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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-0941
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 1 386-394
Copyright © 2005 by The Endocrine Society

Responses to a Saline Load in Gonadotropin-Releasing Hormone Antagonist-Pretreated Premenopausal Women Receiving Progesterone or Estradiol-Progesterone Therapy

Nina S. Stachenfeld, David L. Keefe and Hugh S. Taylor

John B. Pierce Laboratory and Departments of Epidemiology and Public Health, and Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine and Women and Infants Hospital, Brown University School of Medicine, New Haven, Connecticut 06519

Address all correspondence and requests for reprints to: Dr. Nina S. Stachenfeld, John B. Pierce Laboratory, 290 Congress Avenue, New Haven, Connecticut 06519. E-mail: nstach{at}jbpierce.org.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The effects of estradiol (E2) and progesterone (P4) on fluid and sodium regulation may have important clinical implications with respect to cardiovascular and renal disease as well as reproductive syndromes such as preeclampsia and ovarian hyperstimulation syndrome. We tested the hypothesis that sodium excretion is reduced in response to a sodium load during combined P4-E2 treatment, but P4 administration alone has little effect on sodium regulation. Fifteen women (22 ± 2 yr) used a GnRH antagonist to suppress endogenous E2 and P4 for 9 d; for d 4–9, eight subjects used P4 (200 mg/d), and seven subjects used P4 with E2 (two E2 patches, 0.1 mg/d each). On d 3 and 9, isotonic saline (0.9% NaCl) was infused [120 min at 0.1 ml/kg body weight (BW)·min], followed by 120 min of rest. Compared with GnRH antagonist alone, P[P4] increased from 1.6 ± 0.8 to 9.4 ± 2.3 ng/ml (5.1 ± 2.5 to 29.9 ± 7.3 nmol/liter, P < 0.05) in the P4 treated group, with no change in P[E2]. In the P4-E2 treated group P[P4] increased from 1.6 ± 0.5 to 6.7 ± 0.6 ng/ml (5.1 ± 1.6 to 21.3 ± 1.6 nmol/liter, P < 0.05 and P[E2] increased from 17.9 ± 6.3 to 200 ± 41 pg/ml (65.7 ± 23 to 734.6 ± 150.0 pmol/liter, P < 0.05). Before isotonic saline infusion, renal sodium and water excretion were similar under all conditions, but during isotonic saline infusion, cumulative sodium excretion was lower in the P4-E2 treated women (34.1 ± 5.1 mEq) compared with GnRH antagonist (50.2 ± 11.4 mEq). Sodium excretion was unaffected by P4 treatment (48.0 ± 8.2 and 41.2 ± 5.1 mEq, for GnRH antagonist and P4). Compared with GnRH antagonist alone, P4-E2 treatment increased distal sodium reabsorption and transiently decreased proximal sodium reabsorption, whereas P4 treatment did not alter either distal or proximal sodium reabsorption. Before isotonic saline infusion, the plasma aldosterone (Ald) concentration was greater during P4 treatment (153 ± 25 pg/ml; 3883 ± 1102 pmol/liter) and P4-E2 treatment (242 ± 47 pg/ml; 6373 ± 1390 pmol/liter) than during their respective GnRH antagonist alone treatments [96 ± 13 and 148 ± 47 pg/ml (2598 ± 475 and 3284 ± 973 pmol/liter) for P4 and combined P4-E2, respectively]. Compared with GnRH antagonist alone treatments, preisotonic saline infusion plasma renin activity was greater only with P4-E2 treatment, whereas the plasma atrial natriuretic peptide concentration was lower only with P4 treatment. Isotonic saline infusion suppressed plasma Ald under all conditions, but decreased plasma renin activity only with P4-E2 treatment (average decrease, 1.3 ± 0.5 ng/ml angiotensin I·h; P < 0.05). In summary, we found that P4-E2 treatment decreased sodium excretion via either renin-angiotensin-Ald system stimulation or direct effects on kidney tubules. P4 treatment at these plasma concentrations had no independent effect on the renal response to acute sodium loading. These data suggest that E2 is the more powerful reproductive hormone involved in sodium retention relative to P4, and that estrogen-induced up-regulation of P4 receptors is required for the effects of P4 on sodium regulation.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
ALTHOUGH PROGESTERONE (P4) and estradiol (E2) primarily control reproductive function, receptors for these steroid hormones have been found in nonreproductive sites, such as the brain (1, 2, 3, 4, 5), cardiovascular system (6, 7), and kidney tubules (7, 8). These receptors in renal tubules are involved in the regulation of sodium, potassium, and calcium (9, 10). E2- and P4-related effects on sodium and fluid regulation may have important clinical implications with respect to fluid balance during the normal menstrual cycle, orthostatic hypotension, hypertension, and sodium retention during pregnancy or other high P4-E2 states, such as ovarian hyperstimulation syndrome (OHSS) and preeclampsia.

