Human Renin Gene Research Articles

Metformin modulates microbiota and improves blood pressure and cardiac remodeling in a rat model of hypertension.

Metformin has been attributed to cardiovascular protection even in the absence of diabetes. Recent observations suggest that metformin influences the gut microbiome. We aimed to investigate the influence of metformin on the gut microbiota and hypertensive target organ damage in hypertensive rats. Male double transgenic rats overexpressing the human renin and angiotensinogen genes (dTGR), a model of angiotensin II-dependent hypertension, were treated with metformin (300 mg/kg/day) or vehicle from 4 to 7 weeks of age. We assessed gut microbiome composition and function using shotgun metagenomic sequencing and measured blood pressure via radiotelemetry. Cardiac and renal organ damage and inflammation were evaluated by echocardiography, histology, and flow cytometry. Metformin treatment increased the production of short-chain fatty acids (SCFA) acetate and propionate in feces without altering microbial composition and diversity. It significantly reduced systolic and diastolic blood pressure and improved cardiac function, as measured by end-diastolic volume, E/A, and stroke volume despite increased cardiac hypertrophy. Metformin reduced cardiac inflammation by lowering macrophage infiltration and shifting macrophage subpopulations towards a less inflammatory phenotype. The observed improvements in blood pressure, cardiac function, and inflammation correlated with fecal SCFA levels in dTGR. Invitro, acetate and propionate altered M1-like gene expression in macrophages, reinforcing anti-inflammatory effects. Metformin did not affect hypertensive renal damage or microvascular structure. Metformin modulated the gut microbiome, increased SCFA production, and ameliorated blood pressure and cardiac remodeling in dTGR. Our findings confirm the protective effects of metformin in the absence of diabetes, highlighting SCFA as a potential mediators.

Discovery of Novel 2-Carbamoyl Morpholine Derivatives as Highly Potent and Orally Active Direct Renin Inhibitors.

The renin-angiotensin-aldosterone system (RAAS) plays a key role in the regulation of blood pressure. Renin, the first and rate-limiting enzyme of the RAAS, is an attractive target for the treatment of hypertension and cardiovascular/renal diseases. Therefore, various direct renin inhibitors (DRIs) have been researched over recent decades; however, most exhibited poor pharmacokinetics and oral bioavailability due to the peptidomimetic or nonpeptidomimetic structures with a molecular weight (MW) of >600, and only aliskiren is approved. This study introduces a novel class of DRIs comprised of a 2-carbamoyl morpholine scaffold. These compounds have a nonpeptidomimetic structure and a MW of <500. The representative compound 26 was highly potent despite not occupying S1'-S2' sites or the opened flap region used by other DRIs and exerted a significant antihypertensive efficacy via oral administration on double transgenic mice carrying both the human angiotensinogen and the human renin genes.

Kidney Injury Caused by Preeclamptic Pregnancy Recovers Postpartum in a Transgenic Rat Model.

Preeclampsia (PE) is characterized by the onset of hypertension (≥140/90 mmHg) and presence of proteinuria (>300 mg/L/24 h urine) or other maternal organ dysfunctions. During human PE, renal injuries have been observed. Some studies suggest that women with PE diagnosis have an increased risk to develop renal diseases later in life. However, in human studies PE as a single cause of this development cannot be investigated. Here, we aimed to investigate the effect of PE on postpartum renal damage in an established transgenic PE rat model. Female rats harboring the human-angiotensinogen gene develop a preeclamptic phenotype after mating with male rats harboring the human-renin gene, but are normotensive before and after pregnancy. During pregnancy PE rats developed mild tubular and glomerular changes assessed by histologic analysis, increased gene expression of renal damage markers such as kidney injury marker 1 and connective-tissue growth factor, and albuminuria compared to female wild-type rats (WT). However, four weeks postpartum, most PE-related renal pathologies were absent, including albuminuria and elevated biomarker expression. Only mild enlargement of the glomerular tuft could be detected. Overall, the glomerular and tubular function were affected during pregnancy in the transgenic PE rat. However, almost all these pathologies observed during PE recovered postpartum.

