http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, Early Online: 1–6 ! 2014 Informa UK Ltd. DOI: 10.3109/14767058.2014.951627
ORIGINAL ARTICLE
Suppression of aldosterone and progesterone in preeclampsia J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by University Of Alabama Birmingham on 03/02/15 For personal use only.
Mohammad N. Uddin1,2, Darijana Horvat1, Richard O. Jones1, Madhava R. Beeram2, David C. Zawieja3, Lena Perger4, David C. C. Sprague5, and Thomas J. Kuehl1,2 1
Department of Obstetrics & Gynecology, 2Department of Pediatrics, 3Department of Medical Physiology and 4Division of Pediatric Surgery, Baylor Scott & White Health and Texas A&M Health Science Center College of Medicine, Temple, TX, USA, and 5Texas Bioscience Institute, Temple, TX, USA Abstract
Keywords
Objective: Preeclampsia (preE) is a hypertensive disorder seen in 3–10% of human pregnancies and is diagnosed by de novo onset of hypertension and proteinuria. Several research groups provided evidence for reduced aldosterone (Aldo) and progesterone (Prog) availability in preE. The aim of this study was to determine the levels of Aldo and Prog in preE. Methods: Normal pregnant (NP; n ¼ 39) and preE (n ¼ 30) patients were recruited to have their blood drawn between 21 and 40 weeks of pregnancy. Two groups of rats were used in this study: NP rats (n ¼ 10) and preE rats (n ¼ 10), which were given weekly injections of desoxycorticosterone acetate and 0.9% saline to drink. Aldo and Prog levels were assayed in plasma and urine samples by ELISA kits. Results: In preE patients, the mean Aldo and Prog levels were suppressed (p50.05) compared to NP patients. NP patients exhibited a trend of increased levels of Aldo with an increase in gestational age; however, preE patients had the opposite trend. Both normal and preE patients exhibited a trend of increased levels of Prog with an increase in gestational age. The plasma and urinary Aldo and Prog levels were lower (p50.05) in preE rats compared to NP rats. Conclusions: We have demonstrated using a rat model and patients that both Aldo and Prog are suppressed in preE and thus may be used as biomarkers for preE.
Aldosterone, preeclampsia, pregnancy, progesterone
Introduction Preeclampsia (preE) is a pregnancy disorder characterized by hypertension and proteinuria that occurs in 3–10% of all gestations [1,2]. This syndrome is characterized by the de novo development of hypertension and proteinuria after 20 weeks of gestation and is the leading cause of maternal and fetal morbidity and mortality [1–3]. The specific etiologies of this syndrome remain unknown; however, it seems clear that preE is not a single disorder but a syndrome with multiple pathophysiologic factors and mechanisms [2,4]. PreE often leads to pre-term deliveries and is associated with multiple complications including intrauterine growth restriction [2]. A rat model, which has many of the phenotypic characteristics of human preE, has been developed [5]. It consists of the expansion of the extracellular fluid volume in pregnant rats with the administration of desoxycorticosterone acetate (DOCA) and the replacement of their drinking water with 0.9% saline. The rats develop hypertension, proteinuria and
Address for correspondence: Mohammad Nasir Uddin, PhD, Scott & White Hospital (Building 1) Room 352, 2401 South 31st Street, Temple, TX 76508, USA. Tel: 254-724-3624. Fax: 254-724-1046. E-mail: [email protected]
History Received 24 April 2014 Revised 10 July 2014 Accepted 01 August 2014 Published online 28 August 2014
intrauterine growth restriction. The hematocrits of these animals, as is the case in humans, are elevated compared to those of normal pregnant (NP) rats [5]. An alteration of the renin–angiotensin system (RAS) in this rat model has been observed [6]. The renin–angiotensin–aldosterone system (RAAS) is a hormone system that regulates blood pressure (BP) [7]. When blood volume is low, the kidneys secrete renin directly into circulation [7]. Plasma renin then converts angiotensinogen released by the liver to angiotensin I [7]. Angiotensin I is then converted to angiotensin II (Ang II) by the angiotensinconverting enzyme found in the lungs [7]. Ang II causes blood vessels to constrict, which results in increased BP [7]. Ang II also stimulates the secretion of the hormone aldosterone (Aldo) from the adrenal cortex [7]. Aldo causes the kidneys to increase the reabsorption of sodium and water into the blood, which increases the fluid volume in the body, which also increases BP [7]. Progesterone (Prog) is a steroid hormone made from cholesterol that is essential for successful reproduction [8]. During the menstrual cycle, it is produced by the corpus luteum (in the ovaries) and stimulates the secretory activity of the endometrium [8]. After 6–8 weeks of pregnancy, Prog production by the corpus luteum declines and placental trophoblasts take over as the main source of Prog production [9]. Aldo is produced by the enzyme
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aldosterone synthase and competes with cortisol and Prog for the mineralocorticoid receptor (MR), thus affecting sodium reabsorption and maternal volume expansion [10]. Bussen et al. found that while serum Aldo levels and plasma renin activity were significantly lower in preE patients, there were no differences in patients with a severe preE syndrome known as HELLP compared to NP patients [11]. In pregnant women who developed preE, the RAAS was stimulated in the first trimester [12]. In the second trimester, RAAS remained stimulated; however in the early third trimester, when preE was diagnosed, plasma renin activity and urine Aldo excretion decreased [12]. Plasma Prog concentrations increased from weeks 12–24 and 24–36 of pregnancy in normotensive women [13]. In hypertensive women, Prog increased from week 12 to week 24, but not between weeks 24 and 36 [13]. There was no difference in the plasma Prog concentration between the normotensive and hypertensive groups in week 12 and 24, but in week 36, Prog concentration was higher in the normotensive group [13]. This meant that estrogen-Prog ratio was high in the hypertensive women in week 36 [13]. In this study, we evaluated the levels of Aldo and Prog in plasma of preE patients and normal pregnancy. We also measure the urinary and plasma levels of Aldo and Prog in a rat model of preE. The purpose of the study is to compare plasma Aldo and Prog levels in normal and Pre pregnancies with regard to gestational age.
