1

A LONG AFFAIR WITH RENAL TUBULES

Early Days in Vienna

I was born in Vienna in 1927, the only son of a middle class Austrian family. My

father taught German and French literature in high school. My mother, who spent her

early childhood in Rumania, spoke French fluently, and loved literature and music. I

recall my father as a kind and romantic soul. His grandparents labored as farmers in

lower Austria, near the Czech border. I enjoy fond memories of spending childhood

summers in his family’s small village. My father wrote and published poetry and

inspired a lasting interest in history. My mother grew up in a well-to do business family

that became impoverished after the First World War. She promoted my interest in music

and encouraged me to study the piano, which I practiced until starting medical school.

Although I grew up in modest financial circumstances during the post-World War

I economic depression in Austria, I remember a happy childhood. The unconditional love

and support of my parents, to whom I remain indebted, allowed me to flourish

academically amidst economic hardships.

I attended an excellent parochial preparatory school and entered public High

School in 1938. I remember a superb English professor in my third year of high school

grade who inspired my interest in learning and “adopting” English as my second

language. I continued studying and reading English, but my linguistic confidence was

badly shaken by my almost total failure to understand a movie performance of Henry V

given in the original Shakespearian English. Unfortunately, the Second World War

curtailed my high-school education. Almost daily air raids, changing teachers and

merging classes of students hampered our education. I have always regretted the lack of

a rigorous high school education. Despite these hardships, I maintain some fond

memories of these times. I made several friendships that outlasted time and distance, and

I recall many unforgettable and shared opera and concert performances in Vienna. Since

then, classical music has remained an essential part of my life.

2

In retrospect, my decision to start medical school in 1946 was a bit haphazard,

and, as I recall, motivated by my respect for two uncles in my family that were

physicians. The subsequent training in medicine at the University in Vienna was totally

different from that in the USA, especially at Cornell Medical College and Yale where I

subsequently worked. I began as one of approximately 1800 entering medical students,

because everybody with a high school diploma was admitted. If you were fortunate and

passed all oral exams - often chancy ordeals - one could finish in five years. We had

access to very few textbooks, instruction in practical patient care was very limited and

the professional future obviously bleak. Despite these major educational shortcomings,

we had several excellent and truly inspiring teachers. I remember three of them that I

credit with having had a profound influence on my later basic science career.

The first of these memorable teachers was Franz von Bruecke. He was highly

educated, came from an established academic family and remains in my memory a truly

fascinating lecturer. He had received a broad scientific education, held the chair of the

Department of Pharmacology for many years and presented superb lectures deeply

grounded in physiology. While he focused on pharmacology I remember his lectures as

dealing with regulated physiological events potentially modified by disease and

modulated by drugs. As no other teacher, Franz von Bruecke convinced me at this very

early state of my career of the importance of physiology as the foundation of medicine.

My plan at the end of medical school in 1951 was to pursue a career in internal

medicine. An individual who played an important role during that period was a young

assistant in the First Department of Internal Medicine, Professor Erwin Deutsch. His

research interests included not only the mechanisms of renal insufficiency, but also the

pathophysiology of blood clotting disorders. I remember him as a very formal individual,

rather demanding and austere, but highly respected by all of us who came to know him.

He got the point across to us that a scientific basis of internal medicine was essential for

clinical care and treatment. He also insisted that everybody who wanted to become an

academic internist had to spend a minimum of two years in a basic science department. In

view of my enthusiasm and respect for Franz v.Bruecke, I decided to fulfill this

requirement by working in the Department of Pharmacolgy. I did not know at that time,

3

but joining the Pharmacology Department was, with a one year intermission, the end of

my clinical career.

My interest in the kidney started early. As a third year medical student I became

fascinated with the kidney by reading Homer Smith’s Porter Lectures, published in 1943

( ). One of the senior Viennese internists, Prof. Eduard Hueber, whose wife was

American and provided a small circle of physicians in the Clinic with new books from

the United States, lent me this book. I attended one of Prof. Hueber’s seminars and he

was kind enough to lend me Smith’s “arrival”. The encounter with Smith’s writing had a

decisive influence on the direction of my early research interests. Briefly, Homer Smith

was for at least three decades, from the nineteen thirties to his death in the early nineteen

sixties, one of the preeminent renal physiologists and chairman of the Physiology

Department at New York University. He is credited with making renal clearance

techniques a major tool for quantifying and analyzing renal function. He realized that

inulin could be used for accurate measurement of the glomerular filtrate, and that the

comparison of filtered with excreted solutes allowed insights into major transport

properties of renal tubules. Smith also showed that noninvasive clearance methods could

be used for measurements of renal blood flow, for quantification of reabsorptive and

secretory processes and for studying the effects of drugs, hormones and metabolic

manipulations on renal tubules and renal circulation. Smith also deserves credit for

introducing renal clearance methods to the study of a wide variety of derangements of

kidney function. Moreover, he inspired an entire school of nephrologists to apply

clearance methods to study renal function in renal failure and hypertension. As an aside,

I should mention that I was so enthralled by Smith’s writing that I asked him somewhat

later to send me a copy of his encyclopedic major opus, the Kidney ( ). I thanked him

many years later in New York, offering reimbursement, but he had all but forgotten his

gift.

My literary curiosity turned out to be yet another important and decisive factor in

my formative years in Vienna. Browsing through a medical bookstore, I ran quite

accidentally across a small monograph entitled Modern Methods for Studying Renal

Function. It was authored by a Swiss internist, Professor Otto Spuehler, the head of the

Policlinic of the University Hospital in Zurich. He was the third individual whose

4

influence was important. As a fourth year medical student, I applied for a training

position in his unit, was accepted, and spent 3 months in the Kantonsspital. Professor

Spuehler was a well-established internist who had done seminal work on the pathology

of interstitial nephritis and run a well-organized nephrology division. I was fully

integrated in his unit and profited greatly from the lively discussions dealing not only

with clinical cases, but also with the rapidly expanding field of renal function

methodologies. It was at this time that I began considering a visit to the United States,

because I realized that new approaches for studying kidney function originated in

laboratories in the USA.

Following graduation from Medical School in 1952, I chose to spend time in the

Pharmacology Department. Fortunately, I was given the opportunity to carry out my first

renal clearance study. A guest in the institute, Prof. Sam Rapoport, initiated a project

designed to elucidate the site of action along the nephron where mercurial diuretics

inhibit renal salt transport. He was an experienced senior investigator who previously had

done extensive work on erythrocyte metabolism, and also developed an interesting

approach to locating salt transport along the nephron. Together with H. Klupp, another

member of the Pharmacology Department, we tested the action of two mercury-

containing diuretics during different types of osmotic loading. Making several

assumptions based on the then sketchy knowledge of the precise site of action of renal

tubule solute transport, we tentatively concluded that diuretics act mainly upstream of

those distal tubule segments that dilute the final urine ( ). Many years later, perfusion

studies on mammalian isolated thick ascending limbs of Henle showed that mercurial

diuretics indeed inhibit the apical Na-K-2Cl transporter, at this nephron site upstream of

final urine dilution ( ). This project provided me with a good learning experience and

prepared me quite well for my future research efforts.

Although my stay in the Pharmacology Department at that time was fairly brief, it

was a stimulating experience. This was due to a number of inspiring faculty members in

the Department and the fact that v. Bruecke attracted interesting and motivated

clinicians to spend time doing research in his basic science department. Three

individuals come to mind. I remember Gerhard Werner, a superb neuropharmacologist

who greatly stimulated my research interests. He later had a very prominent academic

5

career at Johns Hopkins. Another was Hans Klupp, who early on took me under his wing,

and Bruno Watschinger, a highly respected nephrologist who almost single handedly

introduced dialysis into Austria during the early postwar years.

