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*
* Department of Physiology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; Division of Nephrology, Department of Internal Medicine, Tri-Service General Hospital, Taipei, Taiwan; and Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry, New Jersey Medical School, Newark, New Jersey
Address correspondence to: Dr. Joost G. Hoenderop, 286 Cell Physiology, Radboud University Nijmegen Medical Centre, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands. Phone: +31-24-3610571; Fax: +31-24-3616413; E-mail: j.hoenderop{at}ncmls.ru.nl
Received for publication June 29, 2006.
Accepted for publication August 8, 2006.
The epithelial Ca2+ channel TRPV5 facilitates apical Ca2+ entryduring active Ca2+ reabsorption in the distal convoluted tubule.In this process, cytosolic Ca2+ remains at low nontoxic concentrationsbecause the Ca2+ influx is buffered rapidly by calbindin-D28K.Subsequently, Ca2+ that is bound to calbindin-D28K is shuttledtoward the basolateral Ca2+ extrusion systems. For addressingthe in vivo role of TRPV5 and calbindin-D28K in the maintenanceof the Ca2+ balance, single- and double-knockout mice of TRPV5and calbindin-D28K (TRPV5–/–, calbindin-D28K–/–,and TRPV5–/–/calbindin-D28K–/–) werecharacterized. These mice strains were fed two Ca2+ diets (0.02and 2% wt/wt) to investigate the influence of dietary Ca2+ contenton the Ca2+ balance. Urine analysis indicated that TRPV5–/–/calbindin-D28K–/–mice exhibit on both diets hypercalciuria compared with wild-typemice. Ca2+ excretion in TRPV5–/–/calbindin-D28K–/–mice was not significantly different from TRPV5–/–mice, whereas calbindin-D28K–/– mice did not showhypercalciuria. The similarity between TRPV5–/–/calbindin-D28K–/–and TRPV5–/– mice was supported further by an equivalentincrease in renal calbindin-D9K expression and in intestinalCa2+ hyperabsorption as a result of upregulation of calbindin-D9Kand TRPV6 expression in the duodenum. Elevated serum parathyroidhormone and 1,25-dihydroxyvitamin D3 levels accompanied theenhanced expression of the Ca2+ transporters. Intestinal Ca2+absorption and expression of calbindin-D9K and TRPV6, as wellas serum parameters of the calbindin-D28K–/– mice,did not differ from those of wild-type mice. These results underlinethe gatekeeper function of TRPV5 being the rate-limiting stepin active Ca2+ reabsorption, unlike calbindin-D28K, which possiblyis compensated by calbindin-D9K.
Ca2+ homeostasis is of crucial importance for many physiologicfunctions, including neuronal excitability, muscle contraction,blood clotting, and bone mineralization. Therefore, the Ca2+balance is tightly controlled through constant regulation ofthree physiologic processes: Intestinal absorption, renal reabsorption,and exchange of Ca2+ from the bone mass (1). Both in intestineand in kidney, Ca2+ enters the interstitium by passive paracellularas well as active (re)absorption (2,3). Active Ca2+ (re)absorptionis critical in this process, because it constitutes the primarytarget for regulation by calciotropic hormones, including 1,25-dihydroxyvitaminD3 [1,25(OH)2D3] and parathyroid hormone (PTH), enabling theorganism to regulate the extracellular Ca2+ concentration onthe body’s demand (4).