Materials and methods Human subjects Pregnant women presenting with preE and women with uncomplicated pregnancies were recruited from the Obstetrics and Gynecology Department of the Scott & White Healthcare in Temple, Texas. This study was approved by the Scott & White Institutional Review Board. PreE patients had BPs4140/90 mmHg that had increased above pre-pregnancy levels and proteinuria (4300 mg/24 h). The clinical status and assessments at the time of admission for maternal symptoms and fetal growth along with gestational age, maternal age and treatments were used to establish severity and type of preE. We recruited 30 preE patients and 39 patients with uncomplicated pregnancies as controls. After obtaining informed written consent, heparinized blood samples were obtained and plasma analyzed for Aldo and Prog levels by ELISA. Measurement of Aldo and Prog The levels of Aldo and Prog in the plasma and urine of the animals and human subjects were measured using commercially available kits (R & D Systems, Minneapolis, MN and Assay Designs, Inc., Ann Arbor, MI). Animal preparation Male (275–325 g) and female (200–250 g) Sprague–Dawley rats were obtained from Charles River Laboratories (Wilmington, MA). These animals were housed in the institutional animal facility and allowed free access to standard rat chow (Lab Diet 5001 Laboratory Rodent Diet) and tap water. Two groups of animals were studied: NP
J Matern Fetal Neonatal Med, Early Online: 1–6
(n ¼ 10) and pregnant rats, which received weekly injections of DOCA and whose drinking water was replaced with 0.9% saline (rat model of preE, n ¼ 10). Systolic BP was measured by the tail-cuff method (IITC Inc., LifeScience Instruments, Woodland Hills, CA, model 59). At 18–20 d of pregnancy, 24h urine was collected in the absence of food. Each animal was housed separately in a metabolic cage. The animals were then sacrificed on days 18–20 and blood samples were taken. Blood and urine samples were analyzed for the measurement of biochemical parameters. The 24-h urinary protein excretion was measured using the Micro BCA Protein Assay Kit (Pierce, Thermo Scientific, Rockford, IL). BP and urinary protein will be used as markers for the hypertensive status of the experimental rats. Statistical methods Heterogeneity of variance on patient characteristics and assay values were examined with Levene’s test of homogeneity of variance. For those measurements with heterogeneous variance, Mann–Whitney U tests were used for comparisons of the two patient groups. Variation in measurements associated with gestational age in normal pregnancies was examined using linear regression methods (Statistica, Statsoft, Tulsa, OK).
Results Human data As expected, there was a significant difference in systolic BP between the two groups when considering age, weight and height. The mean systolic BPs for the preE patients and normal patients, respectively, were 166 ± 11 and 122 ± 10 mmHg (p50.0001). The mean diastolic BPs were 92 ± 12 and 71 ± 9 mmHg (p50.0001). Mean gestational age at delivery was 34.0 ± 3.6 weeks in the preE group and 35.5 ± 2.7 weeks in the control group (p ¼ 0.68) (Table 1). Plasma Aldo levels were suppressed in preE patients In preE patients, the mean Aldo (352.4 ± 111.6 pg/mL) levels were suppressed (p50.001) compared to NP patients (588.9 ± 106.8 pg/mL; Table 1). The levels of Aldo were plotted against gestational age in Figure 1. As shown in Figure 1, NP patients exhibited a trend of increased levels of Aldo with an increase in gestational age; however, preE patients had the opposite trend. Table 1. Systolic and diastolic blood pressure, age, gestational age at blood collection, urinary proteins and plasma aldosterone and progesterone in preeclamptic and normal pregnant patients.
Patient group Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Urinary protein (mg/24 h) Age (years) Gestational age at blood collection (weeks) Plasma aldosterone (pg/mL) Plasma progesterone (ng/mL)
Normal pregnant Preeclamptic (n ¼ 39) (n ¼ 30) p value 122 ± 10
166 ± 11
50.0001
71 ± 9
92 ± 12
50.001
160 ± 24 26.5 ± 4.2 35.5 ± 2.7
457 ± 48 23.5 ± 5.1 34.0 ± 3.6
50.0001 0.07 0.68
618.5 ± 101.6 89.7 ± 14.0
352.4 ± 111.6 50.0001 46.7 ± 8.9 50.0001
DOI: 10.3109/14767058.2014.951627
Aldosterone and progesterone in preeclampsia
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Figure 1. The plasma aldosterone levels in pg/mL of normal pregnant (n ¼ 39) and preE (n ¼ 30) patients by their gestational age in weeks.
Figure 2. The plasma progesterone levels in ng/mL of normal pregnant (n ¼ 39) and preE (n ¼ 30) patients by their gestational age in weeks.
