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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 10 4937-4943
Copyright © 2004 by The Endocrine Society

Peripheral Blood Monocyte Vitamin D Receptor Levels Are Elevated in Patients with Idiopathic Hypercalciuria

Murray J. Favus, Alexander J. Karnauskas, Joan H. Parks and Fredric L. Coe

Department of Medicine, University of Chicago Pritzker School of Medicine, Chicago, Illinois 60637

Address all correspondence and requests for reprints to: Dr. M. J. Favus at University of Chicago Pritzker School of Medicine, 5841 South Maryland Avenue, MC 1027, Chicago, Illinois 60637. E-mail: mfavus{at}medicine.bsd.uchicago.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Idiopathic hypercalciuria (IH) is the most common cause of calcium oxalate nephrolithiasis. Increased intestinal calcium absorption and bone resorption and decreased tubule calcium reabsorption may be caused by elevated serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in some patients but not in those with normal serum 1,25(OH)2D3 levels. Because 1,25(OH)2D3 exerts its biological actions through binding to the cellular vitamin D receptor (VDR), the present study was undertaken to test the hypothesis that VDR levels are elevated in IH patients.

Ten male IH calcium oxalate stone-formers were paired with controls matched in age within 5 yr and lacking a history of stones or family history of stones. Blood was obtained for serum, peripheral blood monocytes (PBMs) were separated from lymphocytes and other mononuclear cells, and PBM VDR content was measured by Western blotting.

The PBM VDR level was 2-fold greater in IH men at 49 ± 21 vs. 20 ± 15 fmol/mg protein, mean ± SD; P < 0.008. Serum 1,25(OH)2D3 levels were not higher than controls (48 ± 14 vs. 39 ± 11 pg/ml; P < 0.068). In conclusion, PBM VDR levels are elevated in IH calcium oxalate stone-formers. The elevation could not be ascribed to increased serum 1,25(OH)2D3 levels. These results suggest that the molecular basis for IH involves a pathological elevation of tissue VDR level, which may elevate intestinal calcium absorption and bone resorption and decrease renal tubule calcium reabsorption. The mechanism for increased VDR in IH patients with normal serum 1,25(OH)2D3 levels is unknown.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
IDIOPATHIC HYPERCALCIURIA (IH) has been implicated in the pathogenesis of calcium oxalate nephrolithiasis. Excess urine calcium raises calcium oxalate supersaturation thereby contributing to crystallization (1, 2). Thiazide diuretic agents lower urine calcium in IH and thereby reduce calcium stone recurrence (3, 4, 5). In IH, a diet reduced in protein and sodium content lowers urine calcium (6, 7, 8, 9) and reduces stone recurrence (6). The source of the excess urine calcium in IH patients is from intestinal calcium hyperabsorption and bone resorption (10, 11, 12, 13). Decreased tubule calcium reabsorption facilitates disposal of the calcium load at a normal serum calcium level (14, 15).

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is the only direct hormonal regulator of the intestinal calcium active transport process (16). Serum 1,25(OH)2D3 levels in IH patients form a continuum from normal to high (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) with a broad range of values (13, 17, 18, 20, 21, 22, 23, 24, 25). Elevated circulating 1,25(OH)2D3 levels may account for the hypercalciuria in some IH patients, but not in those with normal serum 1,25(OH)2D3 levels. This latter group constitutes one third to one half of all IH patients. Their hypercalciuria and increased intestinal calcium absorption are of the same magnitude as observed in those patients with elevated serum 1,25(OH)2D3 levels (17, 18, 19, 20). The mechanism of the increased calcium absorption and enhanced bone resorption in patients with normal serum 1,25(OH)2D3 is unknown.

IH patients who have low bone mass measured by bone densitometry are more likely to have persistent hypercalciuria after an overnight fast (29, 30, 31) than those with normal bone mass. Serum 1,25(OH)2D3 levels of patients with and without reduced bone mass overlap completely (30, 31). Thus, bone abnormalities, in addition to all of the other changes in calcium metabolism in IH, may occur in the presence of normal serum 1,25(OH)2D3.

Genetic hypercalciuric stone-forming (GHS) rats model some behaviors of human IH. GHS rats have increased intestinal calcium transport (32, 33, 34), increased bone resorption (33, 35, 36), reduced renal calcium reabsorption (37), and negative calcium balance during low calcium intake (33). The urine is supersaturated with calcium salts, and kidney stones of calcium salts appear spontaneously (38, 39). By contrast with human IH, serum 1,25(OH)2D3 levels are uniformly normal (32, 33, 34), raising the question about how the phenotype arises. We have reported a 2- to 4-fold increase in vitamin D receptor (VDR) level in GHS rat duodenal mucosa, renal cortex, bone, and splenic monocytes (34, 35, 40). The elevated VDR level, and not the serum 1,25(OH)2D3 level, may account for all of the pathological changes observed in the vitamin D target tissues of GHS rats (34, 40).

