Stop-Flow Studies on Ion
and Water Reabsorption in the Dog
By RICHARD
L.
MALVIN, PH.D.,
AND
WALTER S. WILDE, PH.D.
The stop-flow method has been used to study various aspects of renal tubular transport
mechanisms. According to this method, aldosterone promotes the tubular reabsorption
of sodium in the distal tubule. The method also indicates that the secretion of potassium,
hydrogen and ammonium occurs in a very distal portion of the nephron. The substitution
of anions such as ferrocyanide, sulfate and phosphate for chloride apparently increases
the area of the nephron involved in the secretion of hydrogen and potassium. The stopflow experiments support other evidence that the rate of flow of urine through the loops
of Henle affects the countercurrent multiplier system which, in turn, determines the
ability of the kidneys to elaborate a concentrated urine.
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IT HAS been known for many years that
the kidney reabsorbs a large part of the
salts and water filtered at the glomerulus.
However, only in recent years have some of
the mechanisms controlling secretion and reabsorption been elucidated. The micropuneture technic as developed by Richards' has
been extremely useful in this respect, since it
allows direct analysis of tubular urine. The
stop-flow method, although not as precise as
mieropuncture, has also lent itself well to
renal transport studies.
For the past 2 years, the authors, in collaboration with Drs. Lawrence Sullivan, Arthur Vander and Peter Abbrecht, have used
the stop-flow technic extensively as a tool for
investigating the transport of salt and water
in the nephron. Because of the limitations of
time, we have arbitrarily selected 3 areas of
our research for discussion at this symposium:
(1) the effect of aldosterone on the transport
of sodium; (2) the effect of large impermeant
anions on the transport of potassium; (3) the
effect of osmotic dinresis on the renal concentrating mechanism.
Aldosterons
Recently,2 using the stop-flow technic, we
demonstrated a distal site of action of aldos-
terone. The dashed curve in figure 1 illustrates a typical pattern of the concentration
of sodium from a stop-flow experiment in a
normal dog. The sodium concentration of free
flow urine collected immediately before occlusion was 63 mM per liter. As distal fluid
entered the collector, the concentration of
sodium fell to a low of 5 mM per liter. As
fluid from the loops and proximal tubules
entered the collector, the concentrations of
sodium rose to reach a plateau at the original
free flow concentration. The peak concentration of para-aminohippurate (PAH), not
shown in this figure, indicated the best stopflow proximal sample to be at 10 ml.
In contrast to the normal dog, the lowest
concentration achieved by the distal tubule
of the adrenalectomized dog was only 24 mM
per liter. The distal tubule was apparently
unable to reduce the concentration of sodium
to the minimum value achieved in normal
dogs during stop-flow.
Occasionally, normally low concentrations
of sodium were obtained from the distal tubular urine of the adrenalectomized animal. This
seemed to occur only when the concentration
of sodium in plasma was exceedingly low.
The following series of experiments was designed, therefore, to study the relationship
between the concentrations of sodium in the
plasma and in the distal tubular urine. After
the collection of normal stop-flow urine, the
concentration of sodium in plasma was raised
rapidlv by the intravenous infusion of 2 to 3
From the Department of Physiology, School of
Medicine, University of Michigan, Ann Arbor, Mich.
Supported by Amnerican Heart Association, 56G198;
Life Insurance Medical Research Fund, G-57-46; National Institute of Arthritis and Metabolic Diseases,
U. S. Public Health Service, A-1740 Physiology.
902
Circulation, Volume XXI, May 1960
STOP-FLOW STUDIES ON ION AND WATER RECABSORPTION
PAH
MAXIMUA
TO
35-
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30-
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25
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ACCUMULATED URINE VOLUME IN ml
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16
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Figure 1
Comparison of the distal tubular concentrations
of sodium during stop-flow in a normal and in
an adrenalectomized dog. (Republished by permission of the Proceedings of the Society for Experimental Biology and Medicine.2)
Gm. of sodium chloride in 50 ml. of water,
over a period of 5 minutes. After 10 more
minutes, the occlusion was again performed,
and another collection was made.
Figure 2 demonstrates the relationship between the concentration of sodium in the
plasma and in the distal tubular urine during
ureteral occlusion. It can be seen that during
stop-flow, the distal tubule of the normal animal was capable of lowering the concentration
of sodium almost to zero. Except possibly at
extremely high values, the minimum concentration of sodium appears to be independent
of the concentration of sodium in the plasma.
