The Multiplication Principle As The Basis For.28

MILESTONES IN NEPHROLOGY J Am Soc Nephrol 12: 1566–1586, 2001
Mark A. Knepper, Feature Editor
B.
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with comments by
BART HARGITAY AND S. RANDALL THOMAS
AUTHOR COMMENTARY
Bart Hargitay
White Plains, New York

hargitayb@aol.com
eing the last survivora of the original triumvirate that

B launched the hairpin countercurrent theory of urine
concentration, I thought it might be seemly for me to remi-
nisce about how it came about 50 years ago.
In 1946, I graduated in chemistry at the University in
Budapest (Hungary) and became the lucky recipient of a fel-
lowship offered by the University of Basel (Switzerland). A
few months after my arrival to Switzerland, the Iron Curtain
closed and I decided not to return to Hungary.
On arriving in Basel, I contacted Professor Werner Kuhn,
an outstanding physical chemist, and asked him to accept
me as his graduate student to work on a thesis. Kuhn’s name
remains forever linked to various fields of physical chem-
istry (to name some: the absolute configuration of d and l
amino acids, the random walk theory of rubber elasticity of
polymers, isotope separation by centrifuges and distillation,
etc.). He was a brilliant theoretical physical chemist of the
old mold. He refused to use virgin paper for his calculations
and he literally did all of his groundbreaking work on the
back of used envelopes. While I was in his institute, he did
essentially no experimental work himself, relying on his
students whom he guided constantly with wonderful advice.
The main exchange of ideas took place in a coffeehouse,
where we had a reserved table for one afternoon a week. The
number of graduate students around the table varied
between 4 and 10, and the table in front of us was always
covered with scrap paper on which real science was scrib-
bled. Kuhn was a quiet man and never showed impatience
with collaborators who may not have had anything to report
for many weeks. One could not help but learn a lot in this
coffeehouse, because many different problems were dis-
cussed on every occasion (statistics of polymer configura-
tions, dichroism produced by irradiation of dyes immobi-
a Werner Kuhn died in 1963, Heinrich Wirz in 1993.
Milestones in Nephrology 1567
Werner Kuhn, 1950s B. Hargitay, 1951

lized in polymer matrices, mechanochemistry and muscular
contraction, etc.).
Before settling in Basel, Kuhn did some very fundamen-
tal work in Kiel, separating isotopes in a centrifuge. This
caused him to be fascinated with the effect of countercur-
rents in multiplying a very small single effect to significant
separations. In Basel, he separated isotopes of chlorine using
chloroform in a distillation column. A few years before my
arrival, he enriched sugar in water using an elaborate coun-
tercurrent system using two kinds of semipermeable mem-
branes and phenol as an auxiliary liquid, but no other driving
force. This work with Ryffel suggested the possibility that
some similar mechanism may play a role in the kidney.
My assignment was to find a way to demonstrate in a lab-
oratory setup that membrane action can lead to significant
solute concentration using not more than one membrane and
no auxiliary compounds. I had complete freedom in select-
ing membranes, solutes, and driving forces. It was the prin-
ciple that had to be proved.
My first step was to learn something about kidneys, and I
was lucky to have found Dr. Heini Wirz in the Institute for
Physiology, who was very much interested in helping me to
get the principle proved. He was fascinated and also baffled
by the findings of Walker et al., who established by microp-
uncture that the urine concentrations in the proximal and dis-
tal tubuli of the nephron in rodents essentially were identical
(or even reversed from expectation). He was eager to verify
these results himself and was ready to perform the microp-
unctures on animals. It was known that a correlation existed
between the dryness of the environment in which animals
lived and the length of the papilla in their kidneys. For rea-
sons to become clear as experiments progressed, he later
moved from the rat to the Syrian hamster, a rodent that lives
in the desert. In this animal, the single papilla actually pro-
trudes far into the renal pelvis. I was fascinated by the tech-
nique of withdrawing liquid samples from the tubuli accessi-
ble from the surface of the kidney. Micromanipulators and
glass capillaries were used under the microscope.
The analysis of the samples as small as 107 ml was a
thorny problem. I decided to determine the osmotic pressure
rather than the concentration of any particular chemical com-
pound. Making use of the fact that ice is birefringent, its melt-
ing can be detected under the polarizing microscope in the
very glass capillary used for micropuncture. The melting
point depression is a measure of the osmotic pressure of the
solution. Thus, Wirz was satisfied to verify Walker’s findings.
About a year after embarking on my work, I became very
discouraged by a paper written by Kuhn, deriving the theo-
retical limits of permeability for semipermeable membranes.
1568 Journal of the American Society of Nephrology
The results of his speculation convinced me that it would be

