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A Critical Role for Vascular Smooth Muscle in Acute Glucocorticoid-Induced Hypertension

Julie E. Goodwin^*, Junhui Zhang and David S. Geller

Section of Nephrology, Departments of ^* Pediatrics and Medicine, Yale University School of Medicine, New Haven, Connecticut

Correspondence: Dr. David S. Geller, P.O. Box 208029, Section of Nephrology, Yale University School of Medicine, New Haven, CT 06520-8029. Phone: 203-737-5298; Fax: 203-785-4904; E-mail: david.geller{at}yale.edu

Received for publication August 17, 2007. Accepted for publication February 4, 2008.

	Abstract

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Although glucocorticoid (GC)-induced hypertension has commonly been attributed to promiscuous activation of the mineralocorticoid receptor by cortisol, thereby promoting excess reabsorption of sodium and water, numerous lines of evidence indicate that this is not the only or perhaps even the primary mechanism. GC induce a number of effects on vascular smooth muscle (VSM) in vitro that may be pertinent to hypertension, but their contribution in vivo is unknown. To address this question, a mouse model with a tissue-specific knockout (KO) of the GC receptor in the VSM was created and characterized. Similar to control mice, KO mice exhibited normal baseline BP and, interestingly, showed normal circadian variation in BP. When dexamethasone was administered, however, the acute hypertensive response was markedly attenuated in KO mice, and there was a trend toward a decreased chronic hypertensive response. These data suggest that the GC receptor in VSM plays a critical role in the acute hypertensive response to GC in vivo.

	Introduction

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Glucocorticoids (GC), whether endogenous, as in Cushing syndrome, or exogenous, via pharmacologic provision, induce hypertension (HTN).^1–3 The lack of understanding of the underlying mechanisms of GC-induced HTN leads to empiric and often suboptimal treatment of this common clinical condition. Although traditionally GC have commonly been believed to increase BP via promiscuous activation of the mineralocorticoid receptor (MR) in the kidney,^4,5 available data argue that this is not the only or perhaps even the primary mechanism underlying this form of HTN.^6–8

In recent years, a wide variety of hypotheses have been suggested to explain how GC may regulate BP. Many of these have focused on vascular effects of GC. There is evidence to confirm the presence of the GC receptor (GR) in vascular smooth muscle (VSM) cells^6,9 and in the vascular endothelium (VE).^6,9,10 In vitro studies in smooth muscle cells have demonstrated that GC induce an upregulation of angiotensin type 1 receptors⁸ and alter Na⁺ and/or Ca²⁺ influx into the cells.⁹ Similarly, in the VE, GC trigger a reduction in nitric oxide release and a concomitant reduction in BP and cholinergic-induced vasodilation.¹¹ The contribution of these findings to the chronic elevation of BP induced by GC, however, is uncertain, because GC cause a variety of relevant effects within the body. To date, there has not been a reliable way to assess the role of VSM GR or VE GR in vivo. As a result, there is much that is still poorly understood about vascular responses to GC.

We previously demonstrated that administration of dexamethasone (DEX) in the drinking water induces a rapid and sustained elevation of BP in wild-type (WT) mice.¹² Furthermore, we showed that this response is mediated via GR and is substantially independent of MR.¹² To investigate the role of VSM in the hemodynamic response to GC, we generated mice in which GR has been specifically removed from VSM using a Cre-loxP approach and assessed basal BP as well as the hemodynamic response of these mice to GC administration.

