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THE EFFECT OF HYPOTHALMO-PITUITARY DISCONNECTION ON THE RENIN-
ANGIOTENSIN SYSTEM IN THE LATE GESTATION FETAL SHEEP
Kai Chen1, Luke C. Carey1, Jinfang Liu1, Nancy K. Valego2,
Stephen B. Tatter3, and James C. Rose1,2
Departments of 1Obstetrics/Gynecology, 2Physiology/Pharmacology, and 3Neurosurgery,
Wake Forest University School of Medicine, Winston-Salem, NC 27157-1066, USA
Running head: HPD & renin-angiotensin system activity in fetal sheep
Correspondence and reprint requests to:
James C. Rose, Ph.D.
Department of Obstetrics and Gynecology
Wake Forest University School of Medicine
Winston-Salem, NC 27157-1066
Phone: 336-716-4615
Fax: 336-716-6937
Email: [email protected]
Articles in PresS. Am J Physiol Regul Integr Comp Physiol (December 16, 2004). doi:10.1152/ajpregu.00560.2004
Copyright © 2004 by the American Physiological Society.
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ABSTRACT
The activity of the renin angiotensin system (RAS) increases significantly in the late gestation
fetal sheep. Fetal cortisol is also increased during this time and it is thought that the increase in
cortisol may modulate the RAS changes. Previous studies have examined the effects of cortisol
infusion on RAS activity, but the effects of blocking the peripartum increase in cortisol
concentrations on the developmental changes in the RAS are not known. Therefore, we utilized
the technique of hypothalamic-pituitary disconnection (HPD), which prevents the cortisol surge
from occurring, to investigate the importance of the late gestation increase in cortisol on the
ontogenic changes in RAS activity. HPD of fetal sheep was performed at 120 days of gestational
age (dGA), and fetuses were delivered between 135-139 dGA. Control fetuses were sham
operated. HPD blocked the late gestation cortisol increase, but did not alter renal renin mRNA,
renal renin or prorenin protein content, nor plasma renin levels as compared to shams. However,
HPD fetuses had increased angiotensin II receptor subtype 1 (AT1) mRNA and protein
expression in the kidney and lungs. Angiotensin II receptor subtype 2 (AT2) expression was not
altered in these tissues at either mRNA or protein level. HPD did not change AT1 or AT2 mRNA
in the left ventricle, but did result in decreased protein levels for both receptors. These studies
demonstrate that blockade of the naturally occurring increase in fetal cortisol concentration in
late gestation is associated with tissue specific alterations in expression of AT1 and AT2
receptors. These changes may impact on fetal tissue maturation, and hence have consequences in
postnatal life.
Key words: HPD, RAS, ovine fetus, cortisol
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INTRODUCTION
Previous studies indicate that all components of the renin angiotensin system (RAS) in
fetal sheep are detectable in the kidney at 0.27 of gestation (65). It has also been demonstrated
that renin, angiotensinogen, angiotensin I and angiotensin II (AII) are present in the fetal
circulation (18,41). During fetal development, both activity and function of the RAS change
significantly. Human and animal studies reveal that renal renin mRNA and protein levels peak
during the perinatal period , while there is an overall increase in RAS activity upon parturition
(7,11,20,39,64). It has also been noted in the sheep and horse fetus that plasma angiotensin
converting enzyme (ACE) concentrations (15,16,37) and AII levels (44) significantly increase
close to term.
AII exerts its effects via specific receptors, classified, on the basis of selective
antagonism by peptidic and non peptidic ligands, as type 1 (AT1) and type 2 (AT2) (9,57). It is
apparent that the expression of both AII receptor subtypes is developmentally regulated
(19,54,60). In the fetal sheep, renal AT1 mRNA expression is low during the first half of
pregnancy, but significantly elevated in the last third of gestation (42,66). In contrast, AT2
receptor expression is maximal by the middle of gestation, and then declines thereafter to almost
undetectable levels close to parturition (17,42,66). Interestingly, it has been noted that
functionality of the fetal RAS alters with these ontogenic changes in receptor subtype expression.
In early gestation, the fetal RAS plays a major role in promoting cellular growth and organ
differentiation (14,19,38,40), while in late gestation it modulates fetal blood pressure, and fluid
and electrolyte homeostasis (41,44,45). The mechanisms regulating these changes are not clear.
