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Regular Article |
a Departments of Biochemistry and Molecular Biology and
b Pathology and Laboratory Medicine, University of Texas-Houston Medical School, Houston, Texas 77030
c Cardiovascular Center, Cornell University Medical College, New York, New York 10021
d Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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decidua, developmental biology, placenta, pregnancy, trophoblast
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In humans the renin-angiotensin system undergoes major changes in response to pregnancy [3, 4]. One of the most striking changes is a 5- to 10-fold rise in the levels of circulating prorenin and renin [5, 6]. Paradoxically, the increased level of angiotensin II does not provoke an increase in blood pressure. Instead, blood pressure is lower throughout most of pregnancy [3]. The physiologic mechanisms leading to decreased responsiveness to angiotensin II and decreased blood pressure during pregnancy are not known but may be linked to the large amounts of prorenin present in the circulation. The source and function of the increased prorenin and renin during pregnancy have been a matter of continuing debate for many years. One potential source of increased maternal prorenin and renin during pregnancy is the kidney, a well-characterized area of renin production under normal physiologic conditions. Extrarenal sources of renin gene expression during human pregnancy include the ovary and the uteroplacental unit [7–14].
The study of renin gene expression in the mouse is complicated by the fact that some mice have 1 renin gene, Ren1, whereas others have 2 closely linked renin genes, Ren1 and Ren2. The amino acid sequences of REN1 and REN2 proteins show approximately 93% identity. Among the differences at the amino acid level are 2 glycosylation sites that are missing in REN2. Thus, REN1 is glycosylated and REN2 is not. The nucleotide sequences of the Ren1 and Ren2 mRNAs are nearly identical, showing a 95% identity [15–17]. Strains of mice carrying 1 or 2 renin genes differ in their tissue-specific pattern of renin gene expression. The Ren1 gene is predominantly expressed in the juxtaglomerular cells of the kidney in mice having 1 or 2 renin genes. Extrarenal sites of Ren1 expression include testes, anterior prostate, and fetal subcutaneous tissue [18]. The Ren2 gene is expressed at relatively low levels in the kidney but is highly expressed in the submaxillary gland. The similarity of REN1 and REN2 proteins and Ren1 and Ren2 transcripts has made it difficult to distinguish these protein and mRNA products of renin genes using conventional techniques.
There have been a few studies on the regulation of renin gene expression during pregnancy [19, 20]. Use of the pregnant mouse allows measurement of renin gene expression from early gestation to term in maternal and uteroplacental tissues. In the present study, we found significant increases in the levels of renin protein in the circulation of pregnant mice. In ICR mice, very high levels of renin gene expression occur at the maternal-fetal interface, first in decidua and subsequently in placentas. In C57Bl/6 mice, little or no renin gene expression occurs in the placentas, but increased renin gene expression occurs in the kidney. These results are the first to clearly show the sites of increased renin gene expression during pregnancy in different mouse strains. The results of these studies have significant implications regarding the role of renin gene expression at the maternal-fetal interface and the source of renin protein in the maternal circulation during pregnancy.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Six- to 10-wk-old ICR and C57Bl/6 female and male mice were mated to their own strains, respectively. For some experiments, C57Bl/6 female mice were mated with ICR males. The day the vaginal plug was observed was designated the 0.5 gestation day. Blood samples and tissues were obtained on different days of pregnancy. Blood (200–300 µl) was obtained by orbital sinus puncture while animals were lightly anesthetized with metaphane. Blood was collected into ice-cold microcentrifuge tubes containing 3 mmol/L EDTA and immediately centrifuged to isolate plasma. Plasma was stored at -20°C. Procedures followed were in accordance with institutional guidelines.
Renin Activity Assays
Total plasma renin concentration was determined as previously described [21]. Total renin concentration was determined by incubation of serum with a limiting amount of trypsin to convert prorenin to renin. Renin activity was determined from the rate of angiotensinogen I (Ang I) generated from angiotensinogen at a substrate concentration close to Km [21]. Ten microliters of plasma was incubated with 10 µl of partially purified mouse substrate (
6000 ng Ang I/ml) for 1 h at 37°C in a total volume of 300 µl of buffer, pH 7.5. The Ang I generated was measured by RIA.
