Effects of endogenous estrogen on renal calcium and phosphate handling in elderly women
I. M. Dick,1,3,4
A. Devine,1,3,4
J. Beilby,2 and
R. L. Prince1,3,4
1School of Medicine and Pharmacology, University of Western Australia; 2The Western Australian Centre for Pathology and Medical Research; 3Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital; and 4Western Australian Institute of Medical Research, Nedlands Western Australia, Australia
Submitted 24 March 2004
; accepted in final form 30 September 2004
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ABSTRACT
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High postmenopausal endogenous estrogen concentrations are an important determinant of preservation of bone mass and reduced fracture in elderly women. Calcium supplementation can also reduce bone loss in these patients, suggesting an interaction between estrogen deficiency and calcium balance. Potential mechanisms of estrogen on calcium transport include direct effects on the bone, the kidney, and the bowel. Previous studies have demonstrated effects of estrogen on renal phosphate handling. We have used a cross-sectional, population-based analysis of biochemical data obtained from ambulant elderly women to determine the association of endogenous estradiol with urine calcium and phosphorus excretion. The subjects were 293 postmenopausal women >70 yr old. Factors associated with renal calcium and phosphate excretion were measured, including the filtered calcium and phosphate load, parathyroid hormone (PTH), estradiol, and sex hormone-binding globulin (SHBG). The free estradiol concentration (FE) was calculated from a previously described formula. A high plasma estradiol concentration (r2 = 0.023, P = 0.01) and a high FE (r2 = 0.045, P = 0.001) were associated with reduced renal calcium excretion. The estradiol and FE effect on renal calcium excretion remained significant after adjusting for calcium filtered at the glomerulus and serum PTH. A high FE was associated with a reduced renal phosphate threshold in univariate analysis (r2 = 0.023, P = 0.010). The effect remained significant after adjustment for serum PTH. The size of the effect of the FE was of the same order of magnitude as the effect of PTH on reducing renal calcium excretion and increasing renal phosphate excretion. These data support in vitro and animal data demonstrating an effect of estradiol on renal calcium and phosphate handling and indicate that, in elderly postmenopausal women, the effect is of a similar magnitude to the well-recognized effects of PTH on these physiologically regulated parameters.
estradiol; calcium homeostasis; phosphate homeostasis; renal tubular calcium reabsorption; osteoporosis
CORRELATION STUDIES EXAMINING the relationship between circulating estrogen concentrations and bone density have produced a significant amount of evidence to suggest that falling concentrations of estrogen at the menopause are associated with low bone density (3, 27, 29). This accelerated loss of bone can result in osteoporotic fracture, which affects
50% of elderly postmenopausal women (11, 12, 17). Reports from the Study of Osteoporotic Fracture study have shown an association between low estradiol concentrations and reduced bone density (32) and an increased rate of hip, but not spine, fracture (7). The EPIDOS (Epidémiologie de lostéoporose) study was also able to show an effect of low estrogen concentrations on hip fracture in elderly French women that was accounted for by differences in body weight, suggesting that fat mass was the source of endogenous estrogen (4). These studies support the hypothesis that endogenous estrogen concentrations in elderly postmenopausal women have an important physiological effect on the skeleton.
In reviews of estrogen effects on mineral metabolism, we (5, 24) have presented evidence that estrogen may have physiologically important direct effects on the kidney in addition to a direct effect on bone. We hypothesize that estrogen deficiency increases renal calcium loss, thus exacerbating bone resorption, and reduces renal phosphate loss. In this investigation, we have carried out a cross-sectional study to evaluate the relative impact of endogenous estrogen production and parathyroid hormone (PTH) on renal calcium and phosphate handling in elderly women.
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MATERIALS AND METHODS
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Study participants.
Two hundred ninety-three women aged >70 yr, living in the Western Australian metropolitan area, were recruited from the electoral roll. Over 98% of individuals in this age group are registered to vote (2). Subjects excluded were those taking hormone replacement therapy, calcium supplement >500 mg/day or any other bone-active agents (25%), or if they had significant current illness (0.8%). Although the subjects entering the study were weighted in favor of those in higher socioeconomic categories, they did not differ from the whole population in health resource utilization. Baseline measurements included age, [years since menopause (YSM)], height, and weight. A quantitative food frequency questionnaire (16) was used to ascertain the average daily calcium and phosphate intake. Informed consent was obtained, and the study was approved by the Human Rights Committee of the University of Western Australia.
Biochemistry.
