Human Renin Gene Research Articles

Transgenic Mice with −6A Haplotype of the Human Angiotensinogen Gene Have Increased Blood Pressure Compared with −6G Haplotype

Hypertension is a serious risk factor for cardiovascular disease, and the angiotensinogen (AGT) gene locus is associated with human essential hypertension. The human AGT (hAGT) gene has an A/G polymorphism at -6, and the -6A allele is associated with increased blood pressure. However, transgenic mice containing 1.2 kb of the promoter with -6A of the hAGT gene show neither increased plasma AGT level nor increased blood pressure compared with -6G. We have found that the hAGT gene has three additional SNPs (A/G at -1670, C/G at -1562, and T/G at -1561). Variants -1670A, -1562C, and -1561T almost always occur with -6A, and variants -1670G, -1562G, and -1561G almost always occur with -6G. Therefore, the hAGT gene may be subdivided into either -6A or -6G haplotypes. We show that these polymorphisms affect the binding of HNF-1α and glucocorticoid receptor to the promoter, and a reporter construct containing a 1.8-kb hAGT gene promoter with -6A haplotype has 4-fold increased glucocorticoid-induced promoter activity as compared with -6G haplotype. In order to understand the physiological significance of these haplotypes in an in vivo situation, we have generated double transgenic mice containing either the -6A or -6G haplotype of the hAGT gene and the human renin gene. Our ChIP assay shows that HNF-1α and glucocorticoid receptor have stronger affinity for the chromatin obtained from the liver of transgenic mice containing -6A haplotype. Our studies also show that transgenic mice containing -6A haplotype have increased plasma AGT level and increased blood pressure as compared with -6G haplotype. Our studies explain the molecular mechanism involved in association of the -6A allele of the hAGT gene with hypertension.

Effects of aliskiren on stroke in rats expressing human renin and angiotensinogen genes.

ObjectivePre-treatment with angiotensin receptor blockers is known to improve neurological outcome after stroke. This study investigated for the first time, whether the renin inhibitor aliskiren has similar neuroprotective effects.MethodsSince aliskiren specifically blocks human renin, double transgenic rats expressing human renin and angiotensinogen genes were used. To achieve a systolic blood pressure of 150 or 130 mmHg animals were treated with aliskiren (7.5 or 12.5 mg/kg*d) or candesartan (1.5 or 10 mg/kg*d) via osmotic minipump starting five days before middle cerebral artery occlusion with reperfusion. Infarct size was determined by magnetic resonance imaging. mRNA of inflammatory marker genes was studied in different brain regions.ResultsThe mortality of 33.3% (7 of 21 animals) in the vehicle group was reduced to below 10% by treatment with candesartan or aliskiren (p<0.05). Aliskiren-treated animals had a better neurological outcome 7 days post-ischemia, compared to candesartan (Garcia scale: 9.9±0.7 vs. 7.3±0.7; p<0.05). The reduction of infarct size in the aliskiren group did not reach statistical significance compared to candesartan and vehicle (24 h post-ischemia: 314±81 vs. 377±70 and 403±70 mm3 respectively). Only aliskiren was able to significantly reduce stroke-induced gene expression of CXC chemokine ligand 1, interleukin-6 and tumor necrosis factor-alpha in the ischemic core.ConclusionsHead-to-head comparison suggests that treatment with aliskiren before and during cerebral ischemia is at least as effective as candesartan in double transgenic rats. The improved neurological outcome in the aliskiren group was blood pressure independent. Whether this effect is due to primary anti-inflammatory mechanisms has to be investigated further.

