Critical Role of the Epithelial Ca2+ Channel TRPV5 in Active Ca2+ Reabsorption as Revealed by TRPV5/Calbindin-D28K Knockout Mice

Dimitra Gkika; Yu-Juei Hsu; Annemiete W. van der Kemp; Sylvia Christakos; René J. Bindels; Joost G. Hoenderop

doi:10.1681/ASN.2006060676

Abstract

The epithelial Ca²⁺ channel TRPV5 facilitates apical Ca²⁺ entry during active Ca²⁺ reabsorption in the distal convoluted tubule. In this process, cytosolic Ca²⁺ remains at low nontoxic concentrations because the Ca²⁺ influx is buffered rapidly by calbindin-D_28K. Subsequently, Ca²⁺ that is bound to calbindin-D_28K is shuttled toward the basolateral Ca²⁺ extrusion systems. For addressing the in vivo role of TRPV5 and calbindin-D_28K in the maintenance of the Ca²⁺ balance, single- and double-knockout mice of TRPV5 and calbindin-D_28K (TRPV5^−/−, calbindin-D_28K^−/−, and TRPV5^−/−/calbindin-D_28K^−/−) were characterized. These mice strains were fed two Ca²⁺ diets (0.02 and 2% wt/wt) to investigate the influence of dietary Ca²⁺ content on the Ca²⁺ balance. Urine analysis indicated that TRPV5^−/−/calbindin-D_28K^−/− mice exhibit on both diets hypercalciuria compared with wild-type mice. Ca²⁺ excretion in TRPV5^−/−/calbindin-D_28K^−/− mice was not significantly different from TRPV5^−/− mice, whereas calbindin-D_28K^−/− mice did not show hypercalciuria. The similarity between TRPV5^−/−/calbindin-D_28K^−/− and TRPV5^−/− mice was supported further by an equivalent increase in renal calbindin-D_9K expression and in intestinal Ca²⁺ hyperabsorption as a result of upregulation of calbindin-D_9K and TRPV6 expression in the duodenum. Elevated serum parathyroid hormone and 1,25-dihydroxyvitamin D₃ levels accompanied the enhanced expression of the Ca²⁺ transporters. Intestinal Ca²⁺ absorption and expression of calbindin-D_9K and TRPV6, as well as serum parameters of the calbindin-D_28K^−/− mice, did not differ from those of wild-type mice. These results underline the gatekeeper function of TRPV5 being the rate-limiting step in active Ca²⁺ reabsorption, unlike calbindin-D_28K, which possibly is compensated by calbindin-D_9K.

Ca²⁺ homeostasis is of crucial importance for many physiologic functions, including neuronal excitability, muscle contraction, blood clotting, and bone mineralization. Therefore, the Ca²⁺ balance is tightly controlled through constant regulation of three physiologic processes: Intestinal absorption, renal reabsorption, and exchange of Ca²⁺ from the bone mass (1). Both in intestine and in kidney, Ca²⁺ enters the interstitium by passive paracellular as well as active (re)absorption (2,3). Active Ca²⁺ (re)absorption is critical in this process, because it constitutes the primary target for regulation by calciotropic hormones, including 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] and parathyroid hormone (PTH), enabling the organism to regulate the extracellular Ca²⁺ concentration on the body’s demand (4).

Active absorption of dietary Ca²⁺ occurs primarily in the proximal small intestine, whereas renal active Ca²⁺ reabsorption is restricted to the distal convoluted tubule (DCT) and the connecting tubule (CNT) (5,6). Ca²⁺ absorption occurs also in bone, where it is crucial for bone formation to achieve adequate bone quality and strength, as well as for osteoclastic bone resorption (7). At the cellular level, active Ca²⁺ (re)absorption implies entry of Ca²⁺ across the luminal membrane through the epithelial Ca²⁺ channels, followed by intracellular buffering, facilitated diffusion by Ca²⁺-binding proteins, and finally extrusion across the basolateral membrane by a Na⁺/Ca²⁺ exchanger and/or a plasma membrane Ca²⁺ pump. Ca²⁺ influx occurs through two highly Ca²⁺-selective members of the transient receptor potential (TRP) cation channel family, TRPV5 and TRPV6, which constitute the gatekeepers of active Ca²⁺ (re)absorption in kidney and intestine, respectively (8,9). Indeed, ablation of TRPV5 (TRPV5^−/−) in mice impairs renal Ca²⁺ reabsorption, resulting in robust hypercalciuria (10). As a consequence, TRPV5^−/− mice develop compensatory dietary Ca²⁺ hyperabsorption in the intestine. Furthermore, the structure of the bones in these mice is significantly disturbed, showing reduced trabecular and cortical bone thickness (11).

After influx through TRPV5 and TRPV6, Ca²⁺ binds to cytosolic proteins to diffuse toward the basolateral surface of the epithelial cell. Two Ca²⁺-binding proteins, calbindin-D_28K and calbindin-D_9K, are regarded as key components of Ca²⁺ (re)absorption (4). In mammals, calbindin-D_28K is expressed primarily in kidney, whereas calbindin-D_9K is abundantly present in small intestine. Only in mouse kidney are both calbindin-D_28K and calbindin-D_9K expressed in the distal part of the nephron (12). The physiologic importance of calbindin-D_28K in renal Ca²⁺-transporting epithelia is underlined by the consistent coexpression with TRPV5 and their co-regulation by calciotropic hormones, including PTH, 1,25(OH)₂D₃, and also dietary Ca²⁺ (13,14).

The aim of our study was to investigate whether calbindin-D_28K deficiency is critical for active reabsorption in the presence or absence of TRPV5. To this end, single- and double-knockout mice of calbindin-D_28K and TRPV5 (calbindin-D_28K^−/−, TRPV5^−/−, and TRPV5^−/−/calbindin-D_28K^−/−) were generated. These mice were functionally characterized, including measurements of expression of the Ca²⁺ transporter proteins at mRNA and protein levels.