Abstract P212: Kidney Injury Caused By Preeclamptic Pregnancy Improves Postpartum In A Transgenic Rat Model

Preeclamptic pregnancies involve mild renal injuries. However, there has been evidence that women with a history of preeclampsia (PE) have an increased risk to develop kidney disease in association to high blood pressure later in life. This study aims to characterize renal injury during pregnancy and postpartum in an established transgenic rat model for PE. Female Sprague-Dawley rats transgenic for the human angiotensinogen gene develop a PE phenotype in pregnancy, including cardiac remodeling, when mated with male rats harboring the human renin gene. Postpartum, blood pressure restores but cardiac remodeling persists. We hypothesize that PE during pregnancy mediates kidney injuries but does not fully restore after ending of the high blood pressure (postpartum). The renal alterations were analyzed by histological staining, gene expression and urine analysis. PE rats have elevated mean arterial blood pressure during pregnancy (PE d19 148.4 ± 20.7 mmHg) compared to normotensive values in control animals (WT d19 105.3 ± 4.6 mmHg). During PE increased expression of kidney injury marker 1 ( Kim-1/18S : PE d21 4.14 ± 3.26; WT d21 0.08 ± 0.02), neutrophil gelatinase associated lipocalin 2 ( Ngal/18S : PE d21 1.64 ± 0.83; WT d21 0.59 ± 0.28) and connective-tissue growth factor ( Ctgf/18S: PE d21 1.51 ± 0.46; WT d21 0.92 ± 0.32) were detected. Kidneys of PE rats showed mild glomerular (PAS-positive glomerular area PE d21 86.2 ± 4.4%; WT d21 79.1 ± 3.2%) and tubular changes during PE pregnancy resulting in albuminuria (albumin/creatinine ratio PE d19 2193.8 ± 1878.2; WT d19 290.4 ± 252.0). However, 4 weeks after pregnancy (approx. 2 years in humans) most of the PE related renal damages were absent including albuminuria and elevated expression of biomarkers ( Kim-1/18S : PE d50 0.09 ± 0.05; WT d50 0.23 ± 0.13; Ctgf/18S : PE d50 0.78 ± 0.25; WT d50 0.8 ± 0.25; Ngal/18S : PE d50 0.37 ± 0.17; WT 50 0.47 ± 0.11). Only mild enlargement of glomerular tuft area (PE d50 7523.8 ± 418.7 μm 2 ; WT d50 7058.4 ± 198.8 μm 2 ) was detected. Overall, the glomerular and tubular injuries are present during pregnancy in this transgenic PE rat. Most restore postpartum, speculating long-term kidney failure observed in humans is associated to hypertension and additional cardiovascular events.

Statins Reverse Postpartum Cardiovascular Dysfunction in a Rat Model of Preeclampsia.

Preeclampsia is associated with increased cardiovascular long-term risk; however, the underlying functional and structural mechanisms are unknown. We investigated maternal cardiac alterations after preeclampsia. Female rats harboring the human angiotensinogen gene [TGR(hAogen)L1623] develop a preeclamptic phenotype with hypertension and albuminuria during pregnancy when mated with male rats bearing the human renin gene [TGR(hRen)L10J] but behave physiologically normal before and after pregnancy. Furthermore, rats were treated with pravastatin. We tested the hypothesis that statins are a potential therapeutic intervention to reduce cardiovascular alterations due to simulated preeclamptic pregnancy. Although hypertension persists for only 8 days in pregnancy, former preeclampsia rats exhibit significant cardiac hypertrophy 28 days after pregnancy observed in both speckle tracking echocardiography and histological staining. In addition, fibrosis and capillary rarefaction was evident. Pravastatin treatment ameliorated the remodeling and improved cardiac output postpartum. Preeclamptic pregnancy induces irreversible structural changes of cardiac hypertrophy and fibrosis, which can be moderated by pravastatin treatment. This pathological cardiac remodeling might be involved in increased cardiovascular risk in later life.

In silico analysis of human renin gene-gene interactions and neighborhood topologically associated domains suggests breakdown of insulators contribute to ageing-associated diseases.

