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Cell and Transport Physiology
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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 and Joost G. Hoenderop
JASN November 2006, 17 (11) 3020-3027; DOI: https://doi.org/10.1681/ASN.2006060676
Dimitra Gkika
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Yu-Juei Hsu
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Annemiete W. van der Kemp
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Sylvia Christakos
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René J. Bindels
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Joost G. Hoenderop
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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-D28K. Subsequently, Ca2+ that is bound to calbindin-D28K is shuttled toward the basolateral Ca2+ extrusion systems. For addressing the in vivo role of TRPV5 and calbindin-D28K in the maintenance of the Ca2+ balance, single- and double-knockout mice of TRPV5 and calbindin-D28K (TRPV5−/−, calbindin-D28K−/−, and TRPV5−/−/calbindin-D28K−/−) 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-D28K−/− mice exhibit on both diets hypercalciuria compared with wild-type mice. Ca2+ excretion in TRPV5−/−/calbindin-D28K−/− mice was not significantly different from TRPV5−/− mice, whereas calbindin-D28K−/− mice did not show hypercalciuria. The similarity between TRPV5−/−/calbindin-D28K−/− and TRPV5−/− mice was supported further by an equivalent increase in renal calbindin-D9K expression and in intestinal Ca2+ hyperabsorption as a result of upregulation of calbindin-D9K 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-D9K and TRPV6, as well as serum parameters of the calbindin-D28K−/− 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-D28K, which possibly is compensated by calbindin-D9K.

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-D28K and calbindin-D9K, are regarded as key components of Ca2+ (re)absorption (4). In mammals, calbindin-D28K is expressed primarily in kidney, whereas calbindin-D9K is abundantly present in small intestine. Only in mouse kidney are both calbindin-D28K and calbindin-D9K expressed in the distal part of the nephron (12). The physiologic importance of calbindin-D28K 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-D28K deficiency is critical for active reabsorption in the presence or absence of TRPV5. To this end, single- and double-knockout mice of calbindin-D28K and TRPV5 (calbindin-D28K−/−, TRPV5−/−, and TRPV5−/−/calbindin-D28K−/−) 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-D28K−/− mice were provided by Dr. Michael Meyer (Physiologisches Institut, Ludwig Maximilians Universität München, Munich, Germany) (15). Cross-breeding of TRPV5−/−/calbindin-D28K+/+ with TRPV5+/+/calbindin-D28K−/− mice resulted in offspring that were heterozygous for both TRPV5 and calbindin-D28K (TRPV5+/−/cal-bindin-D28K+/−). This heterozygous offspring displayed the wild-type phenotype and subsequently was intercrossed to obtain TRPV5−/−/calbindin-D28K−/− 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-D28K): 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-D28K, calbindin-D9K, 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-D28K polyclonal antibody (1:10,000; Sigma, St. Louis, MO), a rabbit anti–calbindin-D9K 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-D28K−/−, and TRPV5−/−/calbindin-D28K−/− 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-D28K−/− 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-D28K−/− 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-D28K−/− 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).

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Table 1.

Characteristics of TRPV5 and calbindin-D28K 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-D9K mRNA expression levels were increased in TRPV5−/− and TRPV5−/−/calbindin-D28K−/− mice that were fed the 0.02 (wt/wt) Ca2+ diet compared with wild-type mice. Furthermore, renal calbindin-D9K expression was similar in calbindin-D28K−/− compared with wild-type mice. Exposure of the mice to the high-Ca2+ diet resulted in downregulation of renal calbindin-D9K mRNA in TRPV5−/− and TRPV5−/−/calbindin-D28K−/− mice (Figure 1A). Conversely, dietary Ca2+ content did not affect renal expression of calbindin-D28K mRNA, which was significantly reduced in TRPV5−/− compared with wild-type mice (Figure 1B). In duodenum, TRPV5−/− and TRPV5−/−/cal-bindin-D28K−/− mice that were fed the 0.02% (wt/wt) Ca2+ diet demonstrated an upregulation of calbindin-D9K and TRPV6 mRNA expression in comparison with wild-type mice. On the same Ca2+ diet, duodenal calbindin-D9K and TRPV6 expression remained unchanged in calbindin-D28K−/− compared with wild-type mice. The high-Ca2+ diet reduced intestinal calbindin-D9K and TRPV6 mRNA expression in all mouse strains (Figure 2).

