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(Hypertension. 1997;30:667.)
© 1997 American Heart Association, Inc.

Articles

Sexual Dimorphism of Sodium-Sensitive Renal-Wrap Hypertension

Joseph R. Haywood; Carmen Hinojosa-Laborde

From the Departments of Pharmacology and Physiology, University of Texas Health Science Center, San Antonio.

Correspondence to Joseph R. Haywood, PhD, UTHSCSA-Pharmacology, 7703 Floyd Curl Dr, San Antonio, TX 78284-7764. E-mail haywood{at}uthscsa.edu

	Abstract

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Abstract One-kidney, figure-8 renal wrap or sham operation was performed on male and female rats to determine if a difference existed in the expression of hypertension between the sexes. Animals were prepared with radiotelemetry transmitters to monitor mean arterial pressure and heart rate continuously throughout an 8-week study. Dietary sodium content was changed during the post–renal-wrap period from normal (100 µmol/g chow) to high (2000 µmol/g chow) to low (<2 µmol/g chow) to assess sodium sensitivity. Both male and female rats experienced an increase in arterial pressure after the renal-wrap procedure; however, the hypertension was significantly attenuated in the females. High salt caused a further increase in both groups of rats that was again attenuated in the female rats. Low sodium diet reduced arterial pressure in all groups of rats. Heart rate was significantly elevated in the female rats during all dietary interventions. The data were fit to a cosine function to analyze circadian rhythm changes. An increase in the light/dark difference for arterial pressure in the hypertensive rats during high salt diet resulted in an increase in the circadian amplitude (range of the rhythm). In the normotensive rats fed a high salt diet, the arterial pressure acrophase (peak of rhythm) was delayed while the heart rate acrophase was shifted to an earlier time. These studies showed that female rats are protected against one-kidney, figure-8 renal-wrap hypertension and are less sensitive to the effects of sodium.

Key Words: circadian rhythm • blood pressure • heart rate • hypertension, one kidney, figure-8 renal wrap • sex differences • hypertension, sodium-dependent

	Introduction

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Differences between males and females in the expression of hypertension have been well documented. In the clinical literature, premenopausal women have a consistently lower occurrence of high blood pressure than men that no longer exists after menopause.¹ In experimental animals, the sexual dimorphism in the manifestation of high blood pressure occurs in both genetic and induced models of hypertension. Females have lower resting arterial pressure in salt-sensitive spontaneously hypertensive rats,² Dahl salt-sensitive rats,³ deoxycorticosterone-salt–sensitive rats,⁴ and the TGR(mREN2)27 renin transgenic rat.⁵ The mechanism responsible for the difference in blood pressure is unknown; however, sex hormones are clearly involved in the process.

These sex differences have often been demonstrated with tail-cuff measurements of systolic pressure or short-term indwelling arterial catheters used to monitor arterial pressure. The development of a radiotelemetry system has permitted blood pressure measurement for extended periods of time, allowing long-term interventions such as changes in diet. Furthermore, the 24-hour recording of arterial pressure and heart rate has provided previously difficult to obtain information about cardiovascular regulation during the light/dark cycles. The goal of the present study was to determine if the sexual dimorphism that occurs in other models of hypertension applies to the sodium-dependent one-kidney, figure-8 renal-wrap model. In addition, sodium intake was changed during the experiment to assess the sodium sensitivity of male and female animals.

	Methods

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Age-matched male and female Sprague-Dawley rats were obtained from Harlan Sprague Dawley Inc (Indianapolis, Ind). All animals were maintained in Laboratory Animal Resources at the University of Texas Health Science Center (UTHSC) on a 14-hour light/10-hour dark cycle in a temperature-controlled environment. These studies were approved by the UTHSC Institutional Animal Care and Use Committee. Animals were maintained in accordance with the American Physiological Society Guiding Principles for the Use of Animals in Research and Teaching and the FASEB Statement of Principles for the Use of Animals in Research and Teaching. The UTHSC laboratory animal facility is fully accredited by AAALAC International.

Animal Care and Maintenance
Animals were anesthetized with methoxyflurane (Mallinckrodt Veterinary), and the flexible catheter attached to the radio transmitter (Data Sciences) was inserted directly into the abdominal aorta so as to rest just below the renal arteries. After awakening, the rats were housed individually in stainless steel cages and were permitted ad libitum access to a normal sodium diet (100 µmol/g chow; Teklad). Body weight and ad libitum water intake were monitored daily in each rat. Vaginal smears were performed daily to monitor phases of the estrus cycle in the female rats.