First Visit to the United States

The opportunity to spend time in the United States arose in 1951 through the help

of one of my father’s old friends who had emigrated to the USA in the early l920’s. I

was accepted as an intern at Milwaukee Hospital. My rotating internship in Milwaukee

was a period of hard work and included many sleepless nights, but it provided me with

insights into several areas of clinical medicine that proved valuable for my subsequent

teaching in physiology. Although my clinical activities kept me busy, I did find time to

read and to write a brief review on sodium and water metabolism that was published in

the Marquette Medical Review ( ). An important event at the end of this year was my

marriage to Ilse Riebeth, the beginning of a wonderful union that lasted 56 years.

During my internship at Milwaukee, I applied to twelve institutions for

postdoctoral training and received several positive responses. My first and only visit was

to the Physiology Department at Cornell University Medical College in New York

headed by Robert Pitts. I had the good fortune to be invited to join his department. This

was probably the most important event in my early career and the beginning of a long and

fruitful period of activities in his department. I remained with Pitt’s laboratory for sixteen

years, from 1952 until 1968.

Robert Pitts, together with Homer Smith (with whom Pitts had trained), and

Robert Berliner were at that time considered the most prominent leaders in renal

physiology ( ). Pitts and his associates had successfully defined not only the mechanisms

of renal acid excretion but also clarified key aspects of tubule transport of sodium,

ammonium, the mechanism of action of diuretics and the renal effects of adrenal steroids.

Pitts’ earlier contributions to physiology included seminal studies in neurophysiology

defining the brainstem sites of the central control of respiration.

Robert Pitts was a superb investigator. He was able to define and focus on

important problems, design direct and incisive experiments and write concise papers.

6

Differing from present investigators, he did a substantial amount of his laboratory work

himself, washing his own glassware (!), designing and building his own equipment,

including a flame photometer and later an amino acid analyzer. He could be quite stern,

was a hard taskmaster and bravely battled several chronic ailments. To me he was fair

and eminently generous. After only a short period of apprenticeship, he encouraged and

supported my scientific independence. I will always remember him as an example of a

deeply committed investigator with the mission of a physiologist devoted to probing the

complex workings of the kidney. He was the main mentor of my professional life.

My beginnings in Pitts’ laboratory were rather inauspicious. Although I had

ardently hoped to investigate some aspects of renal electrolyte transport, I was first

assigned to study the renal excretion and distribution of several dextrans. ( ) In this first

study at Cornell, Henry Lauson, a very kind and patient senior member of the

department, was my coauthor. Dextran compounds were of interest to the National

Research Council in view of their possible clinical use as plasma substitutes. I had initial

problems with the then accepted method of analysis, and had to modify a fairly

complicated alternative method of analysis. The results were not very exciting. As

expected, dextran fractions and volume distribution varied inversely with molecular

weight, and their mode of excretion was compatible with graded glomerular filtration

without significant tubule reabsorption or secretion. A positive aspect of the study was

that I became well acquainted with the use of renal clearance methods.

My second project brought me closer to electrolytes but still not to their handling

by the kidney. Pitt’s laboratory had a longstanding interest in the renal regulation of acid

base balance. However, it was realized that an acid challenge might, in addition to an

appropriate renal response, also involve compensatory extrarenal buffering. Prior to my

arrival in the department, Pitts and his associates had already examined possible ion

redistribution during metabolic acid base disturbances in nephrectomized animals. Their

results demonstrated significant participation of cell buffering of acid or base load ( ).

Together with Larry Berger, a postdoctoral fellow from Mt. Sinai Hospital (where he

later became a well known nephrologist), we embarked on the study of how respiratory

acidosis and alkalosis might induce shifts in the body’s electrolyte distribution. Our

experiments involved measurements of distribution of markers for extracellular fluid

7

and analysis of several extracellular ions. We showed that acute respiratory acidosis led

to the release of both bicarbonate and potassium from cells. Respiratory alkalosis had the

opposite effect. Thus, coordinated extrarenal shifts of electrolytes are part of the body’s

compensatory acid base defense mechanisms ( ).

Later I participated in three renal clearance studies, two of them dealing with the

effects of adrenal steroids, and one defining the tubule mechanisms of iodide transport (

). A recurring theme in the first two studies was to separate direct renal actions of

mineralocorticoids from indirect effects mediated by their kaliuretic actions. An

additional aspect was to assess the role of sodium metabolism modifying adrenal steroid

effects. In a study in which I collaborated with R. F. Pitts and his experienced technician

Martha Macleod, we observed that mineralocorticoids significantly increase the renal

reabsorption of bicarbonate and that these effects depended on the hormone-induced

sustained loss of potassium. Steroid stimulation of renal bicarbonate transport could also

be prevented by curtailing sodium intake, supporting the view that luminal sodium is a

key prerequisite for effective potassium secretion ( ). We also argued that adrenal

cortical stimulation and depletion of cell potassium participate in the well-documented

renal stimulation of bicarbonate reabsorption in chronic respiratory acidosis ( ).

Another renal clearance study dealt with the effects of adrenal steroids and

potassium depletion on the kidney’s ability to elaborate an osmotically concentrated

urine. ( ) Dr. Lozano and I used an approach that involved measurements of maximal

urinary osmolality during the progression of osmotic diuresis. The theoretical

background of this approach followed the suggestion of Smith that osmotically

concentrated urine can be visualized to consist of an isosmotic component minus the

amount of water abstracted from it to generate hypertonic urine ( ). We could show that

this moiety, TcH20, was consistently reduced in animals treated with mineralocorticoids

as osmotic diuresis increased over a wide range of urine flow rates. Despite the

administration of vasopressin, urine even became hypotonic as osmotic diuresis

progressed. In contrast to the effects of mineralocorticoids, hydrocortisone was

ineffective. The observed mineralocorticoid effect supported the view that tubule fluid

reaching the site of final water abstraction failed to be rendered isosmotic or hypertonic.

From what was known about the sites of fluid transport along the nephron, we concluded

8

that diminished water permeability along the distal tubule and the collecting duct

accounted for the loss of urinary concentrating ability. Similar to the effects of adrenal

steroids on bicarbonate transport, the mineralocorticoids’ effect on concentrating ability

was also mediated by the steroid-induced renal loss of potassium, since repletion of

potassium completely prevented the mineralocorticoids’ effect on urinary concentrating

capacity.

Finally, I was also involved in a renal clearance study on the mechanism of iodide

handling. Little was known about how iodide might be excreted by the kidney, but we

speculated that it might display similar excretion patterns as urea. Indeed, in collaboration

with M. MacLeod and F. Kavaler we showed that iodide excretion closely resembled

the pattern of renal urea handling ( ). By varying urine flow rate, we showed that iodide

reabsorption was essentially determined by the degree of tubule fluid reabsorption and

thus its concentration difference across the tubule epithelium. We also concluded that the

bulk of iodide reabsorption occurred along the proximal tubule because its excretion was

independent of urine flow rate during water diuresis, a finding strongly suggesting that

iodide reabsorption was limited to nephron sites proximal of final water reabsorption.

Absence of a transport maximum during elevation of plasma iodide concentration

provided further proof for the absence of an active tubule reabsorption.

Transition Period- Micropuncture and Single Renal Tubules

The early fifties were historically an interesting and challenging period in renal

research. It was a time when exploration of renal tubule function was largely based on

clearance studies, an approach that rested on the comparison between filtered and

excreted moieties of solutes. It became apparent that the interpretation of such clearance

data had limitations, especially for substances that might be simultaneously reabsorbed

and secreted. Importantly, information based on clearance data alone provided only

limited information about the localization along the nephron of transport processes, their

nature and their cell mechanisms. At that time, it became evident that further progress of

renal transport physiology required more data on single nephron function.