Materials and Methods

Animal Experiments

TRPV5^−/− mice were generated as described previously (10). Calbindin-D_28K^−/− mice were provided by Dr. Michael Meyer (Physiologisches Institut, Ludwig Maximilians Universität München, Munich, Germany) (15). Cross-breeding of TRPV5^−/−/calbindin-D_28K^+/+ with TRPV5^+/+/calbindin-D_28K^−/− mice resulted in offspring that were heterozygous for both TRPV5 and calbindin-D_28K (TRPV5^+/−/cal-bindin-D_28K^+/−). This heterozygous offspring displayed the wild-type phenotype and subsequently was intercrossed to obtain TRPV5^−/−/calbindin-D_28K^−/− mice. Genotypes were determined by PCR analysis using specific primers for Trpv5 (gene for TRPV5) as described previously (10,16) and for Calb1 (gene for calbindin-D_28K): Two sense primers 5′-tgcagcggctagtttgagagtg-3′ to detect the wild-type allele and 5′-tgactaggggaggagtagaag-3′ to detect the null allele in combination with a common antisense primer 5′-gcaagtaactaatggcatcg-3′. At the age of 4 wk, mice were fed ad libitum two diets that contained either 0.02 or 2% (wt/wt) Ca²⁺ for 5 wk and subsequently placed in metabolic cages (Techniplast, Buggiate, Italy), which enabled 24-h collection of urine. At the end of the experiment, blood samples were taken and the mice were killed. Subsequently, kidney and duodenum tissue was sampled. Urine and serum Ca²⁺ concentrations were analyzed using a colorimetric assay kit (Roche, Mannheim, Germany). Serum PTH was measured using an immunoradiometric assay (Immutopics Inc., San Clemente, CA). Serum vitamin D levels were determined by an [I¹²⁵]1,25(OH)₂D₃ RIA assay (IDS Inc., Fountain Hills, AZ). The animal ethics board of Radboud University Nijmegen approved all animal experimental procedures.

Real-Time Quantitative PCR Analysis

Renal and duodenal mRNA expression levels of calbindin-D_28K, calbindin-D_9K, TRPV5, and TRPV6 were quantified by real-time quantitative PCR as described previously (17), using the ABI Prism 7700 Sequence Detection System (PE Biosystems, Rotkreuz, Switzerland). The expression level of the housekeeping gene hypoxanthine-guanine phosphoribosyl transferase was used as an internal control to normalize differences in RNA extractions and reverse transcription efficiencies.

Immunoblotting

Total kidney and duodenum lysates of all mouse groups were prepared as described previously (17). Briefly, protein concentrations of the homogenates were determined by the Bio-Rad protein assay (Bio-Rad, München, Germany), and 10 μg of each sample was loaded on 12 or 16.5% (wt/vol) SDS-PAGE gels and blotted to polyvinylidene difluoride nitrocellulose membranes (Immobilon-P, Millipore Corp., Bedford, MA). Blots were incubated with a rabbit anti–calbindin-D_28K polyclonal antibody (1:10,000; Sigma, St. Louis, MO), a rabbit anti–calbindin-D_9K polyclonal antibody (1:5000; Swant, Bellinzona, Switzerland), or a rabbit β-actin polyclonal antibody (1:20,000; Sigma) at 4°C for 16 h. Subsequently, blots were incubated with a goat anti-rabbit peroxidase-labeled secondary antibody (1:10,000; Sigma). Immunoreactive protein was detected by the chemiluminescence method (Pierce, Rockford, IL). The immunopositive protein bands were scanned and the pixel density was determined by using the Molecular Analyst Software of Bio-Rad Laboratories (Hercules, CA).

In Vivo⁴⁵Ca²⁺ Absorption Assay

Ca²⁺ absorption was assessed by measuring serum ⁴⁵Ca²⁺ at early time points after oral gavage as described previously (10). Briefly, mice were fasted 16 h (overnight) before the test and a ⁴⁵Ca²⁺ solution was administrated by oral gavage. Blood samples were obtained at indicated time intervals, and serum (10 μl) was analyzed by liquid scintillation counting. Differences in serum Ca²⁺ concentration were calculated from the ⁴⁵Ca²⁺ content in the samples and the specific activity of the administrated ⁴⁵Ca²⁺.

Statistical Analyses

Values are expressed as means ± SEM. Statistical significance (P < 0.05) between groups was determined by one-way ANOVA. In case of significance, the Tukey-Kramer multiple comparisons test was applied. All analyses were performed using the Statview Statistical Package Software (Power PC, version 4.51; Berkeley, CA).

Results

Serum Parameters

Wild-type, 4-wk-old TRPV5^−/−, calbindin-D_28K^−/−, and TRPV5^−/−/calbindin-D_28K^−/− mice were fed a diet that contained 0.02 or 2% (wt/wt) Ca²⁺ for 5 wk. All mice strains were fertile and had similar average litter sizes (Table 1). Furthermore, serum analysis showed that TRPV5^−/− and TRPV5^−/−/calbindin-D_28K^−/− mice that were on the 0.02% (wt/wt) Ca²⁺ diet exhibit increased PTH and 1,25(OH)₂D₃ levels compared with wild-type mice. In contrast, serum PTH and 1,25(OH)₂D₃ levels in calbindin-D_28K^−/− mice were not significantly different from those of wild-type mice. The increased PTH and 1,25(OH)₂D₃ levels were normalized in TRPV5^−/− and TRPV5^−/−/calbindin-D_28K^−/− mice that were fed the high-Ca²⁺ diet. Serum Ca²⁺ levels were not significantly altered between the mice genotypes, regardless of the dietary treatment (Table 1).

View this table:

Table 1.

