471
Clinical Science (1991)81, 471-476
Urinary excretion of digoxin-likeimmunoreactive factor and
arginine-vasopressinin hyper- and hypo-thyroid rats
FELIX VARGAS, MARIA J. BAZ, JUAN
AND
DE D. LUNA*, JESUS ANDRADE, ESTEBAN JODAR
JOSE M. HARO
Departamentode Bioquimica y *Departamentode Bioestadistica, Facultad de Medicina, Granada, Spain
(Received 17 October 1990/13 May 1991; accepted 30 May 1991)
SUMMARY
INTRODUCTION
1. Urinary excretion of digoxin-like immunoreactive
factor and arginine-vasopressin and other parameters
related to salt and water metabolism were studied in
hyper- and hypo-thyroid rats after different tests.
2. Urinary excretion of arginine-vasopressin was
increased in hyperthyroid and reduced in hypothyroid
rats with respect to controls, in response to water deprivation o r a hypertonic saline load.
3. Control and hypothyroid rats showed the highest
urinary excretion of digoxin-like immunoreactive factor
after a hypertonic saline load. However, hyperthyroid rats
had the highest urinary levels of digoxin-like immunoreactive factor under normal conditions.
4. From these results it is suggested that: (a)hyper- and
hypo-thyroid rats exhibit hyper- and hypo-responsiveness
of arginine-vasopressin secretion to osmotic stimuli,
respectively; (b) an unidentified digoxin-like immunoreactive factor measured in unextracted rat urine may be
related to diuresis and natriuresis in control and hypothyroid rats; however, dissociation between this factor
and natriuresis is observed in hyperthyroid rats.
Thyroid disorders have important effects on renal
function and salt and water metabolism [l].In the rat,
hypothyroidism causes a decrease in glomerular filtration
rate [2-41 and in effective plasma flow [3,5], together with
a defect in tubular sodium reabsorption [3, 41 and alterations in urine-concentrating ability [4, 51. Thus hypothyroidism in rats is considered to be a disease that
produces water and sodium loss. Although the effects of
hypothyroidism on renal function have been studied
extensively, there is less information concerning the effect
of hyperthyroidism, in which a tendency toward sodium
retention is seen.
T h e impairment of sodium reabsorption in the hypothyroid kidney is attributed to a decrease in Na+-K+ATPase activity, which has been shown to occur when the
plasma thyroxine (TJ concentration is decreased [3, 61.
Several studies suggest that renal sodium excretion might
be regulated by an endogenous digitalis-like factor with
putative natriuretic properties [7]. Its activity as an
inhibitor of the Na+-K+ pump makes it similar to digitalis
compounds; furthermore, a number of observations also
suggest that endogenous digitalis-like factor may crossreact with digoxin-binding antibodies [8]. Although this
issue remains controversial, alterations in levels of
endogenous digoxin-like immunoreactive factor (DLIF)
have been observed in several diseases with abnormal salt
and water balance [9, lo]. However, the possible role of
DLIF in the changes produced by thyroid abnormalities
in salt and water metabolism has not been evaluated.
Arginine-vasopressin (AVP)has only been measured in
plasma from hypothyroid humans [ l l ] and rats [12, 131,
with scarce and conflicting results. We therefore determined the urinary excretion of AVP, a better index of AVP
production, after osmotic stimulus in hyper- and hypothyroid rats.
Key words: arginine-vasopressin, digoxin-like immunoreactive factor, electrolytes, hyperthyroidism, hypothyroidism, urinary excretion.
Abbreviations: AVP, arginine-vasopressin; DLIF, digoxinlike immunoreactive factor; T3, tri-iodothyronine; T4,
thyroxine; UAv,V, urinary excretion of argininevasopressin; U,,,, V, urinary excretion of digoxin-like
immunoreactive factor; U, V, urinary excretion of potassium; U,,V, urinary excretion of sodium; UV, urine
volume.
Correspondence:Dr F. Vargas, Departamento de Bioquimica,
Facultad de Medicina, E-18012 Granada, Spain.
