CME Slides Forum - M. Burnier

TUBULAR SODIUM HANDLING AND HYPERTENSION

M. Burnier, Lausanne, Switzerland

Chair: R.J. Johnson, Gainesville, USA

P. Zucchelli, Bologna, Italy

Prof M. Burnier
Head Division of Nephrology and Hypertension Consultation
CHUV
Lausanne, Switzerland

I have to talk about the tubular sodium handling and hypertension and I want to go into a lot of details into the pathogenesis of hypertension but you know that there are genetic factors and Rick Johnson told you that this accounts for about 40% probably in identical twins. There are also environmental factors and what makes the complexity is that not only are there multiple genes but there are gene interactions but there are also genes controlling the environmental factors and there maybe gene interactions between the environmental factors. Now, one of the very disputed issues in hypertension is really the role of salts. I probably guess that as a nephrologist you’re all convinced that salt is not very good for your blood pressure but still in the literature this issue still remains quite controversial.

Now, to make a long story short we all as nephrologists are really convinced that the kidney is the central organ in the control of blood pressure because it actually controls the extracellular volume, it also controls blood pressure and this depends on the salt intake, on the water intake but the kidney is also responsible to regulate finely, every day, the tubular sodium and the water excretion and that’s probably its major role in the control of blood pressure besides also its role in hormonal control like renin secretion.

Let me just remind you how the sodium is handled by the kidney, you know that very well but when the sodium is filtered, 60-70% of the sodium is reabsorbed in the proximal segment of the nephron. Then a little part, 20-30% is reabsorbed in the thick ascending limb of Henle. In the distal tubule 7% is reabsorbed and finally the fine-tuning is done is in the distal late collecting tubule, collecting duct where 2-3% of the sodium is reabsorbed. From the 100% of which is filtered only less than 1% will be excreted. Of course there is always a big dispute on the respective role of the proximal tubule and of the distal tubules and of course this is very difficult to settle because the proximal convoluted tubule is very difficult to investigate, at least in humans. And for this I will show you a series of studies that we performed and which were based on a technique which is called a lithium clearance technique because lithium has been used now for almost 20 years as a marker of sodium reabsorption in the proximal segment and in fact there is abundant literature suggesting that except in extreme conditions of very severe dehydration lithium can be reabsorbed in the distal segment but normally it’s reabsorbed in parallel to water and sodium in the proximal tubule, so this allows us to investigate a little bit what occurs in the proximal segment.

Now, what happens if you eat low salt and then go to a high salt diet on a normal kidney? So that’s what we experienced first just to demonstrate what’s going on if you go from a low salt to a very high salt diet in normal subjects. Normally your blood pressure doesn’t change, you inhibit your renin, this is very well known normally GFR does not move very much although in men we have seen very often a slight increase in GFR but if you have a normal kidney when you eat a lot of salt, you decrease your reabsorption in the proximal tubule and you decrease your distal reabsorption of sodium leaving more sodium to get out because you have to stay in balance, that’s very important, if you do not get in balance you’ll blow like Bibendum, Mister Michelin.

One important thing is that the renin is important in regulation and I’ll show again what you saw this morning. I think this is a very crucial relationship that we need to refresh every time. Normally, you can adapt your sodium excretion without changing very much your blood pressure and that’s the normal pressure natriuresis relationship. To remind you how important the renin-angiotensin system is that if you give an ACE inhibitor or if you perfuse angiotensin II at a continuous level, then you need to modify your blood pressure to get rid of the excess of salt if you increase salt intake. So what people have shown you this morning this is the typical pressure natriuresis response of a salt resistant dog in this case but they could be a subject and when you need to increase your blood pressure you become salt sensitive that means you need to adapt to increased blood pressure to get rid of the excess of salt.

Now, what about the tubular sodium handling in experimental hypertension.

I mean we’ve been working on this hypothesis for quite a long time and 10-20 years ago with our colleague Jerome Buller we investigated different types of hypertensive animals and ten years later I did the same thing in a small population of hypertensive patients and what we tried to look at is the relationship at the fractional excretion of sodium and the capacity to let sodium get out from the proximal tubules and what we showed is that in a normotensive subject or in a normotensive animal, if you increase the sodium intake, so you increase the fraction of sodium excretion, you adapt by reducing the reabsorption of sodium that means by increasing the fractional excretion of lithium.