Active absorption of dietary Ca2+ occurs primarily in the proximalsmall intestine, whereas renal active Ca2+ reabsorption is restrictedto the distal convoluted tubule (DCT) and the connecting tubule(CNT) (5,6). Ca2+ absorption occurs also in bone, where it iscrucial for bone formation to achieve adequate bone qualityand strength, as well as for osteoclastic bone resorption (7).At the cellular level, active Ca2+ (re)absorption implies entryof Ca2+ across the luminal membrane through the epithelial Ca2+channels, followed by intracellular buffering, facilitated diffusionby Ca2+-binding proteins, and finally extrusion across the basolateralmembrane by a Na+/Ca2+ exchanger and/or a plasma membrane Ca2+pump. Ca2+ influx occurs through two highly Ca2+-selective membersof the transient receptor potential (TRP) cation channel family,TRPV5 and TRPV6, which constitute the gatekeepers of activeCa2+ (re)absorption in kidney and intestine, respectively (8,9).Indeed, ablation of TRPV5 (TRPV5–/–) in mice impairsrenal Ca2+ reabsorption, resulting in robust hypercalciuria(10). As a consequence, TRPV5–/– mice develop compensatorydietary 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 cytosolicproteins to diffuse toward the basolateral surface of the epithelialcell. Two Ca2+-binding proteins, calbindin-D28K and calbindin-D9K,are regarded as key components of Ca2+ (re)absorption (4). Inmammals, calbindin-D28K is expressed primarily in kidney, whereascalbindin-D9K is abundantly present in small intestine. Onlyin mouse kidney are both calbindin-D28K and calbindin-D9K expressedin the distal part of the nephron (12). The physiologic importanceof calbindin-D28K in renal Ca2+-transporting epithelia is underlinedby the consistent coexpression with TRPV5 and their co-regulationby calciotropic hormones, including PTH, 1,25(OH)2D3, and alsodietary Ca2+ (13,14).
The aim of our study was to investigate whether calbindin-D28Kdeficiency is critical for active reabsorption in the presenceor absence of TRPV5. To this end, single- and double-knockoutmice 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+ transporterproteins at mRNA and protein levels.
Animal Experiments
TRPV5–/– mice were generated as described previously(10). Calbindin-D28K–/– mice were provided by Dr.Michael Meyer (Physiologisches Institut, Ludwig MaximiliansUniversität München, Munich, Germany) (15). Cross-breedingof TRPV5–/–/calbindin-D28K+/+ with TRPV5+/+/calbindin-D28K–/–mice resulted in offspring that were heterozygous for both TRPV5and calbindin-D28K (TRPV5+/–/cal-bindin-D28K+/–).This heterozygous offspring displayed the wild-type phenotypeand subsequently was intercrossed to obtain TRPV5–/–/calbindin-D28K–/–mice. Genotypes were determined by PCR analysis using specificprimers 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 antisenseprimer 5'-gcaagtaactaatggcatcg-3'. At the age of 4 wk, micewere fed ad libitum two diets that contained either 0.02 or2% (wt/wt) Ca2+ for 5 wk and subsequently placed in metaboliccages (Techniplast, Buggiate, Italy), which enabled 24-h collectionof urine. At the end of the experiment, blood samples were takenand the mice were killed. Subsequently, kidney and duodenumtissue was sampled. Urine and serum Ca2+ concentrations wereanalyzed using a colorimetric assay kit (Roche, Mannheim, Germany).Serum PTH was measured using an immunoradiometric assay (ImmutopicsInc., San Clemente, CA). Serum vitamin D levels were determinedby an [I125]1,25(OH)2D3 RIA assay (IDS Inc., Fountain Hills,AZ). The animal ethics board of Radboud University Nijmegenapproved 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-timequantitative PCR as described previously (17), using the ABIPrism 7700 Sequence Detection System (PE Biosystems, Rotkreuz,Switzerland). The expression level of the housekeeping genehypoxanthine-guanine phosphoribosyl transferase was used asan internal control to normalize differences in RNA extractionsand reverse transcription efficiencies.
Immunoblotting
Total kidney and duodenum lysates of all mouse groups were preparedas described previously (17). Briefly, protein concentrationsof the homogenates were determined by the Bio-Rad protein assay(Bio-Rad, München, Germany), and 10 µg of each samplewas loaded on 12 or 16.5% (wt/vol) SDS-PAGE gels and blottedto polyvinylidene difluoride nitrocellulose membranes (Immobilon-P,Millipore Corp., Bedford, MA). Blots were incubated with a rabbitanti–calbindin-D28K polyclonal antibody (1:10,000; Sigma,St. Louis, MO), a rabbit anti–calbindin-D9K polyclonalantibody (1:5000; Swant, Bellinzona, Switzerland), or a rabbit-actin polyclonal antibody (1:20,000; Sigma) at 4°C for16 h. Subsequently, blots were incubated with a goat anti-rabbitperoxidase-labeled secondary antibody (1:10,000; Sigma). Immunoreactiveprotein was detected by the chemiluminescence method (Pierce,Rockford, IL). The immunopositive protein bands were scannedand the pixel density was determined by using the MolecularAnalyst Software of Bio-Rad Laboratories (Hercules, CA).