Estradiol was measured by radioimmunoassay (Orion Diagnostica, Espoo, Finland) with a quoted analytical sensitivity of 5 pmol/l. Only 10% of the subjects values were below this concentration. The interassay coefficient of variation (CV) was 6.6% at a mean of 101 pmol/l, calculated from a sample of 17 individuals, and 7.2% at a mean of 48 pmol/l, calculated from a sample of 14 individuals. The intra-assay CV was 5.1% at a mean of 103 pmol/l (n = 10) and 7.5% at a mean of 49 pmol/l (n = 10). The biological variation and intra-assay variation of samples collected at 0 and 4 mo in 17 individuals was 17.2%. Sex hormone-binding globulin (SHBG) was measured using an immunochemiluminometric assay (Immulite, Los Angeles, CA), and the intra- and interassay CVs were 7.1 and 6.8%, respectively, at 24 nmol/l. The biological variation and intra-assay variation of samples collected at 0 and 4 mo in 21 individuals was 29%. The free estradiol concentration (FE) was calculated using a previously described formula (30): FE = [total estradiol]/[(3.14 x 108 x SHBG) + (4.21 x 104 x albumin) + 1]. This method of estimating free estradiol concentration has been shown to be accurate when referenced against an equilibrium dialysis method in postmenopausal women (28).
Blood and urine samples were collected after a 10-h overnight fast. Plasma calcium, phosphate, creatinine, alkaline phosphatase, total protein, albumin, sodium, chloride, and bicarbonate and fasting spot urine creatinine, calcium, and phosphorus concentrations were measured using routine methods (BM/Hitachi 747 Analyzer; Boehringer Mannheim, Mannheim, Germany). The anion gap was calculated as the difference between plasma sodium and potassium and chloride and bicarbonate concentrations. Plasma globulin concentration was calculated as the difference between total protein and albumin concentrations. The ultrafilterable calcium concentration was calculated using an iterative procedure described by Nordin et al. (22). This formula utilizes empirically derived constants that describe the relation between total calcium concentration and albumin, globulin, the anion gap, and bicarbonate concentrations to calculate the ionized calcium concentration. From this calculation of ionized calcium, ultrafilterable calcium was calculated as the ionized calcium fraction plus the complex calcium fraction. Serum intact PTH was measured using an immunochemiluminometric method (31) with intra- and interassay CVs of 3.6 and 6.2%, respectively, a method that does not have the nonlinearity problems of other assays at high PTH concentrations. Renal tubular phosphate reabsorption was expressed as the renal phosphate threshold, which is the ratio of the maximum rate of renal tubular reabsorption of phosphate to the glomerular filtration rate and was calculated using a standard formula (19). Creatinine clearance was estimated from plasma creatinine concentration, age, and weight by use of the Cockcroft-Gault equation (6).
Statistical analysis.
All statistical procedures were performed with SPSSPC for Windows version 11.5 (SPSS, Chicago, IL). The log10 for urine calcium excretion was calculated to normalize the distribution of this variable and was used in subsequent analyses. Linear regression was used to determine the association of urine calcium excretion and renal phosphate threshold with baseline demographics and biochemistry. Multiple regression was used to determine the relation between urine calcium excretion and the free estradiol concentration after adjustment for creatinine clearance calculated using the Cockcroft-Gault equation, PTH concentration, and plasma calcium concentration. Multiple regression was used to determine the association of the renal phosphate threshold with the free estradiol concentration after adjustment for creatinine clearance and PTH concentration. The free estradiol concentration and PTH were then categorized into three equal groups. Univariate ANOVA was used to calculate urine calcium excretion and renal phosphate threshold in the PTH groups after adjustment for the free estradiol concentration group and plasma calcium, and in the free estradiol groups after adjustment for PTH group and plasma calcium. A P value = 0.05, using a two-tailed test, was considered significant.
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RESULTS
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The values for baseline demographics and calcium and phosphate biochemistry, kidney-related biochemistry, and ovarian-related biochemistry are detailed in Table 1. All biochemical values were within the range expected for this aged population.
Urinary calcium excretion.
In univariate analysis, the urine calcium excretion was negatively correlated with the serum estradiol and positively correlated with SHBG and albumin concentrations (Table 2), and therefore negatively correlated with the free estradiol concentration (Fig. 1A). In addition to the relationship with measures of circulating estrogen concentration, the urine calcium excretion was also significantly negatively correlated with PTH concentration (Fig. 1B) and creatinine clearance calculated by the Cockcroft-Gault formula and was positively correlated with plasma calcium concentration but not the ultrafilterable calcium (Table 2). The association of the free estradiol concentration with urine calcium excretion was examined after adjustment for creatinine clearance and PTH concentration and plasma calcium concentration in a multiple linear regression model (Table 3). Plasma calcium concentration was used in this analysis because it was more strongly correlated than ultrafilterable calcium concentration with urine calcium excretion (Table 2). In this model, the association between urine calcium excretion and the free estradiol concentration remained significant (partial r = 0.20, P = 0.001). The number of years since menopause and age did not alter this association. Similar results were obtained if the serum estradiol concentration was used instead of the free estrogen index (partial r = 0.15, P = 0.01). After adjustment for creatinine clearance and PTH and plasma calcium concentrations, a one-standard-deviation (1-SD) increase of 0.4 pmol/l in the free estradiol concentration would be associated with a 1.17 µmol/l glomerular filtrate (GF) decrease in urinary calcium excretion. A 1-SD increase in PTH concentration of 1.6 pmol/l would be associated with a similar decrease in urinary calcium excretion of 1.13 µmol/l GF after adjustment for creatinine clearance, free estradiol concentration, and plasma calcium concentration. The free estradiol concentration and PTH independently explain 4 and 3.2%, respectively, of the variance in urinary calcium excretion.

