Professor Eugenie Lumbers

The (pro)renin receptor ((P)RR) is a multi-functional protein that can be proteolytically cleaved and released in a soluble form (s(P)RR). Recently, the (P)RR and s(P)RR have become of interest in pregnancy and its associated pathologies. This is because the (P)RR not only activates tissue renin angiotensin systems, but it is also an integral component of vacuolar-ATPase, activates the wingless/integrated (Wnt)/ß-catenin and extracellular signal regulated kinases 1 and 2/mitogen-activated protein kinase signalling pathways, and stabilises the ß subunit of pyruvate dehydrogenase. Additionally, s(P)RR is detected in plasma and urine, and maternal plasma levels are elevated in pregnancy complications including fetal growth restriction, preeclampsia and gestational diabetes mellitus. Therefore, s(P)RR has potential as a biomarker for these pregnancy pathologies. Preliminary functional findings suggest that s(P)RR may be important for regulating fluid balance, inflammation and blood pressure, all of which contribute to a successful pregnancy. The (P)RR and s(P)RR regulate pathways that are known to be important in maintaining pregnancy, however their role in the physiological context of pregnancy is poorly characterised. This review summarises the known and potential functions of the (P)RR and s(P)RR in pregnancy, and how their dysregulation may contribute to pregnancy complications. It also highlights the need for further research into the source and function of s(P)RR in pregnancy. Soluble (P)RR levels could be indicative of placental, kidney or liver dysfunction and therefore be a novel clinical biomarker, or therapeutic target, to improve the detection and treatment of pregnancy pathologies.

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.

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.

Suboptimal nutrition during pregnancy is recognised as a significant modifiable determinant in the development of chronic disease in offspring in later life. The current study aimed: (i) to assess the dietary intakes of pregnant Indigenous Australian women against national recommendations and (ii) to investigate the associations between maternal nutrition during pregnancy and the growth of the offspring, including kidney development in late gestation in the Gomeroi gaaynggal cohort (n = 103). Maternal dietary intake in the third trimester was assessed using the Australian Eating Survey Food Frequency Questionnaire. Estimated fetal weight (EFW) and kidney size were obtained by ultrasound. Birth weight was retrieved from hospital birth records. Of the five key nutrients for optimal reproductive health (folate, iron, calcium, zinc and fibre), the nutrients with the highest percentage of pregnant women achieving the nutrient reference values (NRVs) were zinc (75.7%) and folate (57.3%), whereas iron was the lowest. Only four people achieved all NRVs (folate, iron, calcium, zinc and fibre) important in pregnancy. Sodium and saturated fat intake exceeded recommended levels and diet quality was low, with a median score of 28 out of 73 points. After adjusting for smoking and pre-pregnancy body mass index, only maternal intake of retinol equivalents and the proportion of energy from nutrient-dense or energy-dense, nutrient-poor (EDNP) foods were associated with fetal growth. EFW decreased by 0.13 g and birth weight decreased by 0.24 g for every µg increase in maternal dietary retinol intake. Interestingly, EFW, but not actual birth weight, was positively associated with percentage energy from nutrient dense foods and negatively associated with percentage energy from EDNP foods. Dietary supplement usage was associated with increased birthweight, most significantly iron and folate supplementation. Current dietary intakes of pregnant Australian women from this cohort do not align with national guidelines. Furthermore, current findings show that maternal retinol intake and diet composition during pregnancy can influence fetal growth, but not fetal kidney growth in late gestation. Strategies that aim to support and optimise nutrient intakes of Indigenous pregnant women are urgently needed. Future studies with long-term follow-up of the children in the current cohort to assess renal damage and blood pressure are imperative.

