Importance of the (Pro)renin Receptor in Activating the Renin-Angiotensin System During Normotensive and Preeclamptic Pregnancies.

PURPOSE OF REVIEW: For a healthy pregnancy to occur, a controlled interplay between the maternal circulating renin-angiotensin-aldosterone system (RAAS), placental renin-angiotensin system (RAS) and intrarenal renin-angiotensin system (iRAS) is necessary. Functionally, both the RAAS and iRAS interact to maintain blood pressure and cardiac output, as well as fluid and electrolyte balance. The placental RAS is important for placental development while also influencing the maternal circulating RAAS and iRAS. This narrative review concentrates on the (pro)renin receptor ((P)RR) and its soluble form (s(P)RR) in the context of the hypertensive pregnancy pathology, preeclampsia. RECENT FINDINGS: The (P)RR and the s(P)RR have become of particular interest as not only can they activate prorenin and renin, thus influencing levels of angiotensin II (Ang II), but s(P)RR has now been shown to directly interact with and stimulate the Angiotensin II type 1 receptor (AT1R). Levels of both placental (P)RR and maternal circulating s(P)RR are elevated in patients with preeclampsia. Furthermore, s(P)RR has been shown to increase blood pressure in non-pregnant and pregnant rats and mice. In preeclamptic pregnancies, which are characterised by maternal hypertension and impaired placental development and function, we propose that there is enhanced secretion of s(P)RR from the placenta into the maternal circulation. Due to its ability to both activate prorenin and act as an AT1R agonist, excess maternal circulating s(P)RR can act on both the maternal vasculature, and the kidney, leading to RAS over-activation. This results in dysregulation of the maternal circulating RAAS and overactivation of the iRAS, contributing to maternal hypertension, renal damage, and secondary changes to neurohumoral regulation of fluid and electrolyte balance, ultimately contributing to the pathophysiology of preeclampsia.

ACE2: a key modulator of the renin-angiotensin system and pregnancy.

Angiotensin-converting enzyme 2 (ACE2) is a membrane-bound protein containing 805 amino acids. ACE2 shows approximately 42% sequence similarity to somatic ACE but has different biochemical activities. The key role of ACE2 is to catalyze the vasoconstrictor peptide angiotensin (ANG) II to Ang-(1-7), thus regulating the two major counterbalancing pathways of the renin-angiotensin system (RAS). In this way, ACE2 plays a protective role in end-organ damage by protecting tissues from the proinflammatory actions of ANG II. The circulating RAS is activated in normal pregnancy and is essential for maintaining fluid and electrolyte homeostasis and blood pressure. Renin-angiotensin systems are also found in the conceptus. In this review, we summarize the current knowledge on the regulation and function of circulating and uteroplacental ACE2 in uncomplicated and complicated pregnancies, including those affected by preeclampsia and fetal growth restriction. Since ACE2 is the receptor for SARS-CoV-2, and COVID-19 in pregnancy is associated with more severe disease and increased risk of abnormal pregnancy outcomes, we also discuss the role of ACE2 in mediating some of these adverse consequences. We propose that dysregulation of ACE2 plays a critical role in the development of preeclampsia, fetal growth restriction, and COVID-19-associated pregnancy pathologies and suggest that human recombinant soluble ACE2 could be a novel therapeutic to treat and/or prevent these pregnancy complications.

The (pro)renin receptor and soluble (pro)renin receptor in choriocarcinoma.

This study aimed to determine if the (pro)renin receptor (ATP6AP2) changes the cellular profile of choriocarcinomas from cytotrophoblast cells to terminally syncytialised cells and ascertain whether this impacts the invasive potential of choriocarcinoma cells. Additionally, we aimed to confirm that FURIN and/or site 1 protease (MBTPS1) cleave soluble ATP6AP2 (sATP6AP2) in BeWo choriocarcinoma cells and determine whether sATP6AP2 levels reflect the cellular profile of choriocarcinomas. BeWo choriocarcinoma cells were treated with ATP6AP2 siRNA, FURIN siRNA, DEC-RVKR-CMK (to inhibit FURIN activity), or PF 429242 (to inhibit MBTPS1 activity). Cells were also treated with forskolin, to induce syncytialisation, or vehicle and incubated for 48 h before collection of cells and supernatants. Syncytialisation was assessed by measuring hCG secretion (by ELISA) and E-cadherin protein levels (by immunoblot and immunocytochemistry). Cellular invasion was measured using the xCELLigence real-time cell analysis system and secreted sATP6AP2 levels measured by ELISA. Forskolin successfully induced syncytialisation and significantly increased both BeWo choriocarcinoma cell invasion (P < 0.0001) and sATP6AP2 levels (P = 0.02). Treatment with ATP6AP2 siRNA significantly inhibited syncytialisation (decreased hCG secretion (P = 0.005), the percent of nuclei in syncytia (P = 0.05)), forskolin-induced invasion (P = 0.046), and sATP6AP2 levels (P < 0.0001). FURIN siRNA and DEC-RVKR-CMK significantly decreased sATP6AP2 levels (both P < 0.0001). In conclusion, ATP6AP2 is important for syncytialisation of choriocarcinoma cells and thereby limits choriocarcinoma cell invasion. We postulate that sATP6AP2 could be used as a biomarker measuring the invasive potential of choriocarcinomas. Additionally, we confirmed that FURIN, not MBTPS1, cleaves sATP6AP2 in BeWo cells, but other proteases (inhibited by DEC-RVKR-CMK) may also be involved.

