1,25(OH)2D3 stimulates Mg2+ uptake into MDCT cells: modulation by extracellular Ca2+ and Mg2+

Gordon Ritchie1, Dirk Kerstan1, Long-Jun Dai1, Hyung Sub Kang1, Lucie Canaff2, Geoffrey N. Hendy2, and Gary A. Quamme1

1 Department of Medicine, University Hospital, University of British Columbia, Vancouver, British Columbia V6T 1Z3; and 2 Departments of Medicine, Physiology and Human Genetics, McGill University and Royal Victoria Hospital, Montreal, Quebec, Canada H3A 1A1


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The distal convoluted tubule plays a significant role in renal magnesium conservation. Although the cells of the distal convoluted tubule possess the vitamin D receptor, little is known about the effects of 1alpha ,25-dihydroxyvitamin D [1,25(OH)2D3] on magnesium transport. In this study, we examined the effect of 1,25(OH)2D3 on distal cellular magnesium uptake and the modulation of this response by extracellular Ca2+ and Mg2+ in an immortalized mouse distal convoluted tubule (MDCT) cell line. MDCT cells possess the divalent cation-sensing receptor (CaSR) that responds to elevation of extracellular Ca2+ and Mg2+ concentrations to diminish peptide hormone-stimulated Mg2+ uptake. Mg2+ uptake rates were determined by microfluorescence in Mg2+-depleted MDCT cells. Treatment of MDCT cells with 1,25(OH)2D3 for 16-24 h stimulated basal Mg2+ uptake in a concentration-dependent manner from basal levels of 164 ± 5 to 210 ± 11 nM/s, representing a 28 ± 3% change. Pretreatment with actinomycin D or cycloheximide abolished 1,25(OH)2D3-stimulated.Mg2+ uptake (154 ± 18 nM/s), suggesting that 1,25(OH)2D3 stimulates Mg2+ uptake through gene activation and protein synthesis. Elevation of extracellular Ca2+ inhibited 1,25(OH)2D3-stimulated Mg2+ uptake (143 ± 5 nM/s). Preincubation of the cells with an antibody to the CaSR prevented the inhibition by elevated extracellular Ca2+ of 1,25(OH)2D3-stimulated Mg2+ uptake (202 ± 8 nM/s). Treatment with an antisense CaSR mRNA oligodeoxynucleotide also abolished the effects of extracellular Ca2+ on 1,25(OH)2D3-responsive Mg2+ entry. This showed that elevated extracellular calcium modulates 1,25(OH)2D-mediated responses through the CaSR. In summary, 1,25(OH)2D3 stimulated Mg2+ uptake in MDCT cells, and this is dependent on de novo protein synthesis. Elevation of extracellular Ca2+, acting via the CaSR, inhibited 1,25(OH)2D3-stimulated Mg2+ entry. These data indicate that 1,25(OH)2D3 has important effects on the control of magnesium entry in MDCT cells and these responses can be modulated by extracellular divalent cations.

1alpha ,25-dihydroxyvitamin D; calcium/magnesium-sensing receptor; adenosine 3',5'-cyclic monophosphate measurements; intracellular magnesium determinations; magnesium uptake; fluorescence


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THE HORMONALLY ACTIVE METABOLITE of vitamin D, 1,25-dihydroxyvitamin D [1,25(OH)2D3], has an important role in regulating mineral metabolism at the level of the intestine, bone, and kidney (1, 32, 51). It is clear that 1,25(OH)2D3 increases intestinal calcium and magnesium absorption and regulates bone formation, but the actions of 1,25(OH)2D3 on the kidney are not as well characterized (1, 31, 51). On balance, clinical and experimental observations support the notion that 1,25(OH)2D3 increases calcium and magnesium reabsorption within the kidney (1, 19, 25, 26, 33, 36, 38, 45). Nevertheless, some studies reported little, if any, affect on renal divalent cation conservation (9, 13, 30, 39, 40). This discrepancy may be due to associated vitamin D-induced changes in plasma calcium and magnesium concentrations that affect renal divalent cation handling. An extracellular Ca2+/Mg2+-sensing receptor (CaSR) that modulates calcium and magnesium reabsorption in the loop of Henle and distal convoluted tubule has been identified along the length of the kidney tubule (27, 43). The distal convoluted tubule plays an important role in renal magnesium conservation as it has final control of urinary magnesium excretion (43). We have shown that elevated extracellular Ca2+ or Mg2+ inhibits hormone-mediated Mg2+ uptake in a distal convoluted tubule cell line (3). Our evidence is that hypercalcemia and hypermagnesemia diminish distal reabsorption, in part, through CaSR inhibition of peptide hormone receptor-mediated responses (3). In the present study, we investigated whether 1,25(OH)2D3 stimulates Mg2+ uptake into distal convoluted tubule cells and whether elevated extracellular Ca2+ or Mg2+ may inhibit these responses. Our notion is that 1,25(OH)2D3 plays a role in magnesium conservation at both the intestinal and renal level and that the latter is modulated by the prevailing extracellular Ca2+ and Mg2+ concentrations through the CaSR. Accordingly, increases in 1,25(OH)2D3 may lead to enhanced magnesium conservation, no change, or increased urinary excretion, depending on the magnitude of changes in plasma calcium and magnesium concentration.