But when we investigated hypertensive animals or hypertensive humans what we found is that they did not have the same relationship suggesting that the proximal tubule was not adapting correctly to the change in sodium intake and they were not capable of getting rid of the sodium from the proximal tubules. In fact, in line with what Rick Johnson said this morning we even investigated people with white-coat hypertension and even though they had a comparable fractional excretion of sodium to normotensive subjects they already had a defect in proximal excretion of sodium. So with this we went on and more recently there were still some people working on this hypothesis and show that for example in a spontaneously hypertensive rat there is also an increase of sodium reabsorption in the proximal tubule. This is the blood pressure here on the left increasing with age in spontaneously hypertensive rats, these are normotensive controlled and these are denervated spontaneously hypertensive rats and if you look here at the sodium secretion at 3, 6 and 12 weeks, you see that the fractional excretion of sodium is really reduced and mainly the proximal excretion of sodium is very much reduced in this model of spontaneously hypertensive rat and if you denervate the kidney well, you can actually increase the sodium excretion overall but also from the proximal tubule and then blood pressure remains normal at least during the initial weeks, afterwards blood pressure tends to increase for other reasons.

In the last years in Lausanne of course, we have been working with Bernard Rossier and his group a lot on the epithelial sodium channel and you know that this channel is not in the proximal tubule, it is in the distal tubule and it’s especially in the distal nephron, it’s also present in the colon, it is present in the skin and there it is responsible for the reabsorption of sodium and the control of sodium homeostasis, control of extracellular volume and blood pressure.

One interesting thing that may become very important for the future is that this ENaC is also present on the tongue and may actually control the salt taste and that’s maybe an issue for the future when we talk about salt sensitivity. There are also aldosterone and responsive epithelium, which has also some ENaC in the lung and in the ear, but I won’t talk about that. What we know is that this epithelial sodium channel is actually regulated by sodium and as you see here for the 3 subunits if you go from low sodium to the high sodium, you will see on the low sodium where you need to reabsorb a lot of sodium in the distal tubules, you have expression of the ENaC really on the surface, on the luminal side. Whereas if you put the animals on the high salt diet, then you have a down regulation of the subunit and its present for example, the beta and the gamma is present in the cytoplasm rather than on the luminal side.

Now, what you know and you have also seen this morning is that we know that mutation in these ENaC genes can cause either the Liddle syndrome which is a hypertension model or a recessive pseudohypoaldosterone type 1 which is a low or salt wasting disease and in this case you have an increased sodium reabsorption in the distal tubule and here you have a decreased sodium reabsorption in the distal nephron. And we know that mutations in any of these subunits can actually affect blood pressure levels.

Interestingly with Bernard Rossier and his group we tried to reproduce the Liddle mice, the Liddle syndrome that we see very rarely in humans and tried to reproduce it in mice and that’s what we’ve been able to reproduce and we produced a transgenic LL mice who’s an heterozygous mice and to investigate also the role of salt and renin in this model.

When we looked at plasma potassium concentration it was normal, plasma pH and bicarbonate were normal, so even if they had a Liddle mutation these animals on a normal salt diet did not have the phenotype of the Liddle mice or the Liddle syndrome.

However, when we put these same mice on a high salt intake then suddenly you see the appearance of hypokalemia, lower chloride concentration, metabolic alkalosis.

These animals develop the phenotype of hypertension similar to the Liddle syndrome only when they’ve had a high salt diet but very interestingly that these animals, these mice, you have different mice, some mice have only one renin and can suppress their renin on a high salt diet and some animals have two renin genes, one in the sub maxillary gland and one in the kidney and these animals cannot suppress their renin on a high salt diet.

And what is interesting is that in this model of Liddle mutation you see that the blood pressure increases mainly in those who had a high renin, if you had a low renin actually, there was hardly any difference in blood pressure during the Liddle mutation. So that means that having a mutation is one thing but you need to have a high salt and you have to have a non-suppressed renin. That’s not the only factor, you need more than just having a mutation.

In fact, when you look at the blood pressure on the high salt, on the low salt or on the normal salt, these mice look totally normal so everybody disgarded these mice, they’re not interesting. Well, except that if you increase angiotensin II or inject angiotensin II what you see is that the response of the blood pressure response to angiotensin II is very much increased in these heterozygous mice. So what happened is that they maintained their blood pressure because they are very sensitive to angiotensin II.

Remember that these ones are normotensive but look if you look at the cardiac weight index, they develop cardiac hypertrophy. In the absence of hypertension they just receive salt and mineralcorticoid and of course, a lot of people would tell you that’s the demonstration that aldosterone or mineralcorticoids cause hypertrophy and we have a lot of things like that in the literature. In 2-renin gene of course, they develop also hypertrophy because they are hypertensive.

The nice thing is that with these same animals, if we put them on a low salt diet, they do not develop cardiac hypertrophy. They receive the same amount of DOCA but they don’t receive the salt and they have no cardiac hypertrophy. Interestingly they also have no kidney hypertrophy. They had a kidney hypertrophy when they received DOCA and high salt which disappears when you give a low salt diet, which means that salt is also not only modulating blood pressure but the consequences of blood pressure.

