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Genistein Attenuates the Hypertensive Effects of Dietary NaCl in Hypertensive Male Rats

Department of Cell Biology (T.M.C., N.P., L.N., S.R., J.P., J.M.W.), University of Alabama, Birmingham, Alabama 35294; and Department of Physiology (J.T.C.), Meharry Medical College, Nashville, Tennessee 37208

Address all correspondence and requests for reprints to: J. Michael Wyss, Ph.D., Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0006. E-mail: jmwyss{at}uab.edu.

	Abstract

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Diets high in polyphenols may protect estrogen-depleted women and rats from hypertension, but there is little evidence for this beneficial effect in males. On a polyphenol-free diet, ovariectomized spontaneously hypertensive rats (SHRs), high dietary NaCl increases arterial pressure, and this effect is greatly blunted by a soy-based diet. High NaCl diets also elevate arterial pressure in male SHRs, and pilot studies indicated that soy polyphenols blunt this effect. The present studies tested the hypothesis that genistein (the primary polyphenol in soy) reduces NaCl-sensitive hypertension in young, male stroke-prone SHRs (SHR-SP, a very NaCl-sensitive strain of SHR). Seven-week-old male SHR-SPs were placed on polyphenol-free diets with or without normal dietary amounts of genistein [0.06% (wt/wt)] and containing high (4%), moderate (2%), or basal (0.7%) NaCl. SHR-SP on the genistein-free diet displayed a dose-related increase in arterial pressure in response to dietary NaCl, and dietary genistein blunted this response. Ganglionic blockade with hexamethonium reduced arterial pressure to similar levels in all six groups, suggesting that the antihypertensive effects of genistein are influenced by the autonomic nervous system. We further hypothesized that genistein, like estrogen, would improve insulin sensitivity and lipid profiles. Thus, in study 2, 7-wk-old male SHR-SP were placed on high (6%) or basal (0.7%) NaCl diets with or without genistein (0.06%). Dietary genistein reduced plasma insulin and insulin resistance in SHR-SP on a high NaCl diet and decreased plasma cholesterol and triglycerides in SHR-SP on the basal NaCl diet. Thus, in male SHR-SP, dietary genistein blunts NaCl-sensitive hypertension, and these effects may be regulated, in part, by the autonomic nervous system and/or metabolic mechanisms.

	Introduction

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HYPERTENSION CONTRIBUTES TO cardiovascular disease [a leading cause of death in the United States (1)], and about 30% of hypertensive adults display salt-sensitive hypertension, i.e. their arterial pressure increases in direct relation to dietary NaCl intake (2, 3, 4). Dietary polyphenols, such as those from soy, appear to decrease these hypertensive effects and have several other health benefits in rats and humans. For example, in rats, polyphenol-containing diets are associated with decreases in blood pressure and serum total cholesterol and increases in bone mineral density and bone formation rate (5, 6, 7, 8). In humans, soy-containing diets appear to lower blood pressure and reduce deaths from heart disease, and these beneficial effects of dietary polyphenols on the cardiovascular system may be related to the estrogenic or nonestrogenic effects of polyphenols (9, 10, 11). The primary polyphenol in soy, i.e. genistein, is often used as a tyrosine kinase inhibitor for in vitro experiments; however, in vivo and at physiological concentrations (<5 µM) genistein lacks appreciable ability to directly inhibit tyrosine kinase and, rather, has commonly been assumed to act through its biochemical property as a relative high-affinity agonist of the estrogen receptor (ER)-β (12). Genistein supplementation improves endothelial function in aortic rings from ovariectomized adult Sprague Dawley rats (13) and induces endothelial-dependent relaxation of aorta rings that are preconstricted with phenylephrine (14, 15, 16).

