1-112
Deoxycorticosterone Hypertension in the
Intact Weanling Rat Without Salt Loading
Carmen Rodriguez-Sargent, Ivette Torres-Negron,
Jose L. Cangjano, and Manuel Martinez-Maldonado
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Deoxycorticosterone (DOC) hypertension in the rat is generally induced in rats at an age of
approximately 3 months. Both uninephrectomy and a high sodium diet are necessary, however,
to induce DOC hypertension. Considering the inability of the developing kidney to adequately
excrete a sodium load, we studied the possibility that DOC alone might induce hypertension
when treatment is initiated in rats at the age of 21 days. The contribution of volume expansion
as a factor mediating the pressor response to DOC was assessed in rats given a high sodium diet
instead of DOC. Systolic blood pressure increased in DOC-treated rats within 3 weeks.
Although systolic blood pressure also increased in rats on a high sodium diet, the increase was
transient and of a lesser magnitude than that observed in DOC-treated rats. The rise in blood
pressure in both groups of rats was associated with suppression of plasma renin activity and
aldosterone concentration. Furthermore, extracellular fluid volume was similarly increased in
DOC-treated rats and rats given a high sodium diet. Consistent with these data, DOC-treated
rats showed an exaggerated natriuretic response to acute saline loading as compared with a
vehicle-treated control group. Discontinuation of DOC treatment after 5 weeks led to
normalization of all variables studied including blood pressure. Yet, when DOC was continued
for 8 weeks, stopping treatment did not lower blood pressure despite normalization of the
renin-angiotensin-aldosterone system and the natriuretic response to saline loading. In contrast,
discontinuation of the high sodium diet after 8 weeks normalized blood pressure. These data
show induction of DOC hypertension in the developing rat without reduction of renal mass or
high sodium intake. Furthermore, the data suggest that volume expansion might participate in
the initiation but not in the maintenance of hypertension in this experimental model.
{Hypertension 1990;15(suppl I):I-112-I-116)
D
eoxycorticosterone (DOC)-salt hypertension is a widely studied model of mineralocorticoid-induced low renin hypertension
associated with transient extracellular fluid volume
expansion.1-2 This model of hypertension is generally induced in 3-month-old uninephrectomized rats
fed a high sodium diet. Both uninephrectomy and
chronic sodium loading exacerbate steroid-induced
From the Veterans Administration Hospital and University of
Puerto Rico School of Medicine, San Juan, Puerto Rico.
Supported by grants of the Merit Review System of the
Veterans Administration Central Office and by a grant-in-aid of
the Puerto Rico Chapter of the American Heart Association.
Submitted by I. Torres-Negr6n in partial fulfillment of the PhD
degree requirements of the Department of Pharmacology and
Toxicology, University of Puerto Rico School of Medicine, San
Juan, Puerto Rico.
This manuscript from a Guest Editor was given to J. Carlos
Romero, also a Guest Editor of the supplement, for review by
expert referees, for editorial decision, and final disposition.
Address for reprints: Carmen Rodriguez-Sargent, PhD,
Research Service (151), Veterans Hospital, One Veterans Plaza,
San Juan, PR 00927-5800.
fluid volume expansion and, thus, help produce
sustained hypertension.
Although increased susceptibility to DOC-salt
hypertension3 and to chronic hypertonic sodium
loading per se4 have been demonstrated in the
prepubertal rodent, the induction of irreversible
mineralocorticoid hypertension in the absence of
chronic sodium loading and reduction of renal mass
has not been previously described in the rat. Considering the relative inability of the kidneys to
adequately excrete a salt load during the neonatal
and weanling periods of development,5'6 administration of DOC at this age might result in a greater
degree of volume expansion than that observed in
the mature rat.
