Endogenous Aldosterone Contributes to Acute Angiotensin II-Stimulated Plasminogen Activator Inhibitor-1 and Preproendothelin-1 Expression in Heart But Not Aorta

Abstract

To test the hypothesis that angiotensin (Ang) II induces profibrotic gene expression through endogenous aldosterone, we measured the effect of 4 h infusion (600 ng/kg · min) of Ang II on tissue mRNA expression of plasminogen activator inhibitor 1 (PAI-1), preproendothelin-1 (ppET-1), TGF-β, and osteopontin in wild-type (WT), aldosterone synthase-deficient (AS^−/−), and AS^−/− mice treated with aldosterone (either 500 ng/d for 7 d or 250 ng as a concurrent 4 h infusion). Ang II increased aldosterone in WT (P < 0.001) but not in AS^−/− mice. Aldosterone (7 d) normalized basal aldosterone concentrations in AS^−/− mice; however, there was no further effect of Ang II on aldosterone (P = NS). Basal cardiac and aortic PAI-1 and ppET-1 expression were similar in WT and AS^−/− mice. Ang II-stimulated PAI-1 (P < 0.001) and ppET-1 expression (P = 0.01) was diminished in the heart of AS^−/− mice; treatment with aldosterone for 4 h or 7 d restored PAI-1 and ppET-1 mRNA responsiveness to Ang II in the heart. Ang II increased PAI-1 (P = 0.01) expression in the aorta of AS^−/− as well as WT mice. In the kidney, basal PAI-1, ppET-1, and TGF-β mRNA expression was increased in AS^−/− compared with WT mice and correlated with plasma renin activity. Ang II did not stimulate osteopontin or TGF-β expression in the heart or kidney. Endogenous aldosterone contributes to the acute stimulatory effect of Ang II on PAI-1 and ppET-1 mRNA expression in the heart; renin activity correlates with basal profibrotic gene expression in the kidney.

Endogenous aldosterone contributes to the acute stimulatory effect of angiotensin II on the profibrotic factors plasminogen activator inhibitor-1 and prepro-endothelin-1 in murine heart.

Recent studies highlight the important contribution of aldosterone to cardiovascular morbidity and mortality. In rodents on a high-sodium diet, aldosterone induces an inflammatory response, characterized by cellular infiltration and up-regulation of inflammatory genes, followed by fibrosis (1,2,3,4). Mineralocorticoid receptor (MR) antagonism reduces oxidative stress, nuclear factor-κB activation, inflammation, and fibrosis during activation of the renin-angiotensin-aldosterone system (RAAS) (3,5,6). Likewise, aldosterone synthase inhibition or adrenalectomy attenuates cardiac and renal damage in these models (7).

Angiotensin (Ang) II stimulates the expression of several profibrotic genes, including (prepro)endothelin (ppET)-1, TGF-β, plasminogen activator inhibitor (PAI)-1, and osteopontin within the kidney, heart, and vasculature (8,9,10,11). Because administration of exogenous Ang II or stimulation of endogenous Ang II also increases aldosterone, it can be difficult to discern the contribution of endogenous aldosterone to the effects of Ang II on gene expression in vivo. MR antagonists decrease the expression of profibrotic genes during activation of the RAAS (5,6,12); however, this does not necessarily implicate endogenous aldosterone in Ang II-stimulated gene expression. Depending on the tissue, MR can be activated by glucocorticoids or transactivation by the Ang II type 1 (AT₁) receptor (13,14). The development of aldosterone synthase-deficient (AS^−/−) mice, generated by targeted gene disruption, provides the opportunity to dissociate the effects of Ang II and aldosterone on mRNA concentration (gene expression) of profibrotic genes in vivo (15,16). We therefore used AS^−/− mice to test the hypothesis that Ang II induces profibrotic gene expression through endogenous aldosterone. To confirm that any effects observed in AS^−/− mice resulted from aldosterone deficiency and not from introduction of the transgene, we studied Ang II effects on AS^−/− mice in the presence and absence of aldosterone administered for either 4 h or 7 d.