	RESULTS

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Specific VSM GR Knockout
Generation of mice with GR knockout (KO) in VSM was achieved in our laboratory through the use of an Sm-22 Cre model, which has been reported to confer approximately 80% excisional efficiency.¹³ Sm-22 Cre+ GR^loxP/loxP and Cre– littermate controls were born in the expected 1:1 ratio. To verify the effectiveness of tissue-specific GR excision, sections of mouse aorta and mouse kidney from Sm-22 Cre+ and Cre– mice were stained with a commercial antibody to GR and counterstained with an antibody to -smooth muscle actin, a VSM-specific antibody¹⁴ (Figure 1A). Control mice showed robust staining of GR in the smooth muscle layers. In contrast, Sm-22 Cre+ GR^loxP/loxP mice demonstrated minimal presence of GR in VSM by double staining, indicating that we had achieved a high level of GR excision from the VSM layer. A similar staining pattern was also observed in the renal arteriole (Figure 1B) and the renal and mesenteric arteries (data not shown). To confirm that excision of GR staining was specific to this layer, we also stained these tissues with the endothelial-specific anti-CD31 (Figure 1C). GR staining was preserved in the vascular endothelial layer, confirming that excision of GR from the vascular wall was specific to the smooth muscle layer.

View larger version (90K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Specific GR deletion from VSM. (A and B) Representative comparisons of VSM GR in Cre– and Sm-22 Cre+ GRloxP/loxP mice. Four-micrometer sections of mouse aorta (A) and mouse kidney arterioles (B) were stained with a commercial antibody to GR shown in red and an antibody to -smooth muscle actin, which is specific to the VSM, shown in green. The merged images show clear co-localization of GR in the VSM layer in Cre– mice. In Cre+ mice, this co-localization is almost entirely lost, indicating specific and highly efficient excision of GR from the VSM. (C) Preservation of the VE in Sm-22 Cre+ GRloxP/loxP mice. Higher power view of 4-µm sections of mouse kidney arterioles, which were stained with a commercial antibody to GR (red) and an antibody to CD31 (green). The merged image demonstrates preservation of GR in the endothelial layer in Sm-22 Cre+ mice. Images shown are representative of four animals per group.

To determine whether there was a BP phenotype in these mice, radiotelemetric catheters were implanted in male Sm-22 Cre+ GR^loxP/loxP mice (n = 7) and gender-matched littermate controls (n = 9), and mice were allowed to recover for 1 wk. Adequacy of recovery was verified by the return of normal diurnal variation of BP for 3 d. There was no difference between the mean arterial pressure (MAP), averaged over 24 h, of Sm-22 Cre+ GR^loxP/loxP and control mice at baseline (107.5 ± 2.4 versus 107.8 ± 1.2 mmHg; P = 0.91). There were also no differences between the mean systolic BP (SBP) of Sm-22 Cre+ GR^loxP/loxP and control mice averaged over 24 h (123.6 ± 2.9 versus 124.1 ± 1.9 mmHg; P = 0.89) or the mean diastolic BP (DBP) averaged over 24 h (94.9 ± 2.7 versus 90.8 ± 1.1 mmHg; P = 0.16). These data suggest that VSM GR does not play a major role in the basal regulation of BP.

A Delayed BP Response to DEX in Sm-22 Cre GR KO Mice
To determine the effect of the Sm-22 Cre GR KO in a model of GC-induced HTN, we administered DEX 15 mg/L in the drinking water and monitored BP continuously for 1 wk. This dosage was used because previous studies in our laboratory have shown that smaller dosages do not reliably produce BP elevation of >10 mmHg after 2 wk of treatment in these mice, a rise in BP that we believed was necessary for further study (data not shown). On the basis of an average mouse weight of 30 g and an average daily water intake of approximately 2 to 3 ml, we estimate that this dosage provided each mouse with approximately 2 mg/kg per d DEX.

A representative tracing from each group is shown in Figure 2, and cumulative data for each group are shown in Figure 3. As we have noted previously,¹² there was a consistent and highly significant rise in BP within hours of the initiation of DEX therapy in control (Cre–) mice. In contrast, the rise in BP was markedly attenuated in Sm-22 Cre+ GR^loxP/loxP littermates. On the first day of DEX treatment, MAP, averaged over 24 h, increased by 5.05 ± 1.1 mmHg in control mice compared with a rise of only 1.9 ± 0.9 mmHg in Sm-22 Cre+ mice (P = 0.041).