To date, there are no experiments reporting the direct effects of vitamin D metabolites on cellular magnesium transport. In the present studies, we used mouse distal convoluted tubule (MDCT) cells to investigate the actions of 1,25-(OH)2D3 on Mg2+ uptake rates. MDCT cells have been extensively used to study cellular mechanisms of calcium and magnesium absorption within the distal tubule (23, 43). Parathyroid hormone (PTH), glucagon, and arginine vasopressin (AVP) stimulate Mg2+ uptake in MDCT cells (16, 17). In a recent study, we showed that MDCT cells also possess a polyvalent cation-sensing mechanism that is responsive to extracellular Mg2+ and Ca2+ (4). We documented the expression of the parathyroid/kidney Ca2+-sensing receptor (i.e., CaSR) in MDCT cells and suggested that it is responsible, either entirely or in part, for the extracellular polyvalent cation-sensing mechanism in these cells (4). We also found that activation of this mechanism with either extracellular Ca2+, Mg2+, or the polyvalent cation neomycin inhibited PTH-, glucagon-, and AVP-stimulated cAMP release (4) as well as hormone-stimulated Mg2+ uptake in MDCT cells (3). These studies indicated that this cation-sensing mechanism in immortalized MDCT cells plays an important role in modulating hormone-mediated intracellular signals in response to changes in extracellular Mg2+ as well as Ca2+. Yang et al. (54) have reported that MDCT cells also express 1,25(OH)2D3- and PTH-responsive 25(OH)D 24-hydroxylase (54), and Friedman and Gesek (22) have shown that 1,25(OH)2D3 accelerates PTH-dependent Ca2+ uptake, suggesting that vitamin D receptors are present in this cell line and play a role in mineral metabolism (22). Accordingly, these cells provide a useful model by which to determine the effects of 1,25(OH)2D3 on Mg2+ handling in the distal convoluted tubule, which is difficult to study in situ or dissect out for functional measurements. The present studies demonstrate that 1,25(OH)2D3 stimulates Mg2+ uptake in MDCT cells, providing evidence that the metabolite might affect magnesium absorption in the intact distal tubule. Moreover, elevation of divalent cations, acting through the CaSR, modulates 1,25(OH)2D3-mediated Mg2+ entry in this cell line.


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Basal DMEM and Ham's F-12 media were from GIBCO Laboratories, Grand Island, NY. FCS was from Flow Laboratories (McLean, VA). Mag-fura 2-acetoxymethyl ester (AM) was obtained from Molecular Probes (Eugene, OR). 1,25(OH)2D3 and other materials were from Sigma (St. Louis, MO).

Cell preparation. Immortalized MDCT cells were kindly provided by Dr. P. A. Friedman. They were cultured in DMEM-Ham's F-12 media, 1:1, supplemented with 10% FCS, 1 mM glucose, 5 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin in a humidified environment of 5% CO2-95% air at 37°C. For cAMP determinations, the MDCT cells were cultured to confluence in 24-well plastic dishes. Sixteen hours before the cAMP measurements were made, the culture medium was changed to one containing 0.2% BSA rather than FCS. For the fluorescence studies, confluent cells were washed three times with PBS containing 5 mM EGTA, trypsinized, and seeded on glass coverslips. Aliquots of harvested cells were allowed to settle onto sterile glass coverslips in 100-mm Corning tissue culture dishes, and the cells were grown to confluence over 4-6 days in supplemented media as described above. The normal media contained 0.6 mM magnesium and 1.0 mM calcium. In the experiments indicated, the cells were cultured in nominally magnesium-free media (<0.01 mM) for 16 h before study (0.2% BSA replaced FCS during this period). Other constituents of the magnesium-free media were identical to those of the complete media.

cAMP measurements. cAMP was determined in confluent MDCT cell monolayers cultured in 24-well plates in DMEM-Ham's F-12 media without serum, as previously reported (4). After addition of various hormones, MDCT cells were incubated at 37°C for 5 min in the presence of 0.1 mM IBMX. cAMP was extracted with 5% trichloroacetic acid, which was removed with ether acidified with 0.1 N HCl. The aqueous phase was dried, dissolved in Tris-EDTA buffer, and then cAMP was measured with a radioimmunoassay kit (Diagnostic Products, Los Angeles, CA).

Cytoplasmic Mg2+ measurements. Coverslips were mounted within a perfusion chamber, and the attached subconfluent cells were incubated with 5 µM mag-fura 2-AM dissolved in pluronic acid F-127 (0.125%; Molecular Probes) for determination of intracellular Mg2+ concentration ([Mg2+]i) in media for 20 min at 37°C. Cells were subsequently washed three times with a buffered salt solution containing (in mM) 145 NaCl, 4.0 KCl, 0.8 Ka2HPO4, 0.2 KH2PO4, 1.0 CaCl2, 5.0 glucose, and 20 HEPES-Tris, at pH 7.4. The MDCT cells were incubated for an additional 20 min to allow for complete deesterification and washed once before measurement of fluorescence.