Levosimendan improves cardiac function and survival in rats with angiotensin II-induced hypertensive heart failure

Calcium-sensitizing agents improve cardiac function in acute heart failure; however, their long-term effects on cardiovascular mortality are unknown. We tested the hypothesis that levosimendan, an inodilator that acts through calcium sensitization, opening of ATP-dependent potassium channels and phosphodiesterase III inhibition, improves cardiac function and survival in double transgenic rats harboring human renin and angiotensinogen genes (dTGRs), a model of angiotensin II (Ang II)-induced hypertensive heart failure. Levosimendan (1 mg kg(-1)) was administered orally to 4-week-old dTGRs and normotensive Sprague-Dawley rats for 4 weeks. Untreated dTGRs developed severe hypertension, cardiac hypertrophy, heart failure with impaired diastolic relaxation, and exhibited a high mortality rate at the age of 8 weeks. Levosimendan did not decrease blood pressure and did not prevent cardiac hypertrophy. However, levosimendan improved systolic function, decreased cardiac atrial natriuretic peptide mRNA expression, ameliorated Ang II-induced cardiac damage and decreased mortality. Levosimendan did not correct Ang II-induced diastolic dysfunction and did not influence heart rate. In a separate survival study, levosimendan increased dTGR survival by 58% and median survival time by 27% (P=0.004). Our findings suggest that levosimendan ameliorates Ang II-induced hypertensive heart failure and reduces mortality. The results also support the notion that the effects of levosimendan in dTGRs are mediated by blood pressure-independent mechanisms and include improved systolic function and amelioration of Ang II-induced coronary and cardiomyocyte damage.

Inhibition of Trophoblast-Induced Spiral Artery Remodeling Reduces Placental Perfusion in Rat Pregnancy

Rats harboring the human angiotensinogen and human renin genes develop preeclamptic features in pregnancy. The preeclamptic rats exhibit a deeper trophoblast invasion associated with a reduced resistance index by uterine Doppler. Doxycycline inhibits matrix metalloproteinase activity. We tested the hypothesis that matrix metalloproteinase inhibition reduces trophoblast invasion with subsequent changes in placental perfusion. Preeclamptic and pregnant control Sprague-Dawley rats were treated with doxycycline (30 mg/kg of body weight orally) from gestational day 12 until day 18. Placental perfusion was assessed using a micromarker contrast agent. The animals were euthanized on day 18 of pregnancy; biometric data were acquired, and trophoblast invasion was analyzed. Doxycycline resulted in intrauterine growth retardation and lighter placentas in both groups. Maternal body weight was not affected. As shown earlier, preeclamptic rats exhibited a deeper endovascular trophoblast invasion. However, doxycycline treatment reduced trophoblast invasion in the preeclamptic rats. The physiological spiral artery remodeling, as assessed by the deposition of fibrinoid and alpha-actin in the spiral artery contour, was significantly reduced by doxycycline. The vascularity index, as assessed by perfusion measurement of the placenta, was reduced after doxycycline treatment in preeclamptic rats. Thus, matrix metalloproteinase inhibition with doxycycline leads to reduced trophoblast invasion and associated reduced placental perfusion. These studies are the first to show that reducing trophoblast-induced vascular remodeling decreases subsequent placental perfusion. Our model allows the study of dysregulated trophoblast invasion and vascular remodeling in vivo to gain important insights into preeclampsia-related mechanisms.

Effects of Circulating and Local Uteroplacental Angiotensin II in Rat Pregnancy

The renin-angiotensin (Ang) system is important during placental development. Dysregulation of the renin-Ang system is important in preeclampsia (PE). Female rats transgenic for the human angiotensinogen gene crossed with males transgenic for the human renin gene develop the PE syndrome, whereas those of the opposite cross do not. We used this model to study the role of Ang II in trophoblast invasion, which is shallow in human PE but deeper in this model. We investigated the following groups: PE rats, opposite-cross rats, Ang II-infused rats (1000 ng/kg per day), and control rats. Ang II infusion increased only circulating Ang II levels (267.82 pg/mL), opposite cross influenced only uteroplacental Ang II (13.52 fmol/mg of protein), and PE increased both circulating (251.09 pg/mL) and uteroplacental (19.24 fmol/mg of protein) Ang II. Blood pressure and albuminuria occurred in the models with high circulating Ang II but not in the other models. Trophoblast invasion increased in PE and opposite-cross rats but not in Ang II-infused rats. Correspondingly, uterine artery resistance index increased in Ang II-infused rats but decreased in PE rats. We then studied human trophoblasts and villous explants from first-trimester pregnancies with time-lapse microscopy. Local Ang II dose-dependently increased migration by 75%, invasion by 58%, and motility by 282%. The data suggest that local tissue Ang II stimulates trophoblast invasion in vivo in the rat and in vitro in human cells, a hitherto fore unrecognized function. Conceivably, upregulation of tissue Ang II in the maternal part of the placenta represents an important growth factor for trophoblast invasion and migration.

