Angiotensinogen Research Articles

Relationship Between Urinary Angiotensinogen and Mortality in Patients with Heart Failure with Reduced Ejection Fraction.

Activation of the reninangi otens in-aldosterone system has an important role in the pathophysiology of heart failure with reduced ejection fraction. While the effects of systemic reninangi otens in-aldosterone system activation on heart failure with reduced ejection fraction are well known, the impact of the local reninangi otens in-aldosterone system on heart failure with reduced ejection fraction is not fully understood because of limited clinical research. This study aimed to investigate the effect of urinary angiotensinogen level, an accepted indicator of local reninangi otens in-aldosterone system activation, on all-cause mortality in patients with heart failure with reduced ejection fraction. This retrospective, single-center study included 60 patients with baseline urinary angiotensinogen data and survival/mortality data at 4 years. Urinary angiotensinogen values were standardized to the urinary creatinine value measured from the same urine sample. The median urinary angio tensi nogen /urin ary creatinine value among all patients (114 μg/g) was used as a cutoff to divide the patients into 2 groups. Mortality data were obtained from the national registry systems or by telephone. Comparison of all-cause mortality in the 2 groups showed that 22 deaths (71%) occurred in the group with a urinary angio tensinogen/urinary creatinine ratio above the median and 10 deaths (35.5%) occurred in the group of patients with urinary angio tensinogen/urinary creatinine equal to or below the median value (P =.005). Our study suggests that urinary angiotensinogen can be used as a new biomarker in the prognosis and follow-up of heart failure patients.

Genetic Polymorphism in Angiotensinogen and Its Association with Cardiometabolic Diseases.

Angiotensinogen (AGT) is one of the most significant enzymes of the renin-angiotensin-aldosterone system (RAAS) which is involved in the regulation and maintenance of blood pressure. AGT is involved in the production of angiotensin I which is then converted into angiotensin II that leads to renal homeostasis. However, various genetic polymorphisms in AGT have been discovered in recent times which have shown an association with various diseases. Genetic polymorphism increases the level of circulating AGT in blood which exaggerates the effects produced by AGT. The associated diseases occur due to various effects produced by increased AGT levels. Several cardiovascular diseases including myocardial infarction, coronary heart disease, heart failure, hypertrophy, etc. are associated with AGT polymorphism. Other diseases such as depression, obesity, diabetic nephropathy, pre-eclampsia, and liver injury are also associated with some variants of AGT gene. The most common variants of AGT polymorphism are M235T and T174M. The two variants are associated with many diseases. Some other variants such as G-217A, A-6G, A-20C and G-152A, are also present but they are not as significant as that of M235T and T174M variants. These variants increase the level of circulating AGT and are associated with prevalence of different diseases. These diseases occur through various pathological pathways, but the initial reason remains the same, i.e., increased level of AGT in the blood. In this article, we have majorly focused on how genetic polymorphism of different variants of AGT gene is associated with the prevalence of different diseases.

Angiotensinogen Gene M235T and T174M Polymorphisms in Diabetic Nephropathy in a Bangladeshi Population

Background: Marker gene polymorphisms linked with the renin-angiotensin-aldosterone system (RAAS) have been broadly studied in diabetic nephropathy (DN) patients considering that RAAS is a potential drug target to slow down kidney disease progression. Objectives: The aim of the present study was to determine the link between M235T and T174M variants of angiotensinogen (AGT) gene and DN. Methods: A total of 93 patients with DN, mean age of 56±8 years, systolic blood pressure (SBP) of 141±14, and diastolic blood pressure (DBP) of 84±7 mm Hg (mean±SD) were investigated, among whom 59 patients had a family history of type 2 diabetes mellitus. A total of 96 healthy subjects served as the control group with no family history of diabetic nephropathy (FHDN) and type 2 diabetes mellitus, a mean age of 47±10 years, SBP of 126±11, and DBP of 76±6 mm Hg. PCR–restriction fragment length polymorphism was employed for genotyping M235T and T174M molecular variants. Results: Genotype frequencies of the variants M235T (χ2=2.038, P=0.361) and T174M (χ2=2.952, P=0.229) did not show any statistically significant association with type 2 diabetic nephropathy (T2DN) compared to the control. Based on FHDN and family history of diabetes mellitus (FHDM), the frequency of genotypes of M235T marker (P=0.360) in FHDN, and (P=0.886) FHDM; T174M marker (P=0.641) in FHDN, and (P=0.425) FHDM also did not show any statistically significant association with T2DN compared to the controls. Conclusion: M235T and T174M variants were not associated with DN in a Bangladeshi population.