9

I was very fortunate indeed that my department head, Prof. Pitts, recognized the

importance of exploring tubule function directly. He allowed me to learn micropuncture

in Prof. Phyllis Bott’s laboratory at the Women’s Medical College of Pennsylvania in

Philadelphia. Professor Bott had participated in the classical investigation of ion and fluid

transport in the mammalian nephron ( ). She kindly agreed to teach me how to puncture

and obtain fluid from single tubules, and how to analyze nanoliter samples of such tubule

fluid. She thought that I should start with the easier (larger) preparation of an amphibian

tubule (Necturus) and suggested resolution of a discrepancy that arose from previous

work regarding the proximal tubule’s ability to acidify tubule fluid ( ). After six months’

training in Philadelphia, I returned to Cornell and was now truly on my own. With

patience and some skill, I now was able to obtain tubule fluid, locate the puncture site by

re-injecting a marker and microdissect after mazeration of amphibian nephron segments.

I also could handle and analyze nanoliter samples of fluid for hydrogen ion and chloride

concentrations. I was also impressed by the large size of the nephrons, compared to their

mammalian counterparts. However, it took some time before I used these newly acquired

skills.

After my return to Cornell, I decided to start an electrophysiological study on the

Necturus kidney. This certainly was a break with my previous research and a somewhat

risky undertaking since I had no previous experience in electrophysiology. Again, my

chairman, Prof. R. Pitts, demonstrated his generosity by financing the purchase of the

necessary equipment for work on single renal tubules. Given the relatively large size of

Necturus tubule cells and the possibility to perfuse the kidneys via the aortic and portal

circulations, it occurred to me that these cells might be large enough to impale with

microelectrodes. I thought that measuring transepithelial and cell electrical potentials

would define some of the driving forces of ions such as sodium, potassium and chloride

not only across the tubule epithelium, but also across the apical and basolateral cell

membranes. Moreover, the dual circulation of amphibian kidneys such as Necturus also

allowed exposing the apical and basolateral cell membranes selectively to solutions of

different compositions. Such probing of the effects of changes in ion composition

during monitoring of cell potentials could also provide insight into the permeabilities of

the two membranes (apical and basolateral) lining tubule cells ( ). I was also influenced

10

by Hans Ussing’s work. He had done pioneering studies on the isolated frog skin and

developed a cell model of ion transport that depended importantly on the assignment of

specific active and passive transport components to the apical and basolateral membranes

of the epithelium ( ). Stimulated by such studies, I attempted to find out how single

tubule cells behave and how specific apical and basolateral transporters interact to

achieve reabsorption or secretion along the tubule. When I started impaling the tubule

lumen and tubule cells with microelectrodes, very little electrophysiological work had

been done on nephrons ( ), and almost nothing was known about the properties of

individual cell membranes in tubules. Similarly, no information was available on the

driving forces acting on ions across the individual cell membranes lining tubule cells

along the nephron. It became evident that such information could not be obtained from

clearance data alone. The time for the direct exploration of single tubule function seemed

ripe.

The experiments on the perfused Necturus kidney were technically demanding,

because the connective tissue covering the kidney surface often led to breaking of the

very fine pipette tips necessary to obtain stable potentials. We found that the tubule

lumen was electrically negative with respect to the peritubular fluid. The transtubule

potential difference was quite variable, and we probably overestimated its magnitude (

). Cell potentials, on the other hand, were more easily reproducible (in the range of -

70mv). The latter was sharply reduced by elevating the peritubular potassium

concentration. Measurements of cell potassium indicated that diffusion potentials of

potassium could fully account for the cell potentials, which could also be lowered by

metabolic inhibition( ). I was not alone in my experiments, since colleagues in A.K.

Solomon’s laboratory ( ) and in Karl K. Ullrich’s Max Planck Institute ( ) had also

explored electrophysiological properties of amphibian tubules ( ). Members of thse

laboratories and I came to know each other well through our common interest, shared our

data freely and I was gratified that some lasting friendships originated at that time

through our common scientific interest. I remember very well Guillermo Whittembury,

whose research interests overlapped with mine to the extent that one of our studies

appeared back to back in the J. General Physiology ( ). Years later, he visited my

laboratory at Yale and I collaborated with him for several months at the Biophysical

11

Institute of IVIC in Caracas. I also enjoyed excellent relationships with A.K. Solomon at

Harvard and with Karl Ullrich from the Max Planck Institute in Frankfurt. I found my

frequent visits to their laboratories very fruitful and satimulating.

Our experiments in the amphibian tubule defined key aspects of proximal tubule

function and opened , at least partially, the “black box” of tubule cells.. We provided

evidence that the proximal tubule actively reabsorbs sodium and that basolateral active

sodium-potassium exchange was responsible for cell negativity and the low cell sodium

concentration. A sizeable sodium concentration gradient, aided by cell negativity,

provided the driving force for sodium entry into proximal tubule cells along a favorable

electrochemical potential difference ( ). Transepithelial chloride reabsorption could be

accounted for by the lumen negativity, a conclusion later confirmed by tracer chloride

flux measurements ( ). The cell model that emerged from these experiments was in

many respects similar to the one proposed by Ussing and his associates for transepithelial

sodium movement in the isolated frog skin ( ).

Return to Austria

My initial scientific career at Cornell was interrupted after 4½ years when I

returned to Vienna to fulfill a promise I had made to Prof. F. Bruecke that I would work

in his institute for a trial period before making a definite decision where to continue my

career. He made a valiant effort to provide space and equipment, but it became apparent

after a few months that an offer to return to Cornell would be very

tempting. Indeed, Robert Pitts soon offered me the position of Assistant Professor in his

department which I accepted. I have never regretted this decision, but it was with a

heavy heart that I left Vienna. I maintained very cordial relationships with my colleagues

in the Pharmacology Department and with Prof. Bruecke, and many years later lectured

annually in the Pathophysiology Department of the Medical School. It was also through

a contact in the Pharmacology Department, Prof. Heistracher, that I met Jurg Graf who

later was a regular guest of ours at Yale. He was responsible, many years later, for a joint

eaching arrangement in Vienna that I thoroughly enjoyed. My activities in the

Pharmacology Department involved a fruitful interaction with several colleagues who

12

had a long-standing interest in mammalian muscle physiology. They had developed a

perfused mammalian muscle preparation in which they investigated relationships

between muscle contraction, lactic acid metabolism and drugs as modified by altered

ionic composition. I gladly accepted their offer to join their team and added cell potential

measurements to their analyses. The experiments confirmed and expanded results

obtained previously in amphibian muscle on the importance of potassium and chloride

determining membrane polarization. In addition, the experiments defined effects of

various ion substitutions on muscle contraction, blood flow and lactic acid production (

). So I “skipped” the kidney for a while, wrote some manuscripts from data obtained

during my stay at Cornell, witnessed the huge influx of Hungarian refugees into Vienna,

and became familiar with some muscle physiology.

Moving to the United States

Two important events took place in the first few years after my return to the

Physiology Department at Cornell University Medical College. In 1958 I was joined by

Erich Windhager, a colleague of mine from Vienna, and three years later Gerhard Malnic

from Sao Paulo became my first postdoctoral fellow. Both of these individuals have

played a major role in my professional life. Erich Windhager had been associated for

several years with A. K Solomon’s Biophysical Laboratory at Harvard. There he was

involved in studying the relationship between sodium and water movement in Necturus

nephrons ( ), and his decision to join forces with me had considerable impact on the

work in our laboratory. We both had spent several years perfusing amphibian tubules but

it occurred to us, and Erich Windhager was very forceful in this decision, that we should

“advance” to the mammalian nephron. Technically this was quite a challenge because it

required some skill involving the preparation of the rat kidney, the identification of

proximal or distal tubules on the often moving kidney surface, followed by collection of

small tubule fluid samples, plus the identification of the site of micropuncture by

reinjection of a plastic material and dissection of the punctured nephron. We did

eventually succeed. Another problem we had to address was the chemical analysis of the

small nanoliter samples of collected tubule fluid. This involved initially the use of

13

radioactive isotopes (Na and inulin) and an electrometric method for chloride. It was also

at that time that we were joined by an excellent research associate, Mrs. Ruth Klose, who

mastered micropuncture quickly and became a very skilful and devoted collaborator in

our experiments.