An evaluation of preterm kidney size and function over the first two years of life.

BACKGROUND: We carried out a study to determine the impact of prematurity on kidney development in the first 2 years of life. METHODS: In this prospective study, extremely preterm neonates (gestation < 28 weeks) were recruited and underwent assessments at 6, 12, and 24 months of age. A cohort of neonates born term were also recruited and followed up for 24 months. The primary outcomes measured in this study were total kidney volume (TKV) and estimated glomerular filtration rate (eGFR); albuminuria and blood pressure measurements (all provided as mean (standard deviation)) were the secondary outcomes. RESULTS: Fifty-three premature and 31 term neonates (control) were recruited. At the age of 24 months (corrected age), infants born preterm had significantly smaller TKV (56.1 (9.4) vs. 64.8 (10.2) mL; P = 0.006). There was no difference in eGFR. These preterm infants were smaller (11.25 (1.53) vs. 12.9 (1.8) kg; P = 0.002) and shorter (83.8 (3.0) vs. 86.3 (3.4) cm; P = 0.02) when compared with the control group. At 6, 12, and 18 months respectively, preterm infants had, relative to their height, significantly smaller kidney volumes (0.54 (0.1) vs. 0.59 (0.1) mL/cm, P = 0.05; 0.61 (0.1) vs.0.71 (0.1) mL/cm, P = 0.003; and 0.67 (0.1) vs.0.76 (0.1) mL/cm, P = 0.006). CONCLUSIONS: Relative to body length, TKV in premature infants is smaller. Since length reflects adult body proportions more accurately than BSA, TKV to height ratio may be a more important measure in the child. Despite smaller TKV (and therefore fewer nephrons), infants born prematurely achieve similar eGFRs in the first 24 months of life, probably due to single-nephron hyperfiltration.

MicroRNA mimics that target the placental renin-angiotensin system inhibit trophoblast proliferation.

In early gestation, the human placental renin-angiotensin system (RAS) is upregulated and plays a role in placental development. Among other functions, signalling through the angiotensin II type 1 receptor (AT1R) initiates proliferation. Many microRNAs (miRNAs) targeting placental RAS mRNAs are downregulated at this time. We propose that in early gestation miRNAs that target the placental RAS are downregulated, allowing for the increased RAS expression and proliferation required for adequate placentation. HTR-8/SVneo cells (an immortalized human trophoblast cell line) were used to assess the effect of nine miRNA mimics (at 0.08, 0.16, 0.32 and 0.64 ng/µL) on trophoblast cell proliferation and predicted RAS target mRNAs. The effect of the miRNA mimics on the rate of cell proliferation was assessed using the xCELLigence real-time cell analysis system over 48 h. Levels of miRNAs and predicted RAS target mRNAs were determined by RT-PCR (qPCR, n = 9/group). Statistically different levels of expression were determined (P < 0.05). All nine miRNA mimics significantly affected the proliferation rates of HTR-8/SVneo cells. Five of the miRNA mimics (miR-181a-5p (predicted to target: renin (REN), angiotensin converting enzyme (ACE)), miR-378 (REN, ACE), miR-663 (REN), miR-483-3p (ACE, ACE2, angiotensinogen (AGT), angiotensin II type 1 receptor (AGTR1)) and miR-514 (AGT)) were associated with a dose-dependent reduction in cell proliferation. Seven of the mimics significantly decreased expression of at least one of their predicted target RAS mRNAs. Our study shows that miRNAs targeting placental RAS mRNAs play a role in controlling trophoblast proliferation. As placentation is largely a process of proliferation, changes in expression of these miRNAs may be partly responsible for the expression of the placental RAS, proliferation and placentation.

The role of oxygen in regulating microRNAs in control of the placental renin-angiotensin system.

