- Email: [email protected]
The Calciuric Response to Dietary Salt of Dahl Salt-Sensitive and Salt-Resistant Male Rats
The Calciuric Response to Dietary Salt of Dahl Salt-Sensitive and Salt-Resistant Male Rats MYRTLE THIERRY-PALMER, PHD; DETRICE D. SHERMAN, BS; NERIMIAH L. EMMETT, PHD; MIN WANG, MD; MOHAMED A. BAYORH, PHD; NAANA DONKOH, BS
ABSTRACT: Background: There are conflicting reports regarding the effect of salt sensitivity on the calciuric response to salt, perhaps because of gender differences and different modes of salt administration. We tested the hypothesis that the calciuric response to dietary salt would not differ for male Dahl salt-sensitive (S) and salt-resistant (R) rats. Method: S and R rats were fed high(80 g/kg) or low- (3 g/kg) salt diets for 3 weeks and urine (24 hour) was collected weekly. Results: Urinary calcium excretion was up to 20-fold greater for S and R rats fed a high-salt diet (P ⬍ 0.001) than for S and R rats fed a low-salt diet and did not differ significantly between S and R rats. S rats, however, excreted calcium in significantly higher urine volumes (P ⬍ 0.001) during high salt
intake and developed hypertension. Plasma parathyroid hormone concentrations of S and R rats did not differ during low salt intake and increased significantly to the same concentration after 3 weeks of high salt intake. Conclusions: We have previously reported that plasma 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D concentrations of male S rats, but not male R rats, were drastically reduced by 3 weeks of high salt intake. These data suggest that salt-induced hypertension and saltinduced alterations in the vitamin D endocrine system of male S rats do not affect the calciuric response to dietary salt. KEY INDEXING TERMS: Sodium chloride; Hypertension; Salt sensitivity; Vitamin D; Parathyroid hormone. [Am J Med Sci 2001;321(5):342–347.]
A
and 1,25-dihydroxyvitamin D [1,25-(OH)2D] and significantly lower levels of 24,25-dihydroxyvitamin D [24,25-(OH)2D] at baseline, compared with the normotensive subjects. In another study, hypertensive subjects with high sodium chloride sensitivity (low plasma renin activity) showed significantly higher urinary calcium excretion in response to sodium chloride infusion than did hypertensive subjects with low sodium chloride sensitivity.3 de la Sierra et al,6 however, have conducted a dietary study with hypertensive subjects and found no effect of salt-sensitivity on the calciuric response to salt. Hughes et al7 have indicated that hypertensive subjects with normal renin levels responded to varying dietary sodium loads in a manner identical to that of normotensive subjects, suggesting no effect of hypertension per se on the calciuric response to dietary salt. Nordin et al2 and Shortt and Flynn8 have suggested that the correlation between urinary calcium and urinary sodium is caused by the effect of filtered sodium load on the tubular reabsorption of calcium. When sodium is the determinant, 100 mmol of sodium takes out approximately 1 mmol of calcium in the urine.2,9 Dahl salt-sensitive (S) and salt-resistant (R) rats are widely studied genetic models of hypertension.10,11 The Dahl salt-sensitive rat, but not the Dahl salt-resistant rat, develops hypertension, hyperlipidemia, and progressive renal disease when
positive correlation has been found between urinary calcium and sodium excretion for adult men and women.1,2 There are conflicting reports, however, regarding the effect of salt sensitivity on the calciuric response to salt. Urinary calcium excretion in response to an infusion of sodium chloride has been shown to be higher for salt-sensitive (low renin) hypertensive subjects.3–5 In 2 studies, lowrenin hypertensive elderly subjects, compared with normotensive elderly subjects, excreted more calcium into urine in response to saline infusion.4,5 The hypertensive subjects were shown to have significantly higher serum levels of parathyroid hormone
From the Departments of Biochemistry (MT-P, DDS, ND), Pharmacology (MW, MAB) and Physiology (NLE), Morehouse School of Medicine, Atlanta, Georgia. Submitted July 24, 2000; accepted November 3, 2000. This work was supported by NASA Grant NCC 9-53 and National Institutes of Health/Research Centers in Minority Institutions Grant RR03034. Presented in part in abstract form at the joint meeting of the American Society for Bone and Mineral Research and the International Bone and Mineral Society, December 1998, San Francisco, CA [Thierry-Palmer M, Pasquali M, Emmett NL, et al. Calcium and pyridinium cross-links excretion of Dahl salt-sensitive and salt-resistant rats fed a high-salt diet. Bone 1998;23(5 Suppl.):S346]. Correspondence: Myrtle Thierry-Palmer, Ph.D, Associate Professor, Department of Biochemistry, Morehouse School of Medicine, 720 Westview Drive, SW, Atlanta, GA 30310-1495 (E-mail: [email protected]).
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Table 1. Selected Components of the Diets Component
Basala
Low Saltb
High Saltb
Calcium (%) Sodium (%) Phosphorus (%) Sodium chloride (%) Vitamin D3 (IU/g)
0.95 0.4 0.67 1
0.86 0.13 0.63 0.3
0.86 3.15 0.63 8
4.5
5.0
5.0
a
Purina LabDiet 5001; PMI Nutrition International, Inc., Brentwood, MO. b Harlan Teklad, Madison, WI.
