The present study determined whether early loss of estrogen influences salt-sensitive changes in blood pressure, renal injury, and cardiac hypertrophy as well as the effects on the circulating renin-angiotensin-aldosterone system (RAAS) in the hypertensive female mRen(2).Lewis strain. Ovariectomy (OVX) of heterozygous mRen(2).Lewis rats on a normal salt (NS) diet (0.5% sodium) increased systolic blood pressure from 137 ± 3 to 177 ± 5 mmHg (P < 0.01) by 15 wk but did not show any changes in cardiac-to-body weight index (CI), proteinuria, or creatinine clearance. Maintenance with a high-sodium (HS) diet (4%) increased blood pressure (203 ± 4 mmHg, P < 0.01), proteinuria (3.5 ± 0.3 vs. 6.4 ± 0.7 mg/day, P < 0.05), and CI (4.0 ± 0.1 vs. 5.2 ± 0.1 mg/kg, P < 0.01) but decreased creatinine clearance (0.89 ± 0.15 vs. 0.54 ± 0.06 ml/min, P < 0.05). OVX exacerbated the effects of salt on the degree of hypertension (230 ± 5 mmHg), CI (5.6 ± 0.2 mg/kg), and proteinuria (13 ± 3.0 mg/day). OVX increased the urinary excretion of aldosterone approximately twofold in animals on the NS diet (3.8 ± 0.5 vs. 6.6 ± 0.5 ng·mg creatinine−1·day−1, P < 0.05) and HS diet (1.4 ± 0.2 vs. 4.5 ± 1.0 ng·mg creatinine−1·day−1, P < 0.05). Circulating renin, angiotensin-converting enzyme, and angiotensin II were also significantly increased in the OVX group fed a HS diet. These results reveal that the protective effects of estrogen apart from the increase in blood pressure were only manifested in the setting of a chronic HS diet and suggest that the underlying sodium status may have an important influence on the overall effect of reduced estrogen.
- angiotensin II
- angiotensin-coverting enzyme
- cardiac hypertrophy
estrogen depletion after ovariectomy (OVX) markedly exacerbates the development and maintenance of hypertension in the congenic mRen(2).Lewis rat maintained on a normal salt (NS) diet (7). The inbred mRen(2).Lewis rat is derived from the original mRen2(27) strain (a model of tissue renin overexpression) backcrossed over nine generations into the Lewis strain. Similar to the mRen2(27) and other hypertensive models, this congenic strain exhibits marked gender differences in the extent of hypertension and proteinuria (8). The overall effect of estrogen depletion essentially abolished the gender difference in the degree of hypertension but not renal injury (proteinuria) between male and female mRen(2).Lewis rats (7, 8). The increase in blood pressure in the OVX mRen(2).Lewis rat was associated with elevated circulating angiotensin II (ANG II) and angiotensin-converting enzyme (ACE) and increased renal excretion of endothelin-1 and 8-isoprotane as well as reduced renal endothelial nitric oxide (NO) synthase (eNOS) by 11 wk of age (7). Estrogen replacement with 17β-estradiol abolished the hypertension but also reduced blood pressure to a level lower than that of sham-operated or intact rats at the end of the study (8). Moreover, treatment with the potent angiotensin type 1 (AT1) receptor antagonist olmesartan in adult OVX mRen(2).Lewis rats essentially normalized the blood pressure (7), further supporting estrogen's role to regulate the activity of the renin-angiotensin-aldosterone system (RAAS) (2, 4, 35). Interestingly, the AT1 antagonist exhibited a prolonged antihypertensive effect in this model that was evident 2 mo after the 4-wk treatment period ended (7).
Typically, estrogen-dependent effects on blood pressure have been demonstrated in female hypertensive models such as the spontaneously hypertensive rat (SHR) or Dahl salt-sensitive (S) or DOCA-salt rats only when maintained on a HS regimen (12, 13, 16). The extent that estrogen may influence the RAAS in these models on a HS diet is currently not known. Hinojosa-Laborde et al. (23) demonstrated that the female OVX Dahl S rat exhibited increased blood pressure when placed on a chronic low-salt diet, suggesting a modulatory effect of estrogen in the setting of an activated RAAS. In contrast, Harrison-Bernard et al. (22) found that a low-sodium diet or AT1 receptor blockade attenuated the increase in blood pressure in female Dahl S rats after OVX. Previous studies (5, 27, 28) in male mRen2(27) rats, the founder strain to the mRen(2).Lewis strain, revealed a blood pressure response to 2% NaCl in drinking water that was attenuated by central AT1 inhibition. The issue of salt sensitivity, estrogen status, and the RAAS in either female mRen2(27) or mRen(2).Lewis strains has not been addressed. Therefore, we determined the influence of estrogen depletion and a chronic HS diet on blood pressure, cardiac hypertrophy, renal injury, and circulating RAAS components in the congenic female mRen(2).Lewis rat.