View larger version (29K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Differential BP response to DEX in Sm-22 Cre+ GRloxP/loxP versus Cre– control. (A and B) Representative BP tracing of a control mouse (A) and an Sm-22 Cre+ GRloxP/loxP mouse (B) in response to DEX 15 mg/L in the drinking water. The top line is SBP, and the bottom line is DBP. Each point represents the time-averaged BP over a 4-h period. Arrows indicate the time period from 8 p.m. to midnight, usually the time of peak circadian BP variation in the nocturnal mouse. Both the Cre+ and Cre– mice demonstrate a clear pattern of circadian variation, with the peak BP generally occurring at or near the period of 8 p.m. to midnight. After the initiation of DEX, there is a clear loss of normal diurnal variation in each of these mice; however, whereas BP rises rapidly and substantially in the Cre– mouse, the rise in BP in the Cre+ mouse is attenuated.

View larger version (16K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Attenuated GC-induced acute BP response in Sm-22 Cre+ GRloxP/loxP mice. Controls (n = 9) and Sm-22 Cre+ GRloxP/loxP mice (n = 11) were treated with DEX 15 mg/L in the drinking water for 1 wk, and BP was monitored continuously by catheter. In selected pairs (n = 3), BP monitoring continued for 2 wk. The change in mean arterial BP averaged over 24 h is indicated. **P < 0.05, ***P < 0.001.

Guytonian models of BP regulation propose that over a period of days to weeks, renal handling of sodium will compensate for extrarenal stimuli altering BP. To determine whether the difference in BP we observed over 1 wk continued over time, we maintained DEX treatment for a second week in three pairs of mice. BP measured at 14 d showed that Sm-22 Cre+ GR^loxP/loxP mice maintained a MAP of 121.0 ± 5.3 mmHg and controls maintained a MAP of 119.2 ± 3.1 mmHg (P = 0.36), suggesting that Sm-22 Cre+ GR^loxP/loxP mice likely attain the same rise in BP observed in the controls with continued DEX therapy.

Cardiac GR Does not Account for the Observed BP Phenotype of Sm-22 Cre+ GR^loxP/loxP Mice
Previous studies that have used Sm-22 Cre in the generation of transgenic mice have suggested that Sm-22 Cre is expressed at high levels in murine cardiac muscle¹⁵; however, in the Sm-22 Cre model we used, only minimal cardiac expression was observed.¹³ To determine whether GC effects on cardiac muscle could underlie the BP phenotype we observed in our model, we assessed GR staining in the cardiac muscle of Sm-22 Cre+ GR^loxP/loxP and control mice and found no significant difference between Cre+ and Cre– littermates (Figure 4A). On Western blot analysis of GR protein from hearts of control and Sm-22 Cre+ GR^loxP/loxP mouse pairs, we found that, if anything, heart GR was slightly increased and certainly not decreased in Sm-22 Cre+ GR^loxP/loxP mice (Figure 4B), suggesting that there was no significant GR deletion from cardiac myocytes in our KO mice. Consistent with this and previous studies,^15,16 we found no differences in heart rate between the two groups either before or after DEX administration (Figure 5).

View larger version (39K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Cardiac GR is not decreased in Sm-22 Cre+ GRloxP/loxP mice. (A) Preserved GR staining in the cardiac myocytes of Sm-22 Cre+ GRloxP/loxP mice. Four-micrometer sections of Sm-22 Cre+ GRloxP/loxP and control mouse heart were stained with a commercial antibody to GR. (B) Western blot of control and Sm-22 Cre+ mouse heart homogenates (n = 3/group) probed for GR. Total heart GR is significantly increased in Sm-22 Cre+ mice. C, control; KO, Sm-22 Cre+. **P < 0.05. Images shown are representative of three mice per group.

