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

Background: Glucocorticoids modulate the RAS and cause hypertension. Results: SNPs in the hAGT promoter form two haplotypes, −6A and −6G. Transgenic mice with haplotype −6A respond to dexamethasone with tissue-specific up-regulation of hAGT, increased plasma AngII, and hypertension. Conclusion: Haplotypes of the hAGT gene govern transcriptional response to dexamethasone. Significance: Polymorphisms in hAGT provide for genetic predisposition to glucocorticoid-induced hypertension. 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.

blood pressure is accompanied by significantly higher hAGT expression in Hap Ϫ6A mice, with no significant change observed in the mAGT gene. The contributions of the SNP-dependent variable transcriptional regulation of the hAGT gene are highlighted by these findings.
In this regard, glucocorticoids are well known regulators of the AGT gene, especially in the liver and adipose tissue (8,9). Increased glucocorticoid levels cause hypertension (10), mechanisms for which could include increased expression of the AGT gene. Contextually, SNPs in the "cis" element of the genes, such as the hAGT, can promote varying affinity for the GR and thus be amenable to modulation by pathophysiological levels of glucocorticoids. Differential gene regulation by endogenous or exogenous glucocorticoids, via activation of the glucocorticoid receptors (GRs), is an area of intense current research. Importantly, we have shown that the polymorphisms in Hap Ϫ6A positively affect the binding of transcriptional regulators, such as the glucocorticoid receptors, to the promoter region of the hAGT gene (3). In this study, we tested the hypothesis that glucocorticoids via activation of GR will preferentially induce the hAGT gene in Hap Ϫ6A TG mice with, consequentially, an increase in blood pressure. We show here that dexamethasone (DEX) increases hAGT gene expression via increased GR binding to the promoter of the hAGT gene in TG mice with Hap Ϫ6A, as compared with the Hap Ϫ6G. Crucially, DEX-dependent induction of the hAGT gene is accompanied by significant blood pressure elevation in TG mice with Hap Ϫ6A, an effect reversed by angiotensin receptor blockade.

MATERIALS AND METHODS
Generation of TG Mice-All animal experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the institutional animal care and use committee at the University of Toledo. The TG mice utilized in this study were generated in our laboratory as described previously by Jain et al. (3). Genotyping analysis of the tail snips, followed by sequencing, was performed to confirm the genetic lineage of these TG mice. As described previously, the TG mice with Hap Ϫ6A have variants Ϫ6A, Ϫ20A, Ϫ217A, Ϫ532T, Ϫ793A, Ϫ1074T, Ϫ1178G, Ϫ1561T, Ϫ1562C, and Ϫ1670A, whereas TG mice with Ϫ6G Hap have variants Ϫ6G, Ϫ20A, Ϫ217G, Ϫ532C, Ϫ793G, Ϫ1074G, Ϫ1178A, Ϫ1561G, Ϫ1562G, and Ϫ1670G. Because hAGT is not cleaved by the mouse renin, we generated double TG mice containing either Hap Ϫ6A or Ϫ6G of the hAGT gene and human renin gene (made available by Dr. Curt Sigmund). For experimental purposes, these TG mice were housed individually. Twelve-week-old double TG mice containing Hap Ϫ6A and Ϫ6G each were divided into two groups (n ϭ 6). For both Haps, experiments were performed either in the presence or absence of DEX.
DEX Treatment-Twelve-week-old male double TG mice containing Hap Ϫ6A or Ϫ6G, along with age-matched C57/ BL6 mice, were divided into two groups (n ϭ 6). TG mice were caged individually. DEX, at a dosage of 2.5 g/ml in drinking water, was administered to the TG mice for 72 h. The low dose of DEX was selected to avoid the effect of DEX on the endogenous renin-angiotensin-aldosterone axis. Complementary experiments were performed with high-dose (12.5 g/ml) and low-dose (0.25 g/ml) DEX, examining effects on blood pressure of TG mice. Animals were maintained in a 22°C room with a 12-h light/dark cycles and received standard chow and DEX or vehicle in drinking water ad libitum. No significant difference was observed in the volume of drinking water or DEX solution consumed by TG mice with either Hap Ϫ6A or Hap Ϫ6G and non-TG C57/BL6 mice.