E2 increases body fluid and sodium retention (11), the result of greater sodium reabsorption probably via direct effects on renal distal tubules (9, 12). The effects of P4 on sodium regulation may be more indirect. P4 competes with aldosterone (Ald) for the mineralocorticoid receptor in the distal tubule (13). The sodium and water retention hormones renin, Ald, and angiotensin II increase concomitant with elevations in P4 (14, 15, 16), so any natriuretic effects of P4 are thought to be transient and to rarely induce significant overall body sodium losses. In addition to renin-angiotensin-Ald system (RAAS) stimulation, increases in the plasma P4 concentration (P[P4]) may influence sodium regulation through inhibition of atrial natriuretic peptide (ANP) synthesis and release from cardiac myocytes (17, 18, 19, 20).

Studies that have examined the effects of P4 on sodium regulation in humans have compared sodium regulatory responses between the early follicular and midluteal phases of the menstrual cycle. Plasma renin activity (PRA) and plasma Ald concentration (P[Ald]) increase during the midluteal phase of the menstrual cycle, although significant increases in sodium and/or water retention are transient (21, 22). The design in which different phases of the menstrual cycle are used to determine the effects of estrogens and P4 on body systems does not permit an evaluation of the independent effects of P4, because there is no time in the menstrual cycle when P4 is elevated without concomitant elevations in E2. However, an evaluation of the independent effects of P4 is warranted because P4 is increased without concomitant increases in estrogens in during nonphysiological situations, such as P4-only oral contraception. Moreover, these studies are relevant to certain disease states, as suggested by the fluid changes in conditions such as preeclampsia and OHSS. Both of these conditions are associated with fluid retention, extravascularization and edema, and ascites in OHSS.

Although P4 may be the primary stimulant for RAAS stimulation during the normal menstrual cycle, a certain level of E2 may be a necessary condition for any P4 effect on sodium regulation. E2 up-regulates P4 receptors in the reproductive system and may also have this effect within the sodium and fluid regulatory system (11, 21, 23). Thus E2 must be suppressed before the P4 effects on sodium regulation can be isolated. Moreover, the interaction between P4 and E2 with the RAAS and ANP has not been examined. Finally, a number of studies have demonstrated that study of the effects of sex hormones on sodium regulation requires that renal segmental tubular responses be taken into account (11, 23). The purpose of the present investigation was to determine P4 effects, with and without E2, on sodium excretion and renal tubular sodium handling in response to an acute salt load. We hypothesized that sodium excretion would be reduced in response to an acute sodium load during combined P4-E2 administration, but P4 administration alone would have little effect on sodium regulation.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
We recruited 16 healthy nonsmoking women from advertisements posted on the campus of Yale University. All subjects were within normal body mass index, ranging from 19–27 kg/m2. A gynecologist interviewed all subjects to attain their medical history and ensure that the women had no contraindication to GnRH antagonist or reproductive hormone administration. Subjects provided written confirmation of a negative Papanicolaou smear and normal physical examination within 1 yr of being admitted to the study. None of the subjects was taking oral contraceptives, and all had regular ovulatory cycles before beginning GnRH antagonist administration. Subjects gave written informed consent to participate in the study, which had prior approval by the human investigation committee of Yale University School of Medicine, and these studies were performed in a manner consistent with guidelines put forth in the Declaration of Helsinki. Before beginning the studies described below, the subjects visited the laboratory for an orientation to the laboratory procedures and to sign the consent form.