Bone morphogenetic protein 4 in perivascular adipose tissue ameliorates hypertension through regulation of angiotensinogen

Perivascular adipose tissue (PVAT), an active endocrine organ, exerts direct effect on vascular tone through paracrine. Activation of PVAT metabolism plays an inhibitory role in atherosclerosis via secreting relaxing factors. The present studies were designed to investigate the role of PVAT metabolism in regulation of hypertension. Apolipoprotein E (ApoE) knockout mice with BMP4 knockout in adipose tissue or brown adipose tissue (aP2-DKO or UCP1-DKO, respectively) were used for exploring the role of impaired PVAT metabolism in hypertension. Vascular function was assessed using wire myography. The potential regulatory factor of vascular function was explored using qPCR and ELISA and further confirmed in perivascular fat cell line. Knockout of BMP4 either in adipose tissue or specifically in BAT aggravates high-fat diet (HFD, 40% fat)-induced hypertension and endothelial dysfunction in ApoE-/- mice. In the meanwhile, deficiency of BMP4 also aggravates Ang II (angiotensin II) -induced hypertension and vascular remodeling in ApoE-/- mice. Moreover, deficiency of BMP4 inhibits NO release and induces ROS production. In vitro system, aortic rings pretreated with PVAT extracts from BMP4-DKO mice showed increased vasoconstriction and reduced endothelial-dependent relaxation compared with the controls. We further demonstrated that PVAT of BMP4-DKO mice expressed higher level of angiotensinogen (AGT) and Ang II compared with the controls. Impaired PVAT metabolism aggravates hypertension, and this effect is dependent on the activation of local renin-angiotensin-aldosterone system (RAAS). The results of this study first demonstrate the regulatory role of PVAT metabolism in hypertension.

Polymorphism rs7079 in miR-31/-584 Binding Site in Angiotensinogen Gene Associates with Earlier Onset of Coronary Artery Disease in Central European Population.

Angiotensinogen (AGT) represents a key component of the renin-angiotensin-aldosterone system (RAAS). Polymorphisms in the 3' untranslated region (3'UTR) of the AGT gene may alter miRNA binding and cause disbalance in the RAAS. Within this study, we evaluated the possible association of AGT +11525C/A (rs7079) with the clinical characteristics of patients with coronary artery diseases (CAD). Selective coronarography was performed in 652 consecutive CAD patients. Clinical characteristics of the patients, together with peripheral blood samples for DNA isolation, were collected. The genotyping of rs7079 polymorphism was performed with TaqMan® SNP Genotyping Assays. We observed that patients with the CC genotype were referred for coronarography at a younger age compared to those with the AA+CA genotypes (CC vs. AA+CA: 59.1 ± 9.64 vs. 60.91 ± 9.5 (years), p = 0.045). Moreover, according to the logistic regression model, patients with the CC genotype presented more often with restenosis than those with the CA genotype (p = 0.0081). In conclusion, CC homozygotes for rs7079 present with CAD symptoms at a younger age compared with those with the AA+CA genotype, and they are more prone to present with restenosis compared with heterozygotes.

Characteristic of the uncontrolled hypertension course in patients of the aral sea region with uncomplicated hypertensive crisis

Abstract Purpose To study the features of the course of uncontrolled arterial hypertension (AH) in the Aral Sea region with the identification of the prevalence of various genotypes of the angiotensinogen (AGT) gene in patients with uncomplicated hypertensive crisis. Methods The genetic part included 94 patients with uncomplicated hypertensive crisis, an average age of 55.3±8.6 years, 54 (58.3%)women and 40 men (41.7%). The control group consisted of 50 healthy women and men, an average age of 52.7±6.4 years in equal proportion. The genetic study was carried out by real-time PCR with the determination of the prevalence of genotypes and alleles of genetic polymorphisms AGT 521 C>T, AGT 704 T>C. Results Patients with uncomplicated hypertensive crisis were characterized by LV hypertrophy, thickening of the intima-media thickness of the common carotid artery, and kidney damage. Henken's test showed that patients with AH require a significantly higher concentration of table salt to determine the threshold of taste sensitivity than in the control group: 0.51±0.17% versus 0.21±0.14% concentration of NaCl. The distribution of 704T>C genotypes of the AGT gene polymorphism was as follows: CC genotype 43.6%, CT genotype 45.8%, TT genotype 10.6% of cases, χ2=32.777, p=0.000; C allele – 66.5%, T allele - 33.5% of cases, χ2=40.894, p=0.000. Among healthy individuals, the distribution by genotypes showed a significant predominance of the CC genotype: CC 76%, CT 24%, TT genotype was not detected in the presented sample, χ2=67.92, p=0.000; C-allele 88%, T-allele 12%, χ2=115.52, p=0.000. The above data of the group of patients and healthy people corresponded to the theoretical Hardy-Weinberg equilibrium. The distribution of 521T>C genotypes of the AGT gene polymorphism among AH patients was as follows: CC genotype – 79.8%, CT genotype – 16%, TT genotype – 4.2% of cases, χ2=139.8, p=0.000; C-allele – 87.8%, T-allele – 12.2%, χ2=214.5, p=0.000. Among healthy individuals, a significant predominance of the CT genotype was shown: CC genotype – 40%, CT genotype – 60% of cases, χ2=42.0, p=0.000; C-allele – 70%, T-allele – 30%, χ2=32.0, p=0.000. The above data of the group of patients and healthy people did not correspond to the theoretical Hardy-Weinberg equilibrium Conclusion Patients with uncomplicated hypertensive crisis, living in the aral sea region, are characterized by severe damage of target organs, a high threshold of taste sensitivity to table salt. Based on genetic models of inheritance, a significant prevalence of the C-allele and CC genotype 704 T>C AGT gene polymorphism in healthy individuals was revealed, which characterizes the C-allele and CC genotype as protective against AH in people of the Aral Sea region. Significantly greater accumulation of the C-allele 521 C>T polymorphism of the AGT gene among AH patients is a factor of predisposition to a complicated course of AH in people of the aral sea region. Funding Acknowledgement Type of funding sources: None.