Plasma Prog levels were suppressed in preE patients The plasma Prog levels of 46.7 ± 8.9 ng/mL in preE patients were significantly (p50.0001) suppressed compared to normal pregnancy of 87.0 ± 14.1 ng/mL (Table 1). As shown in Figure 2, the levels of Prog were increased with an increase in gestational age for both preE and NP patients, but Prog is less in preE. This suggests that Prog may be less in preE before symptoms begin, perhaps as a marker of placental perfusion. Early in pregnancy, Prog comes primarily from the corpus luteum, but as pregnancy progresses, there is a shift to the placenta. By about 10–12 weeks of pregnancy, sufficient Prog comes from the placenta so that the ovaries are no longer needed. This suggests that Prog at 20 weeks could be primarily
from the placenta and might be marker for placental production. Animal data As listed in Table 2, BP rose in the preE rats compared to NP (p50.05). PreE rats (149.5 ± 9.6 mg/24 h) had a significantly (p50.05) higher protein excretion when compared with NP rats (123.1 ± 6.2 mg/24 h; Table 2). The mean number of pups for the preE rats (11.7 ± 0.9) was significantly (p50.05) lesser than that for the NP rats (14.4 ± 0.8; Table 2). Total pup and placental weights for preE rats (11.89 ± 6.5 gm) were significantly (p50.05) lower than NP rats (20.51 ± 8.2 g).
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Table 2. Blood pressure, urinary protein excretion, number of pups and plasma and urinary aldosterone and progesterone in the two groups of animals.
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Animal group
Normal pregnant (n ¼ 10)
Preeclamptic (n ¼ 10)
Baseline BP (mmHg) 106.2 ± 5.1 93.3 ± 9.4 Final BP (mmHg) 95.9 ± 2.8 135.0 ± 5.4 Urinary protein 123.1 ± 6.2 149.5 ± 9.6 excretion (mg/24 h) Number of pups 14.4 ± 0.8 11.75 ± 0.9 Total pup and placental 20.51 ± 8.2 11.89 ± 6.5 weights (gm) Plasma aldosterone (pg/mL) 578.5.2 ± 152.4 379.55 ± 89.6 Urinary aldosterone 86.7 ± 10.7 52.0 ± 5.2 (ng/24 h) Plasma aldosterone/ 34.1 ± 5.2 47.5 ± 4.5 renin ratio Plasma progesterone 31.5 ± 4.8 18.7 ± 3.2 (ng/mL) Urinary progesterone 6.5 ± 0.8 3.5 ± 0.7 (ng/24 h)
p value 0.070 50.0001 0.033 0.032 0.007 0.008 50.0001 50.0001 50.0001 50.0001
Plasma Aldo levels were suppressed in a rat model of preE Table 2 lists the plasma Aldo levels were significantly (p50.05) lower in preE rats (379.5 ± 89.6 pg/mL) compared to NP rats (578.5 ± 152.4 pg/mL). The urinary Aldo levels were also significantly (p50.05) lower in preE rats (52.0 ± 5.2 pg/mL) compared to NP rats (86.7 ± 10.7 pg/ mL). The plasma Aldo to renin ratio of the preE rats was significantly (p50.05) higher than the ratio of NP rats. Plasma Prog levels were suppressed in a rat model of preE As listed in Table 2, the plasma Prog levels were significantly (p50.05) lower in preE rats (18.6 ± 2.1 ng/mL) compared to NP rats (31.5 ± 2.9 ng/mL). The urinary Prog levels were also significantly (p50.05) lower in preE rats (3.5 ± 0.4 ng/mL) compared to NP rats (6.5 ± 0.5 ng/mL).
Discussion In this study, we have shown in patients that both Aldo and Prog were significantly suppressed in preE patients compared to those with normal pregnancies. It has also been shown that in normal pregnancy Aldo levels are increased with the increasing gestational age, while for the preE patients, we found the opposite trend. However, Prog levels were increased with the increase of gestational age both for the preE and normal pregnancy. We also measured both the plasma and urinary levels of Aldo and Prog in a rat model of preE, which mimicked the human preE. We found similar trends in levels of plasma and urinary Aldo and Prog in the rat model compared to NP rat. We also have shown that plasma Aldo/renin ratio in rats is high in preE compared to NP. These data are similar to those of previous studies on human patients [10,12,14]. Others have documented the suppression of the levels of both plasma Aldo and renin activity in preE patients regardless of retention of intravascular plasma volume compared to normal pregnancy [10,15,16]. Symonds et al. demonstrated that there is a
relatively higher Aldo availability for the given levels of renin [15]. The intravascular volume depletion is supposed to decrease the placental perfusion with subsequent placental ischemia and maternal hypertension. Normally, Aldo binds with high affinity to the MR to stimulate renal sodium reabsorption and potassium excretion [10]. Cortisol, the active glucocorticoid, can also bind to the MR with the same affinity as Aldo even though it is much more abundant in plasma than Aldo [10]. The third hormone known to bind to the MR is Prog, which has even a higher affinity for the MR than Aldo [10]. Aldo, cortisol and Prog are competing with each other to bind to the MR. Lower concentrations of plasma Aldo and Prog in preE upset the balance of the MR to stimulate the Na+/K+ pump. During pregnancy, Prog concentrations increase much more than those of Aldo and reach levels up to 400–700 nmol/L for 1–6 nmol/L for Aldo [10]. Several polymorphisms of the aldosterone synthase gene, CYP11B2, have been associated with elevated BP [10]. The polymorphism R173K was statistically less frequent in preE patients compared to NP patients while the polymorphism 344T/C was more frequent in preE patients [10,17]. Furthermore, the mutation V386A was exclusively observed in preE [16]. The polymorphisms of the aldosterone synthase gene are expected to result in reduced protein activity that may in turn play a role in the development of preE. All of the RAAS components are found in the placenta on the maternal side where there are two major renin-producing systems during pregnancy, the renal and the uteroplacental systems [18]. However, Herse et al. reported that AT1 receptor expression was 10-fold higher in placenta than in decidua both in normal and preE pregnancies [18]. Brown et al. showed that while preE women had significantly (p50.01) lower Aldo levels compared to NP women, there were no differences in Aldo between mild preE and severe preE [14]. However, they did find that women with severe preE had significantly (p50.01) lower renin compared to mild preE in addition to overall lower levels compared to NP women [14]. The preE was considered