CME Slides Forum - A.Y. Bagrov

A joint Congress by ERA-EDTA and ISN

CARDIOTONIC STEROIDS (ENDOGENOUS DIGITALIS)

Alexei Y. Bagrov, Baltimore, USA

Chair: Nathan Levin, New York, USA

Stanley Shaldon, Fontvieille, Monaco

bagrov

Dr Alexei Y. Bagrov
Laboratory of Cardiovascular Science
National Institute on Aging, NIH
Baltimore, MD, USA

Hello thank you very much. So the subject of my presentation today is endogenous cardiotonic steroids or endogenous digitalis.

So this is all about regulation of the Na/K-ATPase or sodium pump, the main function of which, the classical one is transmembrane transport of sodium and potassium. What is marked in red is alpha subunit of the ATPase which is distributed in a tissue specific fashion and represented by 3, 4 actually isoforms which represent specific receptors for cardiotonic steroids and digitalis.

Evidence accumulated over the last 15 years have demonstrated that besides it classical transporting functions Na/K-ATPase is also implicated in generation of variety cell signalling cascade which actually makes the sodium pump a pretty unique regulatory system.

So today we’re going to concentrate on endogenous digitalis, why? In the kidney the sodium pump plays an especially important role because it’s one of the main sodium transporters in the renal tubuli. In the ‘70s and in the ‘80s in several laboratories,

but this use was crystallised by Hugh De Wardener, a theory has emerged, a concept of natriuretic hormone according to which in salt sensitive hypertension when sodium retention and plasma volume expansion occur, a putitative digitalis like natriuretic hormone is being liberated somewhere which is supposed to inhibit ATPase in the kidney and thus promote natriuresis but as very frequently happens under pathophysiological conditions the side effect of this response becomes more important, it inhibits sodium pump in the vascular smooth muscle and which activates sodium calcium exchange and contributes to hypertension. All these views were able to be crystallised because in the ‘70s and ‘80s the community came to the understanding of the importance of excessive dietary salt intake in the pathogenesis of hypertension.

A major breakthrough in the development of use of this hormone was discovery of endogenous oubain in 1991 by John Hamelin and very many very exciting discoveries were made with oubain but oubain was absolutely and is actually inactive a inhibitors of -- renal ATPase. Today we’re going to talk about another compound marinobufagenin this is a hormone which unlike digitalis has a 6-member lactone ring which was purified by Swiss chemists from toads and frogs in the 30s and we and later others demonstrated that it is present in human and mammalian tissues as well. Unlike oubain, marinobufagenin is a pretty effective inhibitor of renal Na/K-ATPase. In vitro at reasonably low concentrations it induces vasoconstriction in human arteries and when measured using competitive immunoassay, its concentration is substantially elevated in patients with essential hypertension.

So to study the possible physiological role of marinobufagenin and endogenous oubain we turned to a classical model, to Dahl salt sensitive rats. On high salt these animals retain sodium and there that’s a classical model of salt-sensitive hypertension. So what we did? We put these animals on 8% salt intake and we compared profiles of renal excretion of endogenous oubain, marinobufagenin and blood pressure. Excretion of oubain exhibited a transient peak response and then it returned down, red line. Marinobufagenin exhibited a sustained increase with parallel elevation in the arterial pressure.

The next question to be answered was to find out whether there was an association or there was a causative link between the two: marinobufagenin and oubain. So during week 1 of salt loading we blocked response of oubain by its neutralisation with a specific antibody and when oubain was blocked red bars versus blue bars, excretion of marinobufagenin was decreased and blood pressure practically didn’t increase in response to high salt diet.
Next we compared, we looked at changes of endogenous oubain in the brain and noticed that this big response of urinary oubain corresponds to an increase in oubain levels in the pituitary which made us think that oubain in this particular model may act as a urohormone which stimulates another player marinobufagenin.

At the stage of established hypertension we’re talking about 3 weeks of high salt intake, administration of oubain antibody does not affect blood pressure in Dahl sensitive rats and when we give marinobufagenin, antibody blood pressure decreases by approximately 30 mmHg.
So what could be a factor which links central oubain with peripheral marinobufagenin? We measured results coming from --- in a laboratory in Ottawa and from our group they indicated that this could be central and peripheral renin-angiotensin system.