These observations have led us to hypothesize that in some patients a primary increase in tissue VDR level gives rise to IH as in the GHS rats. In IH patients with elevated serum 1,25(OH)2D3 levels, VDR level is expected to be increased, as 1,25(OH)2D3 induces receptor homologous up-regulation (41, 42). It is not known, however, whether VDR level is elevated in human IH when increased serum 1,25(OH)2D3 levels are not present. In the present study, VDR was measured in peripheral blood monocytes (PBMs) obtained from male IH calcium oxalate stone-formers and same-sex age-matched normal control subjects to test whether a primary increase of VDR can cause IH.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Study population

Ten men over age 18 yr with the diagnosis of IH were recruited from the University of Chicago Kidney Stone Clinic (Chicago, IL). Clinical assessment of all pertinent radiographs, stone history, stone analyses, procedures for stones, and general medical and family history had been obtained for clinical purposes. All formed calcium oxalate stones. Three 24-h urine collections for calcium, phosphate, creatinine, sodium, potassium, uric acid, chloride, oxalate, citrate, sulfate, ammonium ion, and pH were obtained as outpatients on their usual unregulated diet in the absence of any medical treatments. Corresponding sera were obtained between 0700 and 0900 h, 12 h in the postabsorptive state, for calcium, phosphate, magnesium, sodium, potassium, chloride, and total CO2 content. All subjects met the criteria for IH defined as normocalcemia and urine calcium greater than 300 mg/24 h, greater than 140 mg calcium/g creatinine, or greater than 4 mg/kg body weight for either sex (43) while eating their usual diet. Other known causes of hypercalciuria were absent, such as sarcoidosis, malignant neoplasm, hyperthyroidism, vitamin D intoxication, rapidly progressive osteoporosis, chronic metabolic acidosis, Paget’s disease of bone, or stones associated with other drugs such as acetazolamide.

The IH subjects were each pair-wise matched by race and age within 5 yr to a healthy control subject. The controls were without stone disease, family history of stone or bone disease, other systemic illness, or need to take medications that may influence mineral metabolism. Single 24-h urine collections, fasting blood, and PBMs were obtained for all of the biochemical measurements, and VDR was measured in the IH subjects. Every attempt was made to obtain blood and urine samples from each IH subject and his age-matched control subject within 2 wk. IH patients and controls were not selected for serum 1,25(OH)2D3 levels, and controls were not selected for urine calcium excretion.

IH subjects and volunteers were excluded if there was renal impairment, concomitant active medical conditions, dietary requirements that would not permit variation in calcium intake, use of medications that could not be discontinued, or medications that may alter calcium metabolism such as glucocorticoids, thiazide or loop diuretics, anticonvulsants, heparin, large doses of antacids, vitamin D in doses greater than 4,000 U/d, greater than 10,000 U/d vitamin A, fluoride, bisphosphonates, or calcitonin.

The University of Chicago Institutional Review Board and the University of Chicago General Clinical Research Center approved the study protocol, and IH subjects and volunteers provided written informed consent.

Study protocol and methods

Laboratory tests. Routine urine and serum measurements were made as described elsewhere (43). Serum intact PTH was measured in duplicate aliquots using a commercially available immunoradiometric assay kit (Quest Diagnostics, Inc., Teterboro, NJ). The assay uses human PTH 1–84 as standard. The lower limit of detection is 0.10 pmol/liter (1 pg/ml); intra- and interassay coefficients of variation (CVs) are 5 and 7–11%, respectively. Normal values range from 1.04–6.25 pmol/liter (10–60 pg/ml). Serum 1,25(OH)2D3 was measured in 1.0 ml of serum using a modification of the radioreceptor assay described by Reinhardt et al. (44), which measures levels in human sera. The competitive binding assay uses VDR protein, which was purchased from commercial sources. Sensitivity of the assay is in the 2.4–7.2 fmol (1–3 pg)/assay tube range, and overall sample recovery is 65–75%. Intra- and interassay CVs range from 7–12% and 11–19%, respectively. The range of normal values for 1,25(OH)2D3 for pre- and postmenopausal women and adult men in our laboratory is 58–130 pmol/liter (24–54 pg/ml).

PBM isolation. After an overnight fast, 20 ml of blood was collected (10 ml in EDTA) and subjected to a low-speed centrifugation at 1500 x g for 5 min to separate plasma from the cellular elements. The plasma was removed and frozen for subsequent analysis. The cellular fraction from the sample was washed with 5.0 ml PBS containing 0.3% BSA and 0.6% Na citrate. The supernatant was removed, and the buffy coat was cooled at 4 C for 20 min. A suspension of Dynabeads (Dynal, Lake Success, NY) was washed in ice-cold PBS in 0.3% BSA. In the presence of the Dynabeads, the buffy coat was brought to the initial volume with PBS containing 2% fetal calf serum and incubated at 4 C for 60 min. Monocytes were separated from other cellular elements by their binding to magnetizable polystyrene beads coated with mouse IgG2 monoclonal antibody RM052 specific for the CD14 plasma membrane antigen (45) (Dynabeads M-450 CD14, Dynal A.S., Oslo, Norway). The cells attached to beads were separated in the presence of a magnet for 20 min, the supernatant removed, and the cells washed twice with PBS containing 0.3% BSA and twice with PBS.