severe if the women had systolic BP4170 mmHg, diastolic BP4110 mmHg, proteinuria4500 mg/d, thrombocytopenia, abnormal liver function, epigastric pain, neurological disturbances or renal impairment [14]. Women with preE had a plasma Aldo/renin ratio higher than NP patients which we have also shown in the preE rat model [14]. There is limited literature available on the effects of Prog on extravillous trophoblast (EVT) invasion. Given that Prog can affect the functions of syncytial trophoblast and decidual cells, and it is found in high concentrations at the maternal– fetal interface, it is reasonable to hypothesize that Prog can also play a role in regulating EVT invasion and thus vascular remodeling. Decidualization and recruitment of uterine natural killer cells, both of which occur during the decidualassociated first step of the remodeling process, are regulated by Prog [19,20]. A previous study has demonstrated an antiapoptotic effect of Prog on the EVT cell line model HTR8/ SVneo [21], whereby maintenance of a high number of HTR8/SVneo cells promoted their infiltration into maternal tissues [21]. Taken together, these observations indicate that Prog could act to promote EVT invasion. In contrast, the
Aldosterone and progesterone in preeclampsia
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DOI: 10.3109/14767058.2014.951627
action of Prog on decidual cells is generally believed to promote the formation of a barrier against excessive EVT invasion [22–24], possibly by inhibiting matrix metalloproteinase (MMP) activity [25]. In a study using primary cytotrophoblast (CTB), Prog treatment was found to decrease MMP-9 secretion [26]. These observations suggest that Prog hinders EVT invasion. Chen et al. recently studied the effects of Prog on EVT migration, invasion and proliferation using the cell line HTR8/SVneo as a model of invasive trophoblast. They found that Prog acts as a direct negative regulator of trophoblast invasion, inhibiting cell migration, MMP-2 and -9 activities and proliferation [27]. Other studies have looked at a human extravillous CTB cell line derived from first trimester chorionic villus tissue and the effect the cardiotonic steroid marinobufagenin (MBG), that is shown to be elevated in preE, has on the cells in vitro caused CTB impairment [28,29]. MBG inhibited CTB cell proliferation, migration, invasion and activated apoptosis in CTB cells [28,29]. Prog is involved in regulation of CTB and increases in NP. Decreased Prog results in abnormal placentation. Ang II stimulates the secretion Aldo from the adrenal cortex, so an increase in Ang II causes an increase in Aldo. We found a decrease in plasma Ang II and an increase in placental Ang II in our preE rat model [6]. We also found a decrease in plasma renin and an increase in placental renin in our preE rat model [6]. In summary, the RAS components are suppressed in our preE rat model compared to NP. There are controversial studies that found elevated Prog levels in preE women due to a higher activity of pregnenolone sulfatase and larger store of endogenous cholesterol [11]. Other controversial studies found Prog concentrations in preeclamptic women unchanged compared to normotensive pregnancies [11]. There is long-term regulation of placental Prog synthesis [8]. The contribution of the newly formed syncytiotrophoblasts to Prog synthesis during the first few weeks of pregnancy appears to be relatively insignificant [8]. The activation of placental Prog synthesis correlates with a transition of the organ from one of low blood supply and relative anoxia to one of high blood supply and ample oxygen, oxygen being an essential substrate for the cholesterol sidechain cleavage reaction [8]. Long-term regulation of placental Prog synthesis following this initial activation is exemplified by the increase in maternal plasma Prog levels by approximately 8-fold from week 14 to term [8]. Placental mass increases during this time, so part of the increase in Prog comes from having more secretory tissue [8]. Our study agrees with the findings of Siddiqui et al. [30] who reported that Ang II type 1 receptor agonistic autoantibody-induced soluble Fms-like tyrosine kinase-1 increases were novel pathogenic mechanisms underlying decreased aldosterone production in preE.
Declaration of interest The authors report no conflict of interest. Funding for this work was provided by Scott, Sherwood and Brindley Foundation and Department of Obstetrics and Gynecology (MNU) and the Noble Centennial Endowment for Research in Obstetrics and Gynecology (TJK).
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Author contribution statement M. N. U. conceptualized and designed the study, performed the experiments, carried out data interpretation, drafted and revised the article critically and approved the final manuscript as submitted; D. H. performed the experiments and revised the final manuscript as submitted; R. O. J. and L. P. recruited the patients and revised the manuscript for submission; M. R. B., D. C. Z. and D. C. C. S. critically reviewed the manuscript and approved the final manuscript as submitted; and T. J. K. performed the statistical analysis and critically reviewed the manuscript and approved the final manuscript as submitted.
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20. Pijnenborg R, Vercruysse L, Hanssens M. The uterine spiral arteries in human pregnancy: facts and controversies. Placenta 2006;27: 939–58. 21. Liu J, Matsuo H, Laoag-Fernandez JB, et al. The effects of progesterone on apoptosis in the human trophoblast-derived HTR-8/SV neo cells. Mol Hum Reprod 2007;13:869–74. 22. Bischof P, Meisser A, Campana A, Tseng L. Effects of decidua-conditioned medium and insulin-like growth factor binding protein-1 on trophoblastic matrix metalloproteinases and their inhibitors. Placenta 1998;19:457–64. 23. Staun-Ram E, Shalev E. Human trophoblast function during the implantation process. Reprod Biol Endocrinol 2005;3: 56–67. 24. Szekeres-Bartho J, Halasz M, Palkovics T. Progesterone in pregnancy; receptor-ligand interaction and signaling pathways. J Reprod Immunol 2009;83:60–4. 25. Dai B, Cao Y, Liu W, et al. Dual roles of progesterone in embryo implantation in mouse. Endocrine 2003;21:123–32.