The first mammalian micropuncture study that we undertook had to do with the

exploration of the nature of sodium chloride transport in the proximal tubule. At the time

of these studies, it was known from the only previous mammalian micropuncture study

that a large fraction of the glomerular filtrate was reabsorbed along the proximal tubule.

Moreover, it was postulated on the basis of indirect evidence that sodium ions were

actively transported out of the tubule and that both water and chloride followed passively

( ). This view was challenged in the late fifties ( ) and an alternative hypothesis

presented. It was presumed that the colloid oncotic pressure difference between tubule

fluid and peritubular capillaries provides the main driving force for the bulk of fluid and

sodium retrieval from tubule fluid across the proximal tubule, and not active sodium

movement. Our first micropuncture study was designed to clarify this issue ( ). Our

experiments demonstrated the existence of active transport of sodium directly by

showing that sodium ions can be reabsorbed against a sizeable concentration difference

when poorly reabsorbable nonelectrolytes, for instance mannitol, were present in

proximal tubule fluid. In addition, evidence was obtained that water movement was

strictly dependent on solute transport. These studies were later expanded to include

perfusion of single proximal tubules, in which we excluded a major contribution of the

colloid oncotic pressure as a major driving force for proximal tubule sodium and fluid

transport. In these experiments we could also define the absolute magnitude of the

concentration difference against which sodium ions could move: below a lumen sodium

concentration of 100 mEqu/l sodium reabsorption ceased, and fluid entered the lumen as

progressively more mannitol was added to prevent changes in osmotic driving force ( ).

In a follow-up study, we further explored the kinetics of sodium and fluid

transport along the nephron. Besides expanding our observations to the superficial distal

tubule segments available for puncture from the kidney surface, we also evaluated the

transport behavior of tubules after intravenous loading with sodium chloride ( ). First,

we could show that proximal and distal transport patterns differed significantly. The

14

fraction of filtered sodium remains constant along the proximal tubule and never reaches

a transport maximum. In contrast, in the distal tubule transport capacity is sharply

limited and does not increase when sodium delivery to the distal tubule rises during

sodium chloride loading. Second, we demonstrated a marked flow-dependence of

proximal tubule sodium transport: i.e proximal reabsorption rate increases with enhanced

fluid delivery without any change in the luminal concentration of sodium. Fractional

sodium reabsorption along the proximal tubule also remains constant in conditions of

spontaneous fluctuations of glomerular filtration. This striking phenomenon of flow-

dependent proximal tubule transport, glomerulo-tubular balance, had also been observed

almost simultaneously by Lassiter et al ( ) and been reported in numerous subsequent

studies ( ). It attracted great interest over the following years and several mechanisms

have been proposed subsequent to its discovery ( ).

The Venture into Renal Potassium Transport

My collaboration with Erich Windhager was interrupted in the early sixties

because visa requirements made it necessary for him to leave the United States for two

years. Fortunately he spent this time period in Copenhagen, where he collaborated with

Hans Ussing in a study that discovered and defined paracellular ion movement in the

isolated frog skin as a key element of transepithelial fluid and electrolyte transport ( ).

It was at that time that I was joined by Gerhard Malnic from Sao Paulo. He was, as I soon

discovered, also of Austrian origin, very talented, insightful and hard working. With his

coming, we started a collaboration and close friendship that has now lasted half a century.

After his return to his home country, he was a major force in establishing renal research

in Brazil.

The decision to explore the mechanism of renal potassium transport seemed quite

timely in the early sixties, because it seemed ideally suited for exploration by

micropuncture techniques. Berliner and Kennedy, later joined by Orloff, as well as

Mudge and his associates, had done a series of renal clearance studies suggesting that

potassium excretion was governed by two simultaneous processes: extensive

reabsorption of potassium in proximal nephron segments, followed by potassium

secretion in more distally located nephron segments. These authors also showed that the

15

presumed “distal” potassium secretion was dependent on the presence of sodium in the

lumen. Moreover, such potassium secretion was also thought to be modulated by

changes in acid-base balance, adrenal hormones and diuretics ( ). We thought that this

thesis, entirely based on renal clearance methods, lent itself ideally to be tested by

puncturing, collecting and analyzing fluid samples along the nephron. In addition, we

hoped that such studies on single nephrons would also provide insights into the cell

mechanism of tubule potassium transport.

The first problem we encountered was to find a method for measuring both

potassium and sodium in nanoliter samples of collected fluid. Again I was fortunate.

Looking for interesting papers in the library of Cornell Medical College, I came upon,

quite by accident, a paper published in a review of a symposium on Neurophysiology at

Rockefeller University. The paper was authored by Paul Muller who had developed what

looked like a fairly simple method for measuring sodium and potassium simultaneously

in single nodes of Ranvier to test sodium and potassium shifts during nerve stimulation

and recovery ( ). I contacted Paul Muller, who at that time worked in the one of the

basic science departments of the Eastern Pennsylvania Institute of Pennsylvania in

Philadelphia, to enlist his help. He was very generous and assisted us in building the

ultramicro--flame photometer in the machine shop at Cornell, and this apparatus had the

sensitivity and reproducibility that we had hoped for. The design of the device was

simple: it consisted of small platinum loop on which the nanoliter tubule fluid was

deposited followed by its mechanical positioning into an oxygen flame. The emission

was appropriately filtered and displayed on a polygraph borrowed from the student

laboratory, and allowed simultaneous estimates of potassium and sodium

concentrations.Its use took some skill and patience. I was pleased a modified and

improved Muller photometer was adopted by two major laboratories, that of Karl

Ullrich at the Max Planck Institut in Frankfurt, and by Francois Morel at Saclay.

Eventually we employed the helium-glow photometer and models of atomic absorption

spectrophotometers to measure sodium and potassium in tubule fluid, but I look back

with some nostalgia at the time when we used this simple apparatus which had become so

essential for our research.

16

Our experiments provided answers to several questions. First, how was potassium

transported along the nephron? We could confirm that potassium was extensively

reabsorbed along the proximal tubule as well as along the loop of Henle, and showed

conclusively that it was secreted along the later sections of the distal convoluted tubule.

Such secretion was present even under conditions in which urinary excretion rates of

potassium were only a very modest fraction of filtered potassium. This was a rather

roundabout way of excreting potassium, since failure to reabsorb filtered potassium could

under most conditions fully account for the amount of potassium in the final urine. We

found that secretion was sharply accentuated even under those conditions in which

potassium excretion exceeded the filtered amount Secretion was clearly the important

element of potassium excretion.

Second, what was the functional role of distal tubule transport in potassium in

regulating external potassium balance? Our experiments provided strong direct evidence

for the marked flexibility of distal tubule potassium secretion. Distal tubule secretion of

potassium was positively related to potassium intake. Of interest was the finding that

potassium secretion along the distal tubule could be completely abolished and replaced

by reabsorption when animals were given a low potassium diet ( ). The presence of

potassium reabsorption along nephron segments downstream of the distal tubule was

evidenced by comparing the amounts of potassium at the late distal and urinary levels.

Such reabsorption was later confirmed ( ) and provided a satisfactory answer to the

question of the source of potassium for its medullary recycling ( ).

The second question we addressed was to define the physiological role of distal

tubule potassium secretion in the regulation of urinary potassium excretion. From the

observation that potassium delivery to the early distal tubule varied but little during

many manipulations that drastically altered excretion, we established the distal nephron

as the principal site for the regulation of potassium excretion ( ).

A third question concerned the role of sodium ions in potassium excretion.

