AGT Levels Research Articles

Unveiling the Power of Mitochondrial Fission and Fusion: A Five-Gene Signature for Personalized Prognosis in Gastric Cancer.

Mitochondrial fission and fusion play important roles in tumorigenesis, progression and therapy. Dysregulation of these processes may lead to tumor progression, and regulation of these processes may provide novel strategies for cancer therapy. The involvement of genes related to mitochondrial fission and fusion (MD) in gastric cancer (GC) remains poorly understood. The aim of this study was to establish an MD gene signature for GC patients and to investigate its association with prognosis, tumor microenvironment and treatment response in GC. We use the TCGA-GC database as the cohort, focusing specifically on genes associated with MD. We conducted identification and consistency clustering analysis of differentially expressed genes in MD, conducted MD gene mutation and copy number variation analysis, as well as correlation and functional enrichment analysis between MD gene cluster classification and immune infiltration. TCGA-GC and GSE15459 were used to construct training and validation cohorts for the model. We used various statistical methods, including Cox and Lasso regression, to develop the model. We validated the model using bulk transcriptome and single- cell transcriptome datasets (GSE13861, GSE26901, GSE66229, and GSE13450). We used GSEA enrichment, CIBERSORT algorithm, ESTIMATE, and TIDE to gain insight into the annotation of MD signature and the characterization of the tumor microenvironment. OncoPredict was used to analyze the relationship between the PRG signature and the drug sensitivity. We validated the expression of several key genes in MD signature on GC cell lines using quantitative real-time PCR (qRT-PCR). These MDs-related subtypes exhibited different prognosis and immune filtration patterns. A five-gene signature, comprising AGT, HCFC1, KIFC3, NOX4, and RIN1, was developed. There was a clear distinction in overall survival between low- and high-risk patients. The analyses showed further confirmation of the independent prognostic value of the gene signature. There was a notable correlation between the MD signature, immune infiltration and drug susceptibility. The expression levels of AGT, HCFC1, KIFC3, NOX4 and RIN1 mRNA were all increased in these GC cells. The MD signature has the capacity to significantly contribute to the prediction of personalized outcomes and the advancement of novel therapeutic strategies tailored for GC patients.

Association of Novel Urinary Protein Biomarkers with Persistent Albuminuria in Patients with Sickle Cell Disease

Introduction: Standard assessments for kidney disease, albuminuria and decline in estimated glomerular filtration rate (eGFR), occur well after substantial functional and structural damage have ensued. Chronic kidney disease (CKD) is common in patients with sickle cell disease (SCD). With its association with increased morbidity and mortality, identification of novel biomarkers that could predict early development of CKD offers opportunities to modify risk factors in hopes of attenuating kidney function loss and decreasing mortality risk. Methods: The participants of this exploratory study represent a subset of patients with HbSS or HbSβ0 thalassemia enrolled in a prospective cohort study to assess the natural history of kidney disease in SCD. We excluded patients with diabetic nephropathy, cancer, connective tissue disease, other glomerular diseases, known infection with hepatitis (B or C) or HIV, end-stage kidney disease on dialysis and those who had undergone bone marrow transplantation. Participants were enrolled during routine clinic visits. Urine levels of clusterin [CLU; also referred to as apolipoprotein J], retinol-binding protein 4 [RBP4], alpha-1-microglobulin [A1M], interleukin-18 [IL-18], beta-2 microglobulin [B1M], insulin-like growth factor-binding protein 7 [IGFBP7], tissue inhibitor of metalloproteinases-2 [TIMP2], angiotensinogen [AGT], podocalyxin, and cysteine-rich with EGF-like domains 2 [CRELD2] were measured using quantitative ELISA assay. All values of urine biomarkers were normalized to urine creatinine. Persistent albuminuria (PA) was defined based on presence of at least 2 of 3 spot urine albumin/creatinine ratio (ACR) values ≥30 mg/g of creatinine or a single value ≥100 mg/g. Glomerular filtration rate was estimated using the 2009 creatinine-based Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula. Medians are presented with the interquartile range [IQR]. Associations between ACR and eGFR with each urine biomarker were assessed using linear regression. Levels of urinary biomarkers were compared using the Wilcoxon-Mann-Whitney test. Results: Thirty-seven subjects, 13 (35.2%) with PA (ACR - 147mg/g [IQR: 136.4, 223]), and 24 (64.9%) without PA (non-PA) (ACR - 12 mg/g [IQR: [6, 20]) were evaluated. Subjects with PA were older, had higher blood urea nitrogen (BUN), blood aspartate transaminase, and lower eGFR (Table 1). Urinary levels of CLU, RBP4, A1M, and AGT normalized to urine creatinine were significantly higher in subjects with PA vs. those with non-PA (Table 2). In univariate analysis, significant associations were identified between A1M and ACR (p=0.035) as well as between AGT and ACR (p<0.0001) (Table 2). In multivariable analyses, only AGT remained significantly associated with ACR (p=0.0003). Conclusion: The higher urinary levels of RBP4 and A1M, both low molecular weight proteins freely filtered in the glomerulus and reabsorbed by kidney proximal tubule cells, in subjects with PA suggests the presence of tubular injury. Higher levels of clusterin, a ubiquitously expressed glycoprotein that is rapidly upregulated upon acute kidney damage and present in de-differentiated kidney tubule cells, also indicates tubular injury in subjects with PA. Urinary AGT, the only precursor of all angiotensin peptides, is independently associated with ACR in this cohort. Higher levels of AGT indicates increased intrarenal renin-angiotensin system activity in subjects with PA. More studies are required to evaluate the association of these biomarkers with persistent albuminuria in SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Augmented Urinary Excretion of Angiotensinogen Precedes Albuminuria in Human Young Adults with Overweight and Mice with High Fat Diet‐Induced Type 2 Diabetes

