Reduced Uterine Perfusion Pressure During Pregnancy in
the Rat Is Associated With Increases in Arterial Pressure
and Changes in Renal Nitric Oxide
Barbara T. Alexander, Salah E. Kassab, M. Todd Miller, Sean R. Abram, Jane F. Reckelhoff,
William A. Bennett, Joey P. Granger
Downloaded from http://hyper.ahajournals.org/ by guest on June 17, 2017
Abstract—A reduction in nitric oxide (NO) synthesis has been suggested to play a role in pregnancy-induced hypertension.
We have recently reported that normal pregnancy in the rat is associated with significant increases in whole-body NO
production and renal protein expression of neuronal and inducible NO synthase. The purpose of this study was to
determine whether whole-body and renal NO production is reduced in a rat model of pregnancy-induced hypertension
produced by chronically reducing uterine perfusion pressure starting at day 14 of gestation. Chronic reductions in uterine
perfusion pressure resulted in increases in arterial pressure of 20 to 25 mm Hg, decreases in renal plasma flow (⬍23%)
and glomerular filtration rate (⬍40%), but no difference in urinary nitrite/nitrate excretion relative to control pregnant
rats. In contrast, reductions in uterine perfusion pressure in virgin rats resulted in no significant effects on arterial
pressure. Renal endothelial (⬍4%) and inducible (⬍11%) NO synthase protein expression did not decrease significantly
in the chronically reduced uterine perfusion pressure rats relative to normal pregnant rats; however, significant
reductions in neuronal NO synthase were observed (⬍30%). The results of this study indicate that the reduction in renal
hemodynamics and the increase in arterial pressure observed in response to chronic decreases in uterine perfusion
pressure in pregnant rats are associated with no change in whole-body NO production and a decrease in renal protein
expression of neuronal NO synthase. (Hypertension. 2000;37:1191-1195.)
Key Words: preeclampsia 䡲 hypertension, pregnancy 䡲 glomerular filtration rate 䡲 renal blood flow 䡲 plasma
䡲 endothelium 䡲 nitric oxide
P
reeclampsia is a multisystemic disorder of pregnancy
estimated to affect 5% to 10% of all pregnancies in the
United States.1,2 Although preeclampsia is one of the leading
causes of maternal death and the main contributor of prenatal
morbidity, the mechanisms responsible for this disorder are
unclear.1-3 Preeclampsia develops during pregnancy and
ceases after delivery, implicating the placenta as a primary
cause.1,4 Manifestations generally associated with preeclampsia include an increased responsiveness to vasoconstrictors,
increases in arterial pressure, decreases in glomerular filtration rate (GFR) and renal plasma flow (RPF), proteinuria, and
vascular endothelial damage.1,2,5
The initiating event in preeclampsia is suggested to involve
reduced placental perfusion, which leads to maternal endothelial cell dysfunction.1,3,4 The factors involved in mediating
the hypertension during preeclampsia are unknown and may
involve a delicate balance of vasoconstrictors and vasodilators of which nitric oxide (NO) may play an important
role.1,4,6 Evidence indicates that NO plays an important role
in mediating physiological changes during normal pregnan-
cy.6,7 Increases in regional blood flow, RPF, and GFR during
pregnancy are attenuated by systemic NO synthesis inhibition.8,9 Urinary excretion of cGMP, a second messenger of
NO, and nitrite/nitrate, metabolites of NO, are increased
during normal pregnancy.10 Therefore, as NO may play an
important role in normal pregnancy, NO deficiency may play
an important role in preeclampsia.2,6 It is unclear, however,
whether NO production is reduced during preeclampsia
because it is difficult to accurately assess the NO system in
humans. For example, measurement of whole-body NO in
clinical settings has yielded variable results caused by difficulties in controlling for nitrate intake.2 In addition, wholebody NO production may not be an accurate measure of the
NO system activity in specific tissues such as the kidneys.11,12
During normal pregnancy in the rat, increases in wholebody NO production and changes in renal hemodynamics are
associated with increases in renal protein expression of both
neuronal (nNOS) and inducible (iNOS) nitric oxide synthase
isoforms.13 Although the NO system has been well characterized during normal pregnancy in the rat, information
Received July 20, 2000; first decision September 11, 2000; revision accepted September 29, 2000.
