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Modulation of renal Ca²⁺ transport protein genes by dietary Ca²⁺ and 1,25-dihydroxyvitamin D₃ in 25-hydroxyvitamin D₃-1-hydroxylase knockout mice

Department of Cell Physiology, University Medical Centre Nijmegen, 6500 HB Nijmegen, Netherlands; and
^* Genetics Unit, Shriners Hospital for Children, Montreal, Quebec, Canada

¹Correspondence: 160 Cell Physiology, Nijmegen Centre for Molecular Life Sciences, University Medical Centre Nijmegen, P.O. Box 9101, NL-6500 HB Nijmegen, Netherlands. Express mail: M850 Room 07.048, Centrale Ontvangst Goederen, Geert Grooteplein Zuid 30, NL-6525 GA Nijmegen, Netherlands. E-mail: r.bindels{at}ncmls.kun.nl

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Pseudovitamin D-deficiency rickets (PDDR) is an autosomal disease characterized by hyperparathyroidism, rickets, and undetectable levels of 1,25-dihydroxyvitaminD₃ (1,25(OH)₂D₃). Mice in which the 25-hydroxyvitamin D₃-1-hydroxylase (1-OHase) gene was inactivated presented the same clinical phenotype as patients with PDDR and were used to study renal expression of the epithelial Ca²⁺ channel (ECaC1), the calbindins, Na⁺/Ca²⁺ exchanger (NCX1), and Ca²⁺-ATPase (PMCA1b). Serum Ca²⁺ (1.20±0.05 mM) and mRNA/protein expression of ECaC1 (41±3%), calbindin-D_28K (31±2%), calbindin-D_9K (58±7%), NCX1 (10±2%), PMCA1b (96±4%) were decreased in 1-OHase^-/- mice compared with 1-OHase^+/- littermates. Feeding these mice a Ca²⁺-enriched diet normalized serum Ca²⁺ levels and expression of Ca²⁺ proteins except for calbindin-D_9K expression. 1,25(OH)₂D₃ repletion resulted in increased expression of Ca²⁺ transport proteins and normalization of serum Ca²⁺ levels. Localization of Ca²⁺ transport proteins was clearly polarized in which ECaC1 was localized along the apical membrane, calbindin-D_28K in the cytoplasm, and calbindin-D_9K along the apical and basolateral membranes, resulting in a comprehensive mechanism facilitating renal transcellular Ca²⁺ transport. This study demonstrated that high dietary Ca²⁺ intake is an important regulator of the renal Ca²⁺ transport proteins in 1,25(OH)₂D₃-deficient status and thus contributes to the normalization of blood Ca²⁺ levels.—Hoenderop, J. G. J., Dardenne, O., van Abel, M., van der Kemp, A. W. C. M., van Os, C. H., St.-Arnaud, R., Bindels, R. J. M. Modulation of renal Ca²⁺ transport protein genes by dietary Ca²⁺ and 1,25-dihydroxyvitamin D₃ in 25-hydroxyvitamin D₃-1-hydroxylase knockout mice.

Key Words: ECaC • CaT1 • vitamin D • calcium reabsorption

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
1,25-DIHYDROXYVITAMIN D₃ (1,25(OH)₂D₃) is an important hormone in Ca²⁺ homeostasis and bone mineralization (1) . The genomic effects underlying part of the regulatory processes are mediated by the interaction of 1,25(OH)₂D₃ with the nuclear vitamin D receptor (VDR) in a ligand concentration-dependent manner (2) . 1,25(OH)₂D₃ is synthesized from an inactive metabolite by the renal cytochrome P450 enzyme 25-hydroxyvitamin D₃-1-hydroxylase (CYP27B1, hereafter referred to as 1-OHase). The importance of this enzyme is reflected by severe disorders resulting from mutations in the gene coding for 1-OHase, including pseudovitamin D-deficiency rickets (PDDR), also known as vitamin D-deficiency rickets type I (VDDR I) (3) . PDDR is an autosomal recessive disease characterized by growth retardation, failure to thrive, hypocalcemia, osteomalacia, and rickets. Recently, the genetic association of 1-OHase with PDDR has been confirmed by deletion of the 1-OHase gene in mice (4 , 5) .

