A joint Congress by ERA-EDTA and ISN

NON-OSMOTIC SODIUM STORAGE

Jens Titze, Erlangen, Germany

Chair: Nathan Levin, New York, USA

Stanley Shaldon, Fontvieille, Monaco

Dr Jens Titze Medizinische Klinik 4 Nikolaus-Fiebiger Center for Molecular Medicine Erlangen, Germany

Slide 1

Thank You very much. Ladies and Gentlemen. It happens from time to time that we as nephrologists are asked for some advice in the clinic, very infrequently and then mainly by cardiologists or maybe by hepatologists and then what they have done then is that they put a needle in this blood vessel and drew some blood and then found that sodium concentration is too low and then they usually ask us how much salt they should provide the patient to have normal sodium concentration and then we always tell them that they should better restrict salt and perhaps even more restrict water. Most often they don’t believe us and then after 3 days when the sodium concentration is corrected, they sometimes start admiring us a little bit because they believe that we understand something that Walter Cannon called ‘the wisdom of the body’ which is the idea that the body is an equilibrium and if we give them some advice what they actually do is an over simplification because what we do in terms of functionality is that we reduce a 3 compartment model namely with the arteries, the interstitium and the intercellular space to a two compartment model where we say that the concentration between intracellular and extracellular is always the same to maintain constant the internal environment of our body and this is the concept of the equilibrium of the body which was set up by Walter Cannon following Claude Bernard’s brilliant idea of the ‘milieu intérieur’.

Slide 2

So what happens is that we believe that sodium is the major cation of the extracellular space and always acts to hold water in the extracellular volume and the same for potassium which is inside and that the concentration of the two are the same inside and outside to prevent water shifts and we believe that this is always regulated let’s call it physically, automatically and that there is not much regulation there.
So the site of action to control this internal environment is never on site in the interstitium by itself that’s at least what we believe. It’s somewhere else, it’s in the brain and in the kidney.

Slide 3

So what happens if we eat salt?  We throw it into the extracellular volume and what happens now is that the concentration is too high, that must not be so we need to do we must equilibrate the sodium load with water and the first thing that happens is thirst and thereby we mobilise some water and everybody has a meal at McDonald’s, everybody knows that. The next response is  vasopressin which acts in the kidney and we thereby mobilise water and now we have isotonicity, again at the expense of a little bit of expansion of the extracellular volume. If we continue that, we have again concentration increase, more thirst and again correction. The critical threshold of the extracellular volume is achieved and then suddenly we have the lame duck which is volume regulation now by the renin-angiotensin-aldosterone system or nerves or others would say pressure natriuresis or whatever and what happens then is that these are suppressed and that we secrete sodium together with water and that homeostasis or the steady state of the extracellular volume is achieved again.
So it’s a steady state and some say it’s a steady state without a set point and the controllers are never on site, they are in the brain and in the kidney. Some say the brain is more important and others say that the kidney is more important and what is most remarkable is that those who are in favour of the brain do not like the kidney that much and the nephrologists don’t like the brain that much. It doesn’t have much to do with our calculations when we correct hyponatremia.

Slide 4

So if we’re talking about blood pressure regulation, we certainly must talk about the Guyton concept and what you see here is that there is no doubt that sodium retention leads to extracellular volume retention and that again is an automatism. 9 g of salt will lead to 1 l of sodium retention in the extracellular space. Then this will certainly increase cardiac output and then there’s something that Guyton termed circulatory autoregulation which leads somehow to an increase of peripheral resistance and this was 40 years ago and after these 40 years there was a lot of achievement in terms of circulatory autoregulation, a lot of mechanisms identified but our little over simplification is still there and what I would like to talk about is this over simplification.

Slide 5

The question will be whether all sodium retained in the body will always lead to -- water retention?

Slide 6

Coming to that point we must ask ourselves whether the biology of tissues is in line with what we believe to happen in the body and I would like to briefly talk about the salt in the skin.

Slide 7

So if you don’t make dilution studies but really make an ash out of a rat after you induce some sodium retention then measure sodium, potassium and water, you will find that in the skin at a certain threshold and in this case it’s DOCA and the concentrations are not very high with DOCA by the way that further sodium retention does not lead to marked water retention.

Slide 8

So this is experimental proof of sodium free water retention and the question now is, how is it possible to get sodium inside this model without water retention? The idea in the skin, in the interstitium was that there is extracellular matrix with glycosaminoglycans and this extracellular matrix is polyanionic and negatively charged so it could be that the skin is something like a sodium sink that binds sodium in times of dietary sodium excess. If you have a look at these site chains which consist of -- or -- in the skin, you find that they’re negatively charged and what really happens and I cannot go into the details because I don’t have enough time is that if you feed a rat with high salt the glycosaminoglycans content increases and the sulfatation degree of the skin increases so what happens is that the extra cellular matrix gets more negative and thereby, seems to be a sink for sodium which definitely is not an indefinite thing but some of the sodium is bound very close to these glycosaminoglycans. This is termed osmotically inactive sodium storage and we did not coin this term which was done by -- already in the 50s and what we find now that it seems to be that this process is somehow associated with additional sodium retention that leads to even hypertonicity in this interstitial space whereas you find absolutely no change in blood.

