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* Department of Physiology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; Department of
Internal Medicine and
Orthopedics, Erasmus Medical Centre Rotterdam, The Netherlands; and
Research Business Area Medical Chemistry, Schering AG, Berlin, Germany
1Correspondence: 286 Cell Physiology, Radboud University Nijmegen Medical Centre, P.O. Box 9101, Nijmegen, NL-6500 HB, The Netherlands. E-mail: r.bindels{at}ncmls.ru.nl
ABSTRACT
Vitamin D [1,25(OH)2D3] plays a crucial role in Ca2+ homeostasis by stimulating Ca2+ (re)absorption and bone turnover. The 1,25(OH)2D3 analog ZK191784 was recently developed to dissociate the therapeutic immunomodulatory activity from the hypercalcemic side effects of 1,25(OH)2D3 and contains a structurally modified side chain characterized by a 22,23-double bond, 24R-hydroxy group, 25-cyclopropyl ring, and 5-butyloxazole unit. We investigated the effect of ZK191784 on Ca2+ homeostasis and the regulation of Ca2+ transport proteins in wild-type (WT) mice and mice lacking the renal epithelial Ca2+ channel TRPV5 (TRPV5–/–). The latter display hypercalciuria, hypervitaminosis D, increased intestinal expression of the epithelial Ca2+ channel TRPV6, the Ca2+-binding protein calbindin-D9K, and intestinal Ca2+ hyperabsorption. ZK191784 normalized the Ca2+ hyperabsorption and the expression of intestinal Ca2+ transport proteins in TRPV5–/– mice. Furthermore, the compound decreased intestinal Ca2+ absorption in WT mice and reduced 1,25(OH)2D3-dependent 45Ca2+ uptake by Caco-2 cells, substantiating a 1,25(OH)2D3-antagonistic action of ZK191784 in the intestine. ZK191784 increased renal TRPV5 and calbindin-D28K expression and decreased urine Ca2+ excretion in WT mice. Both 1,25(OH)2D3 and ZK191784 enhanced transcellular Ca2+ transport in primary cultures of rabbit connecting tubules and cortical collecting ducts, indicating a 1,25(OH)2D3-agonistic effect in kidney. ZK191784 enhanced bone TRPV6 mRNA levels and 1,25(OH)2D3 as well as ZK191784 stimulated secretion of the bone formation marker osteocalcin in rat osteosarcoma cells, albeit to a different extent. In conclusion, ZK191784 is a synthetic 1,25(OH)2D3 ligand displaying a unique tissue-specific profile when administered in vivo. Because ZK191784 acts as an intestine-specific 1,25(OH)2D3 antagonist, this compound will be associated with less hypercalcemic side effects compared with the 1,25(OH)2D3 analogs currently used in clinical practice.—Nijenhuis, T., van der Eerden, B. C. J., Zügel, U., Steinmeyer, A., Weinans, H., Hoenderop, J. G. J., van Leeuwen, J. P. T. M., Bindels, R. J. M. The novel vitamin D analog ZK191784 as an intestine-specific vitamin D antagonist.
Key Words: TRPV5 • TRPV6 • Ca2+ homeostasis • 1,25(OH)2D3.
THE MAIN PHYSIOLOGICAL function of the active form of vitamin D [1,25(OH)2D3] is to stimulate intestinal and renal Ca2+ (re)absorption and regulate bone Ca2+ turnover (1
, 2)
. In addition, 1,25(OH)2D3 has potent antiproliferative, immunosuppressive, and immunomodulatory activity (3
4
5)
. However, therapeutic administration of 1,25(OH)2D3 frequently has dose-limiting hypercalcemic side effects, increasing the risk of soft-tissue and vascular calcification as well as osteoporosis when administered in a supraphysiological dose (6
, 7)
. Therefore, there has been great effort in identifying new 1,25(OH)2D3 analogs that retain a beneficial therapeutic profile combined with minimal calcemic action. Such analogs would have attractive clinical potential as immunomodulators in hyperproliferative disorders or to treat secondary hyperparathyroidism complicating chronic kidney disease (8)
.