1,25(OH)₂D₃ is synthesized primarily in the kidney by conversion of 25-hydroxy-vitamin D₃ in the mitochondria of the proximal tubules (6) . The kidney is also an important target organ for 1,25(OH)₂D₃ and responsible for the regulation of the extracellular Ca²⁺ concentration by controlling Ca²⁺ excretion from the body. In normal adult animals, a major portion (>95%) of filtered Ca²⁺ in the kidney is reabsorbed, and only a small fraction (2%) equal to the amount of Ca²⁺ absorbed in the intestine is excreted in urine to maintain Ca²⁺ balance. Immunohistochemical studies demonstrated that the VDR is predominantly localized to the distal tubule, which is the site for 1,25(OH)₂D₃-controlled active Ca²⁺ reabsorption (7) . Ca²⁺ reabsorption in these segments consists of passive entry of Ca²⁺ across the apical membrane through the epithelial Ca²⁺ channel (ECaC1), cytosolic diffusion of Ca²⁺ bound to calbindin (calbindin-D_28K and calbindin-D_9K), and active extrusion of Ca²⁺ across the opposite basolateral membrane by the Na⁺/Ca²⁺ exchanger (NCX1) and the plasma membrane Ca²⁺-ATPase (PMCA1b) (8 , 9) .

The recent identification of the apical Ca²⁺ channel allows the development of a comprehensive study of hormonal-dependent active Ca²⁺ transport in the kidney, where the individual contribution of the participating proteins can be underscored (10) . Primary or secondary involvement of these Ca²⁺ transport proteins can be expected in several pathological situations. For example, conditions associated with a disturbed serum Ca²⁺ concentration, such as in PDDR, are certainly of interest due to the conspicuous presence of these Ca²⁺ transport proteins in the kidney; therefore, knowledge of regulation of these proteins provides new insight into Ca²⁺ metabolism under (patho)physiological circumstances.

The aim of the present study was to investigate the regulation of Ca²⁺ transport proteins in the kidney and their role in maintaining Ca²⁺ balance. The recently generated 1-OHase knockout mice were used, which develop a severe hypocalcemia and represent a unique model system for PDDR. Furthermore, these mice allow to dissociate 1,25(OH)₂D₃-dependent from 1,25(OH)₂D₃-independent processes. 1-OHase knockout mice were rescued by 1,25(OH)₂D₃ repletions or high dietary Ca²⁺ intake and the renal expression of ECaC1, calbindin-D_28K, calbindin-D_9K, NCX1, and PMCA1b was subsequently measured at mRNA and protein level by real-time PCR and immunoblotting and immunohistochemistry.

	MATERIALS AND METHODS

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Animals
25-Hydroxyvitamin D₃-1-hydroxylase knockout mice were recently generated by targeted ablation of exon 8 encoding the heme binding domain of the enzyme (4) . 1-OHase knockout mice were genotyped by Southern blot analysis at 3 wk of age directly after the weaning period as described (4) . Initial characterization of the 1-OHase knockout mice demonstrated there are no significant differences between wild-type (1-OHase^+/+) and heterozygous 1-OHase knockout mice (1-OHase^+/-) (4) . The heterozygous mice therefore were used as control animals. 1-OHase^+/- and homozygous 1-OHase (1-OHase^-/-) knockout mice were fed from wk 3 to 8 either a normal diet (1.1% Ca²⁺, 0.8% phosphorus, 0% lactose), a Ca²⁺-enriched diet (2% calcium, 1.25% phosphorus, 20% lactose; Harlan Teklad, Madison, WI), or a normal diet in combination with i.p. injected 1,25(OH)₂D₃ repletions of 500 pg/g body weight daily in wk 3–4 and 100 pg/g daily in wk 5–8. Animals (n=6 in each group) were killed at 8 wk of age, and blood and kidney samples were taken. The animal ethics board of the University of Nijmegen (Nijmegen) and Shriners Hospital for Children (Montreal) approved all animal experimental procedures.