MATERIALS AND METHODS
Heterozygous female mRen(2).Lewis or female Lewis rats were obtained from the Hypertension and Vascular Disease Center Transgenic colony at 4 wk of age, and either a bilateral OVX or sham operation (similar procedure but without ovary removal) was performed. At 5 wk, animals were placed on either a 0.5% or 4% sodium diet (Harlan TEKLAB, Madison, WI) for an additional 10 wk. Rats were housed in metabolic cages (Harvard Bioscience, South Natick, MA) for the assessment of food and water intake as well as urine collection in rooms maintained on a 12:12-h light-dark cycle (lights on 6:00 AM to 6:00 PM) in an Association for Assessment and Accreditation of Laboratory Care-approved facility. Systolic blood pressure (SBP) was measured at weekly intervals in trained rats (mean of 5 determinations/data point) with a Narco Biosystems device (Houston, TX). The heart rate (HR) was determined by a discrete Fourier transform on the pulse wave signal, yielding a frequency with the highest incidence over the sampled region (HR in Hz). Five samples were collected and averaged for each animal at 15 wk of age for HR. At the end of the study, animals were euthanized by decapitation without anesthesia, and the trunk blood and tissues were rapidly collected for analysis. For the hearts, the tissue was blotted, weighed, and snap frozen on dry ice. The cardiac weight index was expressed as milligrams of heart weight per gram of total body weight (HW/BW). These procedures were approved by the Wake Forest University School of Medicine Institutional Animal Care and Use Committee.
The concentration of ANG II was determined in plasma as described by Allred et al. (1). Plasma was obtained from trunk blood collected directly into chilled Vacutainer blood collection tubes containing a mixture of peptidase inhibitors (25 mM EDTA, 0.44 mM o-phenanthroline, 1 mM 4-chloromercuribenzoic acid, 10 μM lisinopril, 10 μM SCH39370, 2 μM amastatin, and 10 μM bestatin). After 20 min on ice, blood samples were centrifuged at 3,000 rpm for 20 min, and plasma was drawn without disturbing the packed cells. Aliquots were stored at −80°C before extraction on Sep-Pak C18 columns (200 mg, Waters, Milford, MA). The sensitivity and specificity for the ANG II radioimmunoassay have been previously described (31). Serum and urinary aldosterone levels were measured by an radioimmunoassay kit (DPC, Los Angeles, CA) and expressed as either nanograms per milliliter or nanograms per milligram of creatinine per day, respectively. Relative changes in plasma angiotensinogen were determined by an immunoblot assay using a COOH-terminally directed and affinity-purified antibody (Hypertension Center no. A2405) that recognized both ANG I and des-ANG I forms of the protein. We applied 0.5 μl of plasma to 10% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA) for 1 h at 120 V in Tris-glycine-SDS. Proteins were transferred onto a polyvinylidene difluoride membrane and blocked for 1 h with 5% nonfat dried milk in 0.1% Tween 20 in Tris-buffered saline for 60 min at room temperature before incubation with the angiotensinogen antibody (2 μg/ml). Densities of the 54- and 62-kDa bands among the four groups were determined by an IMCID imaging system relative to 0.04 μl of nephrectomized rat plasma as an angiotensinsogen standard for each gel. Immunoblots were then resolved with a Pierce Super Signal West Pico Chemiluminescent substrate (Chicago, IL) as described by the manufacturer and exposed to Amersham Hyperfilm ECL (Piscataway, NJ). Urinary albumin was determined by an enzyme immunoassay kit (SPI-BIO), and its excretion was expressed as milligrams per day. Urinary electrolytes were measured in an automated system (NOVA Biomedical Electrolyte Analyzer). Serum 17-β estradiol levels were analyzed by a radioimmunoassay using a kit from Adaltis Italia SPA. Serum and urinary creatinine were determined by a colorimetric assay kit (Bioassay System, Hayward, CA). Creatinine clearance was derived from the daily urine volume × (urine creatinine/serum creatinine) and expressed in milliliters per minute.