In initial experiments, standard hemocytometer methodology yielded peripheral PBM cell counts ranging from 3–10% with complete overlap between the two groups. As anti-CD14 antibody is specific for monocyte-plasma membrane-specific antigens and does not bind to lymphocytes, and the washes of the monocyte-bound Dynabeads removed unbound cellular elements, the final monocyte preparation was virtually devoid of lymphocytes (<5%) as verified by differential white blood cell counting by light microscopy.

VDR assay. The monocyte-enriched fraction was homogenized in hypertonic buffer (0.3 M KCl, 10 mM Tris, 1 mM EDTA, 10 mM molybdate, and 50 ng/ml aprotinin) and sonicated, and the Dynabeads were removed. The VDR-rich cytosol fraction was added to 5x Laemelli buffer, boiled 5 min, and stored at –80 C for subsequent determination of VDR by Western blotting. Sixty micrograms of monocyte homogenate were loaded onto each lane, and proteins were resolved by 10% SDS-PAGE. A standard curve was constructed from four concentrations of recombinant human (rh)VDR (Calbiochem, San Diego, CA). The separated proteins were transferred onto Immobilon-P membranes (Millipore Corp., Bedford, MA) by electroblotting overnight. Blocking was accomplished using Tris-buffered saline and Tween 20 (2.5% TBST; 2.0 M Tris-HCl, pH 7.5; 5 M NaCl; and 0.05% Tween 20) with 5% nonfat milk powder for 2 h. Membranes were then incubated in the presence of rabbit antirat VDR polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:200 dilution and 5% milk powder for 60 min. The membranes were washed and incubated in secondary antibody, donkey antirabbit IgG-horseradish peroxidase (Santa Cruz Biotechnology), at 1:3000 in 5% milk powder for 60 min. The membranes were then washed, and the intensity of the bands was enhanced using chemiluminescence mediated by horseradish peroxidase catalysis of luminol (Amersham Searle, Arlington Heights, IL) followed by exposure to Kodak Biomax-Ml film (Eastman Kodak Co., Rochester, NY). The VDR bands were quantified using scanning laser densitometry. PBM protein concentration was determined by the Bradford assay (Bio-Rad, Hercules, CA). VDR concentration was expressed as femtomoles of VDR per milligram of monocyte protein. Each of the samples was run two to three times on separate gels, with values falling within the calculated interassay CV. The mean of each value was used in the final analysis.

Statistical analysis

Results are shown as the mean ± SD. Student’s t tests, multivariable regression analysis, and linear regressions used standard software (Systat, Chicago, IL). Confidence ellipses were calculated to include 1 SD.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Characterization of the VDR assay

Standard rhVDR (46) identified a 48,000 VDR band in the PBM preparations. A band of VDR was detected in the intact buffy coat before removal of monocytes. A more intense VDR band was present in the monocyte-enriched fraction (Fig. 1Go). A very faint VDR band was present in the monocyte- depleted buffy coat. Cell counts showed no remaining monocytes in the buffy coat after treatment with CD-14-covered magnetic beads. Thus, the monocyte isolation procedure effectively removed almost all PBMs from the buffy coat, wherein the VDR was concentrated in the PBM fraction. Visual inspection using light microscopy revealed that the beads were specifically attached to monocytes. Rare lymphocytes (less than 5% of the total white blood cells) were observed, and none were observed to be attached to beads. Thus, the specificity of the anti-CD14 antibody for monocytes precluded the requirement for lymphocyte-specific markers to detect contamination.



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  		FIG. 1. PBM VDR by Western blotting. PBMs were isolated from 12 ml of blood from a healthy volunteer. The buffy coat was treated with 0.5 ml of a suspension of magnetic beads coated with anti-CD14 antibody to monocyte surface antigen followed by separation of the beads from the remainder of the buffy coat. VDR in aliquots of PBM cytosol protein was separated from other proteins by SDS-PAGE (see Subjects and Methods). Lanes 1–3, rhVDR standard 2.1, 10.4, and 20.8 pmol/lane; lane 4, 200 µg of PBM-depleted buffy coat; lane 5, 200 µg of PBM-rich fraction; lane 6, 100 µg of buffy coat protein before removing monocyte fraction. Arrow indicates migration of rhVDR 48,000.

 
Western blots of serial dilutions of monocyte protein (PBMs isolated from a normal volunteer) revealed dominant bands of VDR at 48,000 with intensities that were linear over the range from 40–240 µg monocyte protein (Fig. 2Go). Thus, the primary antibody recognized PBM VDR in a linear fashion over the protein range studied. The characteristics of the primary antibody and the expression of VDR level as micrograms of PBM protein excluded variation in PBM count as a contributor to any variation in VDR level.



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  		FIG. 2. Immunoblot of serial dilutions of PBM VDR from normal volunteer. Arrow indicates migration of 48,000 rhVDR standard. Lanes 1–7 are serial dilutions of the same monocyte protein preparation loaded (in micrograms protein per lane): 20, 40, 80, 120, 160, 200, and 240, respectively.