RNA isolation and quantitative RT-PCR
Mice were exsanguinated under anesthesia. Kidney tissue was homogenized in Trizol (Gibco, BRL, Grand Island, NY) and total RNA was isolated as specified by the manufacturer. RNA (2 µg) was reverse transcribed using Moloney murine leukemia virus reverse transcriptase (RT, Gibco, BRL). Quantitative RT-PCR reactions were performed using an ABI-prism 7700 sequence detector (Gene-Amp PCR system 9600, PE Biosystems, Foster City, CA). Sequences of PCR primers and fluorescent probes for ECaC1, calbindin-D_9K, calbindin-D_28K, Ca²⁺-ATPase (PMCA1b), Na⁺/Ca²⁺ exchanger (NCX1), and hypoxanthine-guanine phosphoribosyl transferase (HPRT) are depicted in Table 1 (11) . The amplicons were verified by sequencing. The relative expression levels of the target genes were calculated as a ratio to the HPRT gene.

Immunoblotting
Kidney tissue was removed, immediately frozen in liquid nitrogen, and homogenized in phosphate-buffered saline (PBS). All samples (20 µg of protein) were separated on 12% and 16% (w/v) SDS-PAGE gels and blotted to PVDF-nitrocellulose membranes (Immobilon-P, Millipore Corporation, Bedford, MA). Blots were incubated for 16 h with calbindin-D_28K antibody (Sigma, 1:10,000), calbindin-D_9K antibody (Swant, Switzerland, 1:10,000), or NaK-ATPase antibody (kindly provided by Dr. Moller; 1:20,000; ref 12 ). Subsequently, immunoreactive protein was detected using the enhanced chemiluminescence method as described previously (13) .

Immunofluorescence confocal microscopy
Kidney tissue was cut into pieces, placed in 1% (w/v) periodate-lysine-paraformaldehyde fixative for 2 h at room temperature and incubated overnight at 4°C in PBS containing 15% (w/v) sucrose as described previously (14) . Subsequently, kidney samples were frozen in liquid nitrogen and 7 µm frozen sections were cut for different staining procedures. The kidney sections were stained with affinity-purified guinea pig anti-ECaC antiserum (1:1000) as described previously (14) . Sections triple stained for ECaC1 and calbindins were incubated simultaneously for 16 h at 4°C with affinity-purified antiserum against ECaC1, calbindin-D_9K (1:500), calbindin-D_28K (1:500). To visualize ECaC1, calbindin-D_28K, and calbindin-D_9K, sections were stained with affinity-purified goat anti-guinea pig-biotin as described (15) , goat anti rabbit Alexa 594-conjugated anti IgG (1:300), and goat anti-mouse Cy5-conjugated anti-IgG (1:50), respectively (Molecular Probes, Eugene, OR). All negative controls, including sections incubated with preimmune serum, antiserum preabsorbed for 1 h with 10 µg/mL ECaC-GST fusion protein, or conjugated antibodies alone, were devoid of any staining. Sections were dehydrated in methanol and subsequently mounted in Mowiol (Hoechst, Frankfurt, Germany) and visualized by confocal laser scanning microscopy (MRC-1000; Bio-Rad, Richmond, CA) using a Nikon Diaphot microscope (Tokyo, Japan). The ECaC1 antibody used in this study has been extensively characterized but is, so far not reacting on immunoblots (15) . To semiquantify ECaC1, protein expression immunopositive tubules in 10 random microscopic fields were counted for each condition as described (15) .

Biochemical assays
Serum 1,25(OH)₂D₃ levels were measured using a specific RIA (ImmunoDiognostic Systems Ltd., Boldon, UK). Total calcium was measured using a Monarch automated analyzer (4) .