Urinary Angiotensinogen in Patients With Type 1 Diabetes With Microalbuminuria: Gender Differences and Effect of Intensive Insulin Therapy

Angiotensinogen (AOG) is the precursor of peptides of the renin angiotensin system (RAS). Because insulin up-regulates transcriptional factors that normally repress kidney AOG synthesis, we evaluated urinary AOG (uAOG) in patients with type 1 diabetes (T1D) and microalbuminuria who are receiving either intensive or conventional insulin therapy. Urine samples from participants of the Diabetes Control and Complications Trial (DCCT) were used for the following: (i) uAOG/creatinine measurements in 103 patients with microalbuminuria and 103 patients with normoalbuminuria, matched for age, gender, disease duration, and allocation to insulin therapy; and (ii) uAOG/creatinine measurements from patients with microalbuminuria allocated to intensive insulin therapy (n= 58) or conventional insulin therapy (n= 41) after 3 years on each modality. uAOG was higher in patients who started with microalbuminuria than in those with normoalbuminuria (6.65 vs. 4.0 ng/mg creatinine, P< 0.01). uAOG was higher in females than in males with microalbuminuria (11.7 vs. 5.4 ng/mg creatinine, P= 0.015). uAOG was lower in patients with microalbuminuria allocated to intensive insulin therapy than in conventional insulin therapy (3.98 vs. 7.42 ng/mg creatinine, P< 0.01). These differences in uAOG were observed though albumin excretion rate (AER) was not significantly different. In patients with T1D and microalbuminuria, uAOG is increased and varies with gender and the type of insulin therapy independently of AER. This suggests that AOG production is increased in females and it is decreased by intensive insulin therapy. The reduction in uAOG with intensive insulin therapy, by kidney RAS downregulation, may contribute to the known renoprotective action associated with intensive insulin and improved glycemic control.

Abstract 044: Stimulation Of Intrarenal Angiotensinogen Expression And The Development Of Hypertension And Kidney Injury In Angiotensin II-infused Hepatic Angiotensinogen Knockout Mice

Regulation of systemic and local angiotensinogen (AGT) levels is a key determinant of tissue angiotensin II (Ang II) levels and inappropriate AGT augmentation promotes the development of hypertension and tissue injury. Kidney and urinary AGT levels are increased in Ang II-mediated hypertension. Recent studies have demonstrated that circulating hepatocyte-derived AGT (hAGT) enters kidneys sustaining kidney and urinary AGT levels. However, roles of hAGT in blood pressure elevation and kidney injury in Ang II-mediated hypertension have not been delineated. This study tested if hAGT contributes to the development of the pathophysiological events in Ang II-infused mice. A low dose of Ang II (400 ng/kg/min) was infused to male wild type (WT) and hAGT gene knockout (KO) mice (N=9 and 13) for 4 weeks. The control group in each genotype received vehicle (Veh) infusion (N=5 and 6). Western blot confirmed non-detectable levels of hAGT in KO mice. hAGT KO markedly decreased plasma AGT levels (WT+Veh:12.2±0.6 vs. hAGT KO+Veh: 0.8±0.1 μg/ml). Ang II infusion did not elevate plasma AGT levels in either WT and hAGT KO mice. Although hAGT KO mice exhibited a lower baseline of systolic blood pressure (SBP) than WT mice, Ang II-mediated increases in SBP was not attenuated in hAGT KO mice (ΔSBP in WT+Ang II: 30.1±4.4 vs. hAGT KO+Ang II: 26.0±4.2 mmHg). Kidney AGT mRNA levels were increased by Ang II infusion to the same extent in both WT and hAGT mice (WT+Ang II: 1.30±0.04 vs. hAGT KO+Ang II: 1.34±0.06, ratio to control). Likewise, Ang II infusion increased IL-6 mRNA to the same magnitude in both WT and hAGT KO mice. Urinary AGT was sustained in hAGT KO+Veh mice (66±9%) compared to WT+Veh mice. Ang II infusion did not alter urinary AGT levels in both groups. Glomerular mesangial expansion and fibrosis by Ang II infusion were not observed. Ang II infusion developed tubulointerstitial fibrosis in renal cortex and medulla. hAGT KO prevented the fibrosis only in the medulla. These outcomes demonstrate that elevation of SBP, augmentation of intrarenal AGT and IL-6 expression, and the development of renal cortical fibrosis in Ang II-mediated hypertension do not require hAGT. In contrast, hAGT contributes to renal medullary fibrosis which may be due to the lower absolute levels of blood pressure.