Materials and Methods

Animals

All experiments were reviewed and approved by the Vanderbilt University Institutional Animal Care and Use Committee and conducted in accordance with accepted standards of humane animal care. AS^−/− mice were generated on a 129 background (16) and were backcrossed 7 or more generations onto the C57BL6J strain obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were genotyped by real-time PCR (7900HT; Applied Biosystems, Foster City, CA) using Taqman probes for a sequence in the aldosterone synthase gene (Cyp11b2) and for a portion of the gene contained in the neomycin resistance cassette (please see supplemental Table I, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Male mice were studied. All mice were maintained on standard mouse chow (Purina Laboratory Rodent 5001; St. Louis, MO) and water ad libitum and housed in a temperature controlled facility with a 12-h light, 12-h dark cycle.

Baseline measurements and acute Ang IIM infusion protocol

Eight- to 14-wk-old mice were housed in metabolic cages while ingesting normal chow and water ad libitum for 24 h urine collection. One week later (3–5 d before infusion study) mice underwent placement of right jugular vein and left carotid artery catheters. Catheter patency was maintained by flushing daily with 50 μl of 20% heparinized saline. Two days before infusion study, mice were placed into a clean cage without chow at 0900 h. At 1500 h, the first 25 μl of arterial catheter fluid was discarded and 50 μl of arterial blood were collected passively directly into an iSTAT EC8+ cartridge (Heska Corp., Loveland, CO) for determination of blood chemistries.

On the morning of the infusion study, ambulatory blood pressure was measured via the arterial catheter using a tether-and-swivel system (BPA analyzer; Micro-Med, Inc., Louisville, KY). Baseline blood pressure was taken 30 min after connecting the transducer system to permit blood pressure stabilization. Mice were randomized to receive either Ang II (600 ng/kg · min; Calbiochem, La Jolla, CA) or 0.9% sodium chloride solution at a similar rate (1 μl/25 g · min; Harvard Apparatus, Holliston, MA) for 4 h. Previous studies have demonstrated this Ang II dose to increase PAI-1 mRNA expression in both mice and rats (17,18,19).

After infusion, mice were sedated (50 mg/kg pentobarbital iv). The left renal artery was clamped, and blood was passively collected into dipotassium-EDTA (Microvette CB K2E; Sarstedt AG & Co., Numbrecht, Germany), centrifuged at 6000 rpm (2.4 g) for 5 min, and plasma was stored immediately at −80 C. The base of the heart, the first 2 mm of descending aorta, and coronal sections of the kidney were fixed in 4% buffered paraformaldehyde overnight and embedded in paraffin. An additional sample of each tissue was placed in a 30% sucrose gradient for about 1 h before embedding in paraffin for autoradiography. The remainder of the heart, aorta, and kidney were frozen immediately in liquid nitrogen and stored at −80 C until mRNA analysis. Aorta were stripped of adventitial fat while immersed in PBS solution and then collected into RNAlater solution (Ambion, Austin, TX), stored at 4 C overnight, and then transferred to a vial for storage at −80 C.

Aldosterone reconstitution

To ensure that any differences observed in AS^−/− compared with wild-type (WT) mice resulted from a lack of aldosterone rather than from an artifact of gene manipulation, we conducted a reconstitution experiment in which we added back basal concentrations of aldosterone. We repeated Ang II and vehicle infusions in the presence of physiologic aldosterone replacement. Miniosmotic pumps (Alzet model 2001; Alza Corp., Palo Alto, CA) containing either aldosterone (0.021 mg/ml in 2% methyl sulfoxide; Acros Organics, NJ) or vehicle at a rate of 500 ng/d were implanted sc 7 d before the infusion study in AS^−/− mice [AS^−/−-aldosterone (7 d)] anesthetized with pentobarbital 50 mg/kg. This aldosterone dose was chosen to achieve physiological concentrations based on published data (20).

We also administered aldosterone iv (250 ng over 4 h) during the 4-h infusions of Ang II in AS^−/− mice [AS^−/−-aldosterone (4 h)] in additional sets of experiments to mimic the acute increase in aldosterone during Ang II infusion that occurs in WT mice. This aldosterone dose was chosen based on pilot studies.

Blood and urine chemistry

Whole blood obtained 2 d before the infusion was used for determination of blood electrolytes, glucose, hematocrit, and blood gases according to the manufacturer’s instructions (EC8+ cartridge; Heska). Sodium, potassium, chloride, pH, and partial pressure of carbon dioxide (pCO₂) were measured by ion-selective electrode potentiometry. Blood urea nitrogen (BUN) was first hydrolyzed to ammonium by the enzyme urease, and then ammonium ions were measured by ion-selective potentiometry. Plasma glucose was measured amperometrically by glucose oxidase-catalyzed hydrogen peroxide generation. Hematocrit was determined by measured conductivity with correction for electrolyte concentrations. Urine Na⁺ and Cl⁻ were measured by flame photometry (IL 943; Instrumentation Laboratory, Lexington, MA).