impossible to prove the countercurrent hypothesis in the lab-
oratory using hydrostatic pressure as a driving force. (The
reverse osmosis membranes now used in sea water desalina-
tion did not yet exist. In these, the necessary mechanical
strength is imparted by a tough spongy layer that supports an
extremely thin semipermeable membrane.) Therefore, I spent
some time collaborating on other problems, e.g., mechano-
chemistry and learning various new techniques.

Dr. Aaron Katchalsky, on sabbatical from Rehovoth,
came to spend a few months with us in Basel. He brought
with him his interest in and partial solutions of conforma-
tion of polyelectrolytes in solution. He was a brilliant and

extremely enthusiastic scientist. We all learned a lot from
him. After he returned to Israel,b I continued to experiment
with polyelectrolytes and I believe that I was the first to
develop a “working” mechanochemical “muscle” in the lab-
oratory, which we then published jointly with Katchalsky (1).
My experience with polyelectrolytes led me to the solution of
my primary problem: the demonstration of the countercur-
rent principle in the laboratory. Kuhn’s calculations concern-
ing the permeability of semipermeable membranes were
based on the premise that the pore diameter of the membrane
must be smaller than the solute but large enough for solvent
molecules to permeate. My idea then was to outfox this rea-
soning by making use of the Donnan equilibrium of poly-
electrolytes. The polymer molecule is quite large compared
with water and therefore is excluded from a swollen mem-
brane. The small counterions are prevented from permeating
the membrane by the electrostatic attraction exerted by the
excluded polyelectrolyte molecules. However, these counte-
rions are osmotically active and therefore contribute to the
osmotic pressure of the polyelectrolyte solution.
On the basis of these thoughts, I made the following
choices: membrane, regenerated cellulose (cellophane);
polyelectrolyte, sodium polyacrylate; solvent, water. The
driving force to move the solvent through the membrane was
hydrostatic pressure. I constructed a flow-through osmome-
ter to monitor the osmotic pressure at the turning point of
the “hairpin countercurrent” (HPC) system. (I am indebted
to my friend and colleague Alex Silberberg for coining the
term “hairpin” for us.) My aim was simply to demonstrate
that the osmotic pressure of the flowing solution can be
raised above the generating hydrostatic pressure. It worked!
Encouraged by the successful demonstration that the HPC
system could be a mechanism by which the kidney generates
considerable osmotic pressures from body fluids, Kuhn pro-
cured for me a 2-year fellowship from the Fritz Hoffmann
LaRoche Foundation for Furthering Interdisciplinary
Research. Wirz and I were to demonstrate that our findings
in the rodent kidney were compatible with the HPC hypoth-
esis. We developed a method of sectioning frozen rat kidneys
perpendicular to the papilla axis to determine the freezing
point depression in the various tubuli, Henle loop, and col-
lecting ducts, all at the same time. This work had to be done
below 20°C (4°F) in the cold room of the Bürgerspital in
Basel. I still remember the baffled faces of people in the hos-
pital when they saw us emerging in fur coats and gloves
every 20 minutes in July! Again, it worked! We showed that
while the osmotic pressure in the cortex essentially is isoton-
b Aaron Katchalsky was killed by terrorists in 1972.

ic with blood, as we descended toward the tip of the papilla,

the osmotic pressure increased in all ducts in parallel. This is
what our HPC theory had predicted.
In May 1951, I had the privilege of presenting our work
to the Bunsen Gesellschaft in Göttingen (Germany) (2); a
few weeks later, Wirz presented it in Kopenhagen at the
International Conference for Physiology (3). The reaction at
the two meetings was very different. The physical chemists
in Göttingen were enthusiastic (with the exception of Prof.