Angiotensin receptors in the eyes of arterial hypertensive rats

The aim of the present study was to determine whether the eye tissues of arterial hypertensive rats evince expression of angiotensin receptors (AT(1) and AT(2)) as well as the novel Mas receptor, whose endogenous ligand is vasorelaxing Angiotensin (1-7) [Ang (1-7)]. Enucleated eyes from spontaneously hypertensive rats (SHR) and double transgenic rats harbouring human renin and angiotensinogen genes (dTGR) and their normotensive controls were used. Half of the rats were pretreated orally with an Angiotensin II (Ang II) type 1 receptor blocker (ARB). The eyes were snap-frozen in isopentane at -40 degrees and stored at -70 degrees for subsequent reverse transcriptase polymerase chain reaction (RT-PCR) analysis or in vitro autoradiography. The mRNA expression of AT(1a) and AT(2) as well as the novel Mas receptor was detected in all rat groups, being markedly higher in the retina than in the ciliary body. dTGR had significantly more receptors than SHR, but no direct relation to blood pressure level was seen. According to the autoradiography, treatment with ARB blocked a part of AT(1) receptors but had no clear effect on AT(2) receptors. The novel Mas receptor was found by RT-PCR in eye tissue for the first time. Its specific ligand, Ang (1-7), may be involved in the regulation of intraocular pressure--as recently demonstrated by us--and in the pathogenesis of retinal diseases as a counter-regulatory component for the vascular and proliferative actions of Ang II. The results suggest that the density of AT(1) receptors in the eye is independent of the blood pressure level of the animal.

From mother to child--transplacental effect of AT1R-AAin preeclampsia

Preeclampsia is a common hypertensive disorder of pregnancy and a leading cause of maternal and foetal morbidity and mortality worldwide. Preeclampsia requires the presence of the placenta and generally dramatically resolves after placental delivery. The abnormal placentation with failure of trophoblast remodelling of uterine spiral arterioles represents a point where current theories on mechanisms responsible for the condition converge [1]. Various pathways have been proposed to contribute to placental disease. Two major recent concepts, namely, autoimmunity and anti-angiogenesis, have helped us gain new insights into this ‘disease of theories’. Possible mediators leading to impaired angiogenesis in the placenta were recently reviewed [2]. The autoimmunity notion received support with the discovery of autoantibodies directed against the angiotensin (Ang) II Type 1 receptor (AT1R-AA) in the serum of preeclamptic women [3]. AT1R-AA activate the AT1R and induce a cascade of events in various cell types, similar to the actions of Ang II [4]. Increased oxidative stress, increased production of soluble fms-like tyrosine kinase (sFlt-1) in the placenta and initiation of pro-inflammatory and pro-coagulation responses all appear to be key mechanisms resulting from AT1R-AA actions [5]. AT1R-AA were also found in a rodent model for preeclampsia. In that model, the dams of female rats transgenic for the human angiotensinogen gene crossed with male rats transgenic for the human renin gene developed severe (telemetrymeasured) hypertension and albuminuria in the latter half of pregnancy, accompanied by glomerular endotheliosis [6]. Thus, many features of the preeclampsia-related maternal syndrome including hypertension, proteinuria and generalized endothelial dysfunction were attributed to circulating AT1R-AA. Preeclamptic AT1R-AA are IgG3 subclass immunoglobulins that target the structures contained in the second extracellular loop of AT1R and induce agonistic responses similar to Ang II despite a different binding site from the natural ligand [3]. Their actions can be neutralized with the corresponding seven-amino-acid peptide sequence or pharmacologic blockade of the AT1R. Recent studies on the pathogenesis of preeclampsia focused mainly on the features of the maternal syndrome, as preeclampsia can lead to serious maternal and foetal complications. Agonistic antibody-mediated AT1R stimulation has been demonstrated in severe vascular renal allograft rejection and the passive transfer of human IgG containing AT1R-AA induced vascular lesions similar to the human disease. Furthermore, AT1R-AA led to increased blood pressure in otherwise non-rejecting and normotensive transplanted animals [7]. Investigators from the University of Texas in Houston pursued a similar strategy of passive human IgG transfer. They recently showed that AT1R-AA recovered from the circulation of women with preeclampsia induced preeclampsia-like generalized endothelial dysfunction in pregnant mice. Short duration (5 days) of the passive transfer induced an increase in tail-cuff blood pressure measurements. Links to impaired placental angiogenesis was demonstrated with an increase in sFlt concentrations in the serum of these pregnant mice [8]. sFlt is a shorter splice variant of the functional receptor that lacks the transmembrane domain. The shortened version interferes with angiogenesis and thereby placental development. Together with previous f indings implicating overproduction of plasminogen activator inhibitor-1 and tissue factor in human trophoblast cells, their findings provided further evidence that AT1R-AA may have a causative role in abnormal placentation. Some prototypic features of maternal syndrome in terms of swelling of the endothelial cells (glomerular endotheliosis) were found in the antibody-transferred pregnant mice. However, the mouse model differed from the human disease. For instance, electron microscopic analysis revealed subendothelial deposits of immune complexes. Glomerular immune complex deposition is not common in human preeclampsia [9] and it remains to be determined whether or not the mice developed antibodies against human AT1R-AA IgG used in the transfer experiments. The team from Houston now focused on foetal complications. Intrauterine growth restriction (IUGR) can be