Quantitative RT-PCR-The liver and the kidneys were harvested at the end of DEX treatment and snap-frozen in liquid nitrogen. The extracted tissues were stored at Ϫ80 C until utilized for further experiments. RNA was isolated using RNeasy Plus minikit (Qiagen). RNA (1 g) was reverse-transcribed into cDNA using the Revert Aid first strand cDNA synthesis kit (Fermentas), as described in the manufacturer's protocol. Following a 95°C incubation for 10 min, 40 cycles of PCR (95°C for 30 s, 60°C for 30 s), were then performed using 1 l of cDNA, 50 nM PCR primers, and 12.5 l of SYBR Green PCR Master Mix in 25-l reactions. Threshold cycles for three replicate reactions were determined using MxPro-Mx3005P software (version 4.10), and relative transcript abundance was calculated following normalization with mouse GAPDH. Primers for human and mouse AGT and mouse GAPDH were obtained from Super Array Bioscience Corp. (Frederick, MD) or from Integrated DNA Technologies (Coralville, IA). In general, relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample, such as untreated control. This is a widely used method to present relative gene expression by the comparative C T method, also referred to as the 2 Ϫ⌬⌬CT method.
Immunoblot Analysis-Protein extracts were prepared from the liver, kidneys, and plasma collected from the TG and C57/ BL6 mice at the end of the experiment. Protein extracts (25 g) were separated by SDS-PAGE (12% polyacrylamide) and transferred to 0.45-m PVDF membranes (Millipore, Billerica, MA) for 1 h. The membranes were incubated with either hAGT (1:2000) (catalog no. 3249Ϫ1, Epitomics, Burlingame, CA) or mAGT (catalog no. 28101, IBL, Takasaki, Japan) primary antibody. Blots were developed using an infrared imaging system (Odyssey, LI-COR Biosciences, Lincoln, NE). The results for the liver and the kidneys were normalized to mouse ␤-actin (A2228, Sigma) and those for plasma were normalized to mouse albumin (1:10,000) (NB600-41532, Novus Biologicals).
In Vivo Chromatin Immunoprecipitation (ChiP) Analysis-The ChIP assay was performed using the EZ-ChIP assay kit from EMD Millipore. Mice were perfused with normal saline, and the liver and the kidneys were removed, washed, and fixed with 1% formaldehyde for 20 min at room temperature. The DNA was fragmented by sonication, and 10 l of the chromatin solution was saved as input. 5 g of anti-glucocorticoid receptor (anti-GR) or rabbit immunoglobulin G were added to the tubes containing 900 l of sonicated chromatin solution; the mixture was incubated overnight at 4°C. The antibody complexes were captured with the protein A-agarose beads and subjected to serial washes (as described in the manufacturer's protocol). The chromatin fraction was extracted with SDS buffer and reverse cross-linked at 65°C for 4 -6 h. The DNA was then purified as described in the manufacturer's protocol.
The immunoprecipitated DNA (1 l) and the input DNA (1 l) were subjected to 35 cycles (of PCR amplification (denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s) using (a) Ϫ314AGTfor (CTCAGGCTGTCACACAC-CTA) as a forward and Ϫ6AGTrev (TCTTCCCCTGGC-CGGGTCACGAT) as a reverse primer, when GR antibody was used to examine the binding of GR at the Ϫ217 position of the hAGT gene, or (b) Ϫ1757AGTfor (CAGGCACAGTGG-AAACTCTCC) as a forward primer and Ϫ1554AGTrev (AGTAACAAGTCCACCTGGAC) as a reverse primer, when GR antibody was used to examine the binding of GR across the Ϫ1561 and Ϫ1562 polymorphic sites of the hAGT gene. The PCR-amplified products were analyzed on 2% agarose gel. The relative band intensities by densitometry were measured using Quantity One quantitation software from Bio-Rad, and the results were normalized to the band intensities from the respective input DNA. The fraction enriched by rabbit IgG was used as a negative control for nonspecific binding. A primer set that amplified the DNA fragment of the hAGT gene with no known glucocorticoid response element was used as a nonspecific control for GR-enriched DNA fragments.
Measurement of Plasma Angiotensin II-Plasma angiotensin-II levels were determined by an ELISA kit purchased from Ray Biotech, Inc. The angiotensin II concentration in the samples was determined directly from the standard curve according to the manufacturer's protocol as described previously (3).
Blood Pressure Measurement in TG Mice-Blood pressure was measured in the conscious state by radio telemetry as described previously (3). Systolic blood pressure was continuously acquired by implantation of telemetric probe PA-C20 into the aorta via the left carotid artery. After 1 week of recovery from the surgical procedure, blood pressure readings were recorded every 10 min using Data-Dataquest ART software purchased from Data Science International. Baseline blood pressure was collected for 7 days, followed by blood pressure measurements in response to DEX treatment, with and without losartan.