fed a high-salt diet. In light of the indication2,8 that the correlation between urinary calcium and urinary sodium is caused by the effect of sodium load on the tubular reabsorption of calcium, we have used these rat models to test the hypothesis that the calciuric response to dietary salt would not be affected by salt sensitivity. Materials and Methods Animals and Diets. All protocols involving animals were previously approved by the Morehouse School of Medicine Animal Care Committee. Twelve male Dahl salt-sensitive (SS/Jr) and twelve male salt-resistant (SR/Jr) rats (130-150 g, 4-5 weeks old) were obtained from Harlan Sprague Dawley (Indianapolis, IN). The rats were allowed free access to water and a nonpurified diet (Purina LabDiet 5001; PMI Nutrition International, Inc., Brentwood, MO). They were housed in a room with 12-h light/dark cycles and, after 1 week of acclimation, 6 rats of each type were fed either a low- (3g/kg) or high- (80 g/kg) salt diet (Harlan Teklad, Madison, WI) for 3 weeks. Selected components of the diets are listed (Table 1). The rats were placed in metabolic cages on the seventh day of each week for 24-hour urine collection. At that time, water intake was also measured. A second experiment was conducted with 6 male salt-sensitive and 6 male salt-resistant rats (130-150 g, 4-5 weeks old) fed a high-salt diet for 3 weeks, to confirm the findings of the first experiment regarding urinary calcium and sodium excretion during high salt intake. Results from the 2 experiments are pooled in the data presentation. Blood Pressure Measurements. Indirect blood pressure (systolic) was measured before initiation of the diets and weekly by tail cuff plethysmography using a Grass polygraph model 7400 (Grass Instrument Company, Quincy, MA) and a programmed electrosphygmomanometer PE-300 (Narco Biosystem, Inc. Houston, TX). Blood pressure was determined using the median of 3 to 4 successive measurements. Body weights were determined before the dietary treatments began (basal) and weekly thereafter. After 3 weeks of dietary treatment, rats were anesthetized (0.02 halothane in oxygen) and blood was drawn, by heart puncture, into heparinized tubes for determination of plasma electrolytes. Rats were then humanely killed by sodium pentobarbital overdose. Hematocrit and Plasma and Urinary Electrolytes. Hematocrit and plasma potassium, magnesium, sodium, and ionized calcium concentrations were determined on freshly drawn whole blood using a Stat Profile Ultra electrode (Nova Biomedical, Waltham, MA). Urinary sodium and calcium were measured by an Electrolyte 10⫹ Analyzer (Nova Biomedical). Parathyroid Hormone. Plasma parathyroid hormone (PTH) concentrations were measured with a rat intact PTH enzymelinked immunosorbent assay kit (Immutopics, San Clemente, CA), using an automated plate reader (Spectra Max 250; Molec-
THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
Figure 1. Urinary sodium (A) and calcium (B) of S and R rats fed high- (HS) and low-salt (LS) diets. Values are means ⫾ SEM for 10 to 12 rats fed a high-salt diet, 6 rats fed a low-salt diet, and 12 rats at basal except for sodium determination (6 rats). There was significant effect of high salt intake on urinary calcium for S and R rats (P ⬍ 0.001), but no significant effect of salt sensitivity. **, Urinary sodium significantly different between S and R rats, P ⬍ 0.01. ***, Urinary calcium significantly different between S and R rats at baseline, P ⬍ 0.001. ular Devices, Sunnyvale, CA). Day 0 values are for rats fed a low-salt diet. Values for weeks 1 and 2 are for stored plasma from other experiments. Statistical Analysis. A mean ⫾ SEM was calculated for each group. Statistical significance (P ⬍ 0.05) was evaluated using 2-way analysis of variance followed by the Tukey multiple comparison test (SigmaStat; SPSS, Inc., Chicago, IL).
Results Male Dahl salt-sensitive and salt-resistant rats responded to a high-salt diet with increased excretion of sodium. The high-salt diet contained 27 times the sodium content of the low-salt diet and sodium excretion by S and R rats fed a high-salt diet (Figure 1A) was approximately 20-fold higher than that of S and R rats fed a low-salt diet. S and R rats differed significantly in excretion of sodium at weeks 2 and 3 of high salt intake. Urinary calcium excretion was up to 20-fold greater for S and R rats fed a high-salt diet than for S and R rats fed a low-salt diet (Figure 1B) and did not differ between S and R rats fed high- or low-salt diets. The urinary calcium/urinary sodium ratio for 343
Dietary Salt and Urinary Calcium
Table 2. Urinary Calcium/Urinary Sodium of S and R Rats Fed High- and Low-salt Diets Urinary calcium/urinary sodium Diet
S
R
Basal (nonpurified diet) Low salt High salt 1 week 2 weeks 3 weeks
3.4 ⫾ 0.3a 1.6 ⫾ 0.3
1.4 ⫾ 0.1 1.4 ⫾ 0.2
1.3 ⫾ 0.2 1.8 ⫾ 0.2a 1.0 ⫾ 0.1
1.4 ⫾ 0.2 1.2 ⫾ 0.1 1.2 ⫾ 0.1
mmol/100 mmol
Values are mean ⫾ SEM for 10 to 12 rats fed a high-salt diet and 4 to 6 rats fed the nonpurified diet (basal) or a low-salt diet for 3 weeks. Significantly different from R rats, P ⬍0.002 by the MannWhitney test.
