NIH CLINICAL CENTER GRAND ROUNDS
Episode 101409
Time: 1:30:00
Recorded October 14, 2009

Contemporary Clinical Medicine: Great Teachers
The Sea Within Us: Clinical Disorders of Water Homeostasis
Robert W. Schrier, MD
Professor of Medicine, Division of Renal Diseases and Hypertension
University of Colorado Denver, School of Medicine

ANNOUNCER: Discussing Outstanding Science of the Past, Present and Future - this is NIH Clinical Center Grand Rounds.

(Music establishes, goes under VO)

ANNOUNCER: Greetings and welcome to NIH Clinical Center Grand Rounds, recorded October 14, 2009 at America's Clinical Research Hospital, the Clinical Center at the National Institutes of Health in Bethesda, Maryland, an agency of the United States Department of Health and Human Services. Today, a special Contemporary Clinical Medicine: Great Teachers Grand Roounds, featuring Dr. Robert W. Schrier, professor of medicine in the Division of Renal Diseases and Hypertension at the University of Colorado Denver, School of Medicine.

You can see a closed-captioned videocast of this lecture by logging onto http://videocast.nih.gov -- click the "Past Events" link -- or by clicking the "View Videocast" link on the podcast homepage at www.cc.nih.gov/podcast. The NIH CLINICAL CENTER GRAND ROUNDS podcast is a presentation of the NIH Clinical Center, Office of Communications, Patient Recruitment and Public Liaison. For more information about clinical research going on every day at the NIH Clinical Center, log on to http://clinicalcenter.nih.gov.

We take you to the Lipsett Ampitheater at the NIH Clinical Center in Bethesda, Maryland for today's presentation.

COHEN: My name is Jeff Cohen. I would like to welcome everyone to our second great teacher series at medical grand rounds. I also want to mention that at the end of the lecture today there'll be an opportunity to meet our great teacher just outside the auditorium. There will be a reception. And for those who have seen interesting cases, mysterious cases in which it's been a difficult diagnostic dilemma, in April we will be presenting some of these cases to our great teacher, so please send me an e mail if you have interesting cases. Again, Jeff Cohen, or put it on the CME evaluation.

Now I would like to introduce Dr. Mark Nepper, who is the Chief of the laboratory of kidney and electrolyte metabolism in the National Heart, Lung, and Blood Institute, who will introduce our great teacher today.

NEPPER: Thanks Jeff. It's my great, great pleasure to introduce my good friend Robert W. Schrier from the University of Colorado as the next speaker in the great teachers series here in the Grand Rounds. Bob is a nephrologyst who led the department of medicine at the University of Colorado as chairman from 1976 to 2002. He specializes in disorders of salt and water balance, particularly those related to the hormone vasopressin, and you'll hear about that in his lecture today. Bob was born and reared in Indiana and got his undergraduate education at DePauw University. That's the one with the "W", not the one with the "I". So DePauw is in Greencastle and not in Chicago. At DePauw he starred in both baseball and basketball, in fact he was so good at basketball that he was later named to the Indiana basketball hall of fame along with luminaries like Larry Bird, Willie Gardener, Oscar Robertson and John Wooden. He got his M.D in Indiana as well, at the University of Indiana, School of Medicine in 1962. Now Bob has won a host of awards both locally, nationally and internationally and he's taken leadership positions in many organizations including presidencies at the Association of American Physicians, American Society of Nephrology, the American Kidney Foundation and the International Society of Nephrology. He's published over 450 peer-reviewed papers and has edited numerous textbooks, including an absolutely delightful book for students and host officers called The Internal Medicine Case Book: real patients, real answers. This discusses practical aspects of 88 syndromes and diseases common in internal medicine practice, and I recommend you take a look at it if you're not already familiar with it. Bob has trained many of today's leaders in nephrology and internal medicine and has earned recognition for his innovations in teaching. He's noted for his brilliant analytical lectures on salt and water balance disorders. Bob is truly a great teacher and I'm happy to welcome to the lecture Robert W. Schrier, Bob Schrier.

[applause]

SCHRIER: Well, let me say I'm deeply honored to be here. I think it's not an understatement that this is one of the great institutions in the world. And I don't mean just in health, in any area. I was told to have a slide of objectives—that's this slide—and another slide of disclosures, and that's this slide. The title is "the sea within us" because two-thirds of our bodies are water. So maintaining homeostasis of body water balance is extremely important to survival, and yet we have a number of clinical disorders in which there's an excess of water in the body, as featured by hyponatremia. So I want to have you join me for a journey through water homeostasis over quite a long period of time. It was known that there was an anti-diuretic hormone in the posterior pituitary, but what wasn't known is whether or not this had anything to do with the kidney's ability to regulate renal water excretion.

