ISN VIDEO LEGACY PROJECT

DR. MAURICE BURG
INTERVIEWED BY DR. MARK KNEPPER


MK:

Hello Iím Mark Knepper Chief of the Laboratory of Kidney and Electrolyte Metabolism at the National Heart, Lung, and Blood Institute in Bethesda, Maryland. Today itís my pleasure to interview Maurice B Burg about his nearly 50 year history, career rather, in science. Dr Burg is principle investigator in the Laboratory of Kidney and Electrolyte Metabolism National Institutes of Health in Bethesda. Dr Burg has been a leader in the field of renal physiology. Heís known for the development of critical technology that has opened new paths to discovery of many of the physiological principles that comprise our modern understanding of renal function and osmotic regulation. Not only will we hear today about the invention of the isolated perfused tubule technique, for which he is best known, but we will also hear about a host of other innovations basic to this study of kidney physiology. Now this interview is being conducted on May 10th 2005 at the National Institutes of Health in Bethesda, Maryland. The producer is Alice Hardy. Copies of the interview will be distributed to the American Physiological Society for its Eminent Physiology Archives and to the International Society of Nephrology for its Legacy Archive and a copy would also be deposited in the office of the NIH history here in Bethesda. Now sitting on my right is Dr Burg, known to his friends as ďMoeĒ. Letís start at the beginning.
Did you have any early interest in Science?

MB:

Sadly, no. Those were before the days of science fairs and that kind of effort to attract people to science. I did enjoy my science courses in high school but no more so, for example, than English courses and in college my major was psychology which in retrospect was hardly scientific, at least in those days.

MK:

So why did you decide to go to medical school?

MB:

The choices seemed to be a career in medicine and a career in law. My father wanted me to go into law, maybe thatís why I ended up in medicine. Thatís not clear right now. Anyhow I kept my options open in college and I found I was enjoying my premed courses. The die was cast finally when after three years in college I applied to Harvard Medical School and they accepted me and so I ended up in medical school at that point.

MK:

So how did you become interested in kidney physiology then?

MB:

Well, more or less by accident. Being a psychology major it seemed natural to go into psychiatry after medical school but I had classes in medical school in psychiatry and clinics. It all seemed pretty futile at that point so I turned my interest to more traditional kinds of medicine. After medical school, I was an intern at the Beth Israel Hospital in Boston. In those days we had a draft for doctors. The draft was called the Barry Plan and you had an interesting choice at that time. The choices were you could either volunteer to go into the army medical core, in which case you went in as an officer, or you could not volunteer, in which case you would be drafted as a private. Most physicians volunteered as I did, and so while an intern at the Beth Israel Hospital I had my orders to report to Fort Sam Houston for basic training in the army, in the army medical core. However, about two-thirds of the way through the internship I got a telegram from the army, and the telegram said that they had over-enlisted in the Barry Plan that year and if I could find a medical residency they would permit me to take that medical residency before serving in the army. That seemed like a good deal, so I went scurrying around Boston looking for a medical residency at that late time. Where I ended up was at the Boston VA Hospital which you can see some of the personnel here in this first slide. When I first went there for an interview, the interviewer was Dan Holtzman who was the head of cardiology. The Beth Israel had a strong program in cardiology and that was really what interested me at that point, so I was pleased to go into a residency in cardiology. However when I arrived at the VA hospital to begin my cardiac residency, I discovered I was a resident in kidney and endocrine, so I immediately began seeing consults in kidney disease and endocrine disease. Now the Chief of Medicine in the hospital was Maurice Strauss. He was a charismatic person, great teacher. His interest was kidney physiology; he had a kidney physiology program going in the hospital at that time. The two people who were mainly responsible for that were two physicians, Sol Papper and Jack Rosenbaum. The kidney physiology consisted of clearance experiments in veterans and so I was soon involved in infusing veterans with various solutions, of indulin, and other various conditions. Sol went on to become Chief of Medicine in Albuquerque, New Mexico. Jack sadly developed colon cancer, while I was at the VA hospital, and died. He was a very promising kidney physiology at that time and it was very sad for all of us. As a result of my kidney physiology at the VA, there were two papers. This is the second one, which is ďThe Factors Influencing the Diurectic Response to Ingested WaterĒ and by that time there were already published works by ?Bert which indicated that fluid in the early distil tubule should be diluted. So the idea that the free water clearance, which we were studying, might be regulated by sodium delivery to those regions of the kidney were already understood and that was more or less the conclusions we came to in this paper. It wasnít until many, many years later that I had the opportunity to actually look at the mechanisms involved and describe the transport mechanisms in the kidney that were responsible.

MK:

So that was in your hometown in Boston, how did you happen to come to the NIH?

MB:

Halfway through my residency at the VA hospital, again there was a serendipitous occurrence. Maurice Strauss came to me and said he had contacts at the National Institutes of Health and how would like I to become a public health officer and do research at the National Institutes of Health instead of being a medical officer in the army and treating the soldiers. That seemed like a very good deal to me, so I volunteered to go to NIH, which fulfilled my requirement under the Barry plan.

MK:

Why have you stayed at NIH so long after your post-doctoral fellowship there?

MB:

Well I first came to NIH in 1957 and so I was there for two years initially as a fellow then I went back for a third year of residency to the VA. I think by that point I had already decided that I liked research very much. So that during my residency at the VA, I was trying to decide whether I wanted to continue there or whether I wanted to return to NIH, since Jack Orloff who was my mentor at NIH had already mentioned to me that if I wanted to come back I could. Well there was no comparison between the research opportunities at the VA and the research opportunities at the NIH, so I very quickly elected to go back to the NIH. The research was marvelous, the NIH was marvelous, and I never found a reason to leave since then.
 

MK:

So this was the Laboratory of Kidney Electrolyte and Metabolism that you first went to, what was it like when you arrived in the laboratory in 1957.