Epifluorescence microscopy was used to monitor changes in the mag-fura 2 fluorescence of the MDCT cell monolayer. The chamber was mounted on an inverted Nikon Diaphot-TMD microscope with a Fluor ×100 objective, and, under oil immersion, fluorescence within a single cell was monitored over the course of the study. Fluorescence was recorded at 1-s intervals by using a dual-excitation-wavelength spectrofluorometer (Delta-scan, Photon Technologies, Princeton, NJ) with excitation for mag-fura 2 at 335 and 385 nm (chopper speed set at 100 Hz) and emission at 505 nm. Media changes were made without interruption in recording.

The free [Mg2+]i was calculated from the ratio of the fluorescence at the two excitation wavelengths as previously described (15) by using a dissociation constant (Kd) of 1.4 mM for the mag-fura 2-Mg2+ complex. The minimum (Rmin) and maximum (Rmax) ratios were determined for the cells at the end of each experiment by using 20 µM digitonin. Rmax for mag-fura 2 was found by the addition of 50 mM MgCl2 in the absence of Ca2+, and Rmin was obtained by removal of Mg2+ and addition of 100 mM EDTA, pH 7.2. The excitation spectrum of the cellular mag-fura 2 under these conditions was similar to that of free mag-fura 2 in the same solutions.

Antibody block of the CaSR in MDCT cells. In those experiments where the CaSR was blocked with a specific antibody, MDCT cells were Mg2+ depleted for 16 h and then incubated with the antibody for 1.5-2 h at 36°C. A mouse monoclonal antibody (ADD) raised against a peptide comprising residues 214-236 of the CaSR was used (20). The antibody, which was provided by Drs. P. K. Goldsmith and A. M. Spiegel (National Institutes of Health) and K. V. Rogers (NPS Pharmaceuticals), has been extensively characterized with respect to specificity for the parathyroid/kidney CaSR (24). The antibody was diluted to a final concentration of 1.6 µg/ml in a buffer solution containing 0.02% BSA. The control cells were incubated with mouse IgG. The cells were washed twice in an appropriate buffer solution, and intracellular cAMP content or microfluorescence experiments were performed.

Antisense oligodeoxynucleotides designed to the CaSR in MDCT cells. A second approach was used to block extracellular Ca2+/Mg2+ sensing in MDCT cells. Oligonucleotide (ODN) antisense methodology, a strategy designed to bind specifically and efficiently to the complementary sequence of a targeted mRNA, has been extensively used in in vitro systems. Twenty-base pair phosphorothioate-derivatized sense and antisense CaSR ODNs were synthesized on an automated solid-phase synthesizer by using standard phosphoramide chemistry. The sequence of the sense ODN was 5'-GAGAAGGCAGAGCCATGGCATGG-3', and that of the antisense ODN was 5'-CCATGCCATGGCTCTGCCTTCTC-3'. The MDCT cells were incubated in buffer containing 0.1% BSA with the ODN, 1.0 nmol/ml, for 48-72 h before fluorescence measurements were performed. Cationic transfection agents, such as lipofectamine, were avoided because they interfere with Mg2+ transport, presumably because of membrane damage. Protein was extracted from the cells for Western blot analysis.

Western blots. Cells were lysed in triple detergent buffer [50 mM Tris · HCl (pH 8.0), 150 mM NaCl, 0.02% NaN3, 0.1% SDS, 1 mM EDTA, 100 µg/ml polymethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotonin, 0.1% NP-40, and 0.5% sodium deoxycholate] for 5 min at 0°C. The cell lysates were spun at 1,200 g for 2 min at 4°C, and the supernatants were stored at -80°C. Aliquots were electrophoresed through 8% SDS-polyacrylamide gels and blotted onto polyvinylidene difluoride membranes (Bio-Rad). Membranes were rinsed in TBST (10 mM Tris · HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20), blocked with 5% dried milk powder in TBST for 1-2 h, and incubated with ADD, the antibody against the CaSR (24). As a control, immunoblotting was carried out as described above with the antiserum preabsorbed for 1 h with the peptide (10 µg/ml) against which it was raised. Blots were also incubated with a beta -tublin monoclonal antibody (Cedarlane Laboratories, Hornby, ON).

Statistical analysis. Representative tracings of fluorescent intensity ratios are given, and all results are means ± SE where indicated. Significance was determined by Tukey's analysis of variance where indicated, and comparisons among groups of data were made by using Student's t-test. A probability of P < 0.05 was taken to be statistically significant.