In late gestation, activity of the fetal hypothalmo-pituitary adrenal (HPA) axis also
increases significantly, culminating in a cortisol surge close to term. To this extent, it has been
demonstrated that glucocorticoids induce enzyme activity in many fetal tissues in preparation for
delivery and extrauterine life (15). For example, in the sheep and horse fetus, the increase in
plasma angiotensin converting enzyme (ACE) concentration near term closely parallels the
prepartum rise in plasma cortisol levels (16,37). Previous studies also suggest that cortisol may
play an important role in regulating fetal RAS activity in late gestation (15), but the effects of
cortisol infusion on renin levels in late gestation are controversial. Both decreases (52,69) and
increases have been noted (15). Although the data are limited to mRNA levels, cortisol infusions
have also been reported to decrease AT1 (52) and have no effect on AT2 receptor expression in
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the sheep fetus (42). The effects of decreased cortisol concentrations on RAS activity in the late
gestation sheep fetus have not been examined.
Disconnecting the hypothalamus from the pituitary (hypothalamic-pituitary
disconnection, HPD) results in retarded HPA axis development and resultant activity. Most
notably, following HPD there is no surge in fetal plasma cortisol concentrations in late gestation
(2,3,12,72). With this in mind, the present study was designed to examine the importance of
endogenous cortisol in regulating the fetal sheep RAS in late gestation, specifically by assessing
how blocking the naturally occurring increase in cortisol levels close to term affects plasma and
renal renin and prorenin concentrations, and AT1 and AT2 receptor expression in kidney, heart
and lung. We chose these particular tissues as the RAS is purported to play developmental and/or
functional roles in all (5,49,53-55).
MATERIALS AND METHODS
Animal preparation and surgical procedures
Cross-bred pregnant ewes with known insemination dates were obtained from a local
supplier. All procedures were approved by the WFUSM institutional animal care and use
committee. Ewes were housed in individual pens with food and water provided ad libitum. After
5 days of acclimatization, surgery was performed. After surgery, ewes were returned to their
pens where they remained until the fetuses were delivered. A total of 10 fetuses were used; 5
HPD (3 male, 2 female), and 5 controls (3 male, 2 female).
Surgical preparation
Surgeries were performed at approximately 120 days of gestational age (dGA). Polyvinyl
catheters previously filled with sterile saline were inserted into the fetal femoral arteries and
veins and advanced to the descending aorta and inferior vena cava and HPD was performed as
described by Antolovich et al. (1), with slight modifications (72).
Catheters from the fetus and maternal arterial and venous catheters were exteriorized
through a small incision in the maternal flank, placed into a sterile glove and protected by netting
placed around the ewe’s abdomen. When a control operation was performed, all steps of the
surgery were done except the median eminence and pituitary stalk were not separated, the
median eminence tissue was not removed and a piece of latex glove was placed near the intact
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pituitary stalk. Gentamicin and ampicillin were administered to the ewe at the time of surgery
and for the next three days through the maternal venous catheter. Blood samples were taken from
both ewe and fetus every other day to assess fetal and maternal health via blood gas and pH
measurements. Fetal plasma samples were collected following 3 days of post surgery recovery,
and just prior to necropsy for measurement of cortisol. Thyroxine (T4) and triiodothyronine (T3)
were measured immediately before necropsy. Fetuses were delivered by cesarean section
between 135-139 dGA, and tissues (kidney, heart and lung) were collected, processed and stored
at –80ºC until required for assay. Completeness of HPD was confirmed by both visual
examination at the time of necropsy, and by measuring plasma cortisol concentrations.
Cortisol, T4 and T3 measurement
Cortisol was measured by radio-immuno assay (RIA) using a kit from Diagnostic
Systems Laboratories Inc. (Webster, TX) which measures total cortisol. This assay has been
validated for measuring fetal cortisol levels. The minimum detectable amount of cortisol was
0.6 ng/ml. Coefficients of variation were 4.2 % intra-assay 7.0 % inter-assay.
T4 was measured by RIA using a kit from ICN Pharmaceuticals, Inc. (Costa Mesa, CA)
which measures total T4. The minimum detectable amount of T4 was 0.76 ng/dL. Coefficients of
variation were 5.3 % intra-assay 7.9 % inter-assay. T3 was measured by enzyme immunoassay
assay using a kit from Diagnostic Systems Laboratories Inc. (Webster, TX). The minimum
detectable amount of T3 was 0.4 ng/dL. Coefficients of variation were 5.7 % intra-assay 6.7 %
inter-assay.