Tissue Collection and Protein Isolation
Kidneys were obtained from nonpregnant mice and from pregnant mice at different times during pregnancy. Placentas and decidua were obtained at the time when kidneys were collected. Tissues were either used immediately or immediately frozen in liquid nitrogen and stored at -70°C for later use. The collected tissues were homogenized in buffer containing 0.9% NaCl, 0.2% SDS, and 4 mM Tris-HCl with proteinase inhibitors phenylmethysulfonylfluoride and leupeptin on ice and then centrifuged at the 14 000 rpm for 5 min. The protein concentration in the supernatant was determined, and Western blot analysis was performed.
Western Blot Analysis
Plasma (50 µg and 100 µg) was obtained from nonpregnant mice and from pregnant mice at various times. Samples were fractionated in denaturing polyacrylamide SDS gels at 100 V for 1 h. The separated proteins were transferred from gels onto nitrocellulose membranes at 100 V for 1 h. The blot was blocked with 5% milk in TBST (0.05% Tween-20 in Tris-buffered saline) for 1 h at room temperature, incubated with polyclonal rabbit anti-mouse renin at 1:1500 dilution in 1% milk in TBST for 1 h at room temperature, and then washed 3 times for 15-min each in TBST. The blot was then incubated with goat anti-rabbit IgG at 1:2000 dilution in 1% milk in TBST for 1 h at room temperature. Rabbit antibody was detected by an enhanced chemiluminescence detection system (ECL blotting system, Amersham, Piscataway, NJ).
RNA Isolation and Northern Hybridization
Total RNA was isolated using an isolation kit (Trizon; Gibco, Rockville, MD). Total cellular RNA (10 µg/lane) was electrophoresed in 1.0% agarose under denaturing conditions as previously described [22] and transferred to nylon membranes. RNA was cross-linked to the membrane by ultraviolet irradiation and hybridized to specific probes as described previously [16]. The membranes were then subjected to autoradiography for 24–48 h at -70°C after multiple stringent washes.
The 1.4-kilobase (kb) full-length cDNA probe of mouse renin-2 was obtained from American Type Culture Collection (Rockville, MD). The mouse ß-actin cDNA (450 base pairs [bp]) was kindly supplied by Dr. John Schwartz (State University of New York, Albany, NY). Probes were labeled with 
-[32P]dCTP (ICN Radiochemicals, Irvine, CA) to specific activities of >108 cpm/µg by random primer labeling as described previously [23].
Dideoxynucleotide Primer Extension Analysis
A 38-bp primer was designed, and the modified primer extension assay was performed as previously described [18, 24, 25]. The end-labeled 38-mer oligonucleotide primer (15 000 dpm) was hybridized to 100 µg of total RNA in 40 µl of 80% deionized formamide, 12 mM Tris-HCl (pH 7), 0.56 M NaCl. The samples were denatured at 68°C for 15 min and then incubated at 42°C overnight. The hybrids were then precipitated in ethanol and dissolved in 20 µl of 50 mM Tris-HCl (pH 8), 2 mM dithiothreitol, 5 mM MgCl2, 40 mM KCl. Three dNTPs and 1 dideoxynucleotide (ddNTP) were added to a final concentration of 0.2 mM. Fifteen units of reverse transcriptase (Promega, Madison, WI) was added, and the reaction mixture was incubated at 42°C for 90 min. RNA was hydrolyzed by the addition of 50 µl of 0.4 M NaOH and incubation at 42°C for 2 h. Samples were precipitated by the addition of 1 ml of 95% ethanol, 12.5 mM Tris-HCl (pH 7) containing 3 µg of calf thymus DNA/ml, washed twice with 75% ethanol, and analyzed on 20% acrylamide, 7 M urea sequencing gels [26].