Obesity is linked to the development of hypertension and type 2 diabetes (T2D) and both causing chronic kidney disease (CKD). Increased body mass index (BMI) and high fat diet (HFD) contribute to activation systemic renin angiotensin system (RAS). In addition, activation of intrarenal and intratubular RAS has been associated with increased pressure (BP), CKD, and augmentation of urinary excretion of angiotensinogen (uAGT) during T2D. However, whether augmentation of uAGT reflect intrarenal/intratububular RAS activation in subjects with overweight, remains unclear. Here, we tested the hypothesis that increases in uAGT precedes renal dysfunction in mice and human subjects with overweight,MethodsC57BL/6 mice (N = 10) were fed either a normal fat diet (NFD) or HFD (45% Kcal from fat) for 28 weeks. During this period, metabolic phenotype along with urinary albumin, creatinine, reactive oxygen species (ROS) and AGT, were measured every 4 weeks. In addition, for evidence in humans (age 19‐22 years‐old), 11 individuals with overweight and 17 individuals with normal weight, were studied. In these subjects, anthropometric parameters, systolic and diastolic BP, fasting blood glucose (FBG), and urinary mRNA levels of AGT and kidney injury markers IL‐18 and connecting tissue growth factor, were determined. Patients with history of cardiac or cerebrovascular events and endocrine diseases, anti‐hypertensive treatment (ACE inhibitors, ARBs) and/or anti‐diabetic drugs, were excludedResultsIn mice with metabolic alterations overtime, systolic BP significantly increased after 18 weeks in HFD but not NFD mice. Intrarenal ROS increased in HFD mice. These changes paralleled the augmentation uAGT excretion, which occurred in the absence of overt albuminuria. In human subjects with overweight, systolic and diastolic BP, urinary transcripts levels of AGT, renin, and injury markers IL‐18 and CTGF were increased as compared to normal weight individuals. However, no significant changes were observed in proteinuria or albuminuria.ConclusionsOur results indicate that augmentation of uAGT precedes albuminuria in young human adults with overweight and mice during the development of HFD‐induced T2D. These findings emphasize the relevance of uAGT and indicator of intrarenal/intratubular RAS activations and its potential as early biomarker of kidney injury during metabolic diseases as obesity and diabetes.

Salt-contaminated water inducing pulmonary hypertension and kidney damage by increasing Ang II concentration in broilers.

NaCl is the main component of freshwater salinization. High NaCl concentration in drinking water can cause pulmonary hypertension syndrome (PHS) and kidney damage in broilers. To explore the effect of NaCl in drinking water on broilers' kidneys, this study divided 80 chickens into four groups. With the control group fed with pure water, broiler chickens were fed with fresh water (FW, NaCl 1 g/L), low salt-contaminated water (L-SCW, NaCl 2.5 g/L), and high salt-contaminated water (H-SCW, NaCl 5 g/L). The results show that ascites heart index (AHI) and hematocrit (HCT) of broilers increase in L-SCW and H-SCW, the serum blood urea nitrogen and creatinine of broilers increase significantly, the kidney index increases, the kidney sections show vacuolar degeneration and fibrotic degeneration, and the TUNEL results show that the kidneys possess obvious apoptosis. In addition, the detection of RAAS-related genes (AGT gene in the liver, REN in the kidney, ACE in the lung) demonstrates that after using salt-contaminated water, the transcription levels of AGT, REN, and ACE rise significantly, and the concentration of angiotensin II (Ang II) also increases significantly. In order to verify the effect of Ang II on broiler kidneys, this research used exogenous Ang II to treat chicken embryonic kidney (CEK) cells. The results show that the cell activity of CEK decreased with the increase of the concentration of exogenous Ang II. Meanwhile, the flow cytometry assay shows that Ang II could promote the apoptosis of CEK cells. These results indicate that the salt-contaminated water can aggravate PHS and cause kidney damage. The mechanism may be related to the increase of Ang II.