Plasma active renin measurement
Plasma active renin concentration (ARC) was measured as a function of the amount of
angiotensin I (Ang I) generated from angiotensinogen. In order to measure renin concentration
independent of endogenous angiotensinogen, the method was slightly modified from that
described for renin activity. Excess renin substrate (0.5 ml of adult nephrectomized sheep
plasma) was added to each aliquot (0.1 ml) of plasma along with the enzyme inhibitors,
dimercaprol, 8-hydroxyquinoline, and maleate buffer (pH 6.0, to assure a constant pH at the
optimun for renin activity). One ml of this mixture was then incubated at 37 ºC while the rest
was maintained at 4 ºC for one hour. The Ang I generated was measured by RIA kit
(PerkinElmer Life and Analytical Sciences. Boston, MA). All samples from an animal were
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analyzed simultaneously and in duplicate, and all assays included samples from control and HPD
animals. Results are expressed as ng Ang I/ml plasma/hour of incubation.
Tissue active renin concentration (ARC) measurement
Approximately 100 mg of renal cortex was homogenized on ice for 45 seconds in 4 ml of
saline, the homogenate centrifuged at 2100 × g for 10 minutes, and the resultant supernatant
collected. An aliquot was taken for protein determination, while the remainder was frozen at –80
ºC until time of assay. For the assay, samples were diluted with saline containing 5.2 mM BAL
(2, 3 dimercapto-1-propanol), 0.59 mM 8-hydroxyquinoline, and 10 mM disodium EDTA. ARC
was determined as for plasma and is expressed as ng/ mg of protein / hour of incubation.
Renal pro-renin concentration measurement
Pro-renin concentrations were determined by measuring active renin before and after
treatment of kidney homogenate with bovine pancreatic trypsin at a concentration determined to
yield maximum renin activation. Each lot of trypsin was tested by constructing a dose-response
curve with pooled plasma or kidney homogenate. Once the optimal dose of trypsin was
established for each, this dose was used for subsequent assays. Trypsin activation was at 4 ºC
and pH 7.3 for 0.5 hour. The activation was stopped by addition of trypsin inhibitor at room
temperature for 15 minutes. The total renin concentration represented the sum of active and
prorenin.
RNA extraction
Total tissue RNA was extracted using standard procedures recommended by the
manufacturer of Trizol (Gibico BRL, Carlsbad, CA). Briefly, tissues were homogenized in Trizol
reagent (50 mg tissue/1ml Trizol) using a high speed polytron for 30-60 seconds. Chloroform
was added (0.2 ml/1ml Trizol), and the mixture incubated at room temperature for 5 minutes,
before centrifugation at 12000 × g for 15 minutes at 4 ºC. The aqueous phase was transferred to a
fresh tube and the RNA precipitated by the addition of isopropanol (0.5 ml/1 ml Trizol), and
recentrifuged at 7500 × g at 4 ºC for 5 minutes. The isopropanol was removed and the RNA
pellets were allowed to air dry and then redissolved in RNase-free water. RNA concentrations
were determined by absorbance at 260 nm in a spectrophotometer. The integrity of all RNA
samples was determined by electrophoresis on a 1.0 % agarose gel containing 6.6 %
formaldehyde.
Synthesis of antisense RNA probes
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The probe used for sheep renin mRNA was partial sheep renin cDNA from coordinates
117-983 cloned into pGEM-T easy (Promega. Madison, WI) and cut with the restriction enzyme
EcoR1 to linerize the plasmid in preparation for in vitro transcription. The probe used for sheep
AT1 mRNA was partial sheep AT1 cDNA from coordinates 114-783 cloned into pGEM-T easy
(Promega. Madison, WI) and cut with the restriction enzyme SpeI to linerize the plasmid in
preparation for in vitro transcription. The probe used for sheep AT2 mRNA was partial sheep
AT2 cDNA from coordinates 142-921 cloned into pT7/T3 U18 (Ambion. Austin, TX) and cut
with the restriction enzyme Hind III to linerize the plasmid in preparation for in vitro
transcription.
In vitro transcription was performed by adding the following items in this order: 4 µl 5X
transcription buffer, 2 µl 100 mM dithiothreitol, 1 µl RNasin RNase inhibitor, 4 µl ATP, GTP,
and CTP mix (25 mM each), 2.4 µl 100 µM UTP, 5 µl[α-³²P]UTP (3000 Ci/ mmol; Perkin-
Elmer, Boston, MA), and 1 µl SP6 (for renin) or T7 (for AT1 and AT2) polymerase and
incubating for 2 hours at room temperature. 1 µl RQ1 RNase-free DNase was added, and the
reaction was incubated for an additional 15 minutes at 37 ºC to remove the DNA template.