Human placental renin-angiotensin system (RAS) expression is highest in early gestation, at a time when placental oxygen tension is at its lowest (1-3%), and promotes placental development. Some miRNAs predicted to target RAS mRNAs are downregulated in early gestation. We tested the hypothesis that low oxygen suppresses expression of miRNAs that target placental RAS mRNAs, thus increasing concentrations of RAS mRNAs. HTR-8/SVneo cells were cultured in 1, 5 and 20% oxygen for 48 h. Differences in miRNA expression were measured on an Affymetrix miRNA microarray (n = 3/group). Those predicted to target RAS mRNAs, or that were decreased in early gestation, were confirmed by qPCR (n = 9/group). RAS protein levels were assessed by ELISAs or immuno-blotting. Microarray analysis identified four miRNAs predicted to target RAS mRNAs that were differentially expressed between 1 and 5% oxygen. Using qPCR, 15 miRNAs that target the RAS were measured in HTR-8/SVneo cells. Five miRNAs were downregulated in 1% compared with 5% oxygen. Expression of a number of RAS mRNAs (ATP6AP2, AGT, ACE and AGTR1) were increased in either, or both, 1 and 5% oxygen compared with 20% oxygen. AGT protein levels were increased in 1% oxygen compared with 5%. Further validation is needed to confirm that these miRNAs target RAS mRNAs directly and that placental development is partly regulated by oxygen-sensitive miRNAs that target RAS mRNAs. Since placental oxygen tension changes across gestation, changes in expression of these miRNAs may contribute to the transgestational changes in placental RAS expression and the resulting effects on placental development.

Dysregulation of the placental renin-angiotensin system in human fetal growth restriction.

Fetal growth restriction (FGR) is a pregnancy complication wherein the foetus fails to reach its growth potential. The renin-angiotensin system (RAS) is a critical regulator of placental function, controlling trophoblast proliferation, angiogenesis and blood flow. The RAS significantly influences uteroplacental blood flow through the balance of its vasoconstrictive and vasodilatory pathways. Although the RAS is known to be dysregulated in placentae from women with preeclampsia, the expression of the RAS has not yet been studied in pregnancies compromised by FGR alone. This study investigated the mRNA expression and protein levels of RAS components in placentae from pregnancies compromised by FGR. Angiotensin II type 1 receptor (AGTR1) and angiotensin-converting enzyme 2 (ACE2) mRNA levels were reduced in FGR placentae compared with control (P = 0.012 and 0.018 respectively). Neprilysin (NEP) mRNA expression was lower in FGR placentae compared with control (P = 0.004). mRNA levels of angiotensinogen (AGT) tended to be higher in FGR placentae compared with control (P = 0.090). Expression of prorenin, AGT, angiotensin-converting enzyme (ACE) or ACE2 proteins were similar in control and FGR placentae. The renin-AGT reaction is a first order reaction so levels of expression of placental AGT determine levels of Ang II. Decreasing levels of ACE2 and/or NEP by limiting the production of Ang-(1-7), which is a vasodilator, and increasing placental Ang II levels (vasoconstrictor) may result in an imbalance between the vasoconstrictor and vasodilator arms of the placental RAS. Ultimately this dysregulation of the placental RAS could lead to reduced placental perfusion that is evident in FGR.

Female preterm indigenous Australian infants have lower renal volumes than males: A predisposing factor for end-stage renal disease?

AIM: Indigenous Australians have an increased risk of developing chronic kidney disease (CKD). Indigenous women have a higher rate of CKD than men. In a cohort of Indigenous and non-Indigenous preterm neonates, we assessed total renal volume (TRV) (a proxy indicator for nephron number). We hypothesized that there would be no difference in renal volume between these two groups at term corrected (37 weeks gestation). METHODS: Normally grown preterm neonates less than 32 weeks of gestation were recruited and at term corrected dates, the neonates underwent renal ultrasonography (TRV measurements), urine microalbumin-creatinine ratio and serum analysis for Cystatin C measurement for estimated glomerular filtration rate (eGFR) calculation. RESULTS: One hundred and five neonates (38 Indigenous; 67 non-Indigenous) were recruited. Indigenous neonates were significantly more premature and of lower birth weight. At term corrected age, Indigenous neonates had a significantly smaller TRV (18.5 (4.2) vs 21.4 (5.1) cm3 ; P = 0.027) despite no significant difference in body weight. Despite having a smaller TRV, there was no significant difference in eGFR between Indigenous and Non-indigenous neonates (47.8 [43.2-50.4] vs 46.2 [42.6-53.3] ml/min per 1.73 m2 ; P = 0.986). These infants achieve similar eGFR through hyperfiltration, which likely increases their future risk of CKD. There was no difference in microalbumin-creatinine ratio. Female Indigenous neonates, however, had significantly smaller TRV compared with Indigenous male neonates (15.9 (3.6) vs 20.6 (3.6) cm3 ; P = 0.006), despite no difference in eGFR, birth weight, gestational age, and weight at term corrected. CONCLUSION: The difference in TRV is likely to be an important risk factor for the difference in morbidity and mortality from renal disease reported between male and female Indigenous adults.

