ROLE OF THE ADDUCIN GENES IN HUMAN ESSENTIAL HYPER­TENSION AND IN THE PROGRESSION OF RENAL FAILURE

Giuseppe Bianchi, Milan, Italy

Chair: Pierre Corvol, Paris, France

Friedrich C. Luft, Berlin, Germany

Prof G. Bianchi School of Nephrology, Univ. Vita Salute San Raffaele, Division of Nephrology, Dialysis and Hypertension, San Raffaele Hospital Milan, Italy

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Slide 1

Well, thank you Pierre and thank you to all of you for the invitation to speak about adducin here. Well first of all, I think that I should briefly recall how we did identify adducin. As you may know, we have been studying for a long period of time a strain of rats that develops spontaneous hypertension MHS rats compared to controls and here you see the difference that you may see when you measure these different variables in hypertensive. So the arrow up means that it was greater in hypertensive compared to normotensive. Then we tried to compare with humans and of course, in humans we have a very heterogeneous population of patients and you can always find a subset of patients that is comparable to your rats. The question, is what is really just a chance, what is a real finding?

Slide 2

To answer this question we then used the top-down approach and moving from the whole kidney level in which we showed it was possible to transplant hypertension with transplant in the kidney we moved down to the tubular reabsorption and tubular cell and so on and we were able to identify a protein that was adducin. Well, this is adducin and I’m showing this slide for mainly 2 reasons. First we have three subunits, alpha, beta and gamma each map on different chromosomes in rats and humans and these are the mutations we found in rats in white and in yellow in humans. So the first question is, how can really mutation occur and in different sites produce something similar in rats and humans? And the second question is that these are the advantages, as adducin works in the cell as a dimer or tetramer we can use the epistatic interaction among these three loci to understand the role of adducin as a genetic argument because adducin is working in the cell as a dimer of alpha and beta or alpha and gamma. In tubular cells we mainly have alpha, beta. In tubular cells we have alpha, gamma. In podocytes, alpha, beta.

Slide 3

So studying the interaction on these loci we can use this interaction as a genetic argument to assess the role of these. But let’s try to answer the first questions. We tried to answer the first question that says whether the different mutation sites could affect the function of the protein in a different way basically on 2 approaches and then also with congenic rats. First we transfected the cDNA of different mutants or wild rats or mutant and wild human adducin in tubular cells and we found that for these 4 variables the sodium pump activity on the cell surface, the sodium pump endocytosis and focal adhesion sites we got the same direction of changes when we transfected the mutated adducin compared to wild adducin either in rats and in humans. We have not checked this yet. So this implied that at least in some variables we could have the same changes. Of course, we didn’t ask adducin to play piano, so maybe there could be a lot of other differences but this fundamental difference was the same. Moreover, we synthesised by cDNA recombinant technology the different adducin mutants and we studied the interaction with actin, with sodium potassium pump and src in a cell free system and also in these conditions we found the same kind of difference in activity between the mutant adducin and the wild adducin implying that despite the difference in the mutation sites probably the two mutations occurring in rats and humans diverted about 40 million years ago produces some similar changes at the cellular level.

Slide 4

Then I will illustrate in particular this point here and this one here just to give you some precise data. We published this last year. This is a tubular cell, luminal membrane, basolateral membrane and as you know, the sodium pump is the driving mechanism creating the gradient through which sodium is reabsorbed. Pump is synthesised and localised in the membrane and then is removed by the membrane by endocytosis. So we are studying these 2 processes and here in particular we are expressing these type of processes let’s say the removal of the sodium pump on the assumption that if we have a variation in these two processes, we change the number of pump sites on the plasma membrane, as we have already shown and so we can really detect the molecular mechanism underlying this activity. These are some of the proteins involved in endocytosis. I cannot go into detail but basically we found that tubular cells transfected with the mutated adducin either from rats or from humans had a reduction in endocytosis because a hyperphosphorylation of this protein that is the crucial protein that is needed to bind the cytoplasmatic domain with the sodium potassium pump to clathrin and then  to allow endocytosis. This protein was hyperphosphorylated so the usual cyclophosphorylated, dephosphorylated site cannot be correct and in this way we have a reduction in endocytosis. This hyperphosphorylation of the sodium potassium pump was due to the fact that in adducin the mutated adducin was binding less amount of phosphatase activity that is this was responsible of the increase in phosphorylation of the protein. Of course, this reduction in binding of phosphatase activities was also seen in congenic rats. This is the molecular mechanism of endocytosis. This is the interaction in the cell-free system between adducin and src.

