Shane D. Sykes

Fetal sex affects expression of renin-angiotensin system components in term human decidua

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.

Fetal sex and the circulating renin–angiotensin system during early gestation in women who later develop preeclampsia or gestational hypertension

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.

The balance between human maternal plasma angiotensin II and angiotensin 1-7 levels in early gestation pregnancy is influenced by fetal sex

Hypothesis: There are fetal sex-associated differences in the circulating maternal renin–angiotensin system (RAS) in early pregnancy. Methods: Plasma prorenin, angiotensin (Ang) II, Ang 1-7 and angiotensin-converting enzyme (ACE) concentrations were measured at 15 weeks’ gestation in 131 women with uncomplicated pregnancies from the Adelaide SCOPE cohort. Uterine and umbilical artery Doppler sonography was performed at 20 weeks’ gestation. Results: At 15 weeks, women bearing female fetuses had higher maternal Ang II concentrations (p = 0.017) and lower Ang 1-7 to Ang II ratios (p = 0.016) than women bearing males. In women with male fetuses, Ang II positively correlated with birth weight (p = 0.028) and prorenin negatively correlated with placental weight (p = 0.014). Female fetuses had higher umbilical artery resistance indices (p = 0.019) that were related to maternal prorenin concentrations (p = 0.007). Conclusions: In early human pregnancy, the maternal RAS is influenced by fetal sex. The lower Ang 1-7 to Ang II ratios in women with female fetuses may contribute to the lower maternal peripheral microvascular flow as described previously and the lack of any positive effect of Ang II on fetal growth, as seen in women with male fetuses.

Inflammatory and steroid receptor gene methylation in the human amnion and decidua.

Correct timing of parturition requires inflammatory gene activation in the gestational tissues at term and repression during pregnancy. Promoter methylation at CpG dinucleotides represses gene activity; therefore, we examined the possibility that DNA methylation is involved in the regulation of labour-associated genes in human pregnancy. Amnion and decidua were collected at 11-17 weeks of gestation and at term following elective Caesarean delivery or spontaneous labour. Methylation of the inflammatory genes PTGS2, BMP2, NAMPT and CXCL2 was analysed using the Methyl-Profiler PCR System and bisulphite sequencing. Methylation of the glucocorticoid, progesterone and oestrogen receptor genes, involved in the hormonal regulation of gestational tissue function, and the expression of the DNA methyltransferases DNMT1, -3A and -3B were also determined. Variable proportions of inflammatory and steroid receptor gene copies, to a maximum of 50.9%, were densely methylated in both tissues consistent with repression. Densely methylated copy proportions were significantly different between genes showing no relationship with varying expression during pregnancy, between tissues and in individuals. Methylated copy proportions of all genes in amnion and most genes in decidua were highly correlated in individuals. DNMT1 and -3A were expressed in both tissues with significantly higher levels in the amnion at 11-17 weeks than at term. We conclude that the unmethylated portion of gene copies is responsible for the full range of regulated expression in the amnion and decidua during normal pregnancy. Dense methylation of individually variable gene copy proportions happens in the first trimester amnion influenced by sequence context and affected strongly by individual circumstances.

Regulation of the Renin-Angiotensin System Pathways in the Human Decidua.

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.

Circulating and intrarenal renin–angiotensin systems in healthy men and nonpregnant women

The urinary excretion of renin–angiotensin system (RAS) proteins could reflect the activity of the intrarenal RAS. We hypothesized that the rates of excretion of RAS components into human urine are independent of circulating levels of these proteins and reflect the intrarenal RAS. There are no reports of the simultaneous measurement of prorenin, active renin, angiotensinogen (AGT), and angiotensin‐converting enzyme (ACE) excretion in healthy individuals. Therefore, we measured plasma prorenin, ACE, and AGT and urinary renin (uRenin), prorenin (uProrenin), ACE (uACE), and AGT (uAGT) in men and nonpregnant women. Plasma (p) AGT was higher in women then men. Women who were taking estrogen had significantly higher pAGT. In women, pProrenin was negatively correlated with pAGT. There were no correlations between pProrenin, pAGT, and pACE and their urinary counterparts in either men or women. In men, uProrenin/creatinine ratios were lower than in women. There was no effect of estrogen use on urinary excretion of pProrenin, renin, AGT, and ACE. In men, there were significant correlations between uACE/creat and uRen/creat and uAGT/creat; uProrenin/creat and plasma cystatin C levels; and uRenin/creat and uNa/K were also positively correlated. No associations were found in women. In conclusion, urinary excretion of prorenin is sexually dimorphic and is not affected by estrogen use in women. Our data also suggest that the relationship between renal handling of sodium and urinary renin is sexually dimorphic. Since we found no associations between plasma RAS proteins and their urinary counterparts, and the ratio of uProrenin:pProrenin was strikingly different between men and women, levels of urinary RAS proteins in individuals with normal kidney function are most likely the result of tubular secretion, rather than ultrafiltration.