Reduced blood volume decreases cerebral blood flow in preterm piglets.

KEY POINTS: Preterm infants often have poor cardiovascular function that is associated with adverse neurodevelopmental outcomes. Preterm infants may be vulnerable to hypovolaemia due to excessive vasodilatation and leaky capillaries. Following reduction in blood volume, cardiac output and mean arterial pressure were reduced to the same extent in term and preterm piglets. Cerebral blood flow was maintained following blood volume reduction in term but not in preterm piglets. Effective detection and treatment of functional hypovolaemia may reduce the risk of brain injury in preterm infants. ABSTRACT: Preterm infants often have impaired cardiovascular function that may contribute to poor neurodevelopmental outcomes. The study aimed to determine the effects of reduced blood volume on cardiovascular function, including cerebral blood flow, in preterm and term piglets. In preterm (97/115 days) and term piglets, up to 10% of the estimated blood volume was removed. Removal of blood was stopped if MAP dropped below 20 mmHg. Heart rate, cardiac contractility and relaxation, cardiac output, mean arterial pressure (MAP), and cerebral blood flow were measured at baseline and again after blood volume reduction. The volume of blood removed was less in preterm piglets than in term piglets (5.1 ± 1.8 vs. 7.7 ± 0.9 mL kg-1 , mean ± SD, P < 0.001). Cardiac output and MAP decreased to the same extent in term and preterm piglets. Cerebral blood flow decreased in preterm but not term piglets and cerebral vascular conductance increased in term piglets only. Compensatory responses to maintain cerebral blood flow after blood volume reduction are active in term piglets but not in preterm piglets. As a result, even a small reduction in blood volume, or an increase in the capacity of the circulatory system leading to functional hypovolaemia, may lead to a significant reduction in cerebral blood flow and contribute to brain injury in preterm neonates.

Regulation of the human placental (pro)renin receptor-prorenin-angiotensin system by microRNAs.

STUDY QUESTION: Are any microRNAs (miRNAs) that target the placental renin-angiotensin system (RAS) in the human placenta suppressed in early gestation? SUMMARY ANSWER: Overall, 21 miRNAs with predicted RAS mRNA targets were less abundant in early versus term placentae and nine were more highly expressed. WHAT IS KNOWN ALREADY: Regulation of human placental RAS expression could alter placental development and therefore normal pregnancy outcome. The expression of genes encoding prorenin (REN), angiotensinogen, (pro)renin receptor, angiotensin converting enzyme 2, and the angiotensin II type 1 receptor are highest in early gestation, at a time when oxygen tension is at its lowest. Studies have shown that the human placental RAS is sensitive to oxygen, as are some miRNAs that regulate RAS mRNAs. We propose that in early pregnancy, the prevailing low O2 tension, by suppression of levels of miRNAs that target RAS mRNAs, results in increased expression of RAS mRNAs and encoded proteins. As gestation proceeds and the prevailing oxygen tension rises, abundance of these miRNAs increases, and placental RAS mRNA expression is suppressed. STUDY DESIGN, SIZE, DURATION: The expression of miRNAs was compared in human placentae collected in early (10-11 weeks; n = 7) and mid-gestation (14-18 weeks; n = 8) with placenta collected at term (38-40 weeks; n = 8). Expression of placental miRNAs in women with early (29-35.1 weeks; n = 8) or late-onset pre-eclampsia (PE) (>34-weeks gestation; n = 8) and gestational age matched preterm (31.6-35.1 weeks; n = 8) and term normotensive controls were also compared. PARTICIPANTS/MATERIALS, SETTING, METHODS: Agilent Human miRNA microarray v19 was used to detect up to 2006 miRNAs in four placentae from each group. Statistically different levels of expression were determined and refined using predictive modelling. Placental miRNAs predicted to target RAS mRNAs were identified in three databases. Differences detected on the array were confirmed for some miRNAs by semi-quantitative RT-PCR (qPCR, n = 7-8 for all groups). Two differentially expressed miRNAs that were known to target human renal REN mRNA (miR-181a-5p and miR-663) were transfected into human HTR-8/SVneo trophoblast cells to examine their effect on placental REN expression and prorenin levels. MAIN RESULTS AND THE ROLE OF CHANCE: In early gestation placentae, 186 miRNAs were differentially expressed compared with term placentae (109 increased, 77 decreased). Thirty of the differentially expressed miRNAs were predicted to target RAS components. In mid-gestation placentae, 117 miRNAs were differentially expressed compared with term placentae (69 increased, 48 decreased). Of these, 19 had RAS mRNAs as predicted targets. Eight miRNAs that were lower in early gestation and predicted to target RAS mRNAs were confirmed by qPCR. All showed an increase during gestation and could influence the transgestational profile of the human placental RAS. Additionally, on the array, three miRNAs predicted to target RAS mRNAs (miR-892c-3p, miR-378c and miR-514b-3p) were overexpressed in placentae from women with late-onset PE (P = 3.6E-10, P = 1.8E-05, P = 5.3E-06; respectively). miR-663, which suppresses renal REN mRNA expression, was overexpressed in early-onset PE placentae as determined by qRT-PCR analysis (P = 0.014). Transfection of miR-181a-5p and miR-663 into HTR-8/SVneo trophoblast cells suppressed REN mRNA expression (P = 0.05) and prorenin protein production (P = 0.001). LARGE SCALE DATA: Data can be found via GEO accession number GSE109832. LIMITATIONS, REASONS FOR CAUTION: Further validation that the differentially expressed miRNAs do indeed directly target RAS mRNAs and affect placental development and function is required. This study is limited by the small sample size. Therefore independent validation in a larger cohort is required. WIDER IMPLICATIONS OF THE FINDINGS: We propose that suppression of miRNAs that target the placental RAS in early gestation is partly responsible for the increase in RAS expression at this time, in order to promote placental development. Later in pregnancy, we have detected overexpression of several miRNAs in placentae from women with PE. These may prove to be biomarkers for early detection of women at risk of developing PE. Since the placenta produces at least two miRNAs that were found in the kidney to target REN mRNA, and that also target placental REN mRNA, the escape of these miRNAs into the maternal circulation in excess amounts could affect maternal renal REN mRNA production and thereby disturb maternal fluid and electrolyte homoeostasis. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Health and Medical Research Council, Australia (APP1043537). K.G.P. is supported by an Australian Research Council Future Fellowship (FT150100179). C.T.R. is supported by a Lloyd Cox Professorial Research Fellowship from the University of Adelaide. F.Z.M. is supported by a National Heart Foundation Future Leader Fellowship and Baker Heart and Diabetes Institute Fellowship. The authors declare that they have no competing interests.