Effect of salt intake on residual renal function in rats receiving peritoneal dialysis

To assess the effect of salt intake on residual renal function in rats and explore the possible mechanism. SD rats were 5/6-nephrectomized to induce chronic renal failure followed by peritoneal dialysis for 4 weeks (n=18) or without dialysis treatment (control group; n=18). In both groups, the rats were divided into 3 subgroups and were given lowsalt diet (0.02% NaCl), normal salt diet (0.4% NaCl), and high-salt diet (4% NaCl). After 8 and 12weeks, blood pressure and creatinine and sodium levels in the blood, urine, and peritoneal dialysate of the rats were examined. Glomerular sclerosis, tubulointerstitial fibrosis, and protein expression levels of RAS components (ACE-1, AGT, and AT-1) in renal cortical tissue of the rats were evaluated. The residual renal function of the rats all decreased especially in rats with high salt intake for 8and 12 weeks. In peritoneal dialysis group, the rats with high-salt diet showed signficiantly increased renal interstitial fibrosis score (P=0.036), glomerular sclerosis index (P=0.045), systolic blood pressure (P=0.004), diastolic blood pressure (P=0.048), and renal expressions of AGT, ACE-1, and AT1 (P < 0.05) as compared with those with normal salt intake. In the rats fed the same high-salt diet, the renal interstitial fibrosis score, glomerular sclerosis index, diastolic blood pressure increase, and renal AGT and ACE-1 expression levels were significantly lower in the peritoneal dialysis group than in the control group (P < 0.05). A positive correlation was noted between the reduction of residual renal function and sodium intake in the rats. In rats with chronic renal failure, high salt intake promotes the activation of the renal RAS system, increases blood pressure, and agrevates renal fibrosis to accelerate the decline of residual renal function, and peritoneal dialysis partially reduces the damage of residual renal function induced by high-salt diets by removing excessive sodium.

Pro-Fibrotic Phenotype in a Patient with Segmental Stiff Skin Syndrome via TGF-β Signaling Overactivation

Transforming growth factor β (TGF-β) superfamily signaling pathways are ubiquitous and essential for several cellular and physiological processes. The overexpression of TGF-β results in excessive fibrosis in multiple human disorders. Among them, stiff skin syndrome (SSS) is an ultrarare and untreatable condition characterized by the progressive thickening and hardening of the dermis, and acquired joint limitations. SSS is distinct in a widespread form, caused by recurrent germline variants of FBN1 encoding a key molecule of the TGF-β signaling, and a segmental form with unknown molecular basis. Here, we report a 12-year-old female with segmental SSS, affecting the right upper limb with acquired thickening of the dermis evident at the magnetic resonance imaging, and progressive limitation of the elbow and shoulder. To better explore the molecular and cellular mechanisms that drive segmental SSS, several functional studies on patient’s fibroblasts were employed. We hypothesized an impairment of TGF-β signaling and, consequently, a dysregulation of the associated downstream signaling. Lesional fibroblast studies showed a higher phosphorylation level of extracellular signal-regulated kinase 1/2 (ERK1/2), increased levels of nuclear factor-kB (NFkB), and a nuclear accumulation of phosphorylated Smad2 via Western blot and microscopy analyses. Quantitative PCR expression analysis of genes encoding key extracellular matrix proteins revealed increased levels of COL1A1, COL3A1, AGT, LTBP and ITGB1, while zymography assay reported a reduced metalloproteinase 2 enzymatic activity. In vitro exposure of patient’s fibroblasts to losartan led to the partial restoration of normal transforming growth factor β (TGF-β) marker protein levels. Taken together, these data demonstrate that in our patient, segmental SSS is characterized by the overactivation of multiple TGF-β signaling pathways, which likely results in altered extracellular matrix composition and fibroblast homeostasis. Our results for the first time reported that aberrant TGF-β signaling may drive the pathogenesis of segmental SSS and might open the way to novel therapeutic approaches.

IgE-Mediated Systemic Anaphylaxis And Its Association With Gene Polymorphisms Of ACE, Angiotensinogen And Chymase.

BackgroundThe renin-angiotensin system (RAS) protects the circulation against sudden falls in systemic blood pressure via generation of angiotensin II (AII). Previously, we demonstrated that patients with anaphylaxis involving airway angioedema and cardiovascular collapse (AACVS) had significantly increased “I” gene polymorphisms of the angiotensin-converting-enzymes (ACE). This is associated with lower serum ACE and AII levels and was not seen in anaphylaxis without collapse nor atopics and healthy controls.ObjectivesTo examine the angiotensinogen (AGT-M235T) and chymase gene (CMA-1 A1903G) polymorphisms in these original subjects.Method122 patients with IgE-mediated anaphylaxis, 119 healthy controls and 52 atopics had polymorphisms of the AGT gene and chymase gene examined by polymerase chain reactions and gel electrophoresis. Their previous ACE genotypes were included for the analysis.ResultsAGT-MM genes (associated with low AGT levels) were significantly increased in anaphylaxis (Terr’s classification). When combined with ACE, anaphylaxis showed increased MM/II gene pairing (p<0.0013) consistent with lower RAS activity. For chymase, there was increased pairing of MM/AG (p<0.005) and AG/II and AG/ID (p<0.0073) for anaphylaxis consistent with lower RAS activity. A tri-allelic ensemble of the 6 commonest gene combinations for the healthy controls and anaphylaxis confirmed this difference (p=0.0001); for anaphylaxis, genes were predominately MM/AG/II or ID, while healthy controls were DD/MT/AG or GG patterns.ConclusionOur gene polymorphisms show lower RAS activity for anaphylaxis especially AACVS. Animal models of anaphylaxis are focused on endothelial nitric oxide (eNO) which is shown to be the mediator of fatal shock and prevented by eNO-blockade. The interaction of AII and eNO controls the microcirculation in man. High serum AII levels reduce eNO activity, so higher RAS-activity could protect against shock. Our data shows low RAS activity in anaphylaxis especially AACVS, suggesting the influence of these genes on shock are via AII levels and its effects on eNO.

