AMERICAN
Vol. 228,
JOURNAL
OF
No. 2, February
PHYSIOLOGY
1975.
PrinM
in U.S.A.
Secretion
of salt and water
into the medullary
collecting
duct of Ringer-infused
rats
H. SOKSEXBERG
(With the Technical
Assistance of A. Marsden-Potter
Department of Physiology, University of Toronto, Toronto, Ontario, Canada
transport;
extracellular
volume
expansion;
USING THE MICROCATHETERTZATION
technique
(5)it has been
shown that, in rats chronically
ingesting saline, infusion of
saline resulted in complete inhibition
of the normally
observable reabsorption
of sodium from medullary
collecting
duct fluid (9). In contrast, micropuncture
data from salineinfused normal
rats demonstrated
an increase of sodium
reabsorption
in the papillary
collecting
duct (1). The
apparent
discrepancy
between the two studies could be due
to the differences
in technique,
or it could represent
the
operation
of a long-term
response to extracellular
fluid
expansion.
To further investigate
these possibilities,
microcatheterization
was used to study medullary
collecting
duct
transport
in normal rats during acute intravenous
infusion
of Ringer solution.
METHODS
&iXale Sprague-Dawley
rats (weight
range: 220-400
g)
were maintained
on water and Purina
laboratory
chow.
They were anesthetized
with Inactin
(10 mg/100
g body
wt, ip) and kept at a body temperature
near 38” C on a
heated operating
table. Following
cannulation
of trachea
and jugular vein, Ringer solution (NaCl = 130 m?\if, KC1 =
5 mM, NaHCOs
= 20 mM, CaC12 = 5.3 mM) was infused
intravenously
at a rate of 0.25 ml/min
per 100 g body wt.
l
565
Downloaded from http://ajplegacy.physiology.org/ by 10.220.33.2 on June 18, 2017
The right ureter was cannulated
through an incision in the
left flank and connected
to a femoral vein catheter,
thus
returning
any urine produced
by the right kidney to the
circulation.
The left, experimental
kidney was mobilized
through
the same incision,
placed in a Lucite cup, and
covered to prevent drying and cooling of the surface. A
femoral
artery was cannulated
for blood sampling
and
pressure recording.
On completion
of operative procedures,
inulin3H
was added to the infusate to deliver approximately
125 PCi over the course of the experiment.
Collections
of
tubular fluid and urine were begun 60 min later. Total time
of infusion before collections
varied between 1.5 and 2.5 h,
as did the duration
of the subsequent
experiment.
Urine
from the experimental
kidney was collected quantitatively
at 20-min
intervals;
urine from the control
kidney was
collected in the middle of each interval
by briefly opening
the ureterovenous
shunt. Arterial
blood samples (0.05 ml)
were taken at IO-min
intervals.
Methods
of obtaining
samples of collecting
duct fluid and urine from the experimental
kidney
were described
in detail
previously
(9).
Briefly, the left ureter was dissected to the pelvis and cannulated. The papilla tip was exposed by a small incision in
the upper surface of the ureteral
wall, and urine was collected by gentle continuous
suction.
Fine polyethylene
catheters
(OD
= 16-40 pm) were inserted
to varying
distances into different
collecting
ducts, and samples of
fluid were obtained
by controlled
suction. Measured
distance of insertion
was related to medullary
length taken
from a postmortem
sagittal section of the kidney. Sodium
and potassium
concentrations
in plasma and urine were
determined
by flame photometry,
and inulin-3H
was determined by liquid scintillation
counting
in a toluene-based
scintillant.
Urinary
excretions
of sodium
(I&V)
and
were calculated
for each kidney, as was
potassium
(U&)
glomerular
filtration
rate (GFR).
Sodium
and potassium
concentrations
in aliquots
(10 nl) of tubular
fluid were
determined
by an Aminco helium-glow
photometer;
inulin3H (30 nl) was determined
by liquid scintillation
counting.
