Angiotensinogen Expression Research Articles

High-salt intake accelerates functional and histological renal damage associated with renal tissue overexpression of (pro)renin receptors and AT1 receptors in spontaneously hypertensive rats.

This study aimed to investigate the effect of combination of high-salt intake and hypertension on renal functional and histological damage, associated with renal (pro)renin receptor [(P)RR] and AT1 receptor in rats. Wistar Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) received regular rat chow (normal-salt diet 0.9%) or high-salt rat chow (high-salt diet 8.9%) for 6weeks from 6 to 12weeks of age. Systolic blood pressure, serum creatinine and blood urea nitrogen (BUN) were measured. Histological analysis of the kidney was performed. Western blot analysis was performed on the expressions of (P)RR, angiotensinogen and AT1 receptor in the kidney. High-salt intake significantly increased systolic blood pressure in WKYs and especially in SHRs. High-salt intake significantly increased serum creatinine and BUN, and accelerated renal tubulointerstitial fibrosis and glomerular sclerosis in SHRs. High-salt intake significantly enhanced the renal tissue expressions of (P)RR, angiotensinogen and AT1 receptor in SHRs. High-salt intake accelerates functional and histological renal damage associated with renal tissue overexpression of (P)RR and AT1 receptors in SHRs.

Beneficial Effect of TMAO in Spontaneously Hypertensive Heart Failure Rats is Associated with Diuretic and Hypotensive Actions.

There is an ongoing debate weather trimethylamine‐oxide (TMAO), a molecule present in seafood and a derivate of microbiota metabolism, is beneficial or harmful for the circulatory system. Numerous biophysical studies show that TMAO protects proteins exposed to osmotic and hydrostatic stress (HS). We hypothesized that TMAO may benefit the circulatory system by protecting cardiac proteins exposed to HS produced by the contracting heart.Male, 6‐week‐old, Sprague‐Dawley (SD, n=40) and Spontaneously‐Hypertensive‐Heart‐Failure (SHHF, n=18) rats were randomly divided into either control (Water) or TMAO treatment. After 50 weeks, half of SD were given Isoprenaline (s.c.) to increase arterial blood pressure (BP) and produce a mild, Takotsubo‐like cardiomyopathy (ISO). In vitro, cardiac lactic dehydrogenase (LDH) with or without TMAO was exposed to HS (changes in hydrostatic pressure similar, and higher than those present in the heart) for up to 24hrs and was evaluated using fluorescence correlation spectroscopy.After 58 weeks of the treatment survival was 100% in SD‐Water, SD‐TMAO, ISO‐TMAO and 90% in ISO‐WATER. In SHHF‐Water survival was 66% vs 100% in SHHF‐TMAO (p=0.065). In general, TMAO‐treated rats showed higher natriuresis and diuresis. In comparison to SHHF‐Water, SHHF‐TMAO showed significantly lower plasma NT‐proBNP, lower diastolic BP, lower liver angiotensinogen expression and a trend towards lower cardiac fibrosis. In vitro, exposure of LDH to HS with or without TMAO did not affect the protein structure. TMAO does not exert negative effects. In contrast, TMAO reduces mortality in HF rats that is associated with natriuretic, diuretic and hypotensive effects. HS produced by the contracting heart is neutral for cardiac LDH protein structure.Support or Funding InformationSupported by National Science Centre, Poland grant no 2018/31/B/NZ5/00038.

Angiotensinogen promoter methylation predicts bevacizumab treatment response of patients with recurrent glioblastoma.