From the Department of Physiology and Biophysics, Center for Excellence in Cardiovascular-Renal Research Center, University of Mississippi Medical
Center, Jackson.
Correspondence to Joey P. Granger, PhD, Department of Physiology and Biophysics, University of Mississippi Medical Center, 2500 N State St,
Jackson, MS 39216-4505. E-mail [email protected]
© 2000 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
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Hypertension
April 2000
regarding the activity of the NO system in animal models of
pregnancy-induced hypertension (PIH) is lacking. Thus, the
overall goal of this study was to assess whole-body NO
production under fixed nitrate intake conditions in a rat model
of PIH induced by chronic reductions in uterine perfusion
pressure. In addition, because the kidneys play a major role in
the long-term regulation of arterial pressure and because
abnormalities in the renal pressure-natriuresis relation have
been observed in all forms of hypertension examined to date,
another goal of this study was to assess the changes in renal
hemodynamics and renal protein expression of the NOS
isoforms that occur in response to reduced uterine perfusion
pressure (RUPP) in pregnant rats.
Methods
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All studies were performed in timed pregnant Sprague-Dawley rats
purchased from Harlan Sprague-Dawley Inc. Animals were housed 3
to a cage in a temperature-controlled room (23°C) with a 12:12-hour
light/dark cycle. All experimental procedures executed in this study
were in accordance with National Institutes of Health Guidelines for
Use and Care of Animals, and the Animal Care and Use Committee
at the University of Mississippi Medical Center approved all
protocols.
RUPP Model in Rat
Chronic reductions in uteroplacental perfusion in rats, previously
reported by Eder and McDonald,14 reduced uterine perfusion pressure by 35% to 45% during mid to late gestation by placing a silver
clip or a silk ligature around the aorta below the renal arteries. We
used a modification of this model to examine the role of NO in
mediating hypertension during chronic reductions in uterine perfusion pressure. All rats undergoing surgical procedures were anesthetized with 2% isoflurane.8 Pregnant rats entering the RUPP group
underwent the following clipping procedure at day 14 of gestation.
After a midline incision, the lower abdominal aorta was isolated and
a silver clip (0.203-mm ID) was placed around the aorta above the
iliac bifurcation. In preliminary studies, we have found this procedure to reduce uterine perfusion pressure in the gravid rat by ⬇40%.
Because compensation of blood flow to the placenta occurs in
pregnant rats through an adaptive increase in ovarian blood flow, we
also clipped branches of both the right and left ovarian arteries that
supply the uterus by using a silver clip (0.100-mm ID). When the
clipping procedure resulted in total reabsorption of the fetuses, rats
were excluded from data analyses. A total of 15 normal pregnant and
18 RUPP rats were used for data collection in the metabolic studies,
from which 8 normal pregnant and 10 RUPP rats were analyzed for
renal NOS protein expression. All rats undergoing metabolic studies
were surgically instrumented with catheters (PE 50 tubing) in the
carotid artery for blood pressure monitoring. A total of 12 normal
pregnant and 9 RUPP rats were used for renal and systemic
hemodynamic analyses.
The effect of chronic reductions in uterine perfusion pressure on
systemic arterial pressure was also examined in virgin rats. A total of
6 virgin rats underwent the same surgical procedures as in the RUPP
rats described above. Blood pressure in these virgin clipped rats was
compared with control virgin rats (n⫽7).
Measurement of Renal Hemodynamics and
Arterial Pressure in Conscious Rats
Renal hemodynamics and arterial pressures were determined in
conscious control pregnant (n⫽12) and RUPP (n⫽9) animals at day
19 of gestation. Similar surgical procedures for monitoring of blood
pressure were performed in virgin control and virgin clipped rats.
Measurements of renal hemodynamics and arterial pressures in
conscious rats were performed as previously described by the
authors.13
Figure 1. Measurement of MAP in model of RUPP in pregnant
rats. All data are expressed as mean⫾SEM.
