A joint Congress by ERA-EDTA and ISN

THE RENAL EPITHELIAL SODIUM CHANNEL AND HYPERTENSION

David G. Warnock, Birmingham, USA

Chair: Xavier Jeunemaitre, Paris, France

Gerjan Navis, Groningen, Netherlands

Prof David G. Warnock Department of Nephrology University of Alabama at Birmingham Birmingham, AL, USA

Slide 1

Thank you very much. It’s my great pleasure to be here and I thank the organisers of course and the World Congress of Nephrology.

Slide 2

I’m going to start off this session first of all with a tribute to Grant Liddle himself. I will be talking about Liddle’s syndrome and this is a wonderful picture of the man and the irony of course is that as an endocrinologist he was convinced that he was on the track of a new mineralocorticoid and to his great disappointment the syndrome turned out to be a kidney problem of all things my goodness. We became involved because in fact, the original pedigree, the family that Doctor Liddle described was in fact from Alabama. Those of you who know your southern US geography they are actually from the upper corner of lower Alabama which the natives refer to as UCLA. I originally came from California and to me UCLA has a totally different connotation but the family that we’ll talk about came from the upper corner of lower Alabama.

Slide 3

The disorder was described in 1963 in this publication of familial renal disorder simulating primary aldosteronism but with negligible aldosterone secretion. One of those wonderful titles that summarises you know the saving and the critical points to remember about the syndrome and from this low aldo secretion but stimulating primary aldosteronism you can tell why Liddle himself referred to this as pseudoaldosteronism. Remember back in the late 50’s or so Conn’s syndrome the adrenal tumours had been described and this condition fundamentally presents itself as if it were a case of primary aldosteronism Doctor Liddle had an assay for aldo and the aldo levels are exquisitely low hence the interest.

Slide 4

I’ll talk very quickly about the clinical manifestations in the pedigree itself. We’ll touch on the normal renal cell biology and physiology and we’ll talk about the pathophysiology of sodium channel activation. The mutations I will describe this afternoon are in fact activating mutations. Channels by definition Christophe and I were just talking about this channels open and close that’s what they do and you can describe a channel in terms of its open probability as a statistical function. So this condition I think is easily described as one in which the open probability is prolonged. Maybe there are more channels and the channels are open longer, hence ongoing salt reabsorption. We’ll talk a little bit about treatment and future research and I think hopefully what we’ll lead into in the rest of the session this afternoon some glimpses into some genetic components and other clues that might well be worthwhile.

Slide 5

So pseudoaldosteronism is Liddle’s syndrome, it is marked by hypertension of course, hypokalemia, metabolic alkalosis, it looks for all the world like primary aldosteronism but the plasma renin activity is suppressed as are the aldosterone secretion rates. I’m not talking about lower limits of normal, the aldosterone secretion rates are extremely low. To make the diagnosis we prefer looking at 24-hour urinary aldosterone because then you get an integrated view you may be thrown off by a single plasma measurement, so 24-hour urine for aldo that’s the way to go and the condition itself in fact is an autosomal dominant.

Slide 6

As I mentioned as a volume expanded state it’s not surprising to see the plasma renin levels are low and that in fact is the cause of the suppressed aldosterone secretion. In that original publication Doctor Liddle looked at the effects of spironolactone and as I told you -- endocrinologists he was sure he was on the trail of a different, of a novel mineralcorticoid and he was shocked, disappointed and yet he pushed on when he found that spironolactone, a mineralcorticoid receptor blocker had no effect in this condition because in fact, the mineralcorticoid levels are low. He then reported that triamterene, a sodium channel blocker, is beneficial but only if the dietary salt intake was controlled. The quotation from his work is that one might think of this as a disorder in which the renal tubules transport ions with such abnormal facility that the end result simulates that of a mineralcorticoid excess. Remember this is 1963 a long time we were talking about --bladders in those days and that sort of thing but this was long before ENaC or all of the other electrophysiological approaches had become possible.