A Genome-Wide Association Study of Sprint Performance in Elite Youth Football Players

Pickering, C, Suraci, B, Semenova, EA, Boulygina, EA, Kostryukova, ES, Kulemin, NA, Borisov, OV, Khabibova, SA, Larin, AK, Pavlenko, AV, Lyubaeva, EV, Popov, DV, Lysenko, EA, Vepkhvadze, TF, Lednev, EM, Leońska-Duniec, A, Pająk, B, Chycki, J, Moska, W, Lulińska-Kuklik, E, Dornowski, M, Maszczyk, A, Bradley, B, Kana-ah, A, Cięszczyk, P, Generozov, EV, and Ahmetov, II. A genome-wide association study of sprint performance in elite youth football players. J Strength Cond Res 33(9): 2344-2351, 2019-Sprint speed is an important component of football performance, with teams often placing a high value on sprint and acceleration ability. The aim of this study was to undertake the first genome-wide association study to identify genetic variants associated with sprint test performance in elite youth football players and to further validate the obtained results in additional studies. Using micro-array data (600 K-1.14 M single nucleotide polymorphisms [SNPs]) of 1,206 subjects, we identified 12 SNPs with suggestive significance after passing replication criteria. The polymorphism rs55743914 located in the PTPRK gene was found as the most significant for 5-m sprint test (p = 7.7 × 10). Seven of the discovered SNPs were also associated with sprint test performance in a cohort of 126 Polish women, and 4 were associated with power athlete status in a cohort of 399 elite Russian athletes. Six SNPs were associated with muscle fiber type in a cohort of 96 Russian subjects. We also examined genotype distributions and possible associations for 16 SNPs previously linked with sprint performance. Four SNPs (AGT rs699, HSD17B14 rs7247312, IGF2 rs680, and IL6 rs1800795) were associated with sprint test performance in this cohort. In addition, the G alleles of 2 SNPs in ADRB2 (rs1042713 & rs1042714) were significantly over-represented in these players compared with British and European controls. These results suggest that there is a genetic influence on sprint test performance in footballers, and identifies some of the genetic variants that help explain this influence.

A new model of the mechanism underlying lead poisoning: SNP in miRNA target region influence the AGT expression level

BackgroundTo determine if the rs7079 polymorphism located in the 3′ UTR of the angiotensinogen gene (AGT) altered AGT gene expression and the risk of lead poisoning. A case-control study and luciferase reporter gene assay identified a significant association between rs7079 variants and the risk of lead poisoning.ResultsSerum AGT levels were significantly higher in individuals carrying the rs7079 CA genotype, as compared to those carrying the rs7079 CC genotype. The binding of the miRNA mimics miR-31-5p and miR-584-5p to the 3′ UTR of AGT differed based on which rs7079 variant was present, implying that AGT gene expression depends on the rs7079 variant carried.ConclusionsThe rs7079 C to A substitution reduced the binding of miR-31-5p/miR-584-5p to the 3′ UTR of AGT, possibly altering the risk of lead poisoning.

Beraprost Sodium, a Stable Analogue of PGI2, Inhibits the Renin-Angiotensin System in the Renal Tissues of Rats with Chronic Renal Failure

Chronic renal failure (CRF) is a prolonged kidney condition characterized by decreased kidney function that can eventually develop into total kidney failure. The renin-angiotensin system (RAS) helps to regulate the balance between human bodily fluids and electrolytes. The aim of the present study was to investigate the effects of a prostacyclin analogue (beraprost sodium [BPS]) on the expression of key factors associated with local RAS activities in the renal tissues of rats with CRF. After a CRF rat model was successfully established, the levels of BUN, SCr, phosphorus, and calcium were detected by an automatic biochemistry analyzer. Furthermore, the activities of malondialdehyde (MDA) and superoxide dismutase (SOD) in rat renal tissues were measured using a colorimetric method, while the activity of angiotensin-converting enzyme (ACE) was determined by ultraviolet (UV) spectrophotometry. In situ hybridization was employed to determine the expression of angiotensin II type 1 receptor (AT). Finally, the positive expression rates of cells expressing important apoptotic proteins (Bax and Bcl-2) were determined, and the protein and mRNA levels of phosphatidylinositol 3-kinase (AKT) and key factors involved in the RAS (AT1, AT2, angiotensin ACE and angiotensinogen [AGT]) were evaluated by RT-qPCR and western blot analysis. Initial observations revealed that treatment with BPS decreased the levels of BUN, SCr and phosphorus but increased calcium levels in the renal tissues of CRF rats. Additionally, BPS reduced the levels of MDA while increasing the levels of SOD, ACE activity, and AT1 expression in the renal tissues of CRF rats. BPS inhibited glomerular hypertension and hyperfiltration; increased the mRNA and protein levels of AKT and AT2; and decreased the mRNA and protein levels of AT1, AGT, and ACE in the renal tissues of CRF rats. The results of this study demonstrate that BPS, a PGI2 analogue, inhibits the expression of key factors involved in the local RAS, resulting in a delay in the occurrence and development of CRF. The key findings of the present study ultimately highlight the potential of this PGI2 analogue as a promising therapeutic strategy for treating CRF.