Patients with recurrent glioblastoma achieving response to bevacizumab combined with chemotherapy have clinical improvement and prolonged survival. High gene expression of angiotensinogen (AGT) is associated with a poor bevacizumab response. Because AGT expression is epigenetically regulated, we aimed to investigate whether AGT promoter methylation in tumor tissue predicts response to bevacizumab combination therapy in patients with recurrent glioblastoma. The study included 159 patients with recurrent glioblastoma, treated with bevacizumab combination treatment (training cohort, n = 77; validation cohort, n = 82). All patients could be evaluated for treatment response and biomarkers. DNA methylation of 4 CpG sites in the AGT promoter was measured using pyrosequencing. A model for nonresponse was established using logistic regression analysis. In the training cohort, lower methylation of each of the four CpG sites in the AGT promoter was significantly associated with nonresponse (all P < 0.05). Moreover, the mean methylation level of all four CpG sites was associated with an increased likelihood of not achieving response to bevacizumab combination therapy (twofold decrease: odds ratio = 3.01; 95% confidence interval: 1.41–6.44; P = 0.004). We developed a model for nonresponse in the training cohort, where a threshold of mean AGT promoter methylation levels was set to below 12%. The model could predict bevacizumab nonresponse with 96% specificity. Importantly, this predictor was also significantly associated with nonresponse in the validation cohort (P = 0.037). Taken together, our findings suggest that low AGT promoter methylation in tumor tissue predicts nonresponse to bevacizumab combination treatment in patients with recurrent glioblastoma. We have, thus, established and successfully validated a predictor for nonresponse that can be used to identify patients who will not benefit from bevacizumab combination therapy.

Exploration of cardiometabolic and developmental significance of angiotensinogen expression by cells expressing the leptin receptor or agouti-related peptide.

Angiotensin II (ANG II) Agtr1a receptor (AT1A) is expressed in cells of the arcuate nucleus of the hypothalamus that express the leptin receptor (Lepr) and agouti-related peptide (Agrp). Agtr1a expression in these cells is required to stimulate resting energy expenditure in response to leptin and high-fat diets (HFDs), but the mechanism activating AT1A signaling by leptin remains unclear. To probe the role of local paracrine/autocrine ANG II generation and signaling in this mechanism, we bred mice harboring a conditional allele for angiotensinogen (Agt, encoding AGT) with mice expressing Cre-recombinase via the Lepr or Agrp promoters to cause cell-specific deletions of Agt (AgtLepr-KO and AgtAgrp-KO mice, respectively). AgtLepr-KO mice were phenotypically normal, arguing against a paracrine/autocrine AGT signaling mechanism for metabolic control. In contrast, AgtAgrp-KO mice exhibited reduced preweaning survival, and surviving adults exhibited altered renal structure and steroid flux, paralleling previous reports of animals with whole body Agt deficiency or Agt disruption in albumin (Alb)-expressing cells (thought to cause liver-specific disruption). Surprisingly, adult AgtAgrp-KO mice exhibited normal circulating AGT protein and hepatic Agt mRNA expression but reduced Agt mRNA expression in adrenal glands. Reanalysis of RNA-sequencing data sets describing transcriptomes of normal adrenal glands suggests that Agrp and Alb are both expressed in this tissue, and fluorescent reporter gene expression confirms Cre activity in adrenal gland of both Agrp-Cre and Alb-Cre mice. These findings lead to the iconoclastic conclusion that extrahepatic (i.e., adrenal) expression of Agt is critically required for normal renal development and survival.

Suppression of adipocyte differentiation by low-intensity pulsed ultrasound via inhibition of insulin signaling and promotion of CCN family protein 2.

Adipocyte differentiation is regulated by several transcription factors such as the CCAAT/enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptor-γ (PPARγ). Here, we demonstrate that low-intensity pulsed ultrasound (LIPUS) suppressed differentiation into mature adipocytes via multiple signaling pathways. When C3H10T1/2, a mesenchymal stem cell line, was treated with LIPUS (3.0 MHz, 60 mW/cm2 ) for 20 minutes once a day for 4 days during adipogenesis, and both the number of lipid droplets and the gene expression of PPARγ and C/EBPα were significantly decreased. Furthermore, LIPUS treatment decreased the phosphorylation of the insulin receptor and also that of Akt and ERK1/2, which are located downstream of this receptor. Next, we showed that LIPUS suppressed the gene expression of angiotensinogen (AGT), which is an adipokine produced by mature adipocytes, as well as that of angiotensin-converting enzyme1 (ACE1) and angiotensin receptor type 1 (AT1 R) during adipogenesis of pre-adipogenic 3T3-L1 cells. Next, the translocation of Yes-associated protein (YAP) into the nucleus of 3T3-L1 cells was promoted by LIPUS, leading to upregulation of CCN family protein 2 (CCN2), a cellular communication network factor. Moreover, forced expression of CCN2 in 3T3-L1 cells decreased PPARγ gene expression, but it did not increase alkaline phosphatase and osterix gene expression. Finally, gene silencing of CCN2 in C3H10T1/2 cells diminished the effect of LIPUS on the gene expression of PPARγ and C/EBPα. These findings suggest that LIPUS suppressed adipogenesis through inhibition of insulin signaling and decreased PPARγ expression via increased CCN2 production, resulting in a possible decrease of mature adipocytes.