Figure 1.
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Figure 1.

Renal mRNA expression of Ca2+ transporters. Expression of calbindin (CaBP)-D9K (A) and CaBP-D28K (B) mRNA in kidney of wild-type, TRPV5−/−, CaBP-D28K−/−, and TRPV5−/−/CaBP-D28K−/− 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.
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Figure 2.

Duodenal mRNA expression of Ca2+ transporters. Expression of CaBP-D9K (A) and TRPV6 (B) mRNA in duodenum of wild-type, TRPV5−/−, CaBP-D28K−/−, and TRPV5−/−/CABP-D28K−/− 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-D9K protein expression was increased in TRPV5−/− and TRPV5−/−/calbindin-D28K−/− mice that were fed the 0.02% (wt/wt) Ca2+ diet compared with wild-type mice. On the same diet, calbindin-D28K−/− mice expressed wild-type levels of calbindin-D9K protein in kidney. However, an increase in dietary Ca2+ content from 0.02 to 2% (wt/wt) resulted in downregulation of renal calbindin-D9K in wild-type, TRPV5−/−, and TRPV5−/−/calbindin-D28K−/− mice (Figure 3, A and B). Furthermore, renal calbindin-D28K protein abundance was significantly decreased in TRPV5−/− mice in accordance with the downregulated mRNA levels. Variations in dietary Ca2+ did not affect renal calbindin-D28K protein expression in both wild-type and TRPV5−/− mice (Figure 3, C and D). In duodenum, calbindin-D9K protein expression was increased in TRPV5−/− and TRPV5−/−/calbindin-D28K−/− mice compared with wild-type and calbindin-D28K−/− mice, in line with measured mRNA expression levels. Finally, dietary Ca2+ restriction resulted in a significant increase of duodenal calbindin-D9K protein in TRPV5−/− and TRPV5−/−/calbindin-D28K−/− mice, which was consistent with the calbindin-D9K mRNA expression data (Figure 4).

Figure 3.
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Figure 3.

Renal protein expression of CaBP-D28K and CaBP-D9K. Immunoblot of total kidney homogenates from wild-type, TRPV5−/−, CaBP-D28K−/−, and TRPV5−/−/CaBP-D28K−/− mice (n = 6) that were on a 0.02% (wt/wt) and a 2% (wt/wt) Ca2+ diet and probed with anti–CaBP-D9K antibody (A) or anti–CaBP-D28K antibody (C). The intensities of the CaBP-D9K (B) and the CaBP-D28K (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.
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Figure 4.

Duodenal protein expression of CaBP-D9K. Immunoblot analysis of duodenum homogenates from wild-type, TRPV5−/−, CaBP-D28K−/−, and TRPV5−/−/CaBP-D28K−/− mice (n = 6) that were on a 0.02% (wt/wt) and a 2% (wt/wt) Ca2+ diet and probed with anti–CaBP-D9K antibody (A). The intensity of the CaBP-D9K 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-D28K−/− mice compared with wild-type mice. Conversely, Ca2+ excretion was not significantly different in calbindin-D28K−/− 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-D28K−/− mice compared with wild-type and calbindin-D28K−/− mice (Figure 5C).

Figure 5.
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Figure 5.