So Professor Verney at Cambridge, he had the hypothesis that there must be central osmoreceptors that are regulating this antidiuretic hormone that circulates to the kidney in circumstances of water excess and water deprivation. He performed, with his wife, experiments in a single animal. He reasoned that if this is an osmoreceptor, urea infusions in a water-diureasing animal shouldn't do anything because it crosses the membrane. And if it's an osmoreceptor, then hyperontonic sodium chloride, sucrose or mannitol all should give the same antidiuretic effect. And if we infuse it correctly into the carotid over ten seconds, the antidiuresis should be seen sooner than if we infuse it into an ankle vein; and those studies are shown on the next slide. And you can see during the carotid injection, with the three different hypertonic solutions, there was an earlier antidiuresis than during the ankle vein injection. And, not shown in this slide, urea infusion did not alter the water diuresis.

Then Professor Vincent Du Vigneaud synthesized a preparation for assessing the biological properties associated with arginine- vasopressin, which we know now is the antidiuretic hormone. But what was problematic is when one looked at—with the bioassay of this antidiuretic hormone, there was no difference between individuals who had a normal plasma sodium and normal plasma osmolality verses—versus those who had low plasma osmolality. So the focus turned to the kidney in exclusion of antidiuretic hormone or arginine- vasopressin, and there was data suggesting that catacolmilates modulated water transport in the kidney. So very early we decided to take a look at this and see if it could be demonstrated in vivo. And as shown on the left-hand side of the slide, there was indeed a profound antidiuretic effect of beta adrenegic stimulation. But to make sure that it was adh-independent and specifically for an intra renal mechanism, the studies were repeated as shown on the the right-hand side of the slide and hypophysectomized animals that were replaced with glucocorticoid hormone, and the effect was totally abolished. So rather than our hypothesis that this was an interregnal phenomenon, it appeared to be totally dependent on antidiuretic hormone. The mechanism of the water diuresis without the alpha adrenergic stimulation produced similar results. Intact animals, urinary osmolality went down, free water excretion increased. This was totally abolished by hypophysectomy and removal of the central source of vasopressin release. And this occurred totally independent of osmolality. So then the question was: what is, how is the central nervous system sensing these effects that are causing water diuresis, antidiuresis, totally independent of the osmoreceptors of Verney. Well one clue was the mechanism of antidiuretic effect of vagotomy also was abolished by hypophysectomy as shown on this slide. So we turn to experiments where the arterial baroreceptors were denervated and as shown here, arterial baroreceptor denervation totally abolished the antidiuretic effect of beta adrenergic stimulation. And the anti i have these reversed of anti adrenergic stimulation. So this is when the nonosmotic baoreceptor regulation of arginine- vasopressin became very important in our research. I had a very good renal fellow who was involved in most of those experiments, Thomas Berl, and I'm congratulating him here 30 years later when he became president of the American Society of Nephrology.

Then the radio immunoassay was developed, and they confirmed the Verney experiments, as shown in the open circles, that small changes, one to two percent changes, in osmolality, this is a increase in osmolality increased in a linear manner: plasma arginine- vasopressin. But the nonosmotic baroreceptor pathway was much less sensitive, as shown in the solid circles, and it took an eight to ten percent change in blood volume, for example, for these arterial baroreceptors to be activated. And when they were activated, the levels of arginine vasopressin increased to a much greater extent. And we know that vasopressin not only acts on the v2 receptor in the collecting duct but on the v1 receptor in vascular smooth muscle. There were neurophysiological studies out of Japan that showed that a single superoptic neuron where vasopression is synthesized would respond to changes in osmolalty via the osmoreceptors, but would also respond to the nonosmotic baroreceptor pathway. And if a large water load would cause urinary dilution, we call that the reset osmostep. If it doesn't cause urinary dilution and the baroreceptor dominates independent of osmolalty, we call that the syndrome of antidiuretic hormone. Not only are the osmotic effects on the synthesis and release of vasopressin but also there's an effect on thirst and both of these can be overridden by the arterial baroreceptor nonosmotic pathway.