Measure of Urinary Nitrite/Nitrate Excretion
Urinary nitrite/nitrate excretion rates were determined in both control
pregnant (n⫽15) and RUPP (n⫽18) animals fed a low nitrite/nitrate
diet (AIN76, ICN Pharmaceuticals Inc) for 5 days before collection.
Measure of urinary nitrite/nitrate was performed as previously
described by the authors.13
Determination of Urinary Protein Levels
Urinary excretion of protein was measured in both control pregnant
(n⫽15) and RUPP (n⫽18) animals with 24-hour urine collection and
storage of a final sample at ⫺20°C. Protein concentration was
determined with a Sigma Protein Determination kit (P5656, Sigma
Chemical Co).15
Measure of Plasma Estradiol 17-␤ and
Progesterone Levels
Plasma estradiol and progesterone concentrations were determined
by chemiluminescence immunoassay with the Chiron ACS-180
autoanalyzer (Chiron Diagnostics Corp). The assay sensitivity for
estradiol and progesterone was 10 pg/mL and ⬍0.1 ng/mL, respectively. The within-assay precision (%CV) was ⱕ5.0% and the
between-assay precision was ⱕ9.2%.
Isolation of Total Cellular Proteins and Western
Blot Analyses
Kidneys were removed from control pregnant (n⫽8) and RUPP
(n⫽10) rats at day 19 of pregnancy, quick-frozen in liquid nitrogen,
then stored at ⫺80°C. Isolation of total cellular proteins and Western
blot analyses of the renal NOS isoforms were performed as previously described by the authors.13
Statistical Analysis
All data are expressed as mean⫾SEM. Comparisons of control
pregnant rats with RUPP rats and comparisons between virgin
control and virgin clipped rats were analyzed by factorial ANOVA
followed by Scheffé’s test. A value of P⬍0.05 was considered
statistically significant.
Results
Effects of Chronic Reductions in Uterine Perfusion
Pressure on Mean Arterial Pressure and
Protein Excretion
Chronic reductions in uterine perfusion pressure in pregnant
rats resulted in significant increases in arterial pressure
relative to normal pregnant rats. Figure 1 illustrates the
increase in arterial pressure that was present in RUPP animals
as compared with control pregnant animals. Mean arterial
pressure (MAP) averaged ⬇126⫾7 mm Hg (P⬍0.05) in the
RUPP rats at day 19 of pregnancy. This was a significant
increase as compared with an average MAP of
Alexander et al
Nitric Oxide and Pregnancy-Induced Hypertension
Figure 2. Measurement of MAP in virgin rats with chronic
reductions in uterine perfusion pressure. All data are expressed
as mean⫾SEM.
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103⫾4 mm Hg observed in control pregnant rats. In contrast,
reductions in uterine perfusion pressure in virgin rats resulted
in no significant effects on arterial pressure relative to control
virgin rats (Figure 2). MAP averaged ⬇113⫾3 mm Hg in the
clipped virgin rats and 112⫾10 mm Hg in the virgin control
rats.
Although not significant, an increase in urinary protein
excretion was also observed in the RUPP model when
compared with normal pregnancy (108⫾15 and 15⫾2 mg/24
hours, respectively) (Figure 3). Intrauterine growth restriction, another hallmark of human preeclampsia, was also
evident as pup weights were slightly decreased in RUPP
animals relative to control pregnant animals (2.68⫾0.22 and
3.18⫾0.19 g, respectively). In addition, at day 19 of pregnancy, the RUPP model was also associated with decreased
litter size (7.3⫾0.48 and 10.438⫾0.63 pups, respectively,
P⬍0.05) as well as decreased body weight (248.4⫾7.14 and
307.9⫾0.36 g, respectively, P⬍0.05).
Effects of Chronic Reductions in Uterine Perfusion
Pressure on Renal Hemodynamics
The renal hemodynamic changes observed in the RUPP
animals relative to normal pregnant animals are illustrated in
Figure 4. Both GFR (1.29⫾0.42 and 2.21⫾0.14 mL/min,
respectively, P⬍0.05) and RPF (4.57⫾1.17 and 5.91⫾0.67
mL/min, respectively) decreased in the RUPP model relative
to normal pregnancy at day 19, with a significant reduction
occurring in GFR.