Slide 7

Now the pedigree itself, the probane was a young woman named GS if you will identified in the publications. She developed ESRD which in itself is unusual. There are very few of those, I’m aware of one other case in Paris that developed ESRD and both patients were transplanted with the consequence that the condition was ameliorated. So what our modest contribution quite frankly is that we fulfil Cook’s postulates for Liddle. He said it was a kidney problem, we put in a normal kidney that if you will closed the loop.One year after the probane had undergone her successful renal transplantation she spent some time in the general clinical research centre on a high salt, low salt sort of a typical dietary manipulation and at that time we found that her plasma renin would respond appropriately to posture, would respond appropriately to salt restriction, that was not the case before her transplant and before she developed renal failure.

Slide 8

So here’s the probane at the age of 49, a typical blood pressure after transplant, normal serum potassium and urinary aldosterone excretion and this ratio was perfectly normal. In the affected members of her pedigree 18 members who were at risk we identified them as having the genetic mutation later but on this analysis we could say that they had the clinical phenotype, they were obviously hypertensive compared to those who were not affected, their potassiums were lower on average, not much the bicarbonate story.

Slide 9

Notice the kidney function was normal and notice that the aldosterone excretion rates were low compared to normals and there’s a ratio factoring the aldo by the urinary potassium very clearly separated these two. The next step was meeting up with Rick Lifton and telling him that we had a large pedigree that had been successfully phenotyped and we were quite certain that we had about 32 patients at risk, 18 of them were affected and the only regret I have in the whole story is that I didn’t keep the table cloth because we were at lunch and Rick did one of these freehand calculations and he said the LOD score would be 8, when the gene was then discovered the LOD score came out to be 7.92 so Rick is one of these guys that does LOD based -- in his head and what can I say? Anyway to focus on the story here we’re talking about the epithelial sodium channel in the aldosterone responsive segment of the nephron.

Slide 10

The channels themselves had been cloned and in Lausanne Cecilia Canessa and Bernard Rossier used expression cloning to get all three subunits of the channel, those were described, their structure was described and those clones went to Rick’s group where Rick pulled out the human clones and very quickly identified the location of the mutation in this particular pedigree, as well as 4 other cases that he had access to not large pedigrees but they confirmed the finding that this condition is caused by mutations in the sodium channel itself and with a LOD score that really was quite impressive.

Slide 11

Slide 12

So the sodium channel subunits are organised in a very similar fashion, their extracellular domains but the point of interest for us is the cytoplasmic tail and a very, very special proline-rich region that we’ll touch upon. This turns out to be a very important hook for a series of processes through adaptor proteins called Nedd 4 that retrieve the channel that pull the channel out of the membrane when it’s time to turn the channel off. If this proline-rich region is mutated or otherwise affected, then you have problems.

Slide 13

Here’s the wild type β subunit with a mutation and you can see here’s that proline-rich region, this is the normal wild type and here the truncating mutations described in the various pedigrees. This is the Alabama pedigree, other pedigrees were described in which they’re either frameshifts or truncating mutations but the proline-rich region in every instance was abrogated or ameliorated.

Slide 14

This led straightforward then to expression studies introducing mutation into the beta or the gamma subunit and the oocyte expression system and you can see there was more channel activity with these activating mutations and the next series, I’m limited in my time for this presentation because we have such a nice series going forward, so I’m not going to belabour this, it’s not a very complicated system.

Slide 15

but there are 2 adaptor proteins called the Nedd 4 and Nedd 4-2 proteins that interact with each other as well as with the cytosolic tail of the beta and the gamma and probably even the alpha subunits. You’re going to hear a lot more about the notion of these interacting proteins in the next session from Xavier with the WNK kinases and I’m going to let him dwell on that.

Slide 16

But the Nedd 4 proteins now have been I think coupled or associated with hypertension in very interesting ways. We know that these Nedd 4 proteins are involved also with the various kinases that seem to activate sodium channel activity. We know that dietary salt restriction is a very important mechanism by which Nedd 4 expression is affected and so in the instance of salt restriction you really want to keep the channels in the membrane and become salt avid and if you do that the Nedd4 pathways are downregulated.