Extra uterine development of preterm kidneys.

OBJECTIVE: We carried out a study to determine the impact of prematurity on renal development. The primary outcomes measured were nephrinuria and albuminuria; renal volume and glomerular filtration rate were the secondary outcomes. METHODS: Preterm neonates born at less than 28 weeks of gestation, with birth weight between 10th and 90th centile (appropriate for gestational age), were recruited and underwent assessments at 28, 32 and 37 weeks postmenstrual age (PMA). RESULTS: Fifty-three premature neonates and 31 term neonates (control) were recruited. The median gestational age of the premature neonates was 26.4 [24.7-27.4] weeks, with a mean birth weight of 886 (179) g. The mean gestational age of term neonates was 39.1 (1.2) weeks and the mean birth weight was 3406 (406) g. The median age of the term neonates was 6.5 [3.0-12.5] days. The total kidney volume (TKV) almost doubled from 10.3 (2.9) cm3 at 28 weeks PMA to 19.2 (3.7) cm3 at 37 weeks PMA (P = 0.0001). TKV at 37 weeks PMA was significantly smaller compared to term neonates (19.2 (3.7) vs 26.3 (7.0) cm3; P = 0.0001). However, there was no significant difference in estimated glomerular filtration rate (eGFR) between premature neonates (at 37 weeks PMA) and term neonates (control) (43.5 [39.7-48.9] vs. 42.0 [38.2-50.0] mL/min/1.73 m2; P = 0.75). There was a statistically significant decline in nephrin-creatinine ratio and albumin-creatinine ratio from 32 to 37 weeks PMA. CONCLUSIONS: Despite having a smaller renal volume (and fewer nephrons), extremely premature neonates achieve similar eGFRs at corrected term as term-born neonates, likely through single nephron hyperfiltration. Extremely premature neonates also show evidence of glomerular injury.

The intrauterine renin-angiotensin system: Sex-specific effects on the prevalence of spontaneous preterm birth.

Preterm birth (PTB) is the single largest cause of death in infants and young children. The rate of PTB is significantly higher in male infants, particularly those that are born very preterm. Here we present evidence to suggest that the decidual renin-angiotensin system may play a role in inhibiting inflammation and maintaining the integrity of the fetal membranes during pregnancy, and that sex-specific alterations in the intrauterine RAS could contribute to the increased risk of PTB in male babies. Women carrying female fetuses have high levels of expression of decidual prorenin at term. Decidua from 'female' pregnancies also have greater expression of the anti-inflammatory angiotensin (Ang)-(1-7) pathway, than decidua from 'male' pregnancies, and have lower levels of the pro-inflammatory Ang II pathway. We propose that in 'female' pregnancies, the very high levels of decidual prorenin drive the anti-inflammatory Ang-(1-7) pathway, thus reducing the likelihood of PTB. In addition, the high levels of prorenin produced by the decidua in 'female' pregnancies are able to diffuse into the amnion and bind to the PRR. We postulate that PRR/prorenin interactions, possibly through both angiotensin dependent and independent pathways, stimulate the production of ECM proteins, inhibit ECM degradation and prevent apoptosis, thus strengthening the amnion. Thus control of the inflammatory signature and the integrity of the fetal membranes prior to parturition may partly depend on the sexually determined activity of the decidual and amniotic renin-angiotensin system pathways.