Well, there was a question about the delivery of water to the distal diluting segment and how much that was playing a role in water retention and hyponatremia. We know the ability of the the normal kidney to excrete solid free water is enormous. And one can calculate that, as shown on this slide. And if twenty percent of this filtered load per day reaches the distal diluting segment, if given over 24 hours, at least theoretically, one could excrete large amounts of water. And this was somewhat disturbing because all of the hyponatremic disorders, these patients were not drinking a lot of water. In fact here's a study where the average water intake in these hyponatremic patients was only 2.4 liters, so you would have to decrease this 28 liters to less than 2.4 if distal delivery was the major factor causing hyponatremia. And we did studies in hypophysectamized animals trying to decrease distal delivery and see if we could get urinary osmolality in the absence of adh above plasma. Never could achieve that. And that's when we really suggested that the nonosmotic release of vasopressin was what was primary causing of retention and impairing urinary dilution independent of distal delivery to the diluting segment.

Why do we worry about hypoosmolalty? This is a brain cell, and you can see the fluid moving in and out in equilibrium and then with hyponatremia, water will move into the cells and one has an expansion of the brain cells. Now the skull limits expansion to about eight percent, so that if one has acute hyponatremia, one can have severe morbidity and mortality. And here's an imaging of the normal brain and then the hyponatremic brain with brain edema and one can have herniation and cardio respiratory arrest and death. But fortunately there are adaptations to brain edema that occur chronically with hyponatremia. Very early, within one to three hours, ECF moves into the cerebral spinal fluid and then into the circulation. Intracellular osmolality decreases by extrusion of cell potassium, organic solutes and, chronically, the organic osmolytes such as phosphocreatine, myoinositol, and amino acids are lost so that the brain volume returns back to normal. But what we know now, if there is rapid correction of chronic hyponatremia, one can have osmotic demyelination, morbidity and mortality. So there's an adjustment but also a predisposition to dilitarious effects on the brain.

Well what about asympotomatic hyponatramia, which you see clinically all the time—serum sodium 130, 128. Large percentage of patients that come in the hospital with cirrhosis, heart failure, sidh. And what this slide shows is a study out of Belgium using a very sensitive measure of gate, and here is an example serum sodium of 130 versus 139, a marked improvement in gait. The second example is shown here, abnormalities in gait that became more normal when the serum sodium concentration was corrected. So what we call asymptomatic hyponatremia when looked at in a sensitive matter is not asymptomatic.

We also know there's increased risk of falls, with "asymptomatic hyponatremia," and here's a study with 122 asymptomatic chronic hyponatremic patients had a sixty-seven fold odds ratio for falls. In contrast, the normal natremic controls had no increased predisposition to falls. Well, then there was another dilemma, this bilipid membrane around cells didn't seem to be the reason that the rather fast equilibration of fluid across cell membrane. So people suggested there must be water channels. But for decades no one could find any water channels. And then a young hematologist, Peter Agre, at Johns Hopkins was looking for the RH factor. And he found a protein that hadn't been identified before in the red blood cells and since they were permeable to water, he thought maybe this is a water channel. And what he did then was to transvect that protein into frog eggs, and then put the frog eggs for three minutes in hypotonic solution. Normally the frog egg is impermeable to the water, but with this transvected of this water channel, within three minutes, the eggs exploded and in this hypotonic solution. So this is a major contribution, and Dr. Agre received the Nobel prize for this finding. We know now that this is the schematic representation of the structure of aquaporin with the npa motest feeding back on themselves producing this water channel. And this is another schematic representation, and in the membrane, these aquaporin water channels occur as tetramers.

Now one of Dr. Agre's friends said, the proximal tubule of the kidney is very water permeable—maybe these aquaporin one water channels could be found in the proximal tubule. And he found both on the basolateral and apical surface of the proximal tubule, these aquaporin one water channels. But this excited the biomedical community and a group in Japan said, we know water regulation doesn't occur in the proximal tubule by vasopressin, and so let's look in the collecting ducts where arginine- vasopressin works and see if there are other water channels. And here you can see the single molecules of water permeating the aquaporin water channel. This is the Dr. Sei Sasaki and San E. Ishikaw and Sasaki's group actually demonstrated the aquaporin two water channels which are sensitive to regulation by vasopressin, both short-term and long-term. These are the nephron sites involved in the aquaporin water channels, the ion channels, and urea transporters. This aquaporin water is not only in the approximate tubule but also in the descending limb in the luphenry where water moves out and the osmolality increases towards the tip. And we know cortex—the outer medulla, the inner medulla—here's where the osmatic gradient occurs for urinary concentration. And it's very important to have an intact sodium potassium-chloride two transporter in the ascending limb because those ions are transported out in a water impermeable segment of the nephron, again initiating the counter current mechanism which leads to the osmotic gradient. And here in the collecting duct, we see aquaporin two, which is in the cytosol but can traffic to the atypical membrane; aquaporin three which is constitutive in the basolateral side of the membrane; and then, in the inner medulla, aquaporin four, along with two and three, exists. And then there are the urea transporters with recycling, which also contributes to the osmotic gradient for water conservation. Verquin at San Francisco has done a number of knock-out studies and then has compared urinary concentrating mechanism in these various knock-out animals. Here you can see the increase in osmolality normally and, with the aquaporin one knock out, total evolution of the concentrating ability. Less so with aquaporin three, but a very polyuric animal, and less with aquaporin four, even though it's significant because it's in the inner medulla where most of the water has already been reabsorbed. Well, then the vasopressin receptor, the g protein linked receptor was cloned. And many studies, including here at the NIH showing the cell signaling in the principal cell, the collecting duct and we know that adenylyl cyclase is actived, cyclic anp and then protein cynise clase and that leaves the carboxyl terminal of the aquaporin two and that leads to trafficking of these water channels to the apicol urinary membrane which becomes permeable and with the osmotic gradient, the water is transported across the collecting duct membrane into the interstitium and out through the aquaporin three and four. And this apparently involves some deep polymerization of actin, this trafficking effect but there's also a long-term effect where the aquaporin two protein expression is increased by vasopressin, as well as a short-term trafficking effect. And then with suppression of vasopressin, these water channels return to the cytoplasm in these vesicles.