Statistical Analyses-All experiments were conducted with six animals in each group. Data are expressed as the means Ϯ S.E. Statistical significance was assessed using two-way analysis of variance with a Tukey-Kramer post hoc analysis. The significance level was set at (p Ͻ 0.05).

DEX Treatment Promotes Greater GR Binding to the Chromatin of Ϫ6A Hap Mice-The hAGT gene polymorphisms at
Ϫ217 and Ϫ1561 in Ϫ6A HapTG mice have stronger homology with consensus GRE as compared with Hap Ϫ6G (Fig. 1); therefore, we examined the effect of DEX on the binding of GR to the Ϫ217 and Ϫ1561/Ϫ1562 region of the hAGT gene in both TG animals. A ChIP assay was performed using GR-specific antibody on chromatin extracts from hepatic and renal tissues of the TG animals. As shown in Fig. 2, GR binds more strongly to the chromatin obtained from hepatic tissue of TG mice with Hap Ϫ6A, as compared with Hap Ϫ6G. This effect is further increased by DEX treatment, where GR enrichment at these sites increases further (p Ͻ 0.05) only in TG mice containing Hap Ϫ6A. Similar results were observed in chromatin isolated from renal tissues of these TG animals (Fig. 3). These results show increased GR binding to the hAGT promoter in the liver and kidney of TG mice containing Hap Ϫ6A, an effect accentuated by treatment with a GR agonist. The effect of DEX on the chromatin of TG mice containing Hap Ϫ6G was not statistically significant. Our previous transfection assays have shown that DEX treatment slightly increased the promoter activity of reporter construct containing Ϫ6G Hap. However, this small increase in the promoter activity of Ϫ6G Hap may be attributed to co-transfection of GR in these experiments.
DEX Treatment Increases hAGT Expression in Liver and Kidneys of Ϫ6A Hap Mice-Transcriptional effects of the increased GR-GRE interaction were examined by gene expression analysis. Liver is the major source of circulating AGT, and renal production of AGT governs the tissue RAS. Together, plasma AGT and tissue RAS critically regulate the extracellular volume and long term set point of the mean arterial pressure. Liver (Fig.  4, A and C) and renal (Fig. 4 DEX Treatment Increases Plasma hAGT and AngII in Ϫ6A Hap Mice-In order to discern the systemic effects of increased tissue AGT, caused by DEX treatment, we examined hAGT levels in plasma of our TG mice. As seen in Fig. 6, A and B, baseline plasma hAGT is significantly (p Ͻ 0.05) increased in TG mice containing Hap Ϫ6A of the hAGT gene. DEX treat-  FEBRUARY 27, 2015 • VOLUME 290 • NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5751 ment significantly (p Ͻ 0.05) increased plasma hAGT levels in TG mice with Hap Ϫ6A while having no effect on the hAGT levels in plasma of TG mice with Hap Ϫ6G. Importantly, this low-dose DEX treatment had no significant effect on the endogenous mAGT gene in either Hap Ϫ6A (densitometric analyses with normalization to albumin: without DEX ϭ 0.22 Ϯ 0.01 versus with DEX ϭ 0.30 Ϯ 0.06) or Hap Ϫ6G (without DEX ϭ 0.20 Ϯ 0.02 versus with DEX ϭ 0.22 Ϯ 0.03) or in the C57/BL6 mice (without DEX ϭ 0.33 Ϯ 0.03 versus with DEX ϭ 0.42 Ϯ 0.02).

Dexamethasone, Human Angiotensinogen Gene, and Hypertension
Classically, renin secretion by the juxtaglomerular apparatus has been viewed as the rate-limiting step in conversion of AGT to ANGII. However, recent studies confirm a causal link between increased AGT and increased ANGII levels, even in the absence of renin modifications. To this end, we analyzed plasma ANGII levels in our TG mice and found them to be elevated (p Ͻ 0.05) in TG mice with Hap Ϫ6A (Fig. 6C). Moreover, DEX treatment brought about further increase in ANGII levels in Ϫ6A animals only, results corroborating our hypothesis of DEX-induced modulation of the hAGT levels in this Hap (results from individual experiments for AngII levels are provided as a dot plot in supplemental Fig. S3). AngII levels were significantly (p Ͻ 0.05) lower in C57/BL6 mice (145 Ϯ 27 pg/ml) and were not amenable to modulation by our low-dose DEX regimen (with DEX: 179 Ϯ 32 pg/ml).