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1,25(OH)2D3 stimulates Mg2+ entry in MDCT cells. MDCT cells were treated with 1,25(OH)2D3 for 16 h before experimentation. During this time they were incubated in media without magnesium to deplete them of intracellular Mg2+ (16). The cells were then placed in 1.5 mM MgCl2, and [Mg2+]i was monitored by fluorescence. Figure 1 shows a typical result of a fluorescence experiment of MDCT cells pretreated with 1,25(OH)2D3 compared with control cells. The rate of entry into control cells, measured over the initial 500 s, was 164 ± 5 nM/s, n = 6. Pretreatment with 1,25(OH)2D3 increased Mg2+ uptake by 59 ± 4% to 260 ± 15 nM/s, n = 5. Next, we determined that vitamin D-stimulated Mg2+ uptake required a minimum of 3 h for the largest response and was maintained for 24 h. We have previously shown that the channel blocker nitrendipine inhibits Mg2+ uptake into MDCT cells (36). Nitrendipine (50 µM) inhibited Mg2+ uptake to a similar extent in 1,25(OH)2D3-treated cells (25 ± 5 nM/s, n = 4) as in control cells (24 ± 5 nM/s, n = 5), indicating that the vitamin D metabolite 1,25(OH)2D3 stimulated Mg2+ entry and not intracellular Mg2+ release or inhibition of Mg2+ efflux. 1,25(OH)2D3 increased Mg2+ uptake in a concentration-dependent manner, with half-maximal responses (185 ± 3 nM/s) occurring at ~10-9 M (Fig. 2). This study indicates that 1,25(OH)2D3 stimulates Mg2+ entry into MDCT cells.


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Fig. 1.   1,25-Dihydroxyvitamin D [1,25(OH)2D3] stimulates Mg2+ uptake in Mg2+-depleted mouse distal convoluted tubule (MDCT) cells. MDCT cells were cultured in Mg2+-free media (<0.01 mM) for 16 h. Where indicated, 10-7 M 1,25(OH)2D3 was added to the buffer solution from a stock ethanol solution during the 16-h Mg2+-depletion period. Fluorescence studies were performed in buffer solutions in the absence of external magnesium, and, where indicated, MgCl2 (1.5 mM final concentration) was added to observe changes in intracellular Mg2+ concentration ([Mg2+]I). The buffer solutions contained (in mM) 145 NaCl, 4.0 KCl, 0.8 K2HPO4, 0.2 KH2PO4, 1.0 CaCl2, 5.0 glucose, and 10 HEPES-Tris, pH 7.4, with and without 1.5 mM MgCl2. Fluorescence was measured at 1 data point/s with 25-point signal averaging, and the tracing was smoothed according to methods previously described (17).



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Fig. 2.   Concentration dependence of 1,25(OH)2D3 stimulation of Mg2+ entry in MDCT cells. Values are means ± SE for 3-6 cells. MDCT cells were treated with the given 1,25(OH)2D3 concentrations for 16 h before fluorescence determinations. The rate of Mg2+ influx, as determined by d([Mg2+]i)/dt, was measured with the given 1,25(OH)2D3 concentrations by using fluorescence techniques described in the legend for Fig. 1. d([Mg2+]i)/dt values were determined over the first 500 s of fluorescence measurements. Significantly different from control values,*P < 0.05.

Steroid hormones may mediate cellular responses through nongenomic or genomic actions (10). Nongenomic effects of 1,25(OH)2D3 include rapid changes in phosphoinositide metabolism, increases in intracellular Ca2+ concentration, elevation of cAMP levels, and activation of protein kinase C (reviewed in Ref. 10). We have reported that many of these signaling pathways may influence hormone-mediated Mg2+ uptake in MDCT cells (16, 17). Accordingly, we tested the acute (<20 min) effects of vitamin D metabolites on the mean Mg2+ entry rate. The acute treatment of MDCT cells for 20 min with 1,25(OH)2D3 had no effect on Mg2+ uptake (178 ± 17 nM/s, n = 3), which suggested that MDCT cells do not affect transport by nongenomic mechanisms. Genomic mechanisms involve changes in gene expression, requiring some hours before de novo protein synthesis. We determined this by using the transcriptional and translational inhibitors actinomycin D and cycloheximide, respectively. Because of the toxicity of actinomycin D, we measured the effect of the inhibitor for a 3- to 4-h incubation period. 1,25(OH)2D3 was equally effective in stimulating Mg2+ entry after 3 h of incubation as it was at 16 h (Fig. 3A). The MDCT cells were initially Mg2+ depleted by incubating them in magnesium-free media for 16 h before the addition of 1,25(OH)2D3, with and without actinomycin (5 µg/ml). The 1,25(OH)2D3-treated Mg2+-depleted cells were incubated for a further 4-5 h; then, Mg2+ uptake was performed by microfluorescence. Actinomycin D did not alter basal Mg2+ uptake in cells that had been Mg2+ depleted for 16 h (18). In those cells treated with actinomycin D, 10-7 M 1,25(OH)2D3 failed to stimulate Mg2+ entry, indicating that the vitamin D metabolite increases uptake by mechanisms involving transcriptional processes (Fig. 3A). Next, we determined the effect of a translational inhibitor, cycloheximide. Cycloheximide (1.0 µg/ml) was added with 10-7 M 1,25(OH)2D3 for 16 h before fluorescence studies. 1,25(OH)2D3 did not stimulate Mg2+ uptake (155 ± 18 nM/s, n = 4) in those cells treated with cycloheximide, indicating that translational processes were also involved in this response (Fig. 3B). Accordingly, these results indicate that 1,25(OH)2D3 stimulates Mg2+ entry by genomic mechanisms involving de novo synthesis of transport proteins.