Berliner and his associates had already stressed the dependence of potassium excretion

on sodium supply, and suggested direct obligatory coupling of sodium reabsorption to

potassium secretion in apical membranes of distal tubule cells ( ). However, our

experiments did not support this notion because comparison of sodium reabsorption and

17

potassium secretion showed that sodium ions never become limiting for potassium

secretion; sodium reabsorption along the distal tubule remains many times higher than

potassium secretion (and never becomes limiting), for potassium secretion ( ).

Finally, our studies allowed us to begin distinguishing between several

mechanisms of potassium transport in the distal nephron. First, we compared the

magnitude of the transtubular electrical potential with peak potassium concentration

ratios and observed that the magnitude of the transepithelial concentration difference of

potassium could be fully accounted for by the negative distal electrical potential. This

implied a passive component of passive diffusion across the apical membrane. We

proposed that stimulation of sodium reabsorption increases the lumen negative potential,

providing an enhanced electrochemical driving force for passive potassium secretion.

We thought that sodium and potassium movement were linked by the effect of sodium

on the electrochemical driving force for potassium movement across the apical

membrane of distal tubule cells. The notion that sodium ions play a crucial role for

potassium secretion was further demonstrated by exploring the effects of amiloride, a

specific blocker of sodium channels. In collaboration with C. Duarte, we showed later

that this diuretic agent blocked both sodium reabsorption and potassium secretion ( ).

These effects could readily be explained by the reduction of the transepithelial

potential brought about by the well-known inhibitory effect on sodium channels by

amiloride ( ).

An intriguing finding was the relationship between observed transepithelial

potassium concentration differences and tubule negativity. We observed transtubular

concentration ratios of potassium that were consistently below that expected from the

electrical driving force across the epithelium ( ). We interpreted this finding by

proposing that a component of active potassium reabsorption was responsible for the

apparent electrochemical disequilibrium ( ). The molecular nature of such a process was

defined by Doucet and his associates with their identification of an exchange mechanism

in collecting ducts that reabsorbed potassium for hydrogen ions ( ). The potential of the

distal tubule epithelium to reabsorb potassium was later confirmed ( ). After these

initial studies on renal potassium transport, we followed up with investigations into the

effects of acid-base manipulations ( ), disturbances of adrenal function ( ) as well as an

18

evaluation of several diuretics ( ). An important conclusion was the profound effect of

distal tubule flow rate on potassium secretion ( ), especially after inhibition of sodium

and fluid reabsorption along the thick ascending limb of Henle ( ) or following

administration of poorly permeant anions ( ). Some of these observations had clinical

implications, because they clarified the mechanisms underlying the kaliuretic effects of

certain diuretics and acid-base disturbances, all of them involving redistribution and

enhanced fluid absorption from proximal to distal nephron sites ( ).

I look back at my years at Cornell with a sense of gratitude. I received excellent

research training and was encouraged to be independent very early. I also participated

quite actively in the teaching efforts of the department, a fact that was helpful when I

moved to Yale. Finally, Pitts’ department attracted outstanding trainees, mostly from

Europe. This permitted me to make many professional and personal contacts with

talented colleagues. Several come to mind. With Bruno Ochwadt from the Max Planck

Institute in Goettingen, I spent a summer doing micropuncture on an isolated perfused

kidney preparation( ). Through Bruno Ochwadt I met Kurt Kramer, the head of the

Physiology Department and members of his group including Karl Ullrich, Klaus Thurau,

Peter Deetjen, K.H. Gertz and E. Froemter. Several of these individuals later assumed

leadership positions, such as Karl Ullrich as the Director of the Biophysical Max Planck

Institut in Frankfurt, Klaus Thurau in Muenchen where he became chairman of the

Physiology Department, and Peter Deetjen who led the Physiology Department in

Innsbruck. These contacts had far-reaching effects. I visited their Department frequently

and our exchange of ideas over many years was very positive and productive. Other

guests at Cornell with whom I eventually collaborated were Klaus Hierholzer and

Michael Wiederholt from Berlin. Both spent time with me subsequently at Yale, where

we jointly investigated adrenal steroid effects on proximal and distal tubule sodium and

potassium transport ( ).

At Cornell I was also given the opportunity of enlarging my laboratory. Besides

Erich Windhager and Gerhard Malnic, I was joined subsequently by S. Glabman who

explored the topography of ammonium secretion ( ), by J. Strickler who clarified

phosphate transport along the nephron ( ) and by D. Landwehr who extended our studies

on renal tubule sodium transport by identifying several mechanisms that altered the

19

distribution of sodium reabsorption along the tubule ( ). I was also pleased that J.

Schnermann, early in his very successful career on tubulo-glomerular feedback, spent a

year with us ( ). Another individual who joined our laboratory was a young physiologist

from Loewen, Emile Boulpaep, who had been trained as a cardiac electrophysiologist.

Fortunately for us, he became interested in renal epithelial transport and quite

independently continued very successful work defining the permeability properties of

amphibian tubule cells as well as elucidating the role of paracellular ion and fluid

movement in tubule reabsorption ( ). I was fortunate that I could persuade him a few

years later to become a faculty member in the Department of Physiology at Yale.

Move to Yale Medical School

In the fall of 1967, I was approached quite unexpectedly by the Dean of the Yale

University Medical School to consider the chairmanship of the Department of

Physiology. I had never before considered such a career change because I was quite

happy at Cornell and my work was going well. However, two aspects of this possible

offer were intriguing and tempted me. First, the department had recently been rebuilt by

Cuy Hunt, whom I knew and respected when he worked previously in New York at

Einstein Medical College. Second, the new faculty at Yale included two individuals

whose work complemented my research interest. I knew Joe Hoffman from his work on

red cell transport and I had met Peter Curran, a biophysicist who had worked on ion and

water transport in model systems during visits to AK Solomon’s Biophysical Laboratory

at Harvard. From Yale I had met Frank Epstein, the head of nephrology in Internal

Medicine and Mike Kashgarian from the Department of Pathology, who had a

longstanding interest in renal pathophysiology. Thus, the nephrological environment was

outstanding. Third, the chairmanship in basic Science Departments at Yale was time-

limited, and offered the opportunity to return eventually to full-time research. After

several visits to Yale, I decided to accept their offer and started the new position in July

1968. I have never regretted the move.

My five years as chairman of the department were a time of student unrest and

dissent, but the Medical School on the whole was spared the excesses of other

20

institutions. I got along well with the faculty and, aided by Joe Hoffman and Peter

Curran, continued to expand the department by recruiting Dick Tsien, Emile Boulpaep

and John Sachs. When I stepped down, we started a successful Program Project on

membrane transport that is still funded. At the time of its initiation, it included studies on

red cells, amphibian epithelia, liver and kidney. The grant supported a broad range of

interactive research, both at Yale and abroad. It also provided start-up money for new

projects. Examples are Peter Aronson’s initial research on sodium/hydrogen exchange in

brushborder vesicles ( ), the study of canalicular transport function in hepatocyte

couplets by Jim Boyer and Jurg Graf ( ), and our joint electron probe studies with Franz

Beck exploring functional heterogeneity in single collecting duct cells in Klaus Thurau’s

Physiology Department in Munich ( ). Thus, my move to Yale allowed me to profit in

many ways from the continuous interaction with numerous colleagues who shared my

interest in transport physiology. I was offered an excellent opportunity to broaden and

deepen my research.

My forty years at Yale have provided me with an ideal environment to do

research on the topics that I found most fascinating. How do sodium, chloride, potassium

and hydrogen transport interact at different nephron levels and how are they controlled?