Functional characterization of single- and double-knockout mice for TRPV5 and CaBP-D28K. Twenty-four-hour urinary Ca2+ excretion in wild-type, TRPV5−/−, CaBP-D28K−/−, and TRPV5−/−/CaBP-D28K−/− 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-D28K−/− (▴), and TRPV5−/−/CaBP-D28K−/− (○) 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-D28K. This conclusion is based on the following experimental data. First, TRPV5−/−/calbindin-D28K−/− and TRPV5−/− mice showed a comparable hypercalciuria and compensatory Ca2+ hyperabsorption in comparison with wild-type mice. Second, the expression of calbindin-D9K in kidney as well as calbindin-D9K and TRPV6 in duodenum increased equally in TRPV5−/−/calbindin-D28K−/− and TRPV5−/− mice compared with wild-type mice. Third, upregulation of TRPV6 and cal-bindin-D9K in TRPV5−/−/calbindin-D28K−/− 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-D28K−/− 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-D28K−/− and TRPV5−/− mice. The observed hyperabsorption is in line with the upregulation of duodenal calbindin-D9K and TRPV6 expression.

Calbindin-D28K 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-D28K 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-D28K strikingly co-localizes with TRPV5, which constitutes the apical Ca2+ entry mechanism in DCT and CNT (4,19). Taking into account that calbindin-D28K 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-D28K expression levels. This suggested that the impaired TRPV5-mediated Ca2+ influx suppresses the expression of calbindin-D28K. 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-D28K 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-D28K that partly underlies the Purkinje cell–specific expression of cal-bindin-D28K. 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-D28K 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-D28K−/− mice exhibit normal Ca2+ excretion values. In line with our data are two previous studies that showed that genetic ablation of calbindin-D28K 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-D28K−/− 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-D28K 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-D28K−/− mice or mice that lack both the VDR and calbindin-D28K. Furthermore, we showed that the renal Ca2+ leak in TRPV5−/− mice is not increased in the TRPV5−/−/calbindin-D28K−/− 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-D28K−/− mice, our data indicate no significant differences in serum Ca2+, PTH, and 1,25(OH)2D3 levels in calbindin-D28K−/− 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-D9K expression in calbindin-D28K−/− 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-D9K expression. Zheng et al. (22) demonstrated a modest decrease in bone mineral density in calbindin-D28K−/− mice. In addition, detailed structural analysis of teeth and bones showed that mineralization was unaffected in cal-bindin-D28K−/− 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-D28K−/− mice. Theoretically, ablation of calbindin-D28K 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-D28K−/− mice suggests that cal-bindin-D28K deficiency might be compensated for by other renal Ca2+-binding proteins. It is interesting that the specific coexpression of calbindin-D9K and calbindin-D28K in mouse DCT cells hints to a comparable function of calbindin-D9K in Ca2+ reabsorption (13). In the VDR−/− mice, there is a 90% decrease in the level of renal calbindin-D9K compared with wild-type mice (22). Therefore, in mice that lack both VDR and calbindin-D28K, the increased urinary Ca2+ excretion may reflect the loss of compensation by calbindin-D9K (22). However, we cannot exclude the possibility that other molecular mechanisms could compensate for the deficiency of calbindin-D28K or that downstream reabsorptive nephron segments balance an impaired Ca2+ transport capacity of DCT that lack calbindin-D28K.

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-D9K abundance correlated positively with serum 1,25(OH)2D3 levels as consistently shown in various mouse models (17,25,26). Conversely, intestinal calbindin-D9K 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-D9K 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-D28K, 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-D28K 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-D28K−/− mice display normal serum parameters, intestinal Ca2+ absorption, and renal Ca2+ excretion. Ablation of cal-bindin-D28K in TRPV5−/− mice does not aggravate the TRPV5−/− phenotype, indicating that the role of calbindin-D28K possibly can be compensated for by calbindin-D9K.

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-D28K?,” on pages 2954–2956.

  • © 2006 American Society of Nephrology

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Journal of the American Society of Nephrology: 17 (11)
Journal of the American Society of Nephrology
Vol. 17, Issue 11
November 2006
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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
JASN Nov 2006, 17 (11) 3020-3027; DOI: 10.1681/ASN.2006060676

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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
JASN Nov 2006, 17 (11) 3020-3027; DOI: 10.1681/ASN.2006060676
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