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Departments of Biochemistry and * Pharmacology/Toxicology, Morehouse School of Medicine, Atlanta, GA
3To whom correspondence should be addressed. E-mail: thierrm{at}msm.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • salt sensitivity • vitamin D • Dahl rats • sodium chloride
The liver metabolite of vitamin D, 25-hydroxyvitamin D (25-OHD),4 is the precursor to the hormone, 1,25-dihydroxyvitamin D [1,25-(OH)2D], which is synthesized in the kidney. Plasma 25-OHD concentrations are lower, and 1,25-(OH)2D higher, in elderly hypertensive females with low plasma renin activity, characteristic of salt-induced hypertension, compared with normotensive elderly and young females (1
). African-Americans, who have a higher rate of hypertension and salt-sensitivity than Caucasian Americans (2
,3
), have been demonstrated to have significantly lower mean plasma 25-OHD concentrations than Caucasian Americans in several studies involving both men and women (4
–10
) and in a report (11
) based on the Third National Health and Nutrition Examination Survey (NHANES III).
A connection between the vitamin D endocrine system and salt-induced hypertension has been shown for the Dahl salt-sensitive (S) rat, a widely studied genetic model of salt sensitivity (12
,13
). High salt intake significantly decreases the plasma 25-OHD concentration of S rats (14
–18
). Blood pressure was shown to be directly correlated (r = 0.97) and plasma 25-OHD concentration indirectly correlated (r = -0.98) with the number of days that S rats were fed a high salt diet (14
). An inverse correlation (r = -0.99) between blood pressure and plasma 25-OHD concentration was shown for S rats (14
). The decrease in plasma 25-OHD concentration during high salt intake was preceded by a decrease in 25-OHD content in the kidneys of S rats. The 25-hydroxyvitamin D content in the kidneys of S rats at d 2 of high salt intake was 81% of that in the kidneys of S rats at d 2 of low salt intake (17
).
Attempts to increase plasma 25-OHD concentration, by administering 25-hydroxycholecalciferol (25-OHD3) via osmotic pumps, were successful in S rats fed a low, but not a high salt diet. This suggested increased clearance or accelerated metabolism of 25-OHD during high salt intake (18
). We report here on the urinary excretion of 25-OHD by S rats.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Protocols involving animals were previously approved by the Animal Care and Use Committee at Morehouse School of Medicine. Guidelines followed were those of the Public Health Service and the revised animal welfare act as regulated by USDA.
Dahl salt-sensitive (SS/Jr) and salt-resistant (SR/Jr) female rats (4–5 wk old, 100–110 g) 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, Brentwood, MO). They were housed in a room with a 12-h light:dark cycle and, after 1 wk of acclimation, were divided into groups of six rats each and fed either a low (3 g/kg) or high (80 g/kg) salt (sodium chloride) diet5 for 2 wk. The rats were placed in metabolic cages at baseline and on d 7 of each week for 24-h urine collection. Urine was stored at -80°C. At the termination of the dietary treatments, rats were anesthetized (ketamine/xylazine, 44:10 mg/kg body) and blood was drawn by heart puncture into heparinized tubes. Plasma was obtained by centrifuging the blood at 1500 x g for 10 min. Rats were killed by sodium pentobarbital overdose.
Materials.
The 25-OHD3 standard was purchased from ICN (Costa Mesa, CA). The concentration of 25-OHD3 dissolved in ethanol was determined by ultraviolet spectroscopy at 265 nm using a Lambda 3B spectrophotometer (Perkin-Elmer, Norvalk, CT) and a molar absorption coefficient of 18,200 mol/L-1 cm-1. Organic solvents were analytical or HPLC grade.
Measurement of 25-OHD concentration.
Radiolabeled 25-OHD3, 25-hydroxy[26(27)-methyl-3H]cholecalciferol (Amersham, Arlington Heights, IL), was purified on a 0.46 x 25 cm Zorbax-Sil HPLC column (Agilent Technologies, Wilmington, DE) with dichloromethane/isopropanol (95:5, v/v) at a flow rate of 1 mL/min or on a Zorbax-CN column (Agilent Technologies) with hexane/isopropanol/methanol (94:5:1,v/v/v) at a flow rate of 1.3 mL/min (19
). Synthetic 25-OHD3 served as the standard.