Three-dimensional chromatin architecture and gene-gene interactions impact gene expression. We assembled this information, in silico, for the human renin gene (REN). We searched for chromatin contacts and boundaries and the locations of super-enhancers that are involved in cell specific differentiation. The REN promoter was connected via RNA polymerase II binding to promoters of 12 neighboring genes on chromosome 1q32.1 over a distance of 762,497bp. This constitutes a regulatory archipelago. The genes formed 3 topologically associated domains (TADs), as follows: TAD1: ZC3H11A, SNRPE, LINC00303; SOX13; TAD2: ETNK2, REN, KISS1, GOLT1A; TAD3: PLEKHA6, LINC00628, PPP1R15B, PIK3C2B, MDM4. REN in TAD2, was isolated from its neighboring genes in TAD1 and TAD3 by CTCF-binding sites that serve as insulators. TAD1 and TAD3 genes SOX13 and LINC00628 overlapped super-enhancers, known to reside near nodes regulating cell identity, and were co-expressed in various tissues, suggesting co-regulation. REN was also connected with 62 distant genes genome-wide, including the angiotensin II type 1 receptor gene. The findings lead us to invoke the following novel hypothesis. While the REN promoter is isolated from neighboring super-enhancers in most cells by insulators, these insulators break down with cell age to permit the inappropriate expression of REN in non-kidney cells by using the neighboring super-enhancers, resulting in expression in a wider spectrum of tissues, contributing to aging-related immune system dysregulation, cardiovascular diseases and cancers. Research is needed to confirm this hypothesis experimentally.

Abstract P126: Statines Reduce Cardiac Dysfunction After Preeclamptic Pregnancy

Introduction: Former preeclamptic women have an increased risk of cardiovascular disease in later life. However, the underlying functional and structural mechanisms are still unknown. Objective: We tested the hypothesis that pravastatin has a beneficial effect on the remodeling of the maternal heart after preeclamptic pregnancy and thus reduces the long-term cardiovascular risk. Methods: Female rats harboring the human angiotensinogen gene [TGR(hAogen)L1623] developed a preeclamptic phenotype during pregnancy when mated with male rats carrying the human renin gene [TGR(hRen)L10J], but behaved physiologically normal postpartum. To investigate a possible intervention, we treated preeclamptic dams with pravastatin for four weeks after birth, which was started already during pregnancy. Cardiac structure and function was compared by speckle tracking echocardiography, gene expression analysis, and immune staining of the maternal heart. Results: Due to pravastatin treatment, former preeclamptic rats reduced signs of cardiac hypertrophy (perimeter cardiomyocytes 65.3±0.3 vs. 60.5±0.5 μm), fibrosis (fibronectin 3.9±0.3 vs. 1.8±0.2 %) and improved cardiac microcirculation (CD31+ cells 219.0±4.7 vs. 269.1±8.6). In addition, cardiac output was increased postpartum by statin treatment (ejection fraction 57.2±1.2 vs. 61.0±1.9; global longitudinal strain -14.5±0.6 vs. -19.8±1.0 %; heartrate 355.0±8.7 vs. 328.6±8.3 bpm). Analysis of maternal blood serum showed a reduction of brain natriuretic peptide (BNP 0.39±0.05 vs. 0.33±0.01 ng/ml) and soluble fms-like tyrosine kinase 1 (sFlt1 0.69±0.02 vs. 0.51±0.02 ng/ml) even four weeks after birth. Fetuses at the time of birth also benefited from treatment (brain liver ratio 0.93±0.03 vs. 0.82±0.01; heart body ratio 7.14±0.09 vs. 6.80±0.12). Discussion: Former preeclamptic rats showed significant cardiac hypertrophy in combination with fibrosis and capillary rarefaction. These irreversible structural changes can be reduced by pravastatin treatment. We could not detect any harmful influence of statins on the fetus. Values mean±SEM, PE vs. PE+pravastatin

A polymorphism in intron I of the human angiotensinogen gene (hAGT) affects binding by HNF3 and hAGT expression and increases blood pressure in mice

