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.

Placental deficiency of the (pro)renin receptor ((P)RR) reduces placental development and functional capacity.

The (pro)renin receptor ((P)RR; also known as ATP6AP2) is a multifunctional receptor. The (P)RR activates the tissue renin-angiotensin system (RAS) and is also involved in regulating integral intracellular pathways such as V-ATPase and Wnt/ß-catenin signalling. Given this, the (P)RR may be associated with essential pathways in placentation, however its role within the context of pregnancy remains poorly characterised. The first trimester/extravillous trophoblast cell line, HTR-8/SVneo, underwent an siRNA knockdown where they were incubated for 24 h with a negative control siRNA or siRNA targeting ATP6AP2 mRNA. xCELLigence real-time cell analysis was performed to assess the effect of ATP6AP2 mRNA knockdown on HTR-8/SVneo cell proliferation, migration, and invasion. In subsequent experiments, GFP-encoding lentiviral packaged gene-constructs were used to knockdown (P)RR expression in the trophectoderm of C57/BL6/CBA-F1 mouse blastocysts. Blastocysts were incubated for 6 h with vehicle (no-virus), control virus (non-targeting shRNA and GFP), or (P)RR-knockdown virus ((P)RR shRNA and GFP) before transfer into recipient pseudo-pregnant Swiss CD1 female mice. Fetal and placental tissues were collected and assessed at embryonic age (EA) 10 and 18. (P)RR levels were measured in the labyrinth zone of day 18 placentae and stereological Merz grid analysis was performed to determine the volumetric distribution of trophoblasts, fetal capillaries, and the maternal blood space. We showed that a reduction of ATP6AP2 expression in HTR-8/SVneo cells in vitro, impaired trophoblast proliferation, migration, and invasion. In vivo, decreasing placental labyrinth (P)RR expression adversely effected placental physiology, decreasing placental trophoblast number and total surface area available for exchange, while also increasing maternal blood space. Additionally, decreased (P)RR affected placental efficacy evident by the reduced fetal-placental weight ratio. Our study shows that the (P)RR is necessary for appropriate placental development and function.

Evidence-based circumcision policy for Australia.

The aim was (1) to perform an up-to-date systematic review of the male circumcision (MC) literature and (2) to determine the number of adverse medical conditions prevented by early MC in Australia. Searches of PubMed using "circumcision" with 39 keywords and bibliography searches yielded 278 publications meeting our inclusion criteria. Early MC provides immediate and lifetime benefits, including protection against: urinary tract infections, phimosis, inflammatory skin conditions, inferior penile hygiene, candidiasis, various STIs, and penile and prostate cancer. In female partners MC reduces risk of STIs and cervical cancer. A risk-benefit analysis found benefits exceeded procedural risks, which are predominantly minor, by approximately 200 to 1. It was estimated that more than 1 in 2 uncircumcised males will experience an adverse foreskin-related medical condition over their lifetime. An increase in early MC in Australia to mid-1950s prevalence of 85% from the current level of 18.75% would avoid 77,000 cases of infections and other adverse medical conditions over the lifetime for each annual birth cohort. Survey data, physiological measurements, and the anatomical location of penile sensory receptors responsible for sexual sensation indicate that MC has no detrimental effect on sexual function, sensitivity or pleasure. US studies found that early infant MC is cost saving. Evidence-based reviews by the AAP and CDC support early MC as a desirable public health measure. Although MC can be performed at any age, early MC maximizes benefits and minimises procedural risks. Parents should routinely be provided with accurate, up-to-date evidence-based information in an unbiased manner early in a pregnancy so that they have time to weigh benefits and risks of early MC and make an informed decision should they have a son. Parental choice should be respected. A well-trained competent practitioner is essential and local anaesthesia should be routinely used. Third party coverage of costs is advocated.

Role of the prorenin receptor in endometrial cancer cell growth.