 
Four rhVDR standards 0.26–1.56 fmol (12.5–75 pg) were run on each gel to create a standard curve for each assay against which the subject samples were compared. A representative standard curve is shown in Fig. 3Go. The VDR band intensities from PBM VDR prepared from control and IH subjects fell within the range of intensities of the VDR standards. Interassay CVs across four assays were calculated by treating each of the two middle standards, 0.52 and 1.04 fmol (25 and 50 pg), as unknowns and using the other three standards to create a standard curve. The 0.52-fmol standard was measured as 0.60 ± 0.04 (mean ± SD; n = 4), and the 1.04-fmol standard was measured as 1.18 ± 0.11 fmol (n = 4). The interassay CV was 6.0 and 9.4% for the 25- and 50-pg standards, respectively. PBM extracts from six subjects were run in duplicate in the same VDR assay, and the mean and SDs were used to calculate the intraassay CV. The grand mean intraassay CV was 7.5% and ranged from 2.1–11.6%. The excellent reproducibility of the VDR analyses both within and across assays indicated that loading and blotting and transfer efficiencies varied within an acceptable range. Therefore, VDR levels were quantified without measuring a second PBM-specific protein within each sample.



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  		FIG. 3. Representative VDR standard curve using rhVDR. A standard curve was constructed from the density of each band for rhVDR 0.26, 0.52, 1.04, and 1.56 fmol/lane. Intensity of the bands was determined as described in Subjects and Methods. For this representative curve, the slope is 104.97 x 1000; r = 0.9909; R2 = 0.9818; P = 0.0091.

 
Group comparisons

PBM VDR was readily detected by Western blotting (Fig. 4Go). PBM VDR levels were 2-fold greater in IH compared with age-matched controls, and the difference by paired t test was significant at P = 0.002 (Table 1Go). Analysis using the unpaired t test also revealed a highly significant difference between the two groups (P = 0.014). An example of the differences in intensity of the VDR band between subjects within a single pair is illustrated in Fig. 4Go. Mean serum calcium was lower and 24-h urine calcium excretion higher in the IH patients compared with their controls (Table 1Go). Mean serum 1,25(OH)2D3 levels were not significantly higher in the IH patients by t test; neither were serum PTH, creatinine, magnesium, or phosphate concentrations (Table 1Go).



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  		FIG. 4. Representative immunoblot of PBM VDR in IH patients and controls. Lanes 1–3 and 10, 0.26, 0.52, 1.04, and 2.08 fmol rhVDR standards; lanes 4 and 5, matched control and IH; lanes 6 and 7, matched IH and control; lanes 8 and 9, matched control and IH. Arrow is migration position of VDR. Each lane was loaded with 60 µg monocyte protein.

 

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  		TABLE 1. Biochemical characteristics of patients with IH and control subjects

 
VDR was higher in IH when corrected for serum 1,25(OH)2D3 (adjusted means were 48 and 21 for IH and control, respectively; P = 0.007). The slopes of VDR on 1,25(OH)2D3 were homogeneous between the IH and control groups, and VDR and serum 1,25(OH)2D3 were not correlated (Fig. 5Go, upper left; P = 0.306). The difference in VDR remained significant in a multivariate general linear model that included serum calcium, serum PTH, serum phosphate, serum 1,25(OH)2D3, and urine calcium (P = 0.002), with none of these variables having significant P values as covariates. VDR was inverse to serum calcium (Fig. 5Go, upper right) among IH but not normal subjects (slopes were –55 ± 15 vs. 18 ± 18, IH vs. controls; by ANOVA, slopes were not homogeneous; F = 9.4; P = 0.007). VDR adjusted for serum calcium remained higher in IH than normal subjects (52 vs. 25; P = 0.018). Although suggestive, the relationship of VDR to 24-h urine calcium excretion was not significant (Fig. 5Go, lower left), and the slopes for IH and controls were not different. Finally, VDR did not correlate with serum PTH (Fig. 5Go, lower right). Altogether, PBM VDR of IH patients was higher than among controls, and adjustments for serum 1,25(OH)2D3, serum calcium, PTH, serum phosphate, and urine calcium did not affect this conclusion.



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  		FIG. 5. Values of PBM VDR and related measurements. PBM VDR levels were high in IH (black circles) vs. normal (gray circles) subjects in relation to serum 1,25(OH)2 D3 (upper left). PBM VDR was inversely correlated with serum calcium (upper right) among IH but not control subjects (analysis in Results), as illustrated by the tilt and narrowness of the 1 SD ellipse for the IH subjects. The two slopes were not homogeneous. Although seemingly significant, PBM VDR was not correlated with urine calcium excretion (lower left) or serum PTH levels (lower right) among normal or IH subjects.

