up with the two kinases, a short kinase deficient kidney specific isoform and a long kinase full length which is expressed in all tissues, in kidney of course but also in heart and brain and different tissues, it’s relatively ubiquitous.

The thing is that when trying to explain a little bit the mutation, the mechanism of mutation in WNK transcript is that when you’re looking at the different species especially human and mouse for example you find that the intron 1 is relatively large in both cases, 60 kb in humans, about 30 kb in mice and this long intron favours probably the fact that you might have different regulations between the proximal promoters and the renal promoter which is located on Exon 4 A which is there and there and this type of structure is true in different mammalian species.

What we found in terms of deletions of the PHE two families or Gordon syndrome familial or FHHt families are these large intron 1 deletions corresponding to a 20kb minimal region itself containing conserved regions when you look at all the species differently from intoron1

and so we have entertained some research on C1-C3, C4, C5 to see if each of these domains was corresponding either to an answer element, a repressor element or a insulator element. I’m not going to go through all these details but just to tell you that some of these regions C1 and C5 especially tended to prove in vitro that there was some repressor element in the C1 conservative region and some insulator element in the C5 conservative region.

The hypothesis for the moment is that the mutation leading to Gordon’s syndrome in the WNK1 was corresponding to at least the deletion of these two elements C1 and C5 and that instead of having a proximal promoter and a renal promoter which are independent one from the other deleting this part is going to break this insulator sequence and allow also the proximal promoter to play a role on the renal promoter and vice versa.

At the end of the day when you are looking at the kidney itself and the renal tubule, probably this mutation is going to change the ratio between the long isoform, the kidney specific isoform and then to have some consequences on the expression of WNK 4 and then on the activation of NCC and maybe it’s true also for ENaC and ROMK but we have to take into account that these two isoforms are not expressed in the distal tubule similarly kidney specific isoform only in the DCT, long isoform in the whole tubule but at the lower level.

Very briefly, I would like to show you how polymorphisms on this WNK1 isoform could play a role possibly on blood pressure.

You know by different approaches that finding QTLs for hypertension has been very difficult. It was reviewed two years ago by just the familial studies and -- pairs and you see that you have a lot of spots very difficult to find and to see if these different spots are true or not.

You probably remember this first study performed by the Welcome’s trust with a GWAS analysis where instead of having different loci for complex thread like for example, type 1 diabetes, type 2 diabetes or Crohn’s disease, for hypertension per se it was very, very difficult to find anything and the two GWAS studies which have been recently performed in several thousands of people confirmed this that there is no strong locus corresponding to hypertension. So if we are looking at essential hypertension per se, we are going to find only changes which will explain a few mm of molecular difference from the cases from those who are bearing or not the mutation.

A first effort was performed by Mark Caulfield and Pat Monroe in the Bright study they had more than 700 families and they wanted to tag the WNK gene with 8 SNPs corresponding to the major information in terms of genetic information to that gene. As you see, they had nothing in terms of association which is significant when we are taking essential hypertension as a trait but the tendency for at least this polymorphism to be associated with systolic blood pressure at a very low level. The blood pressure analysis and it has been published recently in PLoS ONE with a much larger effect with the BIRGHT study, with the LOLIPOP study, with the Whitehall 1 and II, HYPEST and ELSA and Olivetti study and you have the number there several thousands there and several hundreds for the other ones and the mean age of these people.

You have the effect of the men SNP of the WNK 1 that has been found on systolic blood pressure, on diastolic blood pressure and you see that you have an odds ratio which is a total relatively positive but again very mild compared to what you can expect of course from a monogenic form of hypertension and the average ratio is approximately 1.1 and a little less than that but it’s true for systolic and diastolic blood pressure.

The interesting part when you are looking in detail on the WNK 1 gene and at the different polymorphisms that are linkage equilibrium that are corresponding to these haplotypes and you see here in blue the effect on systolic blood pressure, in green the effect on diastolic blood pressure and in orange the effect on potassium. You can see that if you’re looking in detail you have some polymorphisms that seem to be associated with blood pressure at least at significant but low levels and interestingly this polymorphism especially this one implies an intron 1 deletion that explains the Gordon’s syndrome. So it will be of course of interest to look more precisely to the effect of this polymorphism.

I’ll end it there just saying that these kinases are relatively complex and they play a role -- in the sensitivity to sodium, potassium and chloride in different epithelia. As I just showed you they interact with different transporters in the distal tubule and probably an important switch between the potassium and the sodium balance at this level. But in addition to that at least for LWNK 1 it’s expressed in other tissues, in heart and vessels, it might have a role in the cardiovascular system but also in the neurological system and especially in the cerebellum where it has been shown to interact with some other transporters or proteins important for the transport of ions in the neurons like synpatotagmin 2.