Introduction: In the placenta, the (pro)renin receptor (ATP6AP2) is localised to the syncytiotrophoblast. ATP6AP2 can activate the placental renin-angiotensin system (RAS), producing Angiotensin II (Ang II) which, acting via the angiotensin II type 1 receptor (AGTR1), is important for placental development and function. ATP6AP2 can also independently stimulate intracellular signalling pathways known to regulate trophoblast syncytialisation. We proposed that ATP6AP2 plays a role in trophoblast syncytialisation. Methods: Primary trophoblast cells were isolated from human placentae and transfected with an ATP6AP2 siRNA, a negative control siRNA or vehicle and allowed to spontaneously syncytialise. Syncytialisation was determined by secretion of human chorionic gonadotrophin (hCG) and by decreased CDH1 (E-cadherin) levels. Expression of RAS mRNAs and proteins were measured by qPCR and immunoblotting, respectively. Results: Primary trophoblast cells spontaneously syncytialised in culture. Syncytialisation did not affect ATP6AP2 mRNA or protein levels. However, the expression of REN, AGT and AGTR1 mRNAs were increased (P = 0.02, P = 0.01 and P = 0.03, respectively). ATP6AP2 siRNA had no effect on syncytialisation. Discussion: In primary trophoblasts, syncytialisation was associated with increased expression of the RAS. hCG was increased during syncytialisation and is known to stimulate REN and possibly AGT, however further experiments are needed to confirm that this was the mechanism via which the RAS was activated. Therefore, syncytialisation of primary trophoblasts may involve hCG-induced RAS activation and downstream activation of signalling pathways and growth factors, which can be stimulated via the interaction of Ang II with AGTR1. Nevertheless, it appears that the (pro)renin receptor is not involved.

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.

Childhood obesity rates are higher among Indigenous compared with non-Indigenous Australian children. It has been hypothesized that early-life influences beginning with the intrauterine environment predict the development of obesity in the offspring. The aim of this paper was to assess, in 227 mother-child dyads from the Gomeroi gaaynggal cohort, associations between prematurity, Gestation Related-Optimal Weight (GROW) centiles, maternal adiposity (percentage body fat, visceral fat area), maternal non-fasting plasma glucose levels (measured at mean gestational age of 23.1 weeks) and offspring BMI and adiposity (abdominal circumference, subscapular skinfold thickness) in early childhood (mean age 23.4 months). Maternal non-fasting plasma glucose concentrations were positively associated with infant birth weight (P=0.005) and GROW customized birth weight centiles (P=0.008). There was a significant association between maternal percentage body fat (P=0.02) and visceral fat area (P=0.00) with infant body weight in early childhood. Body mass index (BMI) in early childhood was significantly higher in offspring born preterm compared with those born at term (P=0.03). GROW customized birth weight centiles was significantly associated with body weight (P=0.01), BMI (P=0.007) and abdominal circumference (P=0.039) at early childhood. Our findings suggest that being born preterm, large for gestational age or exposed to an obesogenic intrauterine environment and higher maternal non-fasting plasma glucose concentrations are associated with increased obesity risk in early childhood. Future strategies should aim to reduce the prevalence of overweight/obesity in women of child-bearing age and emphasize the importance of optimal glycemia during pregnancy, particularly in Indigenous women.

Introduction: Placental development occurs in a low oxygen environment, which stimulates angiogenesis by upregulating vascular endothelial growth factor A (VEGFA), plasminogen activator inhibitor-1 (SERPINE1) and the angiopoietin-2/-1 ratio (ANGPT2/1). At this time, Angiotensin II type 1 receptor (AT1R) is highly expressed. We postulated that the early gestation placental oxygen milieu, by stimulating the angiotensin (Ang) II/AT1R pathway, increases expression of proliferative/angiogenic factors. Methods: HTR-8/SVneo cells were cultured in 1%, 5% or 20% O2 with the AT1R antagonist (losartan) for 48 h. mRNA and protein levels of angiogenic factors were determined by qPCR and ELISA. Angiogenesis and cell viability were assessed by HUVEC tube formation and resazurin assay. Results: Culture in low oxygen (1%) increased angiogenic VEGFA, SERPINE1 and placental growth factor (PGF) mRNA and VEGFA and SERPINE1 protein levels, and reduced anti-angiogenic ANGPT1, endoglin (ENG) and soluble fms-like tyrosine kinase-e15a (sFlt-e15a) mRNA (all P = 0.0001). At 1% oxygen, losartan significantly reduced intracellular VEGFA and SERPINE1 levels and secreted VEGF levels (P = 0.008, 0.0001 and 0.0001). HUVEC tube formation was increased in cells grown in HTR-8/SVneo conditioned medium from 1 to 5% cultures (all P = 0.0001). HUVECs cultured in medium from losartan treated HTR-8/SVneo cells had a reduced number of meshes, branching points and total branching length (P = 0.004, 0.003 and 0.0002). At 1% oxygen, losartan partially inhibited the oxygen-induced increase in cell viability (P = 0.0001). Discussion: Thus, AT1R blockade antagonised the low oxygen induced increase in pro-angiogenic factor expression and cell viability. Our findings highlight a role for an oxygen-sensitive Ang II/AT1R pathway during placentation.

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.

