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Endocrinology Vol. 144, No. 9 3885-3894
Copyright © 2003 by The Endocrine Society

Calcium Transporter 1 and Epithelial Calcium Channel Messenger Ribonucleic Acid Are Differentially Regulated by 1,25 Dihydroxyvitamin D3 in the Intestine and Kidney of Mice

Yurong Song, Xiaorong Peng, Angela Porta, Hitomi Takanaga, Ji-Bin Peng, Matthias A. Hediger, James C. Fleet and Sylvia Christakos

Interdepartmental Nutrition Program (Y.S., J.C.F.), Purdue University, West Lafayette, Indiana 47907; Renal Division and Membrane Biology Program (H.T., J.-B.P., M.A.H.), Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115; and Department of Biochemistry and Molecular Biology (X.P., A.P., S.C.), University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07103

Address all correspondence and requests for reprints to: Dr. Sylvia Christakos, Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, 185 South Orange Avenue, MSB Room E665, Newark, New Jersey 07103. E-mail: christak{at}umdnj.edu; or James C. Fleet, Ph.D., 700 West State Street, Purdue University, West Lafayette, Indiana 47907-2059. E-mail: fleetj{at}cfs.purdue.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We examined the expression of calcium transporter 1 (CaT1) and epithelial calcium channel (ECaC) mRNA in the duodenum and kidney of mice. Intestinal CaT1 mRNA level increased 30-fold at weaning, coincident with the induction of calbindin-D9k expression. In contrast, renal CaT1 and ECaC mRNA expression was equal until weaning when ECaC mRNA is induced and CaT1 mRNA levels fall 70%. Long- and short-term adaptation to changes in dietary calcium (Ca) level and 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] injection strongly regulated duodenal calbindin D9k and CaT1 mRNA. Following a single dose of 1,25(OH)2D3, induction of CaT1 mRNA occurred rapidly (within 3 h, peak at 6 h of 9.6 ± 0.8-fold) and preceded the induction of intestinal Ca absorption (significantly increased at 6 h, peak at 9 h). Neither renal CaT1 nor ECaC mRNA were strongly regulated by dietary calcium level or 1,25(OH)2D3 injection. Our data indicate that CaT1 and ECaC mRNA levels are differentially regulated by 1,25(OH)2D3 in kidney and intestine and that there may be a specialized role for CaT1 in kidney in fetal and neonatal development. The rapid induction of intestinal CaT1 mRNA expression by 1,25(OH)2D3, and the marked induction at weaning, suggest that CaT1 is critical for 1,25(OH)2D3-mediated intestinal Ca absorption.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ADAPTIVE CHANGES IN intestinal calcium absorption and renal calcium reabsorption are regulated by 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], presumably by vitamin D-mediated transcriptional activation through the classical vitamin D receptor (1). Although the mechanism for calcium movement across the epithelial cell in these two tissues is unclear, the facilitated diffusion model described by Bronner et al. (2) has been used to explain both processes. In this model, calcium enters the epithelial cell through a calcium channel at the apical membrane, its diffusion across the cells is facilitated by a calcium-binding protein [i.e. calbindin-D9k in intestine or calbindin-D28k in kidney (3)], and active extrusion of calcium across the basolateral membrane is mediated by a calcium-dependent ATPase (e.g. PMCA1b in the intestine) (4). Early studies indicated that calcium uptake into the enterocytes is a vitamin D-regulated step (5, 6, 7, 8, 9, 10). However, until recently the protein mediating this uptake had eluded scientists.

In the last several years, two epithelial calcium channels found in the apical membranes of renal and intestinal epithelial cells have been proposed as the mediators of calcium uptake during transcellular calcium transport, i.e. epithelial calcium channel (EcaC) (also termed CaT2, TRPV5) (11) and calcium transporter 1 (CaT1) (also termed ECaC2, TRPV6) (12). CaT1 is expressed in both intestine and kidney, and ECaC is present predominantly in the kidney (11, 12, 13). Their location and electrophysiological properties suggest that CaT1 and ECaC may play an important role in active transepithelial calcium transport in intestine and kidney (11, 14, 15). However, the vitamin D dependence of these calcium channels is somewhat controversial. Studies in mice with a mutant, nonfunctioning vitamin D receptor suggested that CaT1 in intestine and ECaC in kidney are not regulated by 1,25(OH)2D3 (16). Other studies using vitamin D receptor (VDR) knockout (KO) mice (17), 1,25(OH)2D3-treated 1{alpha} hydroxylase KO mice (18), and Caco-2 cells (19, 20) suggest that intestinal CaT1 expression is vitamin D dependent. However, the time course of induction of CaT1 expression by 1,25(OH)2D3 and whether there is a temporal relationship between induction of CaT1 expression and intestinal calcium absorption that would suggest a functional interrelationship are not known. Studies in the VDR KO indicate that in the kidney, the level of CaT1 and ECaC are not influenced by the absence of the VDR (17). However, other studies in rats and mice indicated that ECaC could be induced in the kidney by 1,25(OH)2D3 injection (21, 22). Although putative vitamin D-responsive elements (VDRE) have been identified by sequence homology in the promoter region of ECaC (16, 21), few studies have been done to address the mechanisms involved in the regulation of these calcium channels.