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26. Shimonovitz S, Hurwitz A, Hochner-Celnikier D, et al. Expression of gelatinase B by trophoblast cells: down-regulation by progesterone. Am J Obstet Gynecol 1998;178:457–61. 27. Chen JZ, Wong MH, Brennecke SP, Keogh RJ. The effects of human chorionic gonadotrophin, progesterone and oestradiol on trophoblast function. Mol Cell Endocrinol 2011;342:73–80. 28. Uddin MN, Horvat D, Glaser SS, et al. Marinobufagenin inhibits proliferation and migration of cytotrophoblast and CHO cells. Placenta 2008;29:266–73. 29. Uddin MN, Horvat D, Glaser SS, et al. Examination of the cellular mechanisms by which marinobufagenin inhibits cytotrophoblast function. J Biol Chem 2008;283: 17946–53. 30. Siddiqui AH, Irani RA, Zhang W, et al. Angiotensin receptor agonistic autoantibody-mediated soluble Fms-like tyrosine kinase-1 induction contributes to impaired adrenal vasculature and decreased aldosterone production in preeclampsia. Hypertension 2013;61: 472–9.
ORIGINAL ARTICLE
Suppression of aldosterone and progesterone in preeclampsia J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by University Of Alabama Birmingham on 03/02/15 For personal use only.
Mohammad N. Uddin1,2, Darijana Horvat1, Richard O. Jones1, Madhava R. Beeram2, David C. Zawieja3, Lena Perger4, David C. C. Sprague5, and Thomas J. Kuehl1,2 1
Department of Obstetrics & Gynecology, 2Department of Pediatrics, 3Department of Medical Physiology and 4Division of Pediatric Surgery, Baylor Scott & White Health and Texas A&M Health Science Center College of Medicine, Temple, TX, USA, and 5Texas Bioscience Institute, Temple, TX, USA Abstract
Keywords
Objective: Preeclampsia (preE) is a hypertensive disorder seen in 3–10% of human pregnancies and is diagnosed by de novo onset of hypertension and proteinuria. Several research groups provided evidence for reduced aldosterone (Aldo) and progesterone (Prog) availability in preE. The aim of this study was to determine the levels of Aldo and Prog in preE. Methods: Normal pregnant (NP; n ¼ 39) and preE (n ¼ 30) patients were recruited to have their blood drawn between 21 and 40 weeks of pregnancy. Two groups of rats were used in this study: NP rats (n ¼ 10) and preE rats (n ¼ 10), which were given weekly injections of desoxycorticosterone acetate and 0.9% saline to drink. Aldo and Prog levels were assayed in plasma and urine samples by ELISA kits. Results: In preE patients, the mean Aldo and Prog levels were suppressed (p50.05) compared to NP patients. NP patients exhibited a trend of increased levels of Aldo with an increase in gestational age; however, preE patients had the opposite trend. Both normal and preE patients exhibited a trend of increased levels of Prog with an increase in gestational age. The plasma and urinary Aldo and Prog levels were lower (p50.05) in preE rats compared to NP rats. Conclusions: We have demonstrated using a rat model and patients that both Aldo and Prog are suppressed in preE and thus may be used as biomarkers for preE.
Aldosterone, preeclampsia, pregnancy, progesterone
Introduction Preeclampsia (preE) is a pregnancy disorder characterized by hypertension and proteinuria that occurs in 3–10% of all gestations [1,2]. This syndrome is characterized by the de novo development of hypertension and proteinuria after 20 weeks of gestation and is the leading cause of maternal and fetal morbidity and mortality [1–3]. The specific etiologies of this syndrome remain unknown; however, it seems clear that preE is not a single disorder but a syndrome with multiple pathophysiologic factors and mechanisms [2,4]. PreE often leads to pre-term deliveries and is associated with multiple complications including intrauterine growth restriction [2]. A rat model, which has many of the phenotypic characteristics of human preE, has been developed [5]. It consists of the expansion of the extracellular fluid volume in pregnant rats with the administration of desoxycorticosterone acetate (DOCA) and the replacement of their drinking water with 0.9% saline. The rats develop hypertension, proteinuria and
Address for correspondence: Mohammad Nasir Uddin, PhD, Scott & White Hospital (Building 1) Room 352, 2401 South 31st Street, Temple, TX 76508, USA. Tel: 254-724-3624. Fax: 254-724-1046. E-mail: [email protected]
History Received 24 April 2014 Revised 10 July 2014 Accepted 01 August 2014 Published online 28 August 2014
intrauterine growth restriction. The hematocrits of these animals, as is the case in humans, are elevated compared to those of normal pregnant (NP) rats [5]. An alteration of the renin–angiotensin system (RAS) in this rat model has been observed [6]. The renin–angiotensin–aldosterone system (RAAS) is a hormone system that regulates blood pressure (BP) [7]. When blood volume is low, the kidneys secrete renin directly into circulation [7]. Plasma renin then converts angiotensinogen released by the liver to angiotensin I [7]. Angiotensin I is then converted to angiotensin II (Ang II) by the angiotensinconverting enzyme found in the lungs [7]. Ang II causes blood vessels to constrict, which results in increased BP [7]. Ang II also stimulates the secretion of the hormone aldosterone (Aldo) from the adrenal cortex [7]. Aldo causes the kidneys to increase the reabsorption of sodium and water into the blood, which increases the fluid volume in the body, which also increases BP [7]. Progesterone (Prog) is a steroid hormone made from cholesterol that is essential for successful reproduction [8]. During the menstrual cycle, it is produced by the corpus luteum (in the ovaries) and stimulates the secretory activity of the endometrium [8]. After 6–8 weeks of pregnancy, Prog production by the corpus luteum declines and placental trophoblasts take over as the main source of Prog production [9]. Aldo is produced by the enzyme
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aldosterone synthase and competes with cortisol and Prog for the mineralocorticoid receptor (MR), thus affecting sodium reabsorption and maternal volume expansion [10]. Bussen et al. found that while serum Aldo levels and plasma renin activity were significantly lower in preE patients, there were no differences in patients with a severe preE syndrome known as HELLP compared to NP patients [11]. In pregnant women who developed preE, the RAAS was stimulated in the first trimester [12]. In the second trimester, RAAS remained stimulated; however in the early third trimester, when preE was diagnosed, plasma renin activity and urine Aldo excretion decreased [12]. Plasma Prog concentrations increased from weeks 12–24 and 24–36 of pregnancy in normotensive women [13]. In hypertensive women, Prog increased from week 12 to week 24, but not between weeks 24 and 36 [13]. There was no difference in the plasma Prog concentration between the normotensive and hypertensive groups in week 12 and 24, but in week 36, Prog concentration was higher in the normotensive group [13]. This meant that estrogen-Prog ratio was high in the hypertensive women in week 36 [13]. In this study, we evaluated the levels of Aldo and Prog in plasma of preE patients and normal pregnancy. We also measure the urinary and plasma levels of Aldo and Prog in a rat model of preE. The purpose of the study is to compare plasma Aldo and Prog levels in normal and Pre pregnancies with regard to gestational age.