What are the specific transporters of these ions in the apical and basolateral tubule

membranes of cells lining the nephron, and how are they affected by hormones and

metabolic manipulations? Which types of ion channels mediate passive ion movement

across apical and basolateral membranes of tubule cells? What messenger mechanisms

coordinate basolateral with apical membrane transport? A recurrent and quite intriguing

finding was the striking redundancy of transporters for a specific ion movement, a

problem that became apparent with the availability of genetically modified animal

models. Our approach used multiple avenues, ranging from the assessment of overall

renal function by traditional clearance methods, followed by defining net movement of

specific ions in each nephron segment, analysis of membrane events at the cell level.

Whenever possible, we tried to integrate such discrete events at the membrane level with

overall renal function. In the following I briefly survey of main areas of research that we

pursued.

21

Sodium Chloride and Fluid Transport

My association with Roger Green provided an opportunity to clarify the

mechanisms of fluid and sodium transport in the mammalian proximal tubule, the main

site of fluid retrieval in the nephron. Roger had great patience, persistence and superb

technical skills that were needed to carry out the simultaneous perfusion of proximal

tubules and peritubular capillaries. I also remember him as unusually calm and totally

unperturbed by the busy lab activities around him. His experiments addressed key aspects

of proximal tubule transport. He defined active and passive transport components of

fluid transport, assessed the role of tubule flow rate, chloride and bicarbonate gradients,

and, importantly, identified finite lumen hypotonicity as an important driving force of

fluid reabsorption along the proximal tubule( ). The results of the latter study,

defining reflexion coefficients of major ions, were further analyzed with Robert Unwin

and AlanWeinstein ( ).

We further extended our studies on renal tubule sodium transport. For two

reasons, we were interested in the behavior of distal nephron sites of sodium

reabsorption. First, we could show that distal tubule transport capacity was normally

unsaturated ( ). Accordingly, increased delivery of fluid and sodium following inhibition

of transport in the proximal tubule enhanced reabsorption distally, thus curtailing urinary

sodium loss ( ). This explains why proximally acting diuretics, such as carbonic

anhydrase inhibitors, are relatively ineffective in promoting sodium loss.

In perfusion studies of distal tubules, done during one of Gerhard Malnic’s visits

with us, we used native proximal tubule fluid instead of artificial solutions to perfuse

distal tubules, and found that sodium transport was stimulated significantly The nature

of such luminal paracrine transport activation is not known ( ). Such “reserve” capacity

of distal sodium transport has implications for potassium secretion because the latter is

strongly stimulated by enhanced distal sodium reabsorption ( ). We found that

inhibition of sodium reabsorption upstream of the distal tubule site of potassium secretion

always leads to urinary potassium loss and potentially to hypokalemia. An extensive

micropuncture study with M. Hropot on the site and mechanism of action of several

diuretics further underscored the stimulating effect of drug-induced distal sodium

22

delivery on potassium secretion, whereas inhibition of distal sodium channels curtailed

potassium loss ( ).

A very productive collaboration with Hans Oberleithner and Bill Guggino, also

involving Florian Lang and Wenhui Wang, identified the electrically neutral Na-2Cl-K

cotransporter in the early distal tubule of the Amphiuma kidney ( ). This tubule segment

shares functional properties with the mammalian thick ascending limb of Henle’s loop.

Moreover, the large cell size allowed stable microelectrode impalement and measurement

of cell potentials and cell ion activities. Several strategies were used to provide evidence

for such a Na-2Cl-K transport mechanism, later also identified and extensively studied in

the mammalian thick ascending limb of Henle’s loop by Greger and Schlatter ( ). They

involved monitoring cell Cl, Na and K ion activities after inhibiting the cotransporter,

either by monitoring or. cell ion activities after transport inhibition by loop diuretics. The

role of potassium recycling across the luminal membrane in maintaining Na-2Cl-K

cotransport activity was also established ( ). The identification of the Na-2Cl-K

cotransporter also allowed defining its role in the volume control of distal tubule cells in

Amphiuma ( ).

The large cell size of both proximal and distal tubule cells of the amphibian

nephron continued to intrigue us and stimulated several studies. I was very lucky when

Ken Spring joined our laboratory at that time He had developed an ingenious method for

measuring short-circuit currents across proximal tubules and succeeded to insert an axial

electrode into segments of tubule fluid isolated by small oil droplets. Measurements of

intracellular sodium and radioactive Na fluxes complemented these studies, an

experimental true tour de force that to my knowledge has never been repeated. We

suggested a transport model with a saturable luminal entry step of Na into the cell and a

second, active unsaturated transport step across the peritubular cell membrane. This large

flexibility of peritubular active sodium transport was well suited to explain the

remarkable ability of proximal tubule cells to adjust to fluctuation of glomerular

filtration rate and sodium delivery. An interesting observation was the striking

dependence of apical sodium permeability on the luminal sodium concentration, also

observed in other epithelia ( ).

23

Somewhat later I was joined by two exceptionally gifted colleagues, Daniel

Horisberger and Laurent Schild, both members of the Pharmacology Department in

Lausanne. They were part of an extended “Swiss period” in our laboratory that started

with Francoise Chomety and Nicole Fowler, both very devoted and skillful technical

associates who joined my laboratory in the early eighties. Daniel Horisberger, together

with Malcolm Hunter and Bruce Stanton, developed a very useful isolated Amphiuma

kidney tubule preparation ( ). We had hoped that the large cell size of their collecting

duct cells would permit “easy” and stable cell potential measurements to explore the

apical potassium permeability of this distal tubule preparation. To our disappointment,

however, the apical potassium permeability of these cells was found to be quite low.

Although potassium secretion could be stimulated by exposing the animals to a high

potassium environment, such transport stimulation took place almost exclusively by

enhanced flux through the paracellular pathway and not, as we had hoped, involving

apical permeability changes. However, Daniel Horisberger used the Amphiuma

collecting duct preparation for an extensive exploration of the transport properties of the

luminal and basolateral cell membrane of collecting duct cells. He focused on defining

their ionic permeability properties, correlated cell ion activities with transport and cell

potentials, and defined the voltage dependence of ATPase-driven pump currents ( ).

Laurent Schild, in the meanwhile, had joined our efforts to explore the properties

of transepithelial chloride transport in the mammalian proximal tubule. Although there

was good evidence that proximal chloride reabsorption involved passive, paracellular

movement, sporadic evidence suggested an additional transcellular pathway that we

hoped to identify ( ). We joined forces with Peter Aronson’s group that had used apical

membrane vesicles from mammalian proximal tubules to show that both formate and

oxalate exchange for chloride ( ). They suggested that such dual anion exchange

mechanisms operate in parallel with Na/H exchange and account for a significant

component of transcellular chloride reabsorption. We tested and provided supportive

evidence for this novel idea in two mammalian preparations. Laurent Schild

demonstrated the presence of chloride-formate exchange in vitro in isolated perfused

proximal rabbit tubules ( ), and Tong Wang showed its presence in vivo in both

perfused rat and mouse proximal tubules ( ). Our collaborative studies with the Aronson

24

laboratory also defined transcellular and paracellular chloride transport patterns in

acidosis, showing a resetting of the relationship between oxalate-chloride exchange and

bicarbonate fluxes ( ). My interactions with Peter Aronson have continued for many

years and developed into a friendship that I deeply value.

Acid-Base Transport

Besides Gerhard Malnic, with whom I continued over the years to collaborate on

several problems of both acid-base and potassium transport ( ), I enjoyed my association

with several individuals who worked with me in this area. One of these was Yun Lai

Chan who came my laboratory in the early eighties. He had mastered the demanding

technique of simultaneously perfusing mammalian proximal and distal tubules and their

adjoining peritubular capillaries in Karl Ullrich’s Max Planck Institute in Frankfurt. He

used his extraordinary skills to define several factors controlling proximal tubule

bicarbonate transport along the nephron, defining the dependence of bicarbonate

reabsorption on , luminal and peritubular bicarbonate and sodium concentrations, tubule

flow rate and bicarbonate permeability ( ). My collaboration with him had an additional

very beneficial impact, because one of his most gifted collaborators, Tong Wang, joined

our group in 1990 after having spent five years of training in his laboratory . Tong Wang,

now head of an independent laboratory, played a major role not only in the studies

already mentioned on chloride reabsorption, but also contributed to our efforts to define

the cell mechanisms of hydrogen secretion and and bicarbonate reabsorption. To this

end, she defined the contribution of individual hydrogen ion transport mechanisms to

tubule acidification along the nephron, studying several regulatory mechanisms and

sodium-hydrogen exchanger knock-out mice ( ). In her career, Tong Wang has

demonstrated for many years a gift for hard work, outstanding experimental and

analytical skills and great loyalty.