And this is another model, it’s an alpha ENaC transgenic mice. You cannot survive, if you have no alpha subunit of ENaC. So we rescued these mice by transfecting the lungs so they can survive but they have no ENaC alpha subunit in the kidney and these animals have very high aldo, five times to six times higher aldo for their entire life and we actually evaluate that cardiac and renal hypertrophy after one year.

And if you have a model where they have a salt loosing phenotype because it’s an alpha ENaC transgenic mouse which looses, it’s like sodohyperaldosterone is, if they loose salt, they don’t develop cardiac hypertrophy or renal hypertrophy even though they have five times higher aldosterone for their own life, one year life over mice. So this would suggest that salt, at least in the experimental model is very important not only in defining blood pressure but also the consequence of blood pressure but what I’ve shown you is that we have evidence for tubular alteration at the distal segment but also at the proximal segment.

What about clinical hypertension?

Well, in the clinical hypertension the question is the same where is the defect located in the proximal or in the distal tubule or probably in both because we know that it’s not only one gene involved in hypertension.

Now if we look at mutation and polymorphisms that have been associated with hypertension in humans, you’ll find a lot of diseases, relatively rare but where the mutation or the polymorphisms have been located in the distal tubule.

So, there is today more evidence for the distal segment but still there are also some other mutations or polymorphisms for example, for alpha-adducin which goes all the way through the entire tubular segments, there is some new data to become with the dopamine receptor polymorphism which acts really on the proximal tubules and of course, the renin-angiotensin system acts also on the proximal tubules. So for me it’s still not totally said that all the mutations are in the distal tubules, we’ll still have to investigate the role of the proximal tubule.

Now just to show you when we actually measure the fractional excretion of lithium in hypertensive patients with the group of Giuseppe Bianchi in Milan who had some alpha-adducin polymorphisms which is supposed to increase sodium reabsorption in fact, the fractional excretion of sodium was identical but you see that the fractional excretion of lithium indicating how much sodium is coming out of the proximal tubule is significantly reduced which may lead to hypervolemia and of course, renin is low. When they investigated the same patient and published that in the Lancet and they looked at the acute salt sensitivity test which means the blood pressure responds to an acute salt load they could demonstrate that hypertensive patients with the polymorphism of alpha adducin actually had a greater change in blood pressure when they went from a high salt to a low salt diet and they were also more responsive to a diuretic suggesting again that maybe this increased sodium reabsorption is responsible for the hypertension in these patients at least.

Now, we asked like Rick Johnson what are the determinants of salt sensitivity and there are several determinants of salt sensitivity of course, there is renal function, there are hemodynamic and tubular adaptations, neuron or hormonal adaptations, genetic determinants and environmental determinants.

Now, we have seen recently a new gene also proposed as a determinant of salt sensitivity and blood pressure increase with age which is the CYP3A5 and I think this is a recently appeared gene on the list which I think is quite interesting because the CYP3A5 is not only responsible for the metabolism of several drugs but it’s also responsible for the metabolism of cortisol in the distal segment and in this paper published this year they found an association.

And we actually found also an association between the CYP3A5 polymorphism and not the baseline ambulatory blood pressure but the increase in blood pressure with age. You have here 1-3 thirtiles of age and you see people with the polymorphism in CYP3A5 one elial and you can see that with age here in this control, blood pressure was increased and here in the other group it was not increased and it was not increased with time, so we had the impression that this gene may actually contribute at the distal level also in humans in the increase in blood pressure.

Some years ago we made one hypothesis, also another one. The idea was that if you’re salt resistant which is probably what happens in most people, if you increase your sodium intake, you’re able to increase your fractional excretion of lithium because you’re able to adapt normally the proximal tubules. If you’re salt sensitive, it means you have a defect somewhere and you don’t adapt to sodium excretion correctly and we put the hypothesis that salt sensitive hypertensive patients will be those who are not able to increase their fractional excretion of lithium when they go on a high salt diet and what we did is we took 38 untreated hypertensive patients put them on the low salt and on the high salt diet.

This is a delta of fractional excretion of sodium in the three thirtiles and the thirtiles were determined according to the change in blood pressure. We have the first thirtile of people who did not change their blood pressure when they went from low to high salt diet, they stayed perfectly stable. Another group had a small increase and the third group had the highest increase in blood pressure, so these ones are really the salt sensitive. If you look at the way the proximal tubule responds, those who had no change in blood pressure they were actually able to increase the fractional excretion of lithium, so they could leave sodium to get out of the proximal tubule but those who were salt sensitive, they actually continued to retain sodium and they could not adapt correctly. So, is that the primary or secondary defect I don’t know, it’s interesting that uric acid has very often been used like lithium because it’s also reabsorbed in the proximal tubule.