Several studies have demonstrated the injurious
effects of increased dietary sodium intake in prepubertal rat.5-6 The administration of hypertonic saline
to weanling rats, for example, results in the development of hypertension, whereas under the same
conditions, blood pressure does not rise in the
mature rat.6 It has been postulated that the lack of a
Rodriguez-Sargent et al A New Model of Deoxycorticosterone Hypertension
circulating natriuretic hormone in the prepubertal rat
might partially explain both the reduced ability of
developing kidneys to excrete sodium as well as the
hypertensinogenic effects of chronic saline loading
observed in weanling rats.6-7 Based on such observations, it might be anticipated that any manipulation
leading to extracellular fluid volume expansion might
produce greater effects in the weanling than the
mature rat. In the present study, we examined the
possibility that long-term administration of DOC
alone to rats from the age of 21 days might induce
hypertension. We also evaluated long-term changes
in blood pressure when DOC treatment was discontinued in separate groups of rats. The importance of
fluid volume expansion in producing changes in
blood pressure was evaluated through chronic isotonic saline loading in rats of the same age.
Downloaded from http://hyper.ahajournals.org/ by guest on July 12, 2017
Methods
A total of 85 normal Wistar rats were selected
from litters limited in size at birth to 6-8 rats. All
rats were housed in a room with an ambient temperature of 26° C and a 12-hour light/dark cycle. At
an age of 21-23 days, the rats were divided at
random into three age- and sex-matched groups.
Thirty rats were given DOC either in biweekly
subcutaneous injections (15 rats; 35 mg/kg body
wt/wk in oil) or through pelleted DOC implants (15
rats) (Innovative Research of America, Toledo,
Ohio) providing a comparable but constant dose.
Another 30 rats were used as control groups and
received either biweekly subcutaneous oil injections (15 rats) or placebo pellets (15 rats). The
remaining 25 rats were placed on a high salt diet
(0.9% saline, replacing drinking water), but these
rats were not given DOC. Except for the high salt
group, rats were maintained on a standard chow
diet (0.46% sodium) and tap water ad libitum. When
the rats were 35 days old, systolic blood pressure
(SBP) was measured by tail-cuff plethysmography.
At this age, the use of smaller restraining cages and
tail cuffs is necessary to achieve normal and reproducible SBP measurements. Indeed, the earliest age
at which we were able to make SBP measurements
in these rats was at 28 days. SBP was expressed as
the mean value of 10 consecutive measurements.
Half of the rats were then transferred to small
rodent metabolism cages, and after a 48-hour period
of adaptation, three consecutive 24-hour urine samples were collected under oil for the determination
of urine flow rate and urinary concentrations of
sodium and potassium. Food and water intake as
well as body weight were also measured daily. The
intake and urinary excretion of sodium and potassium were then calculated per 100 grams body
weight. Afterwards, a similar 3-day balance study
was performed in the remaining rats from each
group. SBP was then measured weekly throughout
the remainder of the study. At the age of 56 days,
beginning at 8:00 AM and after 45-60 seconds exposure to ether, rats were bled by heart puncture for
1-113
the determination of plasma renin activity, plasma
aldosterone concentration, and hematocrit. Although ether anesthesia has been shown to stimulate plasma renin activity,8 the reproducibility of
renin levels measured in the ether-anesthetized rat
has been previously demonstrated.9 In addition, it
has been shown that changes in renin activity can
be detected despite the use of ether when exposure
is limited to 45-60 seconds.9 After the initial bleeding, an additional 2-day balance study was again
performed in all rats to permit recovery from the
bleeding procedure. On completion of this study,
the group of rats treated with DOC and the control
group were fasted overnight in metabolism cages in
preparation for an acute saline-loading study. The
next morning, bladder emptying was induced in
each rat by ether sniffing, and voided urine was
discarded. The rats were then returned to their
metabolism cages for a 4-hour control urine collection period. On completion of the control collection, bladder emptying was again induced and
voided urine added to respective control collection
samples. The rats were then weighed, and a volume
of isotonic saline equal to 3% of body weight was
administered to each rat by gavage. The rats were
returned to metabolism cages for a 2-hour urine
collection. Afterward, bladder emptying was
induced and voided urine added to the postload
sample. The volume of each sample and urinary
concentrations of sodium and potassium were then
measured. No further treatment was given to half
the rats from each group, and these rats were
allowed to recover for 1 month. The remaining rats
were treated for an additional month. Once treatment was discontinued, these rats were also
allowed to recover for 1 month. During the last
week of the respective recovery periods (ages, 86
days or 115 days), all rats were again bled for the
determination of plasma renin activity, plasma
aldosterone concentration, and hematocrit.
