Clinical Science (1972) 42, 701-709.
WATER AND SODIUM EXCRETION AFTER BLOOD
VOLUME EXPANSION UNDER CONDITIONS OF
CONSTANT ARTERIAL, VENOUS AND PLASMA
ONCOTIC PRESSURES AND CONSTANT HAEMATOCRIT
B. L I C H A R D U S A N D A. N I Z E T
Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava,
Czechoslovakia, and Institute of Medicine, University of Li2ge, Lizge, Belgium
(Received 17 November 1971)
SUMMARY
1. The diuretic and natriuretic responses occurring during expansion of blood
volume by homologous blood transfusion were studied in homologous kidneys transplanted to the neck of hydropenic dogs that had previously been given deoxycorticosterone acetate and antidiuretic hormone. The experimental conditions ensured
constant arterial perfusion pressure, venous pressure, osmotic pressure, haematocrit
and plasma oncotic pressure.
2. Moderate but significant increases in urine output, renal sodium excretion, osmotic clearance and tubular sodium rejection fraction were observed; there were no
significant changes in glomerular filtration rate, renal blood flow, postglomerular
haematocrit and postglomerular plasma protein concentration 20 and 40 min after the
end of blood infusion.
3. As the non-hormonal factors known to modulate sodium excretion underwent
no significant change, the results are compatible with the proposition that a specific
factor (‘natriuretic hormone’) plays a role in the mechanism of natriuresis after blood
volume expansion.
Key words : blood volume expansion, water excretion, sodium excretion.
It has been shown that the increase in urine and sodium excretion during extracellular fluid
volume expansion cannot be prevented either by decreasing glomerular filtration rate or by
increasing the plasma concentrations of mineralocorticoids and antidiuretic hormone (de
Wardener, Mills, Clapham & Hayter, 1961); a ‘natriuretic hormone’ was claimed to be responsible for the natriuresis observed under those conditions. Further experimental evidence
for the existence of such a hormone was the induction of a natriuresis in a non-expanded dog
connected by cross-circulation with a dog with increased extracellular fluid volume (de Wardener et al., 1961;Lichardus & Pearce, 1956,1966; Johnston & Davis, 1966). The interpretation
Correspondence: Professor A. Nizet, Department of Medicine, University of Liege, Boulevard de la Constitution 66, Libge, Belgium.
701
702
B. Lichardus and A . Nizet
of many of the experiments is complicated by the fact that it is not possible to exclude the
interference of several factors that are affected by blood dilution and are known to have a considerable influence on the reabsorption and excretion of sodium and water (Earley & Friedler,
1966; Nizet, Godon & Mahieu, 1968, Nizet, 1972). Lichardus & Pearce (1966) attempted to
eliminate such interference by transfusing to the donor dog a suspension of erythrocytes in
Ringer-Locke solution containing 6% bovine albumin, in a volume equal to 33% of the estimated blood volume; in this way, any possible diuretic and natriuretic effect of blood dilution
in the cross-circulated kidney of the recipient dog was eliminated. The mean peak natriuresis
(+ 97% of control in the cross-circulated kidney) was observed 30 min after completion of intravascular expansion of the donor dog. In spite of the constant renal perfusion pressure, there
was an increase in renal blood flow and glomerular filtration rate in the perfused kidney during
peak natriuresis, although this was not statistically significant. The renal haemodynamic
changes might have contributed to the increase of urine and sodium output and might have
been the result of a circulating vasodilator substance (Lichardus & Pearce, 1966; Bahlman,
McDonald, Ventom & de Wardener, 1967). Bahlman et a/. (1967) also suspected a direct
natriuretic effect of the bovine albumin. Furthermore, basal (or control) sodium excretion in the
cross-circulated kidney was subnormal and this might have distorted the effect of a specific
natriuretic factor. In the present experiments an attempt was made to eliminate the previous
uncertainties and to rule out the possible effect of changes in renal venous pressure by studying
under controlled conditions a kidney totally denervated by transplantation. The results have
been presented at the XXV IUPS Congress in Munich and have been published in abstract form
(Lichardus & Nizet, 1971).