Tanshinone IIA contributes to the pathogenesis of endometriosis via renin angiotensin system by regulating the dorsal root ganglion axon sprouting

AimsOur study was designed to explore the function and mechanism of Tanshinone IIA in alleviating pain syndrome caused by endometriosis (EMs). Main methodsFemale Sprague-Dawley rats went through autotransplantation operation to establish EMs model. The rats were randomly divided into five groups: sham, model, positive, Tanshinone IIA (L) (3 mg/kg/d) and Tanshinone IIA (H) (12 mg/kg/d) group. Volume of ectopic endometrium was measured after 21 days of continuous administration. Serum estradiol (E2) was detected by enzyme linked immunosorbent assay (Elisa). The protein expression of angiotensinogen (AGT), renin (REN), angiotensin converting enzyme (ACE), angiotensin II (ANGII) and angiotensin II type 2 receptor (AT2) in the dorsal root ganglion (DRG) neurons were measured by immunohistochemistry and Western Blotting. The mRNA expression levels of AGT and ANGII were measured by Real-time polymerase chain reaction (PCR). Key findingsTissue measurements showed that tanshinone IIA significantly inhibited the growth of ectopic endometrium. Tanshinone IIA could improve the paw withdrawal threshold thus reducing the mechanical hyperalgesia of EMs rats. Moreover, Tanshinone IIA regulated the DRG renin angiotensin system (RAS) by reducing the protein expression of AGT, REN, ACE, ANGII and AT2 in DRG neurons. Furthermore, Real-time PCR results also showed that the mRNA expression levels of AGT and ANGII in the DRG neurons were decreased. SignificanceThe Tanshinone IIA inhibitory effect on the EMs associated pain in EMs rats might occur through decreasing the expression of E2, ANGII and AT2, thus halting DRG sprouting and promoting hyperalgesia threshold.

Blockade of sodium-glucose cotransporter 2 suppresses high glucose-induced angiotensinogen augmentation in renal proximal tubular cells.

Renal proximal tubular angiotensinogen (AGT) is increased by hyperglycemia (HG) in diabetes mellitus, which augments intrarenal angiotensin II formation, contributing to the development of hypertension and kidney injury. Sodium-glucose cotransporter 2 (SGLT2) is abundantly expressed in proximal tubular cells (PTCs). The present study investigated the effects of canagliflozin (CANA), a SGLT2 inhibitor, on HG-induced AGT elevation in cultured PTCs. Mouse PTCs were treated with 5-25 mM glucose. CANA (0-10 µM) was applied 1 h before glucose treatment. Glucose (10 mM) increased AGT mRNA and protein levels at 12 h (3.06 ± 0.48-fold in protein), and 1 and 10 µM CANA as well as SGLT2 shRNA attenuated the AGT augmentation. CANA did not suppress the elevated AGT levels induced by 25 mM glucose. Increased AGT expression induced by treatment with pyruvate, a glucose metabolite that does not require SGLT2 for uptake, was not attenuated by CANA. In HG-treated PTCs, intracellular reactive oxygen species levels were elevated compared with baseline (4.24 ± 0.23-fold), and these were also inhibited by CANA. Furthermore, tempol, an antioxidant, attenuated AGT upregulation in HG-treated PTCs. HG-induced AGT upregulation was not inhibited by an angiotensin II receptor antagonist, indicating that HG stimulates AGT expression in an angiotensin II-independent manner. These results indicate that enhanced glucose entry via SGLT2 into PTCs elevates intracellular reactive oxygen species generation by stimulation of glycolysis and consequent AGT augmentation. SGLT2 blockade limits HG-induced AGT stimulation, thus reducing the development of kidney injury in diabetes mellitus.