MB:

I think itís best described in terms of the people who were there, which is on the next slide. In those days there werenít very many pictures being taken in the laboratory. Nowadays we take a picture every year with all of the lab members, so this is the closest I can come to picturing the people who were there. The occasion was a dinner given for Bob Berliner many years later in Atlantic City, but many of the principle people I want to talk about are represented in this picture. Central to it all is Berliner and Jack Orloff. Those were the principle people in the laboratory and they set the tone in the laboratory. They were both brilliant critics, very bright people. It was an education just to listen to them and to discuss various aspects of kidney physiology. Even more impressive, they were very widely respected in the kidney community and even feared a little bit, because they were such powerful critics. A memory goes back to Atlantic City. In those days the principle meetings for research scientists in renal physiology were in Atlantic City; the first meeting being the clinical societies at the Hadden Hotel and the second meetings being the Physiology Society at the convention centre. Of course there were sessions at the meetings but what was most important went on in the boardwalk, outside of the meetings. I can recall Berliner holding court on the boardwalk and having very eminent investigators come up to him and to tell him about their ideas and work and to get his view on it, and his view was very important to them than what they should make of it themselves. In the meetings, you would have a ten-minute talk and then five minutes of discussion in which people would ask questions or make criticisms. I think there was a gripping of fear on the young fellowsí faces when Berliner would stand up to ask his question or make his criticism of their work. The laboratory of Kidney Electrolyte Metabolism had begun in 1948. I didnít arrive there until 1957. The first director of the Heart Institute was James Shannon, who was a famous kidney physiologist. When he staffed the Heart Institute, the first laboratory that he staffed, one of the first laboratories, was the laboratory of Kidney and Electrolyte Metabolism, which remains the longest established laboratory in the Heart Institute. Why a kidney laboratory in the Heart Institute? Shannon realized that the most important cardiac diseases involve the kidney: congestive failure, hypertension, salt retention, and so on. He recruited Berliner who was a brilliant young kidney physiologist at that point to head the laboratory. So Berliner was the first lab chief, but he was only lab chief for a couple of years. He went on to become the director of the Heart Institute intramural and Jack Orloff took over as the lab chief at that point. They had very different personalities. Berliner was the quiet sort, you had to sort of press him to get his opinions. Jack was very outspoken, very flamboyant, if you wanted to have a party you would have Jack there as the life of the party. Jack also had a very abrasive wit. He was prone to make lacerating comments to people in public. This was done in a way, that was in a joking way, so that you couldnít really respond to it without being a poor sport and it was generally quite funny to everyone except possibly person who was the brunt of the joke. At that time the principle event which is memorable in the laboratory was our lunches. So we had lunch everyday as a laboratory group and at the lunches there was a paper presented by one of the fellows and Orloff and Berliner, who were the professors, would comment on the papers. This is quite educational. They were very knowledgeable and very good critics and you could learn a lot from it. In retrospect one of the aspects which I wasnít really aware of at the time but Iím aware of now with my new sensibilities were that the lunches were an entirely male affair at the time I was there. All of the fellows were male so that made sense. The women in the laboratory were technicians, Nordica Green and Agnes Preston. Iím not sure they werenít included because they were technicians or because they were women. At that time you may recall that when you published a paper you would have a list of the real authors on the paper and then it would say with the technical assistance of, and it would list the technicians who often were the ones who did the actual work. We no longer do that and I never did it. I think the first woman to ever sit in on one of these conferences was a summer student of mine, Evelyn Groman. There was a bit of resistance to her sitting in and Iím not whether it was because she was a student or whether she was a woman. There was a bit of the old guysí attitude at these conferences and I think that Jack feared he would have to be more discreet with his language if there were women present than if there were not. At that time in the laboratory, transport physiology was at its infancy. The only transporter that was really known and was of interest to us was a sodium potassium ATPAs that had been recently discovered by ?Sko, who you remember recently got the Nobel Prize for that discovery. I think the best transport work in the laboratory was work on red blood cell transport, which was being carried out by Dan Tosdeson when I first arrived there and Joe Hoffman. When Dan left to go onto his very eminent career in medicine and medical administration, Joe Hoffman remained on for many years and learned a lot from Joe. The kidney studies that were going on in the laboratory were largely clearance experiments and those clearance experiments were mostly carried out in dogs. I did one dog clearance experiment in my career and then for whatever reason decided not to do anymore and ended up with other preparations. I was Jack Orloffís fellow, the other fellow at that time was Floyd Rector, whoís also in this picture. Floyd left after my first year in the laboratory to go to Dallas where he had had a very eminent career. The desk I sat at when I first arrived had been occupied by Mack Walser, who went on to become the Professor of Pharmacology at Johns Hopkins University. I became quite close to Floyd during that first year. We shared a lot of interests, we spent a lot of time talking. I can remember the two of us trying to sit down and figure how a kidney tubule could have regulated absorption of sodium based only on the sodium potassium ATPAs, and we didnít come up with any answer to that because, of course, much more is required which is the later work that both of us did. We spent a lot of time there and a lot of memories. One which is particularly striking has to do with the urinary concentrating mechanism. This was an interest of Berlinerís, it was at a time when the countercurrent mechanism had been described by ?Haggedy and Koon and Vertz. So the puzzle at that point was exactly how the transport processes in the tubule segments in the medulla worked in the counter current system. Berlinerís idea was that it operated strictly by transport of salt out of the tubule, leaving water behind, which nowadays we would recognize as the Avian model of Modulary Countercurrent Multiplication. He and Norm Levinsky had some clearance experiments that seemed to support that idea which they submitted as an abstract to the meetings in Atlantic City. However, after they had submitted the abstract, Karl ?Gadschiok arrived at the laboratory and he had already at that point made his seminal discoveries regarding the concentration mechanism and knew that the fluid within the loop of ?Henly, rather than being dilute according to the Berliner theory, was concentrated as we now know. This was not very good news for Norm Levinsky since then he was then faced with presenting an abstract at Atlantic City in which the conclusion was incorrect.

MK:

So as a bright young scientist coming to the lab in 1957 what was your initial project?

MB:

My first project was one that was assigned to me by Jack Orloff. The question was is the sodium potassium ATPAs important for transport in the kidney, and the way of answering that was using a specific inhibitor, which in that case was ?strithanfidum and the preparation that we chose to study was the renal portal circulation of the chicken. This is a sketch I made at the time in order to try to understand it, which I donít go over it in detail but the essence of it is that if you infuse a solution into a vein in one leg of a bird or a chicken in this case, it will go through a renal portal circulation and result in much higher concentrations of what you infuse around the kidney tubules in the kidney on that side than on the other. So you can have a experimental on one side and control on the other side by collecting the fluid separately from the two ureters. But I didnít know at the time but this project had been first offered to Floyd Rector and Rector refused to do it. I was neither knowledgeable enough nor assertive enough to refuse to do it. There were two results form the experiment. The first result was what we published that the sodium potassium ATPAs is very important for absorption in the kidney, and this was a novel discover at that point. The second discovery was that the chicken cloaca where the ureters come out was a very filthy place and you donít want to be working there if you can avoid it.