Angiotensinogen (AGT) is the precursor of one of the most potent vasoconstrictors, peptide angiotensin II. Genome-wide association studies have shown that two A/G polymorphisms (rs2493134 and rs2004776), located at +507 and +1164 in intron I of the human AGT (hAGT) gene, are associated with hypertension. Polymorphisms of the AGT gene result in two main haplotypes. Hap-I contains the variants -217A, -6A, +507G, and +1164A and is pro-hypertensive, whereas Hap-II contains the variants -217G, -6G, +507A, and +1164G and does not affect blood pressure. The nucleotide sequence of intron I of the hAGT gene containing the +1164A variant has a stronger homology with the hepatocyte nuclear factor 3 (HNF3)-binding site than +1164G. Here we found that an oligonucleotide containing +1164A binds HNF3β more strongly than +1164G and that Hap-I-containing reporter gene constructs have increased basal and HNF3- and glucocorticoid-induced promoter activity in transiently transfected liver and kidney cells. Using a knock-in approach at the hypoxanthine-guanine phosphoribosyltransferase locus, we generated a transgenic mouse model containing the human renin (hREN) gene and either Hap-I or Hap-II. We show that transgenic animals containing Hap-I have increased blood pressure compared with those containing Hap-II. Moreover, the transcription factors glucocorticoid receptor, CCAAT enhancer-binding protein β, and HNF3β bound more strongly to chromatin obtained from the liver of transgenic animals containing Hap-I than to liver chromatin from Hap-II-containing animals. These findings suggest that, unlike Hap-II variants, Hap-I variants of the hAGT gene have increased transcription rates, resulting in elevated blood pressure.

58. A peptide antagonist in a genetic rat angiotensinogen × renin model of preeclampsia

Introduction Preeclampsia is a pregnancy-related disorder characterized by hypertension and excess protein excretion in the urine. It is the most common hypertensive disease of pregnancy and affects about 5–8% of pregnancies. The Renin-Angiotensin-System (RAS) is involved in the pathogenesis of the disease; however, treatment with a RAS-blocker is contraindicated due to fetal toxicity. Objective The rat angiotensinogen renin cross model has been described as a rodent model that recapitulates many of the changes observed in human preeclampsia. This experimental rat model of preeclampsia will be used to test the hypothesis that peptide antagonists targeting the RAS system impact both maternal and fetal outcomes. Methods Sprague-Dawley (SD) rats harboring the human angiotensinogen (hAGT) gene [SD-Tg(hAGT) L1623] were mated with male SD rats bearing the human renin (hREN) gene [SD-Tg(hRen) L10J] to produce a model of preeclampsia (PE) in the dams. These pregnant dams developed hypertension on day 13 of pregnancy (plug-recognition was assigned as day 1) and albuminuria. At day 15 of gestation when the animals were already hypertensive, a RAS peptide antagonist was administered via mini-osmotic pumps sc. Results The major finding of the study was that a peptide antagonist targeting RAS ameliorated the preeclamptic phenotype. In the transgenic model, treatment led to a reduction of blood pressure and urinary albumin excretion. The improvement of intrauterine growth retardation (IUGR) indicates an improved fetal development. Discussion RAS peptide antagonist ameliorates the preeclamptic phenotype of the rat model due to the reduction of blood pressure and albuminuria. Furthermore an improvement of the fetal outcome was observed by an amelioration of the IUGR.

Phenotypic dissection of the mouse Ren1d knockout by complementation with human renin

Normal renin synthesis and secretion is important for the maintenance of juxtaglomerular apparatus architecture. Mice lacking a functional Ren1d gene are devoid of renal juxtaglomerular cell granules and exhibit an altered macula densa morphology. Due to the species-specificity of renin activity, transgenic mice are ideal models for experimentally investigating and manipulating expression patterns of the human renin gene in a native cellular environment without confounding renin–angiotensin system interactions. A 55-kb transgene encompassing the human renin locus was crossed onto the mouse Ren1d-null background, restoring granulation in juxtaglomerular cells. Correct processing of human renin in dense core granules was confirmed by immunogold labeling. After stimulation of the renin–angiotensin system, juxtaglomerular cells contained rhomboid protogranules with paracrystalline contents, dilated rough endoplasmic reticulum, and electron-lucent granular structures. However, complementation of Ren1d−/− mice with human renin was unable to rescue the abnormality seen in macula densa structure. The juxtaglomerular apparatus was still able to respond to tubuloglomerular feedback in isolated perfused juxtaglomerular apparatus preparations, although minor differences in glomerular tuft contractility and macula densa cell calcium handling were observed. This study reveals that the human renin protein is able to complement the mouse Ren1d−/− non-granulated defect and suggests that granulopoiesis requires a structural motif that is conserved between the mouse Ren1d and human renin proteins. It also suggests that the altered macula densa phenotype is related to the activity of the renin-1d enzyme in a local juxtaglomerular renin–angiotensin system.