Tubular Deficiency of Heterogeneous Nuclear Ribonucleoprotein F Elevates Systolic Blood Pressure and Induces Glycosuria in Mice

We reported previously that overexpression of heterogeneous nuclear ribonucleoprotein F (Hnrnpf) in renal proximal tubular cells (RPTCs) suppresses angiotensinogen (Agt) expression, and attenuates systemic hypertension and renal injury in diabetic Hnrnpf-transgenic (Tg) mice. We thus hypothesized that deletion of Hnrnpf in the renal proximal tubules (RPT) of mice would worsen systemic hypertension and kidney injury, perhaps revealing novel mechanism(s). Tubule-specific Hnrnpf knockout (KO) mice were generated by crossbreeding Pax8-Cre mice with floxed Hnrnpf mice on a C57BL/6 background. Both male and female KO mice exhibited elevated systolic blood pressure, increased urinary albumin/creatinine ratio, tubulo-interstitial fibrosis and glycosuria without changes in blood glucose or glomerular filtration rate compared with control littermates. However, glycosuria disappeared in male KO mice at the age of 12 weeks, while female KO mice had persistent glycosuria. Agt expression was elevated, whereas sodium-glucose co-transporter 2 (Sglt2) expression was down-regulated in RPTs of both male and female KO mice as compared to control littermates. In vitro, KO of HNRNPF in human RPTCs (HK-2) by CRISPR gRNA up-regulated AGT and down-regulated SGLT2 expression. The Sglt2 inhibitor canagliflozin treatment had no effect on Agt and Sglt2 expression in HK-2 and in RPTCs of wild-type mice but induced glycosuria. Our results demonstrate that Hnrnpf plays a role in the development of hypertension and glycosuria through modulation of renal Agt and Sglt2 expression in mice, respectively.

Potassium Intake Prevents the Induction of the Renin-Angiotensin System and Increases Medullary ACE2 and COX-2 in the Kidneys of Angiotensin II-Dependent Hypertensive Rats.

In angiotensin II (Ang II)-dependent hypertensive rats there is an increased expression of proximal tubule angiotensinogen (AGT), collecting duct renin and angiotensin converting enzyme (ACE), which contributes to intratubular Ang II formation. Ang II acts on Ang II type 1 receptors promoting sodium retention and vasoconstriction. However concurrently, the ACE2-Ang-(1–7) axis and the expression of kallikrein and medullary prostaglandins counteract the effects of Ang II, promoting natriuresis and vasodilation. Human studies demonstrate that dietary potassium (K+) intake lowers blood pressure. In this report we evaluate the expression of AGT, ACE, medullary prorenin/renin, ACE2, kallikrein and cyclooxygenase-2 (COX-2) in Ang II-infused rats fed with high K+ diet (2%) for 14 days. Dietary K+ enhances diuresis in non-infused and in Ang II-infused rats. The rise in systolic blood pressure in Ang II-infused rats was attenuated by dietary K+. Ang II-infused rats showed increased renal protein levels of AGT, ACE and medullary prorenin and renin. This effect was attenuated in the Ang II + K+ group. Ang II infusion decreased ACE2 compared to the control group; however, K+ diet prevented this effect in the renal medulla. Furthermore, medullary COX-2 was dramatically induced by K+ diet in non-infused and in Ang II infused rats. Dietary K+ greatly increased kallikrein immunostaining in normotensive rats and in Ang II-hypertensive rats. These results indicate that a high K+ diet attenuates Ang II-dependent hypertension by preventing the induction of ACE, AGT and collecting duct renin and by enhancing medullary COX-2 and ACE2 protein expression in the kidney.

Association of Polymorphisms in the Genes of Angiotensinogen and Angiotensin Receptors With Risk for Basal Cell Carcinoma.

Basal cell carcinoma (BCC) has been genetically associated with an increased expression of angiotensin-converting enzyme (ACE), an important factor of the renin-angiotensin system which produces vasoconstrictor angiotensin II. Other factors of this system include angiotensinogen (AGT) and angiotensin receptors AGTR1, AGTR2. We investigated the possible association of BCC with genetic variability in the AGT, AGTR1 and AGTR2 genes. DNA samples of 190 Greeks were studied, including 91 patients with BCC and 99 matched healthy controls. Molecular genotyping of patients and controls was performed for the polymorphisms AGT M235T, AGTR1 A1166C and AGTR2 G1675A. The mutant T allele that increases AGT gene expression was detected in two-fold increased frequency in BCC patients in comparison to healthy controls (p <0.001). On the contrary, no significant difference was observed in AGTR1 and AGTR2 variants between patients and controls. Increased expression of AGT may be associated with BCC.

