Gender difference in diet-induced obesity hypertension: implication of renal α2-adrenergic receptors

AJH

2002; 15:143–149

Gender Difference in Diet-Induced Obesity Hypertension: Implication of Renal ␣2-Adrenergic Receptors Gwenn Coatmellec-Taglioni, Jean-Pierre Dausse, Yves Giudicelli, and Catherine Ribie` re Although the pathogenesis of the obesity-related hypertension is not fully understood, prevalence of the cardiovascular complications is much higher in obese men than obese women. In a recent study, we reported that male rats fed a cafeteria diet, while becoming obese, developed hypertension and important changes in their renal ␣2adrenergic receptor subtypes distributions. The aim of the present study was to investigate whether these alterations are sex dependent. After 10 weeks of the cafeteria diet, male and female rats had the same increase in fat pad weight and in plasma leptin levels. However, in contrast to males, females had normal blood pressure (BP). On the basis of radioligand-binding studies using [3H]-RX821002 and confirming our recent observation, an increase in ␣2-adrenergic receptor densities occurred in kidneys of

I

cafeteria-fed male but not female rats. Moreover, in contrast with the situation observed in males, ligand competition studies failed to reveal any change in the renal ␣2Aand ␣2B-adrenergic receptors subtypes distribution in females. Finally, in the cafeteria-fed females reverse transcription-polymerase chain reaction experiments showed unaltered expression of these two ␣2-adrenergic receptor subtypes. These data thus suggest a strong relationship between the sexual dimorphism in the cafeteria diet-induced hypertension and altered expression of the ␣2-adrenergic receptor subtypes in the kidney. Am J Hypertens 2002;15:143–149 © 2002 American Journal of Hypertension, Ltd. Key Words: Blood pressure, cafeteria diet, sex, ␣2adrenergic receptors, obesity.

ncreased body weight is often associated with metabolic disorders as well as increased blood pressure (BP). Although the precise mechanisms whereby obesity increases BP are poorly understood, abnormalities in kidney function that cause a hypertensive shift of pressure natriuresis appear to play a major role in obesity induced-hypertension.1,2 Furthermore, increased activity of the sympathetic nervous system appears to contribute to elevated BP and to cause sodium retention in obesity, mainly by the renal nerves.1,3 The ␣2-adrenergic receptors (␣2-AR) stimulation in the kidney has been shown to increase urine flow rate, osmolar clearance, free water clearance, and sodium excretion.4 – 6 Two different isoforms of ␣2-AR have been described in rat kidney. Only ␣2A-AR is able to increase blood flow, osmolar clearance, and sodium excretion.5,6 Furthermore, experimental studies in animals with pharmacologic probes in vivo6 and radioligand-binding technique in

vitro,7–9 have suggested that hypertension is associated with alterations in the functional characteristics of the ␣2-AR subtypes in the kidney. As an example, we have previously shown that male rats fed a cafeteria diet are overweight, hypertensive, and develop disruption in their renal ␣2-AR subtype distribution9 as do genetically hypertensive rats.7,8 In fact an increased ␣2B-AR subtype density was observed in the kidney of male cafeteria-fed rats, whereas ␣2A-AR subtype was no more detectable by radioligand-binding studies. As the prevalence of vascular disease is greater in obese men than obese women, sex hormones are likely involved in the relative protection against cardiovascular disease in women before menopause.10 It has also been established that renal ␣2-AR subtypes expressions and densities are dependent on sex hormones in rats.7,11 Furthermore in humans, responses to an ␣2-AR agonist differ according to sex.12 The aim of the present study was 1) to determine

Received April 4, 2001. Accepted August 28, 2001. From the Department of Biochemistry and Molecular Biology, Faculte´ de Me´decine de Paris-Ouest, Universite´ Rene´ Descartes, Paris, France. This work was supported by grants from the University Rene´ Descartes. Paris V, France.