View larger version (12K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. No effect of VSM GR KO on the cardiac heart rate response to GC. Mean heart rate is not significantly different in Cre– (n = 11) or Sm-22 Cre+ GRloxP/loxP mice (n = 9) at baseline or during steroid treatment with DEX. GC administration induced a significant fall in heart rate (P < 0.005) that persisted for the duration of GC treatment. There was no significant difference in the heart rate response of the control and Sm-22 Cre+ GRloxP/loxP mice to DEX despite the attenuated BP response in the KO. All mean heart rates were averaged over a 24-h period.

	DISCUSSION

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To understand better the molecular origins of GC-induced HTN, we used emerging technologies in mouse genetics and mouse phenotyping to allow the in vivo study of GC-induced HTN. Here, we studied a mouse model in which GR has been excised specifically from the VSM. Our characterization of this model shows that growth, development, body weight, and baseline HR and BP are similar to controls; however, in response to steroids, the Sm-22 Cre+ GR^loxP/loxP mice have an attenuated response to DEX-induced hypertension which is most pronounced in the first 48 h after GC administration.

An important element of this study is the specificity we achieved with the model that we used. Our data indicate that we have achieved high levels of Cre-mediated recombination from the VSM with preservation of GR in the VE. Previous studies assessing vascular reactivity in response to GC have not reliably been able to dissect out the specific roles of endothelium and the smooth muscle. Experiments performed using vascular endothelial KO models have generally used vessels in which the VE is mechanically stripped and hence all functions of the endothelium are lost.^29,30 In our model, we demonstrated that GR is intact in the VE, so we believe it unlikely that primary vascular endothelial alterations are playing a role in the phenotype we have identified.

It should be noted that Sm-22 CreTg is highly expressed in a number of tissues with large smooth muscle components, such as the uterus and intestine; however, we believe that effects of GR deletion in these tissues are unlikely to explain the BP phenotype we have observed. Although some Sm-22 CreTg RNA expression has been observed in virtually all tissues studied, histologic evidence indicates this is due principally to the ubiquitous presence of vascular tissue in all organs.¹³ Although Sm-22 is highly expressed in cardiac tissue, we found the Sm-22 CreTg model that we used did not cause any discernible effect on GR expression in cardiac myocytes. Given the limited or absent expression of Sm-22 CreTg in extravascular tissues, we believe it is highly likely that the BP phenotype observed in our KO mice in response to DEX is primarily, if not wholly, attributable to the loss of GR from VSM.¹³

In recent years, much attention has been paid to the link between GC and cardiac output and indeed overall heart health; however, there are conflicting results in terms of the reported effects of GC on the cardiovascular system. Bal et al.³¹ observed reduced systolic function, stable cardiac output, and increased end-diastolic volume with compensatory dilation in neonatal rats treated with GC for prevention of lung disease. In a study of neonatal sheep, administration of DEX was found to increase expression of brainstem AT1 receptor mRNA and in turn to increase responsiveness to angiotensin II (AngII) compared with sheep that received exogenous cortisol. The authors concluded that the two GC had distinct programming actions and that prenatal DEX may lead to increased cardiac responsiveness later in life.³² One postnatal study in adrenalectomized adult rats showed a decrease in mean BP and cardiac output in rats that received only mineralocorticoid replacement compared with controls that received both mineralocorticoid and GC replacement.³³ Interestingly, these same rats demonstrated an impairment in urinary concentrating ability with decreased expression of aquaporin-2 and impaired countercurrent multiplication,³³ so it is difficult to sort out the effects of GC deficiency versus volume deficiency. Overall, it is unclear whether GC increase or decrease cardiac output; they may have both effects in particular settings. However, it is apparent that there is a complex interplay among GC, AngII and its receptor, and the cardiovascular response.