Adverse pregnancy outcomes including prematurity and low birth weight (LBW) have been associated with life-long chronic disease risk for the infant. Stress during pregnancy increases the risk of adverse pregnancy outcomes. Many studies have reported the incidence of adverse pregnancy outcomes in Indigenous populations and a smaller number of studies have measured rates of stress and depression in these populations. This study sought to examine the potential association between stress during pregnancy and the rate of adverse pregnancy outcomes in Australian Indigenous women residing in rural and remote communities in New South Wales. This study found a higher rate of post-traumatic stress disorder, depression and anxiety symptoms during pregnancy than the general population. There was also a higher incidence of prematurity and LBW deliveries. Unfortunately, missing post-traumatic stress disorder and depressive symptomatology data impeded the examination of associations of interest. This was largely due to the highly sensitive nature of the issues under investigation, and the need to ensure adequate levels of trust between Indigenous women and research staff before disclosure and recording of sensitive research data. We were unable to demonstrate a significant association between the level of stress and the incidence of adverse pregnancy outcomes at this stage. We recommend this longitudinal study continue until complete data sets are available. Future research in this area should ensure prioritization of building trust in participants and overestimating sample size to ensure no undue pressure is placed upon an already stressed participant.

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.

The intrarenal renin-angiotensin system (iRAS) is implicated in the pathogenesis of hypertension, chronic kidney disease and diabetic nephropathy. Urinary angiotensinogen (uAGT) levels reflect the activity of the iRAS and are altered in women with preeclampsia. Since Indigenous Australians suffer high rates and early onset of renal disease, we hypothesised that Indigenous Australian pregnant women, like non-Indigenous women with pregnancy complications, would have altered uAGT levels. The excretion of RAS proteins was measured in non-Indigenous and Indigenous Australian women with uncomplicated or complicated pregnancies (preeclampsia, diabetes/gestational diabetes, proteinuria/albuminuria, hypertension, small/large for gestational age, preterm birth), and in non-pregnant non-Indigenous women. Non-Indigenous pregnant women with uncomplicated pregnancies, had higher uAGT/creatinine levels than non-Indigenous non-pregnant women (P < 0.01), and levels increased as pregnancy progressed (P < 0.001). In non-Indigenous pregnant women with pregnancy complications, uAGT/creatinine was suppressed in the third trimester (P < 0.01). In Indigenous pregnant women with uncomplicated pregnancies, there was no change in uAGT/creatinine with gestational age and uAGT/creatinine was lower in the 2nd and 3rd trimesters than in non-Indigenous pregnant women with uncomplicated pregnancies (P < 0.03, P < 0.007, respectively). The uAGT/creatinine ratios of Indigenous women with uncomplicated or complicated pregnancies were the same. A decrease in uAGT/creatinine with advancing gestational age was associated with increased urinary albumin/creatinine, as is seen in preeclampsia, but it was not specific for this disorder. The reduced uAGT/creatinine in Indigenous pregnant women may reflect subclinical renal dysfunction which limits the ability of the kidney to maintain sodium balance and could indicate an increased risk of pregnancy complications and/or future renal disease.

Tissue renin-angiotensin systems (RASs) are involved in tissue growth and development as they are important regulators of angiogenesis, cell proliferation and migration. The placental RAS is most highly expressed in early gestation, at a time when the oxygen tension within the conceptus is reduced, and plays a key role in placental growth and development. Similar to the placenta, tumour development relies on proliferation, angiogenesis and invasion in order to grow and metastasize. The RAS is known to be upregulated in a variety of solid tumours, including ovarian, endometrial, cervical, breast and prostate. This review explores the roles of oxygen and microRNAs in regulating the normal expression of the placental RAS, providing insight into regulation of its development as well as the development of disease states in which the RAS is overexpressed. We propose that the placental RAS is downregulated by microRNAs that are suppressed during the physiologically normal ¿hypoxic¿ phase of early placentation. Suppression of these miRNAs allows the placental RAS to stimulate placental growth and angiogenesis. We propose that similar mechanisms may be at play in solid tumours, which are characterised by hypoxia.

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.

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.