MATERIALS AND METHODS
Eight experiments were performed under pentobarbitone anaesthesia (25 mg/kg, intravenously)
in dogs (23-30 kg) that had not received any special pretreatment. Three experiments were excluded from the study because of an intense antinatriuretic state (sodium rejection fraction
between 0.01% and 0.05%) which remained unchanged throughout the study. In each experiment three dogs were used: one, the experimental animal, received a kidney from the second
dog; the third animal served as blood donor. After careful surgical preparation, kidneys with
appropriate segments of aorta and vena cava caudalis were taken from one dog and transplanted by the method of Govaerts (1928) to the carotid artery and jugular vein of another dog
with the help of Payr cannulas. One of the dog's own kidneys was subsequently tied off; the
transplanted kidneyweighed between 23 and 30g. In this way, the kidney was denervated, but no
foreign material such as tubing was interposed between the animal and the transplanted kidney
(Fig. 1). The duration of the renal ischaemia was approx. 1 min. Besides the carotid artery and
jugular vein on one side of the neck, one femoral artery and both femoral veins were cannulated for blood-pressure measurements and for infusion of exogenous creatinine, antidiuretic
hormone (Vasopressin-synth. Lypressin, Sandoz, 30-50 munits h-' kg in 10 ml of RingerLocke solution) and blood. Deoxycorticosteroneacetate in oil solution (15 mg/dog) was injected
into the animals at least 3 h before the beginning of the f3st urine collection period. After kidney
transplantation, an equilibration period of approx. 1h followed, during which renal arterial perfusion pressure in the transplanted kidney was kept constant (systolic pressure of 120 or 130mm
Hg) by means of a Goldblatt clamp, and renal venous pressure was kept constant by tilting the
-'
Natriuresis after blood volume expansion
703
animal. In order to facilitatedirect renal blood flow measurements, the animals were heparinized
(1250 international units/kg, intravenously). After the equilibration period, two or more 20
min urine samples were collected. After stabilization of urine output, infusion of fresh blood
from a donor dog (33% of the estimated blood volume of the experimental animal) was performed within 20 min. The infused blood had been quickly withdrawn from the donor's carotid
artery into a beaker containing heparin (1500 international units/lOO ml of blood) 15 min
before the infusion; the blood was kept in a glass beaker at room temperature. Blood-donor
dogs were selected whose plasma protein concentration and haematocrit matched those of the
experimental animals. No significant differences were found in plasma protein, sodium and
potassium concentrations, plasma osmolarity and haematocrit during the first 40 min after
completion of blood infusion into the recipient dogs.
//
Blood pressure
/ manometers
V. ca va
ca u do I is
-
Reversed
wall of
the vessel
Ligature
in?.
-
1 1
Arter,y
or vein
V. jugularis
f
A.corotis
FIG.1. Scheme of transplantation of the dog kidney to the neck by means of cannula of Payr.The
cannula is a 1 cm long metal tube of the appropriate diameter to fit vessels of various size.
Two 20 min periods of urine sampling followed. Urine and plasma sodium concentrations
were measured by flame photometry, osmolarity by cryoscopy, and glomerular filtration rate
by clearance of exogenous creatinine; renal blood flow was measured directly from the venous
side with a graduated 25-ml pipette twice during each sampling period. Plasma protein concentration was determined by refractometry under periodic control by Kjeldahl's technique;
haematocrit was measured in the Coulter counter. Post-glomerular values were calculated by
Bresler's formula: [Plasma protein concentration (or haematocrit) x loo]/(100- filtration fraction). The results of the urine output 0,osmotic clearance (C0,,), sodium excretion (V,,V),
glomerular filtration rate (GFR)and renal blood flow (RBF) were calculated as min"(100 g
of kidney-'). Results were expressed in three ways: (a) absolute values (Table 1); (b)
differences between pre-expansion period (i.e. the last control period) and post-expansion
periods expressed in percentage of pre-expansion period (Table 1); (c) differences between
pre- and post-expansion periods (Fig. 2). The mean differences expressed in % f SE were tested
by Student's t test and the product moment coefficient of correlation was calculated. However,
B. Lichardus and A . Nizet
704
it was clear on inspection that most of the variables had a very skewed non-normal distribution.
As in most instances the mean was approximately equal to its respective standard deviation,
a simple logarithmic transformation was applied to the individual values to bring them to
normality (Walker & Lev, 1953).
300
-
200
-
- loo =c
.t?
.
.
w"a
-
y = 121.lXO~'3
r = 0.936
Pc 0.01
50-
--
s
-
1
,
3
10
=-
5 r
2
6
.c
E
I
Y
E 0.5
aE
u"
0.1
1.
-
c
I-
t-
and tubular rejection fraction of sodium (TRFNa)(Pc0.05).Changes of the glomerular filtration rate, total renal blood flow, peritubular plasma protein concentration and haematocrit
Natriuresis after blood volume expansion
705
were not significant (Table 1). Fig. 2 presents the correlations between the increases in fractional
tubular sodium rejection after expansion and the corresponding changes in absolute sodium
excretion, osmolar clearance and urine flow (on bi-logarithmic scale). Positive and significant
TABLE
1. Effect of expansion of blood volume on function of transplanted kidney. Mean absolute
control values f SE before. and after expansion of blood volume. ns, not significant; V, urine
output; &., osmoticclearance; U,.V, total renal sodium output. The values for postglomerular
haematocrit and postglomerular proteins were calculated as described in the Materials and
Methods section.