To assess the possibility
of variable quenching,
equal I-olumes of diuretic
urine from the same animals were added
to 1 of 2 aliquots of 42 tubular fluid samples. The average
difference in counts was 0.3 c/b & 3.9 (SD), indicating
no
effect of the added urine. A Clifton nanoliter
osmometer
was used to measure total solute concentration
in tubular
fluid and urine samples. Plasma protein concentration
was
estimated by refractometry.
Precision of measurements
was
as described
previously
(9) Plasma inulin
concentration
SoNmNmRG,
I-3. Secretion uf salt and water info the medullary
collecting duct uf Ringer-infused
rats. Am. J Physiol.
228(2) : 565-568.
I975.-Using
a microcatheterization
technique,
the contribution
of the collecting- duct to the renal response
to extracellular
fluid
volume expansion was studied in anesthetized
rats. During
intravenous
infusion
of Ringer
solution
(0.25 ml/min
per 100 g body
wt), urinary
excretion
of fluid,
sodium,
and potassium
was 365
pl/min
per g kidney
avt (V), 52,6 peq/min
per g kidney
wt (L&V),
and 3.86 peq/min
per g kidney
wt (U,V),
representing
23, 24,
and 655% of filtered
load, respectively.
Analysis
of collecting
duct
fluid
from
cortex
and outer
medulla
indicated
continued
net
reabsorption
of ions and water
in these nephron
segments;
in
contrast,
in inner medulla
net secretion
of Na, K, and fluid into
the collecting duct was demonstrated. Addition
of sodium
and
water
was equivalent
to approximately
10% of filtered
load. It is
concluded
that under
the stress of extreme
intravenous
fluid loading tubular
secretion
of salt and water
into the inner medullary
collecting
duct contributes
importantly
to diuresis
and natriuresis.
The mechanism
of such secretion
remains
undetermined.
renal
electrolyte
potassium
secretion
and C, J. Potter)
566
H.
was calculated for the midpoint
of each fluid collection,
and
the tubular
fluid-to-plasma
concentration
ratio (TF/PI,)
was determined.
In addition,
the fractions of filtered sodium
and potassium remaining
at the collection site were obtained
by dividing
the tubular
fluid-to-plasma
concentration
ratio
for each ion by TF/Pr,.
Linear regression and t test analyses
were used for statistical evaluation
of data.
IO0
80
2.0LL.L
0
too
I
50
0
11
100
0
I
I
I
II
20
40
60
80
100
I
I
I
II
20
40
60
80
IO0
I
oy- (ij+
mu3LE 1. Cumfiarison of average renal funclion z’n control and
experimental kidneys of individual rats
-~-___----.-- -___, -~..-~
~-”
V, pl/min
per g KW*
ExptI
304
300
496
275
322
355
482
386
43.9
48.8
62.0
42,2
38.6
45.6
59.5
44.7
43.1
43.2
73.5
33.0
49.5
51.3
67.6
59.5
3.59
1.63
3.23
3.25
2.88
4.44
4*30
3.92
’ 3.25
2.64
4,lO
2.61
3.64
’ 4.91
5.14
i 4.55
365
A30
48.2
zk2.9
52.6
zt4.8
3-40
AZ.32
j 3.86
1 zt.35
423
222
250
357
446
297
328
Ukv, Et4
min per g KW
Control
IzxptI
zt28
UNJC w-s/
min per g RW
Grams kidney
weight.
U 0 srnr
mosmol/llter
Contr01
I
/ Exptl
Con1 trol
Exptl
294
302
319
329
318
322
342
308
1.68
I.44
1.28
291
312
311
329
311
&8
GFR, ml/
min per g KW
/
317
It5
1.38
1,432I,69
1.82
1.86
2.34
1.59
1.44
1.32
1.51
1.38
I,67
1.48
1.62
I .46
jr.05
;*.07
oool
1
100
0
1
50
0
11
IO0
0
0.90
l
0c
0.80
l
,I
l
l
l
l-
-
I
l
0.40-
l
0
l
l
a
a
0.30
I
100
0
Cortex
1
50
0 Outer medulla
11
100
0
Collecting
20
1
I1
40
60
Inner medulla
duct length @ii
Jl
80
I
100
Urine
1. Individual
values of TF/P inulin (A) and fractions of filtered
Na (B) and K (C) remaining
in tubule in different
collecting
duct segments. Open
circles designate
corresponding
average
urinary
values
for each experimental
animal.