MK:

What happened next?

MB:

Jack wanted me to study the sodium potassium ATPAs. I was not a biochemist; I had no biochemical training, and frankly I didnít understand at all what it was biochemically. It was the confused in my mind with the ATPAs and the mitochondria, I tried reading about it, Jack was not much help to me, and I finally decided that was not the thing I was going to study. I decided instead to study renal transport in vitro that is outside the animal having already tried a clearance experiment on dogs and not having enjoying the experience very much. There were good reasons at that time to look to in vitro preparation. One was that the most impressive epithelial transport studies at that time were the studies in frogís skin that had come out of the Using laboratory. I looked as if that could be carried over in some respects to the kidney because there were already descriptions of experiments on the iron contents of kidney slices, which was carried out by Gilbert Muge at Johns Hopkins. There were also experiments showing that kidneyís tubules could survive in vitro from Chamberís laboratory. So I adopted Mugeís kidney slice technique and did some studies to the effect of ?Walbain on the electrolyte content and other aspects of the kidney slices. In retrospect, none of them were terribly important results.

MK:

And so why did you abandon your kidney slice preparation?

MB:

I realized that when you were studying particularly the kinetics of electrolytes in kidney slices that there were almost impossible problems because the kidney slices were relatively thick preparations, many tubules thick, so there was a diffusion delay for anything that you put into the solutions to get to the surfaces of the tubules. The slices, particularly from the cortex, are thick enough so that the oxygenation of the tubules from the center is not very good and also you have all sorts of different tubules there, which means you have different kinds of cells. So I first set out to try to solve the problem of the thickness of the kidney slices, which led to the next slide which is the preparation of kidney tubules. This had never been done up to that point and I researched a way of doing it by looking at papers in cell culture in which they used enzymes including collaginates to desegregate tissues to started the cells and culture. So I tested collaginates, trips, and various other enzymes to see if there are any that would dissociate the kidney into tubules that would be surviving and ultimately devised a method using collaginates to make tubule preparations such as this shown here from the cortex of rabbit kidneys. The rabbit was chosen mainly because that is the species I had been working with on the kidney slices and itís a good species to work with if you want to study the sodium potassium ATPAs because the ATPAs in the rabbits is sensitive to cardiac-like asides, whereas the ATPAs in ratsí kidneys, for example, is not. I did a lot of studies with the suspension over a couple of years. The tubule suspensions have been widely used since then. It seems that each person who used it after that, starting with my method, invented it fresh and forgotten where theyíd gotten the method from. But I think thatís the way science goes

MK:

Back in those days the cutting edge technique was considered to be micropuncture. Did you consider doing micropuncture?

MB:

Oh yeah. Floyd Rector and I discussed that a lot and I wanted to do micropuncture but I couldnít. I couldnít do it because Berliner wanted to do micropuncture and of course he was the professor and decided what went on in the laboratory. He had assigned Tom Kennedy, who was then in the laboratory to set up micropuncture for him, and when I left the laboratory after my first two years of fellowship there, Tom Kennedy was supposed to be setting up to do the micropuncture. By the time I returned a year later, Kennedy was no longer there; he had gone into administration and there was no micropuncture. Rector, who had gone to Dallas, had started the micropuncture there, and the person who was doing there was James Clapp. After Jim finished his time in Dallas, he came to NIH and heís the one that set up the micropuncture at NIH in Berlinerís laboratory. Berliner chose to do micropuncture in dogs. I guess, in part, because thatís where the clearance experience was and where the knowledge was. I donít think that is particularly a good choice. I donít think itís a very practical preparation for doing micropuncture. So I couldnít do micropuncture. Instead, by the time I left NIH after my first two years of fellowship, I decided that the way to study the kidney tubules was to take them out of the kidney and profuse them in vitro, which of course was a rather audacious idea at that time. It seemed to me that it was possible. The examples I had, which, brought into mind, were first the giant squid axon that Hodgekin and Huxly had studied and had famously published at that time. Now a squid axon is a millimeter in diameter whereas a kidney tubule is only one twentieth of that diameter, so thereís a big size difference. However if youíve looked at the pictures of the ?axial electrodes in the squid axons and all of the marvelous transport studies, that had to be very attractive. Also, I knew that Chamberís had made preparations of tubules from fish and amphibia and that those tubules survived in vitro to at least transport dyes, so they were still alive. So why not try to makeÖdissociate single tubules and profuse them?

MK:

So how did you learn to dissect these tubules?

MB:

Well I tried to figure out how to do that myself. We already had the example of the collagines and so it was natural to start with a kidney preparation in which the tissue had been treated with collagines. Itís relatively easy to dissect tubules out, but I didnít know that at the time and I started with a very cumbersome system, which I was using micromanipulaters to try to do the dissection. It wasnít until we had a visitor to the laboratory, who was Iver Sperber, that I really found out how to dissect. Now Iver Sperber was a famous renal anatomist. He was one who had discovered the portal circulation in the bird and which was the first preparation I used, he had also dissected kidneys and shown all of the tubule segments, which you can see here. Of course these dissections were on dead tissue that were macerated with strong hydrochloric acid, and I was trying to dissect more delicate tubules from living specimens. Sperber came to visit the laboratory to give a lecture, while he was there, he showed me how to dissect tubules, which was with forceps and needles rather than with micromanipulators and it turned out much easier to do it that way. While he was there, Sperber gave a lecture, which was very memorable. While he was lecturing, a messenger kept coming into the room and speaking to Berliner, who would shake his head. At the end of the lecture Berliner announced that President Kennedy had been shot and we all rushed out after that too look at the television and see what had happened to the president. I think those of us who were old enough at the time all remember where we were when we got that news.

MK:

November 22nd 1963.