This congenital diabetes insipidus that occurs in young kids and leads to them drinking 10 to 15 liters of fluid a day to avoid dehydration was studied, and it was found that in the v2 receptor many mutations, shown with the red dots. But that only accounted for 80% of the congenital diabetes insipidus, so another group in holland said maybe the other 15% involve mutations in the aquaporin 2 water channel. And that's shown on this slide that accounts for the other 15% of the congenital diabetes insipidus.

Well, then the next advance, manning and sawyer studied protein antagonist to the v2 receptor for 20 years. It worked in every species they studied except man, where it was an agonist, not an antagonist. And then there was the discovery of the first orally-active non-peptide v2 vasopressin receptor antagonist, and now there are several of those available. And Dr. Yamamura was first author on that paper.

We know now that the vasopressin receptor antagonist go into the deep cleft of the v2 receptor and block the effect of vasopressin. But then the question is why don't they activate the receptor, and this appears to be dependent on the fact that vasopressin links to this hi-a link but the orally active nonpeptide vasopressin receptors do not. So this link for the h1 helix appears to be very important for the antidiuretic effect, and the receptor antagonist do not activate that.

So we have a lot of information over several decades by major discoveries, including those here at this institution. Can we apply that and understand some of the concentrating and diluting defects that occur in clinical medicine? The ability to conserve water when you need to, ability to get rid of water when you need to. We know that primary polydipsia, and it's been called psychogenic water drinking, compulsive water drinking, is associated with the concentrating defect. And the studies who were done by Hugh De Wardner and herxheimer … I just had the opportunity to visit with Professor De Wardener who just had his ninety-fourth birthday. But he told an interesting story, they were drinking 10 liters of water per day for 10 days and he and herxheimer were visiting the Tate museum. And they had their big bottles for urine and their big bottles for water, and somehow they got them mixed up and they decided to terminate that study at that time.

[laughter]

But here's their concentrating with and without vasopressin before drinking the water and afterwards, so there's no question: concentrating defect. Well many of the polyuric studies that have been performed in normal animals have used glucose drinking water. Well that increases solid excretion, that alone will impair concentrating ability. So we developed a model where we knew how much the animals had to eat, and if you put it in 22 milliliters of water or a hundred milliliters of water they would drink it all in order to get that food. And it turned out to be a very nice model of primary polydipsia, and you can see the increase in urine output, the decrease in urinary osmolalty. And this occurred independent of changes in osmolar clearance. And like impatience, polydipsia does not cause hyponatremia. The osmolality goes down to the lower level of normal, but unless you have a diuretic or volume depletion, it should not be/cause overt hyponatremia and this is the exact type of data that you see in humans, with the vasopressin levels obviously suppressed and no effect on kidney function. So here's a pretty good model for primary polydipsia and indeed they have a diminished concentrating ability.

Here's normal with 36 hours of fluid deprivation, this is the last 12 hours, and can you see the ability to decrease urine volume is impaired significantly and that's associated with a decrease in urinary osmolalty. Then the question is now that we know about these water channels, are they involved in this concentrating defect with primary polydipsia? And we can see that despite an elevation and plasma vasopressin, that if anything will elevate aquaporin two. The aquaporin twos were substantially suppressed. It was thought for many years, and if you look in textbooks that there's an increase, that primary polydipsia increases medullary blood flow, washes out the osmotic gradient, and that's why we have a defect in concentrating ability.