Our past studies demonstrated that dietary soy supplementation protects young ovariectomized spontaneously hypertensive rats (SHRs) from salt-sensitive hypertension and aged ovariectomized SHRs from both salt-sensitive and baseline hypertension (17, 18). Furthermore, an initial study indicates that dietary soy can also protect young male SHRs from salt-sensitive hypertension (19). Other studies using soy have suggested that such effects are dependent on the protein contained in soy or are at least the result of an interaction between the soy proteins and polyphenols. The present studies test the hypothesis that in young male stroke-prone spontaneously hypertensive rats (SHR-SPs) on a diet containing neither polyphenols nor soy protein, genistein blunts the hypertensive response to elevated NaCl intake. Furthermore, overactivity of the sympathetic nervous system, (leading to vasoconstriction and the activation of neurohormonal systems) and metabolic changes appear to contribute importantly to NaCl-sensitive hypertension in patients and rodent models of the disease, e.g. renal sympathetic nerve activity is increased in several forms of hypertension (2, 3, 4, 20, 21, 22). The data from experiment 1 indicate that genistein protects against the hypertensinogenic effects of elevated dietary salt, prompting us in experiment 2 to hypothesize that genistein supplementation improves lipid profiles and insulin sensitivity. Thus, the present studies tested the hypothesis that dietary genistein blunts the increase in blood pressure and reduces metabolic disturbances associated with high NaCl intake in this model.

	Materials and Methods

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Male SHR-SPs [bred at the University of Alabama at Birmingham from rats originally procured from the National Institutes of Health (NIH)] were group housed (two to three per cage) in a sound-attenuated room at constant humidity (65 ± 5%), temperature (22 ± 1 C), and light cycle (lights on 0600–1800 h). Rats were allowed ad libitum access to casein-based rat diets and tap water during the experiments. The diets were based on AIN 76A (Harlan Teklad, Madison, WI), and NaCl and/or 0.06% genistein (0.6 mg/g diet; Roche Vitamins Ltd., Basel, Switzerland) were added to the powdered diets, as specified below, and the diets were pelleted. All animals were weighed weekly and food and water intake were monitored daily. All experimental procedures were conducted in accordance with NIH guidelines and were approved by the University of Alabama at Birmingham’s Institutional Animal Care and Use Committee.

Experimental protocol 1
Seven-week-old male SHR-SPs were placed on phytoestrogen-free casein-based diets containing high (4%), moderate (2%), or basal (0.7%) NaCl, with or without 0.06% (wt/wt diet) genistein (G) supplementation for 9 wk (4% NaCl, n = 8; 4% NaCl + G, n = 12; 2% NaCl, n = 7; 2% NaCl + G, n = 8; 0.7% NaCl, n = 7; 0.7% NaCl + G, n = 11). Systolic blood pressure and heart rate were measured in conscious, restrained rats for the initial 7 wk (from 8 to 14 wk of age) using a tail-cuff system (Narco Bio-Systems, Inc., Houston, TX). Systolic blood pressure and heart rate are the most reliable measure obtained from tail-cuff plethysmography. The biweekly measurements were taken between 13 and 17 h, as previously reported (23).

Direct arterial blood pressure measurements were made to validate the longitudinal tail-cuff blood pressure measurements and to assess the effects of ganglionic blockade. Rats were anesthetized (after the initial 9 wk on the diets), and SILASTIC brand (Dow Corning Corp., Midland, MI) femoral artery catheters were implanted, and the rats were singly housed thereafter. At least 24 h later, direct arterial pressure and heart rate measurements were taken in conscious, freely moving rats in the home cage (17). Data obtained for direct arterial pressure are presented as mean arterial pressures. After recording a stable baseline resting arterial pressure (BioPac, Inc., Goleta, CA) for at least 20 min, ganglionic blockade was initiated by an intraarterial infusion of hexamethonium (10 mg/kg body weight in 150 µl 0.9% sodium chloride), and arterial pressure was recorded for an additional 20 min. The peak depressor response to ganglionic blockade with hexamethonium is accepted as a consistent measure of neurogenic pressor activity in conscious rats (24, 25, 26). Data for 10 min before and 10 min after hexamethonium infusion were analyzed to assess the participation of the autonomic nervous system in the antihypertensive effects of genistein. After 24 h, blood was withdrawn, and to test the hypothesis that the effects of genistein involve the renin-angiotensin-aldosterone system, plasma rennin activity and aldosterone levels were determined (27). Furthermore, analyses of plasma nitric oxide (NO) (28) was measured and used as an index of vascular oxidative stress.