Statistical analysis
The data are expressed as the mean ± SE. Overall statistical significance was determined by analysis of variance. In the case of significance (P<0.05), individual groups were compared by contrast analysis according to Scheffé.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Inactivation of both alleles of the 1-OHase gene (1-OHase^-/-) resulted in severe hypocalcemia with plasma Ca²⁺ concentrations as low as 1.2 mM, in contrast to the heterozygous littermates, which exhibit normal plasma Ca²⁺ concentrations (Table 2 ). Supplementation of hypocalcemic 1-OHase^-/- mice with 1,25(OH)₂D₃ or a high dietary Ca²⁺ intake normalized the plasma Ca²⁺ concentration.

View this table: [in this window] [in a new window]   Table 2. The effect of normal diet, Ca2+-enriched diet and 1,25(OH)2D3-supplemented diet on serum Ca2+ concentration in 1-OHase knockout micea

To determine the molecular mechanism responsible for the severe hypocalcemia in 1-OHase^-/- mice, renal expression of ECaC1, calbindin-D_28K, calbindin-D_9K, NCX1, and PMCA1b in 1-OHase^-/- and 1-OHase^+/- knockout mice was examined. Using quantitative real-time PCR, a twofold decrease in ECaC1 mRNA levels was observed in kidneys of 1-OHase^-/- compared with 1-OHase^+/- mice (Fig. 1 A). Down-regulation of ECaC1 was accompanied by a decrease in calbindin-D_28K (3-fold) and calbindin-D_9K (2-fold) mRNA (Fig. 1B, C ). The high dietary Ca²⁺ diet restored the low ECaC1 and calbindin-D_28K mRNA levels in the 1-OHase^-/- mice. In contrast, the expression was decreased in the heterozygous littermates by the high Ca²⁺ diet (Fig. 1A, B ). Calbindin-D_9K mRNA was decreased in kidney of 1-OHase^-/- mice whereas this transcript was not up-regulated by high dietary Ca²⁺ intake (Fig. 1C ). Subsequently, regulation of the basolateral extrusion transporters was studied. The most important Ca²⁺ extrusion mechanism in the distal part of the nephron is NCX1, and this exchanger was severely down-regulated in kidneys from 1-OHase^-/- mice. Again, similar to ECaC1 and calbindin-D_28K, this transcript was up-regulated by dietary Ca²⁺ intake whereas Ca²⁺ supplementation had no effect on the expression of the exchanger in 1-OHase^+/- (Fig. 1D ). mRNA encoding PMCA1b is not significantly reduced in kidneys of 1-OHase^-/- mice (Fig. 1E ). High dietary Ca²⁺ intake, however, resulted in a significant increase in expression. Supplementation of the 1-OHase^-/- mice with 1,25(OH)₂D₃ significantly increased mRNA expression levels of all the Ca²⁺ transport molecules measured including ECaC1, calbindin-D_28K, calbindin-D_9K, NCX1, and PMCA1b (Fig. 1) .

View larger version (31K): [in this window] [in a new window]   Figure 1. Gene expression patterns in kidney of 1-OHase-/- (filled bars) and 1-OHase+/- (open bars) mice on normal diet, Ca2+-enriched diet, and the 1,25(OH)2D3-supplemented diet. Renal expression of ECaC1 (A), calbindin-D28K (B), calbindin-D9K (C), NCX1 (D), and PMCA1b (E) assessed by real-time PCR analysis is calculated as a ratio to the HPRT RNA level and expressed relative to levels of 1-OHase+/- mice on a normal diet. Values are presented as means ± SE (n=6). *P < 0.05, significant from 1-OHase+/- mice on a normal diet. #P < 0.05, significant from 1-OHase-/- mice on a normal diet.