Endometrial cancer is the most diagnosed gynecological malignancy. Despite numerous scientific advances, the incidence and mortality rate of endometrial cancer continues to rise. Emerging evidence suggests a putative role of the (pro)renin receptor ((P)RR), in the ontogenesis of endometrial cancer. The (P)RR is implicated in breast cancer and pancreatic carcinoma pathophysiology by virtue of its role in proliferation, angiogenesis, fibrosis, migration and invasion. Thus, we aimed to investigate the functional role of the (P)RR in human endometrial cancer. We employed an siRNA-mediated knockdown approach to abrogate (P)RR expression in the endometrial epithelial cell lines; Ishikawa, AN3CA and HEC-1-A and examined cellular proliferation and viability. We also carried out a sophisticated proteomic screen to explore potential pathways via which the (P)RR is acting in endometrial cancer physiology. These data confirmed that the (P)RR is critical for endometrial cancer development, contributing to both its proliferative capacity and in the maintenance of cell viability. This is likely mediated through proteins such as MGA, SLC4A7, SLC7A11 or DHRS2, which were reduced following (P)RR knockdown. These putative protein interactions/pathways, which rely on the presence of the (P)RR, are likely to contribute to endometrial cancer progression and could therefore, represent several novel therapeutic targets for endometrial cancer.

Renin-angiotensin system (RAS) enzymes and placental trophoblast syncytialisation.

Placental renin-angiotensin system (RAS) components; prorenin, angiotensinogen, and angiotensin (Ang) II type 1 receptor (AT1R) are upregulated during syncytialisation. This study examined whether angiotensin-converting enzyme (ACE), ACE2 and neprilysin (NEP) are also altered during syncytialisation. Two in vitro models of syncytialisation were used: forskolin-treated BeWo cells and spontaneously syncytialising primary human trophoblast cells. Term placentae and primary trophoblasts had the highest levels of ACE, ACE2 and NEP mRNA. In primary trophoblasts, ACE mRNA levels significantly increased with syncytialisation, ACE2 and NEP mRNA levels decreased. ACE, ACE2 and NEP protein levels and ACE2 activity did not change. Syncytialisation of primary trophoblasts decreased soluble (s)ACE and sNEP but not sACE2 levels. In primary trophoblasts, the balance between the enzymes controlling the two opposing pathways of the RAS was maintained. These findings were unable to be reproduced in BeWo cells. Future studies exploring placental levels of these enzymes in pregnancies complicated by placental insufficiency are warranted.

Systems analysis shows that thermodynamic physiological and pharmacological fundamentals drive COVID-19 and response to treatment.

Why a systems analysis view of this pandemic? The current pandemic has inflicted almost unimaginable grief, sorrow, loss, and terror at a global scale. One of the great ironies with the COVID-19 pandemic, particularly early on, is counter intuitive. The speed at which specialized basic and clinical sciences described the details of the damage to humans in COVID-19 disease has been impressive. Equally, the development of vaccines in an amazingly short time interval has been extraordinary. However, what has been less well understood has been the fundamental elements that underpin the progression of COVID-19 in an individual and in populations. We have used systems analysis approaches with human physiology and pharmacology to explore the fundamental underpinnings of COVID-19 disease. Pharmacology powerfully captures the thermodynamic characteristics of molecular binding with an exogenous entity such as a virus and its consequences on the living processes well described by human physiology. Thus, we have documented the passage of SARS-CoV-2 from infection of a single cell to species jump, to tropism, variant emergence and widespread population infection. During the course of this review, the recurrent observation was the efficiency and simplicity of one critical function of this virus. The lethality of SARS-CoV-2 is due primarily to its ability to possess and use a variable surface for binding to a specific human target with high affinity. This binding liberates Gibbs free energy (GFE) such that it satisfies the criteria for thermodynamic spontaneity. Its binding is the prelude to human host cellular entry and replication by the appropriation of host cell constituent molecules that have been produced with a prior energy investment by the host cell. It is also a binding that permits viral tropism to lead to high levels of distribution across populations with newly formed virions. This thermodynamic spontaneity is repeated endlessly as infection of a single host cell spreads to bystander cells, to tissues, to humans in close proximity and then to global populations. The principal antagonism of this process comes from SARS-CoV-2 itself, with its relentless changing of its viral surface configuration, associated with the inevitable emergence of variants better configured to resist immune sequestration and importantly with a greater affinity for the host target and higher infectivity. The great value of this physiological and pharmacological perspective is that it reveals the fundamental thermodynamic underpinnings of SARS-CoV-2 infection.