Abstract P2048: Epigenetic Modification By Angiotensin II Mediates Augmentation Of Intrarenal Angiotensinogen Expression

Inappropriately activated intrarenal renin-angiotensin system, including proximal tubular angiotensinogen (AGT) augmentation, contributes to the progression of hypertension and associated kidney injury. It has been demonstrated that interleukin 6 (IL-6) is required for angiotensin II (Ang II)-induced AGT augmentation in renal proximal tubular cells (PTC). However, molecular mechanisms remain unelucidated. Recent studies suggest that Ang II downregulates cardiac class II histone deacetylases (HDAC), which exhibit protective effects against hypertension related tissue injury. Furthermore, HDAC9 (a class II HDAC) has been shown to act as an epigenetic repressor of proximal tubular AGT. Accordingly, the present study investigated the hypothesis that Ang II decreases HDAC9 levels, thereby enabling IL-6 to promote AGT expression in PTC. Ang II (400 ng/kg/min) was chronically perfused to male mice (N=7) for 14 days to investigate the regulation of intrarenal HDAC9. Moreover, cultured human PTC (hPTC) were used to create wild-type, HDAC9 overexpression, and HDAC9 knockdown by shRNA models and were treated with 100 nM Ang II, 10 ng/ml IL-6, or both for 24 hours. Chronic Ang II infusion elevated blood pressure and suppressed renal cortical HDAC9 levels. A decrease in HDAC9 was also observed in Ang II-treated hPTC. Treatment of hPTC with both Ang II and IL-6 increased AGT mRNA levels (1.50±0.10, ratio to the control, N=6), whereas individual treatments with Ang II or IL-6 did not alter AGT expression levels as previously demonstrated. This upregulation of AGT expression by Ang II and IL-6 co-stimulation was attenuated by the overexpression of HDAC9 in hPTC (1.10±0.08, ratio to the control). Importantly, AGT expression was enhanced by IL-6 without Ang II (2.31±0.08 by IL-6 and 2.27±0.15 by Ang II+IL-6, ratio to the control) in cells receiving the HDAC9 gene knockdown, suggesting that HDAC9 down-regulation mediates IL-6-induced AGT upregulation in hPTC. These results indicate that Ang II-induced epigenetic modification, such as the suppression of intrarenal HDAC9, results in increased accessibility of IL-6-derived transcription factors to the AGT promoter and consequent augmentation of intrarenal AGT expression in Ang II-dependent hypertension.

Dysregulation of the placental renin-angiotensin system in human fetal growth restriction.

Fetal growth restriction (FGR) is a pregnancy complication wherein the foetus fails to reach its growth potential. The renin-angiotensin system (RAS) is a critical regulator of placental function, controlling trophoblast proliferation, angiogenesis and blood flow. The RAS significantly influences uteroplacental blood flow through the balance of its vasoconstrictive and vasodilatory pathways. Although the RAS is known to be dysregulated in placentae from women with preeclampsia, the expression of the RAS has not yet been studied in pregnancies compromised by FGR alone. This study investigated the mRNA expression and protein levels of RAS components in placentae from pregnancies compromised by FGR. Angiotensin II type 1 receptor (AGTR1) and angiotensin-converting enzyme 2 (ACE2) mRNA levels were reduced in FGR placentae compared with control (P = 0.012 and 0.018 respectively). Neprilysin (NEP) mRNA expression was lower in FGR placentae compared with control (P = 0.004). mRNA levels of angiotensinogen (AGT) tended to be higher in FGR placentae compared with control (P = 0.090). Expression of prorenin, AGT, angiotensin-converting enzyme (ACE) or ACE2 proteins were similar in control and FGR placentae. The renin-AGT reaction is a first order reaction so levels of expression of placental AGT determine levels of Ang II. Decreasing levels of ACE2 and/or NEP by limiting the production of Ang-(1-7), which is a vasodilator, and increasing placental Ang II levels (vasoconstrictor) may result in an imbalance between the vasoconstrictor and vasodilator arms of the placental RAS. Ultimately this dysregulation of the placental RAS could lead to reduced placental perfusion that is evident in FGR.