472
F. Vargas et al.
The present study was designed to evaluate the renal
handling of sodium and water in hyper- and hypo-thyroid
rats treated for identical periods. As the kidney is able to
adjust to the disturbances that may result from thyroid
diseases [l], urine collected from control and rats with
thyroid dysfunction was studied under normal conditions
and after marked changes in urine water and sodium
concentration. These changes were produced by water
deprivation and by isotonic and hypertonic saline loads.
Moreover, we also examined the possible role of a
putative natriuretic hormone, measured as DLIF, in the
changes induced in urinary sodium excretion.
METHODS
Animal preparation
Male Wistar rats initially weighing 100-150 g were
maintained on standard chow and tap water except where
stated. The animals were divided into three groups:
control, hyperthyroid and hypothyroid rats ( n= 10 in
each group). Hyperthyroidism was induced by subcutaneous injection of T4 (Merck; 200 p g day-' kg-',
dissolved in 0.5 mol/l NaOH isotonic saline). Hypothyroidism was induced by the continuous administration of
0.02% (w/v) methimazole (Sigma)via the drinking water.
The effectiveness of these treatments was assessed by
comparing rectal body temperature, serum T4 concentration, serum tri-iodothyronine (T3)concentration, heart
rate and the final body weight of control and treated rats.
Rectal body temperature was measured with a small lubricated probe connected to a thermometer (Yellow Springs
Instruments, Yellow Springs, OH, U.S.A.). Blood pressure
and heart rate were recorded as described below.
Experimental protocol
Five weeks after treatment with T4 or methimazole,
control, hyperthyroid and hypothyroid rats were placed
in individual metabolic cages with food and tap water
provided ad libitum. After a 48 h adaptation period, four
tests were performed. Urine samples were taken after: (a)
24 h under normal conditions, (b) 24 h of water deprivation with free access to food, (c) 5 h of an intraperitoneal
isotonic saline [0.9% (w/v) NaCl] load and 5 h of an intraperitoneal hypertonic saline [3% (w/v) NaCl] load.
The water deprivation test was performed on the day
after 24 h under normal conditions. A 48 h recovery
period in the metabolic cages between the water deprivation test and the isotonic saline load was allowed. Isotonic
and hypertonic saline solutions were administered at a
rate of 3 m1/100 g body weight, with an interval of 24 h
between each saline load. During the 5 h of urine collection, rats were deprived of food and water. Before and
after each urine collection, light suprapubic pressure was
applied to ensure emptying of the bladder. The following
parameters were measured in urine samples: urine volume
( U V ) , urinary sodium (U,,V), potassium (U,V), AVP
( U,,,V)
and digoxin-like immunoreactive factor
(U,,,, V ) excretion, and urinary osmolality ( Uos,,,).
After the urinary studies had been completed, the
carotid artery was cannulated. After a 24 h recovery
period, mean arterial pressure and heart rate were
recorded directly (Bell and Howell type 4 transducer
connected to a two-channel Devices MX2 recorder).
When blood pressure had stabilized (30 min), blood
samples from the carotid catheter were taken for determination of serum electrolyte, T3and T4concentrations and
serum osmolality.
Cannulation procedures
Rats were anaesthetized with sodium pentobarbital
(Nembutal, Serva, Heidelberg, Germany; 40 mg/kg intraperitoneally). A polyethylene catheter (PE-10 connected
to PE-SO), containing 100 units of heparin (Leo, Madrid,
Spain) in isotonic sterile NaCl solution, was inserted into
the right carotid artery for either direct blood pressure
measurements or for extracting blood samples. The
catheter was placed subcutaneously and exteriorized at
the dorsum of the neck.
Analytical procedures
DLIF was quantified in unextracted urine samples by
r.i.a. using a commercially available kit (Digoxin MAIA
kit; Biodata S.p.A., Milan, Italy) in which the sensitivity
was enhanced as previously described [lo]. The lower
limit of detection for urine was 83 pg/ml, with intra- and
inter-assay coefficients of variation of 4.9% and 7.7%,
respectively. AVP was measured by r i a . in unextracted
urine; the intra- and inter-assay coefficients of variation
were 6.8% and 10.5%, respectively. Serum T4 and T3
levels were determined with r i a . kits purchased from
Diagnostic Products Corporation (Los Angeles, CA,
U.S.A.). Serum and urinary sodium and potassium
concentrations and osmolality were measured on the day
by flame photometry (Corning Instruments 435,
Halstead, Essex, U.K.) with lithium as the internal
standard and by freezing point depression (Automatic
Osmometter, Osmette A; Precision Systems Inc.,
Sudbury, MA, U.S.A.), respectively.