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Fig. 3.   1,25(OH)2D3-stimulated Mg2+ uptake requires transcriptional/translational processes. Values are means ± SE. A: MDCT cells were Mg2+ depleted for 16 h, and 10-7 M 1,25(OH)2D3 was then added with and without actinomycin D (5.0 µg/ml) as indicated for 4-5 h before fluorescence determinations. Mg2+ uptake was determined with microfluorescence according to methods described in the legend for Fig. 1. B: MDCT cells were treated with 10-7 M 1,25(OH)2D3 for 16 h before the determination of Mg2+ uptake. In those cells indicated, cycloheximide (Cyclo; 1.0 µg/ml) was added with 1,25(OH)2D3 16 h before Mg2+ uptake and cAMP determinations. Actinomycin D and cyloheximide were added from stock solutions of DMSO; the final DMSO concentration did exceed 0.01% (vol/vol), which had no effect on Mg2+ uptake. Significantly different from control values, *P < 0.01.

Acute effects of polyvalent cations on 1,25(OH)2D3-stimulated Mg2+ uptake. We have shown that elevation of extracellular polyvalent cations, neomycin, Mg2+, and Ca2+ inhibit PTH-, glucagon-, and AVP-mediated cAMP formation and glucagon- and AVP-stimulated Mg2+ entry but not basal uptake rates in MDCT cells (3, 4). MDCT cells were treated with 10-7 M 1,25(OH)2D3 for 16 h before the addition of polyvalent cations and determination of Mg2+ uptake. Acute (5-min) addition of 50 µM neomycin or elevation of 5.0 mM extracellular Mg2+ or 5.0 mM Ca2+did not affect Mg2+ uptake into 1,25(OH)2D3-treated, Mg2+-depleted cells (Fig. 4). Accordingly, the acute activation of the CaSR does not alter 1,25(OH)2D3-stimulated Mg2+ entry in the cells pretreated with the vitamin D metabolite 16 h before experimentation.


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Fig. 4.   Summary of the acute effects of cation-sensing receptor (CaSR) activation on 1,25 (OH)2D3-stimulated Mg2+ uptake. Values are means ± SE for 3-5 cells. Measurement d([Mg2+]i)/dt was performed in Mg2+-depleted cells treated with or without 1,25(OH)2D3 for 16 h before performance of fluorescence. Mg2+ uptake was determined with 1.5 mM extracellular Mg2+ in the absence or presence of 50 µM extracellular neomycin (Neo); 5.0 mM Mg2+ or 5.0 mM Ca2+ was added 5 min before determination of Mg2+ uptake. In the studies that tested high levels of extracellular Mg2+, 5.0 rather than 1.5 mM MgCl2 was used. The studies were performed as described in the legend for Fig. 1. The Mg2+ uptake rate was determined over 500 s after addition of 1.5 or 5.0 mM Mg2+. Significantly different from control values,*P < 0.05.

Chronic effects of elevated extracellular Ca2+ on 1,25(OH)2D3-stimulated Mg2+ uptake. Next, we determined the effects of chronic elevation of extracellular Ca2+ on 1,25(OH)2D3-stimulated Mg2+ uptake. A buffer solution containing high CaCl2 (5.0 mM) was added with 1,25(OH)2D3 for 16-20 h before fluorescence measurements were made. In support of our earlier findings, high extracellular Ca2+ for 16-20 h did not alter basal Mg2+ uptake (165 ± 7 nM/s, n = 4) (Fig. 5). However, high extracellular Ca2+ inhibited 1,25(OH)2D3-stimulated Mg2+ uptake (166 ± 6 nM/s, n = 4). Accordingly, the addition of high extracellular Ca2+ with 1,25(OH)2D3 diminished vitamin D-mediated Mg2+ entry. These findings suggest that signaling via the CaSR may alter 1,25(OH)2D3-induced synthesis of proteins involved in Mg2+ transport.


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Fig. 5.   Chronic activation of CaSR-sensing inhibits 1,25(OH)2D3-stimulated Mg2+ uptake. Values are means ± SE for 3-5 cells. MDCT cells were treated with 10-7 M 1,25(OH)2D3 with and without 5.0 mM CaCl2 for 16 h before microfluorescence measurements. Mg2+ uptake was determined with 1.5 mM extracellular Mg2+ in the absence or presence of 5.0 mM Ca2+ as indicated. We have previously shown that high extracellular Ca2+ does not affect basal Mg2+ uptake; i.e., the entry pathway is selective for Mg2+ (43). The studies were performed as described in the legend for Fig. 1. The Mg2+ uptake rate was determined over 500 s after addition of 1.5 mM MgCl2. Significantly different from control values,*P < 0.01.