Experimental Protocol
After 2 to 4 weeks of control measurements of arterial pressure and heart rate had been taken, the male and female rats were divided into two groups. Sham operations, which consisted of a unilateral nephrectomy (n=6 males and n=6 females), were performed on one group. The other group was subjected to renal wrap combined with unilateral nephrectomy (n=7 males and n=8 females).⁶ The four groups of rats were maintained on the normal sodium diet for 2 weeks then changed to a high sodium intake (2000 µmol/g chow; Teklad) for 2 weeks. Finally, the animals were changed to a sodium-deficient diet (<2 µmol/g chow; Teklad) for an additional 2 weeks.

Data Acquisition and Analysis
The radio transmitter sent signals to a radio receiver that was attached to the inside of the cage. The Dataquest system (Data Sciences) was used to collect data; the system is designed to cycle from animal to animal. Data were acquired at a rate of 500 Hz for 20 seconds every 10 minutes. The data were averaged in 60-minute blocks for analysis. Mean arterial pressure and heart rate were averaged over a 24-hour period for daily values. In addition, the mean values for 4 to 5 days at the end of each intervention were used to determine differences between the light and dark periods of the 24-hour cycle. For analysis of circadian rhythms, the daily 60-minute values for 4 to 5 days were fitted to a cosine function to determine the acrophase (time of peak mean arterial pressure and heart rate), amplitude (half of the peak-to-trough difference of the rhythmic change), and mesor (midline of the rhythm adjusted to the cosine fit). Circadia software (Behavioral Cybernetics) was used for the analysis.

Data are expressed as mean±SEM. Because of differences in the variance of the data among the groups studied, a natural logarithmic transformation was performed on the data before analysis. The time-course data were analyzed with three-way ANOVA with repeated measures using SuperANOVA software (Abacus Concepts). Subsequent one-way and two-way ANOVAs and post hoc tests were performed when appropriate. Significance was taken at P<.05.

	Results

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The initial body weights were 358±8 g for the males and 255±3 g for the females. During the course of the study, body weight gradually increased to approximately 434±5 g in the males and 281±4 g in the females. Daily vaginal smears revealed that the female rats were experiencing estrous cycles of 4 to 5 days in length. After surgery or change in sodium diet, the duration of the cycle occasionally increased in some rats. Water intake normalized to body weight was significantly elevated during the control period in female rats (Fig 1). After renal wrap, water intake rose in both the males and females and remained elevated compared with sham-operated rats on all three diets. The female hypertensive animals generally drank more water than the male hypertensive rats; the same relationship held for female and male normotensive rats.

View larger version (32K): [in this window] [in a new window]   Figure 1. Water intake expressed as volume/100 g body weight is shown for renal-wrapped (top) and sham-operated (bottom) male and female rats during the course of the experiment. The vertical line to the left represents the time of renal surgery. The time of dietary interventions is shown in the bottom panel. The female rats are indicated by the closed symbols while the male rats are depicted by the open symbols. Results of the three-way repeated measures ANOVA are shown indicating statistical significance for main effects due to hypertensive treatment (Ht), gender (G), and time (T) as well as their interactions.

The renal-wrap procedure increased mean arterial pressure in both male and female rats on a normal sodium diet (Fig 2). The male rats had a higher blood pressure during the first 10 days after the wrap; however, by the end of the 2-week period, there was no difference between the sexes. When placed on a high sodium diet, mean arterial pressure increased further in both the male and female rats that were subjected to renal wrapping. The sham-operated groups of rats also experienced significant increases in arterial pressure on high salt. Two-way repeated measures ANOVA revealed a sex difference on the high sodium diet between renal-wrapped males and females but not between the groups of sham-operated rats. On sodium-deficient diet, the male and female animals overall did not differ. However, renal-wrapped female rats consistently had a higher arterial pressure than the sham-operated females. The change in arterial pressure during the transition from normal to high sodium intake took 9 days to reach the maximum increase in blood pressure in the hypertensive rats. In contrast, the decrease in arterial pressure associated with the transition from high to low sodium intake took approximately 35 hours in these rats.

View larger version (41K): [in this window] [in a new window]   Figure 2. Mean arterial pressure and heart rate over the course of the study are shown for renal-wrap (left) and sham-operated rats (right). The female rats are depicted by the closed symbols while the males are indicated by the open symbols. The vertical line indicates time of renal wrap or sham operation. The time of changes in dietary sodium intake is shown in the bottom panels. Results of the three-way repeated measures ANOVA are shown in the right panels for mean arterial pressure and heart rate. Main effects associated with the hypertensive treatment (Ht), gender (G), and time (T) are shown.