Statistical analysis
An analysis of the nested design was carried out [14,
151 to compare groups and tests; the design had two fixed
effect factors (group and test) and one random effect
factor (the rat), this factor being nested in the group.
When the different tests for factors and the group-test
interactions were significant, the groups were compared
at different tests using the Bonferroni rule to keep the
overall error as small as possible. The Welch approximation to Student's r-test was performed when the variance
was unhomogeneous under the Bonferroni rule. Comparisons of each biological parameter were carried out
with one-way analysis of variance. When the overall
analysis of variance was significant, we then performed
painvise comparisons by Bonferroni's method.
Digoxin-like immunoreactivefactor and arginine-vasopressin in hyper- and hypo-thyroid rats
thyroidism. There were no significant differences in mean
arterial pressure, serum - sodium and potassium
concentrations and Serum osmolality when hyperthyroid
and hypothyroid
rats were compared with control rats.
__ -
RESULTS
Effects of T, or methimazole administration on biological
parameters in the rat (Table 1)
Animals given T4 or methimazole for 5 weeks gained
significantly less weight than their matched controls
during this period. Heart rate, rectal temperature and
serum T, and T3levels were decreased in hypothyroid rats
and were increased in hyperthyroid rats. Hence rats given
methimazole for 5 weeks developed characteristic
manifestations of hypothyroidism, whereas those
receiving thyroxine for a similar period developed hyper-
U v U,,,, UN,Vand UK V(Tab1e 2)
In hyperthyroid rats UV was increased under normal
conditions and was reduced after the hypertonic saline
load when compared with control rats. In hypothyroid
rats no significant differences were found in UV,except
for a reduction after the isotonic saline load. Obviously,
Table 1. Biological parameters of hypothyroid (treated with 0.02%methimazole in drinking
water), control and hyperthyroid (treated with T,, 200 pg day-' kg- ',subcutaneously) rats
Values are expressed as means k SEM.
Hypothyroid
rats
n
Body wt. (g)
Mean arterial pressure (mmHg)
Heart rate (beats/min)
Rectal temperature ("C)
Serum T4concn. (pg/dl)
Serum T3concn. (ng/dl)
Serum Na+ concn. (mmol/l)
Serum K + concn. (mmol/l)
Serum osmolality (mosm/kg)
10
277.5f24.3
94.3f 1.9
321.0 f7.7
38.1 fO.08
0.5 f 0.04
19.2 k 2.2
142.0 k0.81
4.1 f0.06
288.2 f3.0
Control
rats
1'
<0.01
NS
< 0.05
<0.05
< 0.00 1
< 0.001
NS
NS
NS
P
10
340.7f10.2
102.0f4.5
361.5 f 8.8
38.4f0.08
3.5 f 0.3
48.4 f 6.0
141.8f0.5
4.2 f 0.13
288.2f 1.6
<0.01
NS
<0.01
<0.05
< 0.001
< 0.00 1
NS
NS
NS
Hyperthyroid
rats
10
249.0f31.0
116.8f6.7
471.1 f 16.00
39.2f0.07
34.9 f 8.9
344.0 f 18.00
140.8 k 0.85
4.3 f0.09
284.9f2.4
UNavU K Vand Na+/K+ ratio, in control (C), hyperthyroid (T,) and
hypothyroid (Me) rats
These parameters were measured after normal conditions (NC), water deprivation (WD), an
isotonic saline (0.9% NaCI) load (ISL)and a hypertonic saline (3%NaCl) load (HSL).Values are
expressed as means k SEM ( n= 10, each group). Statistical significance: *P< 0.05, **P< 0.01,
***P< 0.001 compared with control rats.
Table 2. U,, U,,,,
WD
ISL
HSL
221 f 38
161f6
499 fso**
81f11
121 f 4
106f4
115f26**
387 f 53
222 f 33
621 f 52
676 f 32
428 f 33**
1342f 165
1698 k 78
984 f 123*
2087f 171
2121 5 8 1
2722 f79*
1755 f 223**
736 k 43
1485 f 180*
NC
U V ( p 1 h - ' 100 g-l body wt.)