Experimental design

To suppress reproductive function for the duration of the study, the subjects self-administered the GnRH antagonist, ganirelex acetate (250 µg/d; Antagon, Organon, Inc., West Orange, NJ) each day for 9 d (Fig. 1Go, top) (24, 25). Beginning on the fifth day, either oral micronized P4 was given to the women in a dose of 200 mg at bedtime daily (200 mg/d; Prometrium, Solvay Pharmaceuticals, Marrietta, GA; n = 8; 23 ± 2 yr) for 4 d, or oral micronized P4 was given to the women in a dose of 200 mg at bedtime daily along with 17ß-E2 (P4-E2; two transdermal patches delivering 0.1 mg/d each; Vivelle, Ciba Pharmaceuticals, Summit, NJ; n = 7; 20 ± 0 yr; one subject was excluded from the study, see below) for 4 d (Fig. 1Go, top). The subjects changed the patches on the third day of administration, the evening before the experiment. Although this P4 dose led to slightly lower plasma P4 values than those typically seen in the midluteal phase, we chose this dose to minimize symptoms such as semnolence. We considered minimizing such symptoms essential for subject retention and compliance. Moreover, this dose led to significant increases compared with the GnRH antagonist treatment days in both groups (see Results). Group assignment was given alternately as subjects entered the study. Experimental protocols were performed on the third (GnRH antagonist alone) and ninth (GnRH antagonist with hormone) days of GnRH antagonist administration. This design permitted within-subject comparisons concerning hormone effects on sodium and fluid regulation without concern for slow hormone washout between trials.



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  		FIG. 1. A, Protocol for GnRH antagonist and hormone administration. B, Protocol for sodium-loading. BS, Blood sample; U, urine sample; BP, blood pressure; HR, heart rate.

 
Experimental protocol (Fig. 1Go, bottom)

Acute sodium loading was achieved through isotonic saline infusion. The subjects were instructed to maintain their usual diets for 3 d before the protocol while avoiding foods high in sodium. To maintain adequate hydration, the subjects were instructed to drink approximately 25 ml/kg water·d and avoid alcoholic beverages. The subjects were provided meals the night before (between 1900–2200 h) and the morning (1 h before arriving at the laboratory) of the experimental protocol. They were given a dinner providing 13 kcal/kg BW and containing 130–150 mEq sodium and were given breakfast providing 5 kcal/kg BW and containing 40–50 mEq sodium. Subjects were permitted ad libitum water consumption with dinner and drank at least 10 ml/kg BW with the breakfast meal. The meals provided were identical in the two experimental trials.

Isotonic saline infusion protocol (Fig. 1Go, bottom)

To determine renal tubular sodium handling (26, 27), subjects self-administered an oral dose of 300 mg of the renal sodium tracer lithium carbonate at 2200 h on the night before each experiment, which provides a near constant plasma level of lithium by the next morning (26, 27). For each experiment, the subjects arrived at the laboratory at approximately 0700 h, after eating the breakfast and drinking the water provided and having refrained from all caffeinated beverages for the previous 12 h. Upon reporting to the laboratory, the subjects voided their bladders and were weighed to the nearest 10 g on a beam balance. The subjects were then seated in a semirecumbent position for a 60-min control period in an environmental chamber (27 C, 30% relative humidity) to ensure a steady state in plasma volume and constituents. During this control period, a 22-gauge Teflon iv catheter was placed in an antecubital or forearm vein in each arm with a heparin lock (20 U/ml) to maintain catheter patency. A blood pressure cuff was positioned for automatic readings by a sphygnomanometric device (Colin Medical Instruments, Komaki, Japan) to monitor changes in blood pressure. A three-lead electrocardiogram (Colin Medical Instruments) provided continuous heart rate monitoring.

At the end of the control period, a resting blood sample was taken, and a urine sample was collected (0 min). Although subjects did stand to provide urine samples, the blood sample was always drawn before the posture change, allowing for at least 60 min of stable, seated posture between each blood sample throughout the protocol. After the baseline samples, 0.9% NaCl was infused at a rate of 0.1 ml/kg BW·min for 120 min. Blood was sampled at 60 and 120 min during the infusion. After the infusion, the recovery period consisted of a 30-min drinking period during which the subject drank 15 ml/kg BW water, followed by a 90-min rest period. A blood sample was obtained at 60 and 120 min of the recovery period. Urine was collected before the infusion, at 60 min into the infusion, immediately postinfusion (60 and 120 min), and at 60 and 120 min into the recovery period (180 and 240 min). The subjects were weighed at the end of the infusion and at the end of the recovery period. Blood pressure and heart rate were recorded every 15 min throughout the infusion and every 30 min postinfusion.