Clusius of Zurich, who thought that we were competing with
his students who were trying to separate various salts based
on diffusion rates through membranes). In contrast, the
physiologists’ reaction was guarded. They were not ready to

embrace the novel idea (kind of, “cute, but it can’t be so...”).
This attitude changed a few years later!
All of our groundbreaking papers then were published in
German. For me, it was a particular honor that Kuhn put my
name first. In addition to the language barrier, the reason for
the delay in spreading the concept is that my work constitut-
ed a thesis in physical chemistry and therefore was pub-
lished in a journal read by physical chemists (2). The bio-
logical significance of the principle was soon recognized by
Scholander (4), who applied it, among others, to the swim
bladder of deep sea fish, which are able to secrete nitrogen
gas of several hundred atmospheres of pressure. As far as
the application to the human kidney is concerned, it took the
extensive and excellent work of Carl W. Gottshalk to bring
it to the surface (5).
After I left Basel in 1952, Wirz continued work related to
the mechanism proposed by us, resulting in more than a
dozen publications, until about 1960 when he joined indus-
try. Kuhn extended the theory to the “salt kidney”(6) in
which the driving force is salt transport rather than water
transport, which I have dealt with in my thesis.
My motivation for writing this rather personal account of
times long past is to pay homage to the wonderful men who
inspired me, from whom I learned a lot, and who, apart from
being great scientists, also were warm and principled human
beings. I am truly a lucky man to have been associated with
such exceptional people.
References
1. Kuhn W, Hargitay B, Katchalsky A, Eisenberg H: Reversible dilation and con-
traction by changing the state of ionization in high-polymer acid networks.
Nature 165: 515, 1950
2. Hargitay B, Kuhn W: Das Multiplikationsprinzip als Grundlage der Harnkonzen-
trierung in der Niere. Z Electrochem Angew Phys Chem 55: 539–558, 1951
3. Wirz H, Hargitay B, Kuhn W: Lokalisation des Konzentreirungsprozesses in der
Niere durch direkte Kryoscopie. Helv Physiol Pharmacol Acta 9: 196–207, 1951
4. Scholander PF: The woderful net. Sci Am 196: 97–107, 1957
5. Gottschalk CW: Numerous publications starting with Science 128: 594, 1958
6. Kuhn W, Ramel A: Activer Salztransport als möglicher (und wahrscheinlicher)
Einzeleffekt bei der Harnkonzentrierung in der Niere. Helv Chim Acta 92:
628–659, 1959
GUEST COMMENTARY
S. Randall Thomas
INSERM U.467,
Necker Faculty of Medicine
Paris, France
his groundbreaking paper by Bart Hargitay and Werner

T Kuhn developed a new paradigm for the urine concen-
1570 Journal of the American Society of Nephrology
tration mechanism. Because of its appearance in German

soon after the second World War and in a journal of electro-
chemistry, the new hairpin countercurrent hypothesis (HCC)
did not immediately come to the attention of the kidney
community. It went counter to accepted ideas of the time but
was eventually verified thanks to revival of the in vivo
micropuncture technique, most notably at first by Wirz and
colleagues and then definitively in 1959 by Gottschalk and
Mylle (1), who were able to show in several species that

fluid in the long loops is hyperosmotic at the tip of the papil-
la, just as predicted by Hargitay and Kuhn, proving that fluid
first is concentrated as it descends toward the papilla, then
diluted as it returns toward the cortex, and finally becomes

concentrated urine in the collecting ducts as they traverse
the medullary gradient. (For references and a fuller account,
see reference 2.)
Before this work, it was believed that urine was concen-
trated progressively along the nephron by active water reab-
sorption. Although the principle of concentration in coun-
terflowing solutions thanks to permselective membranes
had been introduced earlier, explored mathematically, and
even demonstrated in experimental prototypes, the paper
reprinted here in translation was the first explicit application
of the idea to the architecture of the mammalian medulla. In
this paper, the authors developed the hairpin countercurrent
(HCC) principle mathematically, keeping to general princi-
ples (an arbitrary single effect) but exploring configurations
that seemed directly applicable to the renal medulla. They
also reported here the main result from their companion
cryoscopic study with Wirz characterizing the corticopapil-
lary osmotic gradient in the rat, suggesting that osmolality
is similar in all structures at a given level, and they described
results obtained with the device that they constructed to
demonstrate the countercurrent multiplication principle.
It seemed essential to make this paper available in
English, not only because it is cited in almost every article on
the concentration mechanism but also and mainly because,
both qualitatively and quantitatively, it remains one of the
best step-by-step introductions to the operation of a counter-
current multiplication system. Although the amount of math
may seem daunting, the reader should not be discouraged.
The authors developed their argument in small, logical steps
and gave clear verbal explanations leading to each derivation,
starting with simple ultrafiltration and then passing progres-
sively to a circulating loop, a hairpin loop with no fluid with-
drawal, and finally to a three-tube hairpin loop system with
withdrawal of concentrated solution from the tip. It is this
last arrangement that they applied to Henle’s loop and the
collecting duct in the kidney medulla. At each step in the pro-
gression, they gave helpful numerical examples illustrating
the limits to which such a system can be pushed.
Hargitay and Kuhn left open the question of the exact
nature of the single effect, but they made significant sug-
gestions. They developed their argument using hydrostatic
pressure because that is what they used in their experimen-
tal device demonstrating the principle, but they explicitly
ruled out hydrostatic pressure as the single effect operating
in the medulla. Carefully avoiding any categorical predic-
tion, they nonetheless suggested three possible alternatives:
an electro-osmotic effect, water transport from descending
to ascending limbs of Henle, or salt transport from ascend-
ing to descending limbs (see footnote 4 of their paper). Later
work unequivocally established active salt transport from