Plasma collected at the time the animals were killed was used for hormone assays. Aldosterone was determined using a RIA using ¹²⁵I-aldosterone (MP Biomedicals, Irvine, CA), a primary antibody to aldosterone (National Institute of Diabetes and Digestive and Kidney Diseases, National Hormone and Peptide Program, Torrance, CA) and a secondary antirabbit γ-globulin antibody (Linco Research, St. Charles, MO). Corticosterone was measured using a commercially available RIA kit (ImmuChem double antibody corticosterone kit; MP Biomedicals). Plasma renin activity (PRA) was determined using RIA of 1 h plasma Ang I generation (GammaCoat plasma renin activity ¹²⁵I RIA kit; Diasorin, Stillwater, MN) using 2 μl plasma and excess porcine angiotensinogen (0.4 μmol/liter; Sigma-Aldrich, St. Louis, MO).

AT₁ autoradiography

Heart, kidney, and aorta tissues were processed for Ang binding autoradiography as previously reported (21). Frozen sections (6 μm) were air dried for 1 h and preincubated with a binding buffer (5 mm Na₂EDTA, 0.005% bacitracin, 0.2% BSA in PBS) for 15 min. The slides were incubated in the binding buffer containing 3 μm PD123319 (Sigma, St. Louis, MO), an AT₂ antagonist (for AT₁ binding) or 3 μm Sar¹-Ile⁸-Ang II (for nonspecific binding) at room temperature for 2 h. Thereafter the slides were washed in ice-cold 10 mm Tris · Cl (pH 7.6) four times and then in ice-cold distilled water two times, followed by air drying for 2 h. The slides were exposed to an x-ray film for 1 wk, and the radioactive signal was visualized. Quantitative analysis was performed within the same section using Scion Image (Scion Corp., Frederick, MD).

Relative mRNA expression

Relative mRNA expression was determined using quantitative real-time PCR. Reference to gene expression in the data and figures refers to relative changes in mRNA levels. Cardiac and renal total RNA were extracted using RNA Wiz (Ambion) and RNeasy midikit (QIAGEN, Valencia, CA), and aortic RNA was extracted with RNeasy mini kit (QIAGEN). Reverse transcription was performed using TaqMan reverse transcription kit (Applied Biosystems, Branchburg, NJ). Quantitative real-time PCR was performed on the iCycler iQ multicolor real time PCR detection system (Bio-Rad, Hercules, CA) using iQ SYBR Green Supermix (Bio-Rad) using primers (supplemental Table II) as previously described (11). Experimental cycle threshold (C_t) values were normalized to β-actin measured on the same plate, and fold differences in mRNA concentration were calculated relative to the WT-vehicle group within each tissue using the 2^−ΔΔCt method (22).

Statistics

Data are presented as means ± sem in both text and figures unless otherwise specified. One-way ANOVA was used to compare baseline characteristics, neurohumoral markers, and mRNA expression among groups. Post hoc P values were reported for between-group comparisons. ANOVA results were confirmed using nonparametric methods. Repeated-measures ANOVA was used to compare blood pressures during Ang II or vehicle infusion. Relation between aldosterone concentrations or PRA and gene expression were assessed using Spearman’s rank correlation coefficient (r_s) or multivariate linear regression. All statistical analyses were performed using SPSS for Windows (version 15.0; SPSS, Chicago, IL). A two-tailed P < 0.05 was considered significant.

Results

Baseline characteristics

Mean age was similar (P = NS) between WT and AS^−/− groups at the time of study [12.3 ± 0.4, 12.9 ± 0.4, and 11.3 ± 0.3 wk for WT (n = 17), AS^−/− (n = 21), and AS^−/−-aldosterone (7 d) mice (n = 11)]. WT mice (23.6 ± 0.5 g) were larger than AS^−/− mice (21.8 ± 0.5 g, P = 0.02; F_2,46 = 3.32, P = 0.04); AS^{− /−}- aldosterone (7 d) mice (22.9 ± 0.7 g) were not significantly different in weight compared with AS^−/− mice (P = NS). Relative heart weights were similar (P = NS) among the groups (5.3 ± 0.2, 5.3 ± 0.2, and 4.8 ± 0.3 mg/g body weight for WT, AS^−/−, AS^−/−-aldosterone (7 d), respectively). Relative kidney weight was similar (P = NS) in the AS^−/− (6.5 ± 0.2 mg/g body weight) and AS^−/−-aldosterone (7 d) mice (6.4 ± 0.3 mg/g body weight) compared with WT mice (7.0 ± 0.3 mg/g body weight).