 
Paired comparisons

For each pair, we expressed the difference as patient minus control. By simple t tests, change in VDR differed from 0 (29.5; t = 3.196; P = 0.011), as did the change in serum calcium (–0.069; t = 2.298; P = 0.047). The significant difference in VDR had no correlation with the difference in serum PTH, phosphate, 1,25(OH)2D3, calcium, or 24-h urine calcium excretion. Change in VDR and PTH had an impressive correlation coefficient (–0.525), but the difference was not significant (P = 0.119). Altogether, the paired analysis supports the unpaired analysis in showing an increased PBM VDR level in IH vs. control subjects and that the significantly higher value in IH may not be ascribed to increased serum 1,25(OH)2D3 levels.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In this study of an unselected group of male IH calcium oxalate stone-formers, mean PBM VDR levels were 2-fold higher than same-sex age-matched non-stone-formers. The biological actions of 1,25(OH)2D3 are mediated through the nuclear VDR. Because variances in target tissue VDR level determine the actions of 1,25(OH)2D3 (41), an elevation in VDR may be directly responsible for the increases in intestinal calcium absorption and bone resorption and decreases in renal tubular calcium reabsorption that characterize IH. The present study did not test whether PBM VDR levels in human subjects accurately reflect the VDR levels in intestine, bone, kidney, and other vitamin D target tissues. However, increased 1,25(OH)2D3 actions through increased VDR is supported by observations in GHS rats, in which intestine, bone, kidney, and splenic monocyte VDR levels are increased, and the biological actions of 1,25(OH)2D3 are increased in the presence of normal serum 1,25(OH)2D3 levels (32, 33, 34, 35, 36, 37, 40). Furthermore, the 2-fold increase in VDR level in the IH patients is of a similar magnitude as the 2- to 4-fold increase in VDR that we measured in GHS rat tissues (34, 35, 40). Although our IH subjects were selected for being calcium oxalate stone-formers, the abnormality of VDR is not limited to IH stone-formers, in that one of our controls, who indeed had IH (urine calcium excretion of 535 mg/24 h) but has never formed a stone, had a very high PBM VDR level.

Serum 1,25(OH)2D3 and 25-OH-vitamin D levels are major regulators of intestinal calcium absorption, and serum 1,25(OH)2D3 levels are elevated in some patients with IH (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28). For those patients, elevated VDR levels may be secondary to the elevated 1,25(OH)2D3 through the mechanism of homologous up-regulation (41). Such patients were observed in this study. However, high VDR levels were also found in IH patients with serum 1,25(OH)2D3 levels that overlapped with those of control subjects. This indicates that an elevated serum 1,25(OH)2D3 level is not required for the elevation in VDR, and is reflected in the persistence of higher values for PBM VDR in IH vs. controls after multivariable adjustments for serum 1,25(OH)2D3 levels. The present observations support previous reports that IH patients with normal serum 1,25(OH)2D3 levels have hypercalciuria and increased intestinal calcium absorption that are not different from those with elevated serum 1,25(OH)2D3 levels (17, 19, 20, 29, 31). For example, the study by Kaplan et al. (17) shows the lack of relationship of serum 1,25(OH)2D3 and intestinal calcium absorption in IH, in contrast to the linear relationship between serum 1,25(OH)2D3 and calcium absorption in patients with primary hyperparathyroidism and normal subjects. The data by Kaplan strongly suggest that a factor other than serum 1,25(OH)2D3 levels may be regulating intestinal calcium absorption in IH patients with normal serum 1,25(OH)2D3, and the present study suggests that this factor may be excess VDR. These reports support the current hypothesis that elevated VDR, independent of serum 1,25(OH)2D3 levels could be a pathophysiological basis for IH in such patients.

Endogenous elevations of 1,25(OH)2D3, such as occur during dietary calcium (42) or phosphate (47) restriction, or exogenous administration of 1,25(OH)2D3 (40, 48, 49) up-regulate the VDR level in intestine and other tissues. A hyperresponsiveness of VDR mRNA to 1,25(OH)2D3 has been demonstrated in GHS rats (40), but 1,25(OH)2D3-induced VDR up-regulation and VDR gene expression were not measured in the present study. In addition, VDR is up-regulated by a variety of growth factors that influence cell proliferation (41, 50, 51). However, the potential role of these factors in VDR regulation in vitamin D target tissues in humans and animals is not known. Although an age-dependent decline in intestinal VDR content is well documented (52), subjects in the present study were matched for age. Therefore, an age-related decline in VDR cannot account for the differences between IH stone-formers and controls in the present study.

Serum peripheral blood mononuclear cell fractions have been used previously to measure VDR in humans (48, 53, 54). Monocytes, which compose approximately 3–7% of the total number of peripheral blood mononuclear cells, express VDR that is not dependent upon cell activation. T lymphocytes are more than 90% of mononuclear cells and express the VDR only upon mitogen activation (54). Using a mixed peripheral blood cell preparation, Zerwekh et al. (53) found no relationship between activated peripheral blood T lymphocyte VDR levels and circulating 1,25(OH)2D3 levels in IH patients with normal or elevated serum 1,25(OH)2D3 levels. However, of the six patients with elevated T lymphocyte VDR, four had normal 1,25(OH)2D3 levels. Thus, T lymphocyte VDR level may increase in response to 1,25(OH)2D3, and monocyte VDR level may have been present in some of those IH patients. In the present study, a highly purified preparation of monocytes was isolated using a monoclonal antibody to the CD14 plasma membrane antigen specific for PBMs (45). A potential variable contribution of T lymphocytes to total blood VDR content was thus excluded. Therefore, the apparent differences in VDR levels between the results of the present study and that of Zerwekh et al. may be due to the population of cells used for VDR analysis.