Well the sodium potassium 2 chlorides were not down and medullary osmolality was not washed out. So the defect primarily is associated with that down regulation of aquaporin 2. What about thyroid disorders, we know hypothyroidism can be associated with rather severe hyponatremia. We know that hyperthyroid patients actually have polyuria and polydipsia. So one can produce hypothyroidism and have a post-control by treating the hypothyroid state with thyroxin. Always important to look for potential baroreceptor activity for the nonosmotic release of vasopressin, which modulates aquaporin 2 water channels and just like in humans, the heart rate is less, it can be restored wide thyroid replacement, cardic output also. There was impaired concentrating ability in the hypothyroid state, ability to conserve during fluid restriction which was reversible. Urinary osmolalty also was decreased and was reversible, as was medullary osmolalty.

So this suggested that in contrast to primary polydipsia, there was a defect in the osmotic driving force. So it's very important in that setting to look at the sodium potassium 2 chloride cotransporter which initiates urinary concentrating ability, and indeed it was decreased and reversible with thyroxin. And one can show the same thing with immunoflourescence. Here's control, hypothyroid, hypothyroid plus dyroxin for the sodium potassium 2 chloride expression. And that in addition, in the inner medulla, there's a down regulation of aquaporin 2 and aquaporin 3. And this was independent during the concentration of differences in vasopressin.

So that's the concentrating defect, very much an impairment in the counter current concentrating mechanism, no doubt involving the sodium potassium 2 chloride down regulation. What about the impaired ability to excrete water, which we see clinically in the hyponatremia of advanced hypothyroidism. And one can show that in hypothyroid animals, a marked decrease in urine flow with the same water load and reversible with thyroxin. And if you can't get the urinary osmolalty below plasma, you can't get rid of free water. And that's what happens in this circumstance, and the reason for the hyponatremia, and it's also reversible. And this was in the presence of elevations of plasma vasopressin. And we would expect elevations of aquaporin 2 protein expression in that setting, and that's exactly what happens.

So now with another experimental tool of being able to block the v2 receptor, one can analyze whether or not this effect on aquaporin 2 and water excretion is due to the nonosmotic release of vasopressin. And here can you see with the v2 receptor antagonist, total normalization of ability to excrete water and ability to dilute the urine way below plasma. So the concentrating defect and the urinary dilution defect are due to different mechanisms in the hypothyroid state.

Well what about hyperthyroidism? Again, one can produce a reproducible animal model that's associated with increase in heart rate, increase in cardiac index, increase in blood pressure, increase in pulse pressure and increase in renal blood flow: very similar to what happens in hyperthyroidism. Well, when the hyperthyroid state occurs there's an increase in drinking and eating and also an increase in solid excretion. But if you control the fluid intake and the food intake, you still see increase in solid excretion due to the catabolism, which contributes in part to the polyuria, as shown here with the same amount of fluid and food intake, but there was also a down regulation of aquaporin 2 in the hyperthyroid state.

Well what about glucocorticoid deficiency and excess? Again, hypopituitarism, glucocorticoid insufficiency, one knows, predisposes to hyponatremia. What's the mechanism and is there a defect in Cushing's disease with glucocorticoid excess? And one can do this with adrenalectomy, replacing both hormones: mineralic glucocorticoid hormone compared to only minerlaic glucocorticoid, so you can study the corticoid deficiency. And again, one has to know what's happening systemically, and does it mimic what happens in patients? And you can see the mean arterial pressure is down, cardiac index is down. And interestingly, just in patients, the defect and glucocorticoid deficiencies not only in the heart level but at the peripheral vasculature.Normally this decrease in blood pressure would increase peripheral vascular resistance, so there's a central and a peripheral defect in glucocorticoid hormone deficiency, just like you see in patients. Looking at the concentrating ability, a defect, maximum urinary osmolalty: decreased.

Independent of the changes in plasma osmolality, but again the driving force was diminished and the protein expression of sodium potassium 2 chloride and sodium hydogren ion exchange, are both involved in concentrating ability, are significantly decreased. In addition to this effect on the counter current concentrating mechanism, there was a down regulation of aquaporin 1 or aquaporin 2 and the urea transporter. And one can show that also in these immunohistochemical studies, the urea transporter down regulated in glucocorticoid deficiency, and aquaporin 2 down regulated with the glucocorticoid deficiency. Well what about the impaired water excretion with the glucocortacoid deficiency and does it involve the nonosmotic release of vasopressin? So one can look at glucocorticoid deficiency without and with a vasopressin antagonist, and you can see the normalization by blocking the vasopressin receptor and normalizing the ability to dilute the urine. So again the hemadynamic effects are arterial baroreceptorr nonosmotic release of vasopressin would presumably be incriminated. And if that's the case, one should see the parallel increase in aquaporin 2 and reversal with the v2 vasopressin antagonist. And if one looks at the phosphorylated aquaporin 2, one can see the same thing.