In addition to an attenuation of the acute BP response to GC, our data demonstrate that the Sm-22 CreTg KO mice ultimately do not achieve the same BP as Cre– littermates after 1 wk of treatment with DEX, although they seem eventually to catch up to the controls after 2 wk of therapy. This finding suggests an additional role of VSM GR in the sustained BP response to GC that will need to be studied further. Guytonian models of BP regulation posit that the sustained hypertensive response to GC requires the active participation of the kidney via alteration in renal sodium reabsorption. This renal response could occur via an alteration of GC-mediated changes in renovascular resistance as a result of the presence of GR in the renal vasculature or perhaps via an altered endocrine or neural stimulation of renal sodium handling. Such a response raises the possibility of differential effects of GC on resistance in different vascular beds.

To begin to understand the effects of GC on the kidney, we assessed acute alterations in urinary electrolyte excretion in our mice in response to GC challenge. We focused on acute alterations because the interpretation of net alterations in chronic sodium balance are difficult to measure and the interpretation of these data requires assumptions about intravascular and extravascular volume that may not be valid. High dosages of GC are known to trigger a rapid natriuresis in rodents, which is thought to be due to the rapid elevation of BP that such dosages induce. To our surprise, GC triggered an acute natriuresis not only in our Cre– mice, in which we observed a robust BP response soon after GC administration, but also in Cre+ mice, in which the hypertensive response to GC is blunted; however, the Cre+ mice also demonstrated a statistically significant increase in urine volume, which suggests the possibility that these mice have a more effective pressure natriuresis than controls and that the presence of GR in the VSM may act as an obstacle to pressure natriuresis in the control state. This hypothesis is consistent with the notion of complex effects of GC on systemic vascular resistance and/or renovascular resistance, and further study will be required to understand this phenomenon better.

Previous work on the role of GR in the regulation of BP primarily relied on in vitro model systems, which can highlight cellular processes relevant to GC effects but cannot be used to understand the physiologic basis of complex integrated phenotypes such a BP. For example, there is literature to support the idea that GC increase vascular reactivity to catecholamines,⁸ but the mechanism by which VSM responds to GC is unclear. One possible mechanism that several in vitro studies have suggested is that GC may play a role in coupling of G-proteins and -adrenoceptors in the rat aorta.^34,35 Studies in ex vivo afferent arterioles from endothelin-1 transgenic mice demonstrate lower sensitivity to AngII compared with WT that is directly attributable to increased NO bioavailability.³⁶ In addition, studies in the rat show upregulation of the AngII type 1A receptor gene at the level of both mRNA and protein in rat smooth muscle cells in response to DEX⁷; therefore, we suggest that GC may have direct effects on renovascular resistance, perhaps by preferentially upregulating AngII and ultimately increasing Na and water reabsorption through the actions of aldosterone and ADH. At present, however, we are unable to determine the exact cause for the acute natriuresis observed in the KO group.

Our study is also intriguing with regard to its findings regarding diurnal BP variation. Multiple lines of evidence support an important role of GC in circadian BP rhythms. Humans exhibit diurnal BP variation, with BP peaking soon after the morning cortisol surge, whereas BP peaks in mice and other nocturnal animals soon after the evening GC surge at the time of their awakening. Patients with disordered cortisol secretion, such as those with Cushing syndrome, commonly lose their diurnal BP variation.¹⁸ Our data indicate that GR in VSM plays a critical role in the acute response of BP to systemic GC. The absence of a noticeable effect of VSM GR deletion on circadian BP variation in our mice indicates that this acute effect of GC on VSM tone is not necessary for normal circadian BP variation.

In summary, our study demonstrates that VSM GR plays a critical role in mediating the acute and, perhaps to a lesser extent, the chronic BP response to GC. Furthermore, our data suggest that GC may have direct effects on systemic and renovascular resistance, which result in the observed phenotype. Undoubtedly, additional investigation is needed to elucidate the mechanism of this phenomenon.