Post-traumatic stress disorder symptoms in pregnant Australian Indigenous women residing in rural and remote New South Wales: A cross-sectional descriptive study.

BACKGROUND: Pregnancy can be a stressful time for many women. There is ample evidence of numerous physical and mental health inequities for Indigenous Australians. For those Indigenous women who are pregnant, it is established that there is a higher incidence of poor physical perinatal outcomes when compared with non-Indigenous Australians. However, little evidence exists that examines stressful events and post-traumatic stress disorder (PTSD) symptoms in pregnant women who are members of this community. AIMS: To quantify the rates of stressful events and PTSD symptoms in pregnant Indigenous women. METHODS: One hundred and fifty rural and remote Indigenous women were invited to complete a survey during each trimester of their pregnancy. The survey measures were the stressful life events and the Impact of Events Scale. RESULTS: Extremely high rates of PTSD symptoms were reported by participants. Approximately 40% of this group exhibited PTSD symptoms during their pregnancy with mean score 33.38 (SD = 14.37) significantly higher than a study of European victims of crisis, including terrorism attacks (20.6, SD = 18.5). CONCLUSIONS: The extreme levels of PTSD symptoms found in the women participating in this study are likely to result in negative implications for both mother and infant. An urgent response must be mounted at government, health, community development and research levels to address these findings. Immediate attention needs to focus on the development of interventions to address the high levels of PTSD symptoms that pregnant Australian Indigenous women experience.

Inotropes do not increase cardiac output or cerebral blood flow in preterm piglets.

BACKGROUND: The preterm newborn is at high risk of developing cardiovascular compromise during the first day of life and this is associated with increased risk of brain injury. Standard treatments are volume expansion and administration of inotropes, typically dopamine and/or dobutamine, but there is limited evidence that inotropes improve clinical outcomes. This study investigated the efficacy of dopamine and dobutamine for the treatment of cardiovascular compromise in the preterm newborn using a piglet model. METHODS: Preterm and term piglets were assigned to either dopamine, dobutamine or control infusions. Heart rate, left ventricular contractility, cardiac output, blood pressure, and cerebral and regional blood flows were measured during baseline, low (10 µg/kg/h), and high (20 µg/kg/h) dose infusions. RESULTS: At baseline, preterm piglets had lower cardiac contractility, cardiac output, blood pressure, and cerebral blood flow compared to term piglets. The response of preterm piglets to either dopamine or dobutamine administration was less than in term piglets. In both preterm and term piglets, cardiac output and cerebral blood flow were unaltered by either inotrope. CONCLUSION: In order to provide better cardiovascular support, it may be necessary to develop treatments that target receptors with a more mature profile than adrenoceptors in the preterm newborn.

The prevalence of unique SNPs in the renin-angiotensin system highlights the need for pharmacogenetics in Indigenous Australians.

Genetic differences between ethnic populations affect susceptibility to disease and efficacy of drugs. This study examined and compared the prevalence of single nucleotide polymorphisms (SNPs) in genes of the renin-angiotensin system (RAS) in a desert community of Indigenous Australians and in non-Indigenous Australians. The polymorphisms were angiotensinogen, AGT G-217A (rs5049); AGT G+174A (rs4762); Angiotensin II type 1 receptor, AGTR1 A+1166C (rs5186); angiotensin converting enzyme, ACE A-240T (rs4291), ACE T-93C (rs4292); renin, REN T+1142C (rs5706). They were measured using allelic discrimination assays. The prevalence of REN T+1142C SNP was similar in the two populations; 99% were homozygous for the T allele. All other SNPs were differently distributed between the two populations (P < 0.0001). In non-Indigenous Australians, the A allele at position 204 of ACE rs4291 was prevalent (61.8%) whereas in the Indigenous Australians the A allele was less prevalent (28%). For rs4292, the C allele had a prevalence of 37.9% in non-Indigenous Australians but in Indigenous Australians the prevalence was only 1%. No Indigenous individuals were homozygous for the C allele of AGTR1 (rs5186). Thus the prevalence of RAS SNPs in this Indigenous Australian desert community was different from non-Indigenous Australians as was the prevalence of cytokine SNPs (as shown in a previous study). These differences may affect susceptibility to chronic renal and cardiovascular disease and may alter the efficacy of drugs used to inhibit the RAS. These studies highlight the need to study the pharmacogenetics of drug absorption, distribution, metabolism and excretion in Indigenous Australians for safe prescribing guidelines.