This independence suggests that over the
physiologic range, the maximal concentration
gradient for sodium which the normal animal
can develop and maintain between the plasma
and the distal tubular urine has not been
reached.
The effects of adrenalectomy upon this pattern are striking. Even at very low plasma
coneentrations of sodium, the distal concentrations during ureteral occlusion were abnormally high. During occlusion, there was a
direct relationship between the concentrations
of sodium in the plasmna and in the distal
tubule. This indicates that adrenalectomy has
Circulation, Volume XXIJ May 1960
-
n_
.
o1
*
120
PLASMA
-
e
lio
140
SODIUM
mM
iio
lio
iro
-
Figure 2
Relationship between the concentrations of sodium
in the plasma and in the distal tubular urine
during stop-flow. The vertical axis represents the
concentration of sodium in the particular urine
sample which is acted upon by the maximal number of distal tubules during ureteral occlusion.
reduced the maximal concentration gradient
for sodium which can be maintained across
the distal tubular cells. As the concentration
of sodium in the plasma rises, the minimal
concentration of sodium also rises. Even if
the duration of occlusion is prolonged from 4
to 6 minutes, the peak concentration of sodium
in the distal tubular urine during stop-flow
is not changed, indicating that maximal reabsorption of sodium by the distal tubules occurs within the first 4 minutes of ureteral
occlusion.
The effects of aldosterone on the adrenalectomized dogs are demonstrated in figure 3.
The administration of aldosterone restored the
ability of the distal tubule to lower the concentration of sodium in the distal tubule even
in the presence of an elevated concentration
of sodium in the plasma.
Mode of Action of Aldosterone
Clearance methods indicate that aldosterone
increases the tubular reabsorption of sodium.
This action is manifested in stop-flow analysis
by the ability of aldosterone to increase the
maximal concentration gradient for sodium
which can be developed between the plasma
and the distal tubular urine. Since the mini-
MALVIN, WILDE
904
maximal transport velocity by increasing the
number of carrier sites available for the reabsorption of sodium.
ADRENALECTOMIZED DC)GS
*
O
40E
o
(I)
BEFORE ALDOSTER(ONE
AFTER ALDOSTER ONE
30-
Potassium
20-
-I
en
CX
10r'.
&--o
1
100
.
.
110
120
130
140
PLASMA SODIUM mM/I
150
160
Figure 3
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Effect of aldosterone on distal tubular concentration of sodium.
mal coneentrationi of sodium attain ed in the
distal tubule durinig ureteral ocelus ion is independent of the duration of occlu Sion, this
coneentration must be a steady sta te value;
i.e., the concentration at which sodii im movemnent out of the lumen is equal tI o sodiumi
movemeent inward. Aldosterone co)uld act,
therefore, in 1 of 2 different ways: it could
aetivate carrier systems responsible f or sodium
transport outward or it could deer ease passive back-diffusion of sodium from ir-iterstitial
fluid into distal tubular lumen.
A rough estimate of the rate of b,ack-diffusion of sodium has beenl made in ou r laboratory, using the isotope Na24. The ureteral
occlusion stops filtration so that gi lomerular
substances such as inulin will olot enter the
concentration pattern except as nievv filtrate.
Na24 injected ilitraveinously after t-he occlusion, by crossing the tubular epitheliiLim tranismurally, enters the urinary pattern ahead of
inulin. Precise delinieationi of rates of movemient ilnto the stop-flow pattern is coinplicated
by the colntiniuing deeliine of the precursor
Na24 in the blood plasma during th(e short '2minute period allowed for the Na24 to enter
the tubule lunmeni. The shape of the stop-flow
curves for Na24 seems unehanged afrter adrenalectomy, with no suggestionl of any increased rate of back-diffusion. These preliminary data coupled with the stop-flow data lead
us to believe that aldosteronie inier-eases the
It has been- established that potassiunm is
reabsorbed and secreted by the nephron. The
area involved in the secretion of potassium is
located in a distal region of the mammalian
nephron, which is also a site for the secretiotn
of hydrogen.3 The secretion of ammonia is
also thought to take place in the same distal
area.4 Recently, Pitts, Gurd, Kessler and
Hierholzer have shown that these seeretory
areas do exist at the same distal tubular site.'