Metabolomics in Angiotensin II-Induced Cardiac Hypertrophy

Angiotensin II (Ang II) induces mitochondrial dysfunction. We tested whether Ang II alters the "metabolomic" profile. We harvested hearts from 8-week-old double transgenic rats harboring human renin and angiotensinogen genes (dTGRs) and controls (Sprague-Dawley), all with or without Ang II type 1 receptor (valsartan) blockade. We used gas chromatography coupled with time-of-flight mass spectrometry to detect 247 intermediary metabolites. We used a partial least-squares discriminate analysis and identified 112 metabolites that differed significantly after corrections (false discovery rate q <0.05). We found great differences in the use of fatty acids as an energy source, namely, decreased levels of octanoic, oleic, and linoleic acids in dTGR (all P<0.01). The increase in cardiac hypoxanthine levels in dTGRs suggested an increase in purine degradation, whereas other changes supported an increased ketogenic amino acid tyrosine level, causing energy production failure. The metabolomic profile of valsartan-treated dTGRs more closely resembled Sprague-Dawley rats than untreated dTGRs. Mitochondrial respiratory chain activity of cytochrome C oxidase was decreased in dTGRs, whereas complex I and complex II were unaltered. Mitochondria from dTGR hearts showed morphological alterations suggesting increased mitochondrial fusion. Cardiac expression of the redox-sensitive and the cardioprotective metabolic sensor sirtuin 1 was increased in dTGRs. Interestingly, valsartan changed the level of 33 metabolites and induced mitochondrial biogenesis in Sprague-Dawley rats. Thus, distinct patterns of cardiac substrate use in Ang II-induced cardiac hypertrophy are associated with mitochondrial dysfunction. The finding underscores the importance of Ang II in the regulation of mitochondrial biogenesis and cardiac metabolomics, even in healthy hearts.