CaSR antibody blocks extracellular Ca2+ inhibition of 1,25(OH)2D3-stimulated Mg2+ uptake. If elevation of extracellular Ca2+ inhibits 1,25(OH)2D3-stimulated transport through CaSR-mediated transcriptional processes, then blocking this receptor should abolish the chronic effects of extracellular calcium. To do this, MDCT cells were incubated with a specific CaSR antibody. To substantiate that the CaSR was blocked, we measured the effect of extracellular Ca2+ and Mg2+ on PTH-mediated cAMP formation in control cells and in those pretreated with the CaSR antibody. PTH stimulated intracellular cAMP accumulation by about threefold (72 ± 8 pmol · mg protein-1 · 5 min-1) above control values (25 ± 1 pmol · mg protein-1 · 5 min-1). The presence of 5.0 mM Ca2+ or 5.0 mM Mg2+ abolished PTH-stimulated cAMP formation (33 ± 2 and 29 ± 1 pmol · mg protein-1 · 5 min-1, respectively) (Fig. 6). Preincubation of MDCT cells with the antibody prevented the effect of extracellular Ca2+ (60 ± 5 pmol · mg protein-1 · 5 min-1) or Mg2+ (72 ± 8 pmol · mg protein-1 · 5 min-1) on PTH-sensitive cAMP release (72 ± 8 pmol · mg protein-1 · 5 min-1). Accordingly, the antibody blocked the activation of the CaSR and the inhibition of hormone-mediated cAMP formation. With this knowledge, we determined whether antibody blockade of the CaSR would inhibit the effect of elevated Ca2+ on 1,25(OH)2D3-stimulated Mg2+ uptake. We depleted MDCT cells of Mg2+ for 16 h before experimentation and incubated them with ADD antiserum to the CaSR for 2 h. The cells were then treated with 1,25(OH)2D3 with and without 5.0 mM CaCl2 for an additional 4-5 h before microfluorescence studies were performed. MDCT cells treated with the antibody responded normally to 1,25(OH)2D3 (210 ± 11 nM/s, n = 5) regardless of the presence of elevated extracellular Ca2+ (202 ± 8 nM/s, n = 4) (Fig. 7). This study indicates that extracellular Ca2+ chronically inhibits 1,25(OH)2D3-mediated Mg2+ uptake through the activation of CaSR-mediated mechanisms.


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Fig. 6.   CaSR antibody blocks extracellular polyvalent cation modulation of parathyroid hormone (PTH)-mediated cAMP formation. Values are means ± SE for 10 observations. In the studies indicated, MDCT cells were preincubated with 1.6 µg/ml of a mouse monoclonal antibody (ADD) or 1.6 µg/ml mouse IgG in a buffer solution containing 0.02% BSA and (in mM) 0.5 MgCl2, 1.0 CaCl2, 145 NaCl, 4.0 KCl, 0.8 K2HPO4, 0.2 KH2PO4, 5.0 glucose, and 20 HEPES-Tris, pH 7.4, for 2 h before measurement of intracellular PTH-stimulated cAMP. Where indicated, either 5.0 mM CaCl2 or 5.0 mM MgCl2 was added for 5 min before PTH and cAMP concentrations were measured with radioimmunoassay after a further 5-min incubation period. Significantly different from control values,*P < 0.01.



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Fig. 7.   Specific antibody to the CaSR blocks extracellular Ca2+ inhibition of 1,25(OH)2D3-stimulated Mg2+entry. Values are means ± SE for 3-5 cells. MDCT cells were Mg2+ depleted for 16 h and incubated with CaSR mouse monoclonal antibody ADD (1.6 µg/ml) for 2 h. The cells were then treated with 10-7 M 1,25(OH)2D3 with and without 5.0 mM CaCl2 where indicated, and Mg2+ uptake measurement was performed with microfluorescence. The Mg2+ uptake rate was determined over 500 s after addition of 1.5 mM MgCl2. Significantly different from control values,*P < 0.05.

Antisense ODN to the CaSR blocks extracellular Ca2+ inhibition of 1,25(OH)2D3-stimulated Mg2+ uptake. To substantiate the antibody studies, we used antisense ODN technology to block CaSR receptor expression and its function. The MDCT cells were loaded with sense or antisense CaSR ODNs. Cells with antisense ODN expressed little CaSR as determined by Western analysis (Fig. 8). In the representative experiment shown, the CaSR-to-beta -tublin densitometry ratio was 2.77 for no ODN; 2.6 for sense ODN; and 0.17, for antisense ODN, the last value representing 6.5% of the sense ODN ratio. Extracellular Ca2+ did not inhibit PTH-mediated cAMP formation in MDCT cells treated with antisense ODN to the CaSR (67 ± 1 pmol · mg protein-1 · 5 min-1), whereas the response in cells with sense ODN was similar to that observed with control cells (32 ± 2 pmol · mg protein-1 · 5 min-1) (Fig. 9). Accordingly, antisense ODN blocks expression and function of the CaSR. We used this model to test whether extracellular Ca2+ diminishes 1,25(OH)2D3-stimulated Mg2+ uptake through the CaSR. Mg2+ uptake in sense ODN-treated cells was 115 ± 2% (n = 5) of control cells (Fig. 10). 1,25(OH)2D3 stimulated Mg2+ entry by 189 ± 50% (n = 4) in the sense CaSR ODN-treated cells in the presence of normal extracellular Ca2+ concentration (1.0 mM). Elevated extracellular Ca2+ (5.0 mM) inhibited 1,25(OH)2D3-stimulated Mg2+ uptake (87 ± 27%, n = 6) in these cells. In contrast, 1,25(OH)2D3-stimulated Mg2+ uptake was 172 ± 31% (n = 6) in cells loaded with antisense ODN in the presence of normal Ca2+ and 181 ± 26% (n = 9) in cells incubated with 5.0 mM Ca2+. Accordingly, the antisense CaSR ODN inhibits CaSR expression and mitigates the effect of extracellular Ca2+ on 1,25(OH)2D3-stimulated Mg2+ uptake. These studies, using antibody or antisense ODN technology, clearly indicate that extracellular Ca2+ chronically inhibits 1,25(OH)2D3-mediated Mg2+ uptake through activation of CaSR-mediated mechanisms.