Rat and mouse plasma renin concentrations (PRCs) were determined at pH 6.5 (for rat renin) and pH 8.0 (for mouse renin) by the addition of exogenous angiotensinogen (from nephrectomized rat plasma) and expressed as nanograms ANG I produced per milliliter per hour (3). Serum ACE activity was determined in 20 μl of serum with the synthetic substrate Hip-His-Leu in the presence and absence of the ACE inhibitor lisinopril (10 μM) as previously described (1).
All measurements are expressed as means ± SE computed from an average of 5 determinations/rat for SBP or duplicate values for biochemical data from each rat. Comparisons among intact, OVX, and HS diet-fed rats and combinations were evaluated using ANOVA and Tukey's multiple-comparison test. These analyses were performed with GraphPad Prism IV plotting and statistical software (San Diego, CA). A correlation matrix for nine experimental variables among all the mRen(2).Lewis groups was constructed with the NCSS (Kaysville, UT) statistical package to determine the nonparametric or rank correlation of the data, and the correlations were tested for linearity using a two-tailed Student's t-distribution. The associated scatterplots and linear regression lines with 95% confidence limits were constructed with Prism IV software.
Shown in Fig. 1 are the effects of OVX and HS diet on the development of SBP in mRen(2).Lewis rats over the course of weeks 7 to 15. Compared with sham-operated (intact) mRen(2).Lewis rats, OVX congenics on a 0.5% NS diet exhibited an elevated blood pressure as early as week 8 (164 ± 4 vs. 152 ± 4 mmHg) and remained elevated through week 15. Intact mRen(2).Lewis rats maintained on the 4% HS diet exhibited a continuous increase in blood pressure that plateaued at week 13. By week 15, blood pressures in the mRen(2).Lewis HS group were significantly higher than either intact or OVX mRen2.Lewis NS groups (Fig. 1 and Table 1). Intact female Lewis maintained on the HS diet did not exhibit a significant increase in blood pressure over the 10-wk period (Fig. 1). Estrogen depletion (OVX) and the HS diet resulted in the highest level of blood pressure for mRen(2).Lewis rats (Fig. 1 and Table 1). From the SBP data shown in Fig. 1, the increases in blood pressure due to estrogen depletion and the HS diet appeared to be additive, particularly at the 15-wk time point (see Table 1). We expressed the data as changes in blood pressure from NS to HS diets in intact mRen2.Lewis rats (shamHS − shamNS) versus changes in OVX rats (OVXHS − OVXNS) over the time period of 7–15 wk. As shown in Fig. 1 (inset), there was essentially a linear relationship between estrogen depletion and high sodium intake (slope of 1.05), suggesting that the effects on blood pressure alone were additive in the female mRen2.Lewis strain. Provided that estrogen depletion either positively or negatively influenced the salt-sensitive blood pressure response, we would expect the slope of the line to deviate from 1. The assessment of cardiac hypertrophy expressed as HW/BW also distinguished the effects of estrogen depletion and sodium intake in the mRen(2).Lewis strain. Despite the marked difference in blood pressure, estrogen-depleted rats exhibited a HW/BW value similar to the intact group (Table 1). In contrast, the HS diet alone was associated with significant hypertrophy compared with either intact or OVX rats on a NS diet. The combined effect of estrogen depletion and the HS diet increased heart weight, although this was not statistically different from the HS diet alone. At week 15, the calculated HR data from the blood pressure data were not different among the four congenic groups (Table 1). All four groups of female mRen(2).Lewis rats exhibited essentially identical patterns in weight gain regardless of estrogen status or HS diet from weeks 5 to 15 (data not shown), and the final weights were not different at week 15 (Table 1). The rats were not staged in this study; however, plasma β-estradiol levels were below detectable limits of the assay for the OVX groups and were similar for the intact NS and HS diet-fed rats, respectively (Table 1). Finally, none of the rats died prematurely during the course of the HS diet nor exhibited any signs of morbidity such as weight loss, labored breathing, or pronounced lethargy.