In rats, VDR tissue and monocyte levels measured by Western blotting and saturation binding (47) were highly positively correlated. Therefore, VDR levels determined by the Western blot assays in this study would be expected to correlate with values obtained using standard saturation binding techniques.

Altogether, this work offers the first evidence for an alternative pathway to IH. In some patients, perhaps half, the traditional mechanism of elevated serum 1,25(OH)2D3 levels is sufficient. In at least some of the other patients, whose serum levels of 1,25(OH)2D3 are not abnormal, the results of the present study suggest that elevated VDR abundances create tissue responses otherwise dependent upon elevated 1,25(OH)2D3 levels and produce the clinical syndrome of IH. The present study did not demonstrate intestinal calcium overabsorption directly or include direct measurements of intestinal VDR levels and marks, therefore, only the beginning of a full investigation. But it is sufficient of itself to demonstrate a clear VDR aberration in IH that is not dependent upon elevated serum 1,25(OH)2D3 levels and could create the clinical abnormalities of calcium metabolism that lead to nephrolithiasis and perhaps bone mineral reduction (1, 10, 12, 13, 14, 15, 17, 22, 26, 30, 31, 43).


   Acknowledgments
 
We thank Vrishali Tembe and Yasushi Nakagawa, Ph.D., for their expert technical assistance and advice. We also thank M. Shah for assistance with data management and clinical coordination.


   Footnotes
 
This study was supported by National Institutes of Health (NIH) Grant 1PO1 DK56788, NIH General Clinical Research Center Grant MO1 RR00055, and the University of Chicago Osteoporosis Research Fund.

M.J.F. and A.J.K. contributed equally to this manuscript.

Abbreviations: CV, Coefficient of variation; GHS, genetic hypercalciuric stone-forming; IH, idiopathic hypercalciuria; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; PBM, peripheral blood monocyte; rh, recombinant human; VDR, vitamin D receptor.

Received March 1, 2004.