Characteristics of TRPV5 and calbindin-D_28K single- and double-knockout mice

mRNA Expression of Epithelial Ca²⁺ Transporters

To evaluate the regulation of mRNA expression levels of the Ca²⁺ transporters in kidney and duodenum, we applied quantitative real-time PCR assays. In kidney, calbindin-D_9K mRNA expression levels were increased in TRPV5^−/− and TRPV5^−/−/calbindin-D_28K^−/− mice that were fed the 0.02 (wt/wt) Ca²⁺ diet compared with wild-type mice. Furthermore, renal calbindin-D_9K expression was similar in calbindin-D_28K^−/− compared with wild-type mice. Exposure of the mice to the high-Ca²⁺ diet resulted in downregulation of renal calbindin-D_9K mRNA in TRPV5^−/− and TRPV5^−/−/calbindin-D_28K^−/− mice (Figure 1A). Conversely, dietary Ca²⁺ content did not affect renal expression of calbindin-D_28K mRNA, which was significantly reduced in TRPV5^−/− compared with wild-type mice (Figure 1B). In duodenum, TRPV5^−/− and TRPV5^−/−/cal-bindin-D_28K^−/− mice that were fed the 0.02% (wt/wt) Ca²⁺ diet demonstrated an upregulation of calbindin-D_9K and TRPV6 mRNA expression in comparison with wild-type mice. On the same Ca²⁺ diet, duodenal calbindin-D_9K and TRPV6 expression remained unchanged in calbindin-D_28K^−/− compared with wild-type mice. The high-Ca²⁺ diet reduced intestinal calbindin-D_9K and TRPV6 mRNA expression in all mouse strains (Figure 2).

Figure 1.

Renal mRNA expression of Ca²⁺ transporters. Expression of calbindin (CaBP)-D_9K (A) and CaBP-D_28K (B) mRNA in kidney of wild-type, TRPV5^−/−, CaBP-D_28K^−/−, and TRPV5^−/−/CaBP-D_28K^−/− mice (n = 10) was analyzed by quantitative real-time PCR analysis. Mice were fed a 0.02% (wt/wt; ▪) or 2% (wt/wt; □) Ca²⁺ diet. Values are calculated as a ratio of hypoxanthine-guanine phosphoribosyl transferase (HPRT) expression in relative percentages compared with the wild-type mice on 0.02% (wt/wt) Ca²⁺ diet. Data are means ± SEM. *P < 0.05 versus wild-type on the same diet; ^#P < 0.05 versus the same group on 0.02% (wt/wt) Ca²⁺ diet.

Figure 2.

Duodenal mRNA expression of Ca²⁺ transporters. Expression of CaBP-D_9K (A) and TRPV6 (B) mRNA in duodenum of wild-type, TRPV5^−/−, CaBP-D_28K^−/−, and TRPV5^−/−/CABP-D_28K^−/− mice (n = 10) was assessed by quantitative real-time PCR analysis. Mice were fed a 0.02% (wt/wt; ▪) or 2% (wt/wt; □) Ca²⁺ diet. Values are calculated as a ratio of HPRT expression in relative percentages compared with the wild-type mice on 0.02% (wt/wt) Ca²⁺ diet. Data are means ± SEM. *P < 0.05 versus wild-type on the same diet; ^#P < 0.05 versus the same group on 0.02% (wt/wt) Ca²⁺ diet.

Protein Expression of Epithelial Ca²⁺ Transporters

For validation of whether the changes in renal and duodenal mRNA levels of the Ca²⁺ transporters resulted in altered protein expression, the abundance of the Ca²⁺ transporters was semiquantified by immunoblot analysis. In kidney, calbindin-D_9K protein expression was increased in TRPV5^−/− and TRPV5^−/−/calbindin-D_28K^−/− mice that were fed the 0.02% (wt/wt) Ca²⁺ diet compared with wild-type mice. On the same diet, calbindin-D_28K^−/− mice expressed wild-type levels of calbindin-D_9K protein in kidney. However, an increase in dietary Ca²⁺ content from 0.02 to 2% (wt/wt) resulted in downregulation of renal calbindin-D_9K in wild-type, TRPV5^−/−, and TRPV5^−/−/calbindin-D_28K^−/− mice (Figure 3, A and B). Furthermore, renal calbindin-D_28K protein abundance was significantly decreased in TRPV5^−/− mice in accordance with the downregulated mRNA levels. Variations in dietary Ca²⁺ did not affect renal calbindin-D_28K protein expression in both wild-type and TRPV5^−/− mice (Figure 3, C and D). In duodenum, calbindin-D_9K protein expression was increased in TRPV5^−/− and TRPV5^−/−/calbindin-D_28K^−/− mice compared with wild-type and calbindin-D_28K^−/− mice, in line with measured mRNA expression levels. Finally, dietary Ca²⁺ restriction resulted in a significant increase of duodenal calbindin-D_9K protein in TRPV5^−/− and TRPV5^−/−/calbindin-D_28K^−/− mice, which was consistent with the calbindin-D_9K mRNA expression data (Figure 4).

Figure 3.

Renal protein expression of CaBP-D_28K and CaBP-D_9K. Immunoblot of total kidney homogenates from wild-type, TRPV5^−/−, CaBP-D_28K^−/−, and TRPV5^−/−/CaBP-D_28K^−/− mice (n = 6) that were on a 0.02% (wt/wt) and a 2% (wt/wt) Ca²⁺ diet and probed with anti–CaBP-D_9K antibody (A) or anti–CaBP-D_28K antibody (C). The intensities of the CaBP-D_9K (B) and the CaBP-D_28K (D) immunopositive bands were quantified by densitometry and presented as a ratio to β-actin expression in relative percentages compared with wild-type mice that were fed a 0.02% (wt/wt) diet. Mice were fed a 0.02% (wt/wt; ▪) or 2% (wt/wt; □) Ca²⁺ diet. Data are means ± SEM. *P < 0.05 versus wild-type on the same diet; ^#P < 0.05 versus the same group on 0.02% (wt/wt) Ca²⁺ diet.