When Dahl rats are salt loaded, we observe a parallel increase in pitutary levels of angiotensin 2, adrenocortical levels of AT II and marinobufagenin in the adrenal cortex.

When we pre-treat animals with oubain antibody, levels of oubain following salt administration, they do not rise neither in the pitutary nor in the adrenal cortex and marinobufagenin increase also doesn’t occur.

Last year we finished the study and published it and it was a small group of healthy volunteers, perfectly normotensive people and we observed them 7 days following salt loading and the pattern of responses of two hormones was similar to what we see in Dahl rats namely oubain response was stringent and oubain response was more sustained and levels of marinobufagenin not oubain exhibited a strong positive linear correlation with sodium excretion.

So here is the revised scheme, we just added several boxes and it appears that sodium retention via brain sensor which now appears to be elder sensitive sodium channel, not ENaC something very similar but not exactly the same. Stimulates brain oubain that activates the brain renin-angiotensin system which most likely via the activation of the sympathetic nervous system, activates the adrenocortical renin-angiotensin system and stimulates production of marinobufagenin which is a specific inhibitor of renal isoform of the sodium pump.

So ESRD is a condition which is associated with sustained volume expansion and the salt sensitivity of blood pressure. We compared plasma levels of marinobufagenin and oubain in 53 patients with ESRD and controlled hypertension to age-matched controlled subjects and did the same measurements in rats 4 weeks following 576 nephrectomy and in both cases marinobufagenin rather than oubain became elevated.

Elevation in patients of marinobufagenin was associated with inhibition of the ATPase and the erythrocytes which was actually reversed by monoclonal antibodies against marinobufagenin and levels of marinobufagenin negatively correlated with activity of ATPase which suggests the causative link between the two.

So in the previous slide I mentioned antibody which we used in our further research and maybe you know using antibody in treatment of hypertension may appear a little bit of an eccentric approach but this is becoming a reality in another area in pre-eclampsia. There are more than 50 cases of pre-eclampsia successfully treated with digibind. This is an affinity purified digoxin antibody which recognises endogenous cardiotonic steroids as well and levels of marinobufagenin become dramatically elevated in patients with pre-eclampsia.

So what we did in collaboration with Joe Shapiro’s laboratory in Toledo, Ohio we exposed rats to 5/6 nephrectomy and we didn’t give them antibody but rather we immunised them. So the titre of endogenous antibody was more than 1-10.000. The red bars are nephrectomised animals and green bars are animals which are actively immunised. Effect of blood pressure was minimal if any but what was very interesting was that immunisation of animals improved left ventricular function, decreased heart weight, and decreased levels of markers of oxidative stress and left ventricular myocardium.

But what was the most surprising and unexpected finding for us was that when animals were immunised, we didn’t see any manifestation of cardiac fibrosis which was pretty prominent in nephrectomised animals otherwise. So red bars demonstrate, the left red bar actually of nephrectomised animals, so we see increased levels of type III collagen, pro-collagen and fibronectin and green bars demonstrate that none of those were elevated in immunised animals.

What is even maybe more important is that when we treated naïve control animals with low concentration of marinobufagenin which mimics the uremic levels, we also saw fibrosis in the heart and increased cardiac levels of collagen and pro-collagen.

One of the last things we’re doing in my lab is development of antibody which could have been used to neutralise effects of the substances and we developed an antibody 3E9 which proved to be quite effective in several test systems.
In the last experiment which is still has not been fully analysed we gave nephrectomised animals one shot of this antibody IP and this antibody lowered blood pressure substantially for more than 1 week which was associated with a reduction in heart weight and reduction of carbonlyated protein in the left ventricle and a significant decrease in collagen deposition and surprisingly we have no idea why but this is the case levels of this antibody titre were quite high 1 week after a single administration.

So there are other ways to interfere with endogenous sodium pump inhibitors besides antibodies. Several years ago we compared levels of marinobufagenin in a group of hypertensive subjects with diastolic dysfunction and we noticed that levels of marinobufagenin and ANP correlate. Both marinobufagenin and ANP become dramatically related to ESRD. So ANP is natriuretic and marinobufagenin is natriuretic but MBG is a vasoconstrictor and ANP is a relaxant. So we hypothesised that these two hormones may help each other to excrete sodium but ANP potentially could offset the deleterious vascular effects of marinobufagenin which may also be relevant to its cell signalling.