Statistically
significant
regression
lines
are indicated.
FIG.
into this tubular segment. In contrast, the fraction of filtered
sodium
declined
significantly
from cortex through
outer
medulla
(T = 0.55, P = O.Ol), indicating
continued
Na
reabsorption
from this part of the nephron.
Corresponding
correlations
for inulin and potassium,
although
suggesting
similar reabsorption,
were not statistically
significant+
Since the variability
of the data might cast doubt on the
physiological
reality of the demonstrated
secretion, in two
animals deliberate
attempts were made to collect samples
from the beginning
of the inner medullary
duct, followed
immediately
by a collection from the same duct system close
to the papilla tip. Results of such ‘(paired”
collections
are
depicted for sodium in Fig. 2. There was a rise in the intratubular
amount of Na between the beginning
and end of
each inner medullary
duct system tested. Changes for inulin
and potassium
were similarly
consistent.
To determine
whether alteration
in flow dynamics in the catheterized
duct
could account for this apparent secretion, in three additional
animals ducts were catheterized
with pipettes that could be
wedged either at the beginning
or end of the inner medullary
duct. A set of four samples was then collected from a given
duct system as follows: two samples each were collected at
the beginning
and end of the duct, with the suction set at
either 10 mmHg lower or 10 mmHg higher than the previously determined
optimum.
Sequence
of collections
was
varied for each duct system. Results are shown in Table 2.
Using the paired t test, statistically
significant
secretion of
Downloaded from http://ajplegacy.physiology.org/ by 10.220.33.2 on June 18, 2017
Intravenous
infusion of Ringer solution resulted in average decreases of hematocrit
and plasma protein concentration to 39.0 & 0.6 (SE) % and 3.3 & 0.1 (SE) g/100 ml,
respectively, compared
to values of 47.5 & 1.6 % and 6.1 =t
0.1 g/ 100 ml for nondiuretic
rats (ref. 9 and unpublished
data). The decreases of both hematocrit
and protein concentration
were apparent
at the beginning
of collections
and showed no further consistent variation
throughout
the
experimental
period. The stability
of the preparation
was
also indicated
by the constancy of arterial
blood pressure
(average = 121 & 5 (SE) mmHg).
A comparison
of average function of control and experimental kidneys of each animal is shown in Table 1. Despite
the differences
in urine collection
times and methods, no
statistically
significant
difference
in function
of the two
tended to remain
kidneys was observed. Renal function
constant throughout
each experiment.
Changes along the collecting
duct system of intratubular
inulin
concentration
and fractional
remainder
of filtered
sodium and potassium are plotted in Fig. 1. The relationship
between the percentage
scales of collecting
duct length in
cortex and outer and inner medulla corresponds
to absolute
length differences of these segments (cortex = 2.0 & 0.035
= 5.6 & 0.1
SE, outer medulla
= 2.9 & 0.1, inner medulla
mm). After inspection
of the data, separate
regression
analyses were performed
for points in cortical and outer
medullary
segments and those in inner medulla,
respectively. Statistically
significant
decrease of inulin concentration (correlation
coefficient,
r = 0.65, P < 0.001) was
seen along the inner medullary
duct system, indicating
net
addition
of fluid to tubular
flow. Similarly,
increases in
fractional
sodium
(r = 0.66, P < 0.001) and potassium
net entry of these ions
( r = 0.60, P < 0.001) demonstrated
SE
l
c
60
RESULTS
2
0A
SONNENBERG
TUBULAR
SECRETION
IN
MEDULL~4RY
COLLECTING
inner medullary
duct ((TF/P,)
- inner medulla
= 3.14 0.0067 X % length; r = 0.42, P < 0.01).
Absolute
tubular loads of ions and water were calculated
from filtered load and remaining
fraction at beginning
and
end of the inner medullary
duct system (Table 3). Corresponding
data from antidiuretic
rats (9) are included
for
comparison.
It is evident that with Ringer infusion a larger
proportion
of the enhanced
filtered load reaches the duct
system at the border between inner and outer medulla.