MB:

You remember it. So I at that point had learned to dissect the tubules. What happened after that was that I had my first fellow arrive, who was Maurice Abramov. Maurice is Belgian. He arrived at that time I was working with the suspensions of kidney tubules from the cortex. I was doing experiments on the kinetics of sodium and of potassium transports in the tubule preparation using radioisotopes of sodium and of potassium. One aspect of that was my first real introduction to kinetics and compartmental analysis was evidently required. At the time, the first mainframe computers were available and we had at NIH a mathematician who knew how to program that for kinetic analysis, Monas Berman. So I went to Monas and he set up a kinetic analysis system with the computers, punched the cards, I think youíre old enough to remember the punchcards and the mainframe computers, and it solved our kinetic problems. Maurice and I used the suspension to unravel some of the aspects that were mysterious in the kinetics and the tissue slice, but still we werenít where we wanted to be because the tubule suspension, the main surface thatís available to the fluid, is on the outside of the tubule, and what weíre really interested in was transepithelial transport that requires access to the lumin of the tubule and requires perfusion of the tubules


MK:

So how did you figure out how to profuse the tubule? Sounds impossible.

MB:

Joe Hoffman was a big influence in that, heís shown here. This is a much later picture taken at the time of our laboratory reunion. Joe was in the lab for a long time and eventually left to become a professor of physiology at Yale. Well, I first needed pipettes to get into the tubule lumen. Joe had a ?microford; he let me use his microford to make tubule pipettes. Itís not easy to poke a perfusion pipette into the end of a piece of tubule that is floating around. I tried holding the tubule in various ways with little suction pipettes around the outside to open it. None of that worked. I finally realized that I needed suction all around the tubule which meant I needed concentric pipettes; the outer pipette to provide the suction, the inner to have the suction pull the tubule over that and to profuse the tubule. At that time the electrical potential in the red blood cell had not yet been measured and Joe wanted to measure that along with Walter Friegang, who was a neurophysiologist at NIH. So they made a special apparatus which held pipettes concentrically in which the inner pipette was intended to hold the red blood cell and the outer pipette was intended to flow sucrose over the cells so that they could use the sucrose gap method to measure potential. It didnít work, and when it didnít work, Joe willed that apparatus to me, which was the holder for concentric pipettes that I first used to profuse tubules and so using that I successfully perfused my first proximal straight tubule from a collagine suspension, as shown next here, and the result was rather dramatic; the tubule exploded. The problem, of course, was that the collagines had removed the basement membrane, which is the main support for the tubule and so it could not withstand the pressure inside and it was obviously necessary to dissect the tubules with the collagines inside. That turned out to be not that difficult from the rabbit kidneys. I used rabbit kidneys for the initial perfusion studies and for a long time thereafter. In retrospect, itís clear that the reason that the rabbit kidneys were so easy to profuse was that, unknown to me at the time, NIH had a special colony of rabbits from which I was getting the rabbits that I used, and that colony of rabbits was pathogen free. Ordinary rabbits from a farm are not pathogen free; they have kidney infection from various parasites and that increases the fibrous content and the tubules are much harder to profuse. So at this point I had tubules that I could dissect and a way of perfusing them.

MK:

You needed to collect the fluid, how did you achieve that?

MB:

There I was lucky. My second fellow was Jared Grantham, who is shown here many years later. Jared is a very capable scientist and investigator. He went on after his fellowship with me to return to Kansas City where he originated, becoming eventually the Chief of Nephrology and has remarkable accomplishments with respect to polycystic kidney disease, in which he started a foundation, he has a lot of studies himself. If anyone is responsible for progress in that field, itís Jared. Underneath this portrait here you can see that there is a pipette at the other end of the tubule. Perfusion pipettes are at the right hand side showing the inner perfusion pipette, or the suction pipette, and at the other hand he simple had one pipette that he sucked the tubule into, had oil in the pipette, and introduced a collection pipette to collect the fluid that has accumulated

MK:

Does Hoffman-Friegang concentric pipette apparatus, did you continue to use that

MB:

No, that wasnít very practical. The thing that it lacked was, although it hold pipettes concentrically, adjusting them one within the other was very tedious and what was obviously needed was a micrometer arrangement so that the inner pipette could be advanced relative to the outer pipette. Gerhard Gebish knew what I was doing at the time and he recommended that I go to Johns Hopkins and visit a laboratory there which was run by Phillip Davies, because Phillip Davies had such an apparatus that he was using for other purposes. So Maurice Sabermont, Jared Grantham, and I went to visit Phillip Davies and sure enough he had an apparatus that could do exactly what we wanted. However, it was a very delicate apparatus that he essentially assembled each time he used it and it didnít seem to me to be very practical for continued use in perfusing tubules. So I wanted to have a more practical arrangement and in order to do that I had a choice, I could have gone to the engineers at NIH and have them design it, but I had no faith that the engineers would design something that was both practical and durable, so I went to the mechanics at the NIH shops and visited with Ken Bowlan. Now Ken was the head of the optical section, so called, of the shop, which is where they had a few of the mechanics, machinists, who were most highly skilled. Ken himself was a watchmaker so this is the sort of thing that would be very good. So I sat down with Ken and with one of the very good machinists in his shop who was Jim White and they designed the apparatus that you can see here, which was the first practical apparatus for perfusing tubules. Jim retired from the shop a while after that and devoted himself entirely to constructing perfusion apparati for all the laboratories around the world, including our own, who wanted to have tubule perfusion studies. Jared used this apparatus, which you see to the right here, in one of his early experiments, to hold the perfusion pipettes and at the other end you see the only existing picture of the of the original Hoffman and Friegang concentric pipette holder, which in this case Jared was using to simply to hold the perfusion pipette to collect the fluid.

MK:

What improvements were made subsequently to the apparatus?