In Situ Hybridization
Placental tissues were fixed in 4% paraformaldehyde-PBS overnight at 4°C and processed for in situ hybridization as described previously [22]. The renin probe was generated from a 1.2-kb cDNA fragment and was labeled with [-35S]UTP (Amersham). Samples were hybridized overnight at 60°C and treated as previously described [27]. Slides were dipped in NTB-2 emulsion (Kodak, Rochester, NY) and exposed overnight at 4°C. After development, the slides were viewed using an BX60 fluorescent microscope (Olympus, Sterling Height, MI) equipped with dark-field optics with a red filter and photographed using a SPOT digital camera (Diagnostics, Sterling Height, MI).
Statistics
All values are expressed as mean ± SEM. Comparisons of total plasma renin concentrations in mice before pregnancy, during pregnancy, and after parturition were tested by ANOVA. The significance of multiple comparisons was assessed by a post hoc test. A value of P < 0.05 was interpreted to mean that observed experimental differences were significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These initial experiments were designed to determine whether maternal renin protein and total renin activity increase during pregnancy in mice as they do in humans. For these studies, we examined 2 genetically different strains of mice: ICR and C57Bl/6. The C57Bl/6 mouse is a commonly used inbred line that possesses 1 copy of the renin gene, Ren1, per haploid genome. The ICR mouse is an outbred strain that possesses 2 closely linked renin genes, Ren1 and Ren2. Maternal blood was taken before pregnancy, during pregnancy, and after delivery. Samples were analyzed by Western blot for renin immunoreactive protein or by enzymatic assay for total renin activity. Total renin activity was determined by an assay based on angiotensin I-generating activity in the presence of excess renin substrate. Western analysis (Figs. 1A and 2A) using monospecific antibody for mouse renin revealed 2 bands of renin immunoreactive protein, one migrating with a molecular weight of approximately 45 000 (P45) and the other with a molecular weight of approximately 35 000 (P35). Western analysis showed that the abundance of renin immunoreactive protein increased significantly during pregnancy in both strains of mice.
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Measurements of total plasma renin concentration were in agreement with those of the Western blot analysis, showing significant increases during pregnancy. Higher activities were observed in pregnant ICR (Fig. 1B) than in pregnant C57Bl/6 (Fig. 2B) mice. The total renin concentrations are plotted as the mean ± SEM of 3 determinations each. The ANOVA and post hoc test were used to assess the significance of the differences between samples from nonpregnant animals (Postcoitum Day 0) and samples derived from pregnant animals at various times during and following gestation. Significant differences are marked with an asterisk. In each case total plasma renin concentration was significantly elevated by Day E9.5, remained relatively constant throughout the remainder of pregnancy, and returned to prepregnancy levels within 2–3 days following parturition (Figs. 1B and 2B). These findings indicate that mice can serve as a relevant mammalian model to determine the source of increased renin protein during pregnancy.
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Renin Gene Expression in the Kidneys in C57Bl/6 Mice
Having determined that the concentration of renin immunoreactive protein and total plasma renin concentration increased in the maternal circulation during pregnancy in mice, we sought to identify possible sources of this increased renin protein. We initially addressed this issue using the inbred line C57Bl/6. Timed pregnancies were arranged and terminated at selected intervals, and RNA and protein were extracted from the kidneys, decidua, and placentas. Northern and Western analyses generally failed to detect renin mRNA and protein in placental tissues (Fig. 3, A and B). However, renin mRNA was abundant in the kidneys and showed a marked increase in response to pregnancy. Renin mRNA levels were elevated by Day E15.5 and remained at a constant high level for the remainder of gestation. Analysis at Days E15.5 and E18.5 showed that renin immunoreactive protein was much more abundant in the kidneys of pregnant animals. Thus, in C57Bl/6 mice renin gene expression was markedly increased in the kidneys during pregnancy.