We assessed the status of the circulating RAAS in the four groups of mRen(2).Lewis rats at the end of the 15-wk experimental period. PRCs for rat renin were distinguished from transgenic mouse renin by performing an activity assay at two different pH optima: pH 6.5 for rats and pH 8.0 for mice (3). Neither estrogen depletion nor the HS diet significantly influenced rat or mouse renin PRCs. However, despite the pronounced increase in blood pressure in the OVX HS diet-fed animals, rat PRC was significantly elevated compared with intact NS diet-fed rats. Estrogen depletion or HS diet alone increased serum ACE activity, and the combination further enhanced ACE activity. Circulating ANG II was significantly higher in the OVX HS diet-fed group, with levels that were approximately twofold greater than those in the intact group (Table 1). Although not statistically significant, serum aldosterone levels increased almost twofold with estrogen depletion in both the NS and HS diet-fed groups (Table 1). Immunoblots using an antibody that recognized both ANG I and des-ANG I forms of the precursor angiotensinogen did not reveal significant changes in the relative densities of plasma angiotensinogen among the four groups (intact: 0.42 ± 0.03, OVX: 0.59 ± 0.13, HS: 0.31 ± 0.06, and OVX-HS: 0.27 ± 0.07, n = 4, P > 0.05).
Table 1 also shows urinary excretion data for sodium, potassium, aldosterone, proteinuria, and urine volume as well as creatinine excretion and clearance at the end of week 15 for all four groups of mRen(2).Lewis rats. Compared with intact rats, estrogen depletion significantly increased aldosterone excretion by almost twofold, and a similar trend was evident for rats maintained on the HS diet. Despite the significant increase in blood pressure and augmented levels of aldosterone, estrogen depletion alone did not elevate urinary protein. In contrast, the HS diet significantly increased protein excretion, and the loss of estrogen exacerbated the degree of proteinuria. Albumin excretion in mRen(2).Lewis rats exhibited a similar trend as that for urinary protein with the highest extent of albuminuria in OVX HS diet-fed rats (Table 1). Estrogen depletion alone did not alter creatinine clearance, but the HS diet significantly reduced clearance in intact and OVX groups by 15 wk of age. Urinary potassium did not differ between the four groups. Urinary sodium was similar for those rats maintained on the NS diet; however, sodium excretion was increased to a similar extent in both sham and estrogen-depleted groups fed the HS diet.
We assessed the relationship between circulating RAAS components and various physiological end points for mRen2.Lewis rats by a correlation matrix [Table 2; ranked correlation values (r > 0.6) and statistical significance are shown by asterisks]. SBP was positively correlated with plasma levels of ANG II (r = 0.71) and circulating ACE activity (r = 0.85). SBP was also highly correlated with the extent of cardiac hypertrophy (r = 0.79) and proteinuria (r = 0.70) but negatively correlated with creatinine excretion (r = −0.69). Plasma ANG II was positively correlated with serum ACE (r = 0.64) but less so compared with rat or mouse PRCs. However, plasma rat renin was highly associated with mouse renin (r = 0.84), suggesting similar regulation of the two renin activities in the mRen2.Lewis strain. Aldosterone excretion (expressed as mg/mg creatinine) and serum aldosterone levels (not shown in Table 2) were not associated with blood pressure, proteinuria, or creatinine excretion in the four groups. The associated scatterplots shown in Fig. 2 also suggest a relationship among circulating levels of ANG II, serum ACE activity, and blood pressure as well as alterations in renal injury and cardiac hypertrophy. These data further support that dysregulation of the RAAS may contribute to pathologies of the mRen2.Lewis strain in a setting of HS and/or estrogen depletion.