On the basis of clearanee studies, the secretion
of cations is believed to be coupled with sodium reabsorption.6 7
Using the stop-flow technic, we have obtaimed data which indicate that the secretion
of potassium, hydrogen and ammonium occur
in a very distal area of the nephroni. Figure 4
shows a normal stop-flow curve oni which
sodium, potassium and hydrogen (titratable
acidity) are plotted. The concentration of both
potassium and hydrogen rose in the early distal samples and reached maximum values eveln
before the coneentration of sodium reached its
nminimum. Potassium concentrations then fell
very sharply to values below those observed
during free flow. This drop occurred in the
area of low coneentrations of sodium. As proxinial fluid enitered the collector, potassium conieentrations returned to free flow values.
We believe that the increase in the concentrations of potassium in the early samples
represents secretion of that cation in the collecting ducts anid perhaps in some part of the
distal tubule. This inerease oceurs in the first
few samples, in urine which must have been
trapped in the collecting ducts. This initerpretation agrees with the findings of Ullrich et
al., who demonstrated that the concentration
of potassium may inerease in the collecting
ducts.s UTnfortuLnately, it is impossible for us
to delineate more precisely the anatomic areas
involved in the secretioni of potassium. The
secretory area may very well extend into the
distal tubule itself.
Circulation, Volunte XXI, May 196O
. .:;*r-.e
905
STOP-FLOW STUDIES ON ION AND WATER REABSORPTION
10'
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Figure 4
I
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VOLUME
Is
16
IN
20
Ml.
Stop-flow pattern for sodium, hydrogen and potassium.
The minimum concentration of potassium
always occurred in a distal area of the stopflow pattern and was often below the concentration in plasma. We regard this as evidenee
for the reabsorption of potassium in an area
of the distal tubule which also reabsorbs
sodium to low levels.
Bott9 has demonstrated that throughout the
proximal tubule of the amphibian nephron, the
concentration of potassium remains at the
level in the plasma. There is little doubt that
the reabsorption of potassium against a chemical gradient does occur. Subjects in potassium
deficiency exerete urinie with concentrations
of potassium lower than that in plasma.10
Since the reduction in the concentration of
potassium in amphibia does not seem to occur
Circulation, Volume XXI, May 1960
in the proximal tubule, it must take place in a
distal area. It is not surprising, then,
that the stop-flow patterns for potassium include a distal area of low coneentrations of
potassium.
more
The Effects of Impermeant Anions
Infusions of relatively impermeant anions,
such as ferrocyanide, sulfate, thiosulfate and
phosphate, retard the reabsorptioii of sodium
in the proximal areas of the nephron because
of their charge and their inability to accompany reabsorbed sodium across the tubular
wall. Thus, it has been suggested that these
anions increase the secretion of hydrogen and
potassium because they deliver greater
amounts of sodium to a distal mechanism
906
Downloaded from http://circ.ahajournals.org/ by guest on June 15, 2017
which reabsorbs sodium in exchange for secreted potassium and hydrogen.6 However,
the data derived from the use of these anions
in stop-flow analysis have led us to conclude
that the effect of these anions upon the secretory mechanism is only part of their total influence upon hydrogen and potassium excretion.
The results of these stop-flow experiments
show that the presence of impermeant anions
within the tubule not only increased the secretion of hydrogen, ammonium and potassium,
but also caused the points of maximum titratable acid and of maximum concentrations of
potassium in the stop-flow patterns to be
shifted proximally. The maximuin concentrations of these substances then appeared in the
area of the patterns which normally contained
the minimum concentrations of potassium. An
area of the nephron, much larger than was
apparent in the control occlusions, seemed t-o
be secreting hydrogen and potassium.
Figure 5 shows the results of 1 experiment
in which a control occlusion was followed by
a second after the infusion of phosphate so
that the plasma phosphate level rose from 1.71
mM per liter to 14.3 mM per liter. The control
patterns for potassium, ammonium and hydrogen all showed a distal secretory peak which
was distal to the sodium minimum. After the
infusion of phosphate, the maximum concentrations of potassium, ammonium and hydrogen were all iniereased. Their maximum concentrations were also all moved more proximally so that they coincided with the minimum for sodium. After infusion of phosphate,
the distal reabsorptive area for potassium was
no longer evident. Similar results are obtained
if the dog is infused with any other relatively
impermeant anion, e.g., thiosulfate, ferrocyanide or sulfate.