When we looked at the multivariate analysis we could not include a lot of parameters because we had only a small number of patients but the three factors which predicted the salt index change in day time ambulatory blood pressure was the age, the capacity to adapt in the proximal tubules and the capacity to change renal plasma flow.

So that goes along with your hypothesis that the renal plasma flow is also very important whether the response of the proximal tubule is secondary to the renal plasma flow can be discussed of course.

Now, before I finish just a small hint into the insulin resistance also. We know that with incident resistance, which is a common pathway for metabolic syndrome, we have a high insulin and this high insulin may be responsible for the vasoconstriction and sodium retention. One thing is very important in the metabolic syndrome is that it is characterised by insulin resistance but the kidney is not insulin resistant, actually the kidney is probably very sensitive to insulin in contrast to the other organs and that’s the reason why blood pressure increases and probably eventually leads to sodium retention and vasoconstriction.

Now how is the behaviour of the fractional excretion of lithium in people with the metabolic syndrome and here the Italian group from Naples actually looked at the large population of 555 patients and he looked at the relationship between the reabsorption of sodium in the proximal tubules and different parameters of the metabolic syndrome and he found actually a close correlation with BMI, with the umbilical circumference, subscapular skinfold suggesting that the parameters of the metabolic syndrome are associated also with an increased reabsorption of sodium in the proximal tubules.

So my conclusion will be that I’m pretty much convinced that renal tubular sodium handling is an important determinant of blood pressure but also of the cardiac and renal complication of hypertension not only of generating hypertension but also leading to complications. I want to get out a bit of the dispute between proximal and distal. I’m pretty convinced that both are important in that both proximal and distal segments of the nephron contribute to the regulation of blood pressure. One does the fine-tuning, the other one has a big bulk. The salt sensitive hypertension is associated with inadequate reabsorption of sodium in the proximal tubules, maybe because of the renal lesion that Rick Johnson described in his hypothesis and that may well be but sometimes we have it in the absence of a decrease in the renal plasma flow. And renal tubular sodium handling is also associated with the parameters of the metabolic syndrome in men and women and that maybe something that we can discuss.

Chairman: We can take a few questions. I have a quick question. Michel I think you’ve elegantly shown the relationship of salt intake and environmental factors and their interactions with genetics here. The question and I remember the study of the Yanomami Indians in the New England Journal where they were on an extremely low salt diet and they had these sky high renin and aldosterone levels with normal blood pressure. The question I have for you is do you think that the modulation by salt intake is working at the receptor level or at the signalling levels or do you have…?

Prof Burnier: Well, we know that sodium is effecting the expression of some receptors like the AT1 receptor if you go from low salt to high salt, you have a change in the numbers not so much in the affinity but in the numbers of receptors and we know that salt is modulating at least for angiotensin II but probably for other receptors as well. Even metabolic factors are also effecting as you said but we have looked for example, at PP gamma is also effecting the expression of AT1 receptor so there is really an implication between the metabolic, the salt and the receptor expression. Whether there are also some post receptor effects, I think this is, well it can well be also. What we have done recently and that we are conducting now is a micro array on the heart for example, and kidney to the response to salt and what we see is that salt is inducing per se a lot of genes that are involved in profibrosis and hypertrophy independently of whatever other things.

Question: Yes if I may add one comment about the general dispute between proximal and distal tubules of sodium absorption, it’s not a dispute of course. Classical physiologists tell us that whatever happens in the proximal tubule can reflect that the unique composition only if the macula densa cells are disrupted, only if there is no compensation at the macula densa cells. So all the data that you have presented can have an impact on blood pressure only if you assume that at the macula densa something is happening that is wrong and therefore when you have more salt rich macula densa cells, they don’t sense the sodium and of course you have a less sodium absorption.

Prof Burnier: I totally agree with you, that’s why I think we could not dissociate the salt and the renin for example, and renin is so important and just measuring renin in the sample is not sufficient. O.k.? You may have low renin but the renin may be still too high for the amount of sodium you have. So I think you know 20-30 years ago, people were discussing about this sodium renin balance and this is how we can appreciate that is very difficult but I’m still convinced that this is one very important issue. You must also say that if you loose sodium from the distal tubule, you should be able to compensate somewhere in the proximal tubule. Well sometimes you do and we’ve seen if you give loop diuretics or diuretics acting on the proximal, sodium is actually also trying to reabsorb more. We’ve seen that in animals.

Question: May I add this comment loop diuretics they do the job because also they impair the sodium–sensore mechanism in macula densa cells so that’s nothing to do with the proximal tubule.