The interacting physiology of COVID-19 and the renin-angiotensin-aldosterone system: Key agents for treatment.

SARS-CoV-2 interacting with its receptor, angiotensin-converting enzyme 2 (ACE2), turns the host response to viral infection into a dysregulated uncontrolled inflammatory response. This is because ACE2 limits the production of the peptide angiotensin II (Ang II) and SARS-CoV-2, through the destruction of ACE2, allows the uncontrolled production of Ang II. Recovery from trauma requires activation of both a tissue response to injury and activation of a whole-body response to maintain tissue perfusion. Tissue and circulating renin-angiotensin systems (RASs) play an essential role in the host response to infection and injury because of the actions of Ang II, mediated via its AT1 receptor. Both tissue and circulating arms of the renin angiotensin aldosterone system's (RAAS) response to injury need to be regulated. The effects of Ang II and the steroid hormone, aldosterone, on fluid and electrolyte homeostasis and on the circulation are controlled by elaborate feedback networks that respond to alterations in the composition and volume of fluids within the circulatory system. The role of Ang II in the tissue response to injury is however, controlled mainly by its metabolism and conversion to Ang-(1-7) by the enzyme ACE2. Ang-(1-7) has effects that are contrary to Ang II-AT1 R mediated effects. Thus, destruction of ACE2 by SARS-CoV-2 results in loss of control of the pro-inflammatory actions of Ang II and tissue destruction. Therefore, it is the response of the host to SARS-CoV-2 that is responsible for the pathogenesis of COVID-19.

"LONG COVID"-A hypothesis for understanding the biological basis and pharmacological treatment strategy.

Infection of humans with SARS-CoV-2 virus causes a disease known colloquially as "COVID-19" with symptoms ranging from asymptomatic to severe pneumonia. Initial pathology is due to the virus binding to the ACE-2 protein on endothelial cells lining blood vessels and entering these cells in order to replicate. Viral replication causes oxidative stress due to elevated levels of reactive oxygen species. Many (~60%) of the infected people appear to have eliminated the virus from their body after 28 days and resume normal activity. However, a significant proportion (~40%) experience a variety of symptoms (loss of smell and/or taste, fatigue, cough, aching pain, "brain fog," insomnia, shortness of breath, and tachycardia) after 12 weeks and are diagnosed with a syndrome named "LONG COVID." Longitudinal clinical studies in a group of subjects who were infected with SARS-CoV-2 have been compared to a non-infected matched group of subjects. A cohort of infected subjects can be identified by a battery of cytokine markers to have persistent, low level grade of inflammation and often self-report two or more troubling symptoms. There is no drug that will relieve their symptoms effectively. It is hypothesized that drugs that activate the intracellular transcription factor, nuclear factor erythroid-derived 2-like 2 (NRF2) may increase the expression of enzymes to synthesize the intracellular antioxidant, glutathione that will quench free radicals causing oxidative stress. The hormone melatonin has been identified as an activator of NRF2 and a relatively safe chemical for most people to ingest chronically. Thus, it is an option for consideration of re-purposing studies in "LONG COVID" subjects experiencing insomnia, depression, fatigue, and "brain fog" but not tachycardia. Appropriately designed clinical trials are required to evaluate melatonin.

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.

Corrigendum: Angiotensin Converting Enzyme 2 (ACE2) in Pregnancy: Preeclampsia and Small for Gestational Age.