All blood samples were analyzed for hematocrit (Hct), hemoglobin (Hb), total protein, plasma osmolality (POsm), plasma concentrations of creatinine (P[Cr]), and serum concentrations of sodium (S[Na+]) and potassium (S[K+]). Blood samples at time zero (preinfusion), 60 min into the infusion, immediately postinfusion, and at 60 and 120 min into the recovery period were analyzed for P[ANP], P[Ald], and PRA. The final blood sample was also analyzed for P[E2] and P[P4]. Volume and urinary osmolality (UOsm), sodium (U[Na+]), potassium (U[K+]), and Cr (U[Cr]) concentrations were measured in all urine samples.

Blood sampling

Blood samples were separated into aliquots. One aliquot was immediately analyzed for Hct and Hb in triplicate by microhematocrit and cyanomethemoglobin, respectively. A second aliquot was transferred to a heparinized tube to be analyzed for POsm, P[Cr], and P[Ald]. A third aliquot, for the determination of S[Na+] and S[K+], was placed into a tube without anticoagulant. The remaining aliquots were placed in tubes containing EDTA for analysis of P[Ald], P[ANP], and PRA. The tubes were centrifuged at 4 C, and the plasma was removed. After centrifugation, the EDTA samples were frozen immediately at –70 C until analysis.

Plasma and urinary sodium and potassium concentrations were measured by flame photometry (model 943, Instrumentation Laboratory, Lexington, MA). Plasma and urinary osmolalities were measured by freezing point depression (3DII; Advanced Instruments, Needham Heights, MA), total protein was determined by refractometry, and P[Cr] was determined by colorimetric assay (Sigma-Aldrich Corp., St. Louis, MO). PRA, P[Ald], P[ANP], P[E2], and P[P4] were measured by RIA. P[ANP] was determined after extraction from plasma on octadecylsilane cartridges (Sep-Pak C18, Waters Associates, Needham, MA), and the eluate was collected with 4% acetic acid and 86% ethanol. Intra- and interassay coefficients of variation for the midrange standards were as follows: PRA (3.7 ng angiotensin I/ml·h), 5.9% and 7.5% (Diasorin, Stillwater, MN); P[ANP] (24.9 pg/ml), 12.1% and 14.6% (ALPCO Diagnostics, Windham, NH); P[Ald] (173 pg/ml), 6.6% and 7.6% (Diagnostic Products, Los Angeles, CA); P[E2] (145 pg/ml), 2.5% and 6.4% (Diagnostic Products); and P[P4] (2.4 ng/ml), 6.6% and 5.5% (Diagnostic Products).

Calculations

Changes in plasma volume were estimated from changes in Hct and Hb concentrations from the baseline sample according to the equation: % {Delta} PV = 100 [([Hbb])/([Hba])][(1 – Hcta·10–2)]/[(1 – Hctb·10–2)]] – 100, where subscripts a and b denote measurements at time a and preinfusion, respectively (28).

Body water handling was determined through the assessment of overall fluid balance and the renal clearance of free water, osmoles, and sodium. The following equations were used to calculate renal function: glomerular filtration rate (GFR) was estimated from creatinine clearance [Uvolume/time (minutes)] x U[Cr])/P[Cr]]; renal clearance of Li+ (CLi+) was used to approximate proximal renal tubular Na+ handling, because filtered Li+ is absorbed almost exclusively by the proximal tubule in the same proportion as Na+ and water (26, 27): proximal fractional Na+ reabsorption (PFRNa+) = 1 – CLi+/GFR) x 100; and distal fractional sodium reabsorption (DFRNa+) = (1 – CNa+/CLi) x 100. The distal and proximal fractional sodium reabsorptions described here were the percentage of sodium absorbed relative to the total sodium delivered to the distal or proximal tubules (26, 27).

All of these renal function measurements were made using the timed urine collections beginning immediately before the start of isotonic saline infusion (time zero), at 60 and 120 min of the infusion, and at 60-min intervals during the recovery period (180 and 240 min). The specific time of the void was recorded and was used for calculations of renal function.