Well, what about Cushing's disease? Here a model of Cushing's disease, a known cause of hypertension, increased renal blood flow, increase in clearance of creatine or GFR, urine flow and urea excretion. If one looked at the ability to conserve water, there was clearly a defect, more loss of weight with glucocorticoid excess and a decrease in maximum urinary osmolalty, but the continuing increase in urea excretion. And if one looks at the water channels, the defect was not due to a down regulation of aquaporin 1, it was not due to a down regulation of aquaporin 2, was not due to a down regulation of aquaporin 3. And so in this study, it appeared that the down regulation and experimental cushing's disease was due to the urea transporter a1 that was associated with the increased urea excretion and impaired osmotic driving force.

What about mineralocorticoid deficiency? Where have you bilateral adrenalectomy. The control gets mineralocorticoid and glucocorticoid replacement and the mineralocorticoid deficiency only gets the glucocorticoid replacement. The question is since these individuals lose sodium just like Addison's disease, is it due to the mineralocorticoid deficiency or is it due to the negative sodium chloride balance? And so, control mineralocorticoid deficiency and mineralocorticoid deficiency plus sodium chloride to avoid the volume depletion. And the rest of the parameters were comparable. Again there's a concentrating defect with mineralocorticoid deficiency, maximum urinary osmolality diminished. And in the third group one could see that the negative sodium balance was abolished because the animals had saline drinking water and avoided this volume depletion in the presence of mineralocorticoid deficiency. And when that was the case there was no longer a defect in sodium potassium 2 chloride even though there was still mineralocorticoid deficiency but not a negative sodium balance. Aquaporin 2 also returned, as did aquaporin 3. And there is data that vasopressin not only modulates aquaporin 2 protein expression, but also aquaporin 3 on the basolateral membrane. And then using the peptide antagonist in mineralicorticoid deficiency, a much earlier study, one could show improved diluting ability compared to the mineralicorticoid deficient animals, without the vasopressin antagonist.

Well, clinically, you see a lot of concentrating defects, so called, acquired nephrogenic diabetes insipidus, this occurs with hypercalcemia, this occurs with hypokalemia, this occurs with lithium, this occurs with acute and chronic renal failure. So the group in orhoos, the Danish group, that had actually worked quite closely with Peter Acre, along with Mark Nepper, they did studies looking at these experimental models of these clinical disorders. And aquaporin 2 was found to be down in lithium, hypokalemia and hypercalcemia, contributing to the acquired diabetes insipidus, and the same was true with their bilateral renal obstruction. But they also had data with unilateral showing the same thing: schemic acute renal failure and chronic renal failure, a down regulation of the aquaporin 2 protein expression.

Well what about cardiac failure? We know we see a lot of hyponatremia in advance heart failure. Heart failure is quite a dilemma because total blood volume is actually increased in heart failure. So why isn't the kidney getting rid of sodium and water, if you expand total blood volume in normals you get rid of sodium and water. In heart failure the kidneys are expanded and you retain sodium and water. And we know the kidney's normal because when you do a left ventricular assist device or a heart transplant, the kidney no longer retains sodium and water. And as Bohr wrote, "we shall never understand anything until we have found some contradiction." So heart failure seemed to be rather contradictory relative to the regulation of sodium and water. But what we know now is it's not the total blood volume but the 15% that's estimated in the arterial circulation that modulates renal water excretion and that one can have an increase in total blood volume if it was more on the venus side, low pressure side of the circulation. But we also know that it couldn't be just a decreasing cardiac output causing arterial under filling because we know in high output failure, we know in pregnancy, we know in cirrhosis: cardiac outputs actually increased. So there must be other ways to unload and activate the arterial baroreceptors, and we'll get to that when we talk about cirrhosis and pregnancy. This is about sodium, which we won't be talking about, but part of that activation, these arterial baroreceptors as we talked about, is a nonosmotic release of vasopressin which not only causes water retention but activates the b 1 receptors on the vascular smooth muscle. With the bioassay, the conclusion was that vasopressin is not involved in hyponatremia. But with the assay, normals suppress vasopressin when the osmolality falls below 280, and in heart failure, independent of diuretics or not, the majority of patients had a detectable vasopressin levels by radioimmunoassay and couldn't have been detected by the bioassay that had a sensitivity up here.