Mean f SE
after expansion
Mean f SE
before expansion
20 min
V (ml min-' 100 g of
kidney-')
1.55 f0.53
40 min
2.88f 1.27
(+ 101.2f 27.7%)
n=5, P<O.O5
&,,,, (ml min-' 100 g of
2-36f0.55
kidney- ')
3-09f 0.71
(+ 32.6f 3.7%)
n=5, P<O.001
~
UN.V (pEq min-' 100 g of
kidney- ')
Glomerular filtration rate
(ml min-I 100 g of
kidney-')
~~~~
~~
n = 5 , P<O.O5
2.99 f 0.79
(+25.0+ 3.5%)
n= 5, P<0*05
~~
138.2f68.5
206.8 f 97.9
(+70.6+ 13.8%)
n=5, P<001
194f 103.1
(+67.4+23*4%)
n= 5, P-;O*O5
1.29f 0.71
1.75f 0.87
(+ 56.6f 20.6%)
(+ 65-2f20.6%)
n=5, P<O.O5
n=5, Px0.05
87.0f 6.4
(+23.0+17-2%)
n=5, ns
78.7f 2.8
(+12*6f15.2%)
n=5, ns
75.3 f 9.7
~
Renal blood flow (ml min100 g of kidney-')
~
3.51 f 1.69
(+ 115*6+37.2%)
~
~~
363.6f 36.0
1-85f093
~~
418.0f48.9
(+ 15.8f 11.8%)
n= 5, ns
(+ 14.0f 14.5%)
408.8f 46.0
n=5, ns
Haematocrit postglomerular (%I
60.72 6 5
68.0k4.9
(+17*8f18.8%)
n=4, ns
64.0 & 3.5
(+9*5+12.8%)
n=4, ns
Postglomerular proteins (g/
100 ml)
8G4f 0.88
9.38f 1.01
(+ 17.8f 15.0%)
n= 5, ns
8.72f 0.80
(+ 12.8 f 11.4%)
n=5, ns
correlations were found only between the differences in the tubular rejection fraction on the
one side and the differences in sodium excretion (P<O-Ol) and in osmotic clearance (P<O.OI)
on the other. The correlation between sodium rejection fraction and urine output was slightly
out of the range of significance after logarithmic transformation (P>O-O6). Table 2 indicates
B. Lichardus and A . Nizet
706
the correlation between the absolute values of these variables during the two post-expansion
periods. Significant values are found for the correlations between TRFN, (as x) and V, UN,V
and COsm(as y ) ; the other correlations are not significant.
TABLE
2. Correlation between absolute values of the variables in the first and second post-expansion clearance
periods (after logarithmic transformation). Abbreviations are as in Table 1.
Glomerular filtration rate
TRFN.
r
V
UN.V
Cosm
TRFN,
0.8118
n = 10
0.9641
n= 10
0.9066
n= 10
-
P
<0.01
<0.001
<0.001
r
-0.3730
n= 10
-0.3263
n = 10
-0.2988
n= 10
Renal blood
flow
Postglomerular
protein
P
r
P
r
P
ns
-0.0180
n= 10
0.2561
n = 10
0.1716
n = 10
0.1487
n = 10
ns
0.3182
n= 10
0.0981
n = 10
0.2504
n= 10
0.1141
n= 10
ns
ns
ns
ns
ns
ns
ns
ns
ns
Postglomerular
haematocrit
r
-0.6043
n= 8
-0.5270
n= 8
-0.4055
n=8
-05526
n=8
P
ns
ns
ns
ns
DISCUSSION
Increasing the blood volume in dogs with fresh heparinized blood significantly enhanced urine
output, osmotic clearance, sodium excretion and tubular rejection fraction of sodium in a
homologous dog kidney transplanted to the carotid artery and jugular vein. Renal arterial
perfusion pressure and renal venous pressure were kept constant and the expansion did not
change the initial haematocrit and the plasma protein concentration. Later in the experiment
postglomerular plasma proteins and haematocrit tended to rise as well as renal blood flow and
glomerular filtration rate. The transplanted kidney showed no detectable functional impairment
as judged by the normal values for glomerular liltration rate (75-3f9.7 ml min-' 100 g of kidney-'), renal blood flow (363.6f36.0 ml min-' 100 g of kidney-') and tubular rejection
fraction (1-29f0.71 %). Transplantation of kidneys by means of a Payr cannula is a suitable
method for acute experiments in which a complete denervation of the organ is required (Reinhardt, Klose, Ellinghaus, Brechtelsbauer & Behrenbeck, 1967). Three experiments in which
there was almost complete renal sodium reabsorption (TRFN, O*Ol-O*OS%)were discarded
from further evaluation; such an antinatriuretic state might have been the result of dehydration.