Slide 17

There are very interesting variants now that have been described with splice sites also a SNP that has been associated with hypertension in the Japanese and also in the Scandinavian population, so the Nedd 4 story I think is a very interesting unfolding one.

Slide 18

So low renin hypertension, our sodium channel mutations are a common cause of human low renin hypertension, human low renin hypertension is very common, sodium channel mutations are very rare, the sort that we’ve described. There are some polymorphisms that have been associated with low renin hypertension in blacks but again these are unusual. However, it’s a very common observation now that in resistant hypertension and severe hypertension on several drugs amiloride or spironolactone, mineralcorticoid receptor blockers may be effective. That implies activation of the sodium channel. Now that spironolactone would work means there’s some sort of mineralcorticoid sensitivity, it’s not like pure Liddle’s syndrome but in view of the multiple pathways now that we understand for regulating sodium channel activity I think this opens up a lot of possibilities in terms of thinking about candidate genes if you will that mutations of which in families could affect sodium channel activity.

Slide 19

The Nedd4 family we mentioned, you’ve heard about the SG kinases there’s a whole fascinating group now of channel activating proteins which affect post-translational modification of the subunits. Any of these could activate the channel and if they weren’t properly regulated you could get what I like to call disregulation of the epithelia sodium channel. Nevertheless if the channel is active, if it’s overactive, if its’ inappropriately active the use of the sodium channel blocker like amiloride will be informative as well as effective.

Slide 20

So Liddle’s syndrome demonstrates the importance of salt balance in blood pressure control, Guyton got it right. Constitutive activation of the sodium channel complex results in volume expansion and renin and aldosterone suppression and chronic hypertension. Amiloride works very well in this condition especially in young children. Adults who’ve had hypertension for years and years have vascular disease and they won’t respond to the pure magic bullet and it’ll take other drugs in addition to control the blood pressure. Salt restriction in this condition is absolutely essential to make any progress.

Slide 21

Slide 22

Now there are other monogenic forms of hypertension that could be mentioned in this same context Liddle’s syndrome with low aldosterone secretion. There’s a form of apparent mineralcorticoid excess with abnormalities in the activity of the 11-β hydroxysteroid dehydrogenase. The cortisol cortisone ratios are changed, there’s a glucocorticoid-remedial aldosteronism and all of these are fascinating and interesting and as a consulting nephrologist something we at least have to be familiar with. In the US now we do have a clear approved lab, we can order and have reported in a medical record something that’s called the Endocrine Hypertension profile from one of the big clinical labs and it allows you to assess the importance of these various components. I’m going to stop at this point will be delighted to take a few questions or whatever the Chairs wish.

Slide 23

Chairman: This paper is now open to discussion. Any questions from the audience?

Question: If I may ask a focus question. Thank you David for this very brief but intense summary of Liddle’s syndrome. I was asking myself about the number of families and the fact that I think for the moment no mutation of the α subunit has been reported even though there’s a py in the men and the possibility of mutation that will effect the channel equivalent of the β and γ. Are you aware of any mutation in the α subunit or why has no mutation been described for the moment?

Prof. Warnock: Ok so the question is the mutations described in Liddle’s syndrome are strictly in the β or the γ subunit and specifically either delete or alter this proline-rich region. To date no mutations have been described with the α subunit that causes an activated phenotype Liddle’s syndrome why is that? Your observation is absolutely correct I’m happy to speculate but I really don’t have a good answer for you and my own interpretation is that it has something to do with the way the channel complex is assembled and that assembly takes place in the interplasmic reticulum. It appears that the α subunit if you will as properly named is the lead dog you know the α that kind of organises all of this. So if the α is not properly folded, not properly delivered to its site then mainly the complex itself can’t be assembled but that’s rank speculation. As you well know there are on the other side of the coin, there are mutations described in the α, β, γ subunit which are loss of function mutations not gain of function mutations and those mutations are described in all three subunits. But it is an interesting peculiarity and I think it tells us something about the assembly of the complex that no one has observed an activating mutation until now in the α subunit at this point and you’re absolutely correct.