a
S rats fed the basal diet was twice that of R rats. The urinary calcium/urinary sodium ratio for S rats fed the low-salt diet, which was lower in calcium and lower in sodium content than the basal diet (Table 1), was equal to that of R rats. The urinary calcium/ urinary sodium ratios at weeks 1 and 3 of high salt intake (Table 2) indicate lack of an effect of salt sensitivity on calcium excretion. At week 2 of the high-salt diet, the ratio for S rats was significantly higher than that for R rats because of lowered excretion of sodium (observed in 2 different experiments). When fed a high-salt diet, S rats excreted the sodium and calcium in higher urine volumes (P ⬍ 0.001) than R rats (Figure 2B) and responded to the high-salt diet with significant increases (P ⬍ 0.001) in blood pressure (Figure 3). After 3 weeks of consuming a high-salt diet, systolic blood pressure of S rats was 182 ⫾ 6 mm Hg, compared with 107 ⫾ 2 for R rats. Intake of the high-salt diet was accompanied by significant increases in water intake and urine volume for both S and R rats, but mean urine volume of S rats was up to 70% higher than that of R rats (Figure 2). S and R rats had similar blood pressures and excreted similar urine volumes when fed a low-salt diet. Hematocrit and concentrations of the major plasma electrolytes (sodium, magnesium, ionized calcium, potassium) did not differ in S and R fed high- or low-salt diets. Plasma PTH concentrations increased significantly (P ⬍ 0.001) in both S and R rats after 3 weeks of high salt intake (Figure 4). The increase occurred between weeks 2 and 3 of high salt intake in R rats and between weeks 1 and 3 in S rats. Discussion Both sodium and calcium excretion by male S and R rats fed a high-salt diet increased approximately 344
Figure 2. Water intake (A) and urine output (B) of S and R rats fed high- (HS) and low-salt (LS) diets. Values are means ⫾ SEM for 10 to 12 rats at baseline or fed a high-salt diet, except for water intake of R-HS at week 2 (6 rats), and 6 rats fed a low-salt diet. There was significant difference in water intake and urine output between S and R rats during high salt intake (P ⬍ 0.001).
20-fold compared with that by male S and R rats fed a low-salt diet. The increased calcium excretion was, perhaps, caused in part by the markedly higher volumes filtered through the kidneys during high salt intake. S rats excreted significantly higher urine volumes than did R rats during high salt intake and became hypertensive. The greater urine volumes required by S rats to excrete the dietary sodium increase the workload on the kidneys and most likely contribute to the development of hypertension in these rats. Diuretics have been extensively used for the treatment of salt-induced hypertension in humans. Despite the significantly higher urine volumes, urinary calcium excretion by S rats was not significantly higher than that by R rats. With the exception of S rats at week 2, urinary calcium/urinary sodium ratios (approximately 1 mmol calcium per 100 mmol sodium) during high salt intake were similar to those shown for humans by Nordin et al.2 These data support the suggesMarch 2001 Volume 321 Number 5
Thierry-Palmer et al
Figure 3. Blood pressure of S and R rats fed high- (HS) and low-salt (LS) diets. Values are means ⫾ SEM (n ⫽ 6). There was significant effect of salt sensitivity (P ⬍ 0.001) and of time (P ⬍ 0.001) on blood pressure during high salt intake.
tion2,8 that the correlation between urinary calcium and urinary sodium is caused by the effect of the filtered sodium load on the tubular reabsorption of calcium, when sodium is the determinant. The calciuric response to salt has been previously shown for healthy humans1-2,12-15 and rats.16-20 These data indicate that the risk factor for calcium loss, and thus bone loss, during high salt intake is equal for male S and R rats. High salt intake, however, is a risk
Figure 4. Plasma PTH of Dahl S and R rats during high salt intake. Concentrations at 0 time are for rats fed a low-salt diet. Values are means ⫾ SEM (n ⫽ 5–9). There was significant effect of time on plasma PTH concentration (P ⬍ 0.001) during high salt intake, but no significant effect of salt sensitivity. ***, Significantly different from weeks 0 and 1 (S and R rats) and week 2 (R rats) (P ⬍ 0.001). *, Significantly different from weeks 0, 1, and 3 and from R rats at week 2 (P ⬍ 0.05). THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
factor for hypertension only for S rats. The data suggest that hypertension per se does not affect the calciuric response to salt of male rats. We have previously characterized the vitamin D endocrine system of male S rats.21-23 An inverse relationship was demonstrated between plasma 25hydroxyvitamin D (25-OHD) concentration and blood pressure when S rats were fed a high-salt diet.21-22 Plasma 25-OHD concentrations of S rats were significantly reduced within 3 weeks to lower than 25%, but plasma 25-OHD concentrations of R rats were unaffected by a high-salt diet. We have also demonstrated significantly reduced plasma 24,25-(OH)2D concentrations in S rats in response to high salt intake, whereas the decrease in plasma 24,25-(OH)2D concentration of R rats in response to high salt intake was transient.21,23 The data reported here suggest that the altered vitamin D endocrine system of hypertensive Dahl salt-sensitive male rats does not affect urinary calcium excretion in response to high salt intake. This may be related to lack of an increase in plasma 1,25-(OH)2D concentration in response to high salt intake21. It should also be noted that neither exogenous 25-OHD3 nor 24,25-(OH)2D3 attenuated the blood pressure increase of S rats during high salt intake.22,23 Further studies are necessary to determine what physiological processes, if any, are affected by the markedly lower concentrations of plasma 25-OHD and 24,25(OH)2D in S rats during high salt intake. Plasma PTH concentrations of S and R male rats did not differ during low salt intake and increased significantly to the same level for the 2 groups of rats after 3 weeks of high salt intake. Kurtz et al24 and Ozono et al25 found no significant changes in blood ionized calcium or serum 1,25-(OH)2D and PTH concentrations of human subjects during 1 week of altered salt intake (oral). We also found no significant changes in plasma PTH concentration during the first week of high salt intake and no significant changes in blood ionized calcium and plasma 1,25-(OH)2D21 concentrations after 3 weeks of high salt intake. Although some studies indicate higher serum PTH concentration during high salt intake,26,27 other studies have shown that calcium homeostasis at very high sodium intake is not critically dependent upon PTH.7,13-15 This study supports the latter view, because increases in urinary calcium excretion by S and R rats preceded increases in plasma PTH concentration. Perhaps the increase in plasma PTH concentration after 3 weeks of high salt intake serves to increase mobilization of calcium from bone. Several studies suggest a link between high salt intake and bone loss.1-2,14,16-19,28 Zemel et al,29 in a 14-day dietary study, have shown that PTH concentrations rose in hypertensive (saltsensitive) black subjects when they were fed a highsalt/low-calcium diet, but not when they were fed a high-salt/high-calcium diet. 345