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Fig. 8.   Antisense oligodeoxynucleotides (ODN) to the CaSR diminish receptor expression in MDCT cells. Western analysis of the CaSR was performed in MDCT cells treated with sense or antisense ODNs at concentrations of 1 nmol/ml for 36-42 h before experimentation. Cell extracts (50 µg protein) were subjected to SDS-PAGE on a 4-12% gradient gel. The blot was stained with CaSR mouse monoclonal antibody, and an identical blot was stained with beta -tubulin antibody. Lane 1, control MDCT cells (no ODN); lane 2, sense ODN (CaSR sense); lane 3, antisense ODN (CaSR antisense). The CaSR bands were demonstrated to be specific after the blot was stained with the same antiserum preincubated with the peptide against which it was raised (data not shown). Western blots were performed twice with similar results.



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Fig. 9.   Antisense CaSR ODN diminish CaSR inhibition of PTH-mediated cAMP formation. Values are means ± SE for 6 individual observations. In the studies indicated, MDCT cells were loaded with sense or antisense ODN 72 h before measurement of intracellular PTH-stimulated cAMP. Where shown, 5.0 mM CaCl2 was added for 5 min before PTH and cAMP concentrations were measured with radioimmunoassay after a further 5-min incubation period. Significantly different from control values,*P < 0.01.



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Fig. 10.   Antisense ODN to the CaSR diminishes extracellular Ca2+ inhibition of 1,25(OH)2D3-stimulated Mg2+entry. Values are means ± SE for 4-8 cells. MDCT cells were loaded with sense (S) or antisense (AS) ODN designed to the CaSR for 72 h before fluorescence studies. The cells were Mg2+ depleted for 16 h, treated with 10-7 M 1,25(OH)2D3 for 3-4 h with 5.0 mM CaCl2. The Mg2+ uptake rate was determined over 500 s after addition of 1.5 mM MgCl2.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The role of 1,25(OH)2D3 in renal magnesium reabsorption. Little is known about the effects of 1,25(OH)2D3 on tubular magnesium transport. On balance, urinary clearance data from clinical observations and experimental studies indicate that 1,25(OH)2D3 may stimulate renal magnesium handling (1, 13, 25, 26, 30, 33, 36, 38, 39, 45). There have been no micropuncture studies performed to localize the actions of 1,25(OH)2D3 on magnesium transport. The data concerning the effects of 1,25(OH)2D3 on renal calcium absorption are more substantial but inconclusive (5, 7-9, 31, 49). The most convincing data demonstrating that 1,25(OH)2D3 may have a direct effect on calcium transport were obtained by using isolated cells. Bindels et al. (5) and Van Baal et al. (50) showed that 1,25(OH)2D3 increased calbindin D28k protein levels and stimulated transcellular calcium absorption in primary cultures of rabbit distal connecting tubule and cortical collecting duct cells. The maximal transport response occurred at ~48 h posttreatment, suggesting that the response involved initiation of transcriptional processes (5). The response to 1,25(OH)2D3 was independent of PTH and not additive to PTH-stimulated calcium transport. The above-mentioned authors also have biochemical evidence, as they showed that 1,25(OH)2D3 increased calbindin D28k mRNA and protein content without a change in Na+/Ca2+ exchange or Ca2+-ATPase RNA or protein in rabbit distal connecting tubule and cortical collecting duct cell preparations (50). Recently, Hoenderop et al. (29) have identified an apical Ca2+ channel in 1,25(OH2)D3-responsive cells that is responsible for calcium absorption in this segment. They have reported that 1,25(OH)2D3 stimulates Ca2+ transport in rabbit cortical collecting duct cells (29). The second study to clearly show effects of vitamin D metabolites on calcium transport was performed in MDCT cells by Friedman and Gesek (22). These co-workers reported that 1,25(OH)2D3 did not alter basal Ca2+ uptake but accelerated PTH-dependent calcium entry rates. This response was rapid, concentration dependent, significant at 2 h, maximal by 5 h, and mediated by translational processes because it was inhibited by cycloheximide (22). The reasons for the discrepancies between the above-mentioned two reports are not known; examples may be the different cell types or the techniques used to measure calcium transport in the two separate studies. Nevertheless, it is clear that 1,25(OH)2D3 increases calcium-binding protein in distal tubules, suggesting that it has significant actions on basal or hormone-mediated calcium transport. 1,25(OH)2D3 increases divalent cation-binding proteins, such as calbindin D9K or calbindin D28k, in distal tubule cells, including convoluted segment, connecting tubule, and collecting duct cells (34, 35, 46, 48). In a preliminary report, we have shown that 1,25(OH)2D3 stimulates calbindin D9K in the MDCT cell line (14) The calbindin D group is thought to be involved, either directly or indirectly, in epithelial calcium transport (6-9, 21, 31, 49, 53). Experimental evidence also supports the notion that these proteins bind magnesium, and some investigators have postulated that they are involved in epithelial magnesium transport (2, 12, 28).