Materials and methods Human subjects Pregnant women presenting with preE and women with uncomplicated pregnancies were recruited from the Obstetrics and Gynecology Department of the Scott & White Healthcare in Temple, Texas. This study was approved by the Scott & White Institutional Review Board. PreE patients had BPs4140/90 mmHg that had increased above pre-pregnancy levels and proteinuria (4300 mg/24 h). The clinical status and assessments at the time of admission for maternal symptoms and fetal growth along with gestational age, maternal age and treatments were used to establish severity and type of preE. We recruited 30 preE patients and 39 patients with uncomplicated pregnancies as controls. After obtaining informed written consent, heparinized blood samples were obtained and plasma analyzed for Aldo and Prog levels by ELISA. Measurement of Aldo and Prog The levels of Aldo and Prog in the plasma and urine of the animals and human subjects were measured using commercially available kits (R & D Systems, Minneapolis, MN and Assay Designs, Inc., Ann Arbor, MI). Animal preparation Male (275–325 g) and female (200–250 g) Sprague–Dawley rats were obtained from Charles River Laboratories (Wilmington, MA). These animals were housed in the institutional animal facility and allowed free access to standard rat chow (Lab Diet 5001 Laboratory Rodent Diet) and tap water. Two groups of animals were studied: NP
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(n ¼ 10) and pregnant rats, which received weekly injections of DOCA and whose drinking water was replaced with 0.9% saline (rat model of preE, n ¼ 10). Systolic BP was measured by the tail-cuff method (IITC Inc., LifeScience Instruments, Woodland Hills, CA, model 59). At 18–20 d of pregnancy, 24h urine was collected in the absence of food. Each animal was housed separately in a metabolic cage. The animals were then sacrificed on days 18–20 and blood samples were taken. Blood and urine samples were analyzed for the measurement of biochemical parameters. The 24-h urinary protein excretion was measured using the Micro BCA Protein Assay Kit (Pierce, Thermo Scientific, Rockford, IL). BP and urinary protein will be used as markers for the hypertensive status of the experimental rats. Statistical methods Heterogeneity of variance on patient characteristics and assay values were examined with Levene’s test of homogeneity of variance. For those measurements with heterogeneous variance, Mann–Whitney U tests were used for comparisons of the two patient groups. Variation in measurements associated with gestational age in normal pregnancies was examined using linear regression methods (Statistica, Statsoft, Tulsa, OK).
Results Human data As expected, there was a significant difference in systolic BP between the two groups when considering age, weight and height. The mean systolic BPs for the preE patients and normal patients, respectively, were 166 ± 11 and 122 ± 10 mmHg (p50.0001). The mean diastolic BPs were 92 ± 12 and 71 ± 9 mmHg (p50.0001). Mean gestational age at delivery was 34.0 ± 3.6 weeks in the preE group and 35.5 ± 2.7 weeks in the control group (p ¼ 0.68) (Table 1). Plasma Aldo levels were suppressed in preE patients In preE patients, the mean Aldo (352.4 ± 111.6 pg/mL) levels were suppressed (p50.001) compared to NP patients (588.9 ± 106.8 pg/mL; Table 1). The levels of Aldo were plotted against gestational age in Figure 1. As shown in Figure 1, NP patients exhibited a trend of increased levels of Aldo with an increase in gestational age; however, preE patients had the opposite trend. Table 1. Systolic and diastolic blood pressure, age, gestational age at blood collection, urinary proteins and plasma aldosterone and progesterone in preeclamptic and normal pregnant patients.
Patient group Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Urinary protein (mg/24 h) Age (years) Gestational age at blood collection (weeks) Plasma aldosterone (pg/mL) Plasma progesterone (ng/mL)
Normal pregnant Preeclamptic (n ¼ 39) (n ¼ 30) p value 122 ± 10
166 ± 11
50.0001
71 ± 9
92 ± 12
50.001
160 ± 24 26.5 ± 4.2 35.5 ± 2.7
457 ± 48 23.5 ± 5.1 34.0 ± 3.6
50.0001 0.07 0.68
618.5 ± 101.6 89.7 ± 14.0
352.4 ± 111.6 50.0001 46.7 ± 8.9 50.0001
DOI: 10.3109/14767058.2014.951627
Aldosterone and progesterone in preeclampsia
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Figure 1. The plasma aldosterone levels in pg/mL of normal pregnant (n ¼ 39) and preE (n ¼ 30) patients by their gestational age in weeks.