[This corrects the article DOI: 10.3389/fphys.2020.590787.].

FURIN and placental syncytialisation: a cautionary tale.

FURIN is a pro-protein convertase previously shown to be important for placental syncytialisation (Zhou et al. [1]), a process of cell fusion whereby placental cytotrophoblast cells fuse to form a multinucleated syncytium. This finding has been broadly accepted however, we have evidence suggesting the contrary. Spontaneously syncytialising term primary human trophoblast cells and BeWo choriocarcinoma cells were treated with either FURIN siRNA or negative control siRNA or the protease inhibitor, DEC-RVKR-CMK, or vehicle. Cells were then left to either spontaneously syncytialise (primary trophoblasts) or were induced to syncytialise with forskolin (BeWo). Effects on syncytialisation were measured by determining human chorionic gonadotrophin secretion and E-cadherin protein levels. We showed that FURIN is not important for syncytialisation in either cell type. However, in primary trophoblasts another protease also inhibited by DEC-RVKR-CMK, may be involved. Our results directly contrast with those published by Zhou et al. Zhou et al. however, used first trimester villous explants to study syncytialisation, and we used term primary trophoblasts. Therefore, we suggest that FURIN may be involved in syncytialisation of first trimester trophoblasts, but not term trophoblasts. What is more concerning is that our results using BeWo cells do not agree with their results, even though for the most part, we used the same experimental design. It is unclear why these experiments yielded different results, however we wanted to draw attention to simple differences in measuring syncytialisation or flaws in method reporting (including omission of cell line source and passage numbers, siRNA concentration and protein molecular weights) and choice of immunoblot loading controls, that could impact on experimental outcomes. Our study shows that careful reporting of methods by authors and thorough scrutiny by referees are vital. Furthermore, a universal benchmark for measuring syncytialisation is required so that various studies of syncytialisation can be validated.

The (pro)renin receptor ((P)RR) and soluble (pro)renin receptor (s(P)RR) in pregnancy.

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.

Maternal Diet Influences Fetal Growth but Not Fetal Kidney Volume in an Australian Indigenous Pregnancy Cohort.

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.

Angiotensin Converting Enzyme 2 (ACE2) in Pregnancy: Preeclampsia and Small for Gestational Age.

INTRODUCTION: An imbalance in angiotensin (Ang) peptides could contribute to the pathophysiology of preeclampsia (PE) and poor fetal growth. METHODS: We measured maternal plasma levels of Ang peptides and converting enzymes in non-pregnant women (n = 10), in normal pregnant women (n = 59), women delivering small for gestational age babies (SGA, n = 25) across gestation (13-36 weeks) and in women with PE (n = 14) in their third trimester. RESULTS: Plasma ACE, ACE2, and Ang-(1-7) levels, and ACE2 activity were significantly higher in normal pregnant women compared with non-pregnant women; neprilysin (NEP) levels were not changed. In SGA pregnancies, ACE and ACE2 levels were higher in early-mid pregnancy compared with normal pregnant women. In women with PE, plasma ACE, ACE2, NEP, and Ang-(1-7) levels and ACE2 activity were lower than levels in normal pregnant women. CONCLUSION: The higher plasma ACE2 levels and activity in pregnancy could be driving the higher Ang-(1-7) levels. The early gestation increases in ACE and ACE2 levels in SGA pregnancies highlights the possibility that these enzymes could be used as potential early biomarkers of poor fetal growth. In women with PE, the reduced ACE2 and NEP levels at term, could be contributing to the reduction in Ang-(1-7) levels. These findings suggest that dysfunctional relationships between two key enzymes in the circulating RAS are involved in the pathogenesis of PE and SGA. Since soluble ACE2 can prevent binding of the novel coronavirus, SARS-CoV-2, to membrane bound ACE2, the interplay between ACE2 and the coronavirus and its impact in pregnancy requires further investigation.

Cleavage of the soluble (pro)renin receptor (sATP6AP2) in the placenta.