Me
C
T.l
Uom (mosm/kg)
Me
C
T4
U,,V(,umol h - l 100 g-'
body wt.)
Me
C
7.4
+
975 f 54
1011 f 3 0
1534f54*
12f0.8
20 f 0.8**
13 f 1.2
18fO.5
12 f 0.6**
19 3.0
35 f 5.9
14 f3.2*
191 f8.8
198f8.3
130 f8.5***
30 f 1.5
30 f 1.9
66 f 2.3**
2 0 f 1.7*
36 f 1.3
44 f 1.4
19+ 1.0*
41 f 3.3
40f4.1
50 1.7**
76 f 2.6
80 f 3.9
0.64 +0.03*
0.51 fO.01
0.28+0.01**
0.97+0.10*
0.87 f 0.07
0.32 +0.04**
3.82+0.21***
2.62 f0.09
1.62 f 0.07**
l o + 1.0
UKV(pmo1 h-' 100 g-'
body wt.)
Me
C
T4
Na+/K+ ratio
Me
C
T'l
0.33 f0.03
0.32 f0.02
0.31 fO.01
473
+
474
F. Vargas et al.
changes in the opposite direction were found in Uo,, in
all three groups. No clear pattern in U,, V was seen in rats
with thyroid dysfunction when the results of the different
tests were compared with those in control rats. Thus
hyperthyroid rats showed a higher U,,V after 24 h under
normal conditions, and a significant reduction in U,,V in
the remaining tests. No differences were found between
hypothyroid and control rats. Hypothyroid rats showed a
reduction in U, V in all tests.when compared with control
rats, although the differences were not significant under
normal conditions. Hyperthyroid rats showed an elevation after 24 h under normal conditions. The Na+/K+
ratio was higher and lower in hypo- and hyper-thyroid
rats, respectively, when compared with control rats,
except after 24 h under normal conditions. Uo,, was significantly increased in hyperthyroid rats in all tests except
under normal conditions. However, in hypothyroid rats,
only an increased U,,,, was seen after the isotonic saline
load.
significant increase (P<O.OOI) in UAv,,V with respect to
normal conditions or the isotonic saline load in all three
experimental groups, these responses being higher in
hyperthyroid rats and lower in hypothyroid rats with
respect to that observed in control rats.
u,,, VWg. 1)
Diuresis and U,, V
In hyper- and hypo-thyroid rats UAvl,Vdid not significantly differ from that in control rats under normal conditions and after the isotonic saline load. Both water
deprivation and the hypertonic saline load produced a
-2
-
400r(a)
"
***
NC
WD
ISL
In control rats U,,,,V was increased after the isotonic
saline load with respect to normal conditions and water
deprivation, although the greatest increase was recorded
after the hypertonic saline load ( P < O . O O l compared with
normal conditions and water deprivation). This pattern
was not observed in the hyperthyroid group, which
showed the highest U,,,,,V under normal conditions.
Hypothyroid rats also showed the highest U,,,,, V after
the hypertonic saline load, as did control rats.
DISCUSSION
Several studies provide evidence that hypothyroidism
in the rat is accompanied by increased diuresis and natriuresis under basal conditions [5, 13, 161, after water
deprivation [l]or after an isotonic saline load [17, 181.
This tendency toward sodium loss has been implicated as
a mechanism by which hypothyroidism prevents experimental arterial hypertension [ 11. Our results, however
(Table 2), d o not support these findings. The discrepancy
may be due to the different protocols used, as many as
there are authors, with changes in such fundamental
factors as duration of hypothyroidism, the use of
anaesthetic [5] and the use of loads of the same volume as
in control rats [17], without taking into account the
reduced body weight of hypothyroid rats. On the other
hand, other authors, using equally varied protocols, found
no elevation in diuresis or natriuresis [2,3] in hypothyroid
rats.