Accepted June 28, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Coe FL, Parks JH 1997 New insights and treatment of nephrolithiasis: new research venues. J Bone Miner Res 12:522–533[CrossRef][Medline]
  2. Asplin J, Parks J, Lingeman J, Kahnoski R, Mardis H, Lacey S, Goldfarb D, Grasso M, Coe F 1998 Supersaturation and stone composition in a network of dispersed treatment sites. J Urol 159:1821–1825[CrossRef][Medline]
  3. Laerum E, Larsen S 1984 Thiazide prophylaxis of urolithiasis: a double-blind study in general practice. Acta Med Scand 215:383–389[Medline]
  4. Ettinger B, Citron JT, Livermore B, Dolman LI 1988 Chlorthalidone reduces calcium oxalate calculous recurrence but magnesium hydroxide does not. J Urol 139:679–684[Medline]
  5. Borghi L, Meschi T, Guerra A, Novarini A 1993 Randomized prospective study of a nonthiazide diuretic, indapamide, in preventing calcium stone recurrences. J Cardiovasc Pharmacol 22(Suppl 6):S78–S86
  6. Borghi L, Schianchi T, Meschi T, Borghi L, Schianchi T, Meschi T, Guerra A, Allegri F, Maggiore U, Novarini A 2002 Comparison of two diets for the prevention of recurrent stones in idiopathic hypercalciuria. N Engl J Med 346:77–84[Abstract/Free Full Text]
  7. Breslau NA, Brinkley L, Hill KD, Pak CYC 1988 Relationship of animal protein-rich diet to kidney stone formation and calcium metabolism. J Clin Endocrinol Metab 66:140–146[Abstract/Free Full Text]
  8. Allen LH, Oddoye EA, Margen S 1979 Protein-induced hypercalciuria: a longer term study. Am J Clin Nutr 32:741–749[Abstract/Free Full Text]
  9. Phillips MJ, Cooke JNC 1967 Relation between urinary calcium and sodium in patients with idiopathic hypercalciuria. Lancet 1:1354–1357[Medline]
  10. Bordier P, Ryckwart A, Gueris J, Rasmussen H 1977 On the pathogenesis of so-called idiopathic hypercalciuria. Am J Med 63:398–409[CrossRef][Medline]
  11. Pak CYC, Delea CS, Bartter FC 1974 Successful treatment of recurrent nephrolithiasis (calcium stones) with cellulose phosphate. N Engl J Med 290:175–180
  12. Pak CYC, Kaplan R, Bone H, Townsend J, Waters O 1975 A simple test for the diagnosis of absorptive, resorptive, and renal hypercalciurias. N Engl J Med 292:497–500[Abstract]
  13. Coe FL, Favus MJ, Crockett T, Strauss AL, Parks JH, Porat A, Gantt CL, Sherwood LM 1982 Effects of low calcium diet on urine calcium excretion, parathyroid function and serum 1,25(OH)2D3 levels in patients with idiopathic hypercalciuria and in normal subjects. Am J Med 75:25–32
  14. Edwards, NA, Hodgkinson A 1965 Studies of renal function in patients with idiopathic hypercalciuria. Clin Sci 29:327–338[Medline]
  15. Peacock M, Nordin BEC 1968 Tubular reabsorption of calcium in normal and hypercalciuric subjects. J Clin Pathol 21:355–358
  16. Favus MJ 2002 Intestinal absorption of calcium, magnesium, and phosphorus. In Coe FL, Favus MJ, eds. Disorders of bone and mineral metabolism. 2nd ed. Philadelphia: Lippincott Williams Wilkins; 48–73
  17. Kaplan RA, Haussler MR, Deftos LJ, Pak CYC 1977 The role of 1-{alpha},25-dihydroxyvitamin D3 in the mediation of intestinal hyperabsorption of calcium in primary hyperparathyroidism and absorptive hypercalciuria. J Clin Invest 59:756–760
  18. Shen FH, Baylink DJ, Nielsen RL, Sherrard DJ, Ivey JL, Haussler MR 1977 Increased serum 1,25-dihydroxyvitamin D in idiopathic hypercalciuria. J Lab Clin Med 90:955–962[Medline]
  19. Zerwekh JE, Pak CYC 1980 Selective effects of thiazide therapy on serum 1,25-dihydroxyvitamin D and intestinal calcium absorption in renal and absorptive hypercalciurias. Metabolism 29:13–17[CrossRef][Medline]
  20. Gray RW, Wilz DR, Caldas AE, Lemann J 1977 The importance of phosphate in regulating plasma 1,25-(OH)2-vitamin D levels in healthy subjects, in calcium-stone formers and in patients with primary hyperparathyroidism. J Clin Endocrinol Metab 45:299–306[Abstract/Free Full Text]
  21. Van Den Berg CJ, Kumar R, Wilson DM, Heath 3rd H, Smith LH 1980 Orthophosphate therapy decreases urinary calcium excretion and serum 1,25-dihydroxyvitamin D concentrations in idiopathic hypercalciuria. J Clin Endocrinol Metab 51:998–1001[Abstract/Free Full Text]
  22. Broadus AE, Insogna KL, Lang R, Ellison AF, Dreyer BE 1984 Evidence for disordered control of 1,25-dihydroxyvitamin D production in absorptive hypercalciuria. N Engl J Med 311:73–80[Abstract]
  23. Broadus AE, Insogna KL, Lang R A consideration of the hormonal basis and phosphate leak hypothesis of absorptive hypercalciuria. J Clin Endocrinol Metab 58:161–169
  24. Insogna KL, Broadus AE, Dreyer BE, Ellison AF, Gertner JM 1985 Elevated production rate of 1,25-dihydroxyvitamin D in patients with absorptive hypercalciuria. J Clin Endocrinol Metab 61:490–495[Abstract/Free Full Text]
  25. Breslau NA, Preminger G, Adams BV, Otey J, Pak CY 1992 Use of ketoconazole to probe the pathogenetic importance of 1,25-dihydroxyvitamin D in absorptive hypercalciuria. J Clin Endocrinol Metab 75:1446–1452[Abstract]
  26. Broadus AE, Dominguez M, Bartter FC 1978 Pathophysiological studies in idiopathic hypercalciuria: use of an oral calcium tolerance test to characterize distinctive hypercalciuric groups. J Clin Endocrinol Metab 47:751–760[Abstract/Free Full Text]
  27. Battaile P, Bouillon R, Fournier A, Renaud H, Gueris J, Idrissi A 1987 Increased plasma concentrations of total and free 1,25(OH)2D3 in calcium stone formers with idiopathic hypercalciuria. Contrib Nephrol 58:137–142[Medline]