Resveratrol induces mitochondrial biogenesis and ameliorates Ang II-induced cardiac remodeling in transgenic rats harboring human renin and angiotensinogen genes

There is compelling evidence to indicate an important role for increased local renin–angiotensin system activity in the pathogenesis of cardiac hypertrophy and heart failure. Resveratrol is a natural polyphenol that activates SIRT1, a novel cardioprotective and longevity factor having NAD+-dependent histone deacetylase activity. We tested the hypothesis whether resveratrol could prevent from angiotensin II (Ang II)-induced cardiovascular damage. Four-week-old double transgenic rats harboring human renin and human angiotensinogen genes (dTGR) were treated for 4 weeks either with SIRT1 activator resveratrol or SIRT1 inhibitor nicotinamide. Untreated dTGR and their normotensive Sprague–Dawley control rats (SD) received vehicle. Untreated dTGR developed severe hypertension as well as cardiac hypertrophy, and showed pronounced cardiovascular mortality compared with normotensive SD rats. Resveratrol slightly but significantly decreased blood pressure, ameliorated cardiac hypertrophy and prevented completely Ang II-induced mortality, whereas nicotinamide increased blood pressure without significantly influencing cardiac hypertrophy or survival. Resveratrol decreased cardiac ANP mRNA expression and induced cardiac mRNA expressions of mitochondrial biogenesis markers peroxisome proliferator-activated receptor-γ coactivator (PGC-1α), mitochondrial transcription factor (Tfam), nuclear respiratory factor 1 (NRF-1) and cytochrome c oxidase subunit 4 (cox4). Resveratrol dose-dependently increased SIRT1 activity in vitro. Our findings suggest that the beneficial effects of SIRT1 activator resveratrol on Ang II-induced cardiac remodeling are mediated by blood pressure-dependent pathways and are linked to increased mitochondrial biogenesis.

Rapid genotyping of the human renin (REN) gene by the LightCycler® instrument: identification of unexpected nucleotide substitutions within the selected hybridization probe area.

Preeclampsia is a serious disorder affecting nearly 3% of all in the Western world. It is associated with hypertension and proteinuria, and several lines of evidence suggest that the renin-angiotensin system (RAS) may be involved in the development of hypertension at different stages of a preeclamptic pregnancy. In this study, we developed rapid genotyping assays on the LightCycler® instrument to allow the detection of genetic variants in the renin gene (REN) that may predispose to preeclampsia. The method is based on real-time PCR and allele-specific hybridization probes, followed by fluorescent melting curve analysis to expose a change in melting temperature (Tm). Ninety-two mother-father-child triads (n=276) from preeclamptic pregnancies were genotyped for three haplotype-tagging single nucleotide polymorphisms (htSNPs) in REN. All three htSNPs (rs5705, rs1464816 and rs3795575) were successfully genotyped. Furthermore, two unexpected nucleotide substitutions (rs11571084 and rs61757041) were identified within the selected hybridization probe area of rs1464816 and rs3795575 due to aberrant melting peaks. In conclusion, genotyping on the LightCycler® instrument proved to be rapid and highly reproducible. The ability to uncover additional nucleotide substitutions is particularly important in that it allows the identification of potentially etiological variants that might otherwise be overlooked by other genotyping methods.

Rapid Genotyping of the Human Renin (REN) Gene by the LightCycler® Instrument: Identification of Unexpected Nucleotide Substitutions within the Selected Hybridization Probe Area

Preeclampsia is a serious disorder affecting nearly 3% of all in the Western world. It is associated with hypertension and proteinuria, and several lines of evidence suggest that the renin-angiotensin system (RAS) may be involved in the development of hypertension at different stages of a preeclamptic pregnancy. In this study, we developed rapid genotyping assays on the LightCycler® instrument to allow the detection of genetic variants in the renin gene (REN) that may predispose to preeclampsia. The method is based on real-time PCR and allele-specific hybridization probes, followed by fluorescent melting curve analysis to expose a change in melting temperature (Tm). Ninety-two mother-father-child triads (n=276) from preeclamptic pregnancies were genotyped for three haplotype-tagging single nucleotide polymorphisms (htSNPs) inREN. All three htSNPs (rs5705, rs1464816 and rs3795575) were successfully genotyped. Furthermore, two unexpected nucleotide substitutions (rs11571084 and rs61757041) were identified within the selected hybridization probe area of rs1464816 and rs3795575 due to aberrant melting peaks. In conclusion, genotyping on the LightCycler® instrument proved to be rapid and highly reproducible. The ability to uncover additional nucleotide substitutions is particularly important in that it allows the identification of potentially etiological variants that might otherwise be overlooked by other genotyping methods.