A 25-OHD fraction was isolated from 3 mL urine by dichloromethane/methanol liquid-liquid extraction (14
) followed by solid-phase separation on silica cartridges by the method of Reinhardt and Hollis (20
). The fraction was purified by the HPLC methods described above. Radiolabeled 25-OHD3 (800 dpm) was added to the urine samples before purification to determine recovery. Protein binding assay kits, containing rat vitamin D binding protein (Nichols Institute Diagnostics, San Juan Capistrano, CA), were used to assay the 25-OHD peaks. Standard curves were constructed using 25-OHD3 (0.039, 0.078, 0.156, 0.312, 0.625, 1.25, 2.5 ng/tube). The limit of detection of the assay, as designed, was 0.16 nmol/L for urine. The concentration of 25-OHD (nmol/L) was multiplied by the 24-h urine volume to obtain nmol/24 h.
Measurement of 25-OHD binding activity.
Urine was tested for specific binding to 25-OHD3 by modifications of the method of Reinhardt and Hollis (20
) and the assay procedure for 25-OHD of Nichols Institute Diagnostics. Urine samples were diluted with 0.1 mol/L boric acid buffer, pH 8.6 (5–10 fold dilution). Aliquots of the diluted urine samples (0.5 mL) were incubated with radiolabeled 25-OHD3 (0.58 nmol/L, 10,000 dpm) in the presence of ethanol or 200-fold molar excess unlabeled 25-OHD3. Ligands were added in 10 µL ethanol. Incubation was at 4°C for 2 h. A dextran-coated charcoal preparation (0.2 mL) was added to each incubation mixture and incubation was continued for 20 min at 4°C (20
). The dextran-coated charcoal contained 12 g/L charcoal (Sigma Chemical, St. Louis, MO), 1.2 g/L Dextran T-70 (Pharmacia Fine Chemicals, Piscataway, NJ) and 0.5 g/L 
-globulin suspended in 0.1 mol/L boric acid, pH 8.6. The incubation mixtures were centrifuged at 1800 x g for 20 min to separate the bound and free metabolites. The supernatant, which contained the bound metabolite, was decanted into a scintillation vial and counted in Bio-Safe II (Research Products International, Mount Prospect, IL). Duplicate incubations were made for each rat. Binding in the presence of 200-fold excess unlabeled 25-OHD3 (nonspecific binding) was subtracted from total binding to obtain specific binding. Binding activity [pmol/(L · h)] was multiplied by the 24-h urine volume to obtain pmol/h.
Protein.
Urinary protein was measured by the bicinchoninic acid protein assay kit (Sigma), using an automated plate reader (Spectra Max 250, Molecular Devices, Sunnyvale, CA). Protein concentration (mg/L) was multiplied by the 24-h urine volume to obtain mg/24 h.
Statistical methods.
A mean ± SEM was calculated for each group. Data were evaluated using three-way ANOVA followed by the Tamhane’s T2 test (SPSS, Chicago, IL). Effects tested were S vs. R, low salt (LS) vs. high salt (HS), and time. The Mann-Whitney test was used for comparisons of two groups. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The level of 25-hydroxyvitamin D excreted by S rats at wk 2 of high salt intake was five times that of R rats at wk 2 of high salt intake and nine times that of S rats at wk 2 of low salt intake. Because of a higher level of lipid, necessitating additional HPLC chromatography, baseline urine was not assayed for 25-OHD. The calculated 25-hydroxyvitamin D binding activity in the 24-h urine of S rats (Fig. 2
) at wk 2 of high salt intake was twice that in the urine of S rats at wk 2 of low salt intake and > 35 times that in the urine of female R rats fed either low or high salt diets at wk 2. Urinary protein (Fig. 3
) of S rats was significantly affected by salt and by duration of high salt intake. Urinary protein of S rats was significantly higher than that of R rats.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Nykjaer et al. (21
–22
) proposed that megalin, an endocytic receptor, and cubilin, a membrane-associated protein, provide an endocytic pathway for reabsorption of the vitamin D binding protein (DBP) + 25-OHD complex by kidney proximal tubules. A question to be answered is whether altered cubilin + megalin delivery of DBP + 25-OHD to the kidney proximal tubules is the cause of the appreciable urinary excretion of 25-OHD binding protein, presumably DBP, by S rats during low salt intake.