Figure 4.

Duodenal protein expression of CaBP-D_9K. Immunoblot analysis of duodenum homogenates from wild-type, TRPV5^−/−, CaBP-D_28K^−/−, and TRPV5^−/−/CaBP-D_28K^−/− mice (n = 6) that were on a 0.02% (wt/wt) and a 2% (wt/wt) Ca²⁺ diet and probed with anti–CaBP-D_9K antibody (A). The intensity of the CaBP-D_9K immunopositive bands was quantified by densitometry and presented as a ratio to β-actin expression in relative percentages compared with wild-type mice that were fed a 0.02% (wt/wt) diet (B). Mice were fed a 0.02% (wt/wt; ▪) or 2% (wt/wt; □) Ca²⁺ diet. Data are means ± SEM. *P < 0.05 versus wild-type on the same diet; ^#P < 0.05 versus the same group on 0.02% (wt/wt) Ca²⁺ diet.

Functional Analysis of Ca²⁺ (re)Absorption

The various mouse strains were functionally characterized by measurement of their urinary Ca²⁺ excretion and intestinal Ca²⁺ absorption. On both Ca²⁺ diets, urinary Ca²⁺ excretion was increased in TRPV5^−/− and TRPV5^−/−/calbindin-D_28K^−/− mice compared with wild-type mice. Conversely, Ca²⁺ excretion was not significantly different in calbindin-D_28K^−/− compared with wild-type mice. Dietary Ca²⁺ restriction did not affect the amount of Ca²⁺ excreted in the urine (Figure 5A). Subsequently, intestinal Ca²⁺ absorption was investigated by measurement of serum ⁴⁵Ca²⁺ at early time points after oral gavage. On the 0.02% (wt/wt) Ca²⁺ diet, the time curves of ⁴⁵Ca²⁺ absorption did not differ among the four mouse strains (Figure 5B). On the 2% (wt/wt) Ca²⁺ diet, intestinal ⁴⁵Ca²⁺ absorption was significantly reduced in all groups compared with mice that were fed the low-Ca²⁺ diet. However, the amount of ⁴⁵Ca²⁺ absorption remained significantly higher in TRPV5^−/− and TRPV5^−/−/calbindin-D_28K^−/− mice compared with wild-type and calbindin-D_28K^−/− mice (Figure 5C).

Figure 5.

Functional characterization of single- and double-knockout mice for TRPV5 and CaBP-D_28K. Twenty-four-hour urinary Ca²⁺ excretion in wild-type, TRPV5^−/−, CaBP-D_28K^−/−, and TRPV5^−/−/CaBP-D_28K^−/− mice (n = 10) that were fed a 0.02% (wt/wt; ▪) or 2% (wt/wt’ □) Ca²⁺ diet (A). Changes in serum Ca²⁺ (ΔμM) within 10 min after ⁴⁵Ca²⁺ administration by oral gavage in wild-type (♦), TRPV5^−/− (□), CaBP-D_28K^−/− (▴), and TRPV5^−/−/CaBP-D_28K^−/− (○) mice (n = 6) that were fed a 0.02% (wt/wt; B) or 2% (wt/wt; C) Ca²⁺ diet. Data are means ± SEM. *P < 0.05 versus wild-type on the same diet.

Discussion

Our study demonstrates that TRPV5 may constitute a more critical component of active Ca²⁺ reabsorption in kidney than calbindin-D_28K. This conclusion is based on the following experimental data. First, TRPV5^−/−/calbindin-D_28K^−/− and TRPV5^−/− mice showed a comparable hypercalciuria and compensatory Ca²⁺ hyperabsorption in comparison with wild-type mice. Second, the expression of calbindin-D_9K in kidney as well as calbindin-D_9K and TRPV6 in duodenum increased equally in TRPV5^−/−/calbindin-D_28K^−/− and TRPV5^−/− mice compared with wild-type mice. Third, upregulation of TRPV6 and cal-bindin-D_9K in TRPV5^−/−/calbindin-D_28K^−/− and TRPV5^−/− mice was accompanied by an analogous increase in serum PTH and 1,25(OH)₂D₃ levels. Fourth, urinary Ca²⁺ excretion, intestinal Ca²⁺ absorption, expression levels of the epithelial Ca²⁺ transporters, and serum parameters in calbindin-D_28K^−/− mice were not different from those in wild-type mice. Fifth, dietary Ca²⁺ restriction did not influence the Ca²⁺ excretion in the evaluated mice strains, whereas it enhanced intestinal Ca²⁺ absorption in TRPV5^−/−/calbindin-D_28K^−/− and TRPV5^−/− mice. The observed hyperabsorption is in line with the upregulation of duodenal calbindin-D_9K and TRPV6 expression.