Address correspondence and reprint requests to Dr. Catherine Ribie`re, Department of Biochemistry and Molecular Biology, Faculte´ de Me´decine de Paris-Ouest, Universite´ Rene´ Descartes, 45 rue des SaintsPe`res, 75270 Paris Cedex 06, France; e-mail: [email protected]

© 2002 by the American Journal of Hypertension, Ltd. Published by Elsevier Science Inc.

0895-7061/02/$22.00 PII S0895-7061(01)02269-5

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whether a sex dimorphism also occurs in cafeteria-fed rat model of obesity hypertension, and 2) to evaluate the implication of the renal ␣2-AR subtypes expressions and distributions in the onset of hypertension.

Methods Cafeteria Diet Male and female Sprague-Dawley rats with an average weight of 100 g were divided into two groups and maintained at room temperature with a 12-h light– dark cycle. The first group (control) received standard chow (UAR, Epinay sur Orge, France; protein 25%, fat 6%, carbohydrate 69%) ad libitum. The second group was given a cafeteria diet for 10 weeks as previously described.9 The average composition of the diet was protein 15%, fat 48%, and carbohydrate 36%.13 Systolic BP was measured between 10 and 12 AM using the tail– cuff method, with an electrosphygmomanometer (Physiograph MK III, Narco-Bio-Systems Inc., Houston, TX), on unanesthesized, restrained rats warmed to 38°C for 10 min. At least five replicate BP measurements were obtained on 2 consecutive days (at least 10 measurements over 2 days). The average of all measurements for each rat was taken as representative of systolic BP. Two days later, rats were killed by decapitation. All experimental protocols were approved by the University Animal Use and Care Committee. Radioligand-Binding Studies Renal membranes were prepared from whole kidney and radioligand-binding studies were performed as described previously,8 with a specific ␣2-AR antagonist [3H]RX821002 as radiolabeled ligand. Briefly, 300 ␮g of renal membranes were incubated with 1 to 12 nmol/L [3H]RX821002, 1 mmol/L EDTA-K, 100 ␮mol/L 5⬘-guanylylimidodiphosphate, 140 mmol/L sodium chloride, 50 mmol/L Tris-HCl (pH 7.4), in a final volume of 300 ␮L for 45 min at 25°C. Reactions were stopped by dilution with ice-cold incubation buffer and rapid vacuum filtration through Whatman GF/C filters. Filters were washed twice with ice-cold incubation buffer and the radioactivity re-

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tained on filters was quantified by liquid scintillation counting. Nonspecific binding determined in the presence of 20 ␮mol/L phentolamine averaged 10% of the total binding. For competition studies, membranes were incubated with 2.5 nmol/L [3H]-RX821002 (a concentration similar to the ligand-receptor Kd value) and various concentrations (0.1 nmol/L to 1 mmol/L) of guanfacine, a selective ␣2A-AR agonist, or prazosin, selective for the ␣2B-AR for 45 min at 25°C. The resultant competition curves were analyzed using a nonlinear least-square curvefitting program (GraphPAD PRISM; GraphPad Software Inc., San Diego, CA). Protein concentrations were determined according to Bradford,14 using gamma globulin as a standard. Analysis of Renal ␣2-AR mRNA Total RNA from whole kidney was isolated using TRIzol reagent and cDNA was generated using the Superscript II protocol (Life Technologies, Cergy-Poutoise, France). Briefly, mRNA was isolated by incubating 1.5 ␮g of total RNA for 10 min at 70°C in the presence of 0.5 ␮g oligo(dT)12–18. The cDNA synthesis was conducted for 50 min at 42°C with 0.1 mol/L DTT, 10 mmol/L dNTP, and 200 U of Superscript II followed by heat inactivation at 70°C for 15 min. Ten percent of the reverse transcription products were amplified in polymerase chain reaction (PCR) mixture containing 200 ␮mol/L of each dNTP, 0.5 ␮mol/L of each primer, 1 ␮Ci [3H]-dCTP, and 2.5 U HotStarTaq DNA polymerase, using a program temperature control system. After initial activation of the HotStarTaqDNA polymerase for 15 min at 95°C, 26 amplification cycles were performed for 1 min at 94°C, 1 min at 53°C, and 1 min at 72°C for ␤-actin, 30 and 35 cycles for 1 min at 94°C, 1 min at 56°C, and 1 min at 72°C for ␣2B- and ␣2A-AR, respectively. Primers used for amplification were described elsewhere (␤-actin15, ␣2A-AR16, ␣2B-AR8). The PCR products were separated by low melt agarose gel electrophoresis, the bands of interest were cut off the gel, melted, and radioactivity was quantified by liquid scintillation counting. Reported data were corrected for ␤-actin values and expressed as percentage of the control values.