Another 2-day balance study was then performed in
all rats. Additionally, the acute saline-loading study
was repeated in rats previously treated with DOC
and in the control group. Separate groups of six
DOC-treated rats, six rats given a high sodium diet,
and six control rats were also studied for measurement of extracellular fluid volume during respective
treatment regimens. These rats were studied after 1
month of treatment.
Urinary concentrations of sodium and potassium
were measured by flame photometry. Plasma renin
activity was measured by the radioimmunoassay of
angiotensin I generated from endogenous renin substrate per milliliter plasma per hour during sample
incubation at 37° C in the presence of angiotensinase inhibitors. Radioimmunoassay materials were
purchased from New England Nuclear, Boston,
Massachusetts. Plasma aldosterone concentration
was measured by direct radioimmunoassay. using
materials purchased from Abbott Laboratory, San
Juan, Puerto Rico. Cross-reactivity of the antialdo-
1-114
Supplement I
Hypertension
Vol 15, No 2, February 1990
TABLE 1. Systolic Blood Pressure During and After Treatment
With Deoxycorticosterone
DURING
TREATMENT
1 MONTH AFTER
STOPPING TREATMENT
Age (days)
Group
Control
DOC (stopped at
age 56 days)
DOC (stopped at
age 86 days)
High Na+ diet
(stopped at age
86 days)
42
56
86
122±3
124±3
131±3
153±8*t
170±8*t
128±5
169±4*t
180±4*t
191±3*t
145
20-
125±3
189±2't
10-
132±3*
142±3t
151±9
131±9
•Values significantly different (/?<0.001) from corresponding
control value.
tValues significantly different (p<0.01) from corresponding
high Na+ diet value.
^Values significantly different (p<0.05) from corresponding
control value.
Downloaded from http://hyper.ahajournals.org/ by guest on July 12, 2017
sterone antibody has been reported at 100% with
aldosterone and less than 0.01% with DOC. Extracellular fluid volume was measured with [3H]inulin
dilution.10 Each rat was bilaterally nephrectomized
precisely 30 minutes before administration of
[3H]inulin, and this procedure resulted in only minimal loss of peritoneal fluid. Statistical analysis of
the data was done with Student's t test among
groups and paired t analysis within groups. Differences between values were considered statistically
significant when/? values were less than or equal to
0.05. All values were expressed as the arithmetic
mean±SEM. When no differences were observed
between 5 and 8 weeks of DOC-treatment data from
these two groups were pooled.
This protocol was reviewed and approved by
the local institutional Animal Care and Use Committee. All procedures comply with the standards
for the care and use of animal subjects as stated in
Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources,
National Academy of Sciences, Bethesda, Maryland, 1985, pp 3-55).
Results
SBP shown in Table 1 rose gradually in DOCtreated rats and was significantly increased at the
age of 42 days (3 weeks of DOC treatment) as
compared with values observed in the control
group. When DOC treatment was discontinued in
rats at the age of 56 days, blood pressure levels
fell, and 1 month later, blood pressure in this
group of rats (age, 86 days) was similar to that in
control rats. In contrast, when DOC treatment
was prolonged for an additional 1 month, SBP
remained elevated after DOC was discontinued.
Consequently, under these conditions, the DOCinduced rise in SBP was irreversible. In response
to a high salt diet, blood pressure also increased.