To obtain a better understanding of the regulation of CaT1 in intestine, and CaT1 and ECaC in kidney, their developmental expression and 1,25(OH)2D3 regulation under both acute and chronic conditions were examined and compared with calbindin expression. Our findings suggest differential regulation of the expression of the epithelial calcium channels in intestine and kidney and a specialized role for kidney CaT1 in late fetal and neonatal development. In addition, the induction of CaT1 mRNA in intestine by 1,25(OH)2D3 before the induction of calcium absorption and calbindin-D9k expression, as well as the marked induction of intestinal CaT1 expression at weaning, suggest that CaT1 is important for 1,25(OH)2D3-mediated intestinal calcium absorption.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
{gamma}[32P]Deoxy-ATP (300 Ci/mmol), nylon membranes, prestained protein molecular weight markers and electrochemiluminescent detection system were obtained from NEN Life Science Products (Boston, MA). RNAzol was purchased from Tel-Test (Friendswood, TX). Antiserum against purified rat calbindin-D28k was prepared as previously described (23). Antiserum against rat calbindin-D9k was obtained from Swant Swiss Antibodies (Bellinzona, Switzerland). Chemically synthesized 1,25(OH)2D3 was provided by Dr. M. Uskokovic of Hoffmann-La Roche (Nutley, NJ). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified below.

Animals
C57/BL6 mice (Taconic Farms, Germantown, NY) were exposed to a 12-h light/dark cycle. Food and water were given ad libitum. All of the animal experiments we conducted were approved by the Purdue Animal Care and Use Committee or the University of Medicine and Dentistry of New Jersey Animal Care and Use Committee.

Experimental design
Development study.
Timed pregnant, neonatal, and adult mice were fed a standard chow diet (Rodent Laboratory chow 5001, Ralston Purina Co., St. Louis, MO) ad libitum. The 18-d-old fetus and 1-, 3-, 6-, and 8-wk-old mice were killed, and intestine and kidney were harvested for preparation of RNA (n = 3 per group for wk 1, 3, and 6; n = 6 for 8 wk; for fetus, n = 3 pooled intestinal samples of four to five individual intestines and n = 1 pool of 16 fetal kidneys).

Dietary studies.
In the first experiment, 4-wk-old mice were fed either a high-calcium (1% Ca; Teklad diet 92309) or low-calcium (0.02% Ca; Teklad diet 86162) diet for 4 wk. Twenty-four hours before the end of the experiment, mice were housed in metabolic cages and urine was collected. Urine was examined as we have previously described (24). At the end of the experiment, blood was collected and serum was prepared for the analysis of 1,25(OH)2D3 levels and tissues were harvested and RNA isolated for duodenal CaT1, calbindin-D9k and renal CaT1, ECaC, calbindin-D9k, and calbindin-D28k RNA analysis (n = 6 per group for Northern blots; n = 3 per group for PCR and Western blot analysis).

In another study, 90-d-old mice were randomly assigned to one of three AIN-76A-based diets (25) containing a normal [0.5% Ca, 0.4% phosphorus (P)], low-calcium (0.02% calcium and 0.35% P), or high-calcium content diet (2.0% calcium, 20% lactose, 1.25% P) (Research Diets, New Brunswick, NJ) (n = 5 per group). All diets contained 0.055 ìg of vitamin D3 per gram. After 7 d on the experimental diets, tissues were harvested and RNA isolated as described previously (24) for analysis of duodenal and renal calbindin-D9k, CaT1, ECaC, and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA levels by real-time PCR (see below). A second set of mice (n = 6–11 per group) was raised under the same protocol and intestinal calcium absorption from a test dose (2 mmol/liter Ca) was determined using the in situ ligated loops procedure as previously described (24). Blood was collected into heparinized tubes by cardiac puncture and plasma was analyzed for 1,25(OH)2D3 levels as described below.

Vitamin D administration studies.
Sixty-day-old mice were fed a 0.8% strontium, 0.02% calcium, vitamin D-deficient diet (Teklad diet TD 00562) for 7 d to inhibit endogenous renal synthesis of 1,25(OH)2D3 (26). The serum calcium levels of the strontium-fed mice were less than 7 mg/100 ml. In a vitamin D repletion study, vitamin D-deficient mice were randomly separated into two groups and injected with either vehicle or calcitriol three times over the next 48 h [at 48, 24, and 6 h before termination; ip 100 ng/100 g body weight (BW) in 0.1ml of a 9:1 mix of propylene glycol and ethanol, n = 5–8 per group for Northern blot analysis, n = 3 for PCR and for Western blot analysis]. Tissues were harvested and RNA isolated for duodenal CaT1 and calbindin-D9k, as well as renal CaT1, ECaC, and calbindin-D9k and D28k mRNA analysis.