Effects of DEX Treatment on Physiological Parameters, Including Blood Pressure and Tissue Inflammation, in the Two TG Lines-Pathophysiological implications of DEX-induced elevated AGT, with consequential effects on ANGII, were eval-uated at systemic and local levels. RAS increased mean arterial pressure, in the short term by vasoconstriction and in the long term by renal mechanisms; ANGII, also, activated the oxidative cascade, and both effects were AT1 receptor-mediated. We observed that TG mice with Hap Ϫ6A had significantly increased systolic blood pressure (SBP) as compared with Hap Ϫ6G at resting conditions. We next analyzed the effect of DEX on the SBP of both of our TG mice with Hap Ϫ6A and Ϫ6G. Fig.  7B shows that the elevation in SBP (⌬) after treatment with DEX (2.5 g/ml) in TG mice with Hap Ϫ6A was significantly higher as compared with Hap Ϫ6G after 24, 48, and 72 h of data collection (results from individual experiments are shown on a dot plot; supplemental Fig. S4A). Importantly, the DEX-induced increase in SBP did not disrupt the regular diurnal variation (Fig. 7A). Each point in Fig. 7A represents the hourly mean from six male animals taken over a period of 24 h. The hourly mean was taken from the average of six SBP data points at an interval of 10 min each. It is important to note that although DEX increased blood pressure in C57/BL6 mice (without DEX ϭ 94 Ϯ 6 mm Hg versus with DEX ϭ 103 Ϯ 4 mm Hg), the results did not achieve statistical significance.
We further increased the dosage of DEX to 12.5 g/ml in drinking water to investigate whether the DEX-dependent differential regulation of the blood pressure is maintained at higher dosages. We found a steep rise in the SBP of TG mice containing Hap Ϫ6A, raising it to ϳ165 mm Hg, whereas the TG mice with Hap Ϫ6G showed a maximum of ϳ140 mm Hg (Fig. 7C). DEX-dependent changes in SBP were prevented in animals concurrently treated with an AT1 receptor blocker (losartan) (Fig. 7D and dot plot in supplemental Fig. S4C). On the other hand, low-dose DEX had no significant effect on blood pressure in any treated group (supplemental Fig. S4B). These experiments implicate a DEX-dependent increase in AGT and ANGII in bringing about changes in SBP in TG mice with Hap Ϫ6A. In line with historical evidence, complementary studies showed increased (p Ͻ 0.05) levels of oxidative markers (NOX1 (NADPH oxidase)) and attenuation (p Ͻ 0.05) of antioxidant defenses (SOD1 (superoxide dismutase type I)) in animals of the Ϫ6A Hap undergoing DEX treatment (Fig. 8).
CEBP␤ expression is measured as a negative control to ascertain the specificity of haplotype-dependent effects of DEX on the hAGT, in our transgenic lines. As shown in Fig. 8C, hepatic CEBP␤ expression is up-regulated by DEX treatment in all three groups, C57, Hap Ϫ6G, and Ϫ6A TG mice. Importantly, the DEX-induced increase in CEBP␤ is similar in all treated groups (dot plot analysis in supplemental Fig. S5). These results corroborate that the stimulation of hAGT expression observed in response to DEX in Hap Ϫ6A mice is indeed haplotype-dependent. These results are a clear indication of the susceptibility of the Ϫ6A Hap to DEX-induced RAS activation and associated pathophysiologic alterations.

DISCUSSION
Glucocorticoid therapy is the cornerstone of treatment of diseases with chronic inflammation. DEX is widely employed in the clinics and is as frequently associated with pharmacological  side effects, including osteoporosis, secondary infections, diabetes, preeclampsia, and hypertension. Hypertension can be caused by RAS overactivity (11). The AGT gene is frequently linked with hypertension in humans, especially the M235T locus that is in LD with the Ϫ6 site on the hAGT promoter (12,13). The novelty of the current study lies in the observation that low-dose GR agonist has Hap-dependent transcriptional effect on the hAGT gene with consequential activation of the RAS. This translates into increased blood pressure and RAS-associated pathological outcomes in TG mice with the Ϫ6A Hap of the hAGT gene.