In Vivo45Ca2+ Absorption Assay
Ca2+ absorption was assessed by measuring serum 45Ca2+ at earlytime points after oral gavage as described previously (10).Briefly, mice were fasted 16 h (overnight) before the test anda 45Ca2+ solution was administrated by oral gavage. Blood sampleswere obtained at indicated time intervals, and serum (10 µl)was analyzed by liquid scintillation counting. Differences inserum Ca2+ concentration were calculated from the 45Ca2+ contentin the samples and the specific activity of the administrated45Ca2+.
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 comparisonstest was applied. All analyses were performed using the StatviewStatistical Package Software (Power PC, version 4.51; Berkeley,CA).
Serum Parameters
Wild-type, 4-wk-old TRPV5–/–, calbindin-D28K–/–,and TRPV5–/–/calbindin-D28K–/– micewere fed a diet that contained 0.02 or 2% (wt/wt) Ca2+ for 5wk. All mice strains were fertile and had similar average littersizes (Table 1). Furthermore, serum analysis showed that TRPV5–/–and TRPV5–/–/calbindin-D28K–/– micethat were on the 0.02% (wt/wt) Ca2+ diet exhibit increased PTHand 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-typemice. The increased PTH and 1,25(OH)2D3 levels were normalizedin TRPV5–/– and TRPV5–/–/calbindin-D28K–/–mice that were fed the high-Ca2+ diet. Serum Ca2+ levels werenot significantly altered between the mice genotypes, regardlessof the dietary treatment (Table 1).
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 theCa2+ transporters in kidney and duodenum, we applied quantitativereal-time PCR assays. In kidney, calbindin-D9K mRNA expressionlevels were increased in TRPV5–/– and TRPV5–/–/calbindin-D28K–/–mice that were fed the 0.02 (wt/wt) Ca2+ diet compared withwild-type mice. Furthermore, renal calbindin-D9K expressionwas similar in calbindin-D28K–/– compared with wild-typemice. Exposure of the mice to the high-Ca2+ diet resulted indownregulation of renal calbindin-D9K mRNA in TRPV5–/–and TRPV5–/–/calbindin-D28K–/– mice(Figure 1A). Conversely, dietary Ca2+ content did not affectrenal expression of calbindin-D28K mRNA, which was significantlyreduced 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 demonstratedan upregulation of calbindin-D9K and TRPV6 mRNA expression incomparison with wild-type mice. On the same Ca2+ diet, duodenalcalbindin-D9K and TRPV6 expression remained unchanged in calbindin-D28K–/–compared with wild-type mice. The high-Ca2+ diet reduced intestinalcalbindin-D9K and TRPV6 mRNA expression in all mouse strains(Figure 2).
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. 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 duodenalmRNA levels of the Ca2+ transporters resulted in altered proteinexpression, the abundance of the Ca2+ transporters was semiquantifiedby immunoblot analysis. In kidney, calbindin-D9K protein expressionwas increased in TRPV5–/– and TRPV5–/–/calbindin-D28K–/–mice that were fed the 0.02% (wt/wt) Ca2+ diet compared withwild-type mice. On the same diet, calbindin-D28K–/–mice expressed wild-type levels of calbindin-D9K protein inkidney. However, an increase in dietary Ca2+ content from 0.02to 2% (wt/wt) resulted in downregulation of renal calbindin-D9Kin wild-type, TRPV5–/–, and TRPV5–/–/calbindin-D28K–/–mice (Figure 3, A and B). Furthermore, renal calbindin-D28Kprotein abundance was significantly decreased in TRPV5–/–mice in accordance with the downregulated mRNA levels. Variationsin dietary Ca2+ did not affect renal calbindin-D28K proteinexpression in both wild-type and TRPV5–/– mice (Figure 3,C and D). In duodenum, calbindin-D9K protein expression wasincreased 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 increaseof duodenal calbindin-D9K protein in TRPV5–/– andTRPV5–/–/calbindin-D28K–/– mice, whichwas consistent with the calbindin-D9K mRNA expression data (Figure 4).