Table 1. Characteristics of cafeteria-fed rats Males Control Blood pressure (mm Hg) Fat pad weight (g)‡ Plasma leptin (ng/mL) Plasma insulin (ng/mL)

129 27.9 5.6 1.8

⫾ ⫾ ⫾ ⫾

2 2.9 0.7 0.4

Females Cafeteria 154 49.1 16.8 5.4

⫾ ⫾ ⫾ ⫾

6* 5.7* 4.1* 0.8†

Data shown are mean ⫾ SEM of 5 rats. * P ⬍.05; † P ⬍.01 v corresponding control. ‡ Fat pad weight (mesenteric, perirenal, subcutaneous, epididymal or parametrial).

Control 131 22.2 5.7 1.6

⫾ ⫾ ⫾ ⫾

3 1.4 0.8 0.1

Cafeteria 132 42.8 16.4 5.4

⫾ ⫾ ⫾ ⫾

2 8.7* 4.0* 1.0†

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FIG. 1. Saturation curve of [3H]-RX821002, specific binding to renal ␣2-AR from (A) male (‚) control rats and (Œ) cafeteria-fed rats and (B) female (䊐) control rats and (■) cafeteria-fed rats. Data are representative of five separate experiments and each experiment was conducted in duplicate. ␣2-AR ⫽ ␣2-adrenergic receptor.

Results

Materials [ H]-RX821002 (2.29 ⫻ 10 Bq/mmol), was purchased from Amersham Pharmacia Biotech (Orsay, France) and rat insulin and leptin from LINCO (LINCO Research Inc., St. Charles, MO). The DNA molecular weight markers (100-bp ladder) were from Amersham Pharmacia Biotech, oligonucleotides from Eurogentec (Herstal, Belgium), prazosin from Pfizer Inc. (Sandwich, UK), and guanfacine from Sandoz Ltd. (Basel, Switzerland). All other materials were obtained from Sigma Aldrich (Saint Quentin Fallavier, France).

Physiologic Data

Statistical Analysis

Renal ␣2-AR Subtypes

All results were expressed as the mean ⫾ SEM. For binding studies, statistical analysis were performed using ANOVA. Data from DNA amplification were analyzed using Student t test (P ⬍ .05 was considered statistically significant).

In an attempt to better understand the role of the renal ␣2-AR in the onset of hypertension, ␣2-AR densities were studied in the kidney of male and female rats. Representative saturation experiments for [3H]-RX821002 in kidney membranes from control and cafeteria-fed rats are

3

12

Confirming previous data from our laboratory,9 the BP in cafeteria-fed male rats was significantly increased (Table 1). In contrast, in the female rats no difference in the BP was found between control and cafeteria-fed groups. However, fat pad weights were almost doubled after cafeteriadiet feeding whatever the gender. As a consequence, plasma leptin levels were drastically increased in both male and female cafeteria-fed groups and the same also applied for plasma insulin levels.