the thick ascending limbs as the effective driving force in
the outer medulla, but we are still searching for the answer
in the passive inner medulla, thus amply justifying their pru-
dence on this score. The current “passive hypothesis,” (3,4)
based on an external osmole role for urea dumped into the
papillary interstitium from the terminal collecting ducts, is
known to be incompatible with measured tubule permeabili-
ties; in the absence of an alternative, however, it remains the

default explanation. We recently suggested (5) that metabol-
ic production of net osmoles, such as lactate production by
anaerobic glycolysis in the hypoxic inner medulla, could
contribute to the single effect, but the relative importance of

this contribution in different species (herbivores versus car-
nivores versus omnivores) will be unraveled only by contin-
uing the well-established give and take between modeling
and experimental studies that so uniquely characterizes the
history of the “countercurrent mechanism” and that is so well
demonstrated in this paper that really started it all in 1951.
In closing, I must mention that this translation was an
unconventional multilingual collaboration. Tanja Torossi, a
cheerful German/Italian from Berlin, was spending 2 years
in our laboratory and agreed to help me “iron out the
details” in our spare time. Little did we realize that it would
take almost 2 years. She rendered the German into French,
which I then used along with the original and my own rough
translation to make the English translation. It was then
thanks to my friend Dieter Walz in Basel that we discovered
that Bart Hargitay is alive and well in White Plains, the last
member of the Wirz, Hargitay, & Kuhn triumvirate. Dieter,
whose office at the Biozentrum in Basel is only a few steps
from what was Kuhn’s laboratory, wanted to do his thesis
with Werner Kuhn but Kuhn refused because of his failing
health. After kindly reading and checking our translation,
Dieter was able to contact Wirz’s son, Prof. Hans-Jakob Wirz,
who gave us Bart Hargitay’s address. It has been a real pleas-
ure to correspond with Dr. Hargitay during the final stages of
the translation and preparation of this paper for publication.
References
1. Gottschalk CW, Mylle M: Micropuncture study of the mammalian urinary con-
centrating mechanism: Evidence for the countercurrent hypothesis. Am J Physiol
196: 927–936, 1959
2. Valtin H: Carl W. Gottschalk’s contributions to elucidating the urinary concen-
trating mechanism. J Am Soc Nephrol 10: 620–627, 1999
3. Stephenson JL: Concentration of urine in a central core model of the renal coun-
terflow system. Kidney Int 2: 85–94, 1972
4. Kokko JP, Rector FC Jr: Countercurrent multiplication system without active
transport in inner medulla. Kidney Int 2: 214–223, 1972
5. Thomas SR: Inner medullary lactate production and accumulation: A vasa recta
model. Am J Physiol Renal Physiol 279: F468–F481, 2000
are fulf illed if there is no concentration g radient
Journal of the American Society of Nephrology
1572
1573
Milestones in Nephrology
1574
2Translators’ note: At several points, the authors stated dimensions simply

as “cgs”. Conversion and correction of these to current International Units
would involve numerous changes in the text and would change the numeri-
cal values, so we have left these as in the original.
1576
1577
1578
1579
1580
Figure 12: Time course of the measured osmotic pressure

and conductivity in a Na-polyacrylate solution at the ver-
tex of the hairpin countercurrent system (compare with ,
experiment of Table 2).
1582
1583
1584
Summary
It is demonstrated that the principle of multiplication of consecu-
tive separation steps permits concentration of dilute solutions to
high osmotic pressure, making use of a small active pressure in a