Baseline systolic blood pressure was similar (P = NS) in AS^−/− compared with WT [121 ± 4 (n = 18), vs. 127 ± 2 mm Hg (n = 14)] and AS^−/−-aldosterone (7 d) mice [119 ± 5 mm Hg (n = 9)]. Mean arterial pressure (MAP) was significantly lower in AS^−/− (104 ± 3 mm Hg, P = 0.02) and aldosterone-treated AS^−/− mice (104 ± 4 mm Hg, P = 0.04) compared with WT (115 ± 2 mm Hg; P = 0.04). Baseline heart rate was similar among all groups (P = NS).

Plasma potassium (K⁺) and BUN were higher in AS^−/− mice compared with WT (Table 1). Aldosterone, given as a sc infusion 500 ng/d for 7 d before study, normalized K⁺ and BUN in the AS^−/− mice. However, bicarbonate was increased in AS^−/−-aldosterone (7 d) mice compared with the other two groups.

Table 1.

Baseline blood chemistries and urine electrolytes

	WT (n = 10)	AS−/− (n = 14)	AS−/−-aldosterone (n = 11)
Sodium (mmol/liter)	146.9 ± 1.1	144.1 ± 0.8	149.5 ± 1.5a
Potassium (mmol/liter)	4.7 ± 0.2	5.7 ± 0.1b	4.4 ± 0.2a
Chloride (mmol/liter)	115.2 ± 1.1	114.4 ± 1.5	114.4 ± 1.5
Bicarbonate (mmol/liter)	21.2 ± 1.1	21.6 ± 0.7	25.4 ± 0.8a,c
Glucose (mg/dl)	174 ± 8	172 ± 7	159 ± 8
Arterial pH	7.409 ± 0.015	7.427 ± 0.009	7.456 ± 0.010c
Hemoglobin (g/dl)	14.7 ± 0.4	14.2 ± 0.3	14.3 ± 0.4
BUN (mg/dl)	21.0 ± 1.3	27.1 ± 1.6b	19.9 ± 1.6d
Urine sodium (μmol/24 h)	128 ± 76	278 ± 108c	106 ± 60d
Urine potassium (μmol/24 h)	220 ± 138	421 ± 235b	236 ± 107
Urine volume (ml/24 h)	0.71 ± 0.60	1.55 ± 0.38c	1.14 ± 0.58

Values are means ± sem. 

^aP < 0.005 vs. AS^−/−; 

^bP ≤ 0.05 vs. WT; 

^cP < 0.01 vs. WT; 

^dP < 0.01 vs. AS^−/−. 

Effect of Ang II infusion on blood pressure and circulating hormones

Because systolic blood pressure readings can be affected by dampening artifact, MAP response to vehicle or Ang II is presented in Fig. 1, A and B. Ang II significantly (F_6,34 = 9.47, P < 0.0001) increased MAP compared with vehicle in WT (15 min increase of 18.1 ± 1.0 mm Hg during Ang II vs. −2.9 ± 5.5 mm Hg during vehicle, P < 0.001) and AS^−/− (12.6 ± 3.9 mm Hg vs. −1.2 ± 2.2 mm Hg, P = 0.01) (Fig. 1, A and B). Aldosterone (4 h) in conjunction with Ang II produced an increase in MAP in AS^−/− mice that was greater than that obtained after aldosterone administration for 7 d (27.1 ± 2.4 vs. 11.7 ± 4.1 mm Hg in AS^−/−-aldosterone (7 d)-Ang II, P = 0.009).

Figure 1.

Effect of vehicle (A) or Ang II (B) on MAP in WT, AS^−/−, and AS^−/− mice treated with aldosterone (7 d or 4 h). The results of the repeated-measures ANOVA appear in the text. Aldosterone (C) and corticosterone (D) concentrations measured at the end of infusion by treatment group. *, P < 0.001 vs. WT-vehicle; †, P < 0.02 vs. either WT group; ‡, P < 0.05 vs. all groups; §, P < 0.001 compared with WT-Ang II; ∥, P < 0.05 compared with AS^−/−-Ang II, P = 0.07 vs. WT-Ang II.