Slide 5

Well, why did we do src? Well, because it would be too long a story for that. Anyhow we have some phosphorylation of src alone that is incubated with wild adducin, this is in a cell-free system. When we add adducin, mutated adducin we have an increase in this phosphorylation of src. And this selective inhibitor of the adducin is able to not affect the phosphorylation of the wild adducin while it’s affecting the phosphorylation of the wild or the mutated adducin at 10, -10, 11 molar. We devoted a lot of effort in trying to develop a selective inhibitor because we were convinced that only by selective inhibiting in a given molecular mechanism we could perhaps be able to dissect the biological complexity underlying to a given abnormal protein and its complicated interaction with the other proteins.

Slide 6

Then we moved to the congenic rats. Well, this is the typical, congenic strain we have in which we can introgress this portion of DNA from the hypertensive into the normotensive and vice versa. Of course, we are also introgressing not only alpha adducin but a number of other genes and this is the weak point of this approach but as you know, in rats we cannot do the homologous gene replacement.

Slide 7

Well, when we introgress the mutated alpha adducin of hypertensive rats into normotensive ones, we have an increase in blood pressure and this increase in blood pressure is associated to a number of changes in the caveolae of the tubular cells of these rats. These are the normotensive rates, these are the hypertensive rats in which the hypertensive adducin is introgressed. You see we have an increase in sodium potassium pump in the caveolae, an increase in src, an increase in other proteins implying that if, of course, we have the carrier that we have introduced in other genes but in these conditions, we have a variety of changes in the caveolae of tubular cells that could well explain the increase in tubular reabsorption and other gene transcription events. Of course, that compound just mentioned is able to block these changes observed in congenic rats.

Slide 8

This is just an example which you see that these are the normotensive and hypertensive rats and the merging you see between adducin and src or between sodium potassium ATPase and src is yellow is increased in congenic rats compared to the normotensive ones.

Slide 9

So the src this is really a very complicated issue. We have seen this interaction between adducin, src and the sodium potassium pump and if you look at the literature, you see a lot of changes produced by src. It could affect iron transport, cell volume regulation, reactive oxygen species, actin binding, stress fiber, focal adhesion sites, cell matrix interaction and also gene transcription. These different pathways can occur in different cells according to the context. So the crucial question to study the effect of src and the cellular function of src or the organ complication of hypertension is to define the context in which you are studying the phenomenon of interest. I’m not going to go into detail but anyhow most of these changes we have seen when we transfected adducin and most of these cellular changes can be the basis of this abnormality in human hypertension. We have an increase in tubular reabsorption, we have changes in cell volume, in cell proliferation, in capillary rarefication. We have cardio remodelling. Recently adducin has been shown to be involved in capillary growth in matrigel and production of reactive oxygen species. This is not to say that adducin and src are involved in all these mechanisms I’m just saying that it’s just one player when he has his own contest he can do also these kind of things. This is an important point.

Slide 10

Now let’s move to the clinical data. So far, on adducin there have been published about 80 papers linkage and so on. We have 10 linkage papers, 6 positive, 4 negative but in all the 4 negative studies markers 400 kb have been used implying we have more than 8 recombinant hot spots between the adducin locus and these markers and this certainly is not a valid method to assess the role of a marker so these are the only valid ones. Then we also have been studying because this has been done mainly by other authors of course, 19 after 22 association positive when besides blood pressure also factors regulating blood pressure and body sodium were associated and this also cardiovascular complication and then 28 general population ore case-control studies 50 positive and roughly 50 negative. The usual confusion with candidate gene in which some studies are positive, some are negative.

Slide 11

But just going into some more precise detail, if you look at the ability or possible association a between adducin and cardiovascular risk, you find 11 studies, 8 positive and 3 negative. The 8 positive studies when this association has been studied in hypertensive, the 3 negative studies when adducin was studied in a predominantly normotensive population. There is a lot of variation in the magnitude of the risk. You can move from 30% to 600% according to the context. For instance, in women, post menopausal women the increase of stroke is an odds ratio of 6. Let’s just see 2, one positive study, one negative study just to give you what is the so-called evidence based medicine.

Slide 12

This is a study that has not been published yet in full, I have just had the opportunity to see the manuscript. This number of patients were followed for almost 3 years with an excellent control of blood pressure. The TRP carrier was studied in comparison with the mutated with the wild adducin. They found a 42% excess for non-fatal stroke and myocardial infarction and 83% excess of all cause of death. The most important point is there was no difference in blood pressure, cholesterol, smoke and diabetes across the study period. Because when you study one factor, you have to be sure the other factors are not interfering.

Slide 13

This is let’s call a negative study. This study concludes that adducin, mutated adducin is protective for myocardial infarction and what are the bases of this conclusion? They studied 549 consecutive Caucasians up to an age of 75 years. They survived admission to the coronary care unit and 500 Caucasian controls recruited among healthy adult visitors to non-cardiovascular patients. But consider the big difference between the cases in control of smoke, hypertension and diabetes. The question is probably we are open, the carrier of the mutated adducin could have died before because there are a lot of other risk factors or because as we have seen before adducin by itself increases cardiovascular mortality. Anyhow these are the data.