Decidualisation of human endometrial stromal cells is associated with increased expression and secretion of prorenin.

BACKGROUND: In pregnancy, the decidualised endometrium expresses high levels of prorenin and other genes of the renin-angiotensin system (RAS) pathway. In this study we aimed to determined if the RAS was present in endometrial stromal cells and if decidualisation upregulated the expression of prorenin, the prorenin receptor ((P)RR) and associated RAS pathways. Immortalised human endometrial stromal cells (HESCs) can be stimulated to decidualise by combined treatment with medroxyprogesterone acetate (MPA), 17ß-estradiol (E2) and cAMP (MPA-mix) or with 5-aza-2'-deoxycytidine (AZA), a global demethylating agent. METHODS: HESCs were incubated for 10 days with one of the following treatments: vehicle, MPA-mix, a combination of medroxyprogesterone acetate (MPA) and estradiol-17ß alone, or AZA. Messenger RNA abundance and protein levels of prorenin (REN), the (P)RR (ATP6AP2), angiotensinogen (AGT), angiotensin converting enzyme (ACE), angiotensin II type 1 receptor (AGTR1), vascular endothelial growth factor (VEGF), and plasminogen activator inhibitor-1 (PAI-1) were measured by real-time PCR and ELISA's, respectively. Promyelocytic zinc finger (PLZF) and phospho-inositol-3 kinase (PIK3R1) mRNA abundances were also measured. RESULTS: HESCs expressed the prorenin receptor (ATP6AP2), REN, AGT, ACE and low levels of AGTR1. MPA-mix and AZA stimulated expression of REN. Prorenin protein secretion was increased in MPA-mix treated HESCs. E2 + MPA had no effect on any RAS genes. MPA-mix treatment was associated with increased VEGF (VEGFA) and PAI-1 (SERPINE1) mRNA and VEGF protein. CONCLUSIONS: An endometrial prorenin receptor/renin angiotensin system is activated by decidualisation. Since (P)RR is abundant, the increase in prorenin secretion could have stimulated VEGF A and SERPINE1 expression via Ang II, as both ACE and AGTR1 are present, or by Ang II independent pathways. Activation of the RAS in human endometrium with decidualisation, through stimulation of VEGF expression and secretion, could be critical in establishing an adequate blood supply to the developing maternal placental vascular bed.

Roles of the circulating renin-angiotensin-aldosterone system in human pregnancy.

This review describes the changes that occur in circulating renin-angiotensin-aldosterone system (RAAS) components in human pregnancy. These changes depend on endocrine secretions from the ovary and possibly the placenta and decidua. Not only do these hormonal secretions directly contribute to the increase in RAAS levels, they also cause physiological changes within the cardiovascular system and the kidney, which, in turn, induce reflex release of renal renin. High levels of ANG II play a critical role in maintaining circulating blood volume, blood pressure, and uteroplacental blood flow through interactions with the ANG II type I receptor and through increased production of downstream peptides acting on a changing ANG receptor phenotype. The increase in ANG II early in gestation is driven by estrogen-induced increments in angiotensinogen (AGT) levels, so there cannot be negative feedback leading to reduced ANG II production. AGT can exist in various forms in terms of redox state or complexed with other proteins as polymers; these affect the ability of renin to cleave ANG I from AGT. Thus, during pregnancy the rate of ANG I production varies not only because levels of renin change in response to homeostatic demand but also because AGT changes not only in concentration but in form. Activation of the circulating and intrarenal RAASs is essential for normal pregnancy outcome subserving the increased demand for salt and, hence, water during pregnancy. Thus, the complex integration of the secretions and actions of the circulating maternal renin-angiotensin system in pregnancy plays a key role in pregnancy outcome.

Upregulation of the Renin-Angiotensin System Is Associated with Patient Survival and the Tumour Microenvironment in Glioblastoma.

Glioblastoma is a highly aggressive disease with poor survival outcomes. An emerging body of literature links the role of the renin-angiotensin system (RAS), well-known for its function in the cardiovascular system, to the progression of cancers. We studied the expression of RAS-related genes (ATP6AP2, AGTR1, AGTR2, ACE, AGT, and REN) in The Cancer Genome Atlas (TCGA) glioblastoma cohort, their relationship to patient survival, and association with tumour microenvironment pathways. The expression of RAS genes was then examined in 12 patient-derived glioblastoma cell lines treated with chemoradiation. In cases of glioblastoma within the TCGA, ATP6AP2, AGTR1, ACE, and AGT had consistent expressions across samples, while AGTR2 and REN were lowly expressed. High expression of AGTR1 was independently associated with lower progression-free survival (PFS) (p = 0.01) and had a non-significant trend for overall survival (OS) after multivariate analysis (p = 0.095). The combined expression of RAS receptors (ATP6AP2, AGTR1, and AGTR2) was positively associated with gene pathways involved in hypoxia, microvasculature, stem cell plasticity, and the molecular characterisation of glioblastoma subtypes. In patient-derived glioblastoma cell lines, ATP6AP2 and AGTR1 were upregulated after chemoradiotherapy and correlated with an increase in HIF1A expression. This data suggests the RAS is correlated with changes in the tumour microenvironment and associated with glioblastoma survival outcomes.

