Indigenous Australians experience higher rates of renal disease and hypertension than non-Indigenous Australians. Low birth weight is recognized as a contributing factor in chronic disease and has ...been shown to increase the risk of renal failure in adulthood. A smaller kidney volume with fewer nephrons places an individual at risk of hypertension and renal failure. Indigenous Australians have fewer nephrons than non-Indigenous Australians. In this study, intrauterine fetal and kidney growth were evaluated in 174 Indigenous Australian babies throughout gestation in order to record and evaluate fetal growth and kidney size, within a population that is at high risk for chronic illness.
Pregnant women that identified as Indigenous, or non-Indigenous women that were pregnant with a partner who identified as an Indigenous Australian were eligible to participate. Maternal history, smoking status, blood and urine samples and fetal ultrasounds were collected throughout pregnancy. Fetal kidney measurements were collected using ultrasound. Statistical analysis was performed using the Stata 14.1 software package.
15.2% of babies were born prematurely. 44% of the mothers reported smoking in pregnancy. The median birth weight of this cohort was 3,240 g. Male fetuses had higher kidney to body weight ratios than female fetuses (
= 0.02). The birth weights of term neonates whose mothers smoked during pregnancy were lower (327 g,
< 0.001) than the birth weights of term babies from non-smoking mothers. The kidney volumes of babies whose mothers smoked were also smaller (
= 0.02), but were in proportion to body weight.
In this cohort of Indigenous women smoking was associated with both increased number of preterm births and with a reduction in birth weights, even of term infants. Since kidney volume is a surrogate measure of nephron number and nephrogenesis is complete at birth, babies whose mothers smoked during pregnancy must have fewer nephrons than those from non-smoking mothers. Previous studies have shown that glomerular filtration rate is not related to birth weight, thus infants with smaller kidney volumes are hyperfiltering from birth and therefore are likely to be more susceptible to early onset renal disease in later life.
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.
Abstract
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.
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/β-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.
A dysfunctional endometrial renin–angiotensin system (RAS) could aid the growth and spread of endometrial cancer. To determine if the RAS is altered in endometrial cancer, we measured RAS gene ...expression and protein levels in 30 human formalin-fixed, paraffin-embedded (FFPE) endometrioid carcinomas and their adjacent endometrium. All components of the RAS were expressed in most tumours and in adjacent endometrium; mRNA levels of (pro)renin receptor (ATP6AP2), angiotensin II type 1 receptor (AGTR1), angiotensin-converting enzyme (ACE1) and angiotensin-converting enzyme 2 (ACE2) mRNA levels were greater in tumour tissue than adjacent non-cancerous endometrium (P = 0.023, 0.008, 0.004 and 0.046, respectively). Prorenin, ATP6AP2, AGTR1, AGTR2 and ACE2 proteins were abundantly expressed in both cancerous and adjacent non-cancerous endometrium. Staining was most intense in cancerous glandular epithelium. One potential target of the endometrial RAS, transforming growth factor beta-1 (TGFB1), which is essential for epithelial-to-mesenchymal transition, was also upregulated in endometrial cancer tissue (P = 0.001). Interestingly, TGFB1 was strongly correlated with RAS expression and was upregulated in tumour tissue. This study is the first to characterise the mRNA and protein expression of all RAS components in cancerous and adjacent non-cancerous endometrium. The greater expression of ATP6AP2, AGTR1 and ACE1, key elements of the pro-angiogenic/proliferative arm of the RAS, suggests that the RAS plays a role in the growth and spread of endometrial cancer. Therefore, existing drugs that inhibit the RAS and which are used to treat hypertension may have potential as treatments for endometrial cancer.
Abstract 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.
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.
Summary
The renin–angiotensin system (RAS) plays a critical role in placentation and nephrogenesis. Failure to thrive during intrauterine life, possibly related to placental dysfunction and impaired ...expression of the renal RAS, as well as prematurity, results in smaller kidneys at birth and reduced nephron number. The remaining nephrons are therefore hyperfiltering from birth. Hyperfiltration, infections and Type 2 diabetes cause glomerular and tubular fibrosis, leading to further reductions in nephron number.
The intrarenal RAS plays a key role in promoting tubulointerstitial fibrosis. Low birth weight and a high incidence of preterm birth program Indigenous children for early onset renal disease in adult life. Indigenous Australians have 404 000 fewer nephrons than non‐Indigenous Australians. This, coupled with the high incidence of infectious diseases (particularly acute post‐streptococcal glomerulonephritis) and the increasing prevalence of Type 2 diabetes, explains why end‐stage renal disease is of epidemic proportions in Indigenous Australians.
The existence of RAS gene polymorphisms and inflammatory cytokines may further potentiate susceptibility to renal disease in Indigenous Australians.
Tenascin-C (TNC) is an extracellular matrix glycoprotein which is frequently up-regulated in a variety of pathological conditions including chronic inflammation and cancer. TNC has been implicated in ...the modulation of cell migration, proliferation, invasion and angiogenesis. Multiple isoforms of TNC can be generated through the alternative splicing of nine exons located in the fibronectin type III region of the molecule. The profile of isoforms expressed differs between cancers and normal breast, with the fully truncated TNC isoform being predominant in normal and benign tissues and higher molecular weight isoforms induced predominantly in cancer. The addition of extra domains within the fibronectin type III repeat domain greatly affects TNC function with multiple exon combinations available for splicing. Exons 14 and 16 are considered to be tumour-associated and have been shown to affect breast cell line invasion and growth
in vitro
to a greater extent than the full-length TNC isoform. This mini review will provide a summary of the literature to date regarding the expression of TNC isoforms in the breast and also discuss more recent developments in the field regarding exon AD1.
The maternal decidua expresses the genes of the renin-angiotensin system (RAS). Human decidua was collected at term either before labor (i.e. cesarean delivery) or after spontaneous labor. The mRNA ...for prorenin (REN), prorenin receptor (ATP6AP2), angiotensinogen (AGT), angiotensin-converting enzymes 1 and 2 (ACE1 and ACE2), angiotensin II type 1 receptor (AGTR1), and angiotensin 1–7 receptor (MAS1) were measured by quantitative real-time RT-PCR. Decidual explants were cultured in duplicate for 24 and 48 h, and all RAS mRNA, and the secretion of prorenin, angiotensin II, and angiotensin 1–7 was measured using quantitative real-time RT-PCR, ELISA, and radioimmunoassay, respectively. In the decidua collected before labor, REN mRNA levels were higher if the fetus was female. In addition, REN, ATP6AP2, AGT, and MAS1 mRNA abundance was greater in decidual explants collected from women carrying a female fetus, as was prorenin protein. After 24 h, ACE1 mRNA was higher in the decidual explants from women with a male fetus, whereas after 48 h, both ACE1 and ACE2 mRNA was higher in decidual explants from women with a female fetus. Angiotensin II was present in all explants, but angiotensin 1–7 levels often registered below the lower limits of sensitivity for the assay. After labor, decidua, when compared with nonlaboring decidua, demonstrated lower REN expression when the fetus was female. Therefore, the maternal decidual RAS is regulated in a sex-specific manner, suggesting that it may function differently when the fetus is male than when it is female.
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