Unincorporated nucleotides were removed by G-50 sephadex column chromatography (Roche
Molecular Biochemicals, Indianapolis. IN). 1 µl of the purified probe was placed into a
scintillation vial to determine counts per minute. Sense strand RNA used for the standard was
synthesized with linearized plasmid by in vitro transcription similar to the above, however [α-
³²P] UTP and 100 µM UTP were replaced with 25 mM UTP.
RNase protection assay (RPA)
Renin, AT1 and AT2 mRNAs were quantified by RPA (RPA kit III; Ambion, Austin,
TX). Briefly, 20 µg total tissue RNA was mixed with 10 µl hybridization buffer and 100000 cpm
of the renin, AT1 or AT2 probe. Samples were then heated at 95 ºC for 4 minutes and placed in a
48 ºC water bath for overnight hybridization. RNaseA/T1 (1:150 dilution in RNase digestion
buffer) was then added to the samples to digest unhybridized probe and RNA. Digestion was
stopped, and the hybridized RNA precipitated by adding RNase inactivation/precipitation buffer
and incubating for 30 minutes at –20 ºC. Hybridized RNA was pelleted by centrifugation at
14000 × g for 15 minutes. Samples were then run on a 5 % polyacrylamide/8M urea denaturing
gel at 250 V for 1 hour. Gels were then exposed to film (Biomax-MR, Kodak. Cealex, France)
with an intensifying screen.
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Immunoblotting
Western blot analysis for AT1 and AT2 was performed as previously described (51).
Briefly, tissue samples were homogenized in 50 mM tris(hydroxymethyl)aminomethane (Tris),
10 mM EDTA, 150 mM NaCl, 0.1 % (vol/vol) Tween-20, 0.1 % (vol/vol) 2-mercaptoethanol,
0.1 mM phenylmethysulfonyl fluoride, 5 µg/ml leupeptin, and 5 µg/ml aprotinin, pH 7.5, and
sonicated on ice for 15-20 seconds. 1 ml aliquots were centrifuged in a microfuge at 12,000 × g
for 3 minutes, at 4 ºC. The pellet was discarded, and samples of supernatant were diluted in a
sample buffer. Protein concentrations of samples harvested from HPD and control animals were
determined using a modified Bradford method. A standard curve was produced with known
concentration of bovine albumin, and the protein concentrations of samples were determined by
comparing their OD595 nm values with values from the standard curve. Both AT1 and AT2
receptor-specific polyclonal antibodies (Santa Cruz Biotechnology, CA) have been used to detect
the ovine AT1 and AT2 receptors respectively (51). 40 µg of protein per lane were
electrophoresed on a 12 % polyacrylamide gel containing sodium dodecyl sulphate for 1.5 hours
and then blotted onto a polyvinylidene fluoride membrane (Immobilon, Millipore Corp.,
Marlborough, MA) by semidry electrobloting. The blot was blocked overnight at 4oC with 6 %
nonfat milk in 0.05 % Tween-20 Tris-buffered saline (TTBS) and then incubated with the
primary antibody using a 1:2000 (AT1) or 1:6000 (AT2) dilution in 6 % dry milk/TTBS for 2
hours at room temperature. Blots were then rinsed, washed and incubated with a 1:4000 dilution
of monkey anti-rabbit horseradish peroxidase-conjugated antibody in 6 % dry milk/ TTBS for 1
hour at room temperature. Binding of the secondary antibody was detected using a
chemiluminescent system consisting of horseradish peroxidase-hydrogen peroxide oxidation of
luminol (ECL plus, Amersham, Arlington Heights, IL, USA). Blots were then exposed to film
for 5-10 minutes before densitometric analysis.
Densitometry
Films were scanned and analyzed using Quantity One® software (PDI Imageware
Systems Inc. San Diego, CA). Sense RNA standards were used to calibrate the system for RPA
data. Data were converted from optical density readings to pg mRNA/10 µg total RNA for RPA
data. Western blot data are reported in optical density (OD) units.
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Data analysis
Data pertaining to renal renin mRNA, and renal active and pro-renin levels were
compared by Student’s t-test. All other evaluations were made using two-way ANOVA,
followed by Newman-Keuls multiple comparison test. Results are expressed as mean ± SEM,
with p < 0.05 considered significant.
RESULTS
Fetal plasma cortisol, T4 and T3 levels, and fetal health
Plasma cortisol concentrations were not different between HPD and sham fetuses at 120-
125 dGA. However, by 135-139 dGA, plasma cortisol concentrations were significantly elevated
in control fetuses but not HPD fetuses, where 120-125 dGA levels were maintained (Figure 1).
Fetal health, as assessed by arterial blood gas and pH measurements, was normal throughout the
duration of the study in both HPD and control fetuses (Table 1). Although PO2 levels in the HPD
group exhibited a modest decline during gestation, the values remained in the normal range, and
were not different from those in the control group.