MB:

Well once weíre able to dissect and profuse kidney tubules there were lots of studies we wanted to do. One thing was to measure fluid absorption. Initially we used I-131 ?alpeumanism marker, later we used inulin, radioactive, and such things. You were the one who developed the best method which is to use fluorescently labeled markers and get away from the radioactivity altogether. Once we had those measurements we needed to do the mathematics to calculate the fluid absorption rate. Iím not much of a mathematician, so I went to our very good mathematician at NIH who was Clifford Patlack. A visit to Patlackís office was an interesting experience. In his office every surface was covered with piles of manuscripts and papers, no free surfaces at all. The entire office was surrounded by blackboards and the blackboards were entirely filled with equations. You were allowed to erase just enough of the blackboard to put down your problem then Clifford would stand up, go to the blackboard and would derive the equations that were needed for your project. He would write them down; you would take them back and proceed with them. Clifford was very remarkable. He was a mathematician who was also a physiologist. He understood the mathematical requirements of physiologists and you could talk to him directly since he was as interested in the physiology and the applications as in the mathematics itself, which is not true of many mathematicians. Clifford went on to become a professor at Stoneybrook in New York. We needed methods to measure the ions, organic compounds, and others in the collected tubule fluid. There we were very fortunate to have in the institute the laboratory of technical development which was headed by Robert Bullman, remarkable person, very colorful character. He was very creative and inventive. He had in his laboratory Jerry Vierrick, who was the principle person who helped us, and together they developed the radio frequency photometer, that we used for measuring sodium and potassium. Vierrick developed the method for measuring total CO2 by calorimetry. He developed very microflourimeters and colorimeters and you became the guru of the microflourimeter and the microcolorimeter, adapting them to all sorts of assays, which you eventually used. Also being able to take the kidney apart and dissect the different tubule segments opened up all the tubule segments to study. Micropuncture was marvelous for studying the tubules on the surface of the kidney, but there were very important things happening in tubules within the kidney. So we developed methods for studying all of the segments eventually in the kidney. Also we started with rabbits, which were easy to dissect. Not perhaps the most practical animal to study particularly since so much of the physiology study had been done on rats previously. I tried dissecting rats and decided it was too hard and gave it up. You said you wanted to dissect rats, I told you it was impossible, but being a good investigator you didnít listen to me and you realized that getting pathogen free rats would be very important, just as it was with the rabbits, and you succeeded brilliantly with that.

MK:

So a lot of your early studies were on ion transport, which required electrical measurements. How did you achieve those types of measurements?

MB:

Well one of the electrophysiologists and transport physiologists was Jack Dainty. When I presented my work in a meeting that he was at, he criticized it saying that you can not interpret these results without knowing the electric potential. I realized at that point that could be fairly easy to measure since I can use the perfusion pipette as a bridge into the tubule lumen and so I did it that way. Then I ran into another problem, and that is liquid junction potentials, which of I of course had never heard of. At another one of my presentations and meetings, I ran into Jared Diamond, who was a brilliant, young transport physiologist. Jared of course is now the most famous of us all having recently won the Pulitzer Prize for his work, Guns, Germs, and Steel. While he was a very good physiologist, he was also a better ornithologist and evolutionary biologist. His interest was history so heís now in the geography department at UCLA. Anyhow, Jared was a very good electrophysiologist, he explained liquid junction potentials to me and he explained how you measure them and how you adjust for them and so we made those adjustments and did our first measurements of tubule potential. The measurements are not shown here, but the fellow is shown here. This is Leon Isaacson who came to me from South Africa as one of the yearly fellows. This is many, many years later after retirement from being a professor in South Africa. I didnít realize it at the time, but Leon, the time he came to be my fellow, was already much older than I was, so I was instructing older people. Leon used the electrical system to measure the voltage across cortical collecting ducts, which we were studying then. Further advance came when Sandy Helman arrived. Sandy was an electrical engineer, which really was just what we needed. I know youíre overly fond of engineers but this was one that paid off for me. When he left the laboratory, he went on to become a professor of physiology at Indiana, and very good, continued his electrophysiological studies there. His job, as my fellow, was to measure the resistance of the kidney tubules. In order to measure the resistance of the tubules we used cable analysis of which I was totally ignorant, but Sandy being an electrical engineer was very familiar with it. The original concepts having come, I think, from Lord Kelvin with relation to laying the Atlantic telephone cables and applied extensively in neurological research with the squid giant axons and such. Anyhow, Sandy knew the cable equations, he knew how to do the measures and so he was able to measure the electrical potential, mainly in the collecting duct and to look for changes in that. In the course of measuring the electrical resistance, Sandy discovered that there was a big electrical leak between the tubules and the pipettes that were holding them. That problem was solved by Jerry Verrick, whom Iíve mentioned in Bob Olmanís lab, and his solution is shown on the next slide. The solution was to use a resin, a viscous liquid resin called sylgard, and the sylgard coated both the glass and tubule surface making an electrically tight seal. Eventually what had started as a very simple arrangement shown in A here, perfusing the tubules, became more complicated with the sylgard introduced around the tubule in B and finally with the final arrangement is one in which, rather than two pipettes concentrically there are four pipettes concentrically: an outer pipette which has the sylgard, then one to hold the tubule, then one which is the perfusion pipette, and then inside of that a pipette for changing the fluid so that you can change the perfusion fluid going through the lumen in the middle of an experiment. At the other end of the arrangement, the collecting also became more complicated as shown in the next slide. Again, starting with Jared Granthamís simple arrangement in A, then having sylgard laboriously placed around the tubule, which was a little bit tricky in B, then finally another concentric pipette to hold the sylgard around the whole thing. These were more practical arrangements than they appear because you could set them all up and then if you cleaned things out carefully, leave them set up from day to day and week to week, which made perfusion quite practical.

MK:

So there were hundreds or even thousands of papers published with this isolated perfused tubule technique that you invented. What do you regard as your most important discoveries using the perfused tubule technique?

MB:

A few come to mind, there are a lot, but a few come to mind. One was looking at the part of the tubule called part of the proximal tubule and discovering that the transport characteristics of that part of the tubule differed depending on which portion of it you looked at. For example the early portions transported glucose very well and very rapidly, the late portions transported perimenal ?hepiric acid very rapidly and very easily. From that a concept arose, which I never held to and that was the concept of heterogeneity that you could have heterogeneous parts in the same part of the tubule. That was quite striking to me once when I spoke in Japan and there was a banner over our meeting, which was mostly in Japanese and I couldnít read, and in the middle of the Japanese there was the word heterogeneity. It turns out there was no word in Japanese for heterogeneity since they are very homogeneous people. I found the term objectionable as did you because what it meant was that the refinements and transports along the tubule were greater than what the anatomists had recognized and it gave addition control and in fact I think we wrote a review together in which we tried to trash heterogeneity as a term. I think we may have succeeded; I havenít heard it again recently, have you?

MK:

No, not recently.