Statistics

Data are expressed as the mean ± SEM. The variables over time (GnRH antagonist alone tests and hormone intervention tests) were analyzed by conditions (E2and P4-E2 vs. GnRH antagonist alone) using ANOVA for repeated measures. When significant differences were found, orthogonal contrasts tested differences between specific means related to the hypothesis of interest. Differences were considered statistically significant when P < 0.05 (SPSS, Inc., Chicago, IL).

Sample size calculation

The expected differences in sodium excretion between the GnRH antagonist alone and hormone treatments were derived from responses to hormone administration seen in our laboratory (29). We found that E2 administration increased sodium excretion by 15 mEq with an estimated pooled SD for the group of 12 ml (29).

The desired statistical test is two-sided at an {alpha} level of 0.05, with 80% power to detect a difference. Based on our previous work, 80% power is sufficient to detect a significant alteration in sodium excretion. For a two-sided test, Z({alpha})= 1.96, and for 80% power, Z(ß)= 0.84, s = SD, and d = effect size. The formula for calculating sample size for continuous response variables is (30): n = 2[(Z({alpha}) + Z(ß))2 (s)2)/(d)2]. Substituting the values, the calculated sample size is seven subjects in each group.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
One subject reported mild vasomotor symptoms (hot flushes), but no other symptoms related to GnRH antagonist or hormone administration were reported. This subject’s data did not differ in any way from those of the other subjects in the groups, and she did not have symptoms during the testing day. The subjects reported no other adverse effects due to the GnRH antagonist or hormone administration, and the side-effects did not cause any of the subjects to leave the study. One subject in the P4-E2 group dropped out of the study for personal reasons, so all of her data were excluded. The data presented here are from 15 of the 16 subjects recruited, from eight subjects in the P4 group and seven subjects in the combined P4-E2 group.

P4-only group

Body weight was unaffected by P4 administration (61.8 ± 2.8 and 63.9 ± 3.8 kg for GnRH antagonist and P4 respectively), and subjects were of average height (165 ± 3 cm). The baseline plasma P4 concentration increased during P4 [from 1.6 ± 0.8 to 9.4 ± 2.3 ng/ml (5.1 ± 2.5 to 29.9 ± 7.3 nmol/liter); P < 0.05] with little change in P[E2] [17.8 ± 6.8 to 10.2 ± 2.3 pg/ml (65.3 ± 24.9 to 37.4 ± 8.4 pmol/liter)]. P4 administration had little effect on preinfusion blood variables (Table 1AGo). Renal excretory variables were also unchanged at baseline during P4 (Table 2AGo and Figs. 2Go and 3Go), indicating that the subjects were similarly hydrated under both conditions. The only significant changes at baseline were greater P[Ald] and lower P[ANP] during treatment with P4 relative to GnRH antagonist alone (Fig. 4Go; P < 0.05). Baseline cardiovascular variables were unaffected by P4 administration (data not shown).


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  		TABLE 1A. Blood responses before (0 min), during (60 and 120 min), and after isotonic saline infusion (180 and 240 min) during GnRH antagonist administration alone (GnRH ant) and with P4 treatment

 

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  		TABLE 2A. Renal responses before (0 min), during (60 and 120 min), and after isotonic saline infusion (180 and 240 min) during GnRH antagonist administration alone (GnRH ant) and with P4 treatment

 


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  		FIG. 2. Fractional (FENa+; top), distal (DFRNa+, middle), and proximal (PFRNa+; bottom) sodium excretion in response to isotonic saline infusion during treatment with GnRH antagonist alone, GnRH antagonist with P4 (P4; left), and GnRH antagonist, P4, and E2 (P4-E2; right). The distal and proximal fractional sodium reabsorptions described here are the percentage of sodium absorbed relative to the total sodium delivered to the distal tubule. *, Significantly different from GnRH antagonist alone (by ANOVA). Differences were considered statistically significant at P < 0.05. Data are expressed as the mean ± SEM.

 


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  		FIG. 3. Cumulative urine excretion (milliliters per kilogram BW) in response to isotonic saline infusion during administration of GnRH antagonist alone, GnRH antagonist and P4 (P4; left), and GnRH antagonist, P4, and E2 (P4-E2; right). Data are expressed as the mean ± SEM.