A time-course study was conducted whereby the vitamin D-deficient mice were injected with a single dose of 1,25(OH)2D3 (ip 200 ng/100 g BW), and killed at 1, 3, 6, 12, 24, and 48 h after the injection (n = 3 per group). Control animals received an ip injection of 0.1 ml of vehicle (9:1 mix of propylene glycol and ethanol) and were killed 48 h later. In a dose-response study, the vitamin D-deficient mice (n = 5 per group) were injected with one of five levels of 1,25(OH)2D3: 0, 25, 50, 100, or 200 ng/100 g BW). Mice were killed 6 h after the injection. Duodenal scrapings were harvested for isolation of total cellular RNA. Regulation of duodenal CaT1 and calbindin-D9k mRNA by 1,25(OH)2D3 was found to be dose dependent. Peak expression was observed at the dose of 50 ng/100 g BW. Thus, in a separate group of vitamin D-deficient mice, intestinal calcium absorption from a test dose was determined 0, 3, 6, 9, and 16 h after 1,25(OH)2D3 administration (ip 50 ng/100 g BW, n = 5–11 per group) using the in situ ligated loop technique (24).

Quantitative detection of gene expression by real-time PCR
Total RNA was isolated after disruption of the tissues using RNAzol per the manufacture’s directions (Tel-Test). The isolated RNA was reverse transcribed to cDNA as previously described (27).

To determine the number of CaT1, ECaC, calbindin-D9k, and GAPDH cDNA molecules in the reverse-transcribed samples, real-time PCR analysis was performed in the LightCycler system (Roche Molecular Biochemicals, Indianapolis, IN) using conditions and primers described previously (28) with the following modifications: annealing was conducted for 5 sec at 62 C (CaT1, ECaC), 60 C (GAPDH), or 70 C (calbindin-D9k) with a temperature transition rate of 8 C/sec; detection of SYBR green fluorescence was performed for 3 sec at 86 C (CaT1, ECaC), 85 C (GAPDH), or 84 C (calbindin-D9k). Forty cycles of amplification were used for CaT1 and ECaC and 30 cycles were used for GAPDH and calbindin-D9k; primer sequences used for PCR are as follows: CaT1 forward 5'-ATCGATGGCCCTGCGAACT-3', CaT1 reverse 5'-CAGAGTAGAGGCCATCTTGTTGCTG-3'; ECaC forward 5'-ATTGACGGACCTGCCAATTACAGAG-3', ECaC reverse 5'-GTGTTCAACCCGTAAGAACCAACGGTC-3'; calbindin-D9k forward 5'-ATGTGTGCTGAGAAGTCTCCTGCAGAAATG-3', calbindin-D9k reverse 5'-CATTGTGAGAGCTTTTTGAAGAAAGCTTCG-3'; GAPDH forward 5'-TCACCATCTTCCAGGAGCG-3', GAPDH reverse 5'-CTGCTTCACCACCTTCTTGA-3'.

Northern blot analysis
Total RNA was prepared with RNAzol RNA extraction solution per the manufacturer’s directions. RNA was analyzed via Northern blot for calbindin-D9k and calbindin-D28k gene expression as previously described (29, 30). Briefly, the RNA was fractionated on a 1.2% formaldehyde agarose gel and then transferred from the agarose gel to a nylon filter in 20x standard sodium citrate (SSC) (1x SSC = 150 mM sodium chloride and 15 mM sodium citrate, pH 7.0). Membranes were prehybridized at 42 C in Ultrahyb hybridization buffer (Ambion, Inc. Austin, TX) for 1 h. Hybridization was carried out for 16–18 h at 42 C in the same solution containing 3–5 x 106 cpm/ml labeled DNA probes specific for calbindin-D9k or calbindin D28k. The blots were washed twice for 5 min at room temperature in 2x SSC/0.1% sodium dodecyl sulfate, three times for 15 min at 42 C in 0.1x SSC/0.1% sodium dodecyl sulfate and then exposed to Biomax MR-1 film (NEN Life Science Products) at -80 C. The autoradiograms were analyzed by densitometry. All filters were stripped and rehybridized to 32P ß-actin cDNA. The relative OD obtained using the test probes was divided by the relative OD obtained after probing with ß-actin to normalize for sample variation.

Preparation of [32P] cDNA probes
A 1.2-kb mouse calbindin-D28k cDNA and a 170-base mouse calbindin-D9k cDNA were obtained by restriction endonuclease digestion of the respective plasmid preparations as previously described (30). The ß-actin cDNA was purchased from CLONETECH Laboratories Inc. (Palo Alto, CA) and the 18S ribosomal RNA probe was a gift from Ramareddy Guntaka (University of Missouri). 32P-Labeled cDNA probes were prepared using the random primer DNA labeling system (Boehringer Mannheim Biochemicals, Indianapolis, IN) according to the manufacturer’s instructions.

Western blot analysis
For Western blot analysis, postmitochondrial supernatants were prepared and analyzed for protein concentration by the Bradford method (31). Ten micrograms of protein were analyzed for calbindin-D9k or calbindin-D28k protein using a chemiluminescence Western blotting kit (NEN Life Science Products). Western blots were also analyzed for {alpha}-tubulin antibody (Sigma). The {alpha}-tubulin was used to detect any problems with transfer or differences in sample loading. No variations in {alpha}-tubulin were noted in the samples (data not shown).