Calbindin-D_28K contains six high-affinity binding sites for Ca²⁺ and is present predominantly in kidney, intestine (birds only), pancreas, placenta, bone, and brain (4,12). In these tissues, calbindin-D_28K is widely regarded as a key component in cellular Ca²⁺ handling by acting as a cytosolic Ca²⁺ buffer to protect cells against large fluctuations in the intracellular Ca²⁺ concentration (18), as well as a shuttle that facilitates Ca²⁺ diffusion from the luminal to the basolateral surface (4). In mouse kidney, calbindin-D_28K strikingly co-localizes with TRPV5, which constitutes the apical Ca²⁺ entry mechanism in DCT and CNT (4,19). Taking into account that calbindin-D_28K expression is regulated by calciotropic hormones in a similar way as TRPV5 (4,20), both proteins could be functionally linked in the process of active Ca²⁺ reabsorption. Indeed, TRPV5^−/− mice displayed a profound renal Ca²⁺ wasting combined with significant reduction of renal calbindin-D_28K expression levels. This suggested that the impaired TRPV5-mediated Ca²⁺ influx suppresses the expression of calbindin-D_28K. Our previous experiments in primary cultures of rabbit CNT and CCD cells demonstrated that blockage of TRPV5-mediated Ca²⁺ influx by the channel inhibitor ruthenium red downregulates calbindin-D_28K expression, indicating that regulation of the latter protein is highly dependent on the magnitude of the Ca²⁺ influx through TRPV5 (14). Arnold and Heintz (21) showed that Ca²⁺ is important for gene transcription. A Ca²⁺-responsive element was identified in the promoter sequence of calbindin-D_28K that partly underlies the Purkinje cell–specific expression of cal-bindin-D_28K. However, it is not known whether this element is active in kidney or whether additional intracellular signaling molecules are involved. Together, these findings underline the TRPV5-coordinated expression of calbindin-D_28K and suggest that TRPV5 constitutes the rate-limiting step of active Ca²⁺ reabsorption in kidney.

In contrast to TRPV5^−/− mice that displayed a significant hypercalciuria, calbindin-D_28K^−/− mice exhibit normal Ca²⁺ excretion values. In line with our data are two previous studies that showed that genetic ablation of calbindin-D_28K does not modulate Ca²⁺ excretion in mice that are fed a regular rodent diet that contains 1% (22) or 0.02% (wt/wt) Ca²⁺ (23,24). In contrast, Lee et al. (23) and Sooy et al. (24) fed calbindin-D_28K^−/− mice a defined diet that contained 1% (wt/wt) Ca²⁺ and showed a two- to three-fold increase in urinary Ca²⁺ excretion compared with wild-type controls. In addition, compared with vitamin D receptor (VDR) knockout mice, mice that lack both VDR and calbindin-D_28K and are fed a regular diet have significantly higher urinary Ca²⁺ excretion (1.7-fold), more severe hyperparathyroidism, and rachitic skeletal phenotype (22). Ca²⁺ excretion in TRPV5^−/− mice, however, was 10-fold higher than in wild-type mice and, therefore, more severe compared with calbindin-D_28K^−/− mice or mice that lack both the VDR and calbindin-D_28K. Furthermore, we showed that the renal Ca²⁺ leak in TRPV5^−/− mice is not increased in the TRPV5^−/−/calbindin-D_28K^−/− mice. These findings suggest that TRPV5 acts as the gatekeeper in the process of Ca²⁺ reabsorption in the DCT and CNT.

Although previous studies demonstrated increased Ca²⁺ excretion in calbindin-D_28K^−/− mice, our data indicate no significant differences in serum Ca²⁺, PTH, and 1,25(OH)₂D₃ levels in calbindin-D_28K^−/− mice compared with wild-type mice (24). A compensatory intestinal Ca²⁺ hyperabsorption or increased high bone turnover could occur in these knockout mice. In contrast, we found similar intestinal ⁴⁵Ca²⁺ absorption rates as well as intestinal TRPV6 and calbindin-D_9K expression in calbindin-D_28K^−/− and wild-type mice. Previous studies by Sooy et al. (24) and Zheng et al. (22) are in line with our data on intestinal calbindin-D_9K expression. Zheng et al. (22) demonstrated a modest decrease in bone mineral density in calbindin-D_28K^−/− mice. In addition, detailed structural analysis of teeth and bones showed that mineralization was unaffected in cal-bindin-D_28K^−/− mice (24). Consequently, neither a disturbed Ca²⁺ absorption nor an abnormal bone phenotype can account for the excess of urinary Ca²⁺ that was observed in their calbindin-D_28K^−/− mice. Theoretically, ablation of calbindin-D_28K should seriously impair the Ca²⁺ buffering capacity of the TRPV5-expressing cells in DCT and CNT, which in turn should inhibit the activity of TRPV5. However, the lack of a general hypercalciuria in calbindin-D_28K^−/− mice suggests that cal-bindin-D_28K deficiency might be compensated for by other renal Ca²⁺-binding proteins. It is interesting that the specific coexpression of calbindin-D_9K and calbindin-D_28K in mouse DCT cells hints to a comparable function of calbindin-D_9K in Ca²⁺ reabsorption (13). In the VDR^−/− mice, there is a 90% decrease in the level of renal calbindin-D_9K compared with wild-type mice (22). Therefore, in mice that lack both VDR and calbindin-D_28K, the increased urinary Ca²⁺ excretion may reflect the loss of compensation by calbindin-D_9K (22). However, we cannot exclude the possibility that other molecular mechanisms could compensate for the deficiency of calbindin-D_28K or that downstream reabsorptive nephron segments balance an impaired Ca²⁺ transport capacity of DCT that lack calbindin-D_28K.

In this study, we observed that the expression of renal and duodenal Ca²⁺ transporters is regulated by the dietary Ca²⁺ content. However, it is difficult to investigate the direct effects of dietary Ca²⁺ without affecting serum PTH and 1,25(OH)₂D₃ levels. Indeed, dietary Ca²⁺ restriction was accompanied by a compensatory increase in serum PTH and 1,25(OH)₂D₃ levels. Ample studies indicate that Ca²⁺ transporter genes are transcriptionally controlled by circulating 1,25(OH)₂D₃ (4). For instance, renal and intestinal calbindin-D_9K abundance correlated positively with serum 1,25(OH)₂D₃ levels as consistently shown in various mouse models (17,25,26). Conversely, intestinal calbindin-D_9K and plasma membrane Ca²⁺ ATPase expression was suppressed by alterations of dietary Ca²⁺ content in VDR^−/− mice (27). It is interesting that we demonstrated previously that a reduction in the expression of duodenal cal-bindin-D_9K but also TRPV6 can be normalized by a high-Ca²⁺ diet in 1α-OHase^−/− mice, which lack circulating 1,25(OH)₂D₃ (28). Furthermore, dietary Ca²⁺ controls the renal abundance of TRPV5, calbindin-D_28K, and Na⁺/Ca²⁺ exchanger in this latter knockout model (28). Altogether, these findings suggest that the abundance of Ca²⁺ transport proteins can be controlled by vitamin D–dependent and –independent means.