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 -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-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 -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 bymeasurement of their urinary Ca2+ excretion and intestinal Ca2+absorption. On both Ca2+ diets, urinary Ca2+ excretion was increasedin TRPV5–/– and TRPV5–/–/calbindin-D28K–/–mice compared with wild-type mice. Conversely, Ca2+ excretionwas not significantly different in calbindin-D28K–/–compared with wild-type mice. Dietary Ca2+ restriction did notaffect the amount of Ca2+ excreted in the urine (Figure 5A).Subsequently, intestinal Ca2+ absorption was investigated bymeasurement of serum 45Ca2+ at early time points after oralgavage. 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 wassignificantly reduced in all groups compared with mice thatwere fed the low-Ca2+ diet. However, the amount of 45Ca2+ absorptionremained significantly higher in TRPV5–/– and TRPV5–/–/calbindin-D28K–/–mice compared with wild-type and calbindin-D28K–/–mice (Figure 5C).
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+ (µM) 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.
Our study demonstrates that TRPV5 may constitute a more criticalcomponent of active Ca2+ reabsorption in kidney than calbindin-D28K.This conclusion is based on the following experimental data.First, TRPV5–/–/calbindin-D28K–/– andTRPV5–/– mice showed a comparable hypercalciuriaand compensatory Ca2+ hyperabsorption in comparison with wild-typemice. Second, the expression of calbindin-D9K in kidney as wellas calbindin-D9K and TRPV6 in duodenum increased equally inTRPV5–/–/calbindin-D28K–/– and TRPV5–/–mice compared with wild-type mice. Third, upregulation of TRPV6and cal-bindin-D9K in TRPV5–/–/calbindin-D28K–/–and TRPV5–/– mice was accompanied by an analogousincrease in serum PTH and 1,25(OH)2D3 levels. Fourth, urinaryCa2+ excretion, intestinal Ca2+ absorption, expression levelsof the epithelial Ca2+ transporters, and serum parameters incalbindin-D28K–/– mice were not different from thosein wild-type mice. Fifth, dietary Ca2+ restriction did not influencethe Ca2+ excretion in the evaluated mice strains, whereas itenhanced intestinal Ca2+ absorption in TRPV5–/–/calbindin-D28K–/–and TRPV5–/– mice. The observed hyperabsorptionis in line with the upregulation of duodenal calbindin-D9K andTRPV6 expression.
Calbindin-D28K contains six high-affinity binding sites forCa2+ and is present predominantly in kidney, intestine (birdsonly), pancreas, placenta, bone, and brain (4,12). In thesetissues, calbindin-D28K is widely regarded as a key componentin cellular Ca2+ handling by acting as a cytosolic Ca2+ bufferto protect cells against large fluctuations in the intracellularCa2+ concentration (18), as well as a shuttle that facilitatesCa2+ diffusion from the luminal to the basolateral surface (4).In mouse kidney, calbindin-D28K strikingly co-localizes withTRPV5, which constitutes the apical Ca2+ entry mechanism inDCT and CNT (4,19). Taking into account that calbindin-D28Kexpression is regulated by calciotropic hormones in a similarway as TRPV5 (4,20), both proteins could be functionally linkedin the process of active Ca2+ reabsorption. Indeed, TRPV5–/–mice displayed a profound renal Ca2+ wasting combined with significantreduction of renal calbindin-D28K expression levels. This suggestedthat the impaired TRPV5-mediated Ca2+ influx suppresses theexpression of calbindin-D28K. Our previous experiments in primarycultures of rabbit CNT and CCD cells demonstrated that blockageof TRPV5-mediated Ca2+ influx by the channel inhibitor rutheniumred downregulates calbindin-D28K expression, indicating thatregulation of the latter protein is highly dependent on themagnitude of the Ca2+ influx through TRPV5 (14). Arnold andHeintz (21) showed that Ca2+ is important for gene transcription.A Ca2+-responsive element was identified in the promoter sequenceof calbindin-D28K that partly underlies the Purkinje cell–specificexpression of cal-bindin-D28K. However, it is not known whetherthis element is active in kidney or whether additional intracellularsignaling molecules are involved. Together, these findings underlinethe TRPV5-coordinated expression of calbindin-D28K and suggestthat TRPV5 constitutes the rate-limiting step of active Ca2+reabsorption in kidney.