Plasma analysis
Plasma 1,25(OH)2D3 levels were determined by a 125I-1,25(OH)2D3 RIA kit (Nichols Institute Diagnostics, San Juan Capistrano, CA) per the manufacturer’s directions.

Serum calcium
Serum calcium concentrations were measured using a standard calcium chloride solution (Fisher Scientific, Fair Lawn, NJ) by atomic absorption spectrophotometry (32).

Statistical analysis
Data were analyzed using the SAS statistical program by the general linear models procedure (version 8.0, SAS Institute, Cary, NC). When the plots of predicted values vs. residuals demonstrated that the data were not normally distributed, log transformation was conducted before statistical analysis. Comparisons of multiple group means were done using Fisher’s protected least significant differences. Differences between group means with P < 0.05 were considered statistically significant. Data are expressed as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Developmental changes in CaT1, ECaC, and calbindin gene expression
Calbindin-D9k mRNA and protein levels were present in the 18-d fetal intestine similar to what has previously been shown by others (33). However, CaT1 mRNA levels were not expressed significantly in intestine until after birth. From 1–8 wk of age, duodenal CaT1 and calbindin-D9k mRNA were coordinately regulated (Fig. 1Go, A and B). Low levels of both mRNAs were detected at 1 wk postpartum and increased sharply and peaked at weaning (3 wk of age; 32-fold induction from 1–3 wk of age for CaT1, 59-fold higher for calbindin-D9k mRNA). This transition is the point at which active intestinal calcium absorption and intestinal responsiveness to 1,25(OH)2D3 appears (34, 35). By 6 wk of age, duodenal CaT1 and calbindin-D9k mRNA levels fell by 87 and 71%, respectively. In the duodenum, calbindin-D9k protein levels followed a similar pattern as calbindin-D9k mRNA levels [Fig. 1BGo and C; r2 = 0.85; calbindin-D9k protein = 0.79(calbindin-D9k mRNA) + 25.1]. In the kidney, detectable levels of CaT1, ECaC, calbindin-D9k, and calbindin-D28k mRNA were observed in the fetus (Fig. 2Go, A and B). ECaC, calbindin-D9k, and calbindin-D28k mRNA reached the peak level at 3 wk and then declined afterward. CaT1 was maximal at 1 wk of age and equivalent to ECaC expression but decreased to less than 10% ECaC mRNA levels at 3 wk of age and remained low afterward. Renal calbindin-D9k and calbindin-D28k protein levels were induced at 3 wk of age and remained elevated (Fig. 2CGo; relative to 3-wk level calbindin-D9k: fetal = 39; 1 wk = 38 ± 5; 3 wk = 100 ± 10; 6 wk = 93 ± 12; 8 wk = 93 ± 17; calbindin-D28k: fetal = 46; 1 wk = 61 ± 12; 3 wk = 100 ± 10; 6 wk = 96 ± 6; 8 wk = 91 ± 10).



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  		FIG. 1. Developmental changes in the expression of CaT1 and calbindin-D9k in mouse intestine. Mice were raised under standard conditions and killed at 18 d of gestation or 1, 3, 6, or 8 wk after parturition. Intestine was harvested and prepared for the analysis of specific messages or proteins. A, Real-time PCR analysis of CaT1 mRNA. B, Summary of densitometric scans of Northern blot analysis of calbindin-D9k mRNA (after correction for ß-actin expression, gray bar) and Western blot analysis of calbindin-D9k protein (per 20 mg total protein, white bar). Data are expressed relative to the levels seen in 3-wk-old mice (3 wk = 100%). C, Representative Northern blots for calbindin-D9k and ß-actin mRNA and a Western blot for calbindin-D9k protein. Bars in A and B represent the mean ± SEM (n = 3 per group for wk 1 and 3, n = 6 for 8 wk; for fetus, n = 3 pooled samples of four to five individual intestines). **, Significantly different from all other values at P < 0.01; *, significantly different from all other values at P < 0.05; #, significantly different from fetal, 1 wk, and 3 wk at P < 0.01.

 


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  		FIG. 2. Developmental changes in the expression of CaT1 and calbindin-D9k in mouse kidney. Mice were raised under standard conditions and killed at 18 d of gestation or 1, 3, 6, or 8 wk after parturition. Kidneys were harvested and prepared for the analysis of specific messages or proteins. A, Real-time PCR analysis of CaT1 (black bar) and ECaC (white bar) mRNA. B, Summary of densitometric scans of Northern blot analysis of calbindin-D9k (gray bar) and calbindin-D28k (striped bar) mRNA. Data were corrected for ß-actin expression and are presented relative to the levels seen in 3-wk-old mice (3 wk = 100%). C, Representative Northern blots for calbindin-D9k, calbindin-D28k, and ß-actin mRNA and Western blots for calbindin D9k and calbindin-D28k protein. Bars in A and B represent the mean ± SEM (n = 3 per group for wk 1 and 3, n = 6 for 8 wk; for fetus, n = 1 pool of 16 fetal kidneys). **, Significantly different from all other values at P < 0.01; *, significantly different from all other values at P < 0.05; +, significantly different from fetal and 3 wk at P < 0.05; #, significantly different from fetal, 1 wk, and 3 wk at P < 0.05.