Attenuation of accelerated renal cystogenesis in Pkd1 mice by renin-angiotensin system blockade.

The intrarenal renin angiotensin system (RAS) is activated in polycystic kidney disease. We have recently shown in the Pkd1 mouse that Gen 2 antisense oligonucleotide (ASO), which suppresses angiotensinogen (Agt) synthesis, is efficacious in slowing kidney cyst formation compared with lisinopril. The aim of this current study was to determine 1) if unilateral nephrectomy accelerates cystogenesis in Pkd1 mice (as previously shown in cilia knockout mice) and 2) whether Agt ASO can slow the progression in this accelerated cystic mouse model. Adult Pkd1 conditional floxed allele mice expressing cre were administered tamoxifen, resulting in global knockout of Pkd1. Three weeks after tamoxifen injection, mice underwent left unilateral nephrectomy. Mice were then treated with Agt ASO (75 mg/kg per week) or aliskiren (20 mg/kg per day)+Agt ASO or control for 8 wk. Unilateral nephrectomy accelerated kidney cyst formation compared with nonnephrectomized mice. Both Agt ASO and Aliskiren+Agt ASO treatments significantly reduced plasma and urinary Agt levels. Blood pressure was lowest in Aliskiren+Agt ASO mice among all treatment groups, and the control group had the highest blood pressure. All mice developed significant kidney cysts at 8 wk after nephrectomy, but Agt ASO and Aliskiren+Agt ASO groups had fewer kidney cysts than controls. Renal pAkt, pS6 levels, and apoptosis were significantly suppressed in those receiving Agt ASO compared with controls. These results indicate that suppressing Agt using an ASO slowed the progression of accelerated cystic kidney disease induced by unilateral nephrectomy in Pkd1 mice by suppressing intrarenal RAS, mammalian target of rapamycin pathway, and cell proliferation.

Foxd1 is an upstream regulator of the renin-angiotensin system during metanephric kidney development.

BackgroundWe tested the hypothesis that Foxd1, a transcription factor essential for normal kidney development, is an upstream regulator of the renin-angiotensin system (RAS) during ureteric bud (UB)-branching morphogenesis.MethodsUB branching, RAS gene, and protein expression were studied in embryonic mouse kidneys. RAS mRNA expression was studied in mesenchymal MK4 cells.ResultsThe number of UB tips was reduced in Foxd1-/- compared with that in Foxd1+/+ metanephroi on embryonic day E12.5 (14±2.1 vs. 28±1.3, P<0.05). Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) demonstrated that renin, angiotensin I-converting enzyme (ACE), and angiotensin (Ang) II receptor type 1 (AT1R) mRNA levels were decreased in Foxd1-/- compared with those in Foxd1+/+ E14.5 metanephroi. Western blot analysis and immunohistochemistry showed decreased expression of AGT and renin proteins in Foxd1-/- metanephroi compared with that in Foxd1+/+ metanephroi. Foxd1 overexpression in mesenchymal MK4 cells in vitro increased renin, AGT, ACE, and AT1R mRNA levels. Exogenous Ang II stimulated UB branching equally in whole intact E12.5 Foxd1-/- and Foxd1+/+ metanephroi grown ex vivo (+364±21% vs. +336±18%, P=0.42).ConclusionWe conclude that Foxd1 is an upstream positive regulator of RAS during early metanephric development and propose that the cross-talk between Foxd1 and RAS is essential in UB-branching morphogenesis.

RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM GENES POLYMORPHISM AND RELATION WITH VASOPRESSORS IN ESSENTIAL HYPERTENSION AMONG DAGESTAN INHABITANTS