The increase of sodium excretion in the present experiments during blood volume expansion
was in the same range (mean peak increase + 70%) as in the experiments by Lichardus & Pearce
(1966) (mean peak increase + 97%). We suggest that this striking similarity demonstrates again
that dilution of mineralocorticoids or antidiuretic hormone (hormones which were not given
in the previous experiments and were added in considerable excess in the present experiments)
or a pharmacological natriuretic effect of bovine albumin plays only a minor role in the mechanism of volume natriuresis in dogs. The positive correlations beween TRF,, and sodium excretion, and TRFN, and osmotic clearance suggest that decreased sodium reabsorption rather
than increased glomerular filtration rate and renal blood flow caused the increased sodium
Natriuresis after blood volume expansion
707
excretion after expansion of the blood volume with blood. The same conclusion is probably
justified for the mechanism of urine output in the presence of an exogenously increased antidiuretic hormone concentration.
The non-hormonal factors known to modulate sodium excretion (renal arterial and venous
pressures, glomerularfiltration rate, renal blood flow, postglomerular plasma proteins, haematocrit) demonstrated no significantchange. A possible effect of dilution of mineralocorticosteroids
and antidiuretic hormone was avoided as well as any pharmacological effect of heterologous
serum albumin. Another possibility is that natriuresis could have been secondary to an intrarenal redistribution of blood flow, but no evidence of this was obtained in the present experiments. However, it has not been positively demonstrated that the natriuretic response of the
transplanted kidney is not related to some local consequence of homologous blood transfusion,
such as an increase of renal interstitial pressure due to escape of fluid from the vascular space.
During blood volume expansion an increase of volume and hardness of the transplanted kidney
was observed. Expansion of the interstitial space with a consequent increase in interstitial
pressure might have impaired sodium reabsorption. Hebert & Arbus (1971), however,
demonstrated that increased interstitial pressure as estimated from subcapsular pressure was
not the major cause of saline-induced natriuresis as the increases in subcapsular pressure and
sodium excretion did not follow similar time courses. Moreover, some degree of swelling of the
homotransplanted kidney is usually observed in the absence of blood volume expansion, but
sodium rejection does not significantlyincreasein these conditions and remains stable for several
hours (Reinhardt et al., 1967). None of the factors known to modulate renin secretion was
modified in the present experiments. The observed decrease in fractional tubular reabsorption
and the subsequent natriuresis might therefore be a consequence of the intervention of a more
specific humoral factor such as a natriuretic hormone.
Blythe, D’Avila, Gitelman & Welt (1971) claim to have found no natriuretic response in
the recipient non-expanded dogs in cross-perfusion experiments with donors expanded with
blood. It is difficult to comment on these results as some details concerning the methods and
results were not given. It seems that the total natriuresis during the expansion period of approx.
70 min was compared with the pre-expansion control values. With this approach, a small
natriuretic reaction in the first 20 or 40 min might have been obscured. It might be interesting to examine the changes in plasma protein concentration and haematocrit, as an increase
in these variables could have played an antinatriuretic role. It was found in rats, for example
(Pearce, Sonnenberg, Lichardus &Veress, 1970;Ponec & Lichardus,1970), that in cross-circulation experimentsin which the donor was expanded with blood, there was no natriuretic response
in the recipient unless haemoconcentration was prevented in the donor by returning his urine
to the circulation via the jugular vein (Lichardus & Ponec, 1970; J. W. Pearce, personal communication).
Kaloyanides & h e r (1971) perfused an isolated dog kidney from a second dog with and
without expansion of blood volume with equilibrated blood; a natriuresis occurred in the perfused kidney (U,,V) increased from 153 to 345 pEq/min. Our results are thus in agreement
with their finding despite the fact that their experimental protocol differed from ours in several
points ;the most important differences were that they used deoxycorticosteroneacetate escaped
animals pretreated with salt and they did not keep renal perfusion pressure and venous pressure
constant. However, the perfusion pressure showed a tendency to decrease and natriuresis
occurred despite a significant decrease in glomerular filtration rate and renal blood flow. Such
708
B. Lichardus and A . Nizet
contrary changes in renal haemodynamics and natriuresis provide strong evidence to suggest
that a decrease of tubular sodium reabsorption is a predominant factor in the mechanism of
volume expansion natriuresis.
In isovolumic cross-circulation experiments performed in dogs (Nizet, 1970) no contralateral
natriuretic response was observed after unilateral blood transfusion whereas unilateral saline
infusion was immediately followed by massive bilateral natriuresis. It is possible that, during
the phase of blood transfusion, minor contralateral changes in blood concentration might have
been sufficient to mask a more specific humoral effect; it appears from the present experiments
that the changes in fractional sodium excretion elicited by blood volume expansion without
dilution are of small amplitude (less than 1 % of filtered load) compared with the effect on nonhormonal factors such as blood pressure or blood dilution. Very careful control of these nonhormonal factors is a prerequisite of any attempt to detect a more specific effect.
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