The current study assessed whether activation of the novel estrogen receptor GPR30 ameliorates salt-dependent renal damage in intact mRen2.Lewis (mRen2) females. Hemizygous mRen2 were maintained on either a normal salt (NS, 0.5% Na) or high salt (HS, 4% Na) diet for 10 wks (5 to 15 wks of age), and HS animals were treated with the GPR30 agonist G-1 or vehicle for two weeks. Systolic blood pressure markedly increased with HS [149 ± 3 to 219 ± 5 mmHg, P<0.01], but G-1 did not influence pressure [P=0.42]. G-1 and estradiol induced relaxation of pre-constricted mesenteric vessels from NS mRen2, but both responses were attenuated in the HS group. Despite the lack of an effect on blood pressure, G-1 decreased renal hypertrophy, proteinuria, urinary 8-isoprostane excretion, and tubular 4-HNE staining. HS significantly increased GPR30 mRNA [1.01 ± 0.04 vs. 1.59 ± 0.13; P<0.01] and protein [0.60 ± 0.31 vs. 3.99 ± 0.75; P<0.01] in the renal cortex. GPR30 was highly expressed in the brush border of proximal tubules and co-localized with megalin. Finally, megalin expression was reduced by HS and restored with G-1. We conclude that GPR30-mediated beneficial effects in salt-sensitive mRen2 females occurred independent of changes in systolic blood pressure. The failure of G-1 to influence pressure may reflect a salt-induced impairment in GPR30-mediated vasorelaxation. The renoprotective actions of GPR30 may involve attenuation of tubular oxidative stress and activation of megalin-mediated protein reabsorption.

Yamaleyeva LM, Gilliam-Davis S, Almeida I, Brosnihan KB, Lindsey SH, Chappell MC. Differential regulation of circulating and renal ACE2 and ACE in hypertensive mRen2.Lewis rats with early-onset diabetes. Am J Physiol Renal Physiol 302: F1374 -F1384, 2012. First published February 29, 2012 doi:10.1152/ajprenal.00656.2011We examined the impact of early diabetes on the circulating and kidney renin-angiotensin system (RAS) in male and female mRen2.Lewis (mRen2) hypertensive rats. Diabetes (DB) was induced by streptozotocin (STZ; 65 mg/kg) at 11 wk of age for 4 wk without insulin replacement. Systolic blood pressures were not increased in DB males or females compared with controls (CON). Circulating angiotensinconverting enzyme 2 (ACE2) increased ninefold (P Ͻ 0.05) in DB females and threefold (P Ͻ 0.05) in DB males, but circulating ACE and ANG II were higher in the DB groups. Serum C-reactive protein was elevated in DB females but not DB males, and the vascular responses to acetylcholine and estradiol were attenuated in the DB females. Proteinuria, albuminuria, and angiotensinogen excretion increased to a similar extent in both DB females and males. Glomerular VEGF expression also increased to a similar extent in both DB groups. Renal inflammation (CD68 ϩ cells) increased only in DB females although males exhibited greater inflammation that was not different with DB. Cortical ACE2 did not change in DB females but was reduced (30%) in DB males. Renal neprilysin activity (Ͼ75%, P Ͻ 0.05) was markedly reduced in the DB females to that in the DB and CON males. ACE activity was significantly lower in both female (75%, P Ͻ 0.05) and male (50%; P Ͻ 0.05) DB groups, while cortical ANG II and Ang-(1-7) levels were unchanged. In conclusion, female mRen2 rats are not protected from vascular damage, renal inflammation, and kidney injury in early STZ-induced diabetes despite a marked increase in circulating ACE2 and significantly reduced ACE within the kidney.angiotensin; kidney; neprilysin; hypertension; proteinuria ANGIOTENSIN-CONVERTING ENZYME 2 (ACE2), a homolog of ACE, is recognized as an important enzymatic component of the renin-angiotensin system (RAS) that may regulate functional output of this hormonal system (5,7,8,14,28,30,51,50). Although ACE2 was originally characterized for its ability to hydrolyze ANG I to Ang-(1-9), subsequent studies revealed a very high catalytic activity to convert ANG II to Ang-(1-7) (41,44,45,56,58). Indeed, ACE2 inhibition or genetic knockout studies reveal a heightened sensitivity to ANG II-induced increases in blood pressure, as well as an exacerbation of end-organ damage in these experimental models (31,32,46,48,52,60). In contrast to ACE, circulating ACE2 is low to nondetectable in rodents and humans, and tissue sources of the enzyme are more likely to influence the local RAS (12, 40). Diabetic renal injury is generally associated with a reduction in the activity and/or expression of renal tissue ACE2, which may contribute to a deleterious imbalance in the relative expression of ANG I...