The pro renin receptor and the mystic HRP -Is there a role in cardiovascular disease

In 2002, Nguyen et al. cloned the (pro)renin receptor ((P)RR). Two years later, Suzuki, Ichihara and colleagues provided a concept to inhibit the (P)RR through HRP. This decapeptide mimics a sequence of the prorenin prosegment and functions thereby as a decoy peptide. They showed that HRP prevented diabetic nephropathy in rodents and ameliorated renal and cardiac damage in spontaneously hypertensive rats. We tested HRP and the human renin inhibitor aliskiren in transgenic rats overexpressing the human renin and angiotensinogen genes (dTGR). Only aliskiren, but not HRP, was able to ameliorate target organ damage in this model. HRP had also no effect on target organ damage in renovascular hypertensive rats. In vitro studies showed that HRP did not inhibit (pro)renin binding and signaling. More confusing was the fact that HRP bound to cells lacking (P)RR on their surface. We believe that HRP does not act as a competitive antagonist for the (P)RR and promotes its action via an alternative mechanism. Elucidating this mechanism could offer further opportunities, in terms of (pro)renin research.

Prorenin renin and insulin resistance

Recently we showed that fructose feeding in rats induced nonproteolytic activation of prorenin and subsequent angiotensin II production in skeletal muscle. In addition, a pharmacological inhibitor of prorenin/(pro)renin receptor interaction attenuated the development of insulin resistance. However, the inhibitor did not ameliorate the glucose intolerance in transgenic rats overexpressing the human renin gene. This review article summarizes the current knowledge of the effects of the prorenin/(pro)renin receptor system on insulin resistance and its potential as a therapeutic target.

Milk Products Containing Bioactive Tripeptides Have an Antihypertensive Effect in Double Transgenic Rats (dTGR) Harbouring Human Renin and Human Angiotensinogen Genes

Tripeptides isoleucyl-prolyl-proline (IPP) and valyl-prolyl-proline (VPP) act as ACE inhibitors in vitro. Double transgenic rats (dTGR) harbouring human renin and human angiotensinogen genes develop malignant hypertension due to increased angiotensin II formation. The present study was aimed to evaluate possible antihypertensive effect of IPP and VPP in this severe model. Four-week-old dTGR were randomized in three groups to receive: (1) water (control), (2) fermented milk containing IPP and VPP, and (3) IPP and VPP dissolved in water for three weeks. Fermented milk, but not peptides in water, attenuated the development of hypertension in dTGR by 19 mmHg versus the control group (P = .023). In vitro vascular function tests showed that high concentrations of the peptides evinced ACE inhibitory properties. In other hypertension related variables, no significant differences between the treatment groups were found. In conclusion, fermented milk product containing IPP and VPP prevents development of malignant hypertension in an animal model.

CELLULAR EXPRESSION OF RENAL, CARDIAC AND PULMONARY INDUCIBLE NITRIC OXIDE SYNTHASE IN DOUBLE‐TRANSGENIC MICE EXPRESSING HUMAN RENIN AND ANGIOTENSINOGEN GENES

1. Hypertensive mice expressing the human renin (REN) and angiotensinogen (AGT) genes are used as a model for human hypertension. 2. The aim of the present study was to investigate the cellular expression and distribution of inducible nitric oxide synthase (iNOS) using immunohistochemistry in lung, heart and kidney tissues from a model of human hypertension using male and female double-transgenic (h-Ang 204/1h-Ren6) mice and wild-type C57/BI6J mice as controls. 3. In the kidney, the pattern of iNOS expression in various renal microanatomical regions during hypertension was similar to that of age-matched controls, except in the medullary ascending limb (MAL). In hypertension, iNOS expression was downregulated in the MAL. No significant differences in iNOS expression were seen between control or hypertensive mice in various cardiac microanatomical locations. In the lungs of hypertensive mice, iNOS expression was upregulated in bronchial airway epithelium and bronchial and vascular smooth muscle cells, but downregulated in alveolar macrophages, alveolar septa and pulmonary vascular endothelial cells. Expression of iNOS was similar between male and female mice in the kidney, heart and lungs. 4. In conclusion, iNOS regulation in hypertension is complex and depends on the cell type in which it is expressed and the localization of the cell type in the cardiorenal and pulmonary systems.