Figure 3. Measurement of urinary protein excretion in model of
RUPP in pregnant rats. All data are expressed as mean⫾SEM.
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Figure 4. Renal hemodynamic changes in a model of RUPP in
pregnant rats. All data are expressed as mean⫾SEM.
Effects of Chronic Reductions in Uterine Perfusion
Pressure on Urinary Nitrite/Nitrate Levels
Urinary nitrite/nitrate excretion was measured to estimate
whole-body production of NO. At day 19 of pregnancy,
excretion of nitrite/nitrate in the RUPP model (49.8⫾6.4
␮mol/24 hours) did not differ significantly compared with
normal pregnancy (46.4⫾5.3 ␮mol/24 hours) (Figure 5).
Effects of Chronic Reductions in Uterine Perfusion
Pressure on Plasma Levels of Estradiol 17-␤
and Progesterone
No significant changes were observed in plasma hormone
levels in the RUPP model. A slight but nonsignificant
decrease in plasma estradiol 17-␤ was observed in the RUPP
animals relative to normal pregnant animals (49.9⫾6.1 versus
62.4⫾8.9 pg/mL, respectively), whereas a slight but nonsignificant increase in plasma progesterone was observed
(107.7⫾12.9 versus 83.4⫾8.5 ng/mL, respectively).
Effects of Chronic Reductions in Uterine Perfusion
Pressure on Renal Protein Expression of
NOS Isoforms
Comparable levels of renal endothelial NOS (eNOS) protein
expression were found at day 19 of pregnancy in both the
RUPP and control pregnant animals (Figure 6). However,
renal protein expression of both iNOS and nNOS were
decreased at day 19 of pregnancy in the RUPP animals
relative to control pregnant animals (Figure 6). Renal iNOS
expression decreased by 11% in the RUPP model as com-
Figure 5. Nitrite/nitrate excretion rates in model of RUPP in
pregnant rats. All data are expressed as mean⫾SEM.
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April 2000
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Figure 6. Renal protein expression of renal NOS isoforms in rat
model of RUPP: Representative Western blot (A) and quantification of renal NOS isoform protein expression (B). NOS protein
expression levels are normalized relative to renal actin protein
expression levels. Standard error is calculated from ⱖ3 determinations from ⱖ3 independent experiments. All data are
expressed as mean⫾SEM.
pared with normal pregnancy, whereas renal nNOS expression decreased by 31% (P⬍0.05). Therefore, a significant
decrease in renal nNOS expression was observed in the
RUPP rats.
Discussion
One of the leading theories concerning the cause of
pregnancy-induced hypertension suggests a pathway starting
with inadequate trophoblast invasion of maternal spiral arteries leading to decreased placental perfusion. This in turn leads
to placental ischemia, resulting in placental release of factors,
subsequent maternal endothelial dysfunction, and then systemic vasoconstriction.1-4 Chronic reductions in uterine perfusion pressure in pregnant animals has been used by numerous investigators to study potential mechanisms of human
preeclampsia because it initiates the disorder at an early step
in the cascade described above.14,16,17 Elevations in arterial
pressure and proteinuria in response to chronic reductions in
uteroplacental perfusion in pregnant rats has been previously
reported by Eder and MacDonald.14 They reduced the uterine
perfusion pressure by 30% to 35% during mid to late
gestation by placing a silk ligature around the aorta below the
renal arteries. In this study, we used a modification of this
model to examine the role of NO in mediating hypertension
during chronic reductions in uterine perfusion. We placed a
clip on the abdominal aorta to reduce uterine perfusion
pressure by ⬇40 mm Hg in rats at mid-gestation. Because
compensation of blood flow to the placenta occurs in pregnant rats through an adaptive increase in ovarian blood flow,
we also placed silver clips on branches of both the right and
left ovarian arteries that supply the uterus. In this study, we
report that the RUPP model in the conscious rat is characterized by increases in arterial pressure and urinary protein
excretion, decreases in GFR and RPF, and intrauterine
growth restriction. Thus, the RUPP model in the rat has many
features similar to those of humans with PIH.