The first key finding of the study confirms agonist-induced GR binding to the hAGT gene, in vivo, in a Hap-regulated fashion. We show here, for the first time, increased GR receptor binding to the hAGT promoter at the Ϫ217 and Ϫ1561/62 sites of the Ϫ6A Hap. These results are in line with our published in  vitro reports showing GREs in the hAGT gene that bind GR with increased affinity when the former has SNP variants associated with Ϫ6A and Ϫ217A (3, 14) (see our previous in vitro studies (3)). Other investigators have established both, in vitro and in vivo, glucocorticoid-mediated regulation of the AGT gene (15). Glucocorticoids have been shown to increase AGT expression in the liver and adipocytes (16 -18). However, this is the first report characterizing glucocorticoid-induced, Hap-dependent regulation of the hAGT gene, especially in the kidneys. The SNPs at the Ϫ217, Ϫ1561, and Ϫ1562 sites in the Hap Ϫ6A provide greater homology for the GRE consensus sequence and promote stronger GR binding. The ChIP assay clearly demon- FIGURE 7. Blood pressure analysis in TG mice. The SBP of transgenic mice with haplotype Ϫ6A is significantly higher as compared with that of haplotype Ϫ6G after DEX treatment (A) over a 24-h time period. Each point represents the hourly mean from six male animals taken over 24 h. The hourly mean was taken from the average of six SBP data points at an interval of 10 min. B, change in systolic blood pressure over baseline after DEX treatment in Hap Ϫ6A and Ϫ6G mice; readings are averaged at 24, 48, and 72 h post-DEX (2.5 g/ml in drinking water ad libitum) treatment. The difference in the elevation of SBP is further increased upon treatment with 12.5 g/ml DEX (C). The DEX-induced increase in SBP was alleviated by treatment with the angiotensin type 1 receptor blocker losartan (30 mg/kg/day). Results are shown as mean Ϯ S.E. (error bars) (n ϭ 6). *, p Ͻ 0.05 versus Hap Ϫ6G; †, p Ͻ 0.05 versus Hap Ϫ6A in the absence of losartan.
strates increased agonist-induced GR binding to these sites in the Ϫ6A Hap. DEX is a specific agonist for GR and has been widely used as a pharmacological intervention to study the effects of GR activation. Importantly, increased GR binding is not tissue-specific and is observed in both hepatic and renal tissues.
DEX-dependent increased GR binding to the hAGT promoter of the Ϫ6A Hap is reflected in significantly higher hAGT expression in these mice. Hepatic and renal tissues both exhibit higher hAGT expression. Apart from promoting stronger GR binding, we have previously shown that SNPs in the promoter of Hap Ϫ6A create a strong binding site for HNF-1␣ and C/EBP (3). Glucocorticoid treatment also affects the regulation of genes that contain C/EBP binding sites (19,20), by increasing the synthesis of C/EBP␤ and C/EBP␦ via GR in the hepatocytes (21). Also, GR and HNF-1 act cooperatively and synergistically to increase DEX-induced expression of genes regulated by GR (22). Thus, the increased agonist-induced hAGT expression in Ϫ6A TG mice could be contributed by direct GR binding along with favorable interactions with other transcription factors, including C/EBP and HNF-1. It is noteworthy that the Hap-dependent binding of GR, preferentially to the Ϫ6A Hap, is observed in a setting of low-dose DEX treatment. The endogenous mAGT gene does not contain SNPs to modulate differential GR, HNF-1, or C/EBP binding. Contextually, we did not observe differential regulation of the mAGT gene, which lends credibility to the hypothesis of Hap-regulated GR-GRE binding of the hAGT. However, Clouston et al. (23) have previously shown DEX-induced up-regulation of the mAGT gene in their animal model. Their use of a high dose of DEX (20-fold higher) could partly explain the differences in results. We specifically designed our experiments to emulate pathophysiological settings where endogenous levels of glucocorticoids could be elevated in order to detect even small changes in the Hap-depen- DEX-induced tissue hAGT overexpression is the second key finding of the study. Importantly, this effect is brought about by a low dose of DEX. Elevated levels of plasma hAGT accompany agonist-induced increased GR binding to and subsequent hAGT induction in the liver. An immunoblotting assay for measurement of plasma hAGT level demonstrates significant DEX-induced expression of the hAGT protein in TG mice with Hap Ϫ6A, as opposed to no significant change in Hap Ϫ6G. This is not unexpected because the liver is the primary source of circulating AGT, and DEX treatment induces hAGT in the liver of the Ϫ6A TG mice. The catalytic action of renin on the AGT is essential for synthesis of decapeptide AngI that is subsequently cleaved by ACE to generate the potent effector octapeptide AngII. The classical paradigm of the RAS suggests AGT metabolism by renin as the rate-limiting step in AngII synthesis. However, an increase in blood pressure as a consequence of increased plasma AGT has been shown by Kim et al. (24). The concentrations of hAGT levels in the plasma are lower than the Michaelis constant (K m ) for renin (25), implying that the elevation of the plasma hAGT levels leads to an increased plasma AngII concentration. In this regard, our Western blot data show that the plasma AngII levels are significantly elevated in TG mice with Hap Ϫ6A after DEX treatment. Thus, the results underscore the significance of elevated AGT levels that in this case were brought on by differential GR regulation of the hAGT gene.