The present studies with MDCT cells, a widely used model of the distal convoluted tubule, supports the notion that 1,25(OH)2D3 may affect renal magnesium conservation. The hormonally active vitamin D metabolite 1,25(OH)2D3 increases Mg2+ entry rates in MDCT cells in a concentration-dependent fashion (Fig. 2). The response involves de novo protein synthesis, as it was sensitive to actinomycin D and cycloheximide, inhibitors of transcription and translation, respectively (Fig. 3). The evidence supports the notion that 1,25(OH)2D3 stimulates the synthesis of proteins that are involved with Mg2+ entry, perhaps as yet uncharacterized Mg2+ channels (42). As the distal convoluted tubule is the terminal nephron segment providing final control of renal magnesium excretion, the influence of 1,25(OH)2D3 may play an important part in magnesium balance.

Effect of extracellular Mg2+/Ca2+ sensing on 1,25(OH)2D3-mediated Mg2+ uptake. The extracellular CaSR plays an important role in mineral balance by controlling, among other things, parathyroid gland function and renal calcium and magnesium reabsorption (11, 27, 42). The CaSR mediates a host of intracellular signaling pathways. The CaSR has been best characterized in parathyroid gland cells (11). In the parathyroid, CaSR regulates PTH secretion and activates multiple intracellular pathways involving activation of Galpha -coupled proteins and changes in gene expression among others (11). The ways that CaSR activation influences salt and water transport in the kidney are presently being addressed. Wang et al. (52) showed that the elevation of extracellular calcium inhibits NaCl transport in the thick ascending limb by release of cytochrome P-450 metabolites. We have demonstrated in immortalized MDCT cells that the CaSR initiates intracellular Ca2+ transients and inhibits PTH-, glucagon-, and AVP-mediated cAMP formation and magnesium transport, probably by activating Galpha i-proteins (3, 4, 17). Finally, Sands et al. (47) reported that extracellular calcium inhibits AVP-stimulated cAMP induction of water transport in the inner medullary collecting duct, again likely through Galpha i-protein mediation. Accordingly, the CaSR may modulate epithelial cell functions in many cell types by different intracellular signaling pathways.

Elevation of extracellular Ca2+ inhibits 1,25(OH)2D3-stimulated Mg2+ uptake in MDCT cells (Fig. 5). The argument for modulation by the CaSR of 1,25(OH)2D3-induced transcriptional/translational processes involved with Mg2+ transport is persuasive. First, we show that 1,25(OH)2D3-mediated Mg2+ uptake is responsive to actinomycin D and cycloheximide (Fig. 3). The acute, short-term treatment of MDCT cells with 1,25(OH)2D3 does not elicit changes in Mg2+ entry rates, so that a minimum of 3 h was required; most of the present studies were preformed after 16 h of 1,25(OH)2D3 treatment. Second, acute elevation of extracellular Ca2+ does not affect 1,25(OH)2D3-stimulated Mg2+ uptake (Fig. 4). Third, blockade of the CaSR with a specific antibody or prevention of CaSR expression with an antisense ODN abolished the action of high extracellular Ca2+ on 1,25(OH)2D3-responsive Mg2+ transport (Figs. 7 and 10). Taken together, these data indicate that 1,25(OH)2D3-stimulated Mg2+ entry into MDCT cells is modulated by extracellular polyvalent cations. Thus the presence and magnitude of any 1,25(OH)2D3 responses are dependent on the existing divalent cation concentration.

Role of 1,25(OH)2D3 in overall renal magnesium balance. 1,25(OH)2D3 increases intestinal calcium and magnesium absorption and renal conservation, leading to positive divalent cation balance. Elevation of serum calcium and magnesium concentrations, in turn, diminish renal absorption through the actions of the CaSR on electrolytes in the loop and distal convoluted tubule and water absorption in the medullary collecting ducts (27, 41, 42, 44, 47). Activation of the CaSR modulates the excessive absorption of divalent cations, providing a negative-feedback control to effect normal mineral balance. This may also explain the discrepant reports in the literature concerning the effects of vitamin D metabolites on renal magnesium transport.


    ACKNOWLEDGEMENTS

We thank Dr. Peter A. Friedman for providing the MDCT cell line and Drs. Kimberly V. Rogers (NPS Pharmaceuticals), Allen Spiegel, and Paul Goldsmith (Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases) for the ADD antibody.


    FOOTNOTES

H. S. Kang is a Postdoctoral Fellow of the Korean Science and Engineering Foundation, and L. Canaff is the recipient of a Doctoral Fellowship of the Medical Research Council of Canada. This work was supported by Medical Research Council of Canada Grants MT-5793 (to G. A. Quamme) and MT-9315 (to G. N. Hendy) and grants from the Kidney Foundation of Canada (to G. A. Quamme and G. N. Hendy).

Address for reprint requests and other correspondence: G. A. Quamme, Dept. of Medicine, Univ. Hospital, Koerner Pavilion, 2211 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3 (E-mail: quamme{at}interchange.ubc.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 14 August 2000; accepted in final form 13 December 2000.


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