Abstract 124: Angiotensin-(1-12) Immunoneutralization: A New Approach To Hypertension Treatment

There is a critical need to identify new therapeutic targets and treatments, given emerging data documenting the limited effectiveness of current antihypertensive drugs to have a marked impact in preventing target organ damage and cardiovascular death. While monoclonal antibodies (mAb) are revolutionizing the treatment of human diseases, including severe hypercholesterolemia, their applicability to long-term hypertension control remains unexplored. Identification of angiotensin-(1-12) [Ang-(1–12)] as a non-renin-dependent substrate for Ang II generation in rodents and humans led us to engineer a mAb directed against the C-terminus of human Ang-(1–12) [h-Ang-(1-12)] sequence. We utilized a model of hypertension expressing the human angiotensinogen (AGT) gene in the rat genome [TGR(hAGT)L1623 rats] to address the effectiveness of endogenous h-Ang-(1–12) immunoneutralization in controlling the elevated blood pressure and reversing hypertension-induced target organ damage. Acute delivery of the h-Ang-(1–12) mAb (30 mg/Kg, i.v.) in anesthetized hypertensive transgenic rats induced a prompt arterial pressure fall (M ± SE: -18 ± 6/-16 ± 3 mm Hg, n=7; p&lt; 0.001) in the absence of corresponding heart rate changes. The antihypertensive effect of the h-Ang-(1-12) mAb triggered a rise in plasma renin concentrations (from 107 ± 24 to 381 ± 260 ng/mL/h) that was inversely correlated with the fall in mean arterial pressure (r= -0.92, p&lt; 0.001). Prior intravenous delivery of aliskiren fumarate (3-5 mg/Kg, i.v.) while inducing a fall in arterial pressure did not mask the antihypertensive action of the h-Ang-(1-12) mAb. Extension of these acute effects of the h-Ang-(1-12) mAb to awake instrumented transgenic hypertensive rats given consecutive daily doses of the mAb (30 mg/Kg, i.m. x 4 -5 days, n=7) documented a trend for significant statistical drops in daytime blood pressure and no changes in heart rate. We conclude that Ang-(1–12) is an endogenous Ang II forming substrate participating in the control of arterial pressure in a humanized model of high blood pressure and that selective immunoneutralization of this alternate substrate is a promising and novel therapeutic approach to treat hypertension and prevent its adverse cardiovascular sequelae.

Screening Biomarkers of Sudden Coronary Death Based on mRNA Expression Profile of Rat Myocardial Tissues.

To explore the differential expression of messenger RNA (mRNA) in myocardial tissues of rats with sudden coronary death (SCD), and to provide ideas for the forensic identification of SCD. The rat SCD model was established, and the transcriptome sequencing was performed by next-generation sequencing technology. Differentially expressed genes (DEGs) in myocardial tissues of SCD rats were screened by using the R package limma. A protein-protein interaction (PPI) network was constructed by using the STRING database and Cytoscape 3.8.2 on DEG, and hub genes were screened based on cytoHubba plug-in. Finally, the R package clusterProfiler was used to analyze the biological function and signal pathway enrichment of the selected DEG. A total of 177 DEGs were associated with SCD and were mainly involved in the renin-angiotensin system and PI3K-Akt signaling pathway. The genes including angiotensinogen (AGT), complement component 4a (C4a), Fos proto-oncogene (FOS) and others played key roles in the development of SCD. Genes such as AGT, C4a, FOS and other genes are expected to be potential biomarkers for forensic identification of SCD. The study based on mRNA expression profile can provide a reference for forensic identification of SCD.

Inhibition of the Renin-Angiotensin System Fails to Suppress β-Aminopropionitrile-Induced Thoracic Aortopathy in Mice-Brief Report.