T4 and T3 concentrations were not different between HPD (95.12 ± 22.88 and 0.50 ±
0.07 ng/ml) and sham fetuses (87.22 ± 14.01 and 0.36 ± 0.07 ng/ml).
Effect of HPD on plasma and renal renin
HPD did not alter renal renin mRNA expression, where levels in kidney cortex were 5.83
± 1.89 pg and 5.76 ± 1.86 pg/20 µg total RNA in control and HPD fetuses respectively. HPD
also had no effect on renal active renin (1778.6 ± 301.8 in HPD vs. 1919.3 ± 250.2 ng
angiotensin I released mg protein/hour in controls) and pro-renin content (420.4 ± 76.8 HPDs vs.
323.1 ± 69.7 ng angiotensin I released/mg protein/hour in controls). Plasma renin concentrations
in both HPD and control fetuses significantly increased in late gestation (Figure 2). The active
renin to prorenin ratio in renal cortical tissue tended to decrease in HPD fetuses (4.6 ± 0.6 vs 7.0
± 1.2 in controls, p = 0.056) (Figure 3).
The renin mRNA levels in lung and cardiac ventricular tissue were barely detectable by
ribonuclease protection assay and hence are not reported.
Effect of HPD on angiotensin II subtype receptors
AT1 and AT2 mRNA were expressed in all collected tissues (Figure 4). AT1 mRNA
expression was significantly higher than AT2 mRNA expression in all tissues. AT1 mRNA
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levels in the kidney and lung were higher than that in left ventricle. HPD increased the AT1
mRNA level in the kidney and the lung, but had no effect on AT1 mRNA expression in the left
ventricle. Expression of AT2 mRNA was greatest in the left ventricle, followed by the lung and
kidney. In contrast to AT1 mRNA, HPD had no effect on AT2 mRNA expression in all collected
tissues.
Using the AT1 receptor antibody, we detected bands of the expected sizes in all three
tissues, with the major band at 67 kD, as previously reported (32). HPD significantly increased
AT1 protein levels in the kidneys and lung, but was associated with decreased levels in the left
ventricle (Figure 5). AT2 protein was also present in these tissues. Both the band at 44 kD
(considered to be the primary band expressed in fetal kidney) and the band at 78 kD (the primary
band in adult adrenal and fetal arteries) (51) were found in our study. HPD did not affect protein
amount in any tissue at the 44 kD band. While at the 78 kD band, AT2 levels were decreased in
the left ventricle following HPD, and unchanged in the lung and kidney (Figure 6).
DISCUSSION
The purpose of the present study was to determine if the naturally occurring increase in
fetal plasma glucocorticoid levels close to term is responsible for some of the developmental
changes observed in the renin-angiotensin system (7,11,15,16,20,39,44,64). To study this issue,
we utilized the ovine fetus in which hypothalamic communication with the pituitary was
interrupted, as this manipulation abolishes the peripartum increase in fetal plasma cortisol but
leaves the fetal adrenals intact. An alternative approach for blocking the prepartum elevation in
plasma cortisol is fetal adrenalectomy. However, that procedure also removes a source of
aldosterone and catecholamines from the circulation. Thus in order to avoid problematic and
likely confounding issues related to replacement of these hormones, HPD was utilized. We found
that HPD, while preventing the late gestational plasma cortisol surge, did not influence renal
renin mRNA expression, renal renin content or plasma renin concentration. It seems therefore
that the increases in renal renin expression and plasma renin concentrations close to term in fetal
sheep are not regulated by heightened cortisol levels. In contrast, blockade of the increase in fetal
plasma cortisol did alter the expression of angiotensin receptor subtypes in a tissue specific
fashion. Thus, the data suggest that the natural increase in cortisol in late gestation may be an
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important modulator of the effects of angiotensin in the period immediately preceding the
transition from fetal to newborn life.