Conclusion

TRPV5 and calbindin-D_28K are functionally coupled and play an important role in renal Ca²⁺ handling, where TRPV5 constitutes the rate-limiting step of active Ca²⁺ reabsorption in DCT and CNT. In contrast to TRPV5^−/− mice, calbindin-D_28K^−/− mice display normal serum parameters, intestinal Ca²⁺ absorption, and renal Ca²⁺ excretion. Ablation of cal-bindin-D_28K in TRPV5^−/− mice does not aggravate the TRPV5^−/− phenotype, indicating that the role of calbindin-D_28K possibly can be compensated for by calbindin-D_9K.

Acknowledgments

This work was supported by the Dutch Organization of Scientific Research (Zon-Mw 016.006.001, Zon-Mw 902.18.298, NWO-ALW 810.38.004, and NWO-ALW 805.09.042), the Dutch Kidney foundation (C03.6017), and National Institutes of Health grant DK38961 to S.C.

We thank B. Pelkmans for expert technical assistance.

Footnotes

Published online ahead of print. Publication date available at www.jasn.org.
D.G. and Y.-J.H. contributed equally to this work.
See the related editorial, “Who Wins the Competition: TRPV5 or Calbindin-D_28K?,” on pages 2954–2956.

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OpenUrl Abstract/FREE Full Text
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Hoenderop JG, van der Kemp AW, Hartog A, van de Graaf SF, van Os CH, Willems PH, Bindels RJ: Molecular identification of the apical Ca2+ channel in 1,25-dihydroxyvitamin D3-responsive epithelia. J Biol Chem 274 : 8375 –8378, 1999
OpenUrl Abstract/FREE Full Text
↵
Peng JB, Chen XZ, Berger UV, Vassilev PM, Tsukaguchi H, Brown EM, Hediger MA: Molecular cloning and characterization of a channel-like transporter mediating intestinal calcium absorption. J Biol Chem 274 : 22739 –22746, 1999
OpenUrl Abstract/FREE Full Text
↵
Hoenderop JG, van Leeuwen JP, van der Eerden BC, Kersten FF, van der Kemp AW, Merillat AM, Waarsing JH, Rossier BC, Vallon V, Hummler E, Bindels RJ: Renal Ca2+ wasting, hyperabsorption, and reduced bone thickness in mice lacking TRPV5. J Clin Invest 112 : 1906 –1914, 2003
OpenUrl CrossRef PubMed
↵
Nijenhuis T, Hoenderop JG, Loffing J, van der Kemp AW, van Os CH, Bindels RJ: Thiazide-induced hypocalciuria is accompanied by a decreased expression of Ca2+ transport proteins in kidney. Kidney Int 64 : 555 –564, 2003
OpenUrl CrossRef PubMed
↵
Christakos S, Gabrielides C, Rhoten WB: Vitamin D-dependent calcium binding proteins: Chemistry, distribution, functional considerations, and molecular biology. Endocr Rev 10 : 3 –26, 1989
OpenUrl CrossRef PubMed
↵
Hoenderop JG, Dardenne O, van Abel M, van der Kemp AW, Van Os CH, St-Arnaud R, Bindels RJ: Modulation of renal Ca2+ transport protein genes by dietary Ca2+ and 1,25-dihydroxyvitamin D3 in 25-hydroxyvitamin D3–1a-hydroxylase knockout mice. FASEB J 16 : 1398 –1406, 2002
OpenUrl Abstract/FREE Full Text
↵
van Abel M, Hoenderop JG, van der Kemp AW, Friedlaen-der MM, van Leeuwen JP, Bindels RJ: Coordinated control of renal Ca2+ transport proteins by parathyroid hormone. Kidney Int 68 : 1708 –1721, 2005
OpenUrl CrossRef PubMed
↵
Airaksinen MS, Eilers J, Garaschuk O, Thoenen H, Konnerth A, Meyer M: Ataxia and altered dendritic calcium signaling in mice carrying a targeted null mutation of the calbindin D28k gene. Proc Natl Acad Sci U S A 94 : 1488 –1493, 1997
OpenUrl Abstract/FREE Full Text
↵
Dardenne O, Prud’homme J, Arabian A, Glorieux FH, St-Arnaud R: Targeted inactivation of the 25-hydroxyvitamin D3–1alpha-hydroxylase gene (CYP27B1) creates an animal model of pseudovitamin D-deficiency rickets. Endocrinology 142 : 3135 –3141, 2001
OpenUrl CrossRef PubMed
↵
Renkema KY, Nijenhuis T, van der Eerden BC, van der Kemp AW, Weinans H, van Leeuwen JP, Bindels RJ, Hoenderop JG: Hypervitaminosis D mediates compensatory Ca2+ hyperabsorption in TRPV5 knockout mice. J Am Soc Nephrol 16 : 3188 –3195, 2005
OpenUrl Abstract/FREE Full Text
↵
Guo Q, Christakos S, Robinson N, Mattson MP: Calbindin D28K blocks the proapoptotic actions of mutant presenilin 1: Reduced oxidative stress and preserved mitochondrial function. Proc Natl Acad Sci U S A 95 : 3227 –3232, 1998
OpenUrl Abstract/FREE Full Text
↵
Hoenderop JG, Willems PH, Bindels RJ: Toward a comprehensive molecular model of active calcium reabsorption. Am J Physiol Renal Physiol 278 : F352 –F360, 2000
OpenUrl Abstract/FREE Full Text
↵
Hoenderop JG, Muller D, Van Der Kemp AW, Hartog A, Suzuki M, Ishibashi K, Imai M, Sweep F, Willems PH, Van Os CH, Bindels RJ: Calcitriol controls the epithelial calcium channel in kidney. J Am Soc Nephrol 12 : 1342 –1349, 2001
OpenUrl Abstract/FREE Full Text
↵
Arnold DB, Heintz N: A calcium responsive element that regulates expression of two calcium binding proteins in Purkinje cells. Proc Natl Acad Sci U S A 94 : 8842 –8847, 1997
OpenUrl Abstract/FREE Full Text
↵
Zheng W, Xie Y, Li G, Kong J, Feng JQ, Li YC: Critical role of calbindin-D28k in calcium homeostasis revealed by mice lacking both vitamin D receptor and calbindin-D28k. J Biol Chem 279 : 52406 –52413, 2004
OpenUrl Abstract/FREE Full Text
↵
Lee CT, Huynh VM, Lai LW, Lien YH: Cyclosporine A-induced hypercalciuria in calbindin-D28k knockout and wild-type mice. Kidney Int 62 : 2055 –2061, 2002
OpenUrl CrossRef PubMed
↵
Sooy K, Kohut J, Christakos S: The role of calbindin and 1,25-dihydroxyvitamin D3 in the kidney. Curr Opin Nephrol Hypertens 9 : 341 –347, 2000
OpenUrl CrossRef PubMed
↵
Li YC, Bolt MJ, Cao LP, Sitrin MD: Effects of vitamin D receptor inactivation on the expression of calbindins and calcium metabolism. Am J Physiol Endocrinol Metab 281 : E558 –E564, 2001
OpenUrl Abstract/FREE Full Text
↵
Van Cromphaut SJ, Dewerchin M, Hoenderop JG, Stockmans I, Van Herck E, Kato S, Bindels RJ, Collen D, Carmeliet P, Bouillon R, Carmeliet G: Duodenal calcium absorption in vitamin D receptor-knockout mice: functional and molecular aspects. Proc Natl Acad Sci U S A 98 : 13324 –13329, 2001
OpenUrl Abstract/FREE Full Text
↵
Bouillon R, Van Cromphaut S, Carmeliet G: Intestinal calcium absorption: Molecular vitamin D mediated mechanisms. J Cell Biochem 88 : 332 –339, 2003
OpenUrl CrossRef PubMed
↵
van Abel M, Hoenderop JG, van der Kemp AW, van Leeuwen JP, Bindels RJ: Regulation of the epithelial Ca2+ channels in small intestine as studied by quantitative mRNA detection. Am J Physiol Gastrointest Liver Physiol 285 : G78 –G75, 2003
OpenUrl Abstract/FREE Full Text