 
Regulation of CaT1, ECaC, calbindin-D9k, and calbindin-D28k gene expression by diet
Continuous feeding of a low-calcium diet from 4–8 wk of age increased serum 1,25(OH)2D3 from 180 ± 35 pmol/liter on the 1% calcium diet to 760 ± 90 pmol/liter on the 0.02% calcium diet. This increase in circulating 1,25(OH)2D3 increased duodenal CaT1 mRNA by 30-fold relative to the level seen in duodenum of mice fed a 1% calcium diet, and an 8.2-fold increase in calbindin-D9k mRNA was observed (Fig. 3Go). Renal CaT1 and ECaC mRNA levels were only marginally higher on the low-calcium diet (by 50 and 65%, respectively, Fig. 3AGo) and reflected the 100% increase in calbindin-D9k mRNA (Fig. 3BGo). No change was observed in calbindin-D28k mRNA. Calbindin protein levels in intestine and kidney reflected the pattern observed in the levels of mRNA (Fig. 3CGo; relative to level in high-calcium-fed group, intestine: calbindin-D9k; high calcium = 1.0 ± 0.1, low calcium = 4.4 ± 0.1; kidney: calbindin-D9k; high calcium = 1.0 ± 0.1, low calcium = 1.8 ± 0.3; calbindin-D28k; high calcium = 1.0 ± 0.1, low calcium = 1.1 ± 0.1). Urinary calcium to creatinine ratio was reduced by 50% on the low calcium diet, reflecting up-regulation of calcium reabsorption mechanisms (calcium/creatinine: high calcium = 0.13 ± 0.3, low calcium = 0.07 ± 0.02).



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  		FIG. 3. The effect of feeding a low-calcium or high-calcium diet from weaning on the expression of specific proteins and mRNA levels in 60-d-old mice. A, Real-time PCR analysis of CaT1 and ECaC mRNA in intestine (left panel) and kidney (right panel) (n = 3 per group). B, Summary of densitometric scans of Northern blot analysis for calbindin-D9k and calbindin-D28k mRNA in intestine (left panel) and kidney (right panel). Data were corrected for ß-actin expression and are presented relative to the mice fed the high-calcium diet (high calcium = 1; n = 6 per group). C, Representative Northern blots for intestine calbindin-D9k and ß-actin (top left) and kidney calbindin-D9k, calbindin-D28k, and ß-actin (bottom left) and Western blots of renal and intestinal calbindin-D9k and renal calbindin-D28k protein (right). Bars, Mean ± SEM. *, Significantly different from the high-calcium diet at P < 0.05; **, significantly different from the high-calcium diet at P < 0.001.

 
In the short-term feeding study, relative to the values obtained from mice fed a diet with 0.5% calcium, consumption of a 2% calcium diet for 7 d significantly decreased plasma 1,25(OH)2D3 levels by 82% (from 350 ± 62 pmol/liter to 62 ± 10 pmol/liter), fractional calcium absorption by 100% (from 16.5 ± 1.8% to 0 ± 0.4% per 10 min), duodenal CaT1 mRNA by 86%, and duodenal calbindin-D9k mRNA by 52%. Consumption of the low-calcium diet significantly increased plasma 1,25(OH)2D3 levels by 98% (to 691 ± 80 pmol/liter), duodenal calcium absorption (to 66.7 ± 4.4%), CaT1 by 83%, and calbindin-D9k mRNA by 124% relative to the normal diet (Fig. 4Go). The dietary-induced changes in all the parameters measured were significantly correlated with diet-induced changes in plasma 1,25(OH)2D3 levels (e.g. intestinal calcium absorption, r2 =0.78, P < 0.001; CaT1 mRNA, r2 = 0.7, P < 0.001; calbindin-D9k mRNA, r2 = 0.62, P < 0.005). In addition, duodenal CaT1 mRNA and calbindin-D9k mRNA levels were correlated to one another (r2 = 0.74, P < 0.001). Renal CaT1 and ECaC mRNA levels did not change significantly with short-term dietary manipulation (data not shown), but calbindin-D9k mRNA levels were 54% lower on the high-calcium diet and 46% higher on the low-calcium diet, compared with the 0.5% calcium diet.



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  		FIG. 4. The effect of short-term dietary manipulation on duodenal CaT1 mRNA, calbindin-D9k mRNA, serum 1,25(OH)2D3, and calcium absorption in 90-d-old mice. RNA levels were determined by real-time PCR and corrected for GAPDH expression levels. The results for each dietary intervention were expressed relative to the level seen in mice fed the 0.5% calcium diet. Bars, Mean ± SEM for n = 5–6 samples per group. *, Significantly different from the high-calcium diet at P < 0.05.