Cross-linking of lysine residues in elastic and collagen fibers is a vital process in aortic development. Inhibition of lysyl oxidase by BAPN (β-aminopropionitrile) leads to thoracic aortopathies in mice. Although the renin-angiotensin system contributes to several types of thoracic aortopathies, it remains unclear whether inhibition of the renin-angiotensin system protects against aortopathy caused by the impairment of elastic fiber/collagen crosslinking. BAPN (0.5% wt/vol) was started in drinking water to induce aortopathies in male C57BL/6J mice at 4 weeks of age for 4 weeks. Five approaches were used to investigate the impact of the renin-angiotensin system. Bulk RNA sequencing was performed to explore potential molecular mechanisms of BAPN-induced thoracic aortopathies. Losartan increased plasma renin concentrations significantly, compared with vehicle-infused mice, indicating effective angiotensin II type 1 receptor inhibition. However, losartan did not suppress BAPN-induced aortic rupture and dilatation. Since losartan is a surmountable inhibitor of the renin-angiotensin system, irbesartan, an insurmountable inhibitor, was also tested. Although increased plasma renin concentrations indicated effective inhibition, irbesartan did not ameliorate aortic rupture and dilatation in BAPN-administered mice. Thus, BAPN-induced thoracic aortopathies were refractory to angiotensin II type 1 receptor blockade. Next, we inhibited angiotensin II production by pharmacological or genetic depletion of AGT (angiotensinogen), the unique precursor of angiotensin II. However, neither suppressed BAPN-induced thoracic aortic rupture and dilatation. Aortic RNA sequencing revealed molecular changes during BAPN administration that were distinct from other types of aortopathies in which angiotensin II type 1 receptor inhibition protects against aneurysm formation. Inhibition of either angiotensin II action or production of the renin-angiotensin system does not attenuate BAPN-induced thoracic aortopathies in mice.

Hepatic and proximal tubule angiotensinogen play distinct roles in kidney dysfunction, glomerular and tubular injury, and fibrosis progression.

Components of the renin-angiotensin system, including angiotensinogen (AGT), are critical contributors to chronic kidney disease (CKD) development and progression. However, the specific role of tissue-derived AGTs in CKD has not been fully understood. To define the contribution of liver versus kidney AGT in the CKD development, we performed 5/6 nephrectomy (Nx), an established CKD model, in wild-type (WT), proximal tubule (PT)- or liver-specific AGT knockout (KO) mice. Nx significantly elevated intrarenal AGT expression and elevated blood pressure (BP) in WT mice. The increase of intrarenal AGT protein was completely blocked in liver-specific AGT KO mice with BP reduction, suggesting a crucial role for liver AGT in BP regulation during CKD. Nx-induced glomerular and kidney injury and dysfunction, as well as fibrosis, were all attenuated to a greater extent in liver-specific AGT KO mice compared with PT-specific AGT KO and WT mice. However, the suppression of interstitial fibrosis in PT- and liver-specific AGT KO mouse kidneys was comparable. Our findings demonstrate that liver AGT acts as a critical contributor in driving glomerular and tubular injury, renal dysfunction, and fibrosis progression, whereas the role of PT AGT was limited to interstitial fibrosis progression in chronic renal insufficiency. Our results provide new insights for the development of tissue-targeted renin-angiotensin system intervention in the treatment of CKD.NEW & NOTEWORTHY Chronic kidney disease (CKD) is a major unmet medical need with no effective treatment. Current findings demonstrate that hepatic and proximal tubule angiotensinogen have distinct roles in tubular and glomerular injury, fibrogenesis, and renal dysfunction during CKD development. As renin-angiotensin system components, including angiotensinogen, are important targets for treating CKD in the clinic, the results from our study may be applied to developing better tissue-targeted treatment strategies for CKD and other fibroproliferative diseases.

The Genetic Variants in the Renin-Angiotensin System and the Risk of Heart Failure in Polish Patients.

(1) Background: Heart failure (HF) is a complex disease and one of the major causes of morbidity and mortality in the world. The renin-angiotensin system (RAS) may contribute to the pathogenesis of HF. (2) Aim: To investigate the association of RAS key genetic variants, rs5051 (A-6G) in the gene encoding angiotensinogen (AGT), rs4646994 (I/D) in the gene for angiotensin I converting enzyme (ACE), and rs5186 (A1166C) in the gene encoding type 1 receptor for angiotensin II (AGTR1), with the HF risk in the cohort of Polish patients. (3) Methods: The study group consisted of 415 patients that were diagnosed with HF, while the control group comprised of 152 healthy individuals. Genomic DNA were extracted from blood and genotyping was carried out using either PCR or PCR-RFLP for ACE or AGT and AGTR1 variants, respectively. (4) Results: No association has been found between the I/D ACE and heart failure. The HF risk was significantly higher for AG AGT heterozygotes (overdominance: AG versus AA + GG) and for carriers of the G AGT allele in codominant and dominant modes of inheritance. However, the risk of HF was significantly lower in the carriers of at least one C AGTR1 allele (AC or CC genotypes) or in AC AGTR1 heterozygotes (overdominant mode). There was a significant relationship for AGT and HF patients in NYHA Class I-II for whom the risk was higher for the carriers of the G allele, and for the AG heterozygotes. There was also a significant interaction between heterozygote advantage of AGT and BMI increasing the risk for HF. (5) Conclusion: Our results suggest that the A(-6)G AGT polymorphism may be associated with HF in the Polish population and the HF risk seems to be modulated by the A1166C AGTR1 polymorphism.