Heart rate was significantly lower in the male rats over time (Fig 2). On normal sodium diet, there was no difference between males and females during the control period. After surgery, the heart rate decreased in the male rats while remaining relatively constant in the females. The sex difference persisted through the first week of high sodium diet when heart rate fell significantly in the males. However, an increase in heart rate in the males during the second week of high salt diet brought heart rate to a similar level in wrapped male and female rats. When the animals were given a sodium-deficient diet, heart rate transiently increased in all groups, then returned to control levels in all groups except the hypertensive males, in which it decreased significantly.

During the control period, there was a 2–mm Hg difference in mean arterial pressure during the 24-hour light/dark cycle (Fig 3). The development of hypertension on normal sodium intake did not affect the light/dark difference in blood pressure. However, administration of high sodium diet resulted in a significant increase in the light/dark difference in hypertensive rats due to a greater increase in arterial pressure during the dark period. In the sham-operated male and female rats, arterial pressure rose equally in the light and dark periods in the respective groups. Heart rate was significantly increased in all groups during the dark period. High salt intake significantly suppressed the light/dark difference. In contrast, with sodium-deficient diet, the male rats had a greater light/dark difference in heart rate.

View larger version (27K): [in this window] [in a new window]   Figure 3. Difference in mean arterial pressure and heart rate between the light/dark periods is shown for the control period and post–wrap/sham operation normal sodium intake, high sodium intake, and low sodium intake periods. The groups are depicted as follows: male sham, solid bars; female sham, open bars; male wrap, shaded bars; and female wrap, stippled bars. The overall ANOVA tables demonstrate statistical significance for mean arterial pressure and heart rate. Results of post hoc tests are depicted as follows: *high sodium vs control, low sodium, and normal sodium; high sodium vs control and normal sodium; high sodium vs normal sodium and low sodium; and #high sodium vs low sodium. Differences within a diet are also shown by a connecting bar.

Circadian analysis demonstrated differences in the mesor that paralleled the absolute levels of arterial pressure and heart rate during the control period and each of the post–renal-wrap dietary interventions. The amplitude of the mean arterial pressure was significantly increased during high sodium intake; however, the increase was greater in the hypertensive animals (Fig 4A). The amplitude of heart rate was increased during transition from high sodium intake to low sodium intake in the hypertensive rats. The acrophase of the mean arterial pressure was not affected significantly in the hypertensive animals (Fig 4B). However, high salt intake in the sham-operated animals delayed the acrophase approximately 2 hours. In contrast, the heart rate acrophase was shifted approximately 1 hour earlier in the sham-operated rats.

View larger version (50K): [in this window] [in a new window]   Figure 4. The amplitude (A) and acrophase (B) are shown for mean arterial pressure and heart rate during the control period and post–wrap/sham operation normal sodium intake, high sodium intake, and low sodium intake periods. The groups are depicted as follows: male sham, solid bars; female sham, open bars; male wrap, shaded bars; and female wrap, stippled bars. The overall ANOVA tables indicate statistical significance for mean arterial pressure and heart rate. Results of post hoc tests are depicted as: *high sodium vs control, normal sodium, and low sodium; #high sodium vs low sodium; and high sodium vs control and low sodium. Differences within a diet are also shown by a connecting bar.

	Discussion

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This study confirmed previous observations demonstrating the sodium-sensitivity of renal-wrap male rats.⁷ When the hypertensive rats were given a high sodium diet, arterial pressure rose further. In contrast, a sodium-deficient diet caused a decrease in blood pressure to a level that was not different from the prewrap control period. The major finding of the present study was that the development of one-kidney, figure-8 renal wrap hypertension is attenuated in female rats maintained on a normal sodium diet. When the diet was changed to high salt, renal-wrapped females experienced a gradual but attenuated increase in arterial pressure. In both males and females, the time to the peak arterial pressure was 9 days. These findings generally support the observations in other sodium-dependent models such as the Brookhaven salt-sensitive Dahl rats,³ deoxycorticosterone-salt–sensitive rats,⁴ and salt-sensitive spontaneously hypertensive rats² that have shown that females are protected against the development of hypertension.

The continuous blood pressure measurement with radiotelemetry transmitters revealed a light/dark difference in arterial pressure that was similar in normotensive and hypertensive rats until the animals were placed on a high sodium diet. The high salt intake increased both daytime and nocturnal arterial pressures; however, the nocturnal blood pressure increased more in the hypertensive rats. The time of this increase in blood pressure coincides with the feeding and drinking period. Consequently, the animals were receiving the major portion of the salt and volume stimulus during the augmented rise in blood pressure.