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Renin Gene Expression at the Maternal-Fetal Interface in ICR Mice
The ICR mouse is an outbred strain that possesses 2 renin genes, Ren1 and Ren2. The tissue-specific patterns of renin gene expression differ significantly between mice that possess 1 renin gene and those that possess 2 renin genes [20, 25]. For this reason, we examined the pattern of renin gene expression in pregnant ICR mice (Fig. 4A). Timed pregnancies were arranged and terminated at selected intervals, and RNA and protein were extracted from kidneys, decidua, and placentas. Analysis of kidney RNA taken at various times during pregnancy indicated that renin mRNA was abundant and present at a relatively constant level. Similarly, the analysis of kidney protein by Western blot analysis revealed that renin immunoreactive protein was evident on Day E9.5, with the majority of the immunoreactive protein present as the higher molecular weight form, P45 (Fig. 4B). The levels of renin protein remained constant throughout the remainder of gestation. Analyses of RNA from the maternal-fetal interface indicated that renin mRNA was abundant in decidual tissue at Day E9.5 and declined significantly thereafter. Analysis of decidual protein by Western blot (Fig. 4B) indicated that renin immunoreactive protein was evident by Day E9.5, with the vast majority of the protein present as P45. In contrast, the abundance of renin mRNA was very low in placentas throughout midgestation, even though the chorioallantoic placenta is fully formed by Day E11. Elevated placental renin mRNA was evident by Day E15.5 and increased rapidly during the final days of gestation (Fig. 4A). By Day E19.5, very high levels of renin mRNA were present in the placenta. Western analysis indicated that renin immunoreactive protein was produced in proportion to the abundance of renin mRNA (Fig. 4B). The results of these experiments indicate that the maternal-fetal interface, initially decidua and subsequently placentas, is an important extrarenal site of renin gene expression during pregnancy in the outbred ICR strain of mice.
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Expression of Both Renin Genes at the Maternal-Fetal Interface of ICR Mice
The fact that ICR mice show renin gene expression in decidua and placentas and that C57Bl/6 mice (with only Ren1) do not raises the possibility that only the Ren2 gene is expressed at the maternal-fetal interface. Previous studies have shown that only the Ren1 gene is expressed in the kidney. To determine which renin gene is expressed in decidua and placentas of ICR mice, we used a primer extension/ddNTP termination procedure that discriminates between Ren1 and Ren2 mRNAs. The results of this analysis show that Ren2 transcripts are more abundant in ICR decidua and Ren1 transcripts are more abundant in ICR placentas (Fig. 5). Nevertheless, both renin genes are expressed in both tissues at the maternal-fetal interface in ICR mice.
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In situ hybridization studies were conducted to localize sites of renin gene expression within decidual and placental tissues. In ICR mice, high levels of renin gene expression appear first in the decidua, including the mesometrial and antimesometrial compartments (Fig. 6A). With the regression of the decidual compartments, high levels of renin gene expression appeared in the developing placenta and remained high in the mature placenta until term (Fig. 6B). Renin transcripts were initially detected in giant cells of the developing placenta (Fig. 6, C–E) and subsequently became concentrated in the labyrinthine zone of the mature placenta (Fig. 6B). Renin gene expression in the placenta showed a pronounced upregulation at Day E15.5 and continued to increase until term (Fig. 6B). In situ hybridization localized renin transcripts to the labyrinthine region of placentas.
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Placenta as a Significant Extrarenal Source of Maternal Circulatory Renin in Crosses of C57Bl/6 Females with ICR Males
The placenta is a major extrarenal site of renin gene expression late during gestation in ICR mice. To provide additional evidence that the placenta is a significant source of maternal circulating renin during gestation, we mated ICR males with C57Bl/6 females. As a result of this mating, increased renin gene expression should occur from 2 sites by late gestation, the maternal kidneys (C57Bl/6) and the embryo-derived components of hybrid placentas (C57Bl/6 female x ICR male). To test this prediction, pregnant mice were killed at various times during the second half of pregnancy, and RNA and protein were extracted from kidneys and placentas. Increased renin mRNA abundance was apparent in the maternal (C57Bl/6) kidneys by Day E15.5 and continued to increase somewhat through Day E18.5. Analysis of RNA prepared from the hybrid C57Bl/6 x ICR placentas showed that an increase in the abundance of renin mRNA was evident by Day E16.5 and that the abundance of renin mRNA continued to increase until term (Fig. 7A). Corresponding changes in the levels of renin immunoreactive protein were detected by Western analysis of kidney and placental tissue extracts (Fig. 7B). These results show that in matings between C57Bl/6 females and ICR males, there are 2 major sites of enhanced renin gene expression late in pregnancy, the maternal kidneys and the hybrid placentas.