hairpin countercurrent system.
In particular, model experiments are designed in which the sep-
aration step (“single effect”) consists of removing solvent by forc-
ing it through a semipermeable membrane by a small hydrostatic
pressure, until the osmotic pressure difference so generated coun-

terbalances the driving hydrostatic pressure. We have experimen-
tally demonstrated that the small concentration increase can in
fact be multiplied in a hairpin countercurrent system (Figs. 3 and
12). In these experiments, to concentrate a sodium polyacrylate
solution, a water permeable cellophane membrane was used.
A quantitative treatment is developed for this system. This
treatment is also applicable to any hairpin countercurrent system
in which the hydrostatic pressure is replaced by another driving
force, e.g., an electroosmotic effect.
The treatment is extended to systems in which some concen-
trate is withdrawn at the vertex of the hairpin, i.e., the location
where the concentration is highest. In this case, a somewhat dilut-
ed solution returns through the second chamber of the countercur-
rent system. Significant relations are derived which connect the
relative amount of the concentrate withdrawn, , the degree of
concentration, , and the generating pressure p. These relations,
when applied to the kidney, allow us to draw conclusions concern-
ing the magnitude of the single effect operating in Henle’s loop.
Because the operation of a hairpin countercurrent system cre-
ates from a solution a certain volume of concentrate and a related
volume of diluate, it is possible to calculate the reversible work
necessary to achieve this and to compare it with the actual
mechanical energy (pressure volume) expended in the process.
The ratio of the two is the efficiency, , of the system. The calcu-
lation for the system of Fig. 7b yields a maximal efficiency of
86.5% for the relevant case of small pressures. Very intriguing is
the fact that this maximal value is reached at a concentrating fac-
tor of about 3. The efficiency falls off considerably at both higher
and lower degrees of concentration. However, it is remarkable that
at = 100 (a hundredfold concentration), an efficiency of 14% is
still possible. (By the way, the efficiency at higher concentrations
( > 3.2) increases as the pressure is lowered and withdrawal is
increased up to the theoretically permissible limit.)
Very striking morphological similarities exist between the sys-
tem of Fig. 6b and that which presents itself in the kidney, name-
ly Henle’s loop and the associated collecting duct. This morpho-
logical similarity and the analogy of flow patterns strongly suggest
the existence of a mechanism of multiplication of a single effect
in the kidney. While the functional relation of Henle’s loop to the
concentration of urine has not been known until now, the correla-
tion between its degree of development and the potential of urine
concentration was noticed long ago. If we consider Henle’s loop
as a biological manifestation of the hairpin countercurrent system,
many seemingly contradictory experimental findings become
plausible and consistent. The idea that the kidney represents a
HCC is, to our knowledge, in no contradiction with any known
facts in physiology. Using a novel micromethod to determine the
concentration in the rat kidney, we confirmed the predicted topo-
graphical conditions, i.e., that the highest concentration occurs
solely in the tip of the papilla, which of course corresponds to the
vertex of the theoretical model (Fig. 6b).
1586

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MILESTONES IN NEPHROLOGY J Am Soc Nephrol 12: 1566–1586, 2001

Mark A. Knepper, Feature Editor

White Plains, New York

eing the last survivora of the original triumvirate that

Werner Kuhn, 1950s B. Hargitay, 1951

The results of his speculation convinced me that it would be

chemistry and learning various new techniques.

tion of polyelectrolytes in solution. He was a brilliant and

b Aaron Katchalsky was killed by terrorists in 1972.

ic with blood, as we descended toward the tip of the papilla,

in Göttingen were enthusiastic (with the exception of Prof.

physiologists’ reaction was guarded. They were not ready to

his groundbreaking paper by Bart Hargitay and Werner

tration mechanism. Because of its appearance in German

Mylle (1), who were able to show in several species that

diluted as it returns toward the cortex, and finally becomes

work unequivocally established active salt transport from

ties; in the absence of an alternative, however, it remains the

contribute to the single effect, but the relative importance of

2Translators’ note: At several points, the authors stated dimensions simply

Figure 12: Time course of the measured osmotic pressure

high osmotic pressure, making use of a small active pressure in a

pressure, until the osmotic pressure difference so generated coun-

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