Indeed such interaction occurs at the level of the ATPase and low concentration of ANP, L for human AMP 1 nanomol/l desensitises vascular ATPase to marinobufagenin but dramatically enhances sensitivity of ATPase from renal outer medullar from rats. So 1 nanomol of MBG in the presence of ANP inhibits the ATPase by 50%.

In two tissues in vascular smooth muscle and in the kidney too different isoforms of PKG are expressed which are the final stage in ANP initiated cascades. Indeed ANP does two different things with ATPase; it dephosphorylates ANP in the aorta and hyperphosphorylates it in the kidney which translates respectively to enhancement and decrease of marinobufagenin induced inhibition.

So at the very end let us see how this situation may be translated in the in vivo scenario. What we did is we compared responses of two hormones and cGMP and changes in sodium pump activity in the kidney and vasculature in Sprague-Dawley rats perfectly normotensive and in Dahls. These animals received a single IP salt load. So response of marinobufagenin was identical in two strains but in Dahls response of ANP was blunted. So was the response of cGMP.

In Spragues salt loading didn’t change blood pressure but it did in Dahls. Sodium pump was not inhibited in Spragues but was profoundly inhibited in DAHL rats. The picture was the opposite in the kidney, sodium excretion was reduced in Dahls and inhibition of renal Na/K- ATPase was reduced in Dahl rats as well. So change in the balance between action of the two hormones in the ATPase in the kidney and blood vessel we think could contribute to the pathogenesis of salt-sensitive hypertension.

So in conclusion marinobufagenin and other cardiotonic steroids participate in the pathogenesis of salt-sensitive hypertension and ESRD. These hormones represent a potential target for therapy and you should always be reminded that a simple reduction of salt intake is a good way to antagonise those effects.

Question: I have a question for you Doctor Bagrov. You’re using a clinical term for rats, ESRD. I wasn’t aware of the use of it in rat renal physiology or pathology. Would you care to be more precise and define what you mean by ESRD?

Dr. Bagrov: Well, of course, it would be more accurate to say that these are rats subjected to 5/6 nephrectomy which exhibits some of the critical symptoms characteristic to ESRD. To be very specific oxidative stress is everywhere in all the tissues.

Question: Bagrov your work on the heart is really fascinating because this is one of the major complications of ESRD. Did you have a chance to look at the aorta? The reason why I’m asking is that one important component of modulating cardiac pre- and after load is of course, large vessel mechanical properties. Does the same fibrosis that you observed in the heart interstitium occur in the aorta?

Dr. Bagrov: I cannot answer this question yet but you know the yield of vascular smooth muscle from the aorta is minimal, so we begin throwing hearts in grinders immediately after we’re done with experiments but we’re very careful with the vessels because once we lose it, but we’ll have it in a couple of months because we have membranes.

Question: Thank you for your talk I enjoyed it. So marinobufagenin in humans is made only in the brain?

Dr. Bagrov: No oubain is made in the brain. Marinobufagenin it’s likely we see that it is produced by primary cultures in the adrenocortical cells from rats and in two other cell lines which believe me or not which are placental, well not exactly placental but they’re chorionic epithelium. So we see huge levels of marinobufagenin in the placenta and particularly in pre-eclampsia. Two cell lines derive from chorionic epithelium produced and mouse adrenocortical cells so it’s likely to be adrenocortical.

Question: So would you care to speculate as to why for example frogs use it as a poison and it’s evolved to be a substance which controls blood pressure or sodium excretion in humans?

Dr. Bagrov: Actually this was what was supposed to be one of my slides which I threw away so people won’t believe that it was staged. In fact I put the story because of toads and frogs. There was a person a very good medical chemist in Bethesda, Geoffrey Fryer who passed away a year ago and he performed an expedition study in toad toxin they went to various lakes in American countries and they were looking at these inhibitors of the ATPase in the toad skin in different parts of Puerto Rico, Venezuela etc. and they found that the highest levels of sodium pump inhibitory activity are observed in those in toads who migrate from an environment with lower salinity to higher salinity. In toads the skin is analogous to renal tubuli. So next my friend David Lichtenstein from Hebrew university put these toads in a high salinity environment and this stuff was increased so this is I quote natriuretic or a skin uretic hormone for the amphibian.