MB:

Bruce Tune, when he came, started on an ambitious program. Bruce, after his fellowship, went to Stanford where he became the chief of pediatric nephrology. His job was to remove the tubules after they were perfused and to measure the concentration of the transported solute within the tubule cells, and he was able to discover the transport steps that way, through the cell for glucose in the one case, and ?paramuno-hepuric acid in the other case. Recovering the tubule became very fashionable, you had some very nice studies with Soren Neilson with recovered tubules, if I recalled. Then in proximal tubules, the reabsorption of salt turns out to be very dependent on the reabsorption of organic solutes, like the glucose in amino acids that are present. That is something I found when trying to get different perfusion solutions and bath solutions for proximal tubules, the problem being that we knew the rate of fluid absorption in rats from the micropuncture studies, and it was much less in our isolated tubules, which raised the question that maybe theyíre just not very healthy. I think there were two answers to that. One answer was that rabbit tubules donít absorb fluid as rapidly. The second was that our initial perfusion solutions, we didnít have all those nice organic solutes in the lumen that are necessary to get the maximum rate of absorption. Itís very striking if you do the experiment if you start perfusing the tubule with a solution that had no glucose or amino acids in the perfusion solution. The tubule cells are very flat; the minute you put in the glucose and the amino acids, the tubule cells balloon out, become very broad, because of the transport step and what theyíve accumulated within the cells by the transporters in the lumen. Another controversial issue which we were able to address was the electropotential difference across proximal tubules. That had been measured quite well in amphibian proximal tubules and found to be about twenty millivolts, lumen negative, and the same thing was initially found in the rat proximal tubules. However, that was the wrong answer and the problem with an artifact in which the pieces of tissue got stuck in the electrodes; they were used to pierce the tubules. Of course we didnít have that problem since we had an open canella going into the end of tubules, so there was some interest in our results for that measurement, and what we found was that the early parts of the tubule, dependant on the organic solutes, it was just a couple of millivolts negative and the late parts of the proximal tubule was either negative or positive depending contents of the tubule fluid. Shortly after that Abrahad Frumpter in Frankfurt did these studies correctly in the rat proximal tubule and confirmed our results in the isolated perfused tubule. Another issue, which is historical only, is at that time there was a fashion for measuring fluid absorption from tubules by putting oil in them by micropuncture and splitting the oil drop with a fluid that was to be transported and seeing how fast the oil drops came back together, and from that came the concept that the rate of the absorption of fluid was strictly a geometric question and the rate was proportional to the square of the radius of the tubule. I tested that in the isolated perfused tubules, varying the tubule diameter by varying the outflow pressure from the tubule and found there was absolutely no relation to the diameter of the tubule. It was quite controversial at the time but I havenít heard what was then called ďR^2Ē referred to in the last thirty years. The issue was settled at that point. Perhaps the most striking observation that we had was experiments measuring the voltage across ?thickey-cending limbs of Hendleyís loop. I measured it in experiments with Nordica Green, who was my technician at the time, and I know ?Yuhad Coco, who had been a fellow, measured it in Dallas where he was perfusing tubules, and we both had the same reaction because we had all expected that the voltage would be lumen negative in all parts of the tubule and we found a positive voltage. We both went through the same thing, checking the leads, checking the polarity of everything. Finally it turned out that that actually was the voltage and that led to the discovery of the sodium potassium II chloride transports in the lumin of the ?thickee-cending limb, which Nordica and I were able to discover was the transporter that was inhibited by the important diuretics, like ?pherosimad. This is a picture of Nordica who worked with me for many years. When I first came to the laboratory she was working with Ernest Cutlov, who was studying chloride content of muscle cells but when he left the laboratory she became my technician and she was a marvelous person, a marvelous scientist, we accomplished a great deal together. It was a sad day for me when she decided to retire and I could no longer work along with her. Another discovery was make by Dwight MacKinley who was a fellow and later went on to become director of a big pharmaceutical company. Dwight was studying bicarbonate transport in collecting ducts and everybody knew that bicarbonate was simply absorbed by kidney tubules. What Dwight discovered was whether it was absorbed or secreted by the collecting ducts depended on whether the rabbits, from which the collecting ducts were taken, were given alkali solutions or acid solutions. This is now taken for granted and there are many studies of the particular cells which absorb or secrete bicarbonate but at the time it was quite controversial and I remember the experts in the field for many years greeted our discovery, with as far as I could tell, disdain because they felt that it simply wasnít possible that this was going on. Finally some early studies of Jared Grantham having to do with the root of water absorption through collecting ducts and he found when he applied ?baso-presen to the collecting ducts, with a dilute solution in the lumen, that the tubule cells swelled up which was the indication of the location of the channels, which eventually turned out to be water channels in the lumen membrane that are affected by the anti-diuretic hormone. So there are many other things but those are the ones that come to mind.

MK:

So back in the early eighties you decided to set aside the perfuse tubule technique and go on to other things. Why did you decide to stop?

MB:

There were two things. One thing was that you had agreed to stay on at the laboratory at that point and you were doing a much better job of it than I was so I might as well let you do it. The second was that science had moved on and though I was obviously very fond of perfusing tubules there were all sorts of new developments and it was enticing to me to get my feet wet, so to speak, in some of these new areas.

MK:

So what areas in particular did you decide to pursue?

MB:

Decided to study osmotic regulation, which eventually comes from the fact that in the kidney medulla of animals there are very high concentrations of salt and urea, that maybe poisonous so itís a question what harm does it do to the cells, and how do they adjust to it?

MK:

So what prompted you to go in that direction?