Immunosuppression by Mycophenolate Mofetil Mitigates Intrarenal Angiotensinogen Augmentation in Angiotensin II-Dependent Hypertension

Augmentation of intrarenal angiotensinogen (AGT) leads to further formation of intrarenal angiotensin II (Ang II) and the development of hypertensive kidney injury. Recent studies demonstrated that macrophages and the enhanced production of pro-inflammatory cytokines can be crucial mediators of renal AGT augmentation in hypertension. Accordingly, this study investigated the effects of immunosuppression by mycophenolate mofetil (MMF) on intrarenal AGT augmentation. Ang II (80 ng/min) was infused with or without daily administration of MMF (50 mg/kg) to Sprague-Dawley rats for 2 weeks. Mean arterial pressure (MAP) in Ang II infused rats was slightly higher (169.7 ± 6.1 mmHg) than the Ang II + MMF group (154.7 ± 2.0 mmHg), but was not statistically different from the Ang II + MMF group. MMF treatment suppressed Ang II-induced renal macrophages and IL-6 elevation. Augmentation of urinary AGT by Ang II infusion was attenuated by MMF treatment (control: 89.3 ± 25.2, Ang II: 1194 ± 305.1, and Ang II + MMF: 389 ± 192.0 ng/day). The augmentation of urinary AGT by Ang II infusion was observed before the onset of proteinuria. Elevated intrarenal AGT mRNA and protein levels in Ang II infused rats were also normalized by the MMF treatment (AGT mRNA, Ang II: 2.5 ± 0.2 and Ang II + MMF: 1.5 ± 0.1, ratio to control). Ang II-induced proteinuria, mesangial expansion and renal tubulointerstitial fibrosis were attenuated by MMF. Furthermore, MMF treatment attenuated the augmentation of intrarenal NLRP3 mRNA, a component of inflammasome. These results indicate that stimulated cytokine production in macrophages contributes to intrarenal AGT augmentation in Ang II-dependent hypertension, which leads to the development of kidney injury.

Quantitative label-free proteomic analysis of human follicle fluid to identify novel candidate protein biomarker for endometriosis-associated infertility

BackgroundEndometriosis (EM) leads to a decline in fertility, which is characterized by a decrease in the number and quality of follicles, and thus has a negative impact on in vitro fertilization (IVF) outcomes. However, the mechanism of how EM affects oocytes and leads to infertility remains unclear. As a potentially available sample directly related to oocyte growth, follicular fluid (FF) has important research value. Evaluating the association of FF content and EM-associated infertility through proteomics may helpful to explore the possible pathogenesis of EM-associated infertility. MethodsIn the present experimental study, from August 2019 to June 2020, FF samples were obtained as control group (CON-G; n = 10) from women with no one female factor of infertility and were undergoing IVF due to other reasons, 20 women with EM-associated infertility undergoing IVF with no other female factors were distributed into the EM group according to the time for IVF: (i) EM-group 1 (EM-G1, Stage I to Stage III, n = 10); (ii) EM-group 2 (EM-G2, Stage I to Stage III, n = 10). label-free quantitative proteomics (LFQP) technology and parallel reaction monitoring (PRM) approach were combined to aid in identifying and validating FF protein biomarkers for EM-associated infertility. In PRM analysis, another 20 subjects were enrolled as EM-associated infertility group (EM,Stage I to Stage III, n = 10) and controls (CON, n = 10) within the same time and inclusion criteria are the same as previously described. Finally, a potential protein biomarker panel of FF differential expressed proteins to EM-associated infertility was also evaluated by t-test and receiver operating characteristic (ROC) curve and binary Logistic regression models. Results7 significant differential expressed proteins which closely related to EM-associated infertility were found by LFQP technology, among which immunoglobulin lambda variable 7–46 (IGLV7–46), Immunoglobulin heavy constant gamma 2 (IGHG2), glia-derived nexin (GDN) and Inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3) were significantly up-regulated (p < 0.05), while corticosteroid-binding globulin (CBG), angiotensinogen (AGT) and Fetuin-B (FETUB) were significantly down regulated (p < 0.05). Additionally, GDN and AGT was identified as a potential protein biomarker by further PRM analysis for EM-associated infertility according to ROC curve analysis and t-test (p < 0.05), the area under the curve (AUC) for GDN and AGT was 0.78 and 0.69 with optimum sensitivity of 50%, 70% and specificity of 100%, 90%, respectively. According to binary logistic regression and evaluated ROC analysis, the AUC for the combination of GDN and AGT was 0.80. ConclusionsTo the best of our knowledge, this is the first time that elevated GDN protein levels have been found in the FF of patients with EM-associated infertility. Combining LFQP technology and PRM method we found the abnormal of GDN and AGT in FF may be the potential cause of EM-associated infertility which may help to better understand the physiological and pathological mechanism of EM-associated infertility. Further experimental studies are required to confirm their mechanism in EM-associated infertility. The results of this study are also consistent with the previous conclusion that EM is a chronic inflammatory disease. SignificanceTo the best of our knowledge, this is the first time that elevated GDN protein levels have been found in the follicular fluid of patients with EM-associated infertility. Combining LFQP technology and PRM methods we found the abnormal of GDN and AGT protein in FF may be the potential cause of EM-associated infertility which may help to better understand the physiological and pathological mechanism of EM-associated infertility.Clinically, it has been recognized that EM is related to infertility, but the mechanism remains unclear. Our study combines label-free quantitative proteomics technology and parallel reaction monitoring methods to identify and verify the FF protein biomarkers of EM-associated infertility, which provides a good research method for follow-up research.