Close to term, glucocorticoids are known to induce enzyme activity in many fetal tissues
in preparation for delivery and extrauterine life (16), and it has been suggested that the
heightened RAS activity close to parturition may be modulated by changing plasma cortisol
concentrations (15). However, there are conflicting observations in the literature concerning the
effect of cortisol on renin secretion and expression, with some laboratories reporting that
infusions of the steroid suppress renin (52,69) while others note either increases or no significant
change (8,15,68). The explanation for these different effects is not known, but may relate to the
gestational age when the studies were conducted, the dose of steroid given, the duration of the
exposure to steroid or a combination of these variables. However, the lack of any effect on renal
renin expression and plasma renin concentration caused by blocking the age-related increase in
fetal plasma cortisol demonstrates that the peripartum elevation in glucocorticoids is not a
prerequisite for the maturational increase in renin secretion. Indeed, in both control and HPD
animals, plasma renin concentrations were similar shortly after surgery and the renin
concentration increased in parallel as gestation progressed. This pattern is similar to what has
been reported previously (39), and is consistent with the increased renal renin expression in the
fetal kidney in late gestation (7). The lack of effect of elevated levels of endogenous
glucocorticoids on plasma renin levels in the fetus is consistent with similar observations in adult
dogs with hypercortisolemia. Plasma renin activity in these animals is not different from those in
normal dogs (26).
There is some evidence that renin processing in the fetal kidney is influenced by the
absence of the rise in fetal plasma cortisol. A small decline in the active renin concentration and
increase in the pro-renin concentration in the kidney of HPD animals resulted in a small decrease
in the active to pro-renin ratio (p = 0.056) when compared to the ratio in intact animals. This
suggests that the prenatal increase in fetal plasma cortisol may enhance the processing of pro-
renin to renin. While a number of enzymes have been implicated in this processing
(10,23,61,62), the influence of glucocorticoids on the conversion has not been systematically
investigated. Additional work in this area is needed to firmly establish a role for the prenatal
increase in fetal plasma cortisol in regulating the post translational processing of renin.
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Renal AT1 mRNA expression is known to increase rapidly close to term, and then
decrease postpartum (42,43). The fact that this increase parallels the cortisol surge suggests that
cortisol may play a regulatory role. If this were the case, then HPD, in preventing the cortisol
surge, would also inhibit the normal increase in AT1 receptor mRNA expression. Somewhat
surprisingly, in the present studies, HPD significantly elevated AT1 mRNA levels and the
corresponding protein levels in kidney and lung. Our findings therefore imply that the renal AT1
increase in late gestation is not positively regulated by cortisol. On the contrary, it would appear
that cortisol acts to prevent renal AT1 receptor overexpression during this period of
development.
The effect of cortisol on renal AT1 receptor expression appears to vary depending on the
age at exposure. Exposure to increased cortisol levels in early gestation (around 27 dGA) tends
to upregulate renal AT1 expression (34), while treatment in later gestation (120 dGA) depresses
AT1 mRNA expression in fetal sheep (52). Our findings are supportive of the late gestation
observations, in that by preventing the normal increase in cortisol concentrations we
demonstrated uninhibited/increased renal AT1 receptor expression in HPD fetuses. It is apparent
that the effect of cortisol on renal AT1 expression is developmentally regulated.
The differential effects of cortisol on renal AT1 receptor expression in early and late
gestation suggest that receptor function may change as the fetus develops. In early and mid
gestation, before nephrogenesis is complete, AII is known to act, via the AT1 receptor (67) as a
renal growth factor, stimulating proliferation in mesangial (40) and medullary interstitial cells
(31). Interestingly, overexpression of the AT1 receptor during this time results in increased
kidney weight at birth, thus further emphasizing the importance of the receptor in renal
development (58). In later gestation, the role of the AT1 receptor changes to that of mediating
salt and water excretion by the metanephrons, thereby maintaining volume in fetal fluid
compartments, and ensuring normal growth and development over the last third of pregnancy
(65).
It has been demonstrated that the cardiac AT1 receptor is important in mediating cardiac
function and growth during the perinatal period (4,47,48). During fetal life, the cardiac
inotrophic, chronotrophic and growth promoting effects of angiotensin II appear to be mediated
by the cardiac AT1 receptor (47,48). Blockade of the AT1 receptor with losartan attenuates the
rapid growth of the left ventricle that normally occurs in the first three days of life in newborn
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piglets (4). The effects of cortisol infusions on cardiac AT1 receptor mRNA expression have
been described. Cortisol infusion around 120 dGA significantly increases cardiac AT1 receptor
expression (52). Cortisol also significantly increases the heart to body weight ratio, but not the
kidney or lung to body weight ratios in fetal sheep and rats, as well preterm infants (13,27,56).
Our studies in fetal sheep show that the HPD mediated lower cortisol levels are associated with
significantly decreased cardiac AT1 receptor protein expression. This finding is consistent with
those from previous studies, and indicates that the inhibited fetal cardiovascular function
associated with low cortisol levels may be due in part to decreased cardiac AT1 receptor
expression.