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Experiments were conducted to assess the impact of combined kidney and placental renin gene expression on maternal circulating levels of renin immunoreactive protein and total renin activity. We used Western blot analysis to determine maternal circulating levels of renin immunoreactive protein during pregnancy. The results (Fig. 8) showed that the concentrations of both renin immunoreactive species (P45 and P35) increased late in gestation. These observations are in agreement with the direct measurement of total plasma renin concentration, which initially began to rise at approximately Day E9.5 and continued to rise throughout the remainder of gestation reaching the highest values just before parturition (Fig. 8B). These results suggest that the continued rise in maternal circulating renin protein is due to the initial activation of renin gene expression in the maternal kidneys and subsequent activation of renin gene expression in the hybrid placentas during late gestation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The kidney is the most widely recognized source of renin production; synthesis occurs in specialized cells of the juxtaglomerular apparatus. Our studies with C57Bl/6 mice are the first to directly show changes in renal renin gene expression in response to pregnancy. This finding indicates that in C57Bl/6 mice the pregnancy-induced increase in renin protein in the maternal circulation may be derived from the kidneys. Factors regulating this pregnancy-induced increase in renal renin production are not known. The angiotensin resistance that characterizes pregnancy may set up a situation in which kidneys receive physiologic signals to increase renin production in response to the hypotension and volume expansion that accompany pregnancy. Upregulation of renin gene expression in kidneys during pregnancy may be a compensatory effect in response to the hemodynamic and fluid volume changes of pregnancy.
Numerous studies have provided evidence for renin gene expression at specific extrarenal sites during pregnancy. These sites include the ovaries, decidua, and placentas [24]. To evaluate the importance of the maternal-fetal interface as an extrarenal site of renin gene expression in mice, we examined decidual and placental tissues for renin gene expression at various times during pregnancy. Our results clearly show that the maternal-fetal interface is a major extrarenal site of renin gene expression in pregnant ICR mice. The increase in maternal circulating renin protein seen in ICR mice may be secondary to increased renin gene expression at the maternal-fetal interface. In crosses between C57Bl/6 females and ICR males, an initial rise in circulating maternal renin protein may be attributed to increased production by the maternal kidneys of the C57Bl/6 mouse. However, the additional and continued increase of renin protein in the maternal circulation may be secondary to elevated renin gene expression in the hybrid (C57Bl/6 x ICR) placentas, which display a high level of renin gene expression late in gestation. These findings raise the possibility that the renin gene expression we observed in ICR placentas is a significant contributor to the pool of renin protein present in the maternal circulation near term.
Additional evidence that placentas may serve as a significant extrarenal source of maternal circulating renin protein comes from 2 series of transgenic studies. A human renin genomic clone showed pronounced upregulation in transgenic mouse placentas beginning approximately Day 16 of gestation [26]. The production of human renin by the transgenic placentas resulted in the appearance of significant amounts of human renin in the maternal circulation. When the pregnant mouse was transgenic for the human angiotensinogen gene and produced human angiotensinogen, the expression of the human renin gene in transgenic placentas resulted in gestational hypertension. Similar results have recently been reported for transgenic rats carrying the human renin and angiotensinogen genes [28]. These transgenic studies show that the placenta can be a significant contributor to the pool of renin protein in the maternal circulation. A similar situation may exist during pregnancy in humans. In this regard, transgenic mice bearing human renin genes may provide a convenient and valuable animal model to investigate human renin gene expression during pregnancy, although the correct cell- and tissue-specific expression must be validated [21, 29].