 


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  		FIG. 4. PRA (top), P[Ald] (middle), and P[ANP] (bottom) in response to isotonic saline infusion and recovery during treatment with GnRH antagonist alone, GnRH antagonist and P4(P4; left), and GnRH antagonist, P4, and E2 (P4-E2; right). Conversion factors for Systeme International units: Ald, 27.74 pmol/liter; PRA, 0.2778 ng/(liter·sec).*, Significantly different from GnRH antagonist alone (by ANOVA). Differences were considered statistically significant at P < 0.05. Data are expressed as the mean ± SEM.

 
Changes in the blood variables during isotonic saline infusion were similar in the GnRH antagonist and P4 treatment groups (Table 1AGo). Both Hct and Hb decreased in response to isotonic saline infusion, and these changes were similar in magnitude between hormone treatments, so calculated changes in plasma volume over the infusion were also similar (Table 1AGo). Renal responses to isotonic saline infusion indicate no difference between P4 and GnRH antagonist alone (Table 2AGo and Figs. 2Go and 3Go; P < 0.05). Isotonic saline infusion reduced P[Ald] similarly under both hormonal conditions, so P[Ald] remained elevated during P4 compared with the GnRH antagonist alone (Fig. 4Go; P < 0.05). Similarly, P[ANP] increased slightly during isotonic saline infusion under both GnRH antagonist alone and P4 conditions, so P[ANP] remained lower during P4. During recovery, P4 did not alter any blood or renal variables relative to GnRH antagonist alone (Figs. 2–4GoGoGo and Tables 1AGo-2AGo). Despite the maintenance of renal sodium and water retention, P[Ald] remained increased, whereas P[ANP] remained decreased, during P4 treatment relative to those values with GnRH antagonist alone. Finally, there were no hormone treatment by time interactions for any of the blood or renal variables during isotonic saline infusion or recovery, and cardiovascular variables were similar between treatment days (data not shown).

P4-E2 group

As with P4 administration, BW was unaffected by P4-E2 (63.6 ± 3.2 and 63.3 ± 3.2 kg for GnRH antagonist and P4-E2, respectively), and the subjects in this group were also of normal height (166 ± 3 cm). The baseline plasma P4 concentration increased during P4-E2 [from 1.6 ± 0.5 to 6.7 ± 0.5 ng/ml (5.1 ± 1.6 to 21.3 ± 1.6 nmol/liter); P < 0.05], and P[E2] increased [from 17.9 ± 6.3 to 200.1 ± 40.8 pg/ml (65.7 ± 23.1 to 734.6 ± 150.0 pmol/liter); P < 0.05]. As with P4 administration, P4-E2 had little effect on most preinfusion blood variables, with the exception of S[Na+] (Table 1BGo; P < 0.05). Renal excretory variables were unchanged before the infusion between the hormone treatments (Table 1BGo and Figs. 2Go and 3Go; P < 0.05). P[Ald] and PRA were increased before isotonic saline infusion during P4-E2 treatment relative to those during treatment with GnRH antagonist alone (Fig. 3Go; P < 0.05), but P[ANP] was unaffected by P4-E2. Baseline cardiovascular variables were unaffected by P4 administration (data not shown).


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  		TABLE 1B. Blood responses before (0 min), during (60 and 120 min), and after isotonic saline infusion (180 and 240 min) during GnRH antagonist administration alone (GnRH ant) and with P4 with E2 (P4-E2) treatment

 
S[Na+] continued to fall only with P4-E2 throughout isotonic saline infusion (Table 1BGo; P < 0.05). Renal sodium excretion was lower, and DFRNa+ was greater during P4-E2 compared with GnRH antagonist alone through the first 60 min of the infusion (Fig. 2Go; P < 0.05). However, the apparent increase in sodium retention was not associated with greater water retention (Table 2BGo and Fig. 3Go). Relative to GnRH antagonist alone, P4-E2 attenuated the isotonic saline infusion-induced fall in both P[Ald] and PRA, but had no effect on P[ANP] (Fig. 4Go; P < 0.05).