In
addition,
however,
secretion
of both sodium
and water
equivalent
to about 10 % of filtered load and 40 % of total
urinary
excretion
in inner medulla
of infused rats is contrasted to continued
reabsorption
in antidiuretic
animals.
ions and water was found whether
flow was artificially
reduced or increased. Not unexpectedly,
partial obstruction
reduced
tubular
content,
presumably
by enhancing
upstream reabsorption.
This reduction
was not statistically
significant,
however.
In two rats that were allowed to excrete urine from both
kidneys while receiving a doubled intravenous
infusion,
14
pairs of samples from beginning
and end of inner medulla
showed an increase from 0.233 & -020 to 0297 & 0.010
(SE) (P < 0.01) in the fraction of filtered sodium remaining
in the duct, indicating
that urine reinfusion
is not essential
to the secretion.
In contrast to the change in intratubular
amount
of
sodium, concentration
of the ion remained
unaltered
in any
segment of the collecting
duct system, with an average of
Similarly,
tubular
fluid osmo134 & 11 (SD) meq/liter.
lality remained
constant at 291 =t 21 (SD) mosmol/liter,
while
potassium
concentration
tended
to fall along the
20I
40I
60I
Inner medulla
duct length
Collecting
I
80
I
100
I
DISCUSSION
Comparison
of infusion rate and urinary volume produced
by the single excreting
kidney shows that these animals
excreted on average three-fourths
of the infused fluid. Since
hematocrit
and plasma
protein
concentration
did not
change over the experimental
period, extrarenal
routes of
loss, including
visible intraperitoneal
fluid, contributed
to
maintenance
of fluid balance. However,
as in an earlier
series with comparable
infusion rates (lo), expansion
of
intravascular
volume by about 20 % is indicated
by the
initial
decrease
in hematocrit.
Interestingly,
fractional
excretion
of sodium and water in the previous group in
which both kidneys were functioning
was one-half of that
found in the present experiments.
Direct (9, 11) as well as indirect
(4, 6) evidence indicates
that the medullary
collecting
duct plays a decisive role in
the regulation
of urinary
excretion.
However,
secretion of
sodium and water into collecting
duct fluid has not been
previously
demonstrated,
although
such Na entry was
suggested as a mechanism
of natriuresis
in DOCA-escaped
rats during
intravascular
expansion
(8). The possibility
that the observed secretion was an artifact due either to the
surgical
treatment
of the experimental
kidney or to the
microcatheterization
technique
itself is deemed
unlikely
I
Urine
I%)
FIG. 2. Fractions
of filtered
sodium
in paired
collections
of fluid
from beginning
and end of inner medullary
duct. Urinary
values corresponding
in time with collection
pairs are represented
by open
circles.
TABLE
2.
Effect of increased and reduced suction on collecting duct trans@rt at beginning
---~--.
t
Iner
TF
(-1
P In
Suction-End
(TJVP)
-(TF/Phn
6.30
8.35
6.27
5.28
4.76
3.74
8.28
4.89
6.00
4.82
4.59
5.77
% 1 = percent
during
increased
No
Incr Suction-Begin.
VWVK
OIn
y. 1 im
-125
* 155
,160
.203
.256
,161
,212
A78
.225
.218
A79
,582
.380
,493
,511
.831
.733
.701
738
,589
.755
.639
.603
100
100
100
97
96
90
90
93
93
94
95
,187
.OlO
.630
,036
96
1
-172
l
100
*t Significant
length.
or decreased
suction.
TF
(-1
P In
8.88
12 .o
8.00
9.79
7.84
4.71
8.46
5.26
6.46
6.84
8.43
7.32
7 -83’
.57
% 1 im
-.129
,091
.I49
JO0
.121
,217
,138
.209
s 142
.124
,116
,141
.464
.348
,515
.387
.632
,657
.637
,728
.589
,526
.469
.708
.140*
.OlO
,555-p
,035
difference
(P < 0.01,
7
2
15
10
28
10
27
7
31
14
15
-5
13
3
-(TFp- >In
P < 0.02)
and end of inner medulla
D ecr Suction-End
CWWN,
U-F/P)I,
7.77
Deer
-
(WPIK
OI,
y. 1 im
_---
TF
c-1
P In
--
Suction-Begin
(TF/Ph,
@mK
--
W/P)K
OI,
--
%lh
147
,493
100
17.6
.055
.246
7
7.68
8.42
5.28
3.41
6.42
5.70
5.31
6.12
4.85
6.14
,146
A04
.192
*294
.183
.203
* 168
-178
.191
A58
,472
.450
.741.