BP.12.05] A GENETIC VARIANT IN THE DISTAL ENHANCER REGION OF THE HUMAN RENIN GENE AFFECTS RENIN EXPRESSION

Objective: The high heritability of plasma renin activity was confirmed in recent investigations. A variation located near the strong enhancer of the human renin gene (REN), C-5312T, has been shown to have different transcription activity levels depending on its allele: the 5312T allele shows transcription levels that are 45% greater than those of the 5312C allele. The purpose of this study was to confirm the hypothesis that variations in the enhancer region of the REN gene are involved in regulating renal expression of renin. Design and method: Sixty-four subjects with biopsy-proven renal diseases were included in this study (male/female: 35/29, age 41.9 ± 20.9 years, SBP/DBP 123.1 ± 23.7/73.4 ± 14.8 mmHg, s-Cr 0.93 ± 0.63 mg/dl). A genetic variant of REN, C-5312T, was assayed by PCR-RFLP and the TaqMan method. Total RNAs from a small part of the renal cortex were reverse-transcribed and amplified for REN and GAPDH with a real-time PCR system. Results: Logarithmically transformed expression values of the relative ratio of REN to GAPDH (10[Combining Macron]3) were as follows (mean ± SE): CC (26 cases), 0.016 ± 0.005; CT (33 cases), 0.047 ± 0.021 (p = 0.41 vs. CC); TT (5 cases), 0.198 ± 0.194 (p = 0.011 vs. CC, p < 0.031 vs. CT). Thus, significant differences in REN expression were observed among the genetic variants. Conclusions: The results suggest that variants in the enhancer region of the human renin gene have an effect on the expression levels of renin in renal tissue; this observation is in good accordance with the results of the transcriptional assay.

The PPAR-gamma-binding sequence Pal3 is necessary for basal but dispensable for high-fat diet regulated human renin expression in the kidney.

We reported earlier that PPAR-gamma regulates renin transcription through a human-specific atypical binding sequence termed hRen-Pal3. Here we developed a mouse model to investigate the functional relevance of the hRen-Pal3 sequence in vivo since it might be responsible for the increased renin production in obesity and thus for the development of accompanying arterial hypertension. We used bacterial artificial chromosome construct and co-placement strategy to generate two transgenic mouse lines expressing the human renin gene from identical genomic locus without affecting the intrinsic mouse renin expression. One line carried a wild-type hRen-Pal3 in the transgene (Pal3wt strain) and the other a mutated non-functional Pal3 (Pal3mut strain). Human renin expression was correctly targeted to the renin-producing juxtaglomerular (JG) cells of kidney in both lines. However, Pal3mut mice had lower basal human renin expression. Since human renin does not recognize mouse angiotensinogen as substrate, the blood pressure was not different between the strains. Stimulation of renin production with the angiotensin-converting enzyme inhibitor enalapril equipotentially stimulated the human renin expression in Pal3wt and Pal3mut mice. High-fat diet for 10weeks which is known to activate PPAR-gamma failed to increase human renin mRNA in kidneys of either strain. These findings showed that the human renin PPAR-gamma-binding sequence hRen-Pal3 is essential for basal renin expression but dispensable for the cell-specific and high-fat diet regulated renin expression in the kidney.

A Genetic Variant in the Distal Enhancer Region of the Human Renin Gene Affects Renin Expression.