Expression of Ca²⁺ transport proteins was studied at the protein level. First, the abundance of ECaC1 protein was examined. ECaC1 immunopositive tubules in 10 random microscopic fields of kidney cortex were counted semiquantitatively for each animal (15) . Figure 2 depicts the average values obtained for four 1-OHase knockout mice. Inactivating the 1,25(OH)₂D₃ gene in 1-OHase^-/- mice had a major effect on ECaC1 protein expression as indicated by the low number of immunopositive distal tubules in the kidney cortex. Normalization of the 1,25(OH)₂D₃ levels or high dietary Ca²⁺ intake restored ECaC1 expression to levels comparable to those observed in 1-OHase^+/- mice (Fig. 2) . In agreement with the reduced ECaC1 mRNA expression, this Ca²⁺-enriched diet lowered protein expression in 1-OHase^+/- mice. Immunoblotting showed a significant down-regulation of calbindin-D_28K and calbindin-D_9K protein abundance in the 1-OHase^-/- mice (Fig. 3 ). The decrease in calbindin-D_28K protein was recovered by high dietary Ca²⁺ intake whereas calbindin-D_9K expression was not altered. Calbindin protein expression levels were completely restored in 1-OHase^-/- mice after repletion with 1,25(OH)₂D₃. The corresponding Na⁺-K⁺-ATPase bands did not vary significantly in density, which precludes unequal loading as an explanation for the differences (Fig. 3) .

View larger version (32K): [in this window] [in a new window]   Figure 2. A) Immunofluorescence localization of ECaC1 in kidney (depicted in white) of 1-OHase+/- and 1-OHase-/- mice on normal diet, Ca2+-enriched diet and the 1,25(OH)2D3-supplemented diet. B) Semiquantitative results were obtained by counting randomly 10 microscopic fields for ECaC1 immunopositive distal tubular segments in kidney cortex of 1-OHase+/- (open bars) and 1-OHase-/- (filled bars) mice. Values are presented as means ± SE (n=6). *P < 0.05, significant from 1-OHase+/- mice on a normal diet. #P < 0.05, significant from 1-OHase-/- mice on a normal diet. Bar is 25 µm.

View larger version (74K): [in this window] [in a new window]   Figure 3. Effects of normal diet, Ca2+-enriched diet and 1,25(OH)2D3-supplemented diet on calbindin-D28K and calbindin-D9K expression in kidney of 1-OHase-/- and 1-OHase+/- mice. Immunoblots were run with 20 µg of protein per lane of whole-kidney homogenates and probed with anti-calbindin-D28K or anti-calbindin-D9K. Immunoblots were probed with an antibody against Na+-K+-ATPase (12) to exclude the possibility that the difference in calbindin expression was due to unequal loading.

Figure 4 shows representative triple immunofluorescence labeling of distal tubules in mice kidney cortex sections stained for ECaC1 (green), calbindin-D_28K (blue), and calbindin-D_9K (red). Superimposed images showed colocalization of these Ca²⁺ transport proteins in 1-OHase^-/- and 1-OHase^+/- mice, both on a normal diet. Moreover, kidneys of 1,25(OH)₂D₃-repleted mice clearly indicate an increased number of ECaC1 and calbindins immunopositive distal tubules; identical to the normal diet, a remarkable overlap was apparent. High dietary Ca²⁺ intake by 1-OHase^-/- mice increased the amount of immunopositive tubules expressing ECaC1 and calbindin-D_28K; the expression of calbindin-D_9K was not significantly altered and distal tubules were observed that express exclusively calbindin-D_28K, a phenomenon that was never seen in mice fed the control diet or repleted with 1,25(OH)₂D₃ (Fig. 4 , asterisks). Higher magnifications showed that ECaC1 is predominantly localized to the apical domain of distal tubular segments. In these immunopositive tubules, calbindin-D_28K was observed throughout the cytosol. Intriguingly, the immunofluorescence staining of calbindin-D_9K appeared to be more compartmentalized and associated with apical and basolateral membranes (Fig. 5 ).