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FIG. 2. Binding experiments of an ␣2-AR antagonist, [3H]-RX821002, to renal ␣2-AR from female (䊐) control rats and (■) cafeteria-fed rats. A) Guanfacine (selective ␣2A agonist) and (B) prazosine (selective ␣2B antagonist), compete for ␣2-AR antagonist-specific binding. Data are representative of five separate experiments and each experiment was conducted in duplicate. Abbreviation as in Fig. 1.

shown in Fig. 1. Under the conditions used, radioligand bound to renal membranes in a saturable manner and labeled a single class of binding sites in each group. Consistent with our previous report,9 ␣2-AR density was increased in the kidney of male hypertensive cafeteria-fed rats (Fig. 1A). In contrast, in female rat kidneys, neither maximal density (52.72 ⫾ 2.28 fmol/mg protein and 59.64 ⫾ 1.71 fmol/mg protein in control and cafeteria-fed rats, respectively), nor mean Kd value (1.45 ⫾ 0.23 nmol/L and 1.74 ⫾ 0.14 nmol/L in control and cafeteria-fed rats, respectively) were altered after cafeteria feeding (Fig. 1B). In female rats, ␣2-AR subtype distribution was evaluated under the same experimental conditions as those we recently used for male rats9: competition-binding experiments using either guanfacine or prazosin as competitor (Fig. 2). The data were analyzed by computer modeling and the results are summarized in Table 2. This analysis revealed that those binding data best fitted a two-site model for both control and cafeteria-fed female rats (P ⬍ .05). Table 2 also summarized the pKi values for guan-

facine and prazosin, which are comparable to those previously determined in the same tissue.17,18 The analysis further showed that in the kidney of female rats, proportion of ␣2A-AR (with high affinity for guanfacine) and ␣2B-AR (with high affinity for prazosin) remained insensitive to the diet.

␣2-AR Subtype Gene Expressions in Kidney of the Female Rats Fig. 3A shows a representative gel electrophoresis of the PCR products. cDNA amplification yielded amplified products of predicted size (311 bp for ␣2A-AR; 407 bp for ␣2B-AR; 280 bp for ␤-actin). Fragment generated from ␤-actin primer, which was chosen to span two introns, was the only fragment amplified in both groups, suggesting no genomic DNA contamination. Analysis of the ␣2-AR mRNA products normalized to ␤-actin are shown in Fig. 3A. Cafeteria-diet feeding did not significantly modify

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Table 2. Characteristics of ␣2-adrenergic receptor subtypes of female rat kidneys pKi High Affinity Control Cafeteria

pKi Low Affinity

% of High Affinity Binding

Guanfacine

Prazosin

Guanfacine

Prazosin

Guanfacine

Prazosin

7.08 ⫾ 0.41 7.29 ⫾ 0.24

7.64 ⫾ 0.57 7.36 ⫾ 0.30

6.10 ⫾ 0.41 6.07 ⫾ 0.13

5.83 ⫾ 0.57 5.82 ⫾ 0.27

50 ⫾ 6 46 ⫾ 5

44 ⫾ 4 47 ⫾ 5

Data shown are mean ⫾ SEM of 5 experiments.

either ␣2A-AR mRNA or ␣2B-AR mRNA levels in the kidney of the female rats.

Discussion The present study provides clear evidence for a gender difference in the onset of hypertension during cafeteriadiet feeding. After 10 weeks cafeteria-diet feeding, female rats remained normotensive, whereas male rats developed hypertension.9 Despite this difference, however, both female and male rats had increased fat mass and high plasma leptin and insulin levels. Several studies have suggested an important role for

androgens in the pathogenesis of cardiovascular diseases. For example, in male spontaneously hypertensive (SHR) and Dahl salt-sensitive rats, two genetic models of hypertension, elevation in BP was reduced by gonadectomy.7,19,20 Furthermore, Reckelhoff et al21 have shown that the hypertensive shift in pressure natriuresis relationship found in SHR was specifically mediated by testosterone or one androgen metabolite. Thus, in these rat models at least, androgens appear to be key factors in exacerbation of hypertension. A feature commonly shared by genetic models of hypertension is an increase in renal ␣2-AR density,7,8 which was also observed in male cafeteria-fed rats.9 In contrast,