Vitamin D Receptor Deficiency Does Not Affect Blood Pressure and Heart Function.

Vitamin D is thought to play a role in blood pressure regulation, which in turn can influence cardiovascular risk. Several meta-analyses of cohort studies found low serum levels of 25-hydroxyvitamin D to be associated with increased blood pressure or increased cardiovascular morbidity and mortality in the general population. Active vitamin D mediates its function via the vitamin D receptor (Vdr), which is a ligand-activated transcription factor. A suitable model to examine the causal role of vitamin D in blood pressure regulation and heart function is the Vdr knockout (Vdr–/–) mouse. To elucidate the role of vitamin D on blood pressure, heart function, and cardiac myocyte size, we conducted a long-term study using Vdr–/– mice and well-defined diets. Group 1 comprised Vdr–/– mice that received a high-calcium, high-phosphorus rescue diet to prevent hypocalcemia and a rickets phenotype. Groups 2 and 3 included Vdr+/+ mice that were fed either the rescue diet or a control diet containing normal amounts of these minerals. As Vdr is a nuclear factor that regulates transcription, we analyzed the renal mRNA expression and serum concentration of renin and found that the Vdr–/– group had an almost 50% higher renin mRNA expression in the kidney compared to both groups of Vdr+/+ mice. Additionally, serum concentration of renin in Vdr–/– mice was significantly higher than that of Vdr+/+ mice that received the rescue or control diet (+ 17%,+ 32%; P < 0.05). In contrast, renin activity was lower in Vdr–/– mice than in both groups of Vdr+/+ mice (P < 0.05). However, blood pressure, heart rate, cardiac myocyte sizes, and the expression of renal renin receptor, hepatic angiotensinogen and angiotensin II receptor, type 1, in kidney, liver and heart, did not differ between the three groups of mice. Additionally, data from transthoracic echocardiography did not indicate the role of Vdr on heart function, as the left ventricular ejection fraction, fractional shortening, and velocity of blood flow were comparable between the three groups. To conclude, the roles of Vdr and therefore most probably of vitamin D, in blood pressure regulation and heart function, were not confirmed by our findings.

Urinary Angiotensinogen Predicts Renal Disease Activity in Lupus Nephritis.

Aims: A noninvasive indicator of renal histological lesions and disease activity in lupus nephritis (LN) is needed for timely and targeted treatment before overt renal injury. Here, we tested the utility of urinary angiotensinogen (UAGT) to predict renal disease activity in LN. Results: A prospective, three-stage study was performed in patients with LN. In stage I, UAGT was measured in 140 newly diagnosed LN patients. UAGT significantly increased in LN patients, correlating well with kidney angiotensinogen expression and histological activity. Patients with LN class IV exhibited the highest UAGT compared with other histopathological classes of LN. For identifying LN class IV, a particularly aggressive type of LN, UAGT outperformed the conventional clinical measures and improved their performance. In stage II, UAGT was monitored in 61 subjects from stage I for up to 12 months. UAGT decreased after induction therapy and remained low in patients with LN remission during follow-up. For predicting therapy success at month 12, the area under the receiver operating characteristics curve of UAGT reduction at month 4 was 0.83, outperforming that of 24-h proteinuria. In stage III, UAGT was monitored in 12 LN patients before, during, and after the onset of renal flares. An elevation in UAGT predicted recurrence of LN, and a decline in UAGT after a renal flare heralded the remission of disease before conventional clinical measures. Innovation and Conclusion: UAGT in LN is a promising indicator for dynamic surveillance of renal disease activity and prediction of renal flares. Antioxid. Redox Signal. 31, 1289-1301.