Indigenous Australians have high rates of chronic diseases, the causes of which are complex and include social and environmental determinants. Early experiences in utero may also predispose to later-life disease development. The Gomeroi gaaynggal study was established to explore intrauterine origins of renal disease, diabetes and growth in order to inform the development of health programmes for Indigenous Australian women and children. Pregnant women are recruited from antenatal clinics in Tamworth, Newcastle and Walgett, New South Wales, Australia, by Indigenous research assistants. Measures are collected at three time points in pregnancy and from women and their children at up to eight time points in the child's first 5 years. Measures of fetal renal development and function include ultrasound and biochemical biomarkers. Dietary intake, infant feeding and anthropometric measurements are collected. Standardized procedures and validated tools are used where available. Since 2010 the study has recruited over 230 women, and retained 66 postpartum. Recruitment is ongoing, and Gomeroi gaaynggal is currently the largest Indigenous pregnancy-through-early-childhood cohort internationally. Baseline median gestational age was 39.1 weeks (31.5-43.2, n=110), median birth weight was 3180 g (910-5430 g, n=110). Over one third (39.3%) of infants were admitted to special care or neonatal nursery. Nearly half of mothers (47.5%) reported tobacco smoking during pregnancy. Results of the study will contribute to knowledge about origins of chronic disease in Indigenous Australians and nutrition and growth of women and their offspring during pregnancy and postpartum. Study strengths include employment and capacity-building of Indigenous staff and the complementary ArtsHealth programme.

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.

During the first trimester, normal placental development occurs in a low oxygen environment that is known to stimulate angiogenesis via upregulation of vascular endothelial growth factor (VEGF). Expression of the placental renin-angiotensin system (RAS) is highest in early pregnancy. While the RAS and oxygen both stimulate angiogenesis, how they interact within the placenta is unknown. We postulated that low oxygen increases expression of the proangiogenic RAS pathway and that this is associated with increased VEGF in a first trimester human trophoblast cell line (HTR-8/SVneo). HTR-8/SVneo cells were cultured in one of three oxygen tensions (1%, 5% and 20%). RAS and VEGF mRNA expression were determined by qPCR. Prorenin, angiotensin converting enzyme (ACE) and VEGF protein levels in the supernatant, as well as prorenin and ACE in cell lysates, were measured using ELISAs. Low oxygen significantly increased the expression of both angiotensin II type 1 receptor (AGTR1) and VEGF (both P < 0.05). There was a positive correlation between AGTR1 and VEGF expression at low oxygen (r = 0.64, P < 0.005). Corresponding increases in VEGF protein were observed with low oxygen (P < 0.05). Despite no change in ACE1 mRNA expression, ACE levels in the supernatant increased with low oxygen (1% and 5%, P < 0.05). Expression of other RAS components did not change. Low oxygen increased AGTR1 and VEGF expression, as well as ACE and VEGF protein levels, suggesting that the proangiogenic RAS pathway is activated. This highlights a potential role for the placental RAS in mediating the proangiogenic effects of low oxygen in placental development.

Pregnancy outcome is influenced, in part, by the sex of the fetus. Decidual renin messenger RNA (REN) abundance is greater in women carrying a female fetus than a male fetus. Here, we explore whether the sex of the fetus also influences the regulation of decidual RAS expression with a known stimulator of renal renin and cyclic adenosine monophosphate (cAMP). Cyclic adenosine monophosphate had no affect on decidual REN expression, since REN abundance was still greater in decidual explants from women carrying a female fetus than a male fetus after cAMP treatment. Cyclic adenosine monophosphate decreased prorenin levels in the supernatant if the fetus was female (ie, prorenin levels were no longer sexually dimorphic) and altered the fetal sex-specific differences in other RAS genes seen in vitro. Therefore, fetal sex influences the decidual renin-angiotensin system response to cAMP. This may be related to the presence of fetal cells in the maternal decidua.

Males are more likely to be born preterm than females. The causes are unknown, but it is suggested that intrauterine tissues regulate fetal growth and survival in a sex-specific manner. We postulated that prorenin binding to its prorenin/renin receptor receptor (ATP6AP2) would act in a fetal sex-specific manner in human amnion to regulate the expression of promyelocytic zinc finger, a negative regulator of ATP6AP2 expression as well as 2 pathways that might influence the onset of labor, namely transforming growth factor ß1 (TGFB1) and prostaglandin endoperoxide synthase 2 (PTGS2). Our findings demonstrate that there are strong interactions between prorenin, ATP6AP2, and TGFB1 and that this system has a greater capacity in female amnion to stimulate profibrotic pathways, thus maintaining the integrity of the fetal membranes. In contrast, glucocorticoids or other factors independent of the prorenin/prorenin receptor pathway may be important regulators of PTGS2 in human pregnancy.