DEX-induced increased AngII in TG mice with Hap Ϫ6A leads to hypertension. The pathophysiological impact of the differential hAGT regulation by DEX is the third key finding of this study. RAS governs mean arterial pressure via AngII and aldosterone as the principal end effector molecules. Dual effects, vascular and renal, underlie the prohypertensive actions of RAS overactivity. In an acute setting, AngII causes vasoconstriction and increases the total peripheral resistance, thus increasing mean arterial pressure. Angiotensin type 1 receptor activation-dependent signaling brings about the vasoconstriction. Increased peripheral resistance, alone, is insufficient to sustain elevated mean arterial pressure for significant lengths of time because the renal pressure-natriuresis mechanism will be activated, thus promoting salt loss and reduction of elevated mean arterial pressure. However, up-regulation of renal AngII can shift the pressure-natriuresis curve to right and thus contribute to the prohypertensive effects of systemic AngII (26). In this light, significantly increased blood pressure in Hap Ϫ6A animals treated with DEX alludes to the acute effects of AngII. Additionally, DEX-induced up-regulation of the hAGT gene in the kidney of TG mice with Hap Ϫ6A may contribute toward increased blood pressure. TG mice with Ϫ6A Hap exhibit much higher GR binding, plasma hAGT, and plasma AngII after DEX treatment. A reasonable conclusion of DEX-induced, AngIIdependent hypertension can be deduced for these TG mice. The duration of DEX treatment in our study (72 h) also suggests that these prohypertensive effects are primarily of vascular origin. Increased renal AngII, by alterations in the pressure-natriuresis response, could also play a secondary role in elevating blood pressure in the Hap Ϫ6A TG mice. It is important to note that although Hap Ϫ6G TG mice do not show elevated plasma AngII in response to DEX treatment, they do exhibit minor elevation in blood pressure. These seemingly counterintuitive observations could be explained by nonspecific mineralocorticoid receptor activation by DEX. GR agonists activate the mineralocorticoid receptor to varying extents, and DEX is no exception. Despite these nonspecific actions of DEX, elevated plasma hAGT and AngII are the primary contributors of hypertension in Ϫ6A TG mice. These conclusions are supported by the reversal of DEX-induced hypertension in these mice by an angiotensin type 1 receptor blocker. Further evidence of the principal role of AngII in DEX-induced hypertension, in Ϫ6A TG mice, is provided by the analyses of cellular redox markers. Modulatory effects of AngII on the redox pathways are well established (27,28). In line with reports from other investigators, we see increased NOX1 and decreased SOD1 in renal tissues from Ϫ6A animals, TG mice with DEX-induced RAS overactivity.
In conclusion, as modeled in Fig. 9, DEX treatment activates GR and modulates expression of genes containing the GRE. In this regard, SNP-directed Haps govern gene expression by modulating transcription factor binding, such as to the GR. The SNP cluster in the hAGT gene, marked by adenosine at the Ϫ6 site, predisposes to stronger GR binding by creating greater homology with the GRE consensus sequence. This provides for stronger GR-GRE interaction at these sites when challenged with an agonist. DEX is a potent GR agonist, and its treatment results in increased GR binding to Ϫ217 and Ϫ1561/62 sites of the hAGT gene containing SNPS marked by Hap Ϫ6A. Increased tissue and plasma AGT with RAS overactivity are a consequence, as are pathophysiologies like hypertension and inflammation in subjects with the Ϫ6A Hap of the AGT gene. Thus, appropriate patient screening to identify patients with FIGURE 9. Schematic depicting our hypothesis and conclusions; SNPs in the promoter of the hAGT gene with Hap ؊6A increase glucocorticoidinduced promoter activity. Upon exposure to glucocorticoids, the activated GR binds more strongly to the promoter of the hAGT gene in TG mice with Hap Ϫ6A. This augments expression of the hAGT gene in the liver and the kidneys. Plasma AngII levels are elevated as a result of increased hAGT expression, thereby increasing the systolic blood pressure.