Slide 9

So if we have is paradox on of interstitial skin sodium storage, the question certainly is, is that regulated or is it something passive? You can have a look at the rat skin or the rat ear and find some cartilage here and here is interstitial space and what is stained here in brown is the lymphatic system. Here you find a blood capillary and if we ash it, we determine the sodium concentration at about 160-170 mmol/L under normal conditions which is not a surprise because there are still glycosaminoglycans there and if -- directs an x-ray here to this lymph capillary and measures sodium concentrations he also finds in this lymph capillary about 170 which is much more compared to 135 and it’s no question that the anion gap without bicarbonate in this case is much higher because you have these negative charges of the glycosaminoglycans in the matrix.

Slide 10

So are there other measurements confirming that there is hypertonicity in the interstitial space? You must not believe that any nephrologist in this world helps you with these questions. So the help comes from the immunologist in this case and these are data from Gouy who was interested in knockout mice deficient for TonEBP and what they did is they didn’t measure sodium but they measured osmotic pressure or osmolality by osmotic pressure and what they found is that again in the lymph system there seems to be higher osmolality compared to serum and brain under physiological conditions. So what does this mean? That we have some hypertonicity in the interstitial space.

Slide 11

Now I would like to show you what we think is regulation of the interstitium and which has been summarised as lymphatic regulation of blood pressure, we did not say that, that’s what I want to state here. This was the editor who wrote that. He also linked it to some mental disease in the next paper. So I’m aware that it’s a little bit difficult to swallow what comes next but maybe you could be a little bit open minded because we really believe that there’s hypertonicity in the interstitial space and this is a critical feature of sodium retention.

Slide 12

So coming back to the basic concepts of circulation, circulation is very easy to understand if you believe that the interstitium is an equilibrium and that all sodium is readily mobilised from the interstitial space as soon as you start measuring 160 mmol/L with higher salt, 250 mmol of sodium you ask yourself whether there might be some transportation processes that we haven’t identified yet that might help us to mobilise interstitial sodium into the blood stream and the capillary system. We have to do that, it is the lymph capillary systems. So our idea was that there might be a third circulatory system which might play a role in the maintenance of volume and blood pressure homeostasis. If we have a look into the interstitium from low salt to high salt what we always find is that suddenly a lot of macrophages appear. They regularly appear in the areas where you have a lot of acrocan which is the core protein of the glycosaminoglycans with sodium storage. Coincidental with this macrophage infiltration you find that lymph capillaries are growing into the interstitial space.

Slide 13

Now the question is, is it a coincidence or is there a regulatory link? If you start staining these macrophages with VEGF-C, you find that they secrete some granular vascular endothelial growth factor C and --- explained to us yesterday that this is the major growth factor which leads to hyperplasia or to de novo lymphangiogenesis. If you look at the protein expression with high salt diet, you find suddenly that VEGF-C occurs so the macrophages carry VEGF-C into the interstitium and the question now is why do they do that? We must not forget that there’s some increase from 170 -180-200 mmol/l so there’s hypertonicity. There are some sites in the kidney for example in the medulla where we have some osmoprotective genes that protect us from hypertonicity and one of these osmoprotective genes this TonEBP. If we have look at VEGF-C protein and mRNA expression we find that they’re increased and also TonEBP expression is increased.

Slide 14

So what happens? I don’t have enough time to go through the cell culture experiment, is that somehow the macrophages sense hypertonicity in the interstitial space and go outside into the interstitium and then activate TonEBP. TonEBP has two binding sites at the VEGF-C promoter defining the VEGF-C promoter as an osmoprotective gene so we are dealing with hypertonicity and interstitial space, then the macrophage starts secreting VEGFC and the lymph capillaries grow and the job is quite impressive.

Slide 15

Here’s the lymph capillary network and the 3 dimensional resolution with low salt and here you find it with high salt.

Slide 16

So there’s really lymph angiogenesis and the trigger is salt and it’s mediated by the macrophages.
Now the question is, is that important? So our idea was that it’s not so simple that sodium directly goes to the brain and then is excreted but that the microphages regulate that so if you want to find out whether this plays a role or not we must deplete the macrophages and that’s what we did with clodronate and the idea was then that we would have no lymph capillaries and perhaps some sodium retention in this interstitial space and perhaps blood pressure increase

Slide 17

. Here are the data low salt to high salt, here the lymph capillaries and now we have depleted the macrophages and as soon as we deplete the macrophages the lymph capillaries are not there while the concentrations are still high. Now what happens with VEGF-C, VEGF-C is gone at the mRNA and protein level so the regulator really of salt and water balance in this case was the macrophage.