Thereafter the animals were killed, and brain, heart, and kidney weights were measured, and organ to body weight ratios were calculated. The right kidneys from the control and high-NaCl diet groups were immersion fixed in formalin for 24 h. They were then paraffin embedded, cross-sectioned (5 µm thick), and the sections mounted on slides and stained with eosin-hematoxylin. The sections were examined by a pathologist who was not aware of the group designations of the sections or the design of the study. The sections were scored for their relative overall damage and arteriolar thickening.

Experimental protocol 2
The data from study 1 suggested to us that other metabolic effects of genistein (alterations in insulin and glucose and lipid metabolism) might contribute to its protective effects. To test this hypothesis, we used a more severe salt challenge, which allowed us to more rapidly assess the effects of genistein. Thus, 7-wk-old male SHR-SPs were placed on PE-, casein-based diets containing either high (6%) or basal (0.7%) NaCl, with or without 0.06% (wt/wt) G supplementation (n = 8/group) for 4 wk. The rats were anesthetized, and SILASTIC brand femoral artery catheters were implanted, and the rats were singly housed thereafter. After recording arterial pressure and responses to hexamethonium, as above, the animals were rested for 2 d, and then blood was drawn for plasma aldosterone (Coat-A-Count; Diagnostic Products Corp., Los Angeles, CA), glucose (enzymatic glucose-oxidase reaction, GM7 analyzer; Analox USA, Lunenburg, MA), plasma insulin (RIA; Linco Research, St. Charles, MO), and lipid [Vitros DT60 (Ektachem) analyzer; Ortho-Clinical Diagnostics, Rochester, NY] measurements. From these data an index of insulin sensitivity (quantitative insulin sensitivity check index) was derived (29).

Statistical analysis
All data from experiments were evaluated by ANOVA followed by post hoc Tukey’s test to determine the source of main effects and interactions (SPSS, Chicago, IL). The significance criterion for all experiments was P < 0.05.

	Results

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Experiment 1
Throughout experiment 1, the only difference in body weights among the groups of SHR-SPs was a modest decrease in body weight in the group receiving 4% NaCl diet without G (Table 1). After 4 wk on the diet, food intake was not significantly different in the six groups, but daily water intake was significantly increased in the rats on the high-NaCl diets [66 ± 3 ml (4% NaCl-G), 63 ± 3 ml (4% NaCl + G), 48 ± 4 ml (2.0% NaCl-G) 46 ± 4 ml (2.0% NaCl + G), 32 ± 3 ml (0.7% NaCl-G), 31 ± 4 ml (0.7% NaCl + G)]. G did not modify water intake. Rats on the 4% NaCl diet exhibited minor cardiac (evidenced by increased heart to body weight ratios) and renal (evidenced by increased kidney to body weight ratios) hypertrophy. They also displayed increased relative brain weight (brain to body weight ratio) (Table 1). Both effects were prevented by dietary G.

View larger version (20K): [in this window] [in a new window]   FIG. 1. Systolic arterial pressure (mean ± SEM; tail-cuff) of young male SHR-SPs during the initial 7 wk of dietary NaCl excess (n = 7–12 in each group). SHR-SPs were fed a phytoestrogen-free casein-based diet containing basal (0.7%), moderately high (2.0%), or high NaCl (4%) with or without G. Dietary NaCl excess elevated arterial pressure in the control group, but G supplementation blunted this elevation of arterial pressure. G had no effect on arterial pressure in the basal NaCl diet rats. *, P < 0.05 for 4% NaCl, compared with all other groups.

View larger version (30K): [in this window] [in a new window]   FIG. 2. Mean arterial pressures (direct) of young male SHR-SPs after 9 wk of feeding (mean ± SEM; n = 7–12 in each group) in experiment 1, both before and after hexamethonium treatment. Neither dietary NaCl excess nor genistein supplementation affected heart rate in the groups. *, P < 0.05, compared with respective controls; **, P < 0.05, compared with group’s pretreatment control.

The 4% NaCl diet and/or associated increases in arterial pressure caused significant damage to the kidneys in the rats that did not receive the G supplementation. G treatment was associated with significantly less damage in response to excess dietary NaCl (Fig. 3). Specifically, kidneys from the non-G-treated 4% NaCl diet group displayed mild expansion of the mesangial matrix and occasional collapsed glomeruli, marked medial thickening of large and small arterioles, occasional occlusion of the lumina (hyaline arteriolosclerosis), narrowed tubules, and fibrosis in 10–30% of each kidney. In contrast, the G-treated, 4% NaCl diet rats displayed occasional mild thickening of the vessels in a few animals and no other abnormalities. None of the rats on the basal NaCl diets displayed evidence of renal damage.