The intrauterine renin angiotensin system (RAS) is implicated in placentation and labour onset. Here we investigate whether promoter methylation of RAS genes changes with gestation or labour and if it affects gene expression. Early gestation amnion and placenta were studied, as were term amnion, decidua, and placenta collected before labour (at elective caesarean section) or after spontaneous labour and delivery. The expression and degree of methylation of the prorenin receptor (ATP6AP2), angiotensin converting enzyme (ACE), angiotensin II type 1 receptor (AGTR1), and two proteases that can activate prorenin (kallikrein, KLK1, and cathepsin D, CTSD) were measured by qPCR and a DNA methylation array. There was no effect of gestation or labour on the methylation of RAS genes and CTSD. Amnion and decidua displayed strong correlations between the percent hypermethylation of RAS genes and CTSD, suggestive of global methylation. There were no correlations between the degree of methylation and mRNA abundance of any genes studied. KLK1 was the most methylated gene and the proportion of hypermethylated KLK1 alleles was lower in placenta than decidua. The presence of intermediate methylated alleles of KLK1 in early gestation placenta and in amnion after labour suggests that KLK1 methylation is uniquely dynamic in these tissues.

Sudden infant death syndrome (SIDS), neonatal deaths, and deaths from infection are higher among Indigenous Australians. This study aimed to determine the effects of inflammatory responses and exposure to cigarette smoke, two important factors associated with sudden death in infancy, on preterm birth, and birth weight in a cohort of Indigenous mothers. Indigenous Australian women (n = 131) were recruited as part of a longitudinal study while attending antenatal care clinics during pregnancy; blood samples were collected up to three times in pregnancy. Serum cotinine, indicating exposure to cigarette smoke, was detected in 50.4% of mothers. Compared with non-Indigenous women, the cohort had 10 times the prevalence of antibodies to Helicobacter pylori (33 vs. 3%). Levels of immunoglobulin G, antibodies to H. pylori, and C-reactive protein (CRP) were all inversely correlated with gestational age (P < 0.05). CRP levels were positively associated with maternal body mass index (BMI; ¿ = 0.449, P = 0.001). The effects of cigarette smoke (cotinine) and inflammation (CRP) were assessed in relation to risk factors for SIDS: gestational age at delivery and birth weight. Serum cotinine levels were negatively associated with birth weight (¿ = -0.37, P < 0.001), this correlation held true for both male (¿ = -0.39, P = 0.002) and female (¿ = -0.30, P = 0.017) infants. Cotinine was negatively associated with gestational age at delivery (¿ = -0.199, P = 0.023). When assessed by fetal sex, this was significant only for males (¿ = -0.327, P = 0.011). CRP was negatively associated with gestational age at delivery for female infants (¿ = -0.46, P < 0.001). In contrast, maternal BMI was significantly correlated with birth weight. These data highlight the importance of putting programs in place to reduce cigarette smoke exposure in pregnancy and to treat women with chronic infections such as H. pylori to improve pregnancy outcomes and decrease risk factors for sudden death in infancy.

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 10days 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.

The newborn circulating, cardiac and renal renin¿angiotensin systems (RASs) are essential for blood pressure control, and for cardiac and renal development. If cardiac and renal RASs are immature this may contribute to cardiovascular compromise in preterm infants. This study measured mRNA expression of cardiac and renal RAS components in preterm, glucocorticoid (GC) exposed preterm, and term piglets. Methods: Renal and cardiac RAS mRNA levels were measured using real-time polymerase chain reaction (PCR). Genes studied were: (pro)renin receptor, renin, angiotensinogen, angiotensin converting enzyme (ACE), ACE2, angiotensin type 1 receptor (AT1R) and angiotensin type 2 receptor (AT2R). Results: All the genes studied were expressed in the kidney; neither renin nor AT2R mRNA were detected in the heart. There were no gestational changes in (pro)renin receptor, renin, ACE or AT1R mRNA levels. Right ventricular angiotensinogen mRNA levels in females were lower in preterm animals than at term, and GC exposure increased levels in male piglets. Renal angiotensinogen mRNA levels in female term piglets were lower than females from both preterm groups, and lower than male term piglets. Left ventricular ACE2 mRNA expression was lower in GC treated preterm piglets. Renal AT2R mRNA abundance was highest in GC treated preterm piglets, and the AT1R/AT2R ratio was increased at term. Preterm cardiac and renal RAS mRNA levels were similar to term piglets, suggesting that immaturity of these RASs does not contribute to preterm cardiovascular compromise. Since preterm expression of both renal and cardiac angiotensin II-AT1R is similar to term animals, cardiovascular dysfunction in the sick preterm human neonate might be effectively treated by agents acting on their RASs. © 2015, SAGE Publications. All rights reserved.