Additional colleagues have greatly furthered our work on renal tubule acid-base

transport. Members of this team included Gianni Capasso, Robert Unwin, Philippe

Jaeger, Chris Wilcox and Bertil Karlmark, as well as colleagues from Gerhard Malnic’s

laboratory in Sao Paulo, especially Marguerida de Mello-Aires and Antonio Cassola.

25

Their work extended our knowledge of the nephron localization and nature of specific

hydrogen ion transporters, dealt with questions of how the property of lumen buffers

affects hydrogen ion secretion and titratable acid formation, and how metabolic

derangements, especially those of potassium metabolism, and adrenal hormones,

modulate hydrogen transport along the nephron ( ). Another colleague who helped to

explore the regulation of tubule hydrogen ion secretion was John Geibel, who joined the

laboratory in 1986 after we met earlier in Innsbruck. He had demonstrated great

expertise in devising novel micromethods for analyzing tubule fluid, and I was fortunate

to persuade him to join our laboratory. He became an expert in optical cell pH

measurements and we first collaborated with Walter Boron to define apical and

basolateral acid transporters in proximal tubules ( ). In later studies, with Carsten

Wagner, we used cell pH measurements to characterize the proximal and distal tubule

mechanisms by which angiotensin-II modulates hydrogen ion secretion ( ).

Potassium Transport

When Gerhard Malnic and I started our collaboration in the early ninety sixties,

we hardly anticipated that our efforts would continue until the present. However, our

common interest has lasted and we still join forces and remain intrigued by the complex

mechanisms by which the kidney regulates potassium excretion.. Noteworthy was the

development of a novel perfusion technique in Gerhard Malnic’s laboratory that allows

studying potassium movement in a distal tubule “split-drop” preparation independent of

flow-induced transport changes ( ). The use of potassium-sensitive electrodes permits

monitoring of time-dependent changes in lumen potassium concentrations during

experimental manipulations. Such experiments provided information on specific

potassium flux components, such as electroneutral KCl secretion ( ), potassium

secretion mediated by different potassium channels, and by changes in luminal pH and

bicarbonate ( ). Other colleagues contributing to our efforts were Fred Wright, who

defined adaptive changes in distal potassium secretion during the development of

hyperkalemia ( ), and the important stimulatory effects on potassium secretion by distal

tubule sodium were explored by Raja Khuri, Strieder and Michael Wiederholt ( ). My

26

former graduate student, Bruce Stanton, carried out extensive distal tubule perfusions in

which he was able to separate luminal and peritubular factors on potassium secretion (

). It evolved that the effects on distal secretion, for instance inhibition by metabolic

acidosis,. can be drastically modified by changes in luminal sodium delivery, which has

the opposite effect.. The observation that luminal sodium delivery is a key modulator of

potassium secretion was pursued further by Michael Field, who investigated the

mechanism by which three major factors known to modulate potassium excretion

(hyperkalemia, mineralo- and glucocorticoids) alter distal tubule potassium secretion (

). This investigation was timely because there was no agreement about the relative

impact of these agents on potassium excretion. Whereas glucocorticoids stimulate

potassium secretion solely by enhancing flow rate and sodium delivery, aldosterone’s

direct kaliuretic effect is strongly modulated, and dependent on an adequate supply of

sodium in the lumen. The latter rises during hyperkalemia owing to inhibition of sodium

reabsorption along the proximal tubule ( ), a factor contributing to effective potassium

secretion. Direct stimulation of potassium secretion by vasopressin was also observed (

).

The problem of potassium recycling in the renal medulla also attracted our

interest, and was explored by Jacques Diezi and his colleagues ( ). He focused and

extended the observations originating from Jamison’s studies, that potassium is lost

from tubule fluid in the most terminal collecting duct segments and re-enters tubule

elements of the loop of Henle ( ). Jacques Diezi’s work used the exposed renal papilla

in which collecting ducts, segments and blood vessels could be identified and punctured.

He confirmed variable potassium reabsorption and found that sodium depletion

stimulates potassium retrieval, an observation with implications for the stimulation of

aldosterone release in volume depletion ( ). I had also collaborated with Jacques on

problems related to the distribution of sodium transport along the nephron following

unilateral nephrectomy and volume expansion ( ). He and his wife Francoise were

wonderful hosts to me and my family when I later spent a sabbatical year in Lausanne.

Although the collecting duct had been defined earlier as an important site of

action of mineralocorticoids ( ), the cell mechanisms, especially apical and basolateral

transport modes of sodium and potassium, had been incompletely defined when several

27

members of our laboratory began their explorations. Bruce Koeppen used a mammalian

isolated cortical collecting duct preparation in which he employed microelectrode

techniques to define the apical sodium and potassium permeabilities and their modulation

by mineralocorticoids ( ). An interesting observation was his finding that passive

potassium transport across the basolateral membrane of principal tubule cells underwent

drastic changes following mineralocorticoid treatment. Whereas potassium ions, once

taken up into cells by Na-K-ATPase activity, normally extensively recycle across the

peritubular membrane, they can reverse transport direction and enter cells from the

blood. Such potassium uptake is driven by the sharp increase in cell negativity that

follows mineralocorticoid stimulation of electrogenic Na-K ATPase ( ).

Some of our studies continued to explore problems related to electrolyte transport

across the cortical collecting tubule. Mineralocorticoid-related modulation of apical and

basolateral transport components of sodium and potassium was extensively explored by

Steve Sansom and Shigeaki Muto. They succeeded in defining the electrical

characteristics of the two cell types, principal and intercalated, and explored their

response to mineralocorticoids, adrenalectomy and changes in potassium intake. What

became apparent from these studies was that all manipulations that effect changes in

potassium and sodium transport do so by acting simultaneously on apical and basolateral

transport ( ). Thus, mineralocorticoids, or high potassium intake, activate not only the

potassium uptake basolaterally via Na-K-ATPase stimulation, and an increase in

potassium permeability, but they also enhance apical sodium and potassium

permeability.. Importantly, such treatment also leads to marked morphological changes

such as amplification of the basolateral membrane infoldings and increased expression of

Na-K-ATPase transporters. These morphological studies were carried out in

collaboration with Bruce Stanton, Jim Wade, Michael Kashgarian and Dan Biemesderfer

( ). Whereas treatment with a high potassium diet affected mainly the basolateral

membrane of principal cells, intercalated cell morphology remained unchanged. In sharp

contrast, the apical membrane area of intercalated cells greatly increased during

treatment with a low potassium diet, consistent with the demonstrated enhancement of

apical potassium-hydrogen ion exchange ( ).