Aim. Investigation on the prevalence of genotypes polymorphisms of reninangiotensin- aldosterone system and beta2-adrenoreceptors in essential hypertension (EH) of I, II, III grades inDagestan population, and evaluation of the relation of genotype expression with vasopressors levels in blood. Material and methods. Totally, 98 patients included, with the diagnosis “essential hypertension of I, II, III grades”. The genotypes were studied, of polymorphisms Тhr174Мet and Мet235Тhr gene AGT , polymorphism A1166C gene AGTR1 and polymorphisms Arg16Gly and Gln27Glu gene ADRB2 . Test of the mentioned polymorphisms was done with the allele-specific polymerase chain reaction, making to assess the absence or presence of a mutation allele in hetero- and homozygous form. The level of angiotensin (AT) II, endothelin (ET) 1-21 and aldosterone (AS) in serum was measured with the hard-phase immune-enzyme assay. The level of angiotensin-converting enzyme (ACE) was measured with enzymatic method. Statistics was done with the software Statistica v. 6.0 and “Biostat4.03”. Results. EH of I, II, III grades in Dagestan population was associated with significant increase of the prevalences (criteria χ2) of genotypes АА and СС of polymorphism A1166C gene AGTR1 , genotype Arg/Gly of polymorphism Arg16Gly gene ADRB2 , genotype Gln/Gln of polymorphism Gln27Glu gene ADRB2 and genotype ММ of polymorphism Thr174Met gene AGT . In the carriers of the genotypes mentioned, relative risk of EH onset was significantly higher than in controls. The level of vasopressors ATII, ET 1-21, AS and ACE showed a controversial dynamics in Dagestan population. Serum levels of ET 1-21 and ACE were significantly higher or had tendency to increase, but AT II and AS decreased in both cases, comparing to the controls (р<0,05, р<0,01). Most number of significant direct correlations was found in polymorphisms of the genes AGT , AGTR1 and ADRB2 and level of serum ET 1-21. Conclusion. Significant increase of the prevalence of the studied genotypes polymorphisms of genes AGT, AGTR1 and ADRB2 in EH of I, II III grades represents a definite specifics of mutational process involving the mentioned genes inDagestan population. These processes are associated with significant changes in vasopressors levels in serum, that is a witness for pathogenetic relationship of the mentioned polymorphisms and pressoric contour of EH inDagestan population.

Adipocytes do not significantly contribute to plasma angiotensinogen.

Recently, it has been reported that 25% of plasma angiotensinogen (Agt) is derived from fat. Meanwhile, liver-specific Agt knockout (KO) mice have markedly low plasma Agt, which may be due to reduced fat mass. To study the contribution of the fat to plasma Agt, we tested whether increasing fat mass can elevate plasma Agt and blood pressure in liver-Agt KO mice. Epididymal fat mass in liver-Agt KO mice fed a high-fat diet (HFD) was 4.1-fold larger than that in liver-Agt KO mice on a normal-fat diet (NFD). The liver-Agt KO mice on NFD were hypotensive with low levels of plasma Agt (on average, 0.11 vs 2.38 μg/ml). HFD slightly increased plasma Agt (0.17 μg/ml) without increase in blood pressure. To further increase fat mass, liver-Agt KO mice were fed HFD and simultaneously supplemented with low-dose angiotensin II and compared with control mice. Fat mass was comparable between the two groups. However, liver-Agt KO mice had uniformly low plasma Agt (0.09 vs 2.07 μg/ml) and systolic blood pressure (78±12 vs 111±6 mm Hg). In conclusion, adipocyte-derived Agt has essentially no contribution to the plasma concentration and no impact on blood pressure compared to liver-derived Agt.

Abstract P280: Novel Insights into Transcriptional Regulation of the Human Angiotensinogen Gene by High-salt: A Study in Transgenic Mice

Angiotensinogen is the substrate for the entire RAS cascade and polymorphisms leading to its overexpression are linked to hypertension. SNPs in the promoter of the hAGT gene are associated with hypertension. Importantly, these SNPs can further modulate the gene of interest in various physiological/environmental settings like the high-sodium diet. In this regard, the human angiotensinogen (hAGT) gene has polymorphisms in its 2.5Kb promoter that form two haplotype (Hap) blocks: -6A/G (-1670A/G, -1562C/T, -1561T/C) and -217A/G (-532T/C, -793A/G, -1074T/C, and -1178G/A). Hap -6A/-217A is associated with human hypertension whereas Hap -6G/-217G reduces cardiovascular risk. We have engineered transgenic (TG) mice with these haplotypes (Hap -6A: -6A/-217A and Hap -6G: -6G/-217G) so as to examine the transcriptional regulation of the hAGT in an in vivo setting. This study is designed to study the effects of a high-sodium diet on the transcriptional milieu of renal tissues with consequential effects on the hAGT expression in our two haplotypes. Male TG mice were placed on 4% Na + diet for a period of 8 weeks. High-salt diet induces mineralocorticoid receptor (MR) and SGK-kinase expression in both haplotypes, equally. MR has been shown to bind to GRE elements in the hAGT gene. Importantly, MR-binding (ChIP assay) and hAGT induction are significantly (p&amp;lt;0.05) greater in the -6A haplotype males as compared to -6G males. High-salt also increased the expression of transcriptional regulators including CEBPβ and HNF4 (p&amp;lt;0.05) that are independent of haplotype. Complementary ChIP assay confirmed enhanced transcription factor (TF) binding to the chromatin of male -6A TG mice as compared to their -6G counterparts after high-salt diet treatment. Thus, we show here an effect of high-salt on cellular transcriptional apparatus that is haplotype-independent. However, increased TF affinity of the chromatin in -6A TG mice leads to higher salt-induced AGT levels in this haplotype than -6G. These observations could partly account for increased salt-sensitivity of some adult males that, in turn, is governed by the “risk” haplotype. Identifying these individuals with the -6A haplotype will help guide therapeutic lifestyle changes in patients with essential hypertension.