Inadequate maintenance of systemic blood flow in neonates following preterm birth is associated with increased morbidity and mortality, and may be due in part to structural immaturity of the myocardium. Maternal glucocorticoid administration is associated with improved cardiovascular function, and possibly promotes structural maturation of the myocardium. This study assessed the structural maturity of the myocardium in male and female preterm and term piglets, and preterm piglets exposed to a regimen of maternal glucocorticoids as used clinically. In preterm, term and glucocorticoid exposed preterm piglets cardiomyocyte maturity was examined by measuring the proportion of binucleated myocytes and the volumes of single living ventricular cardiomyocytes with fluorescence microscopy. Ventricular apoptosis and proliferation were measured by immunohistochemistry. Preterm piglet hearts had fewer binucleated myocytes, smaller myocytes, and more proliferative and fewer apoptotic nuclei than term hearts. Maternal glucocorticoid treatment resulted in increased binucleation with no increase in myocyte volume, and levels of proliferation and apoptosis that were more similar to the term heart. Atrial weights were increased and in female piglets there was an increase in the ratio of left to right ventricular weight. The observed changes in atrial mass and myocyte structural maturation correlated with changes in cardiac function of isolated hearts of littermates. In conclusion, the association between increased myocardial maturation following glucocorticoid exposure, improved cardiac function in littermates, and clinical improvement in human neonatal cardiac function exposed to antenatal glucocorticoids, suggests that glucocorticoid exposure contributes to improved cardiovascular function in preterm infants by promoting myocardial structural maturity. © 2014 Kim et al.

Preterm delivery increases the risk of inadequate systemic blood flow and hypotension, and many preterm infants fail to respond to conventional inotrope treatments. If the profile of cardiac adrenoceptor subtypes in the preterm neonate is different to that at term this may contribute to these clinical problems. This study measured mRNA expression of ß1, ß2, a1A, a2A and a2B-adrenoceptor subtypes by real time PCR in term (113d), preterm (91d) and preterm piglets (91d) exposed to maternal glucocorticoid treatment. Abundance of ß-adrenoceptor binding sites in the left ventricle was measured using saturation binding assays. Relative abundance of ß1-adrenoceptor mRNA in untreated preterm hearts was ~50% of term abundance in both left and right ventricles (P>0.001). Trends in receptor binding site density measurements supported this observation (P = 0.07). Glucocorticoid exposure increased ß1-adrenoceptor mRNA levels in the right ventricle of preterm hearts (P = 0.008) but did not alter expression in the left ventricle (P>0.1). Relative abundance of a1A-adrenoceptor mRNA was the same in preterm and term piglet hearts (P = >0.1) but was reduced by maternal glucocorticoid treatment (P<0.01); a2A-adrenoceptor mRNA abundance was higher in untreated and glucocorticoid exposed preterm piglet hearts than in term piglets (P<0.001). There was no difference between male and female piglets in mRNA abundance of any of the genes studied. In conclusion, there is reduced mRNA abundance of ß1-adrenoceptors in the preterm pig heart. If this lower expression of ß-adrenoceptors occurs in human preterm infants, it could explain their poor cardiovascular function and their frequent failure to respond to commonly used inotropes. © 2014 Kim et al.

There are fetal sex-specific differences in the balance between angiotensin (Ang) II and Ang-(1-7) in the maternal circulation during pregnancy. To determine whether at 15 weeks' gestation plasma levels of Ang II and Ang-(1-7), as well as levels of prorenin and Ang-converting enzyme (ACE), predicted the development of gestational hypertension (GH) or preeclampsia (PreE) and were associated with estimates of fetal and maternal health, women who later developed GH (n=50) or PreE (n=50) were compared with body mass index-matched controls (n=100). Women who subsequently developed PreE or GH had increased Ang-(1-7) levels at 15 weeks' gestation compared with women with normal pregnancies. When separated by fetal sex, this difference was seen only in women carrying a female fetus. Prorenin and ACE concentrations were not useful biomarkers for the prediction of either PreE or GH at 15 weeks' gestation. Women with a male fetus who developed PreE and women who subsequently developed GH had increased blood pressures at 15 weeks' gestation compared with women with normal pregnancies, suggesting that these women were on an early trajectory for the development of hypertension. We propose that measurement of Ang-(1-7) during early gestation could be useful in predicting, those women who will go on to develop new-onset hypertension in pregnancy.