So the progression, the more arterial under filling due to decrease in cardiac output, the more activation the neurohumor access but we're focusing on the nonosmotic release of vasopressin and the sodium and water retention expands total, total blood volume. Pretreatment hyponatremia, therefore, if this is a protective mechanism for arterial underfilling, should correlate with activation of the renal ansotension system and that's exactly what one sees. And as one has more increased nonosmotic increase in thirst and release of ADH, the pretreatment hyponatremia should be one of the worst prognostic factors for mortality and heart failure, and you are that's exactly the case.

Well, can we block this water retention in hyponatremia? This is an acute study, here's the control, here's the placebo and here's increasing doses of a v2 vasopressin antagonist and rises in serum sodium concentration. And if that's the case, then one should be able to show in the inner medulla up regulation of aquaporin 2 and that's when one sees an experimental heart failure. And one should be able to reverse that with the v2 vasopressin antagonist, and that's what this study shows.

Obviously you can't measure inner medulla aquaporin 2 in patients, but some of that spills off into the urine and so with radioimmunoassay, one can measure aquaporin 2, before and after the v2 vasopressin antagonist. And you can see a significant decrease. Well, what about the v1 receptor of vasopressin? Is it involved in the heart failure? And the answer is we don't know. At least theoretically it could be involved and the combination of v1/v2 vasopressin antagonist might be of valu. And those studies have not been done. The v2 causes water retention, increased preload but the v1 is a potent venoconstrictor also has effects on systemic arterial consistence, coronary constriction, and like angiotension, causes remodeling of cardiomyocytes by increasing synthesis. And with this combination, of increased cardiac free load, after load, myocardial aschemia, increased wall stress, one has left ventricular dilification and hypertrophy. So if you block those would have you a beneficial effect? And that study needs to be performed.

What about cirrhosis? Increased cardiac output, increased total blood volume. And yet if you put a normal liver in, those kidneys no longer retain sodium or water. Or someone who dies of liver disease, you can take those kidneys out and put them into a patient who has a normal liver, but end-stage kidney disease and they work, they don't retain sodium and water. Just like heart failure, but the cardiac outputs in the opposite direction. And that's why it's clear now that arterial under filling, those baroreceptors can be unloaded either by decreasing cardiac output or arterial vasodilatation and the first thing that occurs in cirrhosis is blankly vasodilatation and then a secondary compensatory increase in cardiac output but otherwise looking very similar to the compensatory reponses that occur with low output cardiac failure. And again, this is the sodium story, but we're talking about the water story today. But again very similar as one as more vasodilation, one should have a progressive activation of the neurohumor access and expansion of total blood volume. Again, decades using the bioassay suggesting vasopressin was not involved but with the radioimmunoassay, one can measure the vasopressin levels in the area between point 5 and 4 so where concentration occurs and in these nonexcreters with hyponatremia, with cirrhosis, they had persistently elevated nonosmotic release. Hyposmolic didn't suppress their vasopressin levels. And again, we'll lower the serum sodium and this is with about 23 different parameters, more likely to go into petarenal syndrome which has nearly 100% mortality without liver transplant. Again, the inner medulla—water—bloating does not suppress the aquaporin 2 but the vasopressin receptor antagonist does. Very similar to heart failure. And now we know there are there are clinical studies, these are in hyponatremic cirrhotic patients, this is out of munich, different doses, better effect with the higher dose, and decreasing urinary osmolalty, and correction of the hyponatremia.

But what's causing that arterial vasodilitation? Very imporatnt question. If it's initiating this sodium and water retention and if nitric oxide is involved, if you give levels over seven days of an inhibitor of nitric oxide and reverse the arterial under filling by raising vascular resistance back to normal, what will you do about the water excretion? And here we see the vasopressin levels came back down to control. And the ability, these are controls, the ability to excrete water, normalized in the hyponatremia was corrected. These studies need to be supported by clinical studies in patients.

There's the salt studies published in the New England Journal where cirrhosis, heart failure, and SIDH were looked at. Two duplicate studies—one in the united states, one internationally, and the data was very similar in salt 1, salt 2. Here's placebo, here's the rise with the inhibitor. And when the inhibitor of v2 vasopressin receptor stops, there's a reversal. That's all patients, and the results are exactly the same when you start off with more severe hypontremia. Hyponatremia occurs once it's … and this is a non-peptide orally active that's available clinically. And even though maximal effects are different in cirrhosis, heart failure and SIDH because of the different delivery to the diluting segment, one has effects in all three circumstances, and increasing serum sodium concentration.

Well, if this is the way the body's working, it shouldn't just be in disease and it should occur in pregnancy. We know that pregnant women are vasodialated, but is it early enough to be initiating these arterial underfilling neurohumoral responses? And these are studies where the hemodynamics and hormones were studied before the women got pregnant and then very early in their pregnancies. And it's a very early vasodialated state.