Figure 2. The plasma progesterone levels in ng/mL of normal pregnant (n ¼ 39) and preE (n ¼ 30) patients by their gestational age in weeks.
Plasma Prog levels were suppressed in preE patients The plasma Prog levels of 46.7 ± 8.9 ng/mL in preE patients were significantly (p50.0001) suppressed compared to normal pregnancy of 87.0 ± 14.1 ng/mL (Table 1). As shown in Figure 2, the levels of Prog were increased with an increase in gestational age for both preE and NP patients, but Prog is less in preE. This suggests that Prog may be less in preE before symptoms begin, perhaps as a marker of placental perfusion. Early in pregnancy, Prog comes primarily from the corpus luteum, but as pregnancy progresses, there is a shift to the placenta. By about 10–12 weeks of pregnancy, sufficient Prog comes from the placenta so that the ovaries are no longer needed. This suggests that Prog at 20 weeks could be primarily
from the placenta and might be marker for placental production. Animal data As listed in Table 2, BP rose in the preE rats compared to NP (p50.05). PreE rats (149.5 ± 9.6 mg/24 h) had a significantly (p50.05) higher protein excretion when compared with NP rats (123.1 ± 6.2 mg/24 h; Table 2). The mean number of pups for the preE rats (11.7 ± 0.9) was significantly (p50.05) lesser than that for the NP rats (14.4 ± 0.8; Table 2). Total pup and placental weights for preE rats (11.89 ± 6.5 gm) were significantly (p50.05) lower than NP rats (20.51 ± 8.2 g).
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Table 2. Blood pressure, urinary protein excretion, number of pups and plasma and urinary aldosterone and progesterone in the two groups of animals.
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Animal group
Normal pregnant (n ¼ 10)
Preeclamptic (n ¼ 10)
Baseline BP (mmHg) 106.2 ± 5.1 93.3 ± 9.4 Final BP (mmHg) 95.9 ± 2.8 135.0 ± 5.4 Urinary protein 123.1 ± 6.2 149.5 ± 9.6 excretion (mg/24 h) Number of pups 14.4 ± 0.8 11.75 ± 0.9 Total pup and placental 20.51 ± 8.2 11.89 ± 6.5 weights (gm) Plasma aldosterone (pg/mL) 578.5.2 ± 152.4 379.55 ± 89.6 Urinary aldosterone 86.7 ± 10.7 52.0 ± 5.2 (ng/24 h) Plasma aldosterone/ 34.1 ± 5.2 47.5 ± 4.5 renin ratio Plasma progesterone 31.5 ± 4.8 18.7 ± 3.2 (ng/mL) Urinary progesterone 6.5 ± 0.8 3.5 ± 0.7 (ng/24 h)
p value 0.070 50.0001 0.033 0.032 0.007 0.008 50.0001 50.0001 50.0001 50.0001
Plasma Aldo levels were suppressed in a rat model of preE Table 2 lists the plasma Aldo levels were significantly (p50.05) lower in preE rats (379.5 ± 89.6 pg/mL) compared to NP rats (578.5 ± 152.4 pg/mL). The urinary Aldo levels were also significantly (p50.05) lower in preE rats (52.0 ± 5.2 pg/mL) compared to NP rats (86.7 ± 10.7 pg/ mL). The plasma Aldo to renin ratio of the preE rats was significantly (p50.05) higher than the ratio of NP rats. Plasma Prog levels were suppressed in a rat model of preE As listed in Table 2, the plasma Prog levels were significantly (p50.05) lower in preE rats (18.6 ± 2.1 ng/mL) compared to NP rats (31.5 ± 2.9 ng/mL). The urinary Prog levels were also significantly (p50.05) lower in preE rats (3.5 ± 0.4 ng/mL) compared to NP rats (6.5 ± 0.5 ng/mL).