Basal aldosterone concentrations were markedly decreased in AS^−/− mice, at the lower level of detection for the assay (Fig. 1C). Ang II increased aldosterone in WT (1302 ± 138 vs. 425 ± 93 pg/ml after vehicle, P < 0.001) but not AS^−/− mice (74 ± 16 vs. 58 ± 9 pg/ml after vehicle, P = NS). Aldosterone pretreatment (7 d) normalized basal aldosterone concentrations in AS^−/− mice; however, there was no further effect of Ang II on aldosterone (340 ± 79 vs. 469 ± 38 pg/ml during vehicle, P = NS). Coadministration of aldosterone (4 h) with Ang II in AS ^−/− mice produced aldosterone concentrations higher than that achieved in the Ang II-treated WT mice (1763 ± pg/ml, P = 0.01 vs. WT-Ang II).

Basal corticosterone concentrations were similar after vehicle infusion in the WT, AS^−/−, and AS^−/−-aldosterone (7 d) groups (Fig. 1D). Ang II infusion increased corticosterone compared with vehicle infusion in the WT group but not the AS^−/− and AS^−/−-aldosterone (7 d) groups (P < 0.001).

PRA was markedly elevated in vehicle-treated AS^−/− mice compared with WT (31.92 ± 3.60 vs. 0.64 ± 0.22 10³ ng/ml · h, P < 0.001; F_5,19 = 54.9, P < 0.001) and was decreased after either Ang II infusion (2.52 ± 0.76 10³ ng/ml · h, P < 0.001 vs. AS^−/−-vehicle) or aldosterone (7 d) replacement (2.53 ± 1.07 10³ ng/ml · h, P < 0.001 vs. AS^−/−-vehicle).

Effect of Ang II on mRNA concentration

Ang II increased PAI-1 mRNA expression in the heart of WT but not AS^−/− mice (Fig. 2A; F_6,37 = 13.7, P < 0.001). Aldosterone pretreatment (7 d) or coadministration (4 h) restored Ang II-stimulated PAI-1 mRNA expression in the heart of AS^−/− mice. Cardiac PAI-1 expression correlated with aldosterone concentrations (r_s = 0.46, P = 0.003) and corticosterone concentrations (r_s = 0.45, P = 0.004). In the aorta, Ang II stimulated PAI-1 mRNA concentration in both WT and AS^−/− mice (Fig. 2B; F_6,27 = 3.40, P = 0.01). PAI-1 expression in the kidney was increased in the vehicle-treated AS^−/− mice compared with vehicle-treated WT mice; Ang II decreased renal PAI-1 expression in AS^−/− mice (Fig. 2C; F_6,27 = 3.40, P = 0.02).

Figure 2.

Effect of treatment on relative mRNA expression of PAI-1 in the heart (A), aorta (B), and kidney (C) as measured by RT-PCR (expressed as fold change compared with WT-vehicle, β-actin as internal reference). *, P < 0.05 vs. vehicle in the same genotype; †, P < 0.05 vs. WT-Ang II or AS^−/−-aldosterone (7 d)-Ang II; ‡, P < 0.01 vs. AS^−/−-Ang II; §, P < 0.05 vs. WT-vehicle; ∥, P < 0.05 vs. AS^−/−-vehicle (n = at least 5 in each group.

Ang II stimulated ppET-1 mRNA expression in the heart of WT but not in AS^−/− mice (Fig. 3A; F_6,35 = 3.21, P = 0.01). Pretreatment (7 d) or coadministration (4 h) with aldosterone normalized the Ang II response in AS^−/− mice. Cardiac ppET-1 expression correlated significantly with PAI-1 mRNA expression across treatment groups (r_s = 0.46, P = 0.003). In the aorta (Fig. 3B), there was considerable variability in ppET-1 mRNA expression and no significant effect of treatment group (F_6,28 = 0.62, P = NS). The pattern of renal ppET-1 expression was again similar to that of PAI-1 within the kidney (Fig. 3C; F_6,35 = 3.21, P = 0.02), and renal PAI-1 and ppET-1 mRNA expression correlated (r_s = 0.81, P < 0.001). Because renin stimulates PAI-1 expression in cultured mesangial cells and the kidney through an AT₁ receptor-independent mechanism (23,24) and renin activity is increased in AS^−/− mice, we examined the relationship between PAI-1 and PRA and ppET-1 mRNA expression in the kidney. Renal PAI-1 mRNA expression correlated with PRA (r_s = 0.57, P = 0.004). By linear regression, renal PAI-1 mRNA expression correlated independently with PRA (r = 0.71, P = 0.006) and aldosterone (r = 0.68, P = 0.007) in AS^−/− mice. Renal ppET-1 expression also correlated with PRA (r_s = 0.67, P = 0.001).