Slide 14

Now just a quick look at the kidney. This is a protein excretion in beta adducin null mice in which the protein excretion is lower in null mice either male and female compared to wild mice.

Slide 15

These are the congenic rats in which we have in normotensive rats already an increase in protein excretion compared to hypertensive rats because of some other reason. This is the problem of all the congenic rats, you just pick up a group of genes and fix by brother system and you can also pick up a lot of other genes and in this case we have already shown that we have an increase in thromboxane production that is responsible of this type of focal glomerulosclerosis with increase in protein excretion. But if you introgress the hypertensive adducin into the normotensive strain, you see a reduction of protein excretion and also the histology is much better. While the introduction of beta adducin here impairs the protein increase, the protein excretion compared to this value and also the histology. However again, if you introduce alpha adducin in this strain you have an improvement of the histology and a decrease in the protein excretion implying some interaction between alpha and beta adducin either in the morphology and in protein excretion.

Slide 16

These are 2 human cohorts, one with IgA nephropathy, 328 patients followed for more than 8 years. This is type II diabetic nephropathy, 242 also followed for several years. You see that there is a significant interaction between alpha adducin, alpha and beta adducin, AD1 is alpha and AD2 is beta either in IgA nephropathy and in diabetic II nephropathy. This is the rate of creatinine decay with time and here, for instance, you see a difference of 4 times implying that if these patients go to dialysis in 20 years, these will go in dialysis in 5 years. So I am not going to go into detail also because I want to be on time.

Slide 17

This is just taken by a nice picture from a recent paper in the New England in which these are the proteins involving the slit membranes nephrin, podocin and CD2A that have been associated to spontaneous or let’s say genetic forms of nephrotic syndrome. Well, we are studying the interaction among these proteins in null mice, in podocytes transfected with adducin, in congenic rats and preliminary data seems to suggest that the adducin, the alpha and beta adducin affect the degree of phosphorylation of these proteins and there is a parallelism between the level of phosphorylation of these proteins and the protection towards the development of focal glomerulosclerosis.

Slide 18

That is the conclusion we think that we have some data supporting the role of adducin in humans and in rats but provided that the context is taken into account. Context means biological context, genetic context and environmental context. There is no gene working in the vacuum but always the context must be taken into account.

Slide 19

And finally, this long list of people that have been and you see also the previous speaker that helped us in better confounding the congenic strain and these are also my conflicts of interest because in order to develop congenic rats, maintaining null mice and so on, you need a very strong organisation that cannot be provided by the Italian funding system. We can win the world cup but we don’t have very much money for research. Thank you very much.

Slide 20

Chairman: Thank you very much. We can see if there are any questions. Maybe I can ask you one or two questions. The first one is a fundamental one. If the state of phosphorylation of src is affected by adducin, I suppose that in the case of adducin mutation you must have counter regulation within the cell because src is such an important molecule. So have you looked at the compensatory systems which might operate in a cell where adducin is mutated and where src is hyperphosphorylated?

Prof Bianchi: Yes, as I showed before also for AP2 we have been studying the mechanism of phosphorylation and dephosphorylation because you can assess the phosphorylation potential of a given protein is given by just the balance between the two. Going back to src as you have seen also in congenic rats, perhaps I was too quick in showing the results but the phosphorylated src that you can now detect with very selective antibodies and the phosphorylation of src, as you know is phosphorylated in different sites we have a regulatory site at 5209 tyrosine and the catalytic site of 425 and we have seen an increase in phosphorylation of src at the catalytic site and a decrease in phosphorylation of src at the regulatory site implying that you have a balance of mechanisms keeping the src enzyme open and more prone to phosphorylate the other protein. Also we have studied all the different mechanisms I can tell you that adducin interferes with a very selective domain of src and the interaction is greater with adducing mutate, compared to the wild adducin.

Chairman: Thank you. Maybe I can ask you a more clinical question but it’s a question you know that we all have actually when we are dealing with good candidate genes you have  shown positive, negative what is the next step you are going to see in order to solve or not to solve this important question?

Prof Bianchi: To leave blood pressure because the variability of blood pressure is too high and as you may have seen, we are moving to organ complication but anyhow we have been shown already a few years ago that you can have positive and negative association studies in two populations, one living in North Italy, the other in Sardinia but despite of that you got always the same peculiar biochemical effect of the mutated adducin implying that the association study is too rough as an approach to detect the role of a given gene. Linkage is better but also there you have problems and the context is crucial because there is no gene working throughout the different populations, you must define. I just mentioned that post menopausal women are perhaps the subset of the population in which we constantly find the effect of adducin and on organ complications.

Chairman: Thank you, so probably we’ll move on to the next talk.