Na+/K+ ratio and U,,,,
x
0
VWg. 1)
HSL
s
TI
u,,,,
200
.,
NC
WD
ISL
HSL
Fig. 1. UAv, V ( a ) and U,,,,, V ( b )in control ( o ) ,hyperthyroid ( m ) and hypothyroid ( m ) rats under normal
conditions (NC), after water deprivation (WD) and after
isotonic (ISL) and hypertonic (HSL) saline loads. Values
are means k SEM. Statistical significance: * P < 0.05,
**P< 0.01, ***P< 0.001 compared with control rats;
t P < 0.05, ttP< 0.01 compared with normal conditions.
Our results show that the N a + / K +ratio was higher and
lower in hypo- and hyper-thyroid rats, respectively, when
compared with control rats, except after 24 h under
normal conditions. In this sense, a high Na+/K+ ratio and
a reduction in V,V have also been recognized in hypothyroid rats fed a diet deficient in sodium and potassium
[I91 or after a saline load [2]. Nevertheless, either no
significant differences [6, 181 or contradictory results
have been reported, depending on the method of induction of hypothyroidism [ 171. On the other hand, increases
in kaliuresis related to the T4 dose have been found in
hyperthyroid rats after 24 h of water deprivation [I].
The data on U,,, indicated that concentrating ability
was not impaired in methimazole-treated rats, as has been
previously reported in other models of experimental
hypothyroidism [4, 51. However, hyperthyroid rats
showed an increased concentrating capacity after water
deprivation.
Digoxin-like immunoreactive factor and arginine-vasopressin in hyper- and hypo-thyroid rats
UAvpVwas used as an index of AVP production under
normal conditions and after the different stresses [lo, 201.
Contradictory results have been observed with respect to
plasma AVP levels in hypothyroid rats [12, 13, 211. We
found no significant differences in UAvpV in the three
experimental groups under normal conditions and after
the isotonic saline load. However, hyper-responsiveness
in hyperthyroid and hyporesponsiveness in hypothyroid
rats occurred t o stimuli such as 24 h of water deprivation
or a hypertonic saline load. T h e reduced UAv,V after the
hypertonic saline load in hypothyroid rats agrees with the
data obtained in plasma by Ali et al. [12]. These authors
showed that in propylthiouracil-treated rats under a salt
load (5'/0, 2 m l / l O O g body weight), plasma AVP levels
a r e lower than those i n normal rats throughout t h e time
course of AVP release. However, no data are available (to
our knowledge) on AVP values in hyperthyroid rats under
normal conditions or after stimulation.
Several authors have described an endogenous factor
with digoxin-like immunoreactivity, measurable by
digoxin r.i.a.s in the plasma and urine of humans and
animals [lo, 221. T h e relationship between these factors
and sodium metabolism has been observed in several
situations [lo, 23,241 in humans and animals. The higher
UDLIFVafter the hypertonic saline load in control and
hypothyroid rats agrees with these previous observations.
However, a clear dissociation between UD,,V and U,,V
was observed in hyperthyroid rats. Dissociations have
also been observed between digoxin immunoreactivity
and natriuresis [25] and with other biochemical [26] and
pharmacological [27] properties of digoxin-like factor.
T h e changes in UD,,V in the three experimental
groups were similar t o those observed in diuresis: the
highest UI,LlFV in control and hypothyroid rats was
obtained after the hypertonic saline load, and in hyperthyroid rats, UDUFVwas highest under normal conditions.
In these situations, the highest values of UV were also
observed. T h e s e data agree with a previous report that
showed UDL,FV to b e related to UV in humans [25].
Moreover, it should be recalled that this putative natriuretic factor, in spite of intensive investigation, has not
been clearly identified, and different workers have used
different preparations of samples and antisera t o measure
this factor.
ACKNOWLEDGMENTS
~
We thank Ms Karen Shashok for her help with the English
in this paper.
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