 
Regulation of gene expression and calcium absorption by 1,25(OH)2D3
Duodenal and renal CaT1 mRNA expression was very low in vitamin D-deficient mice, and vitamin D repletion (three injections of 1,25(OH)2D3, 100 ng/100 g BW) significantly increased duodenal CaT1 mRNA levels by 21.5-fold and calbindin-D9k mRNA levels by 10-fold (Fig. 5Go, A and B). Calbindin-D9k protein was induced 11 ± 0.6-fold (Fig. 5CGo). In kidney, CaT1 and ECaC mRNA expression increased by 5.4 ± 0.6-fold and 4.7 ± 0.3-fold, respectively (Fig. 5AGo). Renal calbindin-D9k and D28k mRNA increased 9.7 ± 1.8- and 2.4 ± 0.2-fold, respectively, after 1,25(OH)2D3 injection, and renal calbindin-D9k and D28k protein levels increased 4.9 ± 1.0- and 1.6 ± 0.4-fold, respectively (Fig. 5Go, B and C).



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  		FIG. 5. The effect of repeated administration of 1,25(OH)2D3 on duodenal and renal mRNA levels. Sixty-day-old mice were made vitamin D deplete by feeding a 0.8% strontium diet for 7 d. Mice were then injected with 1,25(OH)2D3 (+D) or vehicle (-D) three times over 48 h (at 48, 24, and 6 h before termination; ip 100 ng/100 g BW per injection). A, Real-time PCR analysis of CaT1 and ECaC mRNA in intestine (left panel) and kidney (right panel) (n = 3 per group). B, Summary of densitometric scans of Northern blot analysis of calbindin-D9k and calbindin-D28k mRNA in intestine (left panel) and kidney (right panel) (n = 5–8 per group). Data were corrected for ß-actin or 18S rRNA expression and are presented relative to the vitamin D-depleted mice (-D = 1.0). C, Representative Northern blots for intestine calbindin-D9k and 18S rRNA (top left) and kidney calbindin-D9k, calbindin-D28k, and ß-actin (bottom left) and Western blots of renal (Kd) and intestinal (Dd) calbindin-D9k and renal calbindin-D28k protein (right). Bars, Mean ± SEM. *, Significantly different from the nontreated group at P < 0.05; **, significantly different from the nontreated at P < 0.001.

 
A single injection of 1,25(OH)2D3 caused tissue- and gene-specific differences in mRNA induction (Fig. 6Go). Duodenal CaT1 mRNA levels were significantly increased at 3 h (P = 0.002) and peaked at 6 h after injection (9.6-fold increase, P < 0.001) (Fig. 6AGo). At 24 h after administration of 1,25(OH)2D3, CaT1 mRNA levels dropped back to the levels seen at 0 h. Vitamin D-induced changes in intestinal calcium absorption efficiency reflected the changes in duodenal CaT1 mRNA; absorption was 20.8 ± 2.8% at 0 h, increased significantly by 6 h (P < 0.05), and peaked at 9 h post injection (48.0 ± 6.6%, P < 0.01, compared with 0 h), and started to drop 16 h after 1,25(OH)2D3 injection (38.6 ± 3.3%) (Fig. 6AGo). Duodenal calbindin-D9k mRNA levels (Fig. 6AGo) gradually increased and peaked at 24 h after one dose of 1,25(OH)2D3 (3.9-fold increase, P < 0.001). Similar changes were also observed for calbindin-D9k protein levels (Fig. 6BGo; relative to 0 h = 1.0 ± 0.2, 1 h = 0.91 ± 0.13, 3 h = 1.18 ± 0.16, 6 h = 1.32 ± 0.18, 12 h = 1.59 ± 0.22, 24 h = 1.82 ± 0.18, 48 h = 1.59 ± 0.29). Serum calcium levels were initially low (6.3 ± 0.6 mg/dl at 0 h) and increased only after the induction of vitamin D-mediated CaT1 gene expression and intestinal calcium absorption (8.6 ± 0.4 mg/dl at 12 h, P < 0.05).



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  		FIG. 6. Effect of a single injection with 1,25(OH)2D3 on gene expression and calcium absorption. A, Intestinal CaT1 mRNA (real-time PCR analysis, 0–16 scale), calbindin-D9k mRNA (percent maximal response at 24 h from Northern blot analysis, 0–160 scale) and fractional calcium absorption from ligated loops of intestine. B, A representative Western blot for calbindin-D9k protein. C, Real-time PCR analysis of renal CaT1 mRNA. D, Renal ECaC mRNA (real-time PCR analysis, 0–12 scale) and calbindin-D9k and calbindin-D28k mRNA (percent maximal response at 48 h from Northern blot analysis, 0–120 scale). Points represent the mean + SEM of n = 3 per time point for mRNA and n = 5–11 per time point for duodenal calcium absorption.

 
In kidney, ECaC and CaT1 mRNA levels were significantly increased only at 12 h after 1,25(OH)2D3 injection (2.5- and 2.2-fold increase, respectively, Fig. 6Go, C and D, P < 0.01). At 24–48 h after injection, renal ECaC and CaT1 mRNA levels dropped back to the levels seen at 0 h. The levels of renal calbindin-D9k and D28k mRNA gradually increased to a maximum at 48 h (4.4-fold, P < 0.001) and 24 h (3-fold, P < 0.001), respectively.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Of the three steps crucial to transcellular renal or intestinal calcium transport (uptake, intracellular movement, extrusion), apical uptake into cells has received the least attention because of the lack of information on the identity of the apical membrane calcium channel. With the discovery of the calcium channels CaT1 and ECaC (11, 12), interest in apical membrane calcium uptake in intestine and kidney has been reinvigorated. However, careful experiments on the regulation of the channels by 1,25(OH)2D3 that also link the expression CaT1 or ECaC to the physiologic process of calcium absorption or excretion have been lacking.