Dietary Salt and Urinary Calcium
We report here that male Dahl S rats, compared with male R rats, did not exhibit significantly higher calcium excretion in response to a salt load over a period of 3 weeks. Kotchen et al,30 however, have shown significantly higher urinary calcium for S rats compared with R rats fed a high-salt diet for 5 days. In the same study, plasma PTH levels were shown to be higher for prehypertensive S rats than for R rats and to decline when S rats were fed a high-salt diet for 5 days.30 The difference between our findings and those of Kotchen et al30 may lie in differing calcium and sodium content of the diets (Table 1), the duration of high salt intake, and differences in assay method (intact PTH in this study and mid-molecule PTH in the earlier study). We confirm the finding of Kotchen et al30 regarding higher excretion of calcium by S rats than by R rats fed Purina chow (Table 2). The high-salt diet of Kotchen et al30 was prepared by adding sodium chloride to Purina chow. In our experiments, rats were switched at baseline to high- and low-salt diets containing lower calcium content (Table 1). It is probable that, as at baseline, S rats would have excreted more calcium than R rats on a high-salt/ higher-calcium diet. The significantly higher urinary calcium/urinary sodium ratio of S rats compared with R rats at baseline (Purina chow) and equal ratios on the low-salt diet suggest greater intestinal absorption of a higher calcium intake by male S compared with male R rats. Galletti et al20 have compared calcium/creatinine and calcium/sodium excretion of spontaneously hypertensive and Wistar-Kyoto female rats and concluded that high natriuresis during a high-sodium diet is associated with high urinary calcium excretion only in hypertensive rats. Preliminary studies in our laboratory indicate a greater calciuric response to salt of female S compared with female R rats. Three studies3–5 indicate higher urinary calcium excretion by salt-sensitive female subjects in response to saline infusion. A gender difference may contribute to conflicting data on the calciuric response to salt. The data reported here indicate that high salt intake is a risk factor for calcium loss and that the risk is equal for male S and R rats. Salt-induced hypertension and salt-induced changes in the vitamin D endocrine system of male S rats did not influence the calciuric response to salt. For male Dahl S and R rats, the correlation between urinary calcium and urinary sodium during high salt intake, therefore, may be caused by the effect of the filtered sodium load on the tubular reabsorption of calcium. Acknowledgments We thank Robin Socci and Blanca Tejada for aid in preparation of this manuscript. 346
References 1. Massey LK, Whiting SJ. Dietary salt, urinary calcium, and bone loss. J Bone Miner Res 1996; 11:731– 6. 2. Nordin BEC, Need AG, Morris HA, et al. The nature and significance of the relationship between urinary sodium and urinary calcium in women. J Nutr 1993;123:1615–22. 3. Galletti F, Ferrara I, Stinga F, et al. Effect of intravenous sodium chloride on renal sodium and calcium handling in hypertensive patients with different sensitivities to sodium chloride. J Hypertens 1993;11(Suppl 5):S194 –5. 4. Imaoka M, Morimoto S, Kitano S, et al. Calcium metabolism in elderly hypertensive patients: possible participation of exaggerated sodium, calcium and phosphate excretion. Clin Exp Pharmacol Physiol 1991;18:631– 41. 5. Morimoto S, Imaoka M, Kitano S, et al. Exaggerated natri-calci-uresis and increased circulating levels of parathyroid hormone and 1,25-dihydroxyvitamin D in patients with senile hypertension. Contrib Nephrol 1991;90:94-8. 6. de la Sierra A, Lluch MM, Coca A, et al. Fluid, ionic and hormonal changes induced by high salt intake in salt-sensitive and salt-resistant hypertensive patients. Clin Science 1996;91:155– 61. 7. Hughes GS, Oexmonn MJ, Margolius HS, et al. Normal vitamin D and mineral metabolism in essential hypertension. Am J Med Sci 1988;296:252–9. 8. Shortt C, Flynn A. Sodium-calcium inter-relationships with specific references to osteoporosis. Nutr Res Rev 1990;3:101– 15. 9. Nordin BEC, Polley KJ. Metabolic consequences of the menopause: a cross-sectional and longitudinal intervention study on 557 normal postmenopausal women. Calcif Tissue Int 1987;41:S1–59. 10. Dahl LK. Salt and hypertension. Am J Clin Nutr 1972;25: 231– 44. 11. Dahl LK, Schackow E. Effects of chronic excess salt ingestion: experimental hypertension in the rat. Can Med Assoc J 1964;90:155– 60. 12. Castenmiller JJM, Mensink RP, van der Heijden L, et al. The effect of dietary sodium on urinary calcium and potassium excretion in normotensive men with different calcium intake. Am J Clin Nutr 1985;41:52–9. 13. Chan ELP, Ho CS, MacDonald D, et al. Interrelationships between urinary sodium, calcium, hydroxyproline and serum PTH in healthy subjects. Acta Endocrinologica 1992;127: 242–5. 14. Lietz G, Avenell A, Robins SP. Short-term effects of dietary sodium intake on bone metabolism in postmenopausal women measured using deoxypyridinoline excretion. Br J Nutr 1997;78:73– 82. 15. McCarron DA, Rankin LI, Bennett WM, et al. Urinary calcium excretion at extremes of sodium intake in normal man. Am J Nephrol 1981;1:84 –90. 16. Chan ELP, Swaminathan R. Calcium metabolism and bone calcium content in normal and oophorectomized rats consuming various levels of saline for 12 months. J Nutr 1998;128:633–9. 17. Goulding A, Campbell D. Dietary NaCl loads promote calciuria and bone loss in adult oophorectomized rats consuming a low calcium diet. J Nutr 1983;113:1409 –14. 18. Goulding A, Campbell DR. Effects of oral loads of sodium chloride on bone composition in growing rats consuming ample dietary calcium. Miner Electrolyte Metab 1984;10:58 – 62. 19. Goulding A, Gold E. Effects of dietary NaCl supplementation on bone synthesis of hydroxyproline, urinary hy-
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20.