Figure 3.

Effect of treatment on mRNA expression of ppET-1 in the heart (A), aorta (B), and kidney (C) as measured by RT-PCR (expressed as fold change compared with WT-vehicle, β-actin as internal reference). *, P < 0.05 vs. vehicle in the same genotype; †, P < 0.05 vs. WT-Ang II or AS^−/−-aldosterone (7 d)-Ang II; ‡, P < 0.05 vs. WT-vehicle; §, P < 0.05 vs. AS^−/−-vehicle; ∥, P = 0.095 vs. AS^−/−-vehicle (n = at least 5 in each group.

Basal TGF-β mRNA concentration was increased in the kidney of vehicle-treated AS^−/− mice compared with WT mice (2.1 ± 0.8, P = 0.03 vs. WT vehicle, see online data supplemental Table II) and correlated with PRA (r_s = 0.44, P = 0.03). Ang II administration to AS^−/− mice did not alter this difference (1.9 ± 0.7-fold change vs. WT vehicle, P < 0.05), but aldosterone administration (1.8 ± 0.9, P = 0.09) or Ang II infusion after aldosterone (1.0 ± 0.2, P = NS) produced TGF-β mRNA levels similar to that of WT vehicle controls. Acute infusion of Ang II did not stimulate osteopontin or TGF-β mRNA expression in the heart or kidney of WT or AS^−/− mice (supplemental Table II). Limited mRNA did not permit quantification of relative TGF-β or osteopontin expression in the aorta.

AT₁ receptor autoradiography

In the heart, AT₁ receptor density was similar among treatment groups (0.67 ± 0.35 in WT-vehicle, 0.59 ± 0.16 in WT-Ang II, 0.70 ± 0.26 in AS^−/−-vehicle, 0.67 ± 0.31 in AS^−/−-Ang II, 0.62 ± 0.15 in AS^−/−-aldosterone (7 d)-vehicle, and 0.57 arbitrary units in AS^−/−-aldosterone (7 d)-Ang II; F_5,10 = 0.03, P = NS).

In the aorta, there was no effect of genotype, acute Ang II infusion, or aldosterone on AT₁ receptor density (0.93 ± .03 in WT-vehicle, 0.65 ± 0.19 in WT-Ang II, 0.69 ± 0.16 in AS^−/−-vehicle, 0.47 ± 0.09 in AS^−/−-Ang II, 0.36 ± 0.14 in AS^−/−-aldosterone (7 d)-vehicle, and 0.81 arbitrary units in AS^−/−-aldosterone (7 d)-Ang II; F_5,18 = 1.8, P = NS).

In the kidney, AT₁ receptor density was similar in vehicle-treated AS^−/− and WT (0.48 ± 0.14 vs. 0.70 ± 0.20) and neither Ang II nor aldosterone (7 d) altered the density (F_5,10 = 0.65, P = NS).

Discussion

MR antagonism decreases the expression of profibrotic genes and cardiac, perivascular, and renal fibrosis during the administration of Ang II or activation of the endogenous RAAS in rodent models (6,12,25). Because the MR may be activated via aldosterone-independent pathways (14), we tested the hypothesis that endogenous aldosterone plays a role in Ang II-induced profibrotic gene expression by comparing the effects of acute Ang II infusion in aldosterone synthase intact and deficient mice. The data indicate that endogenous aldosterone contributes to the acute stimulatory effect of Ang II on PAI-1 expression in the heart but not the aorta. Similarly, endogenous aldosterone contributes to Ang II-stimulated ppET-1 expression in the heart.