View larger version (152K): [in this window] [in a new window]   FIG. 3. Photomicrographs of kidneys sections from rats fed on a diet containing 4% NaCl with (A and C) or without (B and D) dietary genistein to document the protection afforded by genistein in this model. Note the renal cortex tubular dilatation, vessel hypertrophy (HV), and interstitial fibrosis in B, compared with A. Furthermore, note the interstitial fibrosis (e.g. circled in yellow) and hypertrophy of peritubular vessels (e.g. HV) in D, compared with C.

View larger version (22K): [in this window] [in a new window]   FIG. 4. Mean arterial pressures of young male SHR-SPs in experiment 2 (mean ± SEM; n = 8 in each group), both before and after hexamethonium treatment. Neither dietary NaCl excess nor genistein supplementation affected heart rate in the groups. *, P < 0.05, compared with respective controls; **, P < 0.05, compared with group’s pretreatment control.

View larger version (17K): [in this window] [in a new window]   FIG. 5. Effects of elevated NaCl intake on plasma levels of total cholesterol, triglycerides, and insulin in young male SHR-SPs on a phytoestrogen-free diet with or without genistein supplementation. Genistein lowered total cholesterol and triglycerides in rats on a basal (0.7%) salt diet. Genistein prevented the increase in plasma insulin and the reduction in insulin sensitivity associated with excess dietary NaCl intake. *, P < 0.05, compared with all other groups. QUICKI, Quantitative insulin sensitivity check index.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Whereas other reports suggest that blood pressure of male rats may be responsive to soy-based diets, this study indicates that soy’s primary isoflavone, genistein can lead to these effects in hypertensive male rats. The arterial pressure protection afforded to the male SHR is very similar to the effects in young and aged, ovariectomized SHRs (17, 18, 20). In our studies in female SHRs, estrogen treatment or genistein inhibited the hypertensive effects of a high-NaCl diet, but the treatment effects were not additive. This suggests that both estrogen and genistein may affect blood pressure through a common pathway, perhaps related to the estrogen ERβ receptor (30, 31).

Compared with 17β-estradiol, genistein has a very low binding affinity for ER receptors, but its binding affinity to ERβ receptors is only 2.8 times lower than that of 17β-estradiol (12). Furthermore, in both rats and humans consuming a soy-based diet, the circulating plasma concentrations of genistein are approximately 100- to 1000-fold greater than the concentration of 17β-estradiol (32).

Previous studies suggest that estrogen reduces arterial pressure and cardiovascular disease in humans, likely via an ERβ mechanism (see Ref. 3). For instance, in premenopausal women, circulating estrogen appears to reduce hypertension and cardiovascular disease, and these women are reported to be relatively resistant to NaCl-sensitive hypertension (33). Furthermore, in women the risk of developing hypertension, salt-sensitive hypertension, and cardiovascular disease significantly increases after menopause, but age-matched men show no similar increases in the rate of these symptoms (33). Circulating estrogen in men remains stable throughout life, whereas in women it is greatly reduced after menopause (34). Together, this suggests that low levels of estrogen may be protective in men throughout life, but the loss of estrogen at menopause may impair cardiovascular function in women. The findings of the Women’s Health Initiative discouraged use of estrogen therapy (35). Phytoestrogens may offer an effective adjuvant for both sexes.

It is unlikely that the present results are due to a direct inhibition of tyrosine kinase by genistein. Genistein is commonly used as a direct inhibitor of tyrosine kinase (36, 37, 38), but in vivo effects appear to be indirect, except at 10–100 nM circulating concentrations. In vitro, genistein directly inhibits tyrosine kinase but only at concentrations well above 5 µM (38). We did not directly assess plasma concentration of genistein in the present study, but in two parallel studies using the same feeding protocol, plasma genistein concentrations were less than 200 nM (our unpublished data), i.e. well below the concentration that directly inhibits tyrosine kinase in vitro. It remains possible that tissue concentrations reach much higher levels; however, in published studies and our experiments, genistein concentrations in organs does not rise above 1 µM using this feeding regimen (39, 40, 41). Further analyses are needed to test whether genistein concentrations in smaller compartments of organs are high enough to directly inhibit tyrosine kinase.