Methylation of Promoter Regions of Genes of the Human Intrauterine Renin Angiotensin System and Their Expression

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.

Urinary angiotensinogen excretion in Australian Indigenous and non-Indigenous pregnant women

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.

PP025. Measurement of the activity of the circulating and intrarenal renin-angiotensin system (iRAS) in pregnant and non-pregnant subjects.

OBJECTIVES To see if urinary angiotensinogen (uAGT)/creatinine and other urinary components of the RAS could be used to detect renal disease in pregnancy, as renal disease predisposes to preeclampsia. METHODS Plasma and urinary prorenin, ACE and AGT (iRAS) were measured by ELISA. Urinary active renin levels were measured enzymatically (9 males, 10 non pregnant, 61 Australian Indigenous pregnant women). RESULTS No relationships between plasma RAS and iRAS were found. In non-pregnant females plasma AGT levels were inversely related to protein and albumin/creatinine (r=-0.72, P=0.019, n=10; r=-0.65, P=0.042, n=10). In pregnancy, plasma ACE levels were related to protein/creatinine (r=0.29, P=0.036, n=54). Urinary protein/creatinine was not related to iRAS activity (males and non-pregnant females) but in pregnancy was related to prorenin and active renin/creatinine (r=0.45, P=0.02, n=26 r=0.47, P<0.001, n=50). Urinary albumin/creatinine was related to uAGT and active renin/creatinine in pregnancy (r=0.39, P=0.005, n=51; r=0.37, P=0.008, n=51). uACE/creatinine and uAGT/creatinine were related (r=0.52, P<0.001, n=51). CONCLUSION Excretion of components of the iRAS is independent of plasma levels. Not only is uAGT/creatinine related to albumin/creatinine but there are similar relationships with other iRAS components. Measurement of the iRAS in human pregnancy may detect early stage renal disease, endemic in Indigenous Australians.

PP036. The intrarenal renin-angiotensin system and pregnancy outcome.

OBJECTIVES The intrarenal renin angiotensin system (iRAS) may be activated in normal pregnancy. Failure of activation may predispose to preeclampsia. METHODS Urinary angiotensinogen/creatinine (uAGT/creat), albumin and protein/creat were measured in 10 non-pregnant and 17 pregnant non-Indigenous women and 61 Indigenous pregnant women (in whom other components of iRAS were also measured). RESULTS uAGT/creat was higher in pregnancy (18.2±3.2μg/mmol, n=9 vs. 1.1±0.3μg/mmol, P=0.001, n=10). Women with clinical proteinuria and/or preeclampsia had low uAGT/creat (n=3). Hypertensive women had normal high uAGT/creat (n=4). Indigenous pregnant women had higher protein/creat (P=0.01) and lower uAGT/creat (2.9±1.0μg/mmol, P=0.010, n=51) than non-Indigenous pregnant women. Indigenous women were classified based on a uAGT/creat of <2μg/mmol (n=37) or >2.0μg/mmol (n=12). Only low uAGT/creat Indigenous pregnant women had correlations between uAGT/creat and albumin/creat (r=0.367, P=0.027), renin/creat and albumin or protein/creat (r=0.493, P=0.002, r=0.603, P<0.001). uAGT/creat levels fell with gestation (r=-0.329, P=0.047) while Cystatin C increased (r=0.592, P=0.000). CONCLUSION The iRAS is activated in normal pregnancy. This is not the case in women with proteinuria/preeclampsia or in many Indigenous women who have higher urinary protein/creat. Therefore a low uAGT/creat in pregnancy may indicate impaired renal function and be associated with an increased risk of preeclampsia.