Brain renin-angiotensin system in broiler chickens with cold-induced pulmonary hypertension

ABSTRACT1. The relative expression of angiotensinogen (AGT), renin, angiotensin-converting enzyme (ACE) and angiotensin II type 1 receptor (AT1R) was determined using quantitative real-time PCR on tissue from the brain (forebrain, midbrain and hindbrain) to investigate the effect of cold-induced pulmonary hypertension syndrome (PHS) in broilers aged 42 days. Brain angiotensin II (Ang II) and AT1R levels were measured using enzyme immunoassay.2. The right ventricle/total ventricles (RV/TV) ratio of the heart was increased in broilers exposed to cold stress (PHS group) at the end of the experiment.3. ACE and renin transcripts in three parts of the brain were significantly increased in the PHS group at 42 d of age compared to controls while AGT transcript was significantly increased only in the hindbrain of PHS birds. The amount of AT1R transcript did not differ between control and PHS groups.4. The amount of Ang II significantly decreased only in the midbrain of PHS birds compared with controls while the amounts of AT1R were not different between treatments in the three segments of the brain.5. It was concluded that brain gene expression of AGT (in the hindbrain), renin, and ACE was upregulated in broilers with PHS whereas Ang II and AT1R levels were not changed. These results provided evidence of diminished involvement of the renin-angiotensin system in the pathogenesis of chicken pulmonary hypertension.

Sex and salt intake dependent renin-angiotensin plasticity in the liver of the rat.

Epidemiological studies confirm that hypertensive patients respond differently to renin-angiotensin system (RAS) inhibition depending on their gender. The aim of present work is to focus on sex-dependent differences in RAS regulation under conditions of increased salt intake. To investigate RAS, we measured the expression of angiotensinogen (Agt) mRNA, angiotensin receptor type 1 (AT1) mRNA and mitochondria assembly receptor (MasR) in the liver of rats under control conditions and after feeding with a salt diet (2% NaCl). In parallel, vascular endothelial growth factor A (VEGF-A) mRNA was analyzed. Regression analysis revealed sex-dependent differences in the correlation between mRNA expression of AT1 and that of Agt, MasR and VEGF-A in both groups. There was a significant negative correlation between AT1 and Agt mRNA expression in the male control group, but this correlation disappeared in males exposed to a salt diet. In females, AT1 and Agt expression correlated only in the group exposed to the salt diet. In control males, there was a borderline trend to correlation between AT1 and MasR mRNA expression. The correlation between AT1 and VEGF-A mRNA expression was significant only in the control females, however, after exposure to a salt diet, this correlation diminished. We hypothesize that RAS components expression is compensated differently in males and females. The observed loss of compensatory relationships in RAS between AT1 and Agt and AT1 and MasR in male rats under a salt diet can contribute to the differences observed in human with hypertension associated with an unhealthy diet.

Heterologous expression, purification, and functional characterization of recombinant ovine angiotensinogen in the methylotrophic yeast Pichia pastoris.

Angiotensinogen (AGT), a glycosylated plasma noninhibitory serpin, serves as a precursor for angiotensin peptides which regulate blood pressure and electrolyte balance. AGT is specifically cleaved by renin to produce angiotensin-I, the first product of the angiotensin-processing cascade. Ovine angiotensinogen (oAGT) is considered an effective substrate for human renin and consequently finds application in clinical renin assays. In this study, oAGT was cloned into the genome of Pichia pastoris and expressed under the control of alcohol oxidase (AOX1) promoter for high-level production. Compared to the shake flask study, the high cell density cultivation in bioreactor resulted in multifold increase in oAGT titer (420 ± 9.26 mg/L), which is its highest reported titer to date. We purified recombinant oAGT to homogeneity using two chromatography steps. The characterization studies revealed oAGT underwent a two-state transition during thermal denaturation process as assessed by differential scanning fluorimetry, and the melting temperature (Tm ) of the purified oAGT from P. pastoris was 48.3°C. Renin reactivity with recombinant oAGT from P. pastoris (0.51 nM angiotensin-I/min) was slightly lower than the renin reactivity for recombinant oAGT from Escherichia coli (0.67 nM angiotensin-I/min), possibly because of its mannosylated N-glycan content. Enhanced production of functionally active recombinant oAGT using P. pastoris expression system reported in this study envisage the effective utilization of oAGT in clinical studies related to renin in near future.

High glucose promotes breast cancer proliferation and metastasis by impairing angiotensinogen expression.