21.
22.
23.
24.
droxyproline excretion and bone 45Ca uptake in the rat. Horm Metab Res 1988;20:743–5. Galletti F, Rutledge A, Triggle DJ. Influence of high sodium intake on urinary calcium excretion and cardiac calcium channels in spontaneously hypertensive rats. J Hypertens 1991;9(Suppl 6):S366 –7. Thierry-Palmer M, Carlyle KS, Williams MD, et al. Plasma 25-hydroxyvitamin D concentrations are inversely associated with blood pressure of Dahl salt-sensitive rats. J Steroid Biochem Mol Biol 1998;66:255– 61. Thierry-Palmer M, Tewolde TK, Wang M, et al. Exogenous 25-hydroxycholecalciferol does not attenuate salt-induced hypertension. J Steroid Biochem Mol Biol 1998;67: 193–9. Thierry-Palmer M, Tewolde T, Wang M, et al. Relationship between plasma 24,25-dihydroxyvitamin D concentration and high salt intake. J Bone Miner Res 1999;14(Suppl 1):S203. Kurtz TW, Al-Bander HA, Morris Jr RC. “Salt-sensitive” essential hypertension in men. Is the sodium ion alone important? N Engl J Med 1987;317:1043– 8.
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25. Ozono R, Oshima T, Matsuura H, et al. Change in blood pressure during altered sodium intake is not associated with calciotropic hormone level. Clin Sci 1997;93:153–7. 26. Breslau NA, McGuire JL, Zerwekh JE, et al. The role of dietary sodium on renal excretion and intestinal absorption of calcium and on vitamin D metabolism. J Clin Endocrinol Metab 1982;55:369 –73. 27. Coe FL, Firpo JJ, Hollandsworth DL, et al. Effect of acute and chronic metabolic acidosis on serum immunoreactive parathyroid hormone in man. Kidney Int 1975;8:262–73. 28. Zarkadas M, Gougeon-Reyburn R, Marliss EB, et al. Sodium chloride supplementation and urinary calcium excretion in postmenopausal women. Am J Clin Nutr 1989;50: 1088 –94. 29. Zemel MB, Gualdoni SM, Walsh MF, et al. Effects of sodium and calcium on calcium metabolism and blood pressure regulation in hypertensive black adults. J Hypertens 1986;4:S364 – 6. 30. Kotchen TA, Ott CE, Whitescarver SA, et al. Calcium and calcium regulating hormones in the “prehypertensive” Dahl salt sensitive rat (calcium and salt sensitive hypertension). Am J Hypertens 1989;2:749 –53.
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ABSTRACT: Background: There are conflicting reports regarding the effect of salt sensitivity on the calciuric response to salt, perhaps because of gender differences and different modes of salt administration. We tested the hypothesis that the calciuric response to dietary salt would not differ for male Dahl salt-sensitive (S) and salt-resistant (R) rats. Method: S and R rats were fed high(80 g/kg) or low- (3 g/kg) salt diets for 3 weeks and urine (24 hour) was collected weekly. Results: Urinary calcium excretion was up to 20-fold greater for S and R rats fed a high-salt diet (P ⬍ 0.001) than for S and R rats fed a low-salt diet and did not differ significantly between S and R rats. S rats, however, excreted calcium in significantly higher urine volumes (P ⬍ 0.001) during high salt
intake and developed hypertension. Plasma parathyroid hormone concentrations of S and R rats did not differ during low salt intake and increased significantly to the same concentration after 3 weeks of high salt intake. Conclusions: We have previously reported that plasma 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D concentrations of male S rats, but not male R rats, were drastically reduced by 3 weeks of high salt intake. These data suggest that salt-induced hypertension and saltinduced alterations in the vitamin D endocrine system of male S rats do not affect the calciuric response to dietary salt. KEY INDEXING TERMS: Sodium chloride; Hypertension; Salt sensitivity; Vitamin D; Parathyroid hormone. [Am J Med Sci 2001;321(5):342–347.]