Determination of angiotensin-converting enzyme concentration in cardiovascular patients

Cardiovascular diseases (CVDs) are the leading cause of death globally, taking an estimated 17.9 million lives each year, More than four out of five CVD deaths are due to heart attacks and strokes, and one third of these deaths occur prematurely in people under 70 years of age . The circulating Renin Angiotensin Aldosterone system (RAAS) comprises liver - secreted angiotensinogen (AGT) that is enzymatically converted into angiotensin I (Ang I). ACE, as a key component in RAS, converts angiotensin I to angiotensin II, Angiotensin II increase the production of adhesion molecules and chemokines stimulates LDL oxidation and foam cell formation in macrophages, Increased level of the ACE and subsequent ACE activity by raising the production of angiotensin II can lead to atherosclerosis . A number of 100 subjects were involved in this investigation, 100 patients of CVDs in AL-Saddir Hospital and AL Hkem Hospital, in AL- Najaf province, 70 of them with CVDs and the other 30 healthy individuals, were used as control.The study was conducted to find out the importance of Determination of angiotensin-converting enzyme concentration in cardiovascular patients and some clinical and biochemical variables .

ANTISENSE OLIGONUCLEOTIDES TARGETING HEPATIC ANGIOTENSINOGEN DOSE-DEPENDENTLY REDUCE ATHEROSCLEROSIS AND LIVER STEATOSIS IN HYPERCHOLESTEROLEMIC MICE

Objective: Liver-derived angiotensinogen (AGT) is the substrate from which renin and ACE generate angiotensin (Ang) II. Ang II not only increases blood pressure, but also is a major determinant of atherosclerosis in hypercholesteremic mice. Here we investigated the effect of hepatocyte-specific (N-acetylgalactosamine-conjugated) antisense oligonucleotides targeting AGT (GalNAc AGT ASOs) on blood pressure and atherosclerosis in hypercholesterolemic mice. Design and method: Eight-week-old male LDL receptor deficient mice were fed a Western diet for 12 weeks, after receiving vehicle, GalNAc control ASO, or 1, 2.5 or 5 mg/kg of GalNAc AGT ASO on day 1, 3, 5 and 7 in the week prior the start of the diet (n = 6–10/group). Treatment was continued thereafter on a weekly basis. Systolic blood pressure (SBP) was monitored by tail cuff. After 12 weeks, mice were euthanized and plasma was collected. Plasma AGT concentration was measured by ELISA kit, and aortic atherosclerotic lesion area was measured by an en face method. Results: The 3 GalNAc AGT ASO doses decreased plasma AGT from 10.3 ± 0.8 to 1.4 ± 0.1, 0.9 ± 0.1 and 0.7 ± 0.1 g/mL, respectively. These decreases were paralleled by dose-dependent SBP reductions from 114 ± 3 to 96 ± 2, 92 ± 2, and 84 ± 2 mmHg, respectively, and atherosclerotic lesion area reductions from 27.4 ± 0.9 to 15.1 ± 1.4, 10.8 ± 1.2, and 7.7 ± 1.0%, respectively. Yet, the attenuation of liver steatosis (liver triglyceride/weight from 139.7 ± 35.7 to 26.8 ± 4.5, 19.9 ± 1.2 and 36.0 ± 3.6 g/mg, liver total cholesterol/weight from 19.8 ± 1.6 to 8.6 ± 0.9, 6.1 ± 0.6 and 8.2 ± 0.7 g/mg) was identical for each GalNAc AGT ASO dose. GalNAc control ASO did not have effects on plasma AGT, SBP, atherosclerosis lesion area, or liver steatosis vs. vehicle. Conclusions: In hypercholesterolemic mice, inhibition of hepatic AGT reduces systolic blood pressure and atherosclerosis dose-dependently, while liver steatosis was prevented in dose-independent manner.