FIG. 3. A) Ethidium bromide staining of RT-PCR products, using ␣2-AR subtypes or ␤-actin primers in kidney of female control rats (lanes 2, 4, 6) and cafeteria-fed rats (lanes 3, 5, 7). Results are representative of five experiments. DNA size markers, lane 1. B) Expression of ␣2-AR mRNA from kidney of female (open bar) control rats and (solid bar) cafeteria-fed rats. Values are expressed as the percentage of the control rats mRNA determined by liquid scintillation counting and normalized to ␤-actin. Data are given as the mean ⫾ SE (n ⫽ 5). RT-PCR ⫽ reverse transcriptase-polymerase chain reaction; other abbreviation as in Figs. 1 and 2.

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the present study, revealed unaltered ␣2-AR density in the kidney of female rats after cafeteria-diet feeding. To explain this sex-related difference in the renal ␣2-AR sensitivity to cafeteria diet, it could be postulated that change in ␣2-AR density is a consequence of hypertension. However, increased renal ␣2-AR density occurs before BP elevation in the SHR22 and is not observed in acquired forms of hypertension, such as the two kidney-one clip, the one kidney-one clip, and the deoxycorticosterone acetate salt-induced hypertension.23 Thus, sex differences in renal ␣2-AR sensitivity to cafeteria diet rather than being the consequence of hypertension, could play a causal role in the protection against the onset of hypertension in female rats. Involvement of male sex hormones in establishment of the enhanced renal ␣2-AR susceptibility to hypertensive stimuli has been suggested by Gong et al.7 They reported that higher BP is associated with higher renal ␣2-AR density in male than female SHR and Wistar rats and that castration reduces both renal ␣2-AR density and BP, whereas testosterone replacement resulted in opposite effects. In another study from the same laboratory, testosterone was reported to enhance ␣2B-AR subtype expression in the kidney of male SHR and this effect was significantly correlated with BP.11 More recently, in the ␣2B-AR-deficient mice model it was found that these animals were unable to increase their BP because they failed to retain sodium and hence, never reached a salt-loaded state.24 Thus, it appears that when the ␣2B-AR subtype is overexpressed in the kidney, as it does in male and gonadectomized testosterone-treated SHR, this leads to an hypertensive shift in pressure natriuresis and to increased BP. Interestingly, we found such an overexpression of ␣2B-AR in the kidney of male cafeteria fed rats9 who are hypertensive but not in the females who elicit normal BP. From these observations, it can reasonably be hypothesized that the low androgen status characterizing female rats may prevent ␣2B-AR increase and play a protective role against the onset of hypertension, which is observed in males after 10 weeks cafeteria-diet feeding. Furthermore, in the kidney of the male cafeteria-fed rat, the ␣2A-AR was no more detectable by radioligand-binding studies.9 Because ␣2A-AR in the rat kidney are implicated in the osmolar clearance and sodium excretion,5 the alteration in renal ␣2A-AR observed in male cafeteria-fed rats, as in genetic model of hypertension,6 may be involved. On the another hand, because of the well-known cardiovascular protective action of estrogen, it cannot be excluded that these hormones also contribute to maintain at normal levels the BP of female cafeteria-fed rats. In summary, although cafeteria feeding increased the BP in male rats, the female rats are protected against the diet-induced hypertension. However, male as well as female cafeteria-fed rats have significantly higher fat pad weight, plasma insulin and leptin levels. Contrary to the situation in male rat kidneys, ␣2-AR subtype densities and expressions in female rat kidneys remained unchanged

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with the diet. In female rats, the lack of alterations in renal ␣2-AR during cafeteria diet could play a pivotal role in the prevention of BP elevation.

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