Discussion In this study, we have shown in patients that both Aldo and Prog were significantly suppressed in preE patients compared to those with normal pregnancies. It has also been shown that in normal pregnancy Aldo levels are increased with the increasing gestational age, while for the preE patients, we found the opposite trend. However, Prog levels were increased with the increase of gestational age both for the preE and normal pregnancy. We also measured both the plasma and urinary levels of Aldo and Prog in a rat model of preE, which mimicked the human preE. We found similar trends in levels of plasma and urinary Aldo and Prog in the rat model compared to NP rat. We also have shown that plasma Aldo/renin ratio in rats is high in preE compared to NP. These data are similar to those of previous studies on human patients [10,12,14]. Others have documented the suppression of the levels of both plasma Aldo and renin activity in preE patients regardless of retention of intravascular plasma volume compared to normal pregnancy [10,15,16]. Symonds et al. demonstrated that there is a
relatively higher Aldo availability for the given levels of renin [15]. The intravascular volume depletion is supposed to decrease the placental perfusion with subsequent placental ischemia and maternal hypertension. Normally, Aldo binds with high affinity to the MR to stimulate renal sodium reabsorption and potassium excretion [10]. Cortisol, the active glucocorticoid, can also bind to the MR with the same affinity as Aldo even though it is much more abundant in plasma than Aldo [10]. The third hormone known to bind to the MR is Prog, which has even a higher affinity for the MR than Aldo [10]. Aldo, cortisol and Prog are competing with each other to bind to the MR. Lower concentrations of plasma Aldo and Prog in preE upset the balance of the MR to stimulate the Na+/K+ pump. During pregnancy, Prog concentrations increase much more than those of Aldo and reach levels up to 400–700 nmol/L for 1–6 nmol/L for Aldo [10]. Several polymorphisms of the aldosterone synthase gene, CYP11B2, have been associated with elevated BP [10]. The polymorphism R173K was statistically less frequent in preE patients compared to NP patients while the polymorphism 344T/C was more frequent in preE patients [10,17]. Furthermore, the mutation V386A was exclusively observed in preE [16]. The polymorphisms of the aldosterone synthase gene are expected to result in reduced protein activity that may in turn play a role in the development of preE. All of the RAAS components are found in the placenta on the maternal side where there are two major renin-producing systems during pregnancy, the renal and the uteroplacental systems [18]. However, Herse et al. reported that AT1 receptor expression was 10-fold higher in placenta than in decidua both in normal and preE pregnancies [18]. Brown et al. showed that while preE women had significantly (p50.01) lower Aldo levels compared to NP women, there were no differences in Aldo between mild preE and severe preE [14]. However, they did find that women with severe preE had significantly (p50.01) lower renin compared to mild preE in addition to overall lower levels compared to NP women [14]. The preE was considered severe if the women had systolic BP4170 mmHg, diastolic BP4110 mmHg, proteinuria4500 mg/d, thrombocytopenia, abnormal liver function, epigastric pain, neurological disturbances or renal impairment [14]. Women with preE had a plasma Aldo/renin ratio higher than NP patients which we have also shown in the preE rat model [14]. There is limited literature available on the effects of Prog on extravillous trophoblast (EVT) invasion. Given that Prog can affect the functions of syncytial trophoblast and decidual cells, and it is found in high concentrations at the maternal– fetal interface, it is reasonable to hypothesize that Prog can also play a role in regulating EVT invasion and thus vascular remodeling. Decidualization and recruitment of uterine natural killer cells, both of which occur during the decidualassociated first step of the remodeling process, are regulated by Prog [19,20]. A previous study has demonstrated an antiapoptotic effect of Prog on the EVT cell line model HTR8/ SVneo [21], whereby maintenance of a high number of HTR8/SVneo cells promoted their infiltration into maternal tissues [21]. Taken together, these observations indicate that Prog could act to promote EVT invasion. In contrast, the
Aldosterone and progesterone in preeclampsia
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DOI: 10.3109/14767058.2014.951627
action of Prog on decidual cells is generally believed to promote the formation of a barrier against excessive EVT invasion [22–24], possibly by inhibiting matrix metalloproteinase (MMP) activity [25]. In a study using primary cytotrophoblast (CTB), Prog treatment was found to decrease MMP-9 secretion [26]. These observations suggest that Prog hinders EVT invasion. Chen et al. recently studied the effects of Prog on EVT migration, invasion and proliferation using the cell line HTR8/SVneo as a model of invasive trophoblast. They found that Prog acts as a direct negative regulator of trophoblast invasion, inhibiting cell migration, MMP-2 and -9 activities and proliferation [27]. Other studies have looked at a human extravillous CTB cell line derived from first trimester chorionic villus tissue and the effect the cardiotonic steroid marinobufagenin (MBG), that is shown to be elevated in preE, has on the cells in vitro caused CTB impairment [28,29]. MBG inhibited CTB cell proliferation, migration, invasion and activated apoptosis in CTB cells [28,29]. Prog is involved in regulation of CTB and increases in NP. Decreased Prog results in abnormal placentation. Ang II stimulates the secretion Aldo from the adrenal cortex, so an increase in Ang II causes an increase in Aldo. We found a decrease in plasma Ang II and an increase in placental Ang II in our preE rat model [6]. We also found a decrease in plasma renin and an increase in placental renin in our preE rat model [6]. In summary, the RAS components are suppressed in our preE rat model compared to NP. There are controversial studies that found elevated Prog levels in preE women due to a higher activity of pregnenolone sulfatase and larger store of endogenous cholesterol [11]. Other controversial studies found Prog concentrations in preeclamptic women unchanged compared to normotensive pregnancies [11]. There is long-term regulation of placental Prog synthesis [8]. The contribution of the newly formed syncytiotrophoblasts to Prog synthesis during the first few weeks of pregnancy appears to be relatively insignificant [8]. The activation of placental Prog synthesis correlates with a transition of the organ from one of low blood supply and relative anoxia to one of high blood supply and ample oxygen, oxygen being an essential substrate for the cholesterol sidechain cleavage reaction [8]. Long-term regulation of placental Prog synthesis following this initial activation is exemplified by the increase in maternal plasma Prog levels by approximately 8-fold from week 14 to term [8]. Placental mass increases during this time, so part of the increase in Prog comes from having more secretory tissue [8]. Our study agrees with the findings of Siddiqui et al. [30] who reported that Ang II type 1 receptor agonistic autoantibody-induced soluble Fms-like tyrosine kinase-1 increases were novel pathogenic mechanisms underlying decreased aldosterone production in preE.
Declaration of interest The authors report no conflict of interest. Funding for this work was provided by Scott, Sherwood and Brindley Foundation and Department of Obstetrics and Gynecology (MNU) and the Noble Centennial Endowment for Research in Obstetrics and Gynecology (TJK).
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Author contribution statement M. N. U. conceptualized and designed the study, performed the experiments, carried out data interpretation, drafted and revised the article critically and approved the final manuscript as submitted; D. H. performed the experiments and revised the final manuscript as submitted; R. O. J. and L. P. recruited the patients and revised the manuscript for submission; M. R. B., D. C. Z. and D. C. C. S. critically reviewed the manuscript and approved the final manuscript as submitted; and T. J. K. performed the statistical analysis and critically reviewed the manuscript and approved the final manuscript as submitted.
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