Differential effect of two haplotypes of the human angiotensinogen gene on blood pressure in transgenic mice

Angiotensinogen (AGT) gene locus is associated with human essential hypertension and over‐expression of the AGT gene increases blood pressure in transgenic mice. Human AGT gene has A/G polymorphic sites at ‐6 and ‐217 positions in its promoter. The frequency of AA haplotype of the human AGT gene containing ‐6A:‐217A is significantly increased in African‐American hypertensive patients as compared to the AG haplotype‐6A:‐217G. Reporter constructs containing AA haplotype of the AGT gene promoter have increased promoter activity on transient transfection in human liver cells as compared to the AG haplotype. In order to understand the role of AA and AG haplotypes on hAGT expression in an in vivo situation, we have generated double transgenic mice containing either haplotype AA or AG of the hAGT gene (using knock‐in strategy at the HPRT locus) and human renin gene. Our studies show that transgenic mice containing AA haplotype have 1.6 fold increase in the AGT mRNA level in the liver as compared to the transgenic mice containing AG haplotype. In addition, Western blot and immunohistochemistry shows that liver and kidney of transgenic mice containing AA haplotype have increased hAGT protein level as compared to AG haplotype. In addition, blood pressure of three months old male transgenic mice containing AA haplotype is increased by about 8‐10 mmHg as compared to the transgenic mice containing AG haplotype. Supported by NHLBI.

Antidiuretic Effects of Endothelin A Receptor Antagonism

Selective ET‐A receptor antagonism has diuretic and natriuretic effects which may be potentiated by the unopposed activation of ET‐B receptors by endogenous endothelin. A major side effect of selective ET‐A receptor antagonists is fluid retention and edema. We hypothesized that compounds with high selectivity against ET‐A receptors produce fluid retention at concentrations where non‐specific blockade of ET‐B receptors may occur. Incremental doses of the ET‐A receptor antagonist SPP301 (0.003; 0.03; 3 mg/kg; lower dose to target ET‐A receptor, highest dose to target both ET‐A and ET‐B receptors based on a CHO cell assay) were administered i.v. to anesthetized rats (SD, n=6) undergoing saline diuresis (15 mL/kg/h, 1% BSA). Urine was collected from the bladder while blood pressure (BP) and glomerular filtration rate (GFR) were monitored. SPP301 decreased urine output (49; 46; 32 ul/min, p&lt;0.05) and fractional excretion of water (6; 5; 4 %, p&lt;0.05) in a concentration‐dependent manner. Sodium excretion was initially increased but returned to control values after the highest dose of SPP301. GFR was unchanged while BP was reduced by 10 mmHg only by the highest dose of SPP301. Administration of BQ‐788 (ET‐B receptor antagonist, 3mg/kg) after SPP301 3 mg/kg did not further decrease urine output or water excretion and was without effect on GFR. The highest dose of SPP301 caused a decrease of both 24‐hour diuresis (18.3%) and natriuresis (17.6%) with a consequent hematocrit decrease (8.3%) in a hypertensive transgenic model expressing both human renin and angiotensinogen genes. These data indicate that increasing concentrations of SPP301 may also block ET‐B receptors and cause antidiuresis. This effect could explain why fluid retention and edema occur during treatment with selective ET‐A receptor blockers.

Rosuvastatin protects against angiotensin II-induced renal injury in a dose-dependent fashion