Efforts to identify mechanisms controlling renin gene expression have focused primarily on the kidneys, although changes in renal renin gene expression during pregnancy have not previously been reported. Our study is the first to show that both Ren1 and Ren2 are expressed at the maternal-fetal interface of pregnant mice. In a previous study, the expression of human renin transgenes was examined in the placentas of transgenic mice. A 15-kb human genomic clone, including approximately 3 kb of 5'-flanking sequence unexpectedly showed pronounced upregulation in transgenic mouse placentas beginning approximately Day 16 of gestation [28]. Expression of the human renin transgene in decidua was not examined. Because the mice used in these transgenic studies were C57Bl/6, no endogenous mouse renin mRNA was observed in the transgenic placentas. Thus, even though C57Bl/6 mice do not activate their own Ren1 gene during placental development they are capable of activating the human renin transgenes. These findings suggest that cis-acting signals required for activation of the human renin gene during murine placental development reside within the 15-kb genomic clone used in the transgenic studies reported by Murakami and colleagues [26]. It is not known why the C57Bl/6 mice do not activate their endogenous Ren1 genes in their placentas and why ICR mice activate both Ren1 and Ren2 in placentas and decidua. The lack of activation in C57Bl/6 placentas may be due to an absence of essential cis-regulatory elements required for activation or the presence of cis-regulatory elements involved in repression. The fact that the human renin transgenes can be activated in transgenic C57Bl/6 placentas suggests that the required transcriptional machinery is present at the human renin gene locus. Because the human renin gene is upregulated in transgenic mice during pregnancy, it should be possible to identify the cis-regulatory elements and trans-acting factors by using transgenic mouse approaches. In addition, the temporal pattern of renin gene expression in the placenta of the ICR mouse appears to mimic the expression pattern of placental lactogen II. Both renin and lactogen II are expressed in giant cells and show a shift to the labyrinthine region during the latter part of gestation [30–32], suggesting that the 2 genes may be under the similar transcriptional regulation during normal placental development.
It has been proposed that renin gene expression at the human maternal-fetal interface plays a local autocrine/paracrine function [8, 33, 34]. However, the absence of significant renin gene expression at the maternal-fetal interface in many mammals, including the C57Bl/6 mice reported here, suggests that local renin gene expression is not required for a critical function at the maternal-fetal interface. In C57Bl/6 mice, the reduced level of renin gene expression at the maternal-fetal interface appears to be compensated by increased renal expression, which presumably supplies the maternal circulation. The results reported here, along with those of transgenic studies [26, 28] indicate that in some animals placental renin gene expression may contribute to the pool of renin protein in the maternal circulation. These findings suggest that in ICR mice the placenta serves as a significant extrarenal source of prorenin and renin protein to meet a need for elevated levels of renin protein in the maternal circulation. The pattern of renin gene expression we observed in ICR mice bears a striking resemblance to that reported for the synthesis of renin at the maternal-fetal interface during pregnancy in humans [9–14, 24].
Most of the maternal circulating renin protein is of the higher molecular weight form, presumably prorenin. Aside from being the precursor to active renin, prorenin may itself be a powerful vasoactive hormone [8] antagonistic to renin and angiotensin II. Prorenin appears to stimulate production of vasodilatory prostaglandins [34, 35]. Understanding the changes in the renin-angiotensin system and the function of prorenin in normal pregnancy may provide insight into the pathogenesis of hypertensive disorders of pregnancy. Mice may serve as valuable models for exploring the regulation and function of the renin-angiotensin system during pregnancy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by a grant from the National Institutes of Health (HD34130) to R.E.K. and by a National Institutes of Health training grant (T32-HD07324) to Y.X. 
2 Correspondence: Rodney E. Kellems, Department of Biochemistry and Molecular Biology, University of Texas-Houston Medical School, 6431 Fannin, Houston, TX 77030. FAX: 713 500 0652; rodney.e.kellems{at}uth.tmc.edu
Accepted: August 28, 2001.
Received: June 7, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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