View larger version (21K): [in this window] [in a new window]   Figure 4. Localization of Ca2+ transport proteins in kidney of 1-OHase-/- and 1-OHase+/- mice. Effect of normal diet, Ca2+-enriched diet and 1,25(OH)2D3-supplemented diet on the expression pattern of ECaC1 (green), calbindin-D28K (blue) and calbindin-D9K (red). Bar is 100 µm.

View larger version (37K): [in this window] [in a new window]   Figure 5. Immunofluorescence triple staining of a mice kidney cortex section depicting the cellular localization of ECaC1 (green), calbindin-D28K (blue) and calbindin-D9K (red) in distal tubules. A glomerulus is depicted in the image (G). Bar is 25 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The present study demonstrates that the expression of the renal Ca²⁺ transport proteins ECaC1, calbindin-D_28K, calbindin-D_9K and NCX1 is significantly down-regulated in the kidney of 1-OHase^-/- mice, which is in line with a diminished Ca²⁺ reabsorption capacity contributing to the development of hypocalcemia. Furthermore, high dietary Ca²⁺ intake restored the decreased expression of Ca²⁺ transport proteins independent of 1,25(OH)₂D₃ and normalized the serum Ca²⁺ concentration.

Recently two laboratories independently generated 1-OHase knockout mice strains that represent valuable animal models for PDDR since they display undetectable 1,25(OH)₂D₃ concentrations, hypocalcemia, secondary hyperparathyroidism, and failure to thrive (4 , 5) . The 1-OHase^-/- mice developed distinct histological evidence of rickets and osteomalacia. There was a correlative relationship between the expression level of these Ca²⁺ transport proteins and blood Ca²⁺ levels. Normalization of the plasma Ca²⁺ level by 1,25(OH)₂D₃ or Ca²⁺ supplementation was associated with an increase in the expression level of the renal Ca²⁺ transport proteins, confirming the essential role of these proteins in active hormone-mediated Ca²⁺ transport and maintaining Ca²⁺ balance. These findings imply that in addition to intestinal Ca²⁺ absorption, the kidney also plays a crucial role in maintaining body Ca²⁺ homeostasis. This follows from experiments performed with VDR knockout mice. In these hypocalcemic mice, urinary Ca²⁺ excretion is inappropriately high, suggesting renal Ca²⁺ wasting due to disturbed Ca²⁺ reabsorption (11 , 16 , 17) . Taken together, in 1-OHase^-/- and VDR^-/- mice the sustained hypocalcemia is related to defective distal Ca²⁺ reabsorption.

The reduced expression level of Ca²⁺ transport proteins in the 1-OHase^-/- mice was restored by high dietary Ca²⁺ intake and accompanied by normalization of serum Ca²⁺ concentrations. This effect was observed in the absence of 1-OHase-activity. In contrast, the Ca²⁺-enriched diet reduced the expression of Ca²⁺ transport proteins in 1-OHase^+/- mice, which exhibit normal serum vitamin D and Ca²⁺ levels. Under physiological conditions, Ca²⁺ acts via a negative feedback mechanism that eventually leads to suppression of the 1-OHase-activity, which decreases Ca²⁺ reabsorption and expression of Ca²⁺ transport proteins (3) .

Our study with 1-OHase^-/- mice has revealed that Ca²⁺ supplementation can up-regulate gene transcription encoding for Ca²⁺ transporters in the absence of circulating 1,25(OH)₂D₃, but the molecular mechanism of this vitamin D-independent Ca²⁺-sensitive pathway remains elusive. It is likely, however, that Ca²⁺-responsive elements are present in the promoter regions of the ECaC1 and calbindin genes in addition to the identified vitamin D-responsive ones (15 , 18 , 19) . Several elements have been proposed to function as Ca²⁺-sensitive transcriptional regulators, including the serum-responsive element and the cAMP/Ca²⁺-responsive element (20) . Of interest is the identification by Arnold and Heintz of a Purkinje cell expression specific element (PCE1) in the calbindin-D_28K gene that functions as a Ca²⁺-sensitive transcriptional regulatory mechanism (21) . This mechanism may play a role in fine-tuning the Ca²⁺ buffer capacity of Purkinje cells. Since these elements could not be found in the 5' upstream region of the ECaC1 and NCX1 gene, this Ca²⁺-sensitive transcriptional mechanism could only apply to the calbindin-D_28K up-regulation reported here.