28

I have continued a very productive collaboration with Shige Muto since the time

of our initial cooperation in the mid eighties until the present. He is a superb investigator

with great experimental skills and persistence and an expert in the art of working with

isolated cortical collecting tubules. Despite very active clinical involvement, he

continues doing experiments. We have both been intrigued by the interaction between

basolateral transport stimulation and apical permeability changes of sodium and

potassium. Thus, an acute activation of basolateral Na-K-ATPase by a rapid increase of

extracellular potassium leads to a sharp rise in both sodium and potassium conductances (

). The mechanism of such coordinated “cross-talk” between basolateral and apical

membrane transporters is incompletely understood. It may be related to cell calcium

changes that follow cell sodium alterations after pump stimulation ( ). The recent

finding that Na-K-ATPase transporters can interact directly with cell messengers may

be relevant ( ). Another problem we addressed had to do with the puzzling

observation that amiloride, a potent inhibitor of apical sodium channels, does diminish

but not completely block potassium secretion. Continued secretion can be shown to

depend on basolateral sodium-hydrogen exchange that acts as a supplementary source of

sodium for basolateral Na-K-ATPase activity ( ). Such basolateral entry of sodium may

attain significance whenever the supply of sodium from the lumen is severely curtailed

following diminished sodium supply from proximal tubule segments.

I have also greatly profited from my collaboration with Franz Beck from the

Department of Physiology of the University of Munich. He had previously developed

electron probe techniques for measuring cell electrolytes in renal tissue, and we joined

forces to study the uptake of rubidium, widely used as a marker for potassium, into

principal and intercalated cells to gain insight into their role in potassium secretion. We

observed that the basolateral uptake of rubidium following a bolus injection was much

higher in principal cells, consistent with high potassium uptake via Na-K-ATPase.

Moreover, enhanced luminal sodium supply greatly stimulated rubidium uptake into

principal calls but failed to do so in intercalated cells. These in vivo findings fully

support the indirectly derived conclusion that principal cells are the main site of

potassium secretion, and they are also in accord with the much higher Na-K-ATPase

levels detected in basolateral membranes of principal cells ( ).

29

Several colleagues, Takahiro Kubota and Y. Matsumura, with their respective

collaborators, took advantage of the large size of proximal tubule cell in the Necturus

kidney to study cell potentials and potassium activities during changes in electrolyte

environment and acid-base manipulations. Besides characterizing the basolateral ion

permeabilities and Na-K-ATPase contribution to cell potentials and potassium activities,

they provided strong evidence for an important role of bicarbonate ions in maintaining

cell polarization and high cell potassium. This finding is interesting because it may

explain their observation that respiratory acidosis, owing to its elevated bicarbonate

levels, does not lower cell potentials and cell potassium activities ( ). Their observation

also mimics clinical observations that respiratory acid-base disturbances are much less

effective to promote loss of potassium from cells ( ). Our studies in amphibian tubules

were further extended to mammalian proximal tubules by Bruce Biagi, Ducan

Cemerikic and Chris Wilcox and their respective colleagues ( ). They succeeded in

the difficult task of obtaining stable cell potentials that allowed them to characterize the

role of potassium ions in the generation of cell polarity, and to define several factors ,

especially acid-base disturbances, modulating electrical properties of proximal tubule

cells.

Two important events, starting in the mid eighties, greatly affected the activities

of our laboratory. The first was the introduction of patch-clamp techniques which enabled

us to identify and explore potassium channels along the nephron. The second was the

close cooperation with Steve Hebert’s laboratory that enabled us to participate in the

effort to learn more about the structure and function of cloned renal potassium channels,

and compare them with the channels that we had identified in renal tubules. Our efforts

started with the arrival of Malcolm Hunter from England. Malcolm was a well-trained

electrophysiologist, displayed an excellent sense of humor, and was blessed with energy,

skill and, most of all, persistence. We used the “split-open” tubule preparation, initially

obtained from rabbit kidneys. Malcolm was able to obtain inside-out patches, but rarely.

Weeks went by, a rare recording now and then, despair was mixed with gentle

encouragement on my part (we always have great courage for the misery of others ( )),

but Malcolm succeeded. As it turned out, the potassium channel from apical membrane

patches of the cortical collecting tubule was a typical maxi K channel, with very low

30

open-probability that could, however, not account for much potassium secretion ( ).

Later it turned out that this maxi-K channel displays stretch-sensitivity and does play a

role in potassium secretion, especially at high flow rates and following adaptation to high

potassium intake ( ). The low open probability in the split-open preparation that we and

others observed can best be explained by the fact that no tension is applied to the channel,

whereas in vivo physiological stretch by provide an appropriate stimulus. It should be

mentioned that K potassium channels are studied much more easily in the rat collecting

tubule, and that such channels are present in both principal and intercalated cells.

Our studies on renal potassium channels flourished with the arrival of Wenhui

Wang in 1987, one year after Malcolm Hunter left.. Wang (as he wanted to be called

because he thought we could not quite properly pronounce his first name) was trained in

Peter Deetjen’s Physiology Department in Innsbruck, where he collaborated with Hans

Oberleithner. He stayed in our laboratory about six years and established himself as a

superb investigator. He now heads an independent and productive laboratory at Valhalla,

and we have for many years now continued our research alliance. A second potassium

channel in the apical membrane of principal cells, originally identified by Larry

Palmer’s laboratory ( ), with lower single channel conductance but much higher open

probability, attracted our attention and became the topic of our research for many years.

Several investigators, among them Ming Lu, Albert Schwab, Carmel Mc Nicholas,

Gordon MacGregor, Jens Leipziger, Stanley White, Katsumara Kawahara, Antonio

Cassola, Manabu Kubokawa and Adam Zweifach contributed to the characterization of

potassium channels in principal and intercalated tubule cells. Initially we were also

assisted by Robert Henderson, and we continuously profited from the closeness of

Emile Boulpaep and his group that included, over the years, Alan Siegel, Henry Sackin

and Christoph Korbmacher. Their efforts played a major role in characterizing the

properties and regulation of potassium channels in the apical membrane of thick

ascending limbs of Henle’s loop where they control potassium recycling and thus

participate in regulating sodium chloride reabsorption. In addition, similar potassium

channels also are present in cortical collecting tubules, the main site of controlled

potassium secretion. What emerged was that two potassium channels play a key role in

potassium secretion: a low-conductance pH-regulated channel with high open probability

31

that is active under control conditions, and a maxi- potassium channel that mediates

secretion at high flow rates. Both channels are stimulated by high potassium intake and

account for the observation that potassium secretion may continue, albeit at a reduced

rate, whenever the function of one secretory channel is compromised. The fact that at

least two potassium channels participate in the critical process of potassium secretion is

an example of redundancy of transport operations in the kidney that is often encountered.

And can be evaluated by genetic manipulation of specific transport processes.

Steve Hebert cloned a renal potassium channel (ROMK) in 1993, and we soon

discovered that it shared many properties with the low-conductance channel that we

had localized to principal tubule cells ( ). We discussed the possibility of working

together, and from the mid-1990s joined forces until his sudden death in April 2008.

Our developing collaboration was a most satisfying event that resulted in an even closer

scientific companionship when he was appointed chairman of our department at Yale.

Steve brought to our joint studies a fresh point of view, and his enormous energy and

drive, his knowledge of pathophysiology, together with genetic insights, made our

collaboration most satisfying. Our team effort included structure-function studies of

ROMK, locating several phosphorylation sites not only involved in the channels

surface targeting and pH-sensitivity, but also those mediating interaction with CFTR

and a variety of cell messengers ( ). Steve’s laboratory also developed a ROMK

knockout mouse which developed an electrolyte disorder similar to Bartter’s syndrome,

giving us the opportunity to pinpoint the functional lesions along the nephron that

resulted and the compensatory mechanisms that result from the absence of ROMK

channels ( ). I will always look back at my period of cooperation with Steve Hebert

with great gratitude. With him the nephrological community lost a superb investigator,

and I lost a good and loyal friend.

I retired on June 30, 2004 but have remained fairly active. I am still interested in

the cell mechanisms by which renal tubules adapt to changes in potassium balance, and

I: have been associated with some of activities in Dr. W. Wang ‘s laboratory ( ). I have

also followed with great interest the activities of Rick Lifton’s group, especially their

studies on the role of WNK kinases in regulating the coupling between sodium

reabsorption and potassium secretion ( ).

32