In the present study, we determined the effects of estrogen depletion and increased sodium intake on the development of blood pressure and expression of the circulating RAAS in female mRen(2).Lewis rats, a monogenetic strain that overexpresses the mouse renin 2 gene. The mRen2.Lewis strain was derived from the original Ren(2)27 outbred Sprague-Dawley, and the congenic strain does not exhibit the severity of renal damage or incidence of malignant hypertension associated with mRen(2)-27 Sprague-Dawley rats. To our knowledge, no studies to date have determined the interaction of estrogen and salt in either female mRen(2)-27 or mRen2.Lewis rats regarding blood pressure, tissue injury, or the status of the RAAS, although Brosnihan et al. (4) found that estrogen replacement reduced serum ACE activity and improved the vasodilatory actions of Ang-(1-7) in mRen(2)27 rats fed a NS diet after OVX. Similar to our previous findings (7), early estrogen depletion between 4 and 5 wk of age markedly increased the degree of hypertension in the female mRen(2).Lewis strain. We now demonstrate that either intact or estrogen-depleted mRen(2).Lewis rats exhibit profound salt-sensitive hypertension, developing SBP > 60 mmHg higher than littermates maintained on a normal sodium intake. Estrogen depletion clearly increased the salt-dependent response, resulting in the largest increase in blood pressure among the four experimental groups (230 mmHg), as well as increased the extent of renal injury or proteinuria. Biochemical characterization revealed an inability of the mRen(2).Lewis strain to downregulate various RAAS components after either estrogen depletion or the HS diet that is particularly notable given the marked increase in blood pressure. Indeed, the continued or elevated expression of RAAS components including renin, ACE, and ANG II may contribute to the development and/or maintenance of salt-dependent increases in blood pressure, cardiac hypertrophy, and renal injury in this congenic model of renin overexpression, particularly after estrogen depletion.
This is the first study to demonstrate salt-sensitive hypertension in the intact female mRen(2).Lewis strain as well as the influence of estrogen depletion. Wyss and colleagues (16) reported significant increases in blood pressure after high sodium intake in female SHR after OVX, but these animals were restricted to a phytoestrogen-free diet. The increase in blood pressure was attenuated by ganglionic blockade, suggesting an estrogen-dependent influence on the central sympathetic system in the SHR (35). In female Dahl S rats, Otsuka et al. (33) observed salt-sensitive changes in blood pressure only subsequent to OVX. In contrast, Harrsion-Bernard et al. (22) found that OVX increased blood pressure in female Dahl S rats on a NS diet and that either a low-sodium diet, estrogen, or AT1 antagonist reduced blood pressure. The substantial increase in blood pressure with a HS diet observed in female mRen2.Lewis rats is not unexpected because this model exhibits overexpression of the renin gene that likely leads to an enhanced capacity for ANG II formation. Clearly, increased ANG II levels can induce or exacerbate salt-dependent alterations in blood pressure by various mechanisms that include a shift in the pressure-natriuresis curve to a higher pressure, enhancing sympathetic drive, attenuating the baroreflex, increased vascular reactivity, and the exacerbation of renal injury or inflammation. Indeed, adult male mRen2(27) rats on a NS diet, the founder strain to the mRen(2).Lewis, exhibit a pronounced rightward shift in the natriuresis function curve that was partially reversed by RAAS blockade (19, 20).
An unexpected finding in the present study was the sustained or increased expression of the circulating RAAS with HS diet alone or in conjunction with estrogen depletion in the mRen(2).Lewis rat. Both rat and mouse circulating renin activities were maintained on the HS diet and significantly increased after estrogen depletion, the group that exhibited the greatest increase in SBP. Plasma levels of ANG II were significantly elevated in estrogen-depleted rats maintained on the HS diet. Duggan and colleagues (24) observed an increase in plasma ANG II with a HS diet in either NO-inhibited male Wistar-Kyoto rats or naive male SHR. Because circulating renin and ACE were not altered, they concluded that the higher plasma levels of angiotensinogen may contribute to the augmented ANG II (24). The circulating levels of angiotensinogen are close to the Km value for renin, such that small alterations in the substrate may markedly influence the rate of ANG I production. Although estrogen can influence angiotensinogen expression (11, 17, 37), plasma levels of angiotensinogen (ANG I/des-ANG I forms) were not significantly changed after the HS diet or OVX in female mRen(2).Lewis rats. Studies in male Dahl S rats have also suggested a role for the RAAS in superoxide production and hypertension after a HS diet (50), although the influence of estrogen in female Dahl rats has not been addressed.