A
and 1,25-dihydroxyvitamin D [1,25-(OH)2D] and significantly lower levels of 24,25-dihydroxyvitamin D [24,25-(OH)2D] at baseline, compared with the normotensive subjects. In another study, hypertensive subjects with high sodium chloride sensitivity (low plasma renin activity) showed significantly higher urinary calcium excretion in response to sodium chloride infusion than did hypertensive subjects with low sodium chloride sensitivity.3 de la Sierra et al,6 however, have conducted a dietary study with hypertensive subjects and found no effect of salt-sensitivity on the calciuric response to salt. Hughes et al7 have indicated that hypertensive subjects with normal renin levels responded to varying dietary sodium loads in a manner identical to that of normotensive subjects, suggesting no effect of hypertension per se on the calciuric response to dietary salt. Nordin et al2 and Shortt and Flynn8 have suggested that the correlation between urinary calcium and urinary sodium is caused by the effect of filtered sodium load on the tubular reabsorption of calcium. When sodium is the determinant, 100 mmol of sodium takes out approximately 1 mmol of calcium in the urine.2,9 Dahl salt-sensitive (S) and salt-resistant (R) rats are widely studied genetic models of hypertension.10,11 The Dahl salt-sensitive rat, but not the Dahl salt-resistant rat, develops hypertension, hyperlipidemia, and progressive renal disease when
positive correlation has been found between urinary calcium and sodium excretion for adult men and women.1,2 There are conflicting reports, however, regarding the effect of salt sensitivity on the calciuric response to salt. Urinary calcium excretion in response to an infusion of sodium chloride has been shown to be higher for salt-sensitive (low renin) hypertensive subjects.3–5 In 2 studies, lowrenin hypertensive elderly subjects, compared with normotensive elderly subjects, excreted more calcium into urine in response to saline infusion.4,5 The hypertensive subjects were shown to have significantly higher serum levels of parathyroid hormone
From the Departments of Biochemistry (MT-P, DDS, ND), Pharmacology (MW, MAB) and Physiology (NLE), Morehouse School of Medicine, Atlanta, Georgia. Submitted July 24, 2000; accepted November 3, 2000. This work was supported by NASA Grant NCC 9-53 and National Institutes of Health/Research Centers in Minority Institutions Grant RR03034. Presented in part in abstract form at the joint meeting of the American Society for Bone and Mineral Research and the International Bone and Mineral Society, December 1998, San Francisco, CA [Thierry-Palmer M, Pasquali M, Emmett NL, et al. Calcium and pyridinium cross-links excretion of Dahl salt-sensitive and salt-resistant rats fed a high-salt diet. Bone 1998;23(5 Suppl.):S346]. Correspondence: Myrtle Thierry-Palmer, Ph.D, Associate Professor, Department of Biochemistry, Morehouse School of Medicine, 720 Westview Drive, SW, Atlanta, GA 30310-1495 (E-mail: [email protected]).
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Table 1. Selected Components of the Diets Component
Basala
Low Saltb
High Saltb
Calcium (%) Sodium (%) Phosphorus (%) Sodium chloride (%) Vitamin D3 (IU/g)
0.95 0.4 0.67 1
0.86 0.13 0.63 0.3
0.86 3.15 0.63 8
4.5
5.0
5.0
a
Purina LabDiet 5001; PMI Nutrition International, Inc., Brentwood, MO. b Harlan Teklad, Madison, WI.
fed a high-salt diet. In light of the indication2,8 that the correlation between urinary calcium and urinary sodium is caused by the effect of sodium load on the tubular reabsorption of calcium, we have used these rat models to test the hypothesis that the calciuric response to dietary salt would not be affected by salt sensitivity. Materials and Methods Animals and Diets. All protocols involving animals were previously approved by the Morehouse School of Medicine Animal Care Committee. Twelve male Dahl salt-sensitive (SS/Jr) and twelve male salt-resistant (SR/Jr) rats (130-150 g, 4-5 weeks old) were obtained from Harlan Sprague Dawley (Indianapolis, IN). The rats were allowed free access to water and a nonpurified diet (Purina LabDiet 5001; PMI Nutrition International, Inc., Brentwood, MO). They were housed in a room with 12-h light/dark cycles and, after 1 week of acclimation, 6 rats of each type were fed either a low- (3g/kg) or high- (80 g/kg) salt diet (Harlan Teklad, Madison, WI) for 3 weeks. Selected components of the diets are listed (Table 1). The rats were placed in metabolic cages on the seventh day of each week for 24-hour urine collection. At that time, water intake was also measured. A second experiment was conducted with 6 male salt-sensitive and 6 male salt-resistant rats (130-150 g, 4-5 weeks old) fed a high-salt diet for 3 weeks, to confirm the findings of the first experiment regarding urinary calcium and sodium excretion during high salt intake. Results from the 2 experiments are pooled in the data presentation. Blood Pressure Measurements. Indirect blood pressure (systolic) was measured before initiation of the diets and weekly by tail cuff plethysmography using a Grass polygraph model 7400 (Grass Instrument Company, Quincy, MA) and a programmed electrosphygmomanometer PE-300 (Narco Biosystem, Inc. Houston, TX). Blood pressure was determined using the median of 3 to 4 successive measurements. Body weights were determined before the dietary treatments began (basal) and weekly thereafter. After 3 weeks of dietary treatment, rats were anesthetized (0.02 halothane in oxygen) and blood was drawn, by heart puncture, into heparinized tubes for determination of plasma electrolytes. Rats were then humanely killed by sodium pentobarbital overdose. Hematocrit and Plasma and Urinary Electrolytes. Hematocrit and plasma potassium, magnesium, sodium, and ionized calcium concentrations were determined on freshly drawn whole blood using a Stat Profile Ultra electrode (Nova Biomedical, Waltham, MA). Urinary sodium and calcium were measured by an Electrolyte 10⫹ Analyzer (Nova Biomedical). Parathyroid Hormone. Plasma parathyroid hormone (PTH) concentrations were measured with a rat intact PTH enzymelinked immunosorbent assay kit (Immutopics, San Clemente, CA), using an automated plate reader (Spectra Max 250; Molec-
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Figure 1. Urinary sodium (A) and calcium (B) of S and R rats fed high- (HS) and low-salt (LS) diets. Values are means ⫾ SEM for 10 to 12 rats fed a high-salt diet, 6 rats fed a low-salt diet, and 12 rats at basal except for sodium determination (6 rats). There was significant effect of high salt intake on urinary calcium for S and R rats (P ⬍ 0.001), but no significant effect of salt sensitivity. **, Urinary sodium significantly different between S and R rats, P ⬍ 0.01. ***, Urinary calcium significantly different between S and R rats at baseline, P ⬍ 0.001. ular Devices, Sunnyvale, CA). Day 0 values are for rats fed a low-salt diet. Values for weeks 1 and 2 are for stored plasma from other experiments. Statistical Analysis. A mean ⫾ SEM was calculated for each group. Statistical significance (P ⬍ 0.05) was evaluated using 2-way analysis of variance followed by the Tukey multiple comparison test (SigmaStat; SPSS, Inc., Chicago, IL).