In contrast to TRPV5–/– mice that displayed a significanthypercalciuria, calbindin-D28K–/– mice exhibit normalCa2+ excretion values. In line with our data are two previousstudies that showed that genetic ablation of calbindin-D28Kdoes not modulate Ca2+ excretion in mice that are fed a regularrodent 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 showeda two- to three-fold increase in urinary Ca2+ excretion comparedwith wild-type controls. In addition, compared with vitaminD receptor (VDR) knockout mice, mice that lack both VDR andcalbindin-D28K and are fed a regular diet have significantlyhigher 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–/– miceis not increased in the TRPV5–/–/calbindin-D28K–/–mice. These findings suggest that TRPV5 acts as the gatekeeperin the process of Ca2+ reabsorption in the DCT and CNT.
Although previous studies demonstrated increased Ca2+ excretionin calbindin-D28K–/– mice, our data indicate nosignificant differences in serum Ca2+, PTH, and 1,25(OH)2D3levels in calbindin-D28K–/– mice compared with wild-typemice (24). A compensatory intestinal Ca2+ hyperabsorption orincreased high bone turnover could occur in these knockout mice.In contrast, we found similar intestinal 45Ca2+ absorption ratesas well as intestinal TRPV6 and calbindin-D9K expression incalbindin-D28K–/– and wild-type mice. Previous studiesby Sooy et al. (24) and Zheng et al. (22) are in line with ourdata 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 andbones showed that mineralization was unaffected in cal-bindin-D28K–/–mice (24). Consequently, neither a disturbed Ca2+ absorptionnor an abnormal bone phenotype can account for the excess ofurinary Ca2+ that was observed in their calbindin-D28K–/–mice. Theoretically, ablation of calbindin-D28K should seriouslyimpair the Ca2+ buffering capacity of the TRPV5-expressing cellsin DCT and CNT, which in turn should inhibit the activity ofTRPV5. However, the lack of a general hypercalciuria in calbindin-D28K–/–mice suggests that cal-bindin-D28K deficiency might be compensatedfor by other renal Ca2+-binding proteins. It is interestingthat the specific coexpression of calbindin-D9K and calbindin-D28Kin mouse DCT cells hints to a comparable function of calbindin-D9Kin Ca2+ reabsorption (13). In the VDR–/– mice, thereis a 90% decrease in the level of renal calbindin-D9K comparedwith wild-type mice (22). Therefore, in mice that lack bothVDR and calbindin-D28K, the increased urinary Ca2+ excretionmay reflect the loss of compensation by calbindin-D9K (22).However, we cannot exclude the possibility that other molecularmechanisms could compensate for the deficiency of calbindin-D28Kor that downstream reabsorptive nephron segments balance animpaired Ca2+ transport capacity of DCT that lack calbindin-D28K.
In this study, we observed that the expression of renal andduodenal Ca2+ transporters is regulated by the dietary Ca2+content. However, it is difficult to investigate the directeffects of dietary Ca2+ without affecting serum PTH and 1,25(OH)2D3levels. Indeed, dietary Ca2+ restriction was accompanied bya compensatory increase in serum PTH and 1,25(OH)2D3 levels.Ample studies indicate that Ca2+ transporter genes are transcriptionallycontrolled by circulating 1,25(OH)2D3 (4). For instance, renaland intestinal calbindin-D9K abundance correlated positivelywith serum 1,25(OH)2D3 levels as consistently shown in variousmouse models (17,25,26). Conversely, intestinal calbindin-D9Kand plasma membrane Ca2+ ATPase expression was suppressed byalterations of dietary Ca2+ content in VDR–/– mice(27). It is interesting that we demonstrated previously thata reduction in the expression of duodenal cal-bindin-D9K butalso TRPV6 can be normalized by a high-Ca2+ diet in 1-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+ transportproteins can be controlled by vitamin D–dependent and–independent means.
TRPV5 and calbindin-D28K are functionally coupled and play animportant role in renal Ca2+ handling, where TRPV5 constitutesthe rate-limiting step of active Ca2+ reabsorption in DCT andCNT. 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 compensatedfor by calbindin-D9K.
Acknowledgments
This work was supported by the Dutch Organization of ScientificResearch (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 availableat www.jasn.org.
D.G. and Y.-J.H. contributed equally to this work.
See the related editorial, "Who Wins the Competition: TRPV5or Calbindin-D28K?," on pages 2954–2956.
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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.
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Top
Abstract
Introduction
-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). 
-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. 
) 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.
µM) within 10 min after 45Ca2+ administration by oral gavage in wild-type (
), TRPV5–/– (
), 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.