Abstract P198: Increased Urinary Angiotensinogen Reflects Intrarenal Renin-angiotensin System Activation in High Fat Diet Induced-type 2diabetic Mice

Obesity is a risk factor for diabetes mellitus (DM) and patients with DM are often hypertensive and exhibit inappropriate activation of the intrarenal renin-angiotensin system (RAS), which increases the risk for diabetic nephropathy and progression to chronic kidney disease. Enhanced intrarenal AGT levels contribute to increased intrarenal Angiotensin II levels. To examine the role of an activated RAS in DM, we measured urinary AGT along with albuminuria and other metabolic parameters in high fat diet induced-type 2 DM mice. Immediately after weaning, male C57BL/6J mice (N=18) were fed either a normal fat diet (18 % Kcal from fat; CT mice) or high fat diet (42.0 % Kcal from fat; HF mice) for 24 wk. Body weight (BW), food intake, and systolic blood pressure (SBP) by telemetry, were measured weekly. Glucose tolerance test (GTT) and homeostasis model assessment index of insulin resistance (HOMA-IR) were further assayed. At wks 0, 12, and 24 on dietary regimen, measurements of albuminuria and uAGT by ELISA were used as markers of renal injury and intrarenal RAS activation, respectively. Body weight increased by 140% in HF mice compared to 66 % in CT mice (from 18±0.5 to 44±1.4 g and 17±0.6 to 28±0.6 g, respectively) with no differences in daily energy intake (15.02±1.3 kcal/day and 15.4±0.8 kcal/day). Plasma glucose, area under curve (AUC) of GTT, and HOMA-IR were significantly increased in HF mice (121± 6mg/dL; 32,865±6,222; 5±1) compared to CT mice (220± 27mg/dL; 55,549±4,611; 54±9). In HF mice, SBP was augmented by wk 10 compared to CT mice (126±5 vs. 113±1 mmHg; P&lt;0.05). Interestingly, no differences in albuminuria were found between HF and CT mice either at wk 12(50±13 vs. 40±6 ug/day) or at wk 24 (57±7 vs. 43±3 ug/day). However, uAGT excretion was increased by wk 24 in HF mice but not in CT mice ( wk 0 : 4±1.5 vs. 4±1.5ng/day; wk 12 : 4.6±2.3 vs. 2.3±0.9 ng/day; wk 24 : 11.8±2.9 vs. 3.9±0.3 ng/day; P&lt;0.05). During HF diet induced-type 2 DM, the elevation of uAGT reflects the increase of intrarenal RAS which may contribute to the development of renal injury.

Influence of the Expression Level of O6-Alkylguanine-DNA Alkyltransferase on the Formation of DNA Interstrand Crosslinks Induced by Chloroethylnitrosoureas in Cells: A Quantitation Using High-Performance Liquid Chromatography-Mass Spectrometry.

Chloroethylnitrosoureas (CENUs), which are bifunctional alkylating agents widely used in the clinical treatment of cancer, exert anticancer activity by inducing crosslink within a guanine-cytosine DNA base pair. However, the formation of dG-dC crosslinks can be prevented by O6-alkylguanine-DNA alkyltransferase (AGT), ultimately leading to drug resistance. Therefore, the level of AGT expression is related to the formation of dG-dC crosslinks and the sensitivity of cells to CENUs. In this work, we determined the CENU-induced dG-dC crosslink in mouse L1210 leukemia cells and in human glioblastoma cells (SF-763, SF-767 and SF-126) containing different levels of AGT using high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. The results indicate that nimustine (ACNU) induced more dG-dC crosslinks in L1210 leukemia cells than those induced by carmustine (BCNU), lomustine (CCNU) and fotemustine (FTMS). This result was consistent with a previously reported cohort study, which demonstrated that ACNU had a better survival gain than BCNU, CCNU and FTMS for patients with high-grade glioma. Moreover, we compared the crosslinking levels and the cytotoxicity in SF-763, SF-767 and SF-126 cells with different AGT expression levels after exposure to ACNU. The levels of dG-dC crosslink in SF-126 cells (low AGT expression) were significantly higher than those in SF-767 (medium AGT expression) and SF-763 (high AGT expression) cells at each time point. Correspondingly, the cytotoxicity of SF-126 was the highest followed by SF-767 and SF-763. The results obtained in this work provided unequivocal evidence for drug resistance to CENUs induced by AGT-mediated repair of DNA ICLs. We postulate that the level of dG-dC crosslink has the potential to be employed as a biomarker for estimating drug resistance and anticancer efficiencies of novel CENU chemotherapies.