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Fig. 1. A: linear regression of free estradiol concentration with renal calcium excretion. FE, free estradiol concentration; UCE, urinary calcium excretion; GF, glomerular filtrate. B: linear regression of parathyroid hormone (PTH) with renal calcium excretion.
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Univariate ANOVA was used to examine the urine calcium excretion in tertiles of free estradiol concentration after adjustment for PTH. This analysis indicated that a free estradiol concentration >0.63 pmol/l was associated with a 41% reduction in renal calcium excretion compared with a free estradiol concentration of <0.41 pmol/l, independent of PTH concentration (Fig. 2A). This was higher than the 29% reduction in urine calcium excretion between the lowest and highest PTH groups after adjustment for the free estradiol concentration (Fig. 2B).

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Fig. 2. A: association of tertiles of free estradiol concentration with renal calcium excretion adjusted for filtered calcium load and PTH. B: association of tertiles of PTH with renal calcium excretion adjusted for filtered calcium load and free estradiol concentration. Results are means ± 95% confidence interval (CI). **P < 0.005 compared with highest tertile. P values are adjusted for multiple comparisons using the Bonferroni method.
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Urinary phosphate excretion.
In univariate analysis, the renal phosphate threshold was negatively associated with the free estradiol concentration and was positively associated with SHBG and albumin concentrations (Table 4). No significant association with the circulating total estradiol concentration was found. Age was positively correlated with the renal phosphate threshold, and weight, creatinine clearance, and PTH concentration were negatively correlated with the renal phosphate threshold (Table 4). The association of free estradiol concentration and SHBG with the renal phosphate threshold was examined after adjustment for age, creatinine clearance, and PTH in a multiple linear regression model (Table 5). In this model, the association between the renal phosphate threshold and free estradiol concentration was significant (partial r = 0.12, P = 0.035).
Univariate ANOVA was used to examine the renal phosphate threshold in tertiles of free estradiol concentration after adjustment for PTH concentration. This analysis indicated that a free estradiol concentration >0.63 was associated with a 7% reduction in the renal phosphate threshold compared with a free estradiol concentration of <0.41, independent of PTH (Fig. 3A). This compared closely with the 9% reduction in the renal phosphate threshold between the lowest and highest PTH groups after adjustment for the free estradiol concentration (Fig. 3B).

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Fig. 3. A: association of tertiles of free estradiol concentration with renal phosphate threshold adjusted for filtered calcium load and PTH. B: association of tertiles of PTH with renal phosphate threshold adjusted for filtered calcium load and free estradiol concentration. Results are means ± 95% CI. **P < 0.005 and ***P < 0.001 compared with highest tertile. P values are adjusted for multiple comparisons using the Bonferroni method.
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DISCUSSION
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In this study PTH, creatinine clearance, and measures of the calcium load filtered at the glomerulus showed the expected association with renal calcium excretion. The current study supports the hypothesis that estradiol acts on the kidney to increase renal tubular reabsorption of calcium, extending previous concepts of the importance of estrogen effects on calcium metabolism to elderly women many years past menopause. This is demonstrated by the association between a low renal calcium excretion and a high free estradiol concentration even after adjustment for other factors affecting renal calcium excretion such as PTH concentration and the filtered calcium load. This association between free estradiol concentration and renal calcium handling may be a significant factor in the relation between endogenous estrogen concentration and fracture observed in elderly women (8).
The association of calcium excretion observed in this study is weaker if total rather than free estradiol concentration is considered. Furthermore, the association of urinary calcium excretion with SHBG concentration is stronger than that observed for total estradiol concentration. Therefore, an alternative explanation is that SHBG has a direct effect on the renal handling of calcium. However, SHBG and albumin bind approximately the same proportion of circulating estradiol in women (30), and in this study the albumin concentration alone was also a relatively strong predictor of renal calcium handling. Thus the simplest explanation, consistent with the observations made in this study, is that both the circulating estradiol concentration and the degree of binding of estradiol by both SHBG and albumin determine the free estradiol concentration, which regulates renal calcium handling via an effect on estrogen receptors.