INTRODUCTION: The (pro)renin receptor (ATP6AP2) is cleaved and released as soluble ATP6AP2 (sATP6AP2). The sATP6AP2 is detected in plasma and urine and is elevated in women with gestational diabetes and preeclampsia. The source and cleavage pathway of sATP6AP2 in pregnancy is unknown. The syncytiotrophoblast is the major placental secretory layer and is in direct contact with maternal blood. Both FURIN and Site 1 protease (MBTPS1) cleave sATP6AP2 in non-placental cells. We postulated that ATP6AP2 was cleaved by FURIN and/or MBTPS1 and that sATP6AP2 is secreted by the placental syncytiotrophoblast. METHODS: Term primary trophoblast cells were transfected with FURIN siRNA, negative control siRNA or vehicle. In a separate experiment, primary trophoblasts were treated with a pro-protein convertase inhibitor (DEC-RVKR-CMK), an MBTPS1 inhibitor (PF 429242) or vehicle. Trophoblasts were left to spontaneously syncytialise before cells and supernatants were collected and intracellular and extracellular sATP6AP2 levels analysed by immunoblot. RESULTS: sATP6AP2 is secreted by placental trophoblasts. Levels of intra and extra-cellular sATP6AP2 decrease with syncytialisation (P = 0.01 and P = 0.02, respectively), as do FURIN mRNA (P = 0.0003) and protein (P = 0.0007). FURIN siRNA decreased FURIN mRNA and protein levels (both P < 0.0001). Neither FURIN siRNA or PF 429242 affected sATP6AP2 levels. DEC-RVKR-CMK significantly decreased extracellular sATP6AP2 protein levels (P = 0.02). DISCUSSION: Soluble ATP6AP2 is secreted by placental trophoblasts and levels decrease with syncytialisation. DEC-RVKR-CMK, a broad inhibitor of pro-protein convertases reduced extracellular sATP6AP2 levels, but FURIN siRNA and MBTPS1 inhibition had no effect. Hence, a convertase other than FURIN or MBTPS1 is most likely responsible for placental sATP6AP2 secretion.

The (pro)renin receptor (ATP6AP2) does not play a role in syncytialisation of term human primary trophoblast cells.

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.

Programming of Renal Development and Chronic Disease in Adult Life.

Chronic kidney disease (CKD) can have an insidious onset because there is a gradual decline in nephron number throughout life. There may be no overt symptoms of renal dysfunction until about two thirds or more of the nephrons have been destroyed and glomerular filtration rate (GFR) falls to below 25% of normal (often in mid-late life) (Martinez-Maldonaldo et al., 1992). Once End Stage Renal Disease (ESRD) has been reached, survival depends on renal replacement therapy (RRT). CKD causes hypertension and cardiovascular disease; and hypertension causes CKD. Albuminuria is also a risk factor for cardiovascular disease. The age of onset of CKD is partly determined during fetal life. This review describes the mechanisms underlying the development of CKD in adult life that results from abnormal renal development caused by an adverse intrauterine environment. The basis of this form of CKD is thought to be mainly due to a reduction in the number of nephrons formed in utero which impacts on the age dependent decline in glomerular function. Factors that affect the risk of reduced nephron formation during intrauterine life are discussed and include maternal nutrition (malnutrition and obesity, micronutrients), smoking and alcohol, use of drugs that block the maternal renin-angiotensin system, glucocorticoid excess and maternal renal dysfunction and prematurity. Since CKD, hypertension and cardiovascular disease add to the disease burden in the community we recommend that kidney size at birth should be recorded using ultrasound and those individuals who are born premature or who have small kidneys at this time should be monitored regularly by determining GFR and albumin:creatinine clearance ratio. Furthermore, public health measures aimed at limiting the prevalence of obesity and diabetes mellitus as well as providing advice on limiting the amount of protein ingested during a single meal, because they are all associated with increased glomerular hyperfiltration and subsequent glomerulosclerosis would be beneficial.