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[1] ↵
Fukugawa M, Kurokawa K: Calcium homeostasis and imbalance. Nephron 92[Suppl 1] : 41 –45, 2002
OpenUrl CrossRef

[2] ↵
Bindels RJ: Calcium handling by the mammalian kidney. J Exp Biol 184 : 89 –104, 1993
OpenUrl Abstract/FREE Full Text

[3] ↵
Wasserman RH, Fullmer CS: Vitamin D and intestinal calcium transport: Facts, speculations and hypotheses. J Nutr 125[Suppl] : 1971S –1979S, 1995
OpenUrl Abstract/FREE Full Text

[4] ↵
Hoenderop JG, Nilius B, Bindels RJ: Calcium absorption across epithelia. Physiol Rev 85 : 373 –422, 2005
OpenUrl Abstract/FREE Full Text

[5] ↵
Bronner F, Pansu D, Stein WD: An analysis of intestinal calcium transport across the rat intestine. Am J Physiol 250 : G561 –G569, 1986

[6] ↵
Friedman PA, Gesek FA: Cellular calcium transport in renal epithelia: Measurement, mechanisms, and regulation. Physiol Rev 75 : 429 –471, 1995
OpenUrl Abstract/FREE Full Text

[7] ↵
van der Eerden BC, Hoenderop JG, de Vries TJ, Schoenmaker T, Buurman CJ, Uitterlinden AG, Pols HA, Bindels RJ, van Leeuwen JP: The epithelial Ca2+ channel TRPV5 is essential for proper osteoclastic bone resorption. Proc Natl Acad Sci U S A 102 : 17507 –17512, 2005
OpenUrl Abstract/FREE Full Text

[8] ↵
Hoenderop JG, van der Kemp AW, Hartog A, van de Graaf SF, van Os CH, Willems PH, Bindels RJ: Molecular identification of the apical Ca2+ channel in 1,25-dihydroxyvitamin D3-responsive epithelia. J Biol Chem 274 : 8375 –8378, 1999
OpenUrl Abstract/FREE Full Text

[9] ↵
Peng JB, Chen XZ, Berger UV, Vassilev PM, Tsukaguchi H, Brown EM, Hediger MA: Molecular cloning and characterization of a channel-like transporter mediating intestinal calcium absorption. J Biol Chem 274 : 22739 –22746, 1999
OpenUrl Abstract/FREE Full Text

[10] ↵
Hoenderop JG, van Leeuwen JP, van der Eerden BC, Kersten FF, van der Kemp AW, Merillat AM, Waarsing JH, Rossier BC, Vallon V, Hummler E, Bindels RJ: Renal Ca2+ wasting, hyperabsorption, and reduced bone thickness in mice lacking TRPV5. J Clin Invest 112 : 1906 –1914, 2003
OpenUrl CrossRef PubMed

[11] ↵
Nijenhuis T, Hoenderop JG, Loffing J, van der Kemp AW, van Os CH, Bindels RJ: Thiazide-induced hypocalciuria is accompanied by a decreased expression of Ca2+ transport proteins in kidney. Kidney Int 64 : 555 –564, 2003
OpenUrl CrossRef PubMed