Under these conditions, however, blood pressure
remained unchanged in seven of the rats studied,
whereas increased blood pressure was observed in
<
I
T
FIGURE 1. Scatterplot of plasma renin activity during
treatment with deoxycorticosterone (•), high sodium diet
(A), or vehicle (control) (o), and 1 month after treatment
was discontinued regardless of length of treatment
period. *Denote values significantly different (p<0.01)
from corresponding control value.
all rats during DOC treatment regardless of the
duration of treatment. Additionally, the rise in
blood pressure during high salt intake was not only
labile from week to week in any given rat but also
transient in all of the rats studied. In contrast, the
rise in blood pressure in response to DOC was
sustained in all rats during treatment. As shown in
Figure 1, plasma renin activity was lower in rats
given either DOC or high salt diet as compared
with control rats during treatment. Regardless of
the duration of treatment, when treatment was
discontinued for 1 month, plasma renin activity
rose in rats that had previously been given either
DOC or a high salt diet such that renin activity was
similar to values observed in control rats. Plasma
aldosterone concentration (Figure 2) reflected levOURING
TREATMENT
Z
1 MONTH AFTER
STOPPING TREATMENT
20-
1S-
10-
5-
K
FIGURE 2. Scatterplot of plasma aldosterone concentration during treatment with deoxycorticosterone (•), high
sodium diet (A), or vehicle (control) (o), and 1 month
after treatment was discontinued regardless of length of
treatment period. *Denote values significantly different
( p<0.01) from corresponding control value.
Rodriguez-Sargent et al A New Model of Deoxycorticosterone Hypertension
DURING
DOC
CONTROL
TREATMENT
SALT
1-115
1 MONTH AFTER
STOPPING DOC
LOAD
t Jt
C0NTR0L
t
LOAD
t
1
200-
SALT
160-
1
160120-
r
120-
(A
\
z
3
z
80-
/
--*
80-
40-
7
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FIGURE 3. Scatterplot showing urinary sodium excretion before (Control) and after (Salt Load) intragastric
saline loading during treatment with deoxycorticosterone
(•) or vehicle (o). "Denote values significantly different
(p<0.01) from corresponding values in vehicle-treated
rats. ^Denote values significantly different (p<0.001)
from corresponding control value within same group.
els of renin activity under all conditions studied.
Aldosterone concentration was lower in DOC and
high salt groups during treatment as compared
with values in the control gToup. When treatment
was discontinued for 1 month, plasma aldosterone
in all rats previously treated with either DOC (5 or
8 weeks' treatment) or a high salt diet rose to
levels observed in the control group. The results
of acute isotonic saline-loading studies in DOCtreated and control rats during the last week of
respective treatment periods and 1 month after
discontinuing DOC are summarized in Figures 3
and 4. An exaggerated natriuretic response to
intragastric saline loading was observed in rats
given DOC for 5 weeks or for 8 weeks as compared with respective control groups (Figure 3).
Although the kaliuretic response to saline loading
(not shown) tended to be blunted in DOC-treated
rats, this difference was not statistically significant. Independent of the duration of DOC administration, when treatment was suspended and rats
allowed to recover for 1 month, the natriuretic
(Figure 4) and kaliuretic (not shown) responses to
saline loading were similar in rats previously
treated with DOC and the respective control
groups. As shown in Table 2, extracellular fluid
volume was similarly increased in DOC and high
salt diet groups after 1 month of treatment as
compared with values in control groups.
FIGURE 4. Scatterplot showing urinary sodium excretion before (Control) and after (Salt Load) intragastric
saline loading, 1 month after deoxycorticosterone (•) or
vehicle (o) treatment was discontinued regardless of
length of treatment period. fDenote values significantly
different (p<0.01) from corresponding control value
within same group.