BLOOD PRESSURE-INDEPENDENT RENOPROTECTION IN DIABETIC RATS TREATED WITH SMALL INTERFERING RNA TARGETING LIVER ANGIOTENSINOGEN

Objective: Small interfering RNA (siRNA) targeting liver angiotensinogen (AGT) exerts beneficial effects on blood pressure and kidney function, but its effects in diabetes are still unknown. Design and method: To address this, TGR(mREN2)27 rats (a model of angiotensin II-dependent hypertension) were made diabetic with streptozotocin for 18 weeks and treated with vehicle, AGT siRNA, the angiotensin receptor blocker valsartan, the ACE inhibitor captopril, valsartan + siRNA, or valsartan + captopril for the final 3 weeks. Arterial pressure was measured via radiotelemetry. Results: Baseline mean arterial pressure (MAP) was 164 ± 1 mm Hg. Diabetes resulted in albuminuria, accompanied by glomerulosclerosis and podocyte effacement, without a further rise in MAP or a change in glomerular filtration rate. All treatments lowered MAP (by ~50 mm Hg) and cardiac hypertrophy identically. Treatment with siRNA resulted in the largest reduction in AGT, while in combination with valsartan AGT virtually disappeared. Only the dual treatment groups and captopril lowered circulating angiotensin II and aldosterone. No treatment affected renal AGT mRNA expression, confirming the liver-specificity of the siRNA. Yet, siRNA with or without valsartan, and valsartan + captopril, but not valsartan alone, or captopril alone, reduced renal angiotensin I and II. All treatments lowered albuminuria and proteinuria, while only siRNA with or without valsartan improved glomerulosclerosis and podocyte dysfunction. Multiple linear regression confirmed both mean arterial pressure and renal angiotensin II as independent determinants of albuminuria. Conclusions: In conclusion, AGT siRNA exerts renoprotection in diabetic TGR(mREN2)27 rats, and this relies, at least in part, on the suppression of renal angiotensin II formation from liver-derived AGT. Consequently, AGT siRNA may prove beneficial in human diabetic kidney disease.

Dissecting Meta-Analysis in GWAS Era: Bayesian Framework for Gene/Subnetwork-Specific Meta-Analysis.

Over the past decades, advanced high-throughput technologies have continuously contributed to genome-wide association studies (GWASs). GWAS meta-analysis has been increasingly adopted, has cross-ancestry replicability, and has power to illuminate the genetic architecture of complex traits, informing about the reliability of estimation effects and their variability across human ancestries. However, detecting genetic variants that have low disease risk still poses a challenge. Designing a meta-analysis approach that combines the effect of various SNPs within genes or genes within pathways from multiple independent population GWASs may be helpful in identifying associations with small effect sizes and increasing the association power. Here, we proposed ancMETA, a Bayesian graph-based framework, to perform the gene/pathway-specific meta-analysis by combining the effect size of multiple SNPs within genes, and genes within subnetwork/pathways across multiple independent population GWASs to deconvolute the interactions between genes underlying the pathogenesis of complex diseases across human populations. We assessed the proposed framework on simulated datasets, and the results show that the proposed model holds promise for increasing statistical power for meta-analysis of genetic variants underlying the pathogenesis of complex diseases. To illustrate the proposed meta-analysis framework, we leverage seven different European bipolar disorder (BD) cohorts, and we identify variants in the angiotensinogen (AGT) gene to be significantly associated with BD across all 7 studies. We detect a commonly significant BD-specific subnetwork with the ESR1 gene as the main hub of a subnetwork, associated with neurotrophin signaling (p = 4e −14) and myometrial relaxation and contraction (p = 3e −08) pathways. ancMETA provides a new contribution to post-GWAS methodologies and holds promise for comprehensively examining interactions between genes underlying the pathogenesis of genetic diseases and also underlying ethnic differences.

Estrogen Metabolite 2-Methoxyestradiol Attenuates Blood Pressure in Hypertensive Rats by Downregulating Angiotensin Type 1 Receptor.

The therapeutic potential of 2-Methoxyestradiol (2ME2) is evident in cardiovascular disease. Our laboratory has previously demonstrated the mechanism involved in the 2ME2 regulation of angiotensin type 1 receptor (AT1R) in vitro. However, 2ME2 regulation of angiotensin receptors and its effects on blood pressure (BP) and resting heart rate (RHR) are uncertain. In this study, male and female Wistar-Kyoto (WKY) rats infused with angiotensin II (65 ng/min) and male spontaneously hypertensive rats (SHR) were surgically implanted with telemetric probes to continuously assess arterial BP and RHR. In both male and female WKY rats, 2ME2 treatment (20 mg/kg/day for 2 weeks) resulted in a significant reduction of Ang II-induced systolic, diastolic, and mean arterial BP. Moreover, significant weight loss and RHR were indicated in all groups. In a separate set of experiments, prolonged 2ME2 exposure in male SHR (20 mg/kg/day for 5 weeks) displayed a significant reduction in diastolic and mean arterial BP along with RHR. We also found downregulation of angiotensin receptors and angiotensinogen (AGT) in the kidney and liver and a reduction of plasma Ang II levels. Collectively, we demonstrate that 2ME2 attenuated BP and RHR in hypertensive rats involves downregulation of angiotensin receptors and body weight loss.