We found that ACE activity was significantly increased, particularly under HS diet and estrogen-depleted conditions, which may contribute to the higher circulating levels of ANG II. Estrogen depletion alone increased serum ACE activity, and these data confirm previous studies (7, 18, 38) on the estrogen-dependent downregulation of ACE expression in the serum and various tissues from several hypertensive models. In the female mRen(2).Lewis rat, high sodium intake also increased ACE activity and was further enhanced after estrogen depletion. The assessment of tissue ACE (lung, aorta, kidney, and heart) and other enzyme activities (such as ACE2 and neprilysin) that may participate in ANG II metabolism as well as tissue peptide concentrations are in progress to distinguish the circulating versus local RAAS after estrogen depletion and high sodium intake in mRen(2).Lewis rats. However, our data suggest that the normal regulation of the circulating RAAS is disrupted in female mRen(2).Lewis rats, leading to inappropriately sustained or higher levels of circulating renin, ACE, ANG II, and aldosterone. The mechanisms underlying the dysregulation of the RAAS are presently not known. Our preliminary data demonstrate that either a HS diet or estrogen depletion are associated with a marked reduction in eNOS and enhanced mRNA levels of both neuronal NOS (nNOS) and cyclooxygenase (COX)-2 in the kidney (9, 48). COX-2 and nNOS have been linked to the regulation of renin release in several studies (6, 25, 41, 43), and the inability to suppress plasma renin may arise from increased expression after high sodium intake or OVX in this strain. The influence of nNOS and COX-2 inhibition on blood pressure and the expression of RAAS components in the female mRen(2).Lewis rat are the focus of ongoing studies in our laboratory. Alternatively, an enhanced vascular responsiveness to ANG II after the HS diet in the intact or OVX groups may arise from a reduction in eNOS, thus contributing to the reduced clearance and sustained or increased levels of renin in this model.
In addition to the elevated serum ACE activity, we observed a sustained increase in urinary excretion and, although not significant, serum levels of aldosterone after OVX in rats maintained on either diet. The increase in aldosterone is consistent with the recent study by Roesch et al. (36) in that OVX potentiated the ANG II-dependent increase in serum aldosterone; this response was associated with a higher density of adrenal AT1 receptors. Moreover, Clark et al. (10) found that OVX was associated with increased serum aldosterone in aged rats, although the extent of renal injury or function was not determined. The AT1 receptor gene contains a negative regulatory element for estrogen, and AT1 mRNA levels are regulated by either OVX or estrogen replacement in various tissues including the vasculature (32), pituitary (14, 38), brain (39), adrenal gland (47), and kidney (6, 22, 25). Thus, in OVX mRen(2).Lewis rats, increased adrenal ANG II (47) or AT1 receptors may well contribute to the enhanced levels of serum and urinary aldosterone, and additional studies that focus on the adrenal RAAS are required to address this issue. Indeed, a study (49) in the mRen2(27) founder strain revealed greater expression of adrenal renin. However, circulating or urinary aldosterone levels did not correlate with alterations in blood pressure, heart weight, or renal injury among the four groups of mRen(2).Lewis rats. It is notable that despite the increase in blood pressure, estrogen-depleted rats on a NS diet did not exhibit evidence of cardiac hypertrophy or proteinuria, particularly compared with the HS diet-fed group. Although a more complete assessment of cardiac and renal function is certainly warranted, as well as a histological evaluation for tissue injury, our data contrast with a number of both experimental and clinical studies (15, 29, 30, 37, 40, 42) that have demonstrated a protective role of estrogen on the cardiovascular system. Moreover, after menopause, there are higher rates of hypertension, renal disease, and cardiovascular events in women. Continual findings from Women's Health Initiative trials, however, have failed to demonstrate any cardiovascular benefit of hormone replacement therapy (estrogen/progestin or estrogen alone) (44–46). The present results reveal that the protective effects of estrogen apart from the increase in blood pressure in adult mRen(2).Lewis rats were only manifest in the setting of a chronic HS diet. Whether the effects of OVX and HS diet reflect the direct actions of estrogen loss on systems such as the RAAS or increasing blood pressure above a critical threshold level are not known, but we conclude that the underlying sodium status may have an important influence on the overall effect of reduced estrogen or interventions to augment the steroid levels of this hormone.
This work was supported by National Heart, Lung, and Blood Institute Grants HL-56973 and HL-51952 and American Heart Association Grants AHA-151521 and AHA-525586. Unrestricted grants from Unifi (Greensboro, NC) and the Farley-Hudson Foundation (Jacksonville, NC) are also acknowledged.
The technical expertise of Nancy T. Pirro for the angiotensinogen and ACE assays is acknowledged.
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