Results Male Dahl salt-sensitive and salt-resistant rats responded to a high-salt diet with increased excretion of sodium. The high-salt diet contained 27 times the sodium content of the low-salt diet and sodium excretion by S and R rats fed a high-salt diet (Figure 1A) was approximately 20-fold higher than that of S and R rats fed a low-salt diet. S and R rats differed significantly in excretion of sodium at weeks 2 and 3 of high salt intake. Urinary calcium excretion was up to 20-fold greater for S and R rats fed a high-salt diet than for S and R rats fed a low-salt diet (Figure 1B) and did not differ between S and R rats fed high- or low-salt diets. The urinary calcium/urinary sodium ratio for 343
Dietary Salt and Urinary Calcium
Table 2. Urinary Calcium/Urinary Sodium of S and R Rats Fed High- and Low-salt Diets Urinary calcium/urinary sodium Diet
S
R
Basal (nonpurified diet) Low salt High salt 1 week 2 weeks 3 weeks
3.4 ⫾ 0.3a 1.6 ⫾ 0.3
1.4 ⫾ 0.1 1.4 ⫾ 0.2
1.3 ⫾ 0.2 1.8 ⫾ 0.2a 1.0 ⫾ 0.1
1.4 ⫾ 0.2 1.2 ⫾ 0.1 1.2 ⫾ 0.1
mmol/100 mmol
Values are mean ⫾ SEM for 10 to 12 rats fed a high-salt diet and 4 to 6 rats fed the nonpurified diet (basal) or a low-salt diet for 3 weeks. Significantly different from R rats, P ⬍0.002 by the MannWhitney test.
a
S rats fed the basal diet was twice that of R rats. The urinary calcium/urinary sodium ratio for S rats fed the low-salt diet, which was lower in calcium and lower in sodium content than the basal diet (Table 1), was equal to that of R rats. The urinary calcium/ urinary sodium ratios at weeks 1 and 3 of high salt intake (Table 2) indicate lack of an effect of salt sensitivity on calcium excretion. At week 2 of the high-salt diet, the ratio for S rats was significantly higher than that for R rats because of lowered excretion of sodium (observed in 2 different experiments). When fed a high-salt diet, S rats excreted the sodium and calcium in higher urine volumes (P ⬍ 0.001) than R rats (Figure 2B) and responded to the high-salt diet with significant increases (P ⬍ 0.001) in blood pressure (Figure 3). After 3 weeks of consuming a high-salt diet, systolic blood pressure of S rats was 182 ⫾ 6 mm Hg, compared with 107 ⫾ 2 for R rats. Intake of the high-salt diet was accompanied by significant increases in water intake and urine volume for both S and R rats, but mean urine volume of S rats was up to 70% higher than that of R rats (Figure 2). S and R rats had similar blood pressures and excreted similar urine volumes when fed a low-salt diet. Hematocrit and concentrations of the major plasma electrolytes (sodium, magnesium, ionized calcium, potassium) did not differ in S and R fed high- or low-salt diets. Plasma PTH concentrations increased significantly (P ⬍ 0.001) in both S and R rats after 3 weeks of high salt intake (Figure 4). The increase occurred between weeks 2 and 3 of high salt intake in R rats and between weeks 1 and 3 in S rats. Discussion Both sodium and calcium excretion by male S and R rats fed a high-salt diet increased approximately 344
Figure 2. Water intake (A) and urine output (B) of S and R rats fed high- (HS) and low-salt (LS) diets. Values are means ⫾ SEM for 10 to 12 rats at baseline or fed a high-salt diet, except for water intake of R-HS at week 2 (6 rats), and 6 rats fed a low-salt diet. There was significant difference in water intake and urine output between S and R rats during high salt intake (P ⬍ 0.001).
20-fold compared with that by male S and R rats fed a low-salt diet. The increased calcium excretion was, perhaps, caused in part by the markedly higher volumes filtered through the kidneys during high salt intake. S rats excreted significantly higher urine volumes than did R rats during high salt intake and became hypertensive. The greater urine volumes required by S rats to excrete the dietary sodium increase the workload on the kidneys and most likely contribute to the development of hypertension in these rats. Diuretics have been extensively used for the treatment of salt-induced hypertension in humans. Despite the significantly higher urine volumes, urinary calcium excretion by S rats was not significantly higher than that by R rats. With the exception of S rats at week 2, urinary calcium/urinary sodium ratios (approximately 1 mmol calcium per 100 mmol sodium) during high salt intake were similar to those shown for humans by Nordin et al.2 These data support the suggesMarch 2001 Volume 321 Number 5
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Figure 3. Blood pressure of S and R rats fed high- (HS) and low-salt (LS) diets. Values are means ⫾ SEM (n ⫽ 6). There was significant effect of salt sensitivity (P ⬍ 0.001) and of time (P ⬍ 0.001) on blood pressure during high salt intake.