The present study determined whether early loss of estrogen influences salt-sensitive changes in blood pressure, renal injury, and cardiac hypertrophy as well as the effects on the circulating renin-angiotensin-aldosterone system (RAAS) in the hypertensive female mRen(2). Lewis strain. Ovariectomy (OVX) of heterozygous mRen(2). Lewis rats on a normal salt (NS) diet (0.5% sodium) increased systolic blood pressure from 137+/-3 to 177+/-5 mmHg (P<0.01) by 15 wk but did not show any changes in cardiac-to-body weight index (CI), proteinuria, or creatinine clearance. Maintenance with a high-sodium (HS) diet (4%) increased blood pressure (203+/-4 mmHg, P<0.01), proteinuria (3.5+/-0.3 vs. 6.4+/-0.7 mg/day, P<0.05), and CI (4.0+/-0.1 vs. 5.2+/-0.1 mg/kg, P<0.01) but decreased creatinine clearance (0.89+/-0.15 vs. 0.54+/-0.06 ml/min, P<0.05). OVX exacerbated the effects of salt on the degree of hypertension (230+/-5 mmHg), CI (5.6+/-0.2 mg/kg), and proteinuria (13+/-3.0 mg/day). OVX increased the urinary excretion of aldosterone approximately twofold in animals on the NS diet (3.8+/-0.5 vs. 6.6+/-0.5 ng.mg creatinine-1.day-1, P<0.05) and HS diet (1.4+/-0.2 vs. 4.5+/-1.0 ng.mg creatinine-1.day-1, P<0.05). Circulating renin, angiotensin-converting enzyme, and angiotensin II were also significantly increased in the OVX group fed a HS diet. These results reveal that the protective effects of estrogen apart from the increase in blood pressure were only manifested in the setting of a chronic HS diet and suggest that the underlying sodium status may have an important influence on the overall effect of reduced estrogen.

Angiotensin converting enzyme 2 (ACE2) is a key enzyme of the renin-angiotensin system (RAS) that influences the relative expression of angiotensin (Ang) II and Ang-(1–7). Although ACE2 expression increases in normal pregnancy, the impact of ACE2 deficiency in pregnancy has not been elucidated. We determined the influence of ACE2 deficiency on circulating and tissue RAS components, fetal and maternal growth characteristics, and maternal hemodynamics (mean blood pressure (MBP) and cardiac output (CO)) at day 18 of gestation. Gestational body weight gain was lower in the ACE2 knock out (KO) vs C57BL/6 (WT) mice (30.3 ± 4.7 vs 38.2 ± 1.0 g, p<0.001). Fetal weight (0.94 ± 0.1 vs 1.24 ± 0.01 g, p<0.01) and length (19.6 ± 0.2 vs 22.2 ± 0.2 mm, p<0.001) were less in KO. MBP was significantly reduced in WT with pregnancy; it was elevated (p<0.05) in the KO virgin and pregnant mice, and this was associated with an increased CO in both WT and KO pregnant mice (p<0.05). Plasma Ang-(1–7) was reduced in pregnant KO mice (p<0.05). Placenta Ang II levels were higher in KO mice (52.9 ± 6.0 vs 22.0 ± 3.3 fmol/mg protein, p<0.001). Renal Ang II levels were greater in KO virgin mice (30.0 ± 1.7 vs 23.7 ± 1.1 fmol/mg protein, p<0.001). There was no change in the Ang-(1–7) levels in the KO placenta and virgin kidney. These results suggest that ACE2 deficiency and associated elevated placenta Ang II levels impact pregnancy by impairing gestational weight gain and restricting fetal growth.

The role of the endogenous apelin system in pregnancy is not well understood. Apelin's actions in pregnancy are further complicated by the expression of multiple forms of the peptide. Using radioimmunoassay (RIA) alone, we established the expression of apelin content in the chorionic villi of preeclamptic (PRE) and normal pregnant women (NORM) at 36 -38 wk of gestation. Total apelin content was lower in PRE compared with NORM chorionic villi (49.7 Ϯ 3.4 vs. 72.3 Ϯ 9.8 fmol/mg protein; n ϭ 20 -22) and was associated with a trend for lower preproapelin mRNA in the PRE. Further characterization of apelin isoforms by HPLC-RIA was conducted in pooled samples from each group. The expression patterns of apelin peptides in NORM and PRE villi revealed little or no apelin-36 or apelin-17. Pyroglutamate apelin-13 [(Pyr 1 )-apelin-13] was the predominant form of the peptide in NORM and PRE villi. Angiotensin-converting enzyme 2 (ACE2) activity was higher in PRE villi (572.0 Ϯ 23.0 vs. 485.3 Ϯ 24.8 pmol·mg Ϫ1 ·min Ϫ1 ; n ϭ 18 -22). A low dose of ANG II (1 nM; 2 h) decreased apelin release in NORM villous explants that was blocked by the ANG II receptor 1 (AT1) antagonist losartan. Moreover, losartan enhanced apelin release above the 2-h baseline levels in both NORM and PRE villi (P Ͻ 0.05). In summary, these studies are the first to demonstrate the lower apelin content in human placental chorionic villi of PRE subjects using quantitative RIA. (Pyr 1 )-apelin-13 is the predominant form of endogenous apelin in the chorionic villi of NORM and PRE. The potential mechanism of lower apelin expression in the PRE villi may involve a negative regulation of apelin by ANG II. apelin-13; apelin receptor; ACE2; pregnancy; explant PREECLAMPSIA IS A pregnancy-induced hypertensive disorder that occurs in 5-7% of all pregnancies worldwide (54). It is associated with maternal perinatal morbidity and mortality and a high risk of premature birth and fetal growth restriction. Despite continued research, the pathogenesis of preeclampsia is still unknown. The endogenous apelin system is an emerging target for the regulation of cardiovascular homeostasis (22); however, its role in pregnancy is not well understood. The levels of preproapelin mRNA are widespread in human tissues, and high levels were identified in the placenta (38), suggesting a possible placental origin of apelin in pregnancy. Apelin may have a paracrine role in human chorionic villi as it has been identified in cytotrophoblasts, in syncytiotrophoblasts, and in fetal endothelial cells (13,15). A positive correlation between maternal and fetal plasma apelin was observed in full-term normal pregnancies (37). Moreover, a transplacental transfer of apelin with potential impact on fetal growth was suggested (37). Apelin is a ligand for the human G protein-coupled receptor APJ (40). The role of APJ in embryonic development was shown by the existence of cardiovascular developmental defects in APJ-deficient mice (11). In addition, a small litter size and a significant loss of homozygous ani...