This is my last slide to thank my collaborators especially Juliette Hadchouel who is in the room and the people who have done some work especially Céline Delaloy who is now in the United States. Thank you for your patience.

Chairman: Thank you very much for this very interesting talk. I’m sure that there will be questions comments.

Question: The SNPs in intron 1 that you showed us -- with blood pressure are they locating in one of the conservative elements that seems to have an insulator function?

Prof. Jeunemaitre: No this particular SNP is not exactly in the C1 or C5 it’s just a little but further than that. So it has not been expressed in vitro yet from what I know. So the functionality of this polymorphism is not known yet. So it might be also another polymorphism which is a linkage because I’m close to this one which might express the association. I don’t know.

Question: You mentioned that the WNK system determines the balance between sodium and potassium excretion. Doctor Warnock mentioned that you should always check sodium excretion, 24-hour sodium excretion in your patients. These studies that have been done in the Gordon syndrome and also in the study that you just showed in PLoS. Did it look at sodium and potassium excretion?

Prof. Jeunemaitre: So yes at least if I can go back to this one because actually as you mentioned the sodium, potassium excretion at least has been looked and sodium also I’ve discussed that with Doctor Caulfield as you can see it seems but once again we have to be very careful with this kind of association there is a tendency for association with the urinary K with some polymorphism but I wouldn’t bet a lot of money on it.

Question: The reason I bring it up is also because it’s a beautiful system let’s say for say gene environment interaction because of course, excretion strongly relates to intake and you can vary it and do studies on it.

Prof. Jeunemaitre: Yes absolutely. Sodium and potassium are going to play an essential role on aldosterone secretion for example also on blood pressure as mentioned by Doctor Warnock.

Question: Xavier probably you mentioned it but I didn’t get it what is the ethnic background of the people you used for your PLoS study? Is it imaginable that this is not true for another racial background or ethnic backgrounds?

Ok this is a paper which is not from my group but from the Caulfield group and it was mainly Caucasian. All were Caucasians actually. The BRIGHT study that has been performed in the UK only contains Caucasians and the other one also I think I’m sure were Caucasians too. -- from another one males and females and the relative heterogeneity in terms of blood pressure. Actually when you look in detail from one study to another one you see that the linkage or the confidence interval is relatively large from some studies and not always corresponding to the size of the study actually and so there is some heterogeneity but all were performed in Caucasians.

Question: If you think of these findings and you translate this to the clinical situation how can we let’s say in the future use this knowledge in the real clinical situation. I mean if you have a patient with hypertension, how would you use this information?

Prof. Jeunemaitre: It’s really for the genetic research in the population and there is no direct usage for the clinician. It’s very difficult to have a clear view of what would be useful or not. The interesting part it seems that at least this pathway, the WNK pathway regulating the transport of sodium and potassium in the distal tubule may have some importance and such importance that you can find it in the general population after that is that a sufficient motivation to try to have some stimulators or inhibitors of this particular pathway? I think so but you might say well we already have plenty of different drugs playing with transport and that’s enough. I think there will be some originality behind that too because also it’s playing some role as I said before in the cardiovascular system.

Question: Excuse me Xavier did you try any interaction between the genes, other genes involved in the sodium transport? Because we found some interaction between WNK 1 and … ?

Prof. Jeunemaitre: Sure absolutely I didn’t mention your work we haven’t ourselves but yes you’re right.

Question: Xavier one of your slides -- was interposed between some cells. Would you say just a word about the chloride business? How can you explain this hyperchloremic metabolic acidosis that’s associated with this?

Prof. Jeunemaitre: It’s said to be probably in vitro at least that the WNK, especially WNK 4 might have a role on -- in the distal tubule but you’re a better nephrologist than I am and at least it would explain the possible increase in transfer of chloride in the distal tubule. After that the change of the polarity might also have some explanation behind that.

Question: Ok. What do you think if the study’s done in a different ethnic race for example, in the Asian race?

Prof. Jeunemaitre: It’s an important question but for the moment I haven’t seen any studies performed with a sufficient number of groups saying that WNK 1 or WNK 4 might be related to hypertension in Asian for example, or African population. We have tried to replicate with a group in Shanghai these kind of results. One of the difficulties is that for example for the Chinese people the SNPs which were informative for this study were absolutely not informative in the Chinese population because the frequency of it was very low. So they had to choose other polymorphisms and from this other polymorphism some of them show some mild results but again it is too preliminary to be definitive for the moment

Chairman: Ok I think we have to stop here for the sake of time thank you very much again. We’ll now move on.