A major goal of this study was to determine whether
whole-body and renal NO production is reduced in the RUPP
model. Although a deficiency in NO production has been
suggested to play a role in PIH, clinical assessment of
whole-body NO production in preeclampsia has given rise to
varied results caused by differences in dietary intake, exercise, and duration of urine collection.11 In our study, 24-hour
urine collections representing steady-state levels of NO production on animals fed a low nitrite/nitrate diet were measured to provide a more accurate measure of whole-body NO
synthesis. No change in whole-body NO production as
determined by urinary nitrite/nitrate excretion was observed
in the RUPP animals. However, whole-body NO production
may not be indicative of the NO synthesis in specific tissues
such as the kidney.11,12
Because the kidneys play a major role in the long-term
regulation of arterial pressure and abnormalities in the renal
pressure-natriuresis relation have been observed in all forms
of hypertension examined to date, another goal of this study
was to assess renal NO production in the RUPP model. As a
means of investigating the NO system in the kidney, we
examined the renal protein expression of the NOS isoforms in
the RUPP model. We observed no difference in renal eNOS
protein expression at day 19 of pregnancy in the RUPP rats as
compared with control pregnant rats. However, renal iNOS
protein expression in RUPP animals was decreased by 11%
as compared with normal pregnancy and renal nNOS by 31%.
Thus, renal nNOS protein expression in the RUPP rats was
significantly decreased, and this decrease was associated with
a significant decrease in GFR of 40% and a decrease in RPF
of 23%.
Therefore, the significant reductions in renal hemodynamics observed in the RUPP model in the rat were associated
with significant reductions in renal expression of the nNOS
isoform. Recent studies suggest that NO produced by either
eNOS or iNOS does not play a significant role in mediating
the renal hemodynamic changes observed during normal
pregnancy.13,18,19 However, NO generated from nNOS has
been implicated in mediating the increase in hemodynamics
observed during normal pregnancy.13,18,20 Whether the decrease in renal nNOS protein expression is involved in
mediating the decrease in renal hemodynamics in the RUPP
model is unclear. We have recently reported, however, that
inhibition of nNOS with 7-nitroindazole significantly reduces
RPF and GFR in pregnant rats but not virgin rats.20 Therefore,
NO generated by nNOS in the kidney may be involved in the
long-term control of renal hemodynamic changes that occur
during normal gestation, and variations in renal nNOS expression may contribute to changes in renal hemodynamics
during preeclampsia.
Summary
We found that a chronic reduction in uterine perfusion
pressure in the pregnant rat was associated with significant
increases in arterial pressure and decreases in GFR. In
addition, decreases in RPF, proteinuria, and intrauterine
growth restriction were also observed in the RUPP animals.
Thus, the RUPP model in the rat closely resembles many of
the features observed in women with PIH. Although hyper-
Alexander et al
Nitric Oxide and Pregnancy-Induced Hypertension
tension in the RUPP model was not associated with significant reductions in whole-body NO synthesis, as assessed by
urinary excretion of nitrite/nitrate, significant reductions in
renal protein expression of the nNOS isoform were found.
These changes in renal nNOS expression may contribute to
changes in renal hemodynamics and possibly the hypertension observed during chronic reductions in uterine perfusion
pressure in the rat.
Acknowledgments
This work was supported by National Institutes of Health grants
HL-38499and HL-51971. The National Institutes of Health National
Research Service Award HL-10137-01 supported Dr Alexander. The
authors would like to thank Jason N. Herrington and A. Nicole
Rinewalt for excellent technical assistance.
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Reduced Uterine Perfusion Pressure During Pregnancy in the Rat Is Associated With
Increases in Arterial Pressure and Changes in Renal Nitric Oxide
Barbara T. Alexander, Salah E. Kassab, M. Todd Miller, Sean R. Abram, Jane F. Reckelhoff,
William A. Bennett and Joey P. Granger
Downloaded from http://hyper.ahajournals.org/ by guest on June 17, 2017
Hypertension. 2001;37:1191-1195
doi: 10.1161/01.HYP.37.4.1191
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