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  		TABLE 2B. Renal responses before (0 min), during (60 and 120 min), and after isotonic saline infusion (180 and 240 min) during GnRH antagonist administration alone (GnRH ant) and with P4 with E2 (P4-E2) treatment

 
S[Na+] continued to fall through the recovery period during P4-E2 administration, although S[Na+] did not change during recovery with the GnRH antagonist alone (Table 1BGo; P < 0.05). Hct and Hb changes indicated a similar plasma volume response during the recovery period with the two hormone conditions (Table 2BGo). Both P[Ald] and PRA remained elevated during P4-E2 administration through the postisotonic saline infusion period (Fig. 3Go; P < 0.05). Finally, there were no hormone treatment by time interactions for any of the blood or renal variables during or after isotonic saline infusion, and cardiovascular variables were similar between treatment days (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The present investigation indicates that P4 has little effect independent of E2 on overall fluid or sodium excretion during an acute volume and sodium load. The greater P[Ald] during P4 administration was expected due to P4 competition with Ald for the mineralocorticoid receptor, which typically leads to compensatory Ald secretion (13, 31), but the increase in Ald did not impact either overall sodium excretion or distal tubular sodium reabsorption. In contrast, when E2 was administered along with the P4, both overall and distal tubular sodium reabsorption were increased. Taking into account earlier studies that demonstrated that E2 administration increased renal sodium reabsorption in the distal tubule (11), our current data support an important role for E2 in sodium regulation. E2 may increase sodium retention either through direct effects on the sodium regulation system or via up-regulation of P4 receptors that may be required for P4 actions on sodium regulation in the kidney. One important caveat to these findings is that the plasma concentration of P4 achieved in these studies was on the low end of that seen during the midluteal phase of the menstrual cycle and was compared with a very high E2 level similar to that achieved during a midcycle E2 peak. Thus, it is possible that greater levels of P4 alone have independent effects on sodium- or fluid-regulating systems.

PRA and Ald secretion increase during the midluteal phase of the menstrual cycle only when ovulation occurs (16), indicating that a functioning corpus luteum (and the P4 it secretes) is a necessary component of the augmented RAAS activity. PRA and Ald secretion also increase during pregnancy and during ovarian stimulation with E2 and P4 (14). In our study, although P[Ald] increased during P4 stimulation, PRA was not concomitantly increased, indicating that this greater Ald secretion occurred independently of the RAAS. Although our study does not illuminate the mechanism for the greater P[Ald], P4 administration to ovariectomized rats stimulates Ald secretion due to Ald synthase conversion of corticosterone to Ald (15), the same mechanism as that in sodium restriction (32).

The fact that increases in P[Ald] during P4 administration did not lead to greater sodium reabsorption suggests that P4 can interfere with the actions of Ald on the kidney tubule (21). P4 can act as a competitive mineralocorticoid receptor antagonist, thus it could attenuate Ald-mediated sodium reabsorption or even lead to sodium loss. P4 has a lower affinity than Ald for the mineralocorticoid receptor; therefore, the effect of P4 on sodium regulation could be more important when Ald is low and P4 is high, in situations such as acute sodium loading concomitant with elevations in concentrations of P4, the conditions induced by our protocols. The low P[Ald]-high P[P4] would also exist, for example, during chronic sodium loading in the luteal phase (21). In these earlier studies, during both chronic and acute sodium-loading, DFRNa+ increased despite the fall in Ald, suggesting that P4 may play a pivotal role in sodium regulation under these conditions (21).

Our findings also support earlier studies indicating that a high P[P4] is typically associated with lower P[ANP] at rest and during acute sodium loading (33) and dehydration (22). In our subjects, P[ANP] was lower during P4 treatment throughout isotonic saline infusion and during recovery. ANP plays a role in the homeostatic feedback system that regulates sodium balance; that is, sodium- and volume-retaining stimuli increase ANP, which, in turn, antagonizes renin and Ald (17, 18, 19, 20). Taken together with earlier investigations, our findings indicate that P4 may play a key role in the modulation of this system; ANP receptor activity is present in the adrenal glomerulosa cells of female rats (34, 35), and the actions of these ANP receptors are modulated by sex steroids (34, 35, 36, 37).