Less fall in blood pressure because the compensatory rise in cardiac output, but not high enough to avoid the decrease in mean arterial pressure and plasma volume expansion. The renal ansitension system is activated, the nonosmotic release of vasopressin is activated, data not shown. But since vasopressin increases aquaporin 2, if this is the reason for the hypo-osmolalty in pregnancy, then one should be an up regulation of aquaporin 2 protein and rat pregnancy pretty much is identical physiologically to human pregnancy. And you can see first trimester, second trimester, third trimester, highly significant increased expression of aquaporin 2 in pregnancy. And if one gives a v2 vasopressin receptor, you have increase in urine volume, decrease in urinary osmolalty.

Oxytocin is used in pregnancy, and the initial work here at the NIH showed that oxytocin is not working through an oxytocin renal receptor, but through a v2 vasopressin receptor. And that's what one sees here, the oxytocin receptor didn't work, but the v2 receptor did. The antidiuresis was reversed, and the effect on urinary osmolalty was reversed with the v2 receptor antagonist. If that was the case, then one should see a similar effect on aquaporin 2 water channels with oxytocin that one sees with vasopressin, and that's what the data showed. Here's the increase with oxytocin reversal with the v2 receptor antagonist and same way with phosphorylated aquaporin 2. And one can see the effect of trafficking. I don't know if you can see this but the aquaporin 2 going out to the atypical membrane with oxytocin just like vasopressin and reversed with the v2 vasopressin receptor antagonist. And not shown, no effect with oxytocin receptor antagonist. So the arterial underfilling can occur by other means. Nitric oxide appears to be involved in cirrhosis, but estrogen also upregulates nitric oxide. And once one has the arterial underfilling, one has the baroreceptor nonosmotic release of vasopressin, the long-term and short-term effect on aquaporin 2 water permiability, water retention and hyponatremia. And here's where the receptor aqua-ready agents were.

So by all of these discoveries over a decade, one can take them to clinical circumstances, develop models and understand in more detail the molecular mechanisms involved in these concentrating, diluting mechanisms. And Mark told me to put forward some stimulating questions, so I put this slide together. Will correction of hyponatremia with the v2 receptor antagonist improve clinical and subclinical dementia in the 20% of nursing home patients who are hyponatremic? Will it decrease falls and hip fractures that occur more frequently in hyponatremic elderly patients? Will it improve mental cognition in hyponatremic, cirrhotic patients with overt or impending hepatic encephalopathy? Will it improve cognition and quality of life in cardiac failure patients who are not profusing their brains well and are hyponatremic? And will it be of symptomatic value in patients with diuretic-resistant cirrhotic patients with ascites or advanced heart failure? And this is our small group. These two research assistants have been with us for 30 years each, a total of 60 years. You may have seen kadha haporchi on one of the first slides, this is the second generation, malisa, and she's an associate professor. These are instructors that have worked very diligently in these studies, and Dr. Chen who's returned to Taiwan and is an associate professor back there.

And so I'd like to give sincere thanks to the National Institutes of Health whose funding has been very important in these studies over many years. Thank you very much for your attention.

[applause]

NEPPER: I think we have time for just a couple of questions. We're close to the end of the hour, but I'll get things started so polycystic kidney disease is recognized as a really important cause of chronic kidney disease, chronic renal failure as they used to call it and it seems to be associated with, the progression seems to be associated with the v2 receptor. So where do things stand if anywhere with regard to using these non-peptide vasopressin receptor antagonists in the treatment of polycystic kidney disease?

SCHRIER: The group at mayo, in three different models of polycystic kidney disease had shown that causing polyurea by blocking the v2 receptor antagonist slowed down the progression of the polycystic kidney disease. And the theory was that with the polycystant 2, which is an ion channel, that the effects of calcium in suppressing cyclic amp, are abolished and so one has high cyclic amp levels and that increases the secretion of fluid into the system and also proliferation, but since the v2 receptors are only on a collecting duct, you'd have to say that the major defect in renal function is due to cysts that are dry from the collecting duct if indeed the v2 receptor antagonist is specific in patients. And there's a study now with tollvap and the orally vasopressin antagonist placebo versus v2 receptor. And it's hard to do a control study when you're urinating a lot due to the vasopressin receptor antagonist verses placebo. But that study is ongoing, and i think we don't know whether it's going to be whether the animal data will be applicable to patients with polycystic kidney disease. But it's an exciting area.

NEPPER: Ok. I guess no other questions, so I want to again thank Bob for a wonderful lecture.

[applause ]