MB:

Youíre to blame. As you probably recall, Bob Balaban was also in the laboratory. Bob is now the director of Intramural Research at the Heart, Lung, and Blood Institute. At that time he was doing, and still is doing, nuclear magnetic resonance spectrometry to analyze the intracellular compounds and their status, particularly things like ATP. You and he had a project together in which you decided that you would use NMR imaging to image urea in the kidney and that you would do that using Nitrogen-14 imaging. The result was that for technical reasons that didnít turn out to be possible but while you were doing the studies you saw enormous concentrations of a nitrogenous substance other than urea in the kidney medulla and Bob had been previously puzzling over enormous concentrations of concentrations of phosphorus containing compound, in the ?una medulla and putting that together you realized that the compound was glyceral phosphal cholane, which is one of the principal osmolytes in the kidney medullas. I think we all realized a little bit later that you hadnít discovered that, that that had been in fact discovered by Karl Ulrich in the 1950ís. Karl being a really talented German renal physiologist, marvelous person, good friend, sorry to have him retired from research at this point and no longer being able to interact with him. Karl found this compound in the kidney medulla. In a second paper he figured out what it was; he knew what it was doing there. That was something none of us either knew to begin with or remembered, so it was a fresh discovery when you found it. So that showed that there was glyceral phosphal cholane in the kidney medulla. In 1982, Paul ?Yancy and George ?Sumero, who were marine biologists, published a landmark review on the organic osmolytes and how their accumulated incells in response to high salt and high urea, in their case, principally in marine species, but they also, in their review, covered plants. Itís really a universal response. In many of those examples thereís more than one organic osmolyte thatís accumulated, so I decided to see what the other organic osmolytes might be in kidney medullas. For that purpose I was lucky to have, as a fellow at the time, Serena Banyasko, who was Italian, who walked into my laboratory one day and asked if she could volunteer to work in the laboratory, her husband having a different job at the NIH. Sure, I took her as a free volunteer. It wasnít many months along the way until I realized that sheís a superb scientist and found a salary for her to stay on as a post-doctoral fellow at the lab. Anyhow she had done other projects but at that point it became her project to find out what the other organic osmolytes were. So she harvest kidneys from rabbits and rats that were either given a lot of water to make them diuretic and lower the osmolality or were deprived of water to raise the osmolality. Then came the question of how to detect the compounds. We were lucky there that Bob Balaban was willing to give us his NMR spectrometry expertise using proton NMR, and that we had in the institute one of the worldís leading analytical chemists who could do the same thing by mass spectrometry and between them able to define the other organic osmolytes, which were ?sorbital, ?mioenocital, and ?glycene bedene. So we published that all together

MK:

Many of your studies have depended having cell culture models of renal cells. How did you get started with that?

MB:

Iím not the one who started that. The one who started that was Joe Handler. Joe was at the laboratory from almost the beginning of the time I was and for many years he was Jack Orloffís other fellow and collaborating who worked with Jack. Joeís specialty was toad bladders, which Jack made seminal discoveries regarding the role of cyclic AMP in signalling ?invaso-presen in the collecting duct in the toad bladder. After studying the toad bladder, we read a very interesting paper by an investigator named Dayton Misfelt, and what Dayton had done was to take an existing line of tissue culture line from a kidney, which was called MDCK, and to put it onto a porous support and to show that there was a electric voltage that was generated across it, giving the possibility of studying transport in tissue culture. Joe latched onto that technique, he trained himself in tissue culture. He had some of the early tissue culture models of transport and he established tissue culture in our laboratory so that when I wanted to use tissue culture to study the organic osmolyte it was already there. I needed to just learn the technology from Joe. We didnít have at that time any cell lines, which was specifically from inumedulary segments. Nordica Green tried very hard with me to start some cell cultures from inumedulary and other parts of the kidney, starting with the single tubules, and we were not successful. The reason was that that was before it was realized that there are certain genes which have to be expressed to immortalize cells in tissue culture and we simply didnít have that information, but by luck and by hard work, Nordica had succeeded in started a culture from the cells lining the inumedula that are exposed to the same high salt and high urea and we were able to use those cells in our initial studies.

MK:

So getting back then to the organic osmolytes, how did you use the cell cultures to understand a regulation of organic osmolytes?

MB:

Well, we looked in the cell cultures to see if they would accumulate the same organic osmolytes as in the kidney, and it turned out that they did. This was marvelous for us because that mean that although it would be difficult to study the details of their accumulation in vivo, in the cell culture there was a direct route to doing it. The initial measurements of the osmolytes in the cell culture were done with Bob Balaban and his nuclear magnetic resonance spectrometry, but about the same time, Paul Yancy came to the laboratory for sabbatical. I mentioned Paul once already, Paul being the marine biologist who was the expert in this area and who had co-authored the landmark review in it. Paul worked with Bob Balaban to develop a HPLC method for measuring the organic osmolytes. That was quite practical and became the standard after that, so we were able to use that to study their accumulation in the kidney cells. And thatís still the principle method for measuring them. And so we used those cultures to figure out the mechanisms by which the organic osmolytes are accumulated and in those studies had some more very talent post-docs. Two of them were Japanese post-docs. By that time the Japanese scientists were coming to our laboratory to learn kidney physiology. They were Takeshi Nakanishi and Toshiki Noriyama and they were able discover the increased abundance of ?aldos inductes that was responsible for the accumulation of sorbital. Sorena Bonyasko and Shunyu Chida discovered the aldos reductes. Takeshi and Toshiki were responsible for discovering the ?Bdene and ?mioenacetol are accumulated in the cell by transport and there were more transporters when you raise the salt concentration. Then Arlyn Garcia-Perez came as a fellow to the laboratory. Arlyn, who is originally from Cuba and Puerto Rico, had studied in MIT in Michigan in the United States and had picked up some experience in molecular biology along the way and she applied that in studies with the cell cultures in a laboratory to clone the CDNA for aldos reductes, which was responsible for the accumulation of sorbitol, and to eventually show that was regulated on the basis of transcription, which turned out to be the case for all the other organic osmolytes. Then Moo Kwan came along to the laboratory. Moo was actually Joe Handlerís fellow, and Joe and Moo worked along with me and my fellows in looking into the organic osmolytes. With Joe and Moo, we were able to use expression systems in ?Toad-oa sites to clone the CDNAs for the mioenocitol transporter and BDene transporter. Moo when he left the laboratory went along with Joe Handler to Johns Hopkins where Joe had become the chief of nephrology. They continued to study the organic osmolyte there and together they cloned the transcription factor that is responsible for the accumulation, which they named Tony BP. Joe has now returned from Johns Hopkins and is back in our laboratory and is very, very welcome here. Moo moved out of Johns Hopkins and is now a professor at the University of Maryland and we continue to follow each others work very closely since weíve remained very much in parallel. Since then weíve continued to investigate the cycling pathways to Tony BP, and those studies have been mostly conducted by Joan Ferraris whoíd been a fellow in the laboratory for many years and is permanently with us and is our molecular biologist and is responsible for uncovering those signaling pathways, which Iíll not describe in detail, it would require too much time at this point, but itís very exciting to be following that through. We have the advantage of the example in the laboratory, which is you studying systems biology, because it turns out thatís what we are getting into now. Starting the systems biology of signaling, of osmotic stress to the stress response proteins.