The relationship between fetal lung maturation and AT1 receptor expression is, however,
not so clear. It has been demonstrated that fetal sheep lung AT1 receptor mRNA levels are
elevated following maternal malnutrition in early gestation, suggesting that the associated
cortisol increase may be the driving factor (58). In contrast, our data (where cortisol
concentrations were manipulated in late gestation), implies that lower fetal cortisol
concentrations promote lung AT1 expression. These different findings imply that fetal lung AT1
receptor expression is also developmentally regulated.
In contrast to AT1, the function of the tissue AT2 receptor is not clear. In the kidney,
there is evidence that the AT2 receptor may play an important role in nephrogenesis. This is
suggested by the observation that AT2 receptor expression is high in interstitial cells of the
cortex, and in the macula densa during the nephrogenic period (6), and by the finding that
expression is very low in the third trimester, when nephrogenesis is complete (17). With respect
to the effects of glucocorticoids on AT2 expression, it has been demonstrated that exposure to
dexamethasone in early gestation (26 and 28 days of gestation) increases levels in the macula
densa (34), while a late gestation (130 d GA) infusion of cortisol has no effect (52). Our
observation that the lower cortisol concentrations in HPD fetuses did not effect renal AT2
expression close to term is in keeping with the theory that cortisol-induced changes in AT2
receptors play an important role in regulating kidney development in early to middle, but not in
late gestation when the organ has attained functionality.
The AT2 receptor is also thought to be an important factor in cell proliferation and
differentiation, and tissue remodeling and repair (25,36,63). Thus, in the present study, the
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tendency for AT2 expression to be higher in HPD fetal lungs might be indicative of lung
immaturity, instigated by the lower prevailing plasma cortisol concentrations.
Cardiac AT2 protein was detected as two bands, at 44 and 78 kD. In sheep, the 44 kD
band has been reported to predominate in the fetal kidney, while the larger band is more evident
in the mature fetal arteries and adult adrenal (51). The finding that AT2 protein was unchanged
by HPD at the 44 kD band, but decreased at the 78 kD band, suggests that hearts from HPD
fetuses were less mature than those from control counterparts, and implicates the lack of cortisol
as a mediating factor.
Changes in mRNA expression do not always translate to the protein level and vice versa.
In the present study, neither AT1 nor AT2 receptor mRNA expression in left ventricle was
altered by HPD, however patterns of protein expression were. A study in sheep by Moritz and
colleagues demonstrated that AT1 mRNA was elevated following dexamethasone treatment,
while there was no corresponding change in protein levels (34). Post-transcriptional regulation of
the AT1 receptor may result in changes in mRNA expression that do not directly reflect changes
in cell surface receptor number (73). In this situation, it is critical that expression of both mRNA
and protein is quantified to facilitate complete interpretation of data.
We recognize that HPD, in addition to preventing the cortisol surge, may also affect other
hormones and physiological parameters which could influence the RAS. For instance with
respect to fetal blood pressure, any HPD associated decrease should result in increased renal and
plasma renin concentrations, changes which were not observed in our study. Thyroid hormones
have also been implicated in regulating renin and angiotensin receptor expression (28-30,33,59).
However, in the present study we measured both T4 and T3 concentrations and found there to be
no difference between HPD and sham fetuses. Similarly, vasopressin and oxytocin have been
shown to affect renin secretion in adult animals (24,50). Because the concentration of these
peptides is low in fetal sheep (35,46), it seems unlikely that any further reduction possibly
caused by HPD would alter renin secretion and no differences in plasma renin concentration
were found in the HPD animals when compared to the sham operated fetuses. Another hormone
with possible effects on the RAS is growth hormone (GH). GH treatment in humans has been
demonstrated to activate the RAS (22), and in rat astrocytes and GH deficient rats increase AT1
mRNA expression, and AII receptor density respectively (70,71). The effect of HPD on plasma
GH concentrations in the fetal sheep have not been examined, however, evidence in adult
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animals suggests no significant effects are apparent (21). Thus, there does not appear to be
compelling evidence suggesting that other variables potentially altered by HPD would
significantly influence the RAS in fetal sheep.
In summary, our studies demonstrate that HPD induced lower plasma cortisol
concentrations in fetal sheep are associated with tissue specific alterations in expression of the
AT1 and AT2 receptors at the mRNA and corresponding protein levels in kidney and lung. In
contrast, HPD did not alter the peripartum increase in renin expression. These changes may
influence fetal tissue maturation, and hence have consequences in neonatal life. The importance
of the naturally occurring increase in cortisol in regulating aspects of RAS development is
emphasized by our findings.
ACKNOWLEDGEMENTS
The authors acknowledge the helpful discussions with Dr. Jorge Figueroa.