It is unknown whether the increased excretion of 25-OHD and 25-OHD binding activity during high salt intake was caused by an acceleration of the mechanism during low salt intake or by an additional mechanism, such as kidney damage. Female S rats excrete significantly more protein (23
) and urine (16
) than R rats during high salt intake and are hypertensive at wk 2 when fed a diet containing 80 g/kg salt (16
). Johnson et al. (24
) suggested that kidney damage precedes salt-induced hypertension. Both 25-OHD and DBP are excreted in patients with nephrotic syndrome, which is characterized by proteinuria and decreased plasma 25-OHD concentration (25
–27
).
The serum 25-OHD concentration was lower in non-Hispanic African-Americans than in non-Hispanic Caucasians and Mexican-Americans in an NHANES III study, which comprised 18,875 adolescents and adults (11
). It has been suggested that low plasma 25-OHD concentrations of African-Americans are caused in part by reduced epidermal vitamin D photosynthesis, associated with high melanin skin content (28
). Results presented here suggest that lower mean plasma 25-OHD concentrations of African-Americans might also be affected by the lower concentrations in a subset of African-Americans with salt-induced hypertension.
It has been estimated that 56% of the Caucasian hypertensive population and 73% of the African-American hypertensive population are salt sensitive (29
). Also, the apparent racial differences in response to antihypertensive drugs have been found to be due in part to salt sensitivity (30
,31
). A multicenter clinical study (32
) showed that the salt intake of the U.S. population is high and that lowering dietary salt intake can decrease blood pressure in both African-American and Caucasian subjects. High salt intake significantly affects the calcium endocrine system, markedly decreasing plasma 25-OHD and 24,25-(OH)2D concentrations in S rats (14
–18
), significantly increasing plasma parathyroid hormone concentration in young male S and R rats (33
) and young female S rats (16
), and significantly increasing plasma 1,25-(OH)2D concentration in young female S rats (16
). The altered vitamin D endocrine system results in a greater calciuric response to salt by young female S rats compared with young female R rats (16
) and greater calcium excretion by S rats compared with R rats fed a standard diet (16
,33
–36
). If these findings are found to be applicable to human salt sensitivity, the effects of high salt intake on the calcium endocrine system of salt-sensitive individuals would be substantial. Spontaneously hypertensive rats do not have low plasma 25-OHD concentrations (14
,37
), suggesting that decreased plasma 25-OHD concentration might be specific to salt-induced hypertension. Urinary 25-OHD and 25-OHD binding activity might thus serve as markers of salt sensitivity and aid in its diagnosis and in the prevention and appropriate treatment of salt-induced hypertension.
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FOOTNOTES |
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2 Supported by National Institutes of Health/NIGMS S06 GM08248 and NIH/RCMI RR03034. K.G. was supported by NASA NCC9–112.
4 Abbreviations used: DBP, vitamin D binding protein; HS, 80 g/kg sodium chloride diet; LS, 3 g/kg sodium chloride diet; NHANES III, Third National Health and Nutrition Examination Survey; 1,25-(OH)2D, 1,25-dihydroxyvitamin D; 25-OHD, 25-hydroxyvitamin D; 25-OHD3, 25-hydroxycholecalciferol; R, salt-resistant; S, salt-sensitive.
5 Supplied by Harlan Teklad (Madison, WI). Low salt, TD 92101 (g/kg diet): corn, yellow, ground, 311.5; soybean meal, 190.0; corn gluten meal, 50.0; corn oil, 33.0; sodium chloride, 3.0. High salt, TD 92012 (g/kg diet): corn, yellow, ground, 214.7; soybean meal, 201.0; corn gluten meal, 56.0; corn oil, 35.8; sodium chloride, 80.0. Identical components (g/kg diet): wheat, hard, ground, 350.0; alfalfa meal, 30.0; dicalcium phosphate, 14.0; calcium carbonate, 12.0; mineral mix (TD 80318), 1.5; vitamin mix (TD 81125), 3.0; DL-methionine, 1.0; lysine · HCl, 1.0; ethoxyquin (antioxidant), 0.01.
Manuscript received 12 July 2002. Initial review completed 10 September 2002. Revision accepted 17 October 2002.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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