The stop-flow patterns indicate that, in the
presence of the impermeant anions, the apparent secretion of hydrogen and potassium
occurred along the full length of the distal
nephron in which the strong sodium reabsorptive mechanism is located. Ordinarily, chloride
follows the reabsorbed sodium at this site.
MALVIN, WILDE
However, in those animals which are infused
with the sodium salt of the impernmeant anioiis,
niore sodium than chloride is presented to the
distal reabsorptive system, and the amount of
sodium which is reabsorbed exceeds the supply
of chloride. This excess of reabsorbed sodiumover chloride cannot be accompanied by the
infused anion because of its size and immobility. Thus, as sodium is removed, the anionic
charge remaining in the tubule will attract
other available cations to replace the sodium.
The only relatively diffusible cationis available
in quantity are hydrogen and potassiuim, and
they are drawn into the tubule fromi either the
tubular cells or the interstitial fluid. Evidenitly
the potassium reabsorptive mechanism calinot
counteract this inward movement. In effect,
then, a pseudo-exchange mechanism, created
by the combination of the reabsorption of
sodium and the preseniee of imnmobile aniolns,
has been set up in a part of the tubule where
one was not apparent in control experiments.
Countercurrent Multiplier System
It has been established that the ability of
the kidneys to concenltrate urine is dependent
upon a countercurrent multiplier system.11
In this system, the interstitial osmnotic pressure increases along the pyramids, being highest at the tip. In the presenee of antidiuretic
hormone (ADH) this gradient will cause
water to be drawn out of the collecting ducts
and so concentrate the urine. However, under
certain conditions, urine osnmolality may decrease even in the presence of miaximnum ADH.
During osmotic diuresis urine osmolality decreases and approaches plasrma osmolality as
the diuresis becomes more severe.1"
This inability to conieentrate during osmotic
diuresis might be considered to result fromn
increased rate of flow through the collecting
ducts with concomitanit deerease in time allowed for osmotic equilibrium. If this were
true, one would expect urine osmolarity to
increase significantly during stop-flow as additional time is allowed for equilibration.
Figure 6 shows the results of a stop-flow
experiment in which urine osmotic pressure
was measured. Although the osn1otic pressure
Circulation, Volume XXI, May 1960
.- . s |
907
STOP-FLOW STUDIES ON ION AND WATER REABSORPTION
U-
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2 A4 6
ACCUMULATED URINE VOLUME IN ML.
.
Figure 5
Effect of phosphate
on
the
stop-flow
patterns for potassium, ammonium and
did rise in a distal area, the increase was only
about 10 per cent of the control osmolarity
(from 496 to 542 mOsm. per liter). These
results suggest that the osmotic pressure of
the interstitium is higher than urine osmolarity during osmotic diuresis, but only
slightly so. Since urine osmolarity does increase during stop-flow, this is evidence that
Circulation, Volume XXI, May 1960
hydrogen.
during osmotic diuresis the colleeting duct
urine does not reach osmotic equilibrium with
the interstitium.
These results are consistent with the formulations of Hargitay and Kuhn13 that the rate
of urine flow through the loops of Henle is 1
of the determinants of the magnitude of the
countercurrent gradient which thd kidney
ALIN, WILDE
90
1908
ol
LLJ500-
Osmotic Pressure
-
480460=44080-,
No
70 LuJ
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5040E
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I
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1
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1
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I
0 2 4 6 8 10 12 14 16 18 20 22 2426 28 30
ACCUMULATED URINE VOLUME IN ml.
Figure 6
Stop-fiobi pattern fo10r para-ainino,hippiuric acid (PAH), sodium. (Republished by
missioni. of the Amierican Jouirual of
per;
Physiology.'-.)
establish. There mllust be ani optinunllil flow
rate through the loops. Auiv incirease iil flow
above that optimuni would serve to carrv o*f
may
solute auid lesseu the osmyotic gradieiit existing in the renial mnedulla.
Figure 7 shows the results of ani experimiienit
which indicates that this is true durinog osmotic diuresis.14 Catheters were placed in the
left anid right ureters and in the left renal
artery. After a conltrol period, 20 ml. of 20
per cenit maiiuitol was injected inito the left
renial arterv over a 1-min-ute period. One-
miniute urine saiimples were collected simnultaneously from the 2 ureters, anid the samuples
anialyzed for their conieelitratioins of sodiumn.