BackgroundThe high heritability of plasma renin activity was confirmed in recent investigations. A variation located near the strong enhancer of the human renin gene (REN), C-5312T, has been shown to have different transcription activity levels depending on its allele: the 5312T allele shows transcription levels that are 45% greater than those of the 5312C allele. The purpose of this study was to confirm the hypothesis that variations in the enhancer region of the REN gene are involved in regulating renal expression of renin.MethodsSixty-four subjects with biopsy-proven renal diseases were included in this study (male/female: 35/29, age 41.9 ± 20.9 years, SBP/DBP 123.1 ± 23.7/73.4 ± 14.8 mmHg, s-Cr 0.93 ± 0.63 mg/dl). A genetic variant of REN, C-5312T, was assayed by PCR-RFLP and the TaqMan method. Total RNAs from a small part of the renal cortex were reverse-transcribed and amplified for REN and GAPDH with a real-time PCR system.ResultsLogarithmically transformed expression values of the relative ratio of REN to GAPDH (10−3) were as follows (mean ± SE): CC (26 cases), 0.016 ± 0.005; CT (33 cases), 0.047 ± 0.021 (p = 0.41 vs. CC); TT (5 cases), 0.198 ± 0.194 (p = 0.011 vs. CC, p < 0.031 vs. CT). Thus, significant differences in REN expression were observed among the genetic variants.ConclusionThe results suggest that variants in the enhancer region of the human renin gene have an effect on the expression levels of renin in renal tissue; this observation is in good accordance with the results of the transcriptional assay.

The sGC stimulator BAY 41-8543 in a rat model of hypertension-induced heart failure

Hypertension with left ventricular hypertrophy is a major cause of diastolic heart failure (DHF). Due to its high prevalence and high rate of mortality, DHF represents a major challenge in today's cardiovascular medicine; with limited therapeutic options. Soluble guanylate cyclase (sGC) stimulation is emerging as a promising treatment option in DHF, and is currently under investigation in preclinical and clinical studies. The present study investigates the effect of the sGC stimulator BAY 41-8543 in a transgenic rat model of hypertension-induced heart failure. We used 4 week-old male double transgenic rats expression both human renin and angiotensinogen genes (dTGRs). At 7 weeks of age, dTGRs exhibit striking cardiac hypertrophy with fibrosis and inflammation, ventricular arrhythmias and heart failure, which is accompanied with high mortality. We compared vehicle-treated dTGR (receiving 10% transcutol, 20% cremophor, 70% water) to those receiving 3 mg/kg/d BAY 41-8543, and vehicle-treated SD control rats (single oral dose per day for 3 weeks). We performed in vivo echocardiography, hemodynamic monitoring, cardiac electrophysiology studies and blood pressure measurements. Endothelial function was measured in isolated mesenteric arteries. Transcriptional analyses in cardiac tissue were performed using qRT-PCR and gene-microarray. Cardiac tissue was analyzed using histology. Treatment of dTGRs with BAY 41-8543 resulted in 100% survival at week 7, whereas only 24% of vehicle-treated dTGRs survived. Mean arterial pressure in dTGRs was significantly by BAY 41-8543 reduced (197 ± 11 mmHg vehicle vs 133 ± 4 mmHg BAY 41-8543). In addition, BAY 41-8543 significantly decreased in vivo total peripheral resistance and improved endothelium-dependent vasorelaxation of isolated mesenteric arteries. Furthermore BAY 41-8543 prevented fibrosis and inflammation of cardiac tissue. Echocardiography and invasive hemodynamic monitoring revealed BAY 41-8543 significantly increased ejection fraction and cardiac output in dTGR, whereas vehicle-treated had preserved systolic function but reduced diastolic function. In addition, diastolic compliance was significantly enhanced by BAY 41-8543, as shown by myocardial strain analysis and end-diastolic pressure volume relationship (EDPVR); indicative of an improved diastolic function. In vivo programmed electrical stimulation revealed a high ventricular tachycardia induction rate in vehicle-treated dTGRs (46%), which was significantly reduced in BAY 41-8543-treated dTGR (11%). Myocardial gene-microarray analysis showed a reversal of dysregulated genes in dTGR by BAY 41-8543 treatment. Our data demonstrate that BAY 41-8543 improves survival and cardiac performance in a transgenic rat model of hypertension-induced DHF. We postulate that treatment of DHF with sGC stimulators offers a novel therapeutic potential for humans.