These studies also showed that when placed on a high sodium diet, sham-operated male and female rats experienced an increase in arterial pressure. The mild hypertension associated with high salt intake has been previously reported using the same telemetry system.⁸ However, indwelling catheters in conscious animals have not previously shown these direct effects of sodium, probably because measurements are usually made during daytime hours. In contrast to the hypertensive rats, sham-operated animals experienced equivalent increases in blood pressure during the day and at night during high salt intake. These differences may reflect the response to differences in sodium retention between renal-wrapped and sham-operated rats. The attenuated responses in female rats are the result of either less sodium retention or a reduced stimulation by sodium.

Heart rate was consistently higher in female rats. In response to the induction of hypertension, male rats experienced decreased heart rate while heart rate was maintained at control levels in female rats. It is unknown whether this sex difference is related to differences in baroreflex function or control of cardiac output. Further studies are necessary to clarify this question. High salt intake resulted in a further reduction in heart rate in the males and a significant but lesser decrease in females. These decreases are consistent with the heart rate responses to high sodium intake observed by others.² The bradycardic effect was reversed in males at the end of the second week, when heart rate returned to control levels.

Sodium-deficient diet reduced arterial pressure in all groups of rats. Blood pressure in both groups of male rats fell to the same level. The decrease in arterial pressure in female hypertensive animals reached the same level as in the male hypertensive rats; however, it was still significantly higher than the blood pressure of the sham-operated female rats. In contrast to the gradual increase in arterial pressure during the transition from normal to high sodium intake, the fall in blood pressure during the transition from high to low sodium diet was much more rapid. Both males and females reached minimum arterial pressure in approximately 35 hours. The rapid decrease in blood pressure may have been caused by a withdrawal of sympathetic activity, which has been shown to be elevated in this model of hypertension.^{7 8}

Analysis of the circadian changes in arterial pressure and heart rate showed additional effects associated with changes in sodium intake. The mesor, or mean value of the cosine fit of the data, reflected the recorded levels of arterial pressure and heart rate. The range of arterial pressure during a 24-hour period, expressed as the amplitude of the cosine function, increased significantly in the hypertensive rats maintained on high salt. This observation is in agreement with two other reports in sodium-dependent hypertensive rats showing that an increase in amplitude accompanied the rise in arterial pressure with high salt intake.^{9 10} This response was likely related to the greater increase in nocturnal arterial pressure observed in these groups of rats.

High sodium intake also shifted the arterial pressure acrophase in the normotensive rats. This effect has been reported previously in Dahl rats¹⁰ in which both salt-sensitive and salt-resistant animals experienced a significant delay relative to salt-resistant animals. In the same study, the acrophase for heart rate was shifted to an earlier time in the high salt–resistant Dahl rats in a manner similar to that observed in the present study. These responses may be related to the effects of salt on the suprachiasmatic nucleus either through pathways from sodium-sensitive areas of the hypothalamus¹¹ or behavioral influences, since rats are nocturnal animals. The effects on blood pressure and heart rate may be mediated by efferent projections from the suprachiasmatic nucleus to the paraventricular nucleus,¹² an area that is known to have effects on the regulation of the sympathetic nervous system.^{13 14}

In summary, this study demonstrated that there is a sexual dimorphism in the expression of one-kidney, figure-8 renal-wrap hypertension. Female rats are protected in part against the development of hypertension when fed a normal sodium diet. When the animals are given a high sodium diet, there is a further increase in blood pressure in both male and female animals. Over the course of the normal and high salt diets, female rats are generally less sensitive to the effects of sodium on blood pressure. Importantly, salt appears to have a significant effect on the circadian regulation of arterial pressure in normotensive and hypertensive animals, affecting the time and magnitude of peak cardiovascular responses.

	Acknowledgments

 
This work was supported by awards from the National Institutes of Health (HL-36080 to J.R.H. and HL-03153 to C.H.-L.) and a Grant-in-Aid from the American Heart Association (No. 94008450 to C.H.-L.). The authors wish to acknowledge the important contribution of Kimberly Holliday-White for her valuable skills with the telemetry system. Also, we would like to thank Patti Lairsey for her excellent secretarial assistance.

Received March 17, 1997; first decision April 18, 1997; accepted May 7, 1997.

	References

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13. Martin DS, Segura T, Haywood JR. Cardiovascular responses to bicuculline in the paraventricular nucleus of the rat. Hypertension. 1991;18(suppl 1):48-55.

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Haywood, J. R. Articles by Hinojosa-Laborde, C. Search for Related Content PubMed PubMed Citation Articles by Haywood, J. R. Articles by Hinojosa-Laborde, C.