reduction of renal mass or a high sodium diet. The
hypertensinogenic effects of this steroid, however,
were dependent on the length of the treatment
period. Prolonged treatment for 2 months led to a
sustained rise in blood pressure even after DOC
administration was discontinued. Thus, in contrast
to the mature rat in which chronic saline loading
and uninephrectomy are necessary to induce DOC
hypertension, in the weanling rat, DOC treatment
alone led to the development of hypertension. The
low renin-low aldosterone state observed during
steroid treatment was associated with increased
extracellular fluid volume, suggesting the possibility
that such fluid volume expansion might participate
in the initial pressor response to DOC. Additionally, the exaggerated natriuretic response to an
acute intragastric saline load during DOC treatment
is also consistent with expanded extracellular fluid
volume. Thus, possible explanations for our findings are that the developing rat might be more
sensitive to changes in extracellular volume, that
the degree of steroid-induced volume expansion
might be greater in the weanling than in the mature
rat, or that both sensitivity to and degree of volume
TABLE 2.
Extracellular Fluid Volume During Treatment
Group
DOC
High sodium diet
Control
Discussion
These studies demonstrated the induction of
DOC hypertension in the developing rat without
Y
ECFV (ml/100 g body wt)
26.3±1.0*
27.2±1.3*
22.1±0.9
ECFV, extracellular fluid volume; DOC, deoxycorticosteronetreated rats.
•Denote values significantly different (p<0.05) from values in
control (vehicle-treated) rats.
1-116
Supplement I
Hypertension
Vol 15, No 2, February 1990
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change might be involved. Another possibility is
supported by studies demonstrating that, in contrast to the mature rat, during the process of renal
maturation in the developing rat, mineralocorticoids stimulate sodium-potassium-dependent adenosine triphosphatase (Na+,K+-ATPase) activity in
the proximal tubule.11 Such a steroid-induced
increase in renal Na+,K+-ATPase activity in a nephron segment receiving the entire filtered load of
sodium might be expected to result in greater
sodium and water reabsorption than in the adult rat
where proximal tubular Na+,K+-ATPase activity is
not influenced by mineralocorticoids. 12 Thus,
administration of DOC might lead to a more marked
expansion of extracellular fluid volume in the weanling rat than in the mature rat.
We evaluated the possibility that increased susceptibility to steroid-induced hypertension during
development might reflect a greater sensitivity to
extracellular fluid volume expansion. Chronic
saline loading through the administration of a high
sodium diet to weanling rats led to a rise in SBP.
These data suggest that the developing rat might be
more sensitive to extracellular fluid volume expansion as compared with the mature rat in which a
high sodium diet fails to influence blood pressure.
Consequently, the rise in blood pressure during
chronic saline loading in the present study is consistent with the concept of enhanced susceptibility
to hypertensinogenic effects of excess dietary
sodium during development.6 This rise in blood
pressure during high sodium intake, however, was
less in magnitude than that observed in response to
DOC despite a similar low renin-low aldosterone
state as well as a similar increase in extracellular
fluid volume in both groups of rats during the
treatment period. In addition, when the high sodium
diet was discontinued, blood pressure fell to control
levels. Consequently, although volume expansion
can contribute to the initial rise in blood pressure
during DOC treatment, it does not fully account for
the increased sensitivity to DOC in weanling rats.
Extrarenal effects of steroids might contribute to
the increased hypertensinogenic effects of DOC in
the weanling rat. Such extrarenal effects of adrenocortical steroids include enhanced vascular
reactivity,13 increased vascular smooth muscle
sodium permeability,14 as well as elevation of circulating vasopressin levels.15 The possibility of agedependent changes in extrarenal sensitivity to DOC
cannot be evaluated on the basis of the present
study. Regardless of this, we have described a
model of DOC hypertension in which neither saline
loading nor uninephrectomy are required because
of enhanced sensitivity of the developing rat to this
adrenocortical steroid.
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KEY WORDS • hypertension • deoxycorticosterone • renin •
mineralocorticoid • kidney
Deoxycorticosterone hypertension in the intact weanling rat without salt loading.
C Rodriguez-Sargent, I Torres-Negron, J L Cangiano and M Martinez-Maldonado
Hypertension. 1990;15:I112
doi: 10.1161/01.HYP.15.2_Suppl.I112
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