Abstract P046: Interplay of Intronic and Promoter Region Polymorphisms Up-regulate Human Angiotensinogen and Cause Hypertension in Transgenic Mice

Angiotensinogen (AGT) is the substrate for the RAS-cascade and polymorphisms leading to its overexpression are linked to hypertension. We have shown that SNPs in linkage disequilibrium with -6A of the hAGT gene cause increased AGT expression and hypertension. Recently, two genome wide association studies (GWAS) have linked SNP at the +1164 in intron-I with increased blood pressure. The +1164A allele has stronger homology with HNF-3 binding site as compared to +1164G. HNF3 belongs to the family of “pioneer” transcription factors, which enable chromatin access for other tissue-specific transcription factors. Thus, we propose that a haplotype, where multiple SNP-groups occur together, is a better model to predict and study disease correlation with genetic variations than individual SNPs alone. Intronic SNPs could increase chromatin access thus modulating promoter-transcription factor (TF) interactions. To study these possible interactions between the intronic SNPs in the promoter and intron I of the hAGT gene, we have divided this gene in two major haplotypes: haplotype-I (containing -6A &amp; +1164A) and II (containing -6G &amp; +1164G). Reporter construct containing haplotype-I has increased promoter activity as compared to haplotype-II on transient transfection in human liver cells, without (hap-1: 73±2 vs. hap-II: 23.64±3.21 A.U; p&lt;0.05) and with dexamethasone treatment (hap-1: 1174±68vs. hap-II: 333.3±37 A.U; p&lt;0.05). Double transgenic mice (TG) containing human renin gene (hREN) and haplotype-I of the hAGT gene (containing 2.0 Kb of the promoter, all the introns, exons and 3’UTR of the hAGT gene at the HPRT locus) have increased basal and GR induced expression of the hAGT gene in liver, kidney and fat as compared to haplotype-II. Complementary CHIP analysis shows increased TF-chromatin binding in the liver of haplotype-I TG mice for HNF3β (4.35 folds), C/EBPβ (2.1 folds), STAT-3 (3.6 folds) and GR (4.2 folds). Crucially, haplotype-I TG mice have increased (p&lt;0.05) SBP (137±4 mm Hg) when compared to haplotype-II mice (126±3 mm Hg). In conclusion, intronic SNPs regulate hAGT gene expression via complex interplay with SNPs in the gene-promoter and this underscores the need for a haplotype-based approach to genetic variability and disease correlation.

Determinants of Plasma Renin Activity Role of a Human Renin Gene Variant as a Genetic Factor

Determinants of Plasma Renin Activity Role of a Human Renin Gene Variant as a Genetic Factor

Dexamethasone Promotes Hypertension by Allele-specific Regulation of the Human Angiotensinogen Gene

The human angiotensinogen (hAGT) gene has polymorphisms in its 2.5-kb promoter that form two haplotype (Hap) blocks: -6A/G (-1670A/G, -1562C/T, and -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. Hap -6A/-217A has increased promoter activity with enhanced transcription factor binding, including to the glucocorticoid receptor (GR). Glucocorticoid therapy frequently causes hypertension, the mechanisms for which are incompletely understood. We have engineered double transgenic (TG) mice containing the human renin gene with either Hap of the hAGT gene and examined the physiological significance of glucocorticoid-mediated allele-specific regulation of the hAGT gene. We have also studied the consequential effects on the renin angiotensin system and blood pressure. TG mice with Hap -6A and -6G were treated with and without a low dose of a GR agonist, dexamethasone (2.5 μg/ml), for 72 h. We found greater chromatin-GR binding with increased GR agonist-induced hAGT expression in liver and renal tissues of Hap -6A mice. Additionally, dexamethasone treatment increased circulating hAGT and angiotensin II levels in Hap -6A mice, as compared with -6G mice. Importantly, GR agonist significantly increased blood pressure and redox markers in TG mice with Hap-6A of the hAGT gene. Taken together, our results show, for the first time, that glucocorticoids affect hAGT expression in a haplotype-dependent fashion with SNPs in Hap -6A favoring agonist-induced GR binding. This leads to increased expression of the hAGT, up-regulation of the renin angiotensin system, and increased blood pressure and oxidative stress in Hap -6A mice.

Vitamin D depletion aggravates hypertension and target-organ damage.