Recovery of expression of Ca²⁺ transport proteins and normalization of the plasma Ca²⁺ concentration by high dietary Ca²⁺ intake were associated with a dissociation in the localization of the calbindins in kidney cortex. In the 1-OHase mutant mice, calbindin-D_28K was up-regulated by Ca²⁺ and present in more cells than calbindin-D_9K, whose amount and localization was not altered. Most of the distal tubular segments coexpress ECaC1 and calbindin-D_28K, but a subpopulation of distal tubules was identified that expresses calbindin-D_28K. Histochemical studies in mouse and rat kidney revealed only the highest expression of ECaC1 and calbindin-D_28K in most of the distal convoluted tubule (DCT2) and connecting tubule; in these segments, both proteins colocalize (15 , 22) . Prominent NCX1 and PMCA1b immunostaining was exactly congruent with that of ECaC1 immunostaining (22) . Staining of the three latter proteins abruptly disappeared at the transition to the cortical collecting duct, whereas staining of calbindin-D_28K continued along the CCD (22) . These latter tubules do express calbindin-D_28K at higher levels after feeding the mice a Ca²⁺-enriched diet. This raises the intriguing question of whether calbindin-D_28K can exert additional functions in these latter segments besides its known function of Ca²⁺ diffusion facilitation.

It is essential that the number of ECaC1 channels at the plasma membrane is matched by the cytosolic Ca²⁺ buffering capacity of these cells since the activity of ECaC1 is tightly controlled by the ambient Ca²⁺ concentration (23 , 24) . The necessity of sufficient Ca²⁺ buffering is underscored by the conspicuous colocalization of ECaC1, the calbindins, NCX1, and PMCA1b in Ca²⁺ transporting epithelial cells (14 , 22) . The 1,25(OH)₂D₃ responsiveness of ECaC1 and calbindins is in line with previous studies. Until now there has been only limited data available regarding regulation of the basolateral extrusion systems by vitamin D. Similar to ECaC1 and the calbindins, an impressive reduction of NCX1 was observed but no significant down-regulation of PMCA1b was measured. Van Baal et al. concluded that Na⁺/Ca²⁺ exchange is the primary Ca²⁺ extrusion mechanism, whereas only a minor amount of Ca²⁺ in the distal tubular cells is extruded by the plasma Ca²⁺ pump (25) . These findings point to a crucial role of the exchanger in vitamin D-dependent Ca²⁺ reabsorption. The present observation that ECaC1, calbindins, NCX1, and, to a lesser extent, PMCA1b, are synchronically controlled by 1,25(OH)₂D₃ demonstrates the intimate relation between apical Ca²⁺ influx, cytosolic Ca²⁺ diffusion, and basolateral Ca²⁺ extrusion during 1,25(OH)₂D₃-stimulated Ca²⁺ reabsorption.