Other evidence in humans exists for direct effects of estrogen on the kidney to increase calcium reabsorption (23, 26). These data have been supported by a study of estrogen replacement therapy in conjunction with PTH infusion in early postmenopausal women (20). In that study, the effect of estrogen on the kidney was to enhance the tubular reabsorption of calcium in response to PTH. However, falling levels of circulating estrogen across the menopause have no effect on PTH concentration (25) and estrogen replacement therapy, and estrogen withdrawal in elderly women has been shown to have no effect on basal or stimulated PTH secretion, suggesting that estrogen does not modulate renal calcium excretion through regulation of PTH (34). Longitudinal calcium balance studies show that the menopause is associated with a negative change in calcium balance that is attributable to both a decrease in intestinal calcium absorption and renal calcium reabsorption efficiency, resulting in an increased dietary calcium requirement to maintain calcium balance (15). The kidney has estrogen receptors (13), and the human estrogen receptor-
, the subtype found in the kidney, has a Kd of 60 pM, with displacement of iodinated estradiol being demonstrated at low picomolar concentrations of estradiol in in vitro studies (18). In vitro studies using renal tubule cell culture have shown a stimulatory effect of 17
-estradiol on calcium membrane transport (9). Furthermore, 17
-estradiol has been shown to upregulate mRNA and protein expression of the ECaC1 calcium transport channel in the ovariectomized rat (33). These data suggest a biological mechanism for direct calcium conservation in the renal distal tubule by estrogen.
Of great interest is that, in this epidemiological study, the size of the estrogen effect on renal calcium excretion was greater in magnitude than the size of the PTH effect across the range of fasting hormone values obtained. Clearly, the fact that estrogen reproduces the effect of PTH but, unlike PTH, is not directly regulated by calcium has led to the difficulties in detecting the effects of estrogen on renal calcium excretion independent of PTH. It does, however, explain the otherwise puzzling fact that the rise in renal calcium excretion that occurs with the fall in estrogen at the menopause does so without a change in PTH (25).
In the present study, we observed that a higher free, but not total, estradiol concentration was associated with reduced renal phosphate reabsorption after adjustment for PTH. This finding is in agreement with previous studies that have shown an effect of exogenous estrogen to regulate renal phosphate handling in women (1, 26) and animals (10). As discussed above, although the total estradiol concentration was not associated with modulation of renal phosphate handling, the simplest explanation for the effects of SHBG and albumin is that it is their combined effect on the free estradiol concentration that is the physiologically important parameter.
An interesting aspect of this study is that the size of the free estradiol association is comparable to the size of the PTH association across the range of values obtained in this population. This potent effect of free estradiol on renal phosphate handling has not been well recognized in the past and should lead to reconsideration of factors regulating renal phosphate excretion, the principal organ of phosphate regulation in the body.
The estrogen assay used in this study had adequate analytic sensitivity to appropriately categorize 90% of the patients. Furthermore, the biological variation in estradiol and SHBG concentrations in these patients was low, as shown by the low CV of samples collected 4 mo apart. Thus a single measurement of estrogen and SHBG appeared to be an adequate categorization of estrogen status that enabled the observed associations with renal calcium and phosphate biochemistry to be made.
The dietary calcium requirement needed to maintain calcium balance increases with age due to decreased intestinal absorption and renal reabsorption efficiency (14). The observation that estradiol concentration is related to urinary calcium excretion in this population of older women has important implications for the risks of osteoporosis. We have demonstrated in this study that the difference in calcium excretion between the lowest and highest tertile of free estradiol is
5 µmol/l glomerular filtrate. Assuming that these individuals filter
100 l/day, the difference in calcium excretion would be
0.5 mmol, or
10% of the daily renal calcium excretion. If this increase in calcium excretion is not matched by an increase in intestinal calcium absorption, it could be expected to result in bone loss of
0.51.0% per annum (21). We (8) have previously demonstrated in a larger cohort of women from which this study was derived that a low estradiol concentration was associated with an increased fracture risk. This indicates that the increased urinary calcium excretion associated with a low estradiol concentration may be involved in the pathogenesis of osteoporosis and suggests that, in older women many years past the menopause, a low endogenous estradiol concentration may further increase the dietary calcium requirement needed to maintain calcium balance.
In conclusion, the results obtained from this study are consistent with a direct effect of estrogen in elderly free-living women to increase renal calcium reabsorption and decrease renal phosphate reabsorption. In this age group, nonbone effects of estrogen appear to play a significant role in calcium and phosphorus homeostasis.
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GRANTS
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This study was supported by a research grant from the Healthway Health Promotion Foundation of Western Australia and the Australasian Menopause Society.
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FOOTNOTES
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Address for reprint requests and other correspondence: I. M. Dick, School of Medicine and Pharmacology, Univ. of Western Australia, 4th Floor G Block, Sir Charles Gairdner Hospital, Nedlands, WA, Australia 6009 (E-mail: iand{at}cyllene.uwa.edu.au)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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