Chairman: Please first question there.

Chairman: I’m happy to repeat the question go ahead Christophe while we’re waiting for the next gentleman to reach the microphone.

Question: -- and as you know in mice overexpressing β in the lung, they develop cystic fibrosis. Have you noticed any pulmonary phenotype in any of the Liddle patients and if not do you have an explanation why overexpression in mice may cause CFDR whereas Liddle patients are unaffected?

Prof. Warnock: So it has to do with this activation of the sodium channel in CFDR. What is that all about? Both in the native state as well as in the beta subunit rescue model? Yes I’ve seen a fair number of Liddle’s patients but they all had the same mutations so I have to --- my comments by saying with that mutation I think half a dozen of them were very patient and allowed us to stick electrodes up their nose and we did not see an activated current as you would have seen in cystic fibrosis. I have no other explanation you know for that other than I think in the cystic fibrosis phenotype you’re dealing with a life long condition which is real stressed evolution in terms of drying out the secretions and so forth with activation in parallel of the sodium channel but I don’t have a better answer than that for you other than the fact that we haven’t seen high PDs in the nasal --. Yes sir.

Question:Pedersen from Denmark, could I ask you to comment a little bit on the differential diagnostic problems between Liddle’s syndrome on the one hand and low renal essential hypertension on the other?

Prof. Warnock: Right so the question is low renal hypertension is an important to genetically determine and prove that in fact they have Liddle’s syndrome and I will tell you that I’m interested in that and I think the question to ask in the clinic is the family history. If it’s a very clear autosomal dominant trait then your curiosity is --. We go on at that point to do what I would call clinical phenotyping with measurements of the aldosterone and so forth but in terms of the utility of genetic sequencing that’s not something we do in our group. I know Rick Lifton is probably the only reference group that I would turn to with any trust and I’m not just sure that that’s necessary or even available at this point. It’s too bad that we’ve kind of passed the phase of the excitement about this syndrome because there may well be alpha subunit mutations out there that we’re not going to see but I think in terms of treating resistant hypertension the questions to ask yourself when you see that patient in the clinic on 3 drugs and so forth first of all what is the 24-hour urinary sodium excretion? I live in an area, a country where salt intake is quite impressive, breathtaking in fact and so you have to get the salt down, aldosterone, amiloride in fact both together in combination I think are very important, very successful and at some point I will assess the aldosterone secretion rates and if it’s extraordinarily low then I’ll chase after the family in that regard but my clinic is overwhelmed with low renin hypertension and I don’t see very many cases of Liddle’s syndrome to tell you the truth.

Chairman: Dave a very last and brief question about the efficiency of amiloride or other drugs like that in your experience of this large family or other families is there a dose ranging inefficiency of amiloride and what is the follow up after several years?

Prof. Warnock: Ok one of the major problems with amiloride is it is a -guanadinium group that binds to the sodium channel and if the sodium concentration in the luminal compartment is high then it’s a competitive inhibition and so you’ve got a problem there with amiloride. So if you have a controlled salt intake amiloride is not going to be a very successful drug. In adolescents we’ve been very successful with amiloride it is a terrific drug it works quite nicely but you have to pay attention to the salt intake. In adults as I mentioned they have 20, 30, 40 years of vascular disease and the consequences of that amiloride is not sufficient to control the hypertension itself. The other interesting aspect of amiloride I think worth mentioning in this context is that for some reason and it’s independent of potassium -- and I have published this it simulates aldosterone secretion and so using amiloride by itself may not be as effective as using a combination of amiloride and a spironolactone-like agent it turns out to be a better choice. We’ve had great success actually in Gitelman’s syndrome a mild salt wasting syndrome with potassium wasting by using that approach. So the combination of amiloride and spironolactone, watch for the dietary salt, realise in that older patients you’re going to have to be more aggressive in controlling the blood pressure because the consequences of this disorder are simply related to the severe hypertensive phenotype.

Chairman: Thank you very much Ian.