Several pathological symptoms in 1-OHase^-/- and VDR^-/- mice are similar including severe hypocalcemia, hyperparathyroidism and rickets, but there are distinctive differences between both mouse models. Renal expression of Ca²⁺ transport proteins was consistently decreased in 1-OHase^-/- mice whereas different effects were reported in VDR^-/- mice (11 , 26) . Obviously, 1,25(OH)₂D₃ levels are exceptionally elevated in VDR^-/- mice, whereas in 1-OHase^-/- mice its synthesis is impaired (4 , 5 , 11 , 17 , 27) . In addition to the VDR-mediated genomic pathway, many studies describe the existence of another nongenomic pathway regulated by 1,25(OH)₂D₃. There are indications that 1,25(OH)₂D₃ stimulates passive paracellular Ca²⁺ transport in VDR^-/- mice to compensate for the reduction in Ca²⁺ reabsorption (28) . Furthermore, 1,25(OH)₂D₃ has been implicated in the stabilization of mRNA (29) , regulation of voltage-gated and store-operated Ca²⁺ channels (30 , 31) , opening of chloride channels (32) , modulation of protein kinase C activity (33) and activation of mitogen-activated protein kinases (34) , which eventually lead to the onset of rapid or long-term biological responses. Recent studies have indicated that the generation of these rapid responses could be mediated via a putative membrane receptor with ligand binding properties different from those of the nuclear VDR (35 36 37) . As an alternative to a specific membrane receptor for 1,25(OH)₂D₃, it has been suggested that membrane-associated annexin II might serve as a receptor for 1,25(OH)₂D₃-mediated responses (38 , 39) . It is conceivable that these 1,25(OH)₂D₃-mediated nongenomic pathways will influence the expression and activity of Ca²⁺ transport proteins in the kidney and, therefore, the 1-OHase^-/- mouse is an ideal mouse model to study the effect of dietary Ca²⁺ independent of the 1,25(OH)₂D₃ regulation of transcellular Ca²⁺ transport in kidney.

Our study provides new insights into the localization of Ca²⁺ transport proteins resulting in a more comprehensive molecular model for active Ca²⁺ reabsorption in the distal part of the nephron (Fig. 6 ). ECaC1 is predominantly localized along the apical membrane and facilitates the first step in this active transport process. The uniform distribution of calbindin-D_28K throughout the cytosol is in accordance with the postulated physiological role as a cytosolic Ca²⁺ buffer and as a shuttle mechanism between the luminal influx and the basolateral efflux sites (Fig. 6) . The efflux of Ca²⁺ is facilitated mainly by Na⁺/Ca²⁺ exchange and to a lesser extent by the ATP-dependent Ca²⁺ pump (25) . The conspicuous localization of calbindin-D_9K along the basolateral and apical membranes excludes a cytosolic Ca²⁺ shuttle function. Alternatively, calbindin-D_9K could distribute Ca²⁺ quickly along the plane of the plasma membrane after influx through a few scattered Ca²⁺ channels and supply sufficient Ca²⁺ to a small number of extrusion proteins (Fig. 6) .

View larger version (86K): [in this window] [in a new window]   Figure 6. Model of transcellular Ca2+ transport by cells lining the distal part of the nephron. Entry of Ca2+ is facilitated by the apical Ca2+ channel ECaC1. Subsequently, the ion binds to calbindin-D9K (light gray) or directly to calbindin-D28K (dark gray) and diffuses through the cytosol to the basolateral membrane. Ca2+ ions are extruded, directly or via intermission of calbindin-D9K, by a Na+/Ca2+ exchanger (NCX1) and a Ca2+-ATPase (PMCA1b).

In conclusion, the present study demonstrate that high dietary Ca²⁺ intake is an important regulator of the renal Ca²⁺ transport proteins in 1,25(OH)₂D₃-deficient status and thus contributes to the normalization of blood Ca²⁺ levels. The treatment of choice for PDDR patients is long-term replacement therapy with 1,25(OH)₂D₃. Repletion with vitamin D analogs has proved to be beneficial in various clinical situations such as the prevention of rickets during infancy. It will therefore be of interest to compare the benefits of supplemented dietary Ca²⁺ to long-term treatment with vitamin D analogs.

	ACKNOWLEDGMENTS

 
This work was supported by the Dutch Organization of Scientific Research (Zon-Mw 016.006.001, Zon-Mw 902.18.298), Dutch Kidney Foundation (C10.1881), and the Shriners of North America. R.S.-A. is a ‘chercheur-boursier’ from the Fonds de la Recherche en Santé du Québec.

Received for publication March 13, 2002. Revision received May 16, 2002.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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