[12] ↵
Christakos S, Gabrielides C, Rhoten WB: Vitamin D-dependent calcium binding proteins: Chemistry, distribution, functional considerations, and molecular biology. Endocr Rev 10 : 3 –26, 1989
OpenUrl CrossRef PubMed

[13] ↵
Hoenderop JG, Dardenne O, van Abel M, van der Kemp AW, Van Os CH, St-Arnaud R, Bindels RJ: Modulation of renal Ca2+ transport protein genes by dietary Ca2+ and 1,25-dihydroxyvitamin D3 in 25-hydroxyvitamin D3–1a-hydroxylase knockout mice. FASEB J 16 : 1398 –1406, 2002
OpenUrl Abstract/FREE Full Text

[14] ↵
van Abel M, Hoenderop JG, van der Kemp AW, Friedlaen-der MM, van Leeuwen JP, Bindels RJ: Coordinated control of renal Ca2+ transport proteins by parathyroid hormone. Kidney Int 68 : 1708 –1721, 2005
OpenUrl CrossRef PubMed

[15] ↵
Airaksinen MS, Eilers J, Garaschuk O, Thoenen H, Konnerth A, Meyer M: Ataxia and altered dendritic calcium signaling in mice carrying a targeted null mutation of the calbindin D28k gene. Proc Natl Acad Sci U S A 94 : 1488 –1493, 1997
OpenUrl Abstract/FREE Full Text

[16] ↵
Dardenne O, Prud’homme J, Arabian A, Glorieux FH, St-Arnaud R: Targeted inactivation of the 25-hydroxyvitamin D3–1alpha-hydroxylase gene (CYP27B1) creates an animal model of pseudovitamin D-deficiency rickets. Endocrinology 142 : 3135 –3141, 2001
OpenUrl CrossRef PubMed

[17] ↵
Renkema KY, Nijenhuis T, van der Eerden BC, van der Kemp AW, Weinans H, van Leeuwen JP, Bindels RJ, Hoenderop JG: Hypervitaminosis D mediates compensatory Ca2+ hyperabsorption in TRPV5 knockout mice. J Am Soc Nephrol 16 : 3188 –3195, 2005
OpenUrl Abstract/FREE Full Text

[18] ↵
Guo Q, Christakos S, Robinson N, Mattson MP: Calbindin D28K blocks the proapoptotic actions of mutant presenilin 1: Reduced oxidative stress and preserved mitochondrial function. Proc Natl Acad Sci U S A 95 : 3227 –3232, 1998
OpenUrl Abstract/FREE Full Text

[19] ↵
Hoenderop JG, Willems PH, Bindels RJ: Toward a comprehensive molecular model of active calcium reabsorption. Am J Physiol Renal Physiol 278 : F352 –F360, 2000
OpenUrl Abstract/FREE Full Text

[20] ↵
Hoenderop JG, Muller D, Van Der Kemp AW, Hartog A, Suzuki M, Ishibashi K, Imai M, Sweep F, Willems PH, Van Os CH, Bindels RJ: Calcitriol controls the epithelial calcium channel in kidney. J Am Soc Nephrol 12 : 1342 –1349, 2001
OpenUrl Abstract/FREE Full Text

[21] ↵
Arnold DB, Heintz N: A calcium responsive element that regulates expression of two calcium binding proteins in Purkinje cells. Proc Natl Acad Sci U S A 94 : 8842 –8847, 1997
OpenUrl Abstract/FREE Full Text

[22] ↵
Zheng W, Xie Y, Li G, Kong J, Feng JQ, Li YC: Critical role of calbindin-D28k in calcium homeostasis revealed by mice lacking both vitamin D receptor and calbindin-D28k. J Biol Chem 279 : 52406 –52413, 2004
OpenUrl Abstract/FREE Full Text

[23] ↵
Lee CT, Huynh VM, Lai LW, Lien YH: Cyclosporine A-induced hypercalciuria in calbindin-D28k knockout and wild-type mice. Kidney Int 62 : 2055 –2061, 2002
OpenUrl CrossRef PubMed

[24] ↵
Sooy K, Kohut J, Christakos S: The role of calbindin and 1,25-dihydroxyvitamin D3 in the kidney. Curr Opin Nephrol Hypertens 9 : 341 –347, 2000
OpenUrl CrossRef PubMed

[25] ↵
Li YC, Bolt MJ, Cao LP, Sitrin MD: Effects of vitamin D receptor inactivation on the expression of calbindins and calcium metabolism. Am J Physiol Endocrinol Metab 281 : E558 –E564, 2001
OpenUrl Abstract/FREE Full Text

[26] ↵
Van Cromphaut SJ, Dewerchin M, Hoenderop JG, Stockmans I, Van Herck E, Kato S, Bindels RJ, Collen D, Carmeliet P, Bouillon R, Carmeliet G: Duodenal calcium absorption in vitamin D receptor-knockout mice: functional and molecular aspects. Proc Natl Acad Sci U S A 98 : 13324 –13329, 2001
OpenUrl Abstract/FREE Full Text

[27] ↵
Bouillon R, Van Cromphaut S, Carmeliet G: Intestinal calcium absorption: Molecular vitamin D mediated mechanisms. J Cell Biochem 88 : 332 –339, 2003
OpenUrl CrossRef PubMed

[28] ↵
van Abel M, Hoenderop JG, van der Kemp AW, van Leeuwen JP, Bindels RJ: Regulation of the epithelial Ca2+ channels in small intestine as studied by quantitative mRNA detection. Am J Physiol Gastrointest Liver Physiol 285 : G78 –G75, 2003
OpenUrl Abstract/FREE Full Text

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Critical Role of the Epithelial Ca²⁺ Channel TRPV5 in Active Ca²⁺ Reabsorption as Revealed by TRPV5/Calbindin-D_28K Knockout Mice

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