The finding that endogenous aldosterone contributes to Ang II-stimulated cardiac PAI-1 expression in vivo is compatible with studies in cultured cardiomyocytes showing that aldosterone induces PAI-1 mRNA expression within 2 h and in rodent models showing that exogenous aldosterone increases PAI-1 expression (26,27,28). To exclude an effect of the transgene itself on Ang II-stimulated gene expression, we studied AS^−/− mice in the absence and presence of treatment with physiological doses of aldosterone. Aldosterone administered as either 7 d pretreatment or a 4-h cotreatment restored the effect of Ang II on gene expression in the heart. Aldosterone treatment for 7 d corrected the elevation in serum potassium concentration observed in AS^−/− mice and raised the possibility that hyperkalemia, rather than hypoaldosteronism, attenuated Ang II-stimulated gene expression in the heart in this model. However, coadministration of aldosterone and Ang II over a 4-h period also restored this response in the heart, whereas these mice had similar potassium concentrations compared with nonaldosterone-treated mice. In previously published studies, potassium concentrations do not affect MR-dependent inflammatory gene expression or fibrosis in rats (29). Likewise, we reported that MR antagonism attenuates Ang II-stimulated PAI-1 expression in humans through a potassium-independent effect (30).

Prior studies demonstrated tissue-specific effects of Ang II on murine PAI-1 expression with increased expression in the heart compared with other tissues (18). We also observed a more dramatic effect of Ang II in cardiac tissue compared with aorta (11- vs. 3-fold increase, Fig. 2). More importantly, aldosterone did not contribute to Ang II-induced PAI-1 expression in the aorta. This is consistent with data from rat vascular smooth muscle cells (VSMCs), in which aldosterone alone did not increase PAI-1 expression and supraphysiological concentrations of aldosterone were necessary to enhance the effect of Ang II on PAI-1 expression (31).

Our data suggest that endogenous aldosterone facilitates the effect of Ang II on cardiac PAI-1 and ppET-1 mRNA expression, in that restoring aldosterone did not increase basal cardiac expression in AS^−/− mice in the absence of Ang II. One mechanism whereby aldosterone deficiency could attenuate Ang II-stimulated PAI-1 and ppET-1 expression in the heart is by decreasing AT₁ receptor density. For example, aldosterone increases AT₁ receptor mRNA expression and density in the rat heart (32). Aldosterone also increases AT₁ receptor density in rat VSMCs and vessels in a concentration-dependent manner (33). However, this study does not provide evidence of decreased AT₁ receptor density in the heart of AS^−/− mice. Rather, AT₁ receptor density was similar in the heart, aorta, and kidney of WT and AS^−/− mice, as measured by autoradiography. The AT₁ receptor-dependent pressor response to acute Ang II was also similar in WT and AS^−/− and was not modified by 7-d pretreatment with aldosterone, providing evidence that AT₁ sensitivity was not diminished in resistance vessels in AS^−/− mice.

Alternatively, aldosterone may enhance the effect of Ang II on profibrotic gene expression in the heart via synergistic effects on inflammatory signaling pathways (34,35). Both Ang II and aldosterone increase free radical production that in turn reduces nitric oxide bioavailability (36,37,38). In cultured myoctes in vitro and in the mouse heart in vivo, Ang II and aldosterone exert synergistic effects on nicotinamide adenine-dinucleotide phosphate oxidase, nuclear factor-κB, and inflammation. Because nitric oxide reduces ppET-1 and PAI-1 expression (39,40), aldosterone may facilitate effects of Ang II on ppET1 and PAI-1 expression by increasing oxidative stress and decreasing the bioavailability of NO.

Basal corticosterone concentrations were similar in WT and AS^−/− mice. This finding conflicts with a previous report of increased corticosterone concentrations in AS^−/− mice but may reflect differences in background mouse strain, blood collection, or RIA technique (16). Acute administration of Ang II increased serum corticosterone, as well as aldosterone, in WT mice but not AS^−/− mice. Pretreatment with aldosterone (7 d) rendered AS^−/− responsive to the effects of acute Ang II on cardiac PAI-1 mRNA expression and, to a lesser extent, corticosterone secretion. However, coadministration of aldosterone (4 h) rendered AS^−/− responsive to effects of acute Ang II on cardiac PAI-1 expression but not corticosterone, making it unlikely that corticosterone contributed to PAI-1 expression in this model.