.818
,701
.706
.542
100
100
,642
.496
96
97
92
92
93
90
95
93
17.9
10.2
10.6
11.5
5.57
9.86
8.93
5.40
7.44
6.23
.052
.108
.093
A15
.219
.117
.091
l 179
l 135
.163
l 377
.489
,752
.435
543
,306
.518
.494
.507
,592
5
13
12
7
48
7
31
26
18
7
6.10
-44
A79
l OlO
,622
.040
95
1
10.1*
1.3
.lZOf,014
.478t
.041
16
4
between
l
average
l
779
values
at beginning
l
and end of inner
--medulla
Downloaded from http://ajplegacy.physiology.org/ by 10.220.33.2 on June 18, 2017
O.OL0
567
DUCT
568
H.
TABLE
qf
inner
-.
3. Tubular load of ions and wafer at beginning
medullary coILsting duct compared to f&ration
and end
Inner Medulla
Filtered
Beginning
V,
Na,
pI/min
per g KW
peq/min
per g KW
1,460
(910)*
213
UW
K, peq/min
per g KW
5.84
(4.10)
End
A
223
(23.2)
390
(8.5)
+1s7t
(-14.7)T
30.5
(1.7)
51.6
10.4)
+21.1t
C-1.4jt
2.78
(0.84)
3.83
(0.76)
+1.05t
(-0.08)
* Values
in parentheses
are for antidiuretic
rats
significant
change, based on correIation
coefficients.
(from
ref.
9).
t StatisticaIly
collecting
duct in young, saline-infused
rats. While the high
intravenous
infusion
rates in the present
experiments
preclude
direct
comparison
of duct function,
it seems
unlikely
that the difference
in infusion
rate alone could
account for maximal
reabsorption
on the one hand and
actual secretion on the other. It has been found that exposure of the papilla
and alteration
of surrounding
fluid
composition
may affect intratubular
fluid composition
(7).
Since in the present experiments
on adult rats the papilla
remained
within the body of the kidney, and central ducts
were cannulated,
the apparent
discrepancy
could be explained on this basis.
The mechanism
of the observed
secretion
is unclear.
Diffusion
of sodium into the lumen of the collecting
duct
has been suggested as a possible contributing
factor to
natriuresis
(12). Since total solute as well as sodium concentrations along the duct remained
unaltered
in these experiments, however,
it is dificult
to visualize
simultaneous
difiusion of sodium and water in the same direction.
Based
on the increase of interstitial
hydrostatic
pressure with
extracellular
fluid volume expansion
(3), secretion of salt
and water into the collecting
duct might be due to bulk
flow of solution from medullary
interstitium
to duct lumen.
In the absence of information
on the filtration
coefficient
of the duct epithelium
and the magnitude
of pressure
gradients,
such an explanation
remains speculative.
Active
transport of sodium into the lumen followed by diffusion of
water offers an alternate
explanation
for the results. However, active j?;a transport
in opposite directions
at the same
tubular site has not been found previously under any experimental
conditions.
Finally,
active tubular
secretion
of a
substance with consequent
diffusion of Na and fluid in the
same direction
could explain
the present findings.
Such
fluid secretion was shown to be associated with PAH transport in the straight portion of the isolated proximal
tubule
(2). The mechanism
by which sodium and fluid may be
added to collecting duct fluid in the inner medulla therefore
requires further investigation.
This study was supported
Research
Council
of Canada.
This study was published
33: 388, 1974).
Received
for publication
by
Grant
in part
21 February
MA
in abstract
4043
from
form
the
Medical
(Federation
Proc.
1974.