Although earlier studies found that administration of 1,25(OH)2D3 to normal rats with adequate vitamin D status did not increase intestinal CaT1 mRNA levels (12), in mice we found that duodenal CaT1 mRNA level is very responsive to changes in serum 1,25(OH)2D3 caused by diet and is rapidly and robustly induced by 1,25(OH)2D3 injection. We also show that duodenal CaT1 and calbindin-D9k mRNA levels are coordinately up-regulated in response to long- and short-term changes in dietary calcium (Figs. 3Go and 4Go) and that the mRNA changes associated with short-term adaptation to diet are linked to changes in duodenal calcium absorption. Coordinate up-regulation of CaT1 and calbindin-D9k mRNA was also observed at weaning (Fig. 1Go), at the onset of active intestinal calcium transport and intestinal responsiveness to 1,25(OH)2D3 (34, 35). Finally, we found that the rapid induction of duodenal CaT1 mRNA following a single injection of 1,25(OH)2D3 precedes the up-regulation of calcium absorption (Fig. 6AGo). The up-regulation of duodenal calcium absorption preceded the maximal, 1,25(OH)2D3-induced accumulation of either calbindin-D9k mRNA or protein, similar to data previously shown by Spencer et al. (36). However, even though circulating levels of 1,25(OH)2D3 were reduced in the strontium diet-fed mice, calbindin-D9k protein was present in the duodenum before vitamin D injection (see Fig. 6BGo). As such, our data do not eliminate a possible role for calbindin-D9k in transcellular calcium transport. Rather, our data suggest that up-regulation of CaT1 production may be necessary for stimulation of intestinal calcium absorption by 1,25(OH)2 D.

Our data are consistent with findings suggesting regulation of intestinal CaT1 mRNA by 1,25(OH)2D3 using VDR KO mice (17, 24), Na/Pi cotransporter KO mice that have elevated serum 1,25(OH)2D3 levels (37), 1,25(OH)2D3-treated 1{alpha}-hydroxylase KO mice (18), and with findings in 1,25(OH)2D3-treated Caco-2 cells (19, 20). An essential role for CaT1 in transcellular calcium absorption is consistent with the kinetic analysis of the rate of calcium uptake (38) and with mathematical modeling of intestinal calcium absorption conducted by Slepchenko and Bronner (39). However, although our studies suggest that changes in circulating 1,25(OH)2D3 levels should have a dramatic impact on apical membrane calcium uptake, earlier studies showed that vitamin D repletion of vitamin D-deficient animals has only a moderate influence on calcium uptake into brush border membrane vesicles (30–40% increase) (6, 7, 9). In addition, rapid import of calcium across the apical membrane of enterocytes occurs even in vitamin D-deficient chick intestine (10, 40). These data suggest a less prominent role for vitamin D-mediated CaT1 induction in vitamin D-regulated intestinal calcium absorption. Further work on the effect of 1,25(OH)2D3 treatment on duodenal CaT1 protein and activity levels is needed to clarify the importance of vitamin D-mediated CaT1 induction in intestinal calcium absorption.

Previous studies suggested that levels of ECaC or CaT1 in kidney were not influenced by the absence of the VDR or changes in dietary calcium (17, 24). However, other studies have indicated that renal ECaC mRNA was decreased in 1{alpha}-hydroxylase KO mice (22) and that it can be induced by successive 1,25(OH)2D3 injections (21, 22). Our studies show that induction of renal ECaC and CaT1 mRNA by 1,25(OH)2D3 occurs in the kidney and that these changes are coincident with increased calbindin-D9k and calbindin-D28k expression. In addition, extended feeding of a low-calcium diet increased renal expression of these mRNAs and is accompanied by reduced urinary calcium to creatinine ratios, presumably because of increased reabsorption capacity of the renal tubules. However, this simple approach of measuring urinary calcium output only broadly measures the capacity of the kidney cells to reabsorb calcium. Unlike the in situ analysis of calcium absorption from a test dose, in which we control the level of calcium that bathes the enterocytes, we have less control over the filtered load through the kidney. More sophisticated functional analysis of calcium transport in the CaT1- and EcaC-containing renal tubules will be necessary to cement relationships between the level of these proteins and renal calcium transport.