Angiotensin-converting enzyme 2 (ACE2) knockout is associated with reduced fetal weight at late gestation; however, whether uteroplacental vascular and/or hemodynamic disturbances underlie this growth-restricted phenotype is unknown. Uterine artery reactivity and flow velocities, umbilical flow velocities, trophoblast invasion, and placental hypoxia were determined in ACE2 knockout (KO) and C57Bl/6 wild-type (WT) mice at day 14 of gestation. Although systolic blood pressure was higher in pregnant ACE2 KO vs. WT mice (102.3 ± 5.1 vs. 85.1 ± 1.9 mmHg, n = 5-6), the magnitude of difference was similar to that observed in nonpregnant ACE2 KO vs. WT mice. Maternal urinary protein excretion, serum creatinine, and kidney or heart weights were not different in ACE2 KO vs. WT. Fetal weight and pup-to-placental weight ratio were lower in ACE2 KO vs. WT mice. A higher sensitivity to Ang II [pD2 8.64 ± 0.04 vs. 8.5 ± 0.03 (-log EC50)] and greater maximal contraction to phenylephrine (169.0 ± 9.0 vs. 139.0 ± 7.0% KMAX), were associated with lower immunostaining for Ang II receptor 2 and fibrinoid content of the uterine artery in ACE2 KO mice. Uterine artery flow velocities and trophoblast invasion were similar between study groups. In contrast, umbilical artery peak systolic velocities (60.2 ± 4.5 vs. 75.1 ± 4.5 mm/s) and the resistance index measured using VEVO 2100 ultrasound were lower in the ACE2 KO vs. WT mice. Immunostaining for pimonidazole, a marker of hypoxia, and hypoxia-inducible factor-2α were higher in the trophospongium and placental labyrinth of the ACE2 KO vs. WT. In summary, placental hypoxia and uterine artery dysfunction develop before major growth of the fetus occurs and may explain the fetal growth restricted phenotype.

These studies in hypertensive mRen2. Lewis rats underscored the influence of ovarian hormones on BP and tissue injury, as well as the plasticity of this response, apparently due to age and salt status.

Accurate analysis of placental and fetal oxygenation is critical during pregnancy. Photoacoustic imaging (PAI) combines laser technology with ultrasound in real time. We tested the sensitivity and accuracy of PAI for analysis of placental and fetal oxygen saturation (sO) in mice. The placental labyrinth (L) had a higher sO than the junctional zone plus decidua region (JZ+D) in C57Bl/6 mice. Changing maternal O from 100 to 20% in C57Bl/6 mice lowered sO in these regions. C57Bl/6 mice were treated with the NO synthase inhibitor L-N-nitroarginine methyl ester (L-NAME) from gestational day (GD) 11 to GD18 to induce hypertension. L-NAME decreased sO in L and JZ+D at GD14 and GD18 in association with fetal growth restriction and higher blood pressure. Hypoxia-inducible factor 1α immunostaining was higher in L-NAME control mice at GD14. Fetal sO levels were similar between l-NAME and control mice at GD14 and GD18. In contrast to untreated C57Bl/6, L-NAME decreased placental sO at GD14 and GD18 GD10 or GD12. Placental sO was lower in fetal growth restriction in an angiotensin-converting enzyme 2 knockout mouse model characterized by placental hypoxia. On phantom studies, patterns of sO measured directly correlated with those measured by PAI. In summary, PAI enables the detection of placental and fetal oxygenation during normal and pathologic pregnancies in mice.-Yamaleyeva, L. M., Sun, Y., Bledsoe, T., Hoke, A., Gurley, S. B., Brosnihan, K. B. Photoacoustic imaging for quantification of placental oxygenation in mice.