REFERENCES
1. DIEZI, J., P. AZICHOUD, J. &EVES,
AND G. GIEBISCH.
Micropuncture study of electrolyte
transport
across papillary
collecting
duct
of the rat. Am. J. Physiol. 224: 623-634,
1973.
2. GRANTHAM,
J. J., P. B. Q~ALIZZA,
AND R. L. IRWIN.
Net fluid
secretion
in proximal
straight
renal tubules in vitro: role of PXI.
Am. J. Physiol. 226: 191-197,
1974.
3. GWYTON,
A. C, Interstitial
fluid
pressure.
II. Pressure-volume
curves of interstitial
space. Cz’rculation Res. 16 : 452-460,
1965.
4. JAM~SON, R. L., AND F. B. LACY. Evidence
for urinary
dilution
by
the collecting
tubule.
Am. J. Physiol. 223 : 898-902,
1972.
5. JARAUSCH,
K. EL, AND K. J. ULLRXCW.
Zur Technik
der Entnahme
von I-Iarnproben
aus einzelnen
Sammelrohren
der S%ugetierniere
mittels Polygthylen-Capillaren.
gffuegers Arch. 264 : 88-94,
1957.
6. MALNIC,
G., R. XL KLOSE, AND G. GIEBISCH. Micropuncture
study
of distal tubular
potassium
and sodium transport
in rat nephron.
Am, J. Physiol. 221 : 529-547,
1966.
7. SCHULTZ. MT,* AND ‘I‘. SCHNERMANN.
Pelvic urine comDosition
as a
8.
9.
10.
11.
12.
determinant
of inner medullary
solute concentration
and urine
osmolarity.
P~uegers Arch. 334: 154-166,
1972.
SONNENBERG, I-I. Renal response to blood volume expansion
: distal
tubular
function
and urinary
excretion.
Am, J. Physiol. 223 : 916924, 1972.
SONNENBERG,
I-I. MedulIary
collecting-duct
function
in antidiuretic
and in salt- or water-diuretic
rats. Am. J. Physial. 226:
501-506,
1974.
SONNENBERG, I-I., AND S. SOLOMON. Mechanism
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AND T. F. FERRIS.
A comparison
of the segmental
analysis of sodium reabsorption
during
Ringer’s
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J. Clin. Invest. 52 : 23 I S-2323,
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UHLICH,
E., C, A. BALDAMUS, AND K. J. ULLRICH.
Einfluss
t-on
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in den Sammelrohren
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Pyzrfgel-s ,-b-c//. 308 : 11 l-126,
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Downloaded from http://ajplegacy.physiology.org/ by 10.220.33.2 on June 18, 2017
for the following
reasons: first, comparison
of control and
experimental
kidneys
(Table
1) showed
no significant
difference
in either filtration
or excretion,
indicating
that
both kidneys
were functioning
similarlv.
Second,
while
increasing
interference
of the microcathkter
with tubular
flow in progressively
smaller ducts might cause artificially
increased reabsorption
at the deeper sampling sites, such an
effect was not evident in samples taken even farther upstream in the outer medulla. In addition,
deliberate
increase
or decrease in intratubular
flow rate (Table
2) did not
disrupt the secretorv pattern in the inner medulla.
Furthermore, even when samples with enhanced
suction at the
beginning
of the duct were compared
with those during
partial obstruction
at the end of the duct (Table Z), statistically significant
(P < 0.01) Na secretion remained.
It may
be concluded,
therefore,
that secretion of salt and water
into the collecting
duct system of the inner medulla
is a
feature of the renal response to massive Ringer infusion,
contributing
in the present experiments
about 40 % of total
urinary
excretion
(Table
3). The quantitative
aspects of
this secretion must be interpreted
with caution,
however,
since it is obvious that alterations
of intratubular
pressure
may affect upstream transport
(Table 2). It should also be
pointed out that lower infusion rates resulted in inhibition,
but not reversal, of the normal Na and fluid reabsorption
in this nephron segment (9).
The present results are in conflict with those of Diezi
et al. (I), who observed reabsorption
of sodium equal to
2 % of filtered
load per millimeter
of exposed papillarv a
SONNENBERG