BackgroundWe tested the controversial hypothesis that vitamin D depletion aggravates hypertension and target‐organ damage by influencing renin.Methods and ResultsFour‐week‐old double‐transgenic rats (dTGR) with excess angiotensin (Ang) II production due to overexpression of the human renin (hREN) and angiotensinogen (hAGT) genes received vitamin D‐depleted (n=18) or standard chow (n=15) for 3 weeks. The depleted group had very low serum 25‐hydroxyvitamin D levels (mean±SEM; 3.8±0.29 versus 40.6±1.19 nmol/L) and had higher mean systolic BP at week 5 (158±3.5 versus 134.6±3.7 mm Hg, P<0.001), week 6 (176.6±3.3 versus 162.3±3.8 mm Hg, P<0.01), and week 7 (171.6±5.1 versus 155.9±4.3 mm Hg, P<0.05). Vitamin D depletion led to increased relative heart weights and increased serum creatinine concentrations. Furthermore, the mRNAs of natriuretic peptides, neutrophil gelatinase‐associated lipocalin, hREN, and rRen were increased by vitamin D depletion. Regulatory T cells in the spleen and in the circulation were not affected. Ang metabolites, including Ang II and the counter‐regulatory breakdown product Ang 1 to 7, were significantly up‐regulated in the vitamin D‐depleted groups, while ACE‐1 and ACE‐2 activities were not affected.ConclusionsShort‐term severe vitamin D depletion aggravated hypertension and target‐organ damage in dTGR. Our data suggest that even short‐term severe vitamin D deficiency may directly promote hypertension and impacts on renin‐angiotensin system components that could contribute to target‐organ damage. The findings add to the evidence that vitamin D deficiency could also affect human hypertension.

Genetic Variant of C-5434T REN Enhancer on Serum Renin Levels and Binding Pattern of Signal Transducers and Activators Transcription 3.

The human renin gene has been widely known to be involved in essential hypertension (EH) pathogenesis. Genetic variant C-5434T of REN enhancer contributed to renin gene transcription and serum renin regulation. However, the mechanism associated with the transcription level changes remains unknown, and only a few reports exist that discussed serum renin levels on C-5434T of REN. Thus, this study aims to investigate the relationship between genetic variant C-5434T of REN enhancer and serum renin levels in Indonesian hypertensive patients. SNP of C-5434T was genotyped in 56 hypertensive patients by using RFLP. The data showed that serum renin is slightly higher in hypertensive patients with the TT genotype (39 ± 10.3) than patients with the CC genotype (33 ± 10.6) but the difference was not statistically significant (p = 0.35). Here, we also present a docking approach for predicting interaction between genetic variant -5434C/T and STAT3 (Signal Transducers and Activators Transcription 3), the predicted transcription factor that regulates renin gene enhancer. The results showed that STAT3-DNA allele T more favorably binds to DNA than STAT3-DNA allele C. These data suggest that the presence of genetic variant C-5434T has changed the binding pattern of STAT3 to REN enhancer. This is likely to influence STAT3 activity to stimulate the expression of renin gene in producing renin.

Determinants of plasma renin activity: role of a human renin gene variant as a genetic factor.

The plasma renin activity (PRA) is affected by a number of environmental factors. However, significant heritability has been shown for the activity. A hypothesis that a candidate regulatory single-nucleotide polymorphism, C-5312T, of human renin gene should have a significant effect on PRA was elucidated and updating of independent determinants of PRA was attempted.Cross sectional study.Outpatient study.We enrolled consecutive 810 subjects who had consulted our hospitals for lifestyle-related diseases.Genotypes were assayed with genomic DNA for C-5312T. Among the genetic variants, the difference of PRA was evaluated. Monovariate linear regression analysis was performed to test the correlation between PRA and clinical variables. Finally, stepwise multiple regression analysis was performed to evaluate the independent determinants.On comparing 2 genotype groups, CC/CT and T allele homozygote, the geometric means of PRA were 0.778 and 0.941 ng/ml/h, respectively (F = 5.992, P = 0.015). Monovariate linear regression analysis revealed that a number of variables have a significant correlation with the activity, including urinary salt excretion. A stepwise multivariate regression analysis revealed that renin C-5312T variant (TT) is one of the independent determinants of PRA.Thus, for the first time, a human renin gene variant was associated with a significant increase in PRA as a genetic factor and the independent determinants for the activity were updated including genetic factor.