	CONCISE METHODS

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Generation of VSM GR KO Mice
WT BL6 mice homozygous for a floxed GR allele, designated GR^loxP/loxP, were the gift of the laboratory of Dr. Gunther Schutz³⁷ and were mated with WT males (+/+) possessing Sm-22 Cre. By selective breeding, Sm-22 Cre + GR^loxP/loxP homozygotes and Cre– GR^loxP/loxP littermate controls were generated. Determination of mouse genotype was by DNA from mouse tail clipping, which was digested by proteinase K and analyzed by standard PCR. The primers for the floxed GR allele had the following sequence: 5'-GGCATGCACATTACTGGCCTTCT-3' and 5'-CCTTCTCATTCCATGTCAGCATGT-3'. Primers for detection of Cre had the following sequence: 5'-CCGGGCTGCCACGACCAA-3' and 5'-GGCGCGGCAACACCATTTTT-3'. All experiments were performed according to an Institutional Animal Care and Use Committee–approved protocol at Yale School of Medicine, which is consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Immunofluorescence
Aortas and renal arteries were dissected out and fixed in 4% paraformaldehyde for 4 h at room temperature followed by overnight incubation in PBS at 4°C. Whole kidneys were dissected, mounted in OCT, and flash frozen. Sectioned tissue was fixed by incubation in 100% EtOH at 4°C for 30 min followed by 100% acetone at –20°C for 3 min. Sections were rehydrated by incubation in 1x PBS for 5 min at room temperature. Permeabilization was accomplished by incubation in 0.2% Triton-X in PBS for 10 min at room temperature. Blocking solution consisting of 10% serum and 1% BSA in PBS was applied for 30 to 60 min. Tissue sections were then washed 10x quickly in PBS and then incubated in three fresh washes of PBS for 5 to 7 min. Primary antibody (mouse anti-GR at 1:200 and/or rabbit anti-CD31 at 1:1000 and/or mouse anti–-smooth muscle actin with FITC conjugate at 1:200) in 2.5% serum/PBS was applied and incubated 1 to 3 h at room temperature. Sections were again washed 10x in PBS followed by incubation in three fresh washes of PBS. Secondary antibody (goat anti-mouse at 1:200 and/or goat anti-rabbit at 1:1000) in 2.5% serum/PBS was applied and incubated for 30 to 60 min at room temperature. Sections were mounted with Vectashield (Vector Laboratories, Burlingame, CA) and allowed to harden overnight at 4°C.

Western Blotting
Whole hearts were dissected from control and Sm-22 Cre + GR^loxP/loxP mice, cleared of fat and connective tissue, and washed quickly three times in ice-cold PBS to rinse away blood. Whole-heart homogenates were solubilized in lysis buffer with protease cocktail inhibitors and kept on ice. Proteins (20 to 30 µg) were subjected to SDS-PAGE using 7.5% polyacrylamide gels. Proteins were then transferred from the gel to polyvinylidene difluoride microporous membranes that were used for immunoblotting. After transfer, membranes were stained with Ponceau S solution to confirm complete transfer of proteins to blots and verify consistent protein loading among wells. Membranes were incubated with blocking solution (5% nonfat dry milk and 0.1% Tween in PBS) for 1 h at room temperature to block nonspecific protein binding. Membranes were incubated at 4°C overnight with primary antibody (anti-GR at 1:2000) in blocking solution. After several washes, secondary antibody (goat anti-mouse HRP at 1:2000) was added for 1 h at room temperature. Bound antibody on blots was visualized using an enhanced chemiluminescence system, and relative protein quantities were determined by densitometry.