This work was supported by NIH grants HD 11210 and HD17644
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27
Table 1
Gestational Age (days)
<127 128-133 135-139
pH 7.35±0.01 7.36±0.01 7.37±0.01
pCO2(mmHg) 48.4±0.3 50.5±0.5 47.4±1.1
HPD pO2(mmHg) 23.8±0.9 20.5±1.4ª 19.8±0.3ª
Hct(%) 32.5±0.2 33.9±1.4 34.1±1.4
pH 7.34±0.01 7.34±0.01 7.33±0.01
pCO2(mmHg) 48.4±0.7 49.6±0.4 50.7±0.3
Sham pO2(mmHg) 22.4±1.4 20.3±0.6 19.6±0.3
Hct(%) 31.1±1.3 34.2±1.5 36.6±1.3ª
28
Figure 1
Sham
120-125 135-1390
5
10
15
20
25*
Pla
sma
Co
rtis
ol
(ng
/ml)
HP D
120-125 135-1390
10
20
Day of gestation
Pla
sma
Co
rtis
ol(n
g/m
l)
29
Figure 2
Sham
120-125 135-1390
25
50
75
*
PR
C(n
g a
ngi
ote
nsi
nI/m
g/h
our
)
HPD
120-125 135-1390
25
50
75
*
Day of gestation
PR
C(n
g an
gio
tens
inI/m
g/h
our)
30
Figure 3
Sham HPD0.0
2.5
5.0
7.5
10.0
*
Act
ive-
Ren
in/ P
rore
nin
ratio
31
Figure 4
0
10
20
30
40
50
60
70
ShamHPD
AT1 mRNA expression
AT1
mR
NA
(pg/
10 u
g to
tal R
NA
)
0
1
2
3
4
5
6
7
Kidney Lung Left ventricle
AT2 mRNA expression
AT2
mR
NA
(pg/
10 u
g to
tal R
NA
)
*
*
+
+
#
#
#
$
$
32
Figure 5
K id ney
0.0 0
0 .2 5
0 .5 0
0 .7 5
1 .0 0
*
OD
(uni
t/40
ug
pro
tein
)
L u n g
0.0
0 .1
0 .2
0 .3
0 .4*
OD
(uni
t/40
ug
pro
tein
)
L e ft V en tric le
Sham HPD0.0
0 .1
0 .2
0 .3
*OD
(uni
t/40
ug
prot
ein)
33
Figure 6
Kidney-44kD
0.0
0.1
0.2
0.3
0.4
OD
(Uni
t/40
ug
Pro
tein
)
Kidney-78kD
0.0
0.1
0.2
0.3
0.4
OD
(Uni
t/40
ug
Pro
tein
)
Lung-44kD
0.0
0.1
0.2
0.3
0.4
OD
(Uni
t/40
ug
Pro
tein
)
Lung-78 kD
0.0
0.1
0.2
0.3
0.4
OD
(Uni
t/40
ug
Pro
tein
)
Left ventricle-44kD
Sham HPD0.0
0.2
0.4
0.6
OD
(Uni
t/40
ug
Pro
tein
)
Left ventricle-78kD
Sham HPD0.0
0.2
0.4
0.6
*
OD
(Uni
t/40
ug
Pro
tein
)
34
LEGEND
Table 1. Fetal arterial blood gases and pH throughout the experiment.
ªP<0.05 vs <127 group
Figure 1. Plasma cortisol concentrations in sham and HPD fetuses during late gestation.
*p<0.05, 120-125 days vs 135-139 days. (N=8).
Figure 2. Plasma renin concentrations in sham and HPD fetuses. *p<0.05, 120-125 days vs 135-
139 days. (N=8).
Figure 3. Active renin /prorenin ratio in the renal cortex of HPD and sham fetuses. *p=0.056
sham vs HPD (N=7).
Figure 4. AT1 (upper panel) and AT2 (lower panel) mRNA expression in kidney, lung and left
ventricle of HPD and sham fetuses. # p<0.001 AT1 mRNA vs AT2 mRNA; +p<0.05 AT1
mRNA in kidney and lung vs that in left ventricle; *p<0.05 AT2 mRNA in lung and left
ventricle vs that in kidney; $ p<0.05 HPD vs sham.
Figure 5. Effect of HPD on AT1 protein expression in kidney, lung and left ventricle of HPD
and sham fetuses. *p<0.05 HPD vs sham fetuses.
35
Figure 6. Effect of HPD on AT2 protein expression in kidney, lung and left ventricle at both
78kd and 44kd band. *p<0.05 vs sham fetuses.