The soluble (pro)renin receptor promotes a preeclampsia-like phenotype both in vitro and in vivo.

Preeclampsia is classified as new-onset hypertension coupled with gross endothelial dysfunction. Placental (pro)renin receptor ((P)RR) and plasma soluble (P)RR (s(P)RR) are elevated in patients with preeclampsia. Thus, we aimed to interrogate the role (P)RR may play in the pathogenesis of preeclampsia. Human uterine microvascular endothelial cells (HUtMECs, n = 4) were cultured with either; vehicle (PBS), 25-100 nM recombinant s(P)RR, or 10 ng/ml TNF-a (positive control) for 24 h. Conditioned media and cells were assessed for endothelial dysfunction markers via qPCR, ELISA, and immunoblot. Angiogenic capacity was assessed through tube formation and adhesion assays. Additionally, pregnant rats were injected with an adenovirus overexpressing s(P)RR from mid-pregnancy (day 8.5), until term (n = 6-7 dams/treatment). Maternal and fetal tissues were assessed. HUtMECs treated with recombinant s(P)RR displayed increased expression of endothelial dysfunction makers including vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and endothelin-1 mRNA expression (P = 0.003, P = 0.001, P = 0.009, respectively), along with elevated endothelin-1 protein secretion (P < 0.001) compared with controls. Recombinant s(P)RR impaired angiogenic capacity decreasing the number of branches, total branch length, and mesh area (P < 0.001, P = 0.004, and P = 0.009, respectively), while also increasing vascular adhesion (P = 0.032). +ADV rats exhibited increased systolic (P = 0.001), diastolic (P = 0.010), and mean arterial pressures (P = 0.012), compared with -ADV pregnancies. Renal arteries from +ADV-treated rats had decreased sensitivity to acetylcholine-induced relaxation (P = 0.030), compared with -ADV pregnancies. Our data show that treatment with s(P)RR caused hypertension and growth restriction in vivo and caused marked endothelial dysfunction in vitro. These findings demonstrate the significant adverse actions of s(P)RR on vascular dysfunction that is characteristic of the preeclamptic phenotype.

The association of maternal ACE A11860G with small for gestational age babies is modulated by the environment and by fetal sex: a multicentre prospective case-control study.

UNLABELLED: We aimed to determine whether the ACE A11860G genotype is associated with small for gestational age babies (SGA) and to determine whether the association is affected by environmental factors and fetal sex. Overall, 3234 healthy nulliparous women with singleton pregnancies, their partners and babies were prospectively recruited in Adelaide, Australia and Auckland, New Zealand. Data analyses were confined to 2121 Caucasian parent-infant trios, among which 216 were pregnancies with SGA infants and 1185 were uncomplicated pregnancies. Women with the ACE A11860G GG genotype in the combined and Adelaide cohorts had increased risk for SGA [odds ratios (OR) 1.5, 95% confidence interval (CI) 1.1-2.1 and OR 2.0, 95% CI 1.3-3.3, respectively) and delivered lighter babies (P = 0.02; P = 0.007, respectively) compared with those with AA/AG genotypes. The maternal ACE A11860G GG genotype was associated with higher maternal plasma ACE concentration at 15 weeks' gestation than AA/AG genotypes (P < 0.001). When the Adelaide cohort was stratified by maternal socio-economic index (SEI) and pre-pregnancy green leafy vegetable intake, the ACE A11860G GG genotype was only associated with an increased risk for SGA (OR 4.9, 95% CI 1.8-13.4 and OR 3.3, 95% CI 1.6-7.0, respectively) and a reduction in customized birthweight centile (P = 0.006 and P = 0.03) if superimposed on maternal SEI <34 or pre-pregnancy green leafy vegetable intake <1 serve/day. Furthermore, the associations of maternal ACE A11860G with customized birthweight centile observed among Adelaide women with SEI <34 or pre-pregnancy green leafy vegetable intake <1 serve/day were female specific. The current study identified a novel association of maternal ACE A11860G with SGA. More interestingly, this association was modified by environmental factors and fetal sex, suggesting ACE A11860G-environment-fetal sex interactions. Trial Registry Name: Screening nulliparous women to identify the combinations of clinical risk factors and/or biomarkers required to predict pre-eclampsia, SGA babies and spontaneous preterm birth. URL: http://www.anzctr.org.au. REGISTRATION NUMBER: ACTRN12607000551493.