The urinie flow from the left kidney iniereased
almost immediately, reachinig a maximum durinig the third nminiute, then declined relatively
slowly. The conitrol sodiumii conceenitration in
uirine from the left kidney was 39 mEq. per
liter. As the urinie flow iniereased, so did the
coneentrationi of sodiumi. In the second sample
after inijeetionl, the concentrationi of sodiumLl
reached 117 mEq. per liter anid then declined
Circulation, Volume XXI, May 1960
909
STOP-FLOW STUDIES ON ION AND WATER REABSORPTION
to 23 mEq. per liter. A similar pattern was
seen on the right side: as the urine flow increased, so did the concentration of sodium.
Again the concentrations of sodium fell off
more quickly than urine flow. This experiment
suggests that a sudden inerease in flow rate
through the nephron is able to sweep excess
sodium out into the urine. This observation is
consistent with the view that the rate of the
flow of urine through the loop of Hlenle and
the collecting ducts is a determinant of the
countercurrent gradienlt which may be maintained by the kidney.
201
z
61
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3-]
E2
APTFlaY
K
triMrNLA
_¼
1
1201
Left
100
,
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Right Kidney
80-
References
Downloaded from http://circ.ahajournals.org/ by guest on June 15, 2017
1. RICHARDs, A. N., AND XVALKER, A. M.: Method
of collectinig fluid from knownl regions of the
renal tubules of amphibia and of perfusing the
lumen of a single tubule. Am. J. Physiol. 118:
111, 1937.
2. VANDER, A. J., MALVIN, R. L., WILDE, W. S.,
LAPIDES, J., SULLIVAN, L. P., AND MAC.MLURL
RAY, V. M.: Effects of adrenalectomy and]
aldosterone on proxinmal and distal tubular
sodium reabsorption. Proe. Soc. Exper. Biol.
& Med. 99: 323, 1958.
3. BERLINER, R. W., KENNEDY, T. J., AND ORLOFF,
J.: Relationship between acidification of the
urine and potassiumii metabolism. Am. J. Med.
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4. WALKER, A. M.: Anultnoniia forml-ationi in the
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5. PITTS, R. F., GURD, R. S., KESSLER, R. H., AND
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6. BERLINER, R. W., KENN-EDY, T. J., AND HILTON,
J. G.: Renal mechanisms for excretion of potassium. Am. J. Physiol. 162: 348, 1950.
7. PITTS, R. F., AND ALEXANDER, R. S.: The nature
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AND EIGLER, F. W.: uber die Regulation des
Siaure-Basenhaushaltes durch Ionenaustausch in
den Sammelrohren der Saiugetierniere. Pfluigers
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9. BOTT, P. A.: Evidences from the concentrationi
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Circulation, Volume XXI, May 1960
I
U
t-j60n 40-~
z
20
0
0
1
2
4
6
Time in minutes
8
10
Figure 7
Effect of the injection of matnnitol into the renal
artery on the flowv of urine and the concentration
of sodium in the urine.
10.
11.
12.
13.
14.
8th Annual Conifereinee oni the Nephrotic Syndrone. Meteoff, J., Ed. New York, National
Nephrosis Foundation, 1957, p. 39.
FULLER, G. R., MACLEOD, M. B., AND PITTS, R.
F.: Influence of administration of potassium
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WIRZ, H., HARGITAY, B., AND KUHN, W.: Lokalisationi des Konzenitrierunigsprozesses in der
Niere durch direkte Kryoskopie. Helvet.
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WESSEN, L. G., JR., AND ANSLOW, W. P.: Excretioni of sodium anid water during osmotic
diuresis in the dog. Amii. J. Physiol. 153: 465,
1948.
HXRGITAY, B., AND KUHN, W.: Das M-ultiplikationisprinzip als Grundlage der Harmskonzentrieruing in der Niere. Ztschr. Elektrocheimi.
55: 539, 1951.
MALVIN, R. L., AND WILDE, W. S.: Waslhout of
renial countercurrent Na gradient by osml-otic
diuresis. AIm1. J. Physiol. 197: 177, 1959.
Stop-Flow Studies on Ion and Water Reabsorption in the Dog
RICHARD L. MALVIN and WALTER S. WILDE
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Circulation. 1960;21:902-909
doi: 10.1161/01.CIR.21.5.902
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