Our data also indicate that E2 modulates the action of P4 on the ANP-RAAS interaction. We found that P[ANP] increased with P4 administration, but was unaffected when E2 was administered along with P4, suggesting that E2 attenuated the P4-mediated inhibition of ANP (22). Thus, our data did not support an earlier study in rats in which E2 appears to promote ANP inhibition (37). Moreover, in contrast to the effects of P4 treatment, P4-E2 treatment was associated with increases in both PRA and P[Ald], which implies RAAS stimulation. E2 on its own can stimulate the RAAS by enhancing angiotensinogen synthesis, inhibiting angiotensin-converting enzyme activity, and augmenting plasma and tissue concentrations of renin (38, 39). In contrast, the increase in PRA could have been the result of P4 acting as an anti-Ald compound, causing sodium loss and stimulating compensatory increases in PRA. The fact that this response occurred only during P4-E2treatment suggests that E2 may be a necessary condition for this anti-Ald system, perhaps by up-regulating P4 receptors in the adrenal cortex. Finally, certain neurotransmitters and cytokines are known to increase during ovulation induction in premenopausal women (40, 41), and the effects of these substances on sodium-regulating hormones and overall sodium and fluid regulation are unknown.

Despite the greater P[Ald] and PRA, earlier studies have suggested that E2 effects on sodium reabsorption may be independent of the RAAS (9, 12). There is also evidence for independent effects of E2 on the human kidney (11). E2 administration alone increases sodium retention due at least in part to changes in distal tubule sodium reabsorption (11). E2 increases sodium reabsorption in both the proximal and distal tubules of the rabbit nephron (9), and both {alpha} and ß estrogen receptors have been found in kidney tubules in animals (7) and humans (7, 8).

The sodium-loading protocol provided a fluid load of approximately 765–795 ml. Under these fluid-loading conditions, neither blood pressure nor heart rate increased, and there was no increase in the GFR during the infusion or recovery period. This level of plasma volume expansion is sufficient to load low pressure baroreceptors (42), which appeared to lead to autoregulatory functions by the kidney and cardiovascular system to maintain blood pressure at resting levels (43). This autoregulation functioned well under all hormonal conditions. Moreover, due to the greater sodium retention during the P4-E2 treatment, it is likely that both renal and vasodilatory functions were used (6).

The impact of sex hormones on body fluid and sodium regulation has important implications for a number of syndromes for which women are at greater risk, including orthostatic hypotension and neurological consequences from postoperative hyponatremia. In addition, many epidemiological studies indicate that premenopausal women and postmenopausal women taking estrogens have a delayed and less severe manifestation of cardiovascular diseases than do men (44, 45). One mechanism by which estrogens or P4 may impact the cardiovascular system is through regulation of body fluids and sodium. Although many earlier studies have postulated that P4 plays a key role in sodium regulation over the course of the menstrual cycle or during pregnancy, ours is the first to isolate the effects of P4 on this system and indicate that P4 administration in the absence of E2 may have little overall effect on fluid and sodium excretion. The data from our study do not definitively demonstrate whether changes in sodium and water retention during combined P4 and E2 administration were the result of E2 effects or were due to up-regulation of P4 receptors and subsequent P4 effects in the distal tubule or on the hormones that regulate sodium reabsorption by the distal tubule. Our data may also indicate a pivotal role for P4 in sodium regulation under conditions such as sodium loading during pregnancy or in the midluteal phase of the menstrual cycle, when P4 and estrogen are high and Ald is suppressed.


   Acknowledgments
 
We gratefully acknowledge the technical support of Cheryl Leone, Jodi Crimmins, Jennifer Fawcett, and Wendy Calzone, and the cooperation of the volunteer subjects.


   Footnotes
 
This work was supported by National Institutes of Health Grant RO1-HL-62240-01A1.

First Published Online October 14, 2004

Abbreviations: Ald, Aldosterone; ANP, atrial natriuretic peptide; BW, body weight; Cr, creatinine; DFR[Na+], distal fractional sodium reabsorption; E2, estradiol; GFR, glomerular filtration rate; Hb, hemoglobin; Hct, hematocrit; OHSS, ovarian hyperstimulation syndrome; Osm, plasma osmolality; P, plasma concentration; P4, progesterone; PRA, plasma renin activity; RAAS, renin-angiotensin-aldosterone system; S, serum concentration; U, urinary concentration.

Received May 18, 2004.

Accepted September 22, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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