  28. Coe FL, Parks JH, Bushinsky DA, Langman CB, Favus MJ 1988 Chlorthalidone promotes mineral retention in patients with idiopathic hypercalciuria. Kidney Int 33:1140–1146[Medline]
  29. Pacifici R, Rothstein M, Rifas L, Lau KH, Baylink DJ, Avioli LV, Hruska K 1990 Increased monocyte interleukin-1 activity and decreased vertebral bone density in patients with fasting hypercalciuria. J Clin Endocrinol Metab 71:138–145[Abstract/Free Full Text]
  30. Weisinger JR, Alonzo E, Bellorin-Font E, Blasini AM, Rodriguez MA, Paz-Martinez V, Martinis R 1996 Possible role of cytokines on the bone mineral loss in idiopathic hypercalciuria. Kidney Int 49:244–250[Medline]
  31. Ghazali A, Fuentes V, Desaint C, Bataille P, Westeel A, Brazier M, Prin L, Fournier A 1997 Low bone mineral density and peripheral blood monocyte activation profile in calcium stone formers with idiopathic hypercalciuria. J Clin Endocrinol Metab 82:32–38[Abstract/Free Full Text]
  32. Bushinsky DA, Favus MJ 1988 Mechanism of hypercalciuria in genetic hypercalciuric rats. Inherited defect in intestinal calcium transport. J Clin Invest 82:1585–1591
  33. Kim M, Sessler NE, Tembe V, Favus MJ, Bushinsky DA 1993 Response of genetic hypercalciuric rats to a low calcium diet. Kidney Int 43:189–196[Medline]
  34. Li X-Q, Tembe V, Horwitz G, Bushinsky DA, Favus MJ 1993 Increased intestinal vitamin D receptor in genetic hypercalciuric rats: a cause of intestinal calcium hyperabsorption. J Clin Invest 91:661–667
  35. Krieger NS, Stathopoulos VM, Bushinsky DA 1996 Increased sensitivity to 1,25(OH)2D3 in bone from genetic hypercalciuric rats. Am J Physiol 271:C130–C135
  36. Bushinsky DA, Neuman KJ, Asplin J, Krieger NS 1999 Alendronate decreases urine calcium and supersaturation in genetic hypercalciuric rats. Kidney Int 55:234–243[CrossRef][Medline]
  37. Tsuruoka S, Bushinsky DA, Schwartz GJ 1997 Defective renal calcium reabsorption in genetic hypercalciuric rats. Kidney Int 51:1540–1547[Medline]
  38. Bushinsky DA, Grypnas MD, Nilsson EL, Coe FL 1995 Stone formation in genetic hypercalciuric rats. Kidney Int 48:1705–1713[Medline]
  39. Asplin JR, Bushinsky DA, Singharenam W, Riordon D, Parks JH, Coe FL 1997 Relationship between supersaturation and crystal inhibition in hypercalciuric rats. Kidney Int 51:640–645[Medline]
  40. Yao J, Kathpalia P, Bushinsky DA, Favus MJ 1998 Hyperresponsiveness of vitamin D receptor gene expression to 1,25-dihydroxyvitamin D3. A new characteristic of genetic hypercalciuric stone-forming rats. J Clin Invest 101:2223–2232[Medline]
  41. Krishman AV, Feldman D 1997 Regulation of vitamin D receptor abundance. In: Feldman D, Glorieux FH, Pike JW, eds. Vitamin D. 1st ed. San Diego: Academic Press; 179–200
  42. Favus MJ, Mangelsdorf DJ, Tembe V, Coe BJ, Haussler MR 1988 Evidence for in vivo upregulation of the intestinal vitamin D receptor during dietary calcium restriction in the rat. J Clin Invest 98:218–224
  43. Coe FL, Parks JH, Asplin JR 1992 The pathogenesis and treatment of kidney stones. N Engl J Med 327:1141–1152[Medline]
  44. Reinhardt TA, Horst RL, Orf JW, Hollis BW 1984 A microassay for 1,25-dihydroxyvitamin D not requiring high performance liquid chromatography: application to clinical studies. J Clin Endocrinol Metab 58:91–98[Abstract/Free Full Text]
  45. Theodorsen L 1995 Evaluation of monocyte counting with two automated instruments by the use of CD14-specific immunomagnetic Dynabeads. Clin Lab Haematol 17:225–229[Medline]
  46. Sone T, McDonnell DP, O’Malley BW, Pike JW 1990 Expression of human vitamin D receptor in Saccharomyces cerevisiae: purification, properties and generation of polyclonal antibodies. J Biol Chem 265:21997–22003[Abstract/Free Full Text]
  47. Sriussadaporn S, Wong M, Pike JW, Favus MJ 1995 Tissue specificity and mechanism of vitamin D receptor up-regulation during dietary phosphorus restriction in the rat. J Bone Miner Res 10:271–280[Medline]
  48. Merke J, Nawrot M, Hugel U, Szabo A, Ritz E 1989 Evidence for in vivo up-regulation of 1,25(OH)2D3 receptor in human monocytes. Calcif Tissue Int 45:255–256[Medline]
  49. Costa EM, Feldman D 1986 Homologous up-regulation of the 1,25-dihydroxyvitamin D3 receptor in rats. Biochem Biophys Res Commun 137:742–747[CrossRef][Medline]
  50. Mezzetti G, Barbiroli B, Oka T 1987 1,25-Dihydroxycholecalciferol receptor regulation in hormonally induced differentiation of mouse mammary glands in culture. Endocrinology 120:2488–2493[Abstract/Free Full Text]
  51. Krishman AV, Feldman D 1991 Stimulation of 1,25-dihydroxyvitamin D3 receptor gene expression in cultured cells by serum and growth factors. J Bone Miner Res 6:1099–1107[Medline]
  52. Ebeling PR, Sandgren ME, DiMagno EP, Lane AW, DeLuca HF, Riggs BL 1992 Evidence of an age-related decrease in intestinal responsiveness to vitamin D: relationship between serum 1,25-dihydroxyvitamin D3 and intestinal vitamin D receptor concentration in normal women. J Clin Endocrinol Metab 75:176–182[Abstract]
  53. Zerwekh JE, Yu XP, Breslau NA, Manolagas S, Pak CY 1993 Vitamin D receptor quantification in human blood mononuclear cells in health and disease. Mol Cell Endocrinol 96:1–6[CrossRef][Medline]
  54. Bhalla AK, Amento EP, Clemens TL, Holick MF, Krane SM 1983 Specific high-affinity receptors for1,25-dihydroxyvitamin D in human peripheral blood mononuclear cells: presence in monocytes and induction in T-lymphocytes following activation. J Clin Endocrinol Metab 57:1308–1311[Abstract/Free Full Text]



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