To the best of our knowledge, this is the first study to delineate the role of endogenous aldosterone, rather than MR activation, on PAI-1 expression in vivo. This acute study complements prior chronic studies of the effect of Ang II and mineralocorticoids on ppET-1 expression in the heart, aorta, and kidney. Endothelin (ET)_A receptor antagonism attenuates cardiac fibrosis and vascular remodeling in response to chronic Ang II infusion in rodents (9). In addition, ET-1 stimulates aldosterone secretion in adrenal zona glomerulosa cells both independently and synergistically with Ang II via ET_A and ET_B receptors (41,42). During activation of the endogenous RAAS, such as in the transgenic (mREN)27 rat, vascular ppET-1 expression has been reported to be increased and contribute to remodeling in some studies but not others (43,44,45). Schiffrin and coworkers (4,46) demonstrated that chronic treatment with either deoxycorticosterone or aldosterone increases cardiac and vascular ppET-1 mRNA expression and that ET_A receptor antagonism decreases aldosterone-induced cardiac and vascular remodeling in rats ingesting either normal- or high-sodium diets. The findings of the present study indicate that acute increases in endogenous aldosterone also stimulates cardiac ppET-1 expression. Our data do not suggest that endogenous aldosterone influences aortic ppET-1 expression during acute Ang II infusion and normal salt intake. However, the study did not address the role of endogenous aldosterone in stimulating ppET-1 expression in the aorta during chronic administration of Ang II or during high-sodium intake or the possibility of transactivation of the MR by Ang II.

ET-1 promotes natriuresis via ET_B receptors, and interruption of this system produces salt-sensitive hypertension (4,47,48). Other studies have shown that Ang II administration also increases ET-1 expression within the kidney, and ET_A blockade prevents renal podocyte injury and albuminuria in a number of models (43,49). Wong et al. (50) reported that aldosterone increases ppET-1 expression in the kidney of adrenalectomized rats within 1 h of infusion. However, in the kidney, in contrast to the heart and aorta, we found no effect of 4-h Ang II infusion on profibrotic gene expression. Rather, basal PAI-1 and ppET-1 mRNA expression were markedly increased in AS^−/− mice compared with WT mice, and Ang II treatment paradoxically decreased renal PAI-1 and ppET-1 expression in the AS^−/− mice.

TGF-β mRNA expression was also significantly increased in the kidney of AS^−/− mice. Pretreatment with aldosterone attenuated profibrotic gene expression in the AS^−/− mice, although PAI-1 and ppET-1 gene expression remained higher than in WT mice. Because renin stimulates TGF-β and PAI-1 expression in the kidney and VSMCs through an AT₁ receptor-independent pathway (24,51), we hypothesize that increased renin expression contributed to increased PAI-1, ppET-1, and TGF-β expression in the kidney of AS^−/− mice. In support of this hypothesis, we found that PRA was increased in AS^−/− mice, Ang II infusion or pretreatment with aldosterone attenuated PRA, and kidney PAI-1 and ppET-1 expression correlated with PRA. However, 7 d treatment with aldosterone increased PAI-1 expression in the kidneys of AS^−/− mice, even when PRA was suppressed. The net effect was that, whereas PAI-1 expression in the heart correlated with aldosterone concentrations but not PRA, in the kidney of AS^−/−, PAI-1 expression reflected the dual effect of renin and aldosterone. That 4 h treatment with Ang II plus aldosterone, like 4 h treatment with Ang II alone, had no effect on PAI-1 mRNA expression in the kidney of AS^−/− suggests that this duration of aldosterone was insufficient to stimulate PAI-1 expression in the kidney. The role of the renin receptor in the regulation of PAI-1 and/or ppET-1 expression warrants further investigation.

The short duration of these studies does not permit assessment of the contribution of endogenous aldosterone to Ang II-induced target organ damage or protein expression, and long-term studies are warranted to investigate these effects. However, in longer studies, chronic hypertension and sodium retention can confound the interpretation of Ang II and aldosterone effects on gene expression. Although Ang II infusion increases blood pressure, previous studies in rats demonstrate that acute Ang II-induced PAI-1 expression is independent of the hypertensive response (19).

Inappropriately increased aldosterone concentrations contribute to cardiovascular remodeling and renal injury during hypertension and congestive heart failure. MR antagonism decreases oxidative stress, inflammation, and fibrosis during activation of the RAAS; however, MR antagonism is nonspecific for aldosterone. The present study indicates that endogenous aldosterone contributes to the effect of acute elevation of Ang II on PAI-1 and ppET-1 gene expression in the heart but not the aorta. In the kidney, basal PAI-1, ppET-1, and TGF-β gene expression correlated with PRA, suggesting that chronically elevated renin concentrations influence gene expression in the kidney in aldosterone synthase deficiency.