tion2,8 that the correlation between urinary calcium and urinary sodium is caused by the effect of the filtered sodium load on the tubular reabsorption of calcium, when sodium is the determinant. The calciuric response to salt has been previously shown for healthy humans1-2,12-15 and rats.16-20 These data indicate that the risk factor for calcium loss, and thus bone loss, during high salt intake is equal for male S and R rats. High salt intake, however, is a risk
Figure 4. Plasma PTH of Dahl S and R rats during high salt intake. Concentrations at 0 time are for rats fed a low-salt diet. Values are means ⫾ SEM (n ⫽ 5–9). There was significant effect of time on plasma PTH concentration (P ⬍ 0.001) during high salt intake, but no significant effect of salt sensitivity. ***, Significantly different from weeks 0 and 1 (S and R rats) and week 2 (R rats) (P ⬍ 0.001). *, Significantly different from weeks 0, 1, and 3 and from R rats at week 2 (P ⬍ 0.05). THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
factor for hypertension only for S rats. The data suggest that hypertension per se does not affect the calciuric response to salt of male rats. We have previously characterized the vitamin D endocrine system of male S rats.21-23 An inverse relationship was demonstrated between plasma 25hydroxyvitamin D (25-OHD) concentration and blood pressure when S rats were fed a high-salt diet.21-22 Plasma 25-OHD concentrations of S rats were significantly reduced within 3 weeks to lower than 25%, but plasma 25-OHD concentrations of R rats were unaffected by a high-salt diet. We have also demonstrated significantly reduced plasma 24,25-(OH)2D concentrations in S rats in response to high salt intake, whereas the decrease in plasma 24,25-(OH)2D concentration of R rats in response to high salt intake was transient.21,23 The data reported here suggest that the altered vitamin D endocrine system of hypertensive Dahl salt-sensitive male rats does not affect urinary calcium excretion in response to high salt intake. This may be related to lack of an increase in plasma 1,25-(OH)2D concentration in response to high salt intake21. It should also be noted that neither exogenous 25-OHD3 nor 24,25-(OH)2D3 attenuated the blood pressure increase of S rats during high salt intake.22,23 Further studies are necessary to determine what physiological processes, if any, are affected by the markedly lower concentrations of plasma 25-OHD and 24,25(OH)2D in S rats during high salt intake. Plasma PTH concentrations of S and R male rats did not differ during low salt intake and increased significantly to the same level for the 2 groups of rats after 3 weeks of high salt intake. Kurtz et al24 and Ozono et al25 found no significant changes in blood ionized calcium or serum 1,25-(OH)2D and PTH concentrations of human subjects during 1 week of altered salt intake (oral). We also found no significant changes in plasma PTH concentration during the first week of high salt intake and no significant changes in blood ionized calcium and plasma 1,25-(OH)2D21 concentrations after 3 weeks of high salt intake. Although some studies indicate higher serum PTH concentration during high salt intake,26,27 other studies have shown that calcium homeostasis at very high sodium intake is not critically dependent upon PTH.7,13-15 This study supports the latter view, because increases in urinary calcium excretion by S and R rats preceded increases in plasma PTH concentration. Perhaps the increase in plasma PTH concentration after 3 weeks of high salt intake serves to increase mobilization of calcium from bone. Several studies suggest a link between high salt intake and bone loss.1-2,14,16-19,28 Zemel et al,29 in a 14-day dietary study, have shown that PTH concentrations rose in hypertensive (saltsensitive) black subjects when they were fed a highsalt/low-calcium diet, but not when they were fed a high-salt/high-calcium diet. 345
Dietary Salt and Urinary Calcium
We report here that male Dahl S rats, compared with male R rats, did not exhibit significantly higher calcium excretion in response to a salt load over a period of 3 weeks. Kotchen et al,30 however, have shown significantly higher urinary calcium for S rats compared with R rats fed a high-salt diet for 5 days. In the same study, plasma PTH levels were shown to be higher for prehypertensive S rats than for R rats and to decline when S rats were fed a high-salt diet for 5 days.30 The difference between our findings and those of Kotchen et al30 may lie in differing calcium and sodium content of the diets (Table 1), the duration of high salt intake, and differences in assay method (intact PTH in this study and mid-molecule PTH in the earlier study). We confirm the finding of Kotchen et al30 regarding higher excretion of calcium by S rats than by R rats fed Purina chow (Table 2). The high-salt diet of Kotchen et al30 was prepared by adding sodium chloride to Purina chow. In our experiments, rats were switched at baseline to high- and low-salt diets containing lower calcium content (Table 1). It is probable that, as at baseline, S rats would have excreted more calcium than R rats on a high-salt/ higher-calcium diet. The significantly higher urinary calcium/urinary sodium ratio of S rats compared with R rats at baseline (Purina chow) and equal ratios on the low-salt diet suggest greater intestinal absorption of a higher calcium intake by male S compared with male R rats. Galletti et al20 have compared calcium/creatinine and calcium/sodium excretion of spontaneously hypertensive and Wistar-Kyoto female rats and concluded that high natriuresis during a high-sodium diet is associated with high urinary calcium excretion only in hypertensive rats. Preliminary studies in our laboratory indicate a greater calciuric response to salt of female S compared with female R rats. Three studies3–5 indicate higher urinary calcium excretion by salt-sensitive female subjects in response to saline infusion. A gender difference may contribute to conflicting data on the calciuric response to salt. The data reported here indicate that high salt intake is a risk factor for calcium loss and that the risk is equal for male S and R rats. Salt-induced hypertension and salt-induced changes in the vitamin D endocrine system of male S rats did not influence the calciuric response to salt. For male Dahl S and R rats, the correlation between urinary calcium and urinary sodium during high salt intake, therefore, may be caused by the effect of the filtered sodium load on the tubular reabsorption of calcium. Acknowledgments We thank Robin Socci and Blanca Tejada for aid in preparation of this manuscript. 346
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