The present results indicate that dietary genistein can prevent adverse metabolic effects related to dietary NaCl excess in this model. Dietary genistein prevented decreased insulin sensitivity and increased circulating insulin in SHR-SPs on a high-NaCl diet. Furthermore, dietary genistein increased plasma NO in SHR-SPs on both the high and basal NaCl diet. This increase in circulating NO may also contribute to the antihypertensive effect of genistein in the high-NaCl-fed rats. The fact that the increased NO did not decrease arterial pressure in SHR-SPs on the basal NaCl diet is not unexpected because the later rats are not in as challenged a condition as the high-NaCl diet rats. Genistein treatment did not consistently alter circulating renin or aldosterone. These data decrease the likelihood that either hormone plays a major role in the NaCl-sensitive hypertension in this model. The data demonstrate that genistein improves plasma lipid and triglyceride concentrations in the SHR-SPs on the basal NaCl diet, but because plasma lipid concentrations were not significantly different in the groups on the high NaCl diet, any potential beneficial effects remain ambiguous.

Genistein may also act via other routes that we have not tested in this study. Genistein can cause direct vasodilation of resistance vessels (13). Furthermore, genistein may reduce oxidative DNA damage and increases intracellular total glutathione concentration in vascular smooth muscle cells (42), but these effects require micromolar concentrations, suggesting the effects if present are indirect (43).

The present findings support and extend previous reports indicating that soy-based diets provide beneficial effects on cardiovascular and metabolic function. In a human study, consumption of 500 ml of soy milk twice a day by men and women with mild and moderate hypertension reduce systolic, diastolic, and mean arterial pressure (44). In perimenopausal women, daily intake of 20 g of soy protein significantly decreases diastolic arterial pressure (45). A recent randomized, double-blind, placebo-controlled study demonstrated that in osteopenic postmenopausal women, 12 or 24 months of 54 mg genistein/d improves glucose homeostasis (indicated by decreased fasting glucose and insulin levels) without affecting endometrial thickness (46), underscoring the importance of evaluating metabolic effects of genistein. Despite an increasing armamentarium of pharmaceutical agents to treat diabetes and metabolic syndrome, optimal control remains elusive for many. Clearly, genistein will not serve in the place of these drugs, but it might provide complementary effects.

Despite many positive findings, the long-term cardiovascular health benefits of soy isoflavones in humans remain uncertain. Initial studies suggested that soy supplementation reduced cholesterol and blood pressure; however, the American Heart Association recently stated that the use of soy and soy supplements have not been proven consistently beneficial in human trials (47). Thus, greater assessment of the benefits of soy in particular human population groups is needed.

Hypertension causes considerable morbidity and mortality, and more effective methods to reduce hypertension with pharmacological and nonpharmacological treatments are constantly being sought. The current findings provide preclinical evidence that in intact male SHR-SPs soy’s major polyphenol (genistein) can decrease NaCl-sensitive hypertension. This finding suggests that polyphenols may have beneficial effects in both men and women and that the underlying mechanisms may be parallel between the sexes. Together with clinical studies demonstrating the beneficial effects of genistein, these results suggest that genistein may provide a beneficial adjuvant therapy for women and men with NaCl-sensitive hypertension.

	Acknowledgments

 
We thank Mr. Xinhua "Frank" Feng and Mrs. Nancy Brissie for their assistance.

	Footnotes

 
This work was supported by National Institutes of Health (NIH) Grant AT 00477 from the National Center for Complementary and Alternative Medicine (to J.M.W.) and the Office of Dietary Supplements and Grants NS 041071 (to J.M.W. and J.T.C.) and NS 047466 and NS 057098 (to J.M.W.) from the National Institute of Neurological Disorders and Stroke. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Center for Complementary and Alternative Medicine, the Office of Dietary Supplements, or the NIH.

The authors have nothing to disclose.

First Published Online August 2, 2007

Abbreviations: ER, Estrogen receptor; G, genistein; NO, nitric oxide; SHR, spontaneously hypertensive rat; SHR-SP, stroke-prone SHR.

Received February 21, 2007.

Accepted for publication July 23, 2007.

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