Telemetric Measurement of BP and Heart Rate
Radiotelemetric BP catheters were implanted in the aortic arch in 8- to 12-wk-old male Sm-22 Cre + GR^loxP/loxP mice and Cre– controls as described previously,³⁸ and mice were allowed 1 wk to recover from surgery. Adequate recovery was deemed to have occurred when mice demonstrated normal diurnal variation of BP for 3 consecutive days. Mice were housed individually in a controlled environment with 12-h light and dark cycles and had free access to water and standard mouse diet. Measurement of the following six parameters occurred every 1 min using the DataQuest System (Data Sciences, St. Paul, MN): SBP, DBP, mean BP, pulse pressure, heart rate, and activity level. Measurements were averaged over 4-h periods using an Excel program developed specifically for this purpose. After 3 d of a normal BP pattern, mice were treated with DEX in their drinking water at a concentration of 15 mg/L. On the basis of an average water intake of 2 to 3 ml/d per mouse and an average weight of approximately 30 g/mouse, the ingested dosage of DEX was approximately 2 mg/kg per d for each mouse.

Assessment of Mouse Circadian Rhythm
Mice were housed in facilities with a 12-h light-dark cycle such that 7 a.m. to 7 p.m. was the light cycle and 7 p.m. to 7 a.m. was the dark cycle. Data collected using the DataQuest System software were input into an Excel program that calculated time-averaged SBP, DBP, and MAP for 4-h intervals as follows: 8 p.m. to midnight, midnight to 4 a.m., 4 a.m. to 8 a.m., 8 a.m. to noon, noon to 4 p.m., and 4 p.m. to 8 p.m. An additional function in the program calculated the average change in SBP, DBP, and MAP between each pair of consecutive 4-h periods. All mice studied demonstrated a maximum change in BP from the period of 8 p.m. to midnight to 8 a.m. to noon. This difference was calculated for the MAP of each mouse at baseline and for each day of DEX treatment.

Mouse Urine Collection
Eight- to 12-wk-old male Sm-22 Cre + GR^loxP/loxP mice and Cre– controls were housed individually in mouse-specific metabolic cages with free access to water and standard mouse diet. Mice were allowed to acclimate to their new cages for 24 h. On day 2, a 16-h urine collection was started at 5 p.m., shortly before the beginning of the dark cycle. A thin film (50 µl) of mineral oil was used to coat the collection tube and prevent evaporation of urine. On day 3, another 16-h urine collection was started at 5 p.m., and mice were simultaneously treated with DEX 15 mg/L in the drinking water. Cages were cleaned thoroughly in between each urine collection. Urine volumes of <800 µl were excluded from subsequent analysis. Urine electrolytes were determined by the Yale Mouse Phenotyping Core Facility using the COBAS MIRA system (Roche Diagnostics, Nutley, NJ) and mass spectrometry. Each mouse's urine electrolytes were normalized to its own urine creatinine to control for differences in GFR.

Reagents
Dexamethasone phosphate, donkey serum, PBS, and paraformaldehyde were obtained from Sigma Chemicals (St. Louis, MO). Triton-X, acetone, and alcohol were obtained from American Bioanalytical (Natick, MA). Antibody to GR was obtained from Affinity Bioreagents (Golden, CO). Anti-CD31 was courtesy of Dr. Joseph Madri (New Haven, CT). A stock solution of dexamethasone was dissolved in distilled water and prepared fresh before each set of experiments.

Statistical Analysis
Data are expressed as means ± SEM. Statistical differences were determined by paired and unpaired t test or by Wilcoxon signed rank test. P < 0.05 was considered significant.

	DISCLOSURES

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None.

	Acknowledgments

 
This work was supported by a National Kidney Foundation Postdoctoral Research Fellowship Award to J.E.G. We also acknowledge the following support for the Yale Mouse Metabolic Phenotyping Core: U24 DK59635.

We acknowledge and thank Dr. Gunther Schutz of the Deutsches Krebsforschungszentrum (German Cancer Research Center) for his generous gift of mice with a floxed GR allele.

	Footnotes

 
Published online ahead of print. Publication date available at www.jasn.org.

See related editorial, "Glucocorticoid-Mediated Hypertension: Does the Vascular Smooth Muscle Hold All the Answers?’ on pages 1251–1253.

	REFERENCES

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