MK:

Tell us a little more about the damage that urea or sodium chloride at high concentrations causes in cells.

MB:

Well, clearly the cells in the kidneys manage because they survive and they function, but we wanted to look at that in cell culture. We had striking results almost immediately. The background we had for that was that Yancy again and Somero had looked very extensively into the subject on marine organisms. What they had found was that elevating the salt, which is what occurs in cells initially when you elevate the salt concentration outside of them, or elevating the urea, which goes into the cells immediately, has very large effects on the enzomatic activity of proteins and on the structure and function of macromolecules including both proteins and DNA, and that the organic osmolytes helped to protect against those effect. So we started looking at it in cell culture and the one who carried out the initial studies, which were most revealing, was Dietmar Kultz. Dietmar started as a marine biologist as a first post-doctoral fellowship with George Somero in California and when he came to our lab he started studying the cell cultures for ?remalium kidneys. Early on he found that elevating the salt concentration caused an increase in a particular protein called GADD-45, Growth Arrest and DNA Damage Inducible protein-45, which is our first indication of the sort of stress that occurs in the cells with high salt. Those studies were continued by more great talented investigators. Luis Michea, who came as a post-doc from Chile, returned as a professor in a university in Santiago. Natasha Dmitrieva, who came as a post-doc from Russia, from St. Petersburg, very talented scientist whom Iím fortunately enough has agreed to stay on in the laboratory and is one of my most valued collaborators and associates. And in this same picture, Zheng Zhang, who is a post-doc from China. Luis started studies looking at the fate of cells in culture when the salt and urea are elevated, was able to demonstrate apotheosis, which is of cell death Ė a particular form of cell death, and was able delay in the cell cycle, the rate at which the cells were growing. Natasha and Dietmar went on to show that one of the DNA damage responses, which is the activation of a protein called P-53, which is a tumor suppressor protein. They showed that elevating the salt greatly increased the abundance and activity of that, another indication of the kind of damage that was going on. Zheng, together with Natasha, showed that the cells suffered oxidative damage to the proteins when the salt or urea concentrations were elevated. Putting that all together, Dietmar after he left the laboratory and went to the University of Florida, where he was initially, heís now at the University of California in Davis, where heís an associate professor. Dietmar was able to show directly that raising the salt concentration caused the DNA in the cells to be broken up, something which is incredible, and even more incredibly, Natasha later showed that the DNA in the cells in the kidney medulla, while the salt concentration is high, is also broken up. Those discoveries are the basis for studies that we have going on now, which are aimed at knowing exactly why these things occur and knowing exactly how the cells are able to protect themselves from that.

MK:

So in general, looking back, all the people that youíve trained, theyíve all done extremely well in their careers after theyíve left. What factors do you feel have allowed to be so successful as a mentor?

MB:

Youíve exaggerated the case. The people who have done well were very good to begin with; they went out and did well. Iíve had average fellows whoíve done ok and Iíve had other fellows who were not terribly capable and a lot of them did not do very well. So, I can only take a very minor part of the credit. I donít think thereís any particular system of mentoring, which is more successful than any other. What I do think is important is the examples that are set for the fellows by the way things are run in the laboratory. I think the fellows learn by example rather than by being told specifically what they should to do. In managing the laboratory after I became laboratory chief, I wanted very much to have a laboratory that was comfortable as opposed, for example, to a laboratory where things are highly competitive and highly personal. I think weíve succeeded in having a happy and comfortable laboratory where the stresses are scientific rather than personal. I think itís very important that the fellows are appreciated and get credit for all of their good and for their hard work, try very hard to do that. I think they have to be exposed to a laboratory atmosphere where the ethics of science are very much emphasized. They have to learn by example the importance of assigning the right authorship, the importance of being responsible when you review papers, and when you review grants. These are all things which might seem to be very obvious but Iím not sure thatís the case in all laboratory. Iím very pleased that youíve chosen to continue the same way now that youíre Chief in the laboratory. When I say a happy laboratory I donít think we lack scientific criticism; I think that weíre very keen to the science and weíre very self critical and critical of other people, but the criticism is always on the basis of the science and not on the personal basis of the people involved. And finally, I think by example you have to show them that your standards of science are very high that you want to have experiments which are done correctly, which are done reproducibly, and conclusions that will bear up in the present and in the future, and I hope to convey that to the fellows and I hope that some of them have learned that.

MK:

I think they have all learned that. So in retrospect then looking back at these forty-some years in science, how do you feel about your career in science?

MB:

Forty-seven years, and counting. You and many of the people Iíve talked to through the years have heard me gripe about working for the government and gripe about NIH. Forget that. NIH intramural program is a marvelous place to do science, and if the science is what really interests you, this is the place to be. You have a budget, you donít have to worry about grants; you donít have to spend all of your time doing that. You spend your time on the science. There is a certain responsibility there; the government has a big investment in you. I guess the government has invested somewhere between 50 and a 100 million dollars in my science in these forty-seven years. I think that our responsibility at NIH being given this money and not being required to apply for grants is to try to do things that are truly innovative and that might be more difficult to do elsewhere. I think that tubule perfusion projects, I think the organic osmolyte projects fit that picture just as many of the things that youíre doing now. So what I feel is a gratitude to NIH and a satisfaction that Iíve chosen to stay on here. The science has always been very exciting to me, I like the science itself, I like the people. Iíve been involved with the many brilliant people that you interact with at meetings and scientific organizations. Itís marvelous to have enthusiastic bright young fellows to have innovate in your late and to be able to interact with them all of the time, and so my enthusiasm for the science and NIH remains undiminished. Even though Iím well past the age where I could retire comfortably, I have no intention of doing it as long as my health holds up, as long as the science remains as interesting as it has been.

MK:

Well Moe, thank you very much for this marvelous story and continuing story, as you point out, this is extraordinarily important that we record history as it occurs and I think that itís my great pleasure to be involved in this recording archival history project. Thank you very much.

MB:

And thank you for helping me to do it.