We showed earlier that statin treatment ameliorates target-organ injury in a transgenic model of angiotensin (Ang) II-induced hypertension. We now test the hypothesis that rosuvastatin (1, 10, and 50 mg/kg/day) influences leukocyte adhesion and infiltration, prevents induction of inducible nitric oxide synthase (iNOS), and ameliorates target-organ damage in a dose-dependent fashion. We treated rats harboring the human renin and human angiotensinogen genes (dTGR) from week 4 to 8 (n = 20 per group). Untreated dTGR developed severe hypertension, cardiac hypertrophy, and renal damage, with a 100-fold increased albuminuria and focal cortical necrosis. Mortality of untreated dTGR at age 8 weeks was 59%. Rosuvastatin treatment decreased mortality dose-dependently. Blood pressure was not affected. Albuminuria was reduced dose-dependently. Interstitial adhesion molecule (ICAM)-1 expression was markedly reduced by rosuvastatin, as were neutrophil and monocyte infiltration. Immunohistochemistry showed an increased endothelial and medial iNOS expression in small vessels, infiltrating cells, afferent arterioles, and glomeruli of dTGR. Immunoreactivity was stronger in cortex than medulla. Rosuvastatin markedly reduced the iNOS expression in both cortex and medulla. Finally, matrix protein (type IV collagen, fibronectin) expression was also dose- dependently reduced by rosuvastatin. Our findings indicate that rosuvastatin dose- dependently ameliorates angiotensin II-induced-organ damage and almost completely prevents inflammation at the highest dose. The data implicate 3-hydroxy-3-methylglutaryl coenzyme A function in signaling events leading to target-organ damage.

Transcriptional Regulatory Functions of Mammalian AP-Endonuclease (APE1/Ref-1), an Essential Multifunctional Protein

The mammalian AP-endonuclease (APE1/Ref-1) plays a central role in the repair of oxidized and alkylated bases in mammalian genomes via the base excision repair (BER) pathway. However, APE1, unlike its E. coli prototype Xth, has two unique and apparently distinct transcriptional regulatory activities. APE1 functions as a redox effector factor (Ref-1) for several transcription factors including AP-1, HIF1-alpha, and p53. APE1 was also identified as a direct trans-acting factor for repressing human parathyroid hormone (PTH) and renin genes by binding to the negative calcium-response element (nCaRE) in their promoters. We have characterized APE1's post-translational modification, namely, acetylation which modulates its transcriptional regulatory function. Furthermore, stable interaction of APE1 with several other trans-acting factors including HIF-1alpha, STAT3, YB-1, HDAC1, and CBP/p300 and formation of distinct trans-acting complexes support APE1's direct regulatory function for diverse genes. Multiple functions of mammalian APE1, both in DNA repair and gene regulation, warrant extensive analysis of its own regulation and dissection of the mechanisms. In this review, we have discussed APE1's own regulation and its role as a transcriptional coactivator or corepressor by both redox-dependent and redox-independent (acetylation-mediated) mechanisms, and explore the potential utility of targeting these functions for enhancing drug sensitivity of cancer cells.

Activation of Renin-Angiotensin System Induces Osteoporosis Independently of Hypertension

Hypertension and osteoporosis are two major age-related disorders; however, the underlying molecular mechanism for this comorbidity is not known. The renin-angiotensin system (RAS) plays a central role in the control of blood pressure and has been an important target of antihypertensive drugs. Using a chimeric RAS model of transgenic THM (Tsukuba hypertensive mouse) expressing both the human renin and human angiotensinogen genes, we showed in this study that activation of RAS induces high turnover osteoporosis with accelerated bone resorption. Transgenic mice that express only the human renin gene were normotensive and yet exhibited a low bone mass, suggesting that osteoporosis occurs independently of the development of hypertension per se. Ex vivo cultures showed that angiotensin II (AngII) acted on osteoblasts and not directly on osteoclast precursor cells and increased osteoclastogenesis-supporting cytokines, RANKL and vascular endothelial growth factor (VEGF), thereby stimulating the formation of osteoclasts. Knockdown of AT2 receptor inhibited the AngII activity, whereas silencing of the AT1 receptor paradoxically enhanced it, suggesting a functional interaction between the two AngII receptors on the osteoblastic cell surface. Finally, treatment of THM mice with an ACE inhibitor, enalapril, improved osteoporosis and hypertension, whereas treatment with losartan, an angiotensin receptor blockers specific for AT1, resulted in exacerbation of the low bone mass phenotype. Thus, blocking the synthesis of AngII may be an effective treatment of osteoporosis and hypertension, especially for those afflicted with both conditions.