Slide 18

So if we summarise that lymph capillary is low salt, low, high salt, high and then if you deplete the player the regulator this --- receptor lymph capillaries are gone, nothing happens in the blood capillaries and now comes the surprise blood pressure increases further as soon as you block the lymph capillaries and now the question is why? There are two ideas that the blood pressure increases. Here you see low salt here you see high salt and these high salt have lymph capillaries so they might drain sodium more efficiently and as soon as we block the macrophages and lymph capillaries are gone, we have further extracellular volume expansion and a higher blood pressure.

Slide 19

So it could be that you have an internal sodium clearance that is regulated by the macrophages and this seems to coincide with osmotic stress in terms of cell shrinkage which is not a surprise taking into account that the concentration is about 200. Another thing is that if you have them with higher salt, you find increased levels of eNOS in the lymph capillary and the arterial system and as soon as you block the macrophages eNOS expression is gone and that suggests that NO levels are higher which lead to hypertension.

Slide 20

So to summarise this data and first very basically we should take into account that the negatively charged particles are not necessarily only albumin in blood and -- equilibrium perhaps does not really model the biology of tissues because they are negatively charged especially in the interstitium and this I would say I rather Gouy-Chapman modelling we would expect a layer of positive charges very close and much more positive charges and less positive charges the closer we get. The question is whether blood purification can change everything here for example?

Slide 21

Summary two is the skin is much more complex than we thought. There’s a lot of complexity in terms of microcirculation and if you have a look at the microcirculation under the skin, you find a lot of counter-currents which might contribute to local hypertonicity which might trigger macrophage excretion and we don’t know how this third compartment really is regulated.

Slide 22

The first regulatory mechanism suggests that the macrophage senses something in the interstitial space and regulates thereby salt and water balance by an extrarenal mechanism. Thank you very much for your attention.

Slide 23

Question: Jens, to distinguish between the effect of sodium as opposed to sodium and volume we actually have an experiment every time we dialyse somebody where we use a dialysate sodium significantly higher than the patient’s own sodium where we are reducing the volume as we increase the sodium but you’d have to have an end stage kidney disease rat in order to really carry that further. So what happens under those circumstances when you’re adding salt without adding volume or even reducing volume?

Dr. Titze: Well, we only have the animal experiment cell evidence but what you would suggest is high interstitial sodium concentration, high expression of N-acetyl-galactosaminyl transferase, a lot of sulfatation of glycosaminoglycans and what we know in humans now is that if skin sodium content is very high that we have a lot of macrophages in there. So I would think that first of all they will be thirsty no question about that, they will drink. But beyond that I think it’s a prediction I don’t know, but I would predict there will be changes in the extracellular matrix and that it might somehow trigger in the long term an inflammatory process.

Question: Let me just add to that. So it’s not so much a regulation of salt though, you were talking about a regulation of volume that you’re really talking about is that correct?

Dr. Titze: No in the interstitium we think it’s not volume it’s rather hypertonicity

Question: For the body, for the lymph channels that are developed are they looking at volume?

Dr. Titze: Well what we find in the interstitial space is that the volume is rather stable and the concentration increases which happens as soon as you have hypertonicity and you can simulate the same for example with glucose or with mannitol where you do not change experimentally the volume at all but have really the effect of the osmolyte. What is interesting is that this is much more pronounced if you use salt instead of mannitol so it seems to be a real salt triggered effect.

Question: Doctor Titze your whole house depends upon the accuracy of you sodium measurements would you agree?

Dr. Titze: Yes.

Question: You didn’t discuss this but in my opinion it would fall to pieces if one were to query the accuracy of your measurements. I wonder it’s a pity you go on with an assumption which is questionable what is the error of your measurements and how credible is the house that you’ve built upon those measurements? Because basically the error in measurement of sodium balance has always been the puzzle since the flame photometer has been available. I’m not convinced therefore that the house you’ve built on those measurements is necessarily valid without a little more detail the accuracy of your measurement system.

Dr. Titze: So if you’re building a house you can do two things you can simply take care of yourself or you can take care of what others do. What I have shown you in the presentation is that others measure it not with a flame photometer atomic absorption spectrometry but with x-ray and they find the same about 170 mmol/L in the lymph. Then I showed you that away from these electrolyte measurements others have measured osmolality and what you have seen is that the lymphatic system or L cells that residue in the lymphatic system live in an osmolality of 330.

Question: I will accept that osmolality is a much more reliable measurement than the flam photometry much smaller than the --- but you didn’t give us the error of you measurement of sodium.

Dr. Titze: Yes if I just could complete that thought please, two times 170 are roughly 330 so what they found with the osmolality measurements perfectly fits to our sodium measurements. In terms of accuracy the accuracy of our measurements and repeated measurements with the atomic absorption photometer is less than 5% in repeated measurements but in terms of accuracy of the total ashing procedure you never know what happens because you cannot ash the same organism twice. But I would say that at least the atomic absorption measurements are quite reasonable.