A number of investigations have addressed the importance of high glucose in breast cancer, however, the involvement of angiotensinogen (AGT) in this scenario is yet to be defined. Here we set out to analyze the potential pro-tumor effects of high glucose in breast cancer, and understand the underlying molecular mechanism. We demonstrated that high glucose promoted cell proliferation, viability, and anchorage-independent growth of breast cancer cells. In addition, the migrative and invasive capacities were significantly enhanced by high glucose medium. Mechanistically, AGT expression was inhibited by high glucose at both transcriptional and translational levels. High AGT remarkably suppressed proliferation, inhibited viability, and compromised migration/invasion of breast cancer cells. Most importantly, ectopic introduction of AGT almost completely abrogated pro-tumor effects of high glucose. Our study has characterized the pro-tumor properties of high glucose in breast cancer cells, which is predominantly attributed to the suppression of AGT.

Obesity and aging affects skeletal muscle renin-angiotensin system and myosin heavy chain proportions in pre-diabetic Zucker rats.

There is a gap in the knowledge regarding regulation of local renin-angiotensin system (RAS) in skeletal muscle during development of obesity and insulin resistance in vivo. This study evaluates the obesity- and age-related changes in the expression of local RAS components. Since RAS affects skeletal muscle remodelling, we also evaluated the muscle fibre type composition, defined by myosin heavy chain (MyHC) mRNAs and protein content. Gene expressions were determined by qPCR and/or Western blot analysis in musculus quadriceps of 3- and 8-month-old male obese Zucker rats and their lean controls. The enzymatic activity of aminopeptidase A (APA) was determined flourometrically. Activation of renin receptor (ReR)/promyelocytic leukaemia zinc finger (PLZF) negative feedback mechanism was observed in obesity. The expression of angiotensinogen and AT1 was downregulated by obesity, while neutral endopeptidase and AT2 expressions were upregulated in obese rats with aging. Skeletal muscle APA activity was decreased by obesity, which negatively correlated with the increased plasma APA activity and plasma cholesterol. The expression of angiotensin-converting enzyme (ACE) positively correlated with MyHC mRNAs characteristic for fast-twitch muscle fibres. The obesity- and age-related alterations in the expression of both classical and alternative RAS components suggest an onset of a new equilibrium between ACE/AngII/AT1 and ACE2/Ang1-7/Mas at lower level accompanied by increased renin/ReR/PLZF activation. Increased APA release from the skeletal muscle in obesity might contribute to increased plasma APA activity. There is a link between reduced ACE expression and altered muscle MyHC proportion in obesity and aging.

Role of Renin-Angiotensin-System in Human Breast Cancer Cells: Is There a Difference in Regulation of Angiogenesis between Hormone-Receptor Positive and Negative Breast Cancer Cells?

Objective This study examined the role of the RAS in human breast cancer cells to question if there are differences between HR-positive and HR-negative cells with regard to regulation of VEGF. Methods Expression of different RAS components in hormone receptor (HR)-positive and HR-negative breast cancer cells was investigated using RT-PCR. Different stimulation protocols with different RAS inhibitors were used to investigate the effect on VEGF expression. Angiotensin II-dependent expression of VEGF was quantified by real time PCR. In addition, the effect of intrinsic RAS was studied performing siRNA knockdown of angiotensinogen (AGT). Statistical analysis were calculated using IBM SPSS Statistics Version 21. Results Expression of AT 1 R, AT 2 R, AGT and ACE was shown in HR-positive and HR-negative breast cancer cell lines. Extrinsic stimulation with angiotensin II increased VEGF significantly. After treatment with captopril or AT 1 R-inhibitor candesartan, VEGF-expression decreased significantly in HR-positive and HR-negative cell lines. However, inhibition of AT 2 R using PD 123,319 did not show any significant changes of VEGF. After prevention of intrinsic angiotensin II, extrinsic angiotensin II as well as the combination with inhibitors of the receptors caused a significant reduction of VEGF. Surprisingly, the overall effect of the RAS after knockdown of AGT revealed a significant increase of VEGF in HR-positive cells at any time while a significant decrease was observed in HR-negative cells after 144 hours incubation. Conclusion The RAS-dependent regulation of VEGF between HR-positive and HR-negative breast cancer cells seems do be different. These findings provide evidence for a possible future therapeutic strategy.