In the kidney, CaT1 induction is less sensitive to vitamin D treatment than in the duodenum. For example, continuous feeding of a low-calcium diet from weaning significantly induced both renal ECaC mRNA (1.6-fold) and CaT1 mRNA (1.5-fold). However, CaT1 mRNA was induced 30-fold in the intestine of these animals. This finding, as well as the insensitivity of the renal calcium channel mRNA levels to short-term dietary manipulation and 1,25(OH)2D3 injection, and the different time course of 1,25(OH)2D3 responsiveness in intestine and kidney indicate that distinct mechanisms control the accumulation of CaT1 and ECaC mRNA in two traditional vitamin D target tissues. This tissue-specific regulation is not seen for other 1,25(OH)2D3-induced responses, e.g. induction of 24-hydroxylase gene expression (which is involved in 1,25(OH)2D3 catabolism) follows a similar pattern in both kidney and intestine (41, 42). This difference may be due to the different functions of the two 1,25(OH)2D3 target proteins. Possible mechanisms involved in the tissue-specific responses could include tissue-specific methylation patterns in the CaT1 and ECaC promoters that limit renal induction or the existence of repressor elements whose binding factors are expressed only in kidney. Additional work will be necessary to determine whether these or other mechanisms account for tissue-specific differences in vitamin D regulation of the epithelial calcium channels.

To date, putative vitamin D response elements have been identified in the human (21, 43) and mouse (16) ECaC promoter. However, no definitive vitamin D response element has been identified in the CaT1 promoter. We (24) and others (17) have shown that although duodenal CaT1 mRNA levels in VDR knockout mice are only 5% of the level seen in wild-type mouse duodenum, long-term feeding of a 2% calcium-rescue diet suppresses duodenal CaT1 mRNA levels to a similar extent in both VDR KO (82%) and wild-type (74%) mice (24). These data suggest that dietary calcium mediated duodenal CaT1 regulation can occur by a mechanism that is independent of 1,25(OH)2D3 and VDR binding to a traditional VDRE. Further work is clearly needed to understand the mechanism by which 1,25(OH)2D3 regulates duodenal CaT1 gene expression.

In the kidney, as well as the intestine, the induction of epithelial calcium channel mRNA preceded the induction of the calbindins. The exact role of the calbindins in regulated transepithelial calcium movement in intestine and kidney and their relationship to the epithelial calcium channels are not known. In the kidney, the cellular distribution of ECaC is similar to the calbindins; both are localized in the distal tubules (21, 44). Kinetic data have suggested that calbindin-D28k can stimulate calcium transport from the apical membrane (45, 46) and that calbindin-D28k has can act as a cytosolic calcium buffer (47). Although both CaT1 and ECaC effectively increase calcium entry into cells, increases in intracellular calcium can inhibit CaT1 and ECaC activity (48, 49). Inhibition of the ECaC mediated rise in intracellular calcium with chelators can block calcium-mediated inactivation of calcium-induced currents (11). Thus, it is possible that calbindin-D28k, by acting as a calcium buffer, could change the kinetics of decay of the ECaC-induced calcium flux. Similarly, calbindin-D9k may act as a buffer against increases in intracellular calcium resulting from increased CaT1 level and activity, may prolong CaT1 activity, and may thereby increase the efficiency of intestinal calcium absorption. These hypotheses remain to be tested formally.

Although the highest levels of CaT1 mRNA are seen in the duodenum, it is also expressed in the adult mouse kidney, albeit at levels 90% lower than ECaC mRNA. In the fetal and neonatal mouse kidney CaT1 mRNA levels are equivalent to those of ECaC. ECaC and CaT1 are structurally (about 75% homologous at the amino acid level) (50) and functionally similar. The essential difference is that there is faster calcium-dependent inactivation for CaT1 (48, 49). A rapid inactivation may be more critical for calcium absorption in the intestine (in which CaT1 expression is highest), compared with reabsorption in the kidney (in which ECaC is predominantly expressed in adults). However in the neonate, during the period of nephron differentiation, both fast and slow inactivation kinetics may be needed. It will be of interest in future studies to determine whether CaT1 is also expressed during the development of the rat and human kidney.

In summary, our data suggest that 1,25(OH)2D3-induced duodenal CaT1 expression is necessary before induction of intestinal calcium absorption. In addition, differences in the regulatory pattern for 1,25(OH)2D3-induced CaT1 expression in duodenum and kidney and the lack of recognizable VDREs in the promoter region of this gene suggest CaT1 gene expression is controlled by a novel 1,25(OH)2D3-mediated mechanism.


   Acknowledgments
 
The authors thank Candace Langdoc and Zhengtao Zhang for their excellent technical assistance. We also thank Dr. Teh-Li Ho of the University of Arizona Health Sciences Center (Tucson, AZ) for conducting the urinary calcium and serum 1,25(OH)2D3 analysis on samples collected in the 4-wk-long, high/low dietary intervention study.


   Footnotes
 
This work was supported by funds from the National Institutes of Health NIDDK awards DK-54111 (to J.C.F.) and DK-38961 (to S.C.).

Y.S. and X.P. contributed equally to the generation of this manuscript.

Abbreviations: BW, Body weight; CaT1, calcium transporter 1; 1,25(OH)2D3, 1,25 dihydroxyvitamin D3; ECaC, epithelial calcium channel; GAPDH, glyceraldehydes phosphate dehydrogenase; KO, knockout; P, phosphorus; SSC, standard sodium citrate; VDR, vitamin D receptor; VDRE, vitamin D-responsive element.

Received March 11, 2003.

Accepted for publication May 29, 2003.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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