DETERMINING M235T POLYMORPHISM OF ANGIOTENSINOGEN GENE (AGT) IN NORMOTENSIVE AND PREECLAMPTIC/ECLAMPTIC PREGNANT WOMEN

Background: The 235T allele of AGT gene has been reported to be associated with higher AGT level and abnormal remodeling of the uterine spiral arteries, which is an early cause preeclampsia/eclampsia (PE/E). The aim of this study was to: (1) determine the frequencies of 235MM, 235MT and 235TT genotypes of AGT gene in normotensive and preeclamptic/eclamptic pregnant women, and (2) survey the association between AGT M235T polymorphism and PE/E. Patients and methods: 68 preeclamptic pregnant women and 272 normotensive pregnant women were determined M235T genotypes of AGT gene by allele – specific PCR technique with DNA samples extracted from whole blood. Results: The frequencies of 235MM, 235MT and 235TT genotypes in normotensive pregnant women were 1.8%; 15.8% and 82.4%, respectively, the distribution of these genotypes was in Hardy – Weinberg equilibrium. These frequencies in preeclamptic/eclamptic pregnant women were 1.5%; 14.7% and 83.8%, respectively. The frequencies of 235T allele in two groups were 90.3% and 91.2%, respectively. There was no association between the M235T polymorphism and PE/E in all of models, including 235TT/(235MT + 235MM), (235TT + 235MT)/235MM, 235TT/235MM and 235T allele/235M allele. Conclusion: The frequencies 235T allele in both groups were very high. There was no association between M235T polymorphism and preeclampsia/eclampsia. Keywords: Preeclampsia, M235T polymorphism, AGT gene

Effects of soil management in vineyard on soil physical and chemical characteristics

Cover crops in Mediterranean vineyards are scarcely used due to water competition between the cover crop and the grapevine; however, bare soil management through tillage or herbicides tends to have negative effects on the soil over time (organic matter decrease, soil structure and soil fertility degradation, compaction, etc). The objective of this study was to understand how soil management affects soil fertility, compaction and infiltration over time. To this end, two bare soil techniques were compared, tillage (TT) and total herbicide (HT) with two cover crops; annual cereal (CT) and annual grass (AGT), established for 8 years. CT treatment showed the highest organic matter content, having the biggest amount of biomass incorporated into the soil. The annual adventitious vegetation in TT treatment (568 kg dry matter ha-1) that was incorporated into the soil, kept the organic matter content higher than HT levels and close to AGT level, in spite of the greater aboveground annual biomass production of this treatment (3632 kg dry matter ha-1) whereas only its roots were incorporated into the soil. TT presented the highest bulk density under the tractor track lines and a greatest resistance to penetration (at 0.2 m depth). AGT presented bulk density values (upper 0.4 m) lower than TT and penetration resistance in CT lower (at 0.20 m depth) than TT too. The HT decreased water infiltration due to a superficial crust generated for this treatment. These results indicate that the use of annual grass cover can be a good choice of soil management in Mediterranean climate due to soil quality improvement, with low competition and simple management.

Abstract 187: The Absence of Renal ACE Blunts the Renal Response and the Hypertension Induced by Nitric Oxide Synthesis Inhibition

We previously showed that the responses to angiotensin (Ang) II infusion observed in wild type (WT) mice were effectively prevented in mice lacking renal ACE (ACE 3/3 and ACE 10/10 mice) including: hypertension, renal Ang II accumulation, sodium and water retention, and activation of ion transporters in the loop of Henle (NKCC2) and distal nephron (NCC, ENaC, and pendrin). The aim of this study was to test if the absence of renal ACE also protects against hypertension induced by nitric oxide (NO) synthesis inhibition with L-NAME, a low-plasma Ang II hypertension model associated with renal renin-angiotensin system (RAS) activation. In response to L-NAME (0.5 mg/mL in drinking water, 4 wk; n=12-17), systolic blood pressure (SBP) of WT rose from 110±2 to 135±3 mmHg (p&lt;0.01) while SBP in mice lacking renal ACE did not significantly increase: in ACE 10/10, SBP went from 109±3 to 113±2 mmHg (NS), in ACE 3/3 mice from 103±5 to 110±5 mmHg (NS). ACE 10/10 mice exhibited an enhanced natriuretic/diuretic response vs. WT mice: UNaV during the first 24 h of L-NAME was 188±14 μmol in WT and 314±17 μmol in ACE 10/10 (p&lt;0.01). Ang II content [by immunohistochemistry (IHC)] increased from 8±2 to 28±4 % of positive staining/area (p&lt;0.001) in L-NAME-treated WT mice but remained unchanged in ACE 10/10 mice (from 3±1 to 5±1 % of positive staining/area, NS). Consistent with the dissimilar Ang II content, L-NAME increased renal AGT levels by 2.3±0.3 (p&lt;0.01) in WT but not in the ACE 10/10 mice, 0.8±0.1 fold (NS) vs. non-treated animals. The enhanced natriuresis of the L-NAME-treated ACE 10/10 mice was associated with lower NHE3 expression (0.5±0.1 AU) and lower phosphorylation (-P) of NKCC-P (0.48±0.05 AU) and NCC-P (0.5±0.1 AU) vs. equally treated WT (WT defined as 1, p&lt;0.01). In addition, L-NAME-treated WT mice had higher renal macrophage infiltration (82±8 F4/80+ cells/field, by IHC), higher TNF-α content (24±3 pg/mg, by ELISA) and IL-6 (36±2 pg/mg, by ELISA) vs. L-NAME-treated ACE 10/10 (51±3 F4/80+ cells/field, 14±1 pg/mg and 21±1 pg/mg respectively, p&lt;0.01). In conclusion, our results demonstrate that renal ACE plays a fundamental role driving renal responses, hypertension and inflammation induced by NO synthesis inhibition.