The 1,25(OH)2D3 analog ZK191784 was developed in an effort to dissociate the immunomodulatory and hypercalcemic actions of 1,25(OH)2D3 (3)
. This compound contains a structurally modified side chain characterized by a 22,23-double bond, 24R-hydroxy group, 25-cyclopropyl ring, and 5-butyloxazole unit (Fig. 1
). ZK191784 competitively binds to the vitamin D receptor (VDR) with a similar affinity as 1,25(OH)2D3 (3)
. Like 1,25(OH)2D3, ZK191784 inhibited antigen-induced lymphocyte proliferation and cytokine secretion in vitro and exhibited potent immunosuppressive activity in a murine model of contact hypersensitivity. In addition, it exerted a 1,25(OH)2D3-antagonistic effect on the promyelocytic leukemia cell line HL-60. This latter cell model is often used to study the genomic responses of 1,25(OH)2D3 analogs (3
, 9)
, and antagonism of HL-60 differentiation was previously shown with several vitamin D analogs that have 1,25(OH)2D3-antagonistic profiles in vivo (10
11
12
13)
. However, the in vivo effects of ZK191784 regarding Ca2+ homeostasis and regulation of the Ca2+ transport proteins have not been evaluated in detail.
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1,25(OH)2D3-stimulated transcellular Ca2+ (re)absorption involves Ca2+ entry across the luminal membrane via the epithelial Ca2+ channels TRPV5 and TRPV6 (1
, 14
15
16)
. TRPV5 is localized at the luminal membrane of the late distal convoluted tubule (DCT) and connecting tubule (CNT) in kidney. TRPV6 is the homologous epithelial Ca2+ channel localized along the brush-border membrane of the duodenum. After Ca2+ entry across the luminal membrane, Ca2+ bound to Ca2+-binding proteins (calbindins) diffuse to the basolateral membrane of the cell. Ca2+ is finally extruded to the blood compartment by the Na+/Ca2+ exchanger (NCX1) and/or the plasma membrane Ca2+-ATPase (PMCA1b). The stimulatory effect of 1,25(OH)2D3 on the expression of the epithelial Ca2+ channels in kidney and duodenum has been demonstrated and is probably associated with its hypercalcemic side effects (1)
. Furthermore, recent studies showed the expression of TRPV5 and TRPV6 in bone and demonstrated that TRPV5 is exclusively expressed in osteoclasts where it is involved in osteoclastic bone resorption (17
, 18)
.
TRPV5 knockout (TRPV5–/–) mice display profound renal Ca2+ wasting due to impaired active Ca2+ reabsorption in DCT and CNT (19)
. Furthermore, elevated serum 1,25(OH)2D3 levels, intestinal Ca2+ hyperabsorption, and reduced bone thickness were demonstrated in these mice. We showed that additional ablation of 25-hydroxyvitamin-D3-1
-hydroxylase (1-OHase), the renal enzyme responsible for 1,25(OH)2D3 biosynthesis, decreased intestinal TRPV6 expression and Ca2+ absorption in TRPV5–/–/1-OHase–/– mice (20)
. Therefore, hypervitaminosis D in these mice appears to represent a compensatory mechanism in an effort to counteract the significant renal Ca2+ leak. Thus, TRPV5–/– mice constitute an ideal animal model to study the effects of compounds with possible 1,25(OH)2D3-antagonistic actions.
The aim of the present study was, therefore, to evaluate the in vivo effect of the novel 1,25(OH)2D3 analog ZK191784 on Ca2+ and bone homeostasis in WT and TRPV5–/– mice. Animals were treated with ZK191784 for 28 days, after which Ca2+ absorption, Ca2+ excretion,and expression of the Ca2+ transport proteins in intestine, kidney, and bone was determined. In addition, bone morphometry was assessed by detailed microcomputed tomographic analysis. Further characterization of the actions of ZK191784 on intestinal, renal, and osteoblast cell lines was performed to reveal the biological profile of this novel 1,25(OH)2D3 analog.
MATERIALS AND METHODS
ZK191784 treatment in TRPV5+/+ (wild-type) and TRPV5–/– mice
TRPV5–/– mice were generated by targeted ablation of the TRPV5 gene (19)
. TRPV5+/+ (wild-type) mice and TRPV5–/– littermates were housed in a light and temperature-controlled room with ad libitum access to deionized drinking water and standard pelleted chow (0.25% (wt/v) Na; 1.1% (wt/v) Ca). Ten-week-old TRPV5+/+ and TRPV5–/– mice were treated during 28 days with 50 µg/kg/day ZK191784 [(5Z,7E,22E)-(1S,3R,24R)-25-(5-butyloxazole-2-yl)-26,27-cyclo-9,10-secocholesta-5,7,10(19),22-tetraene-1,3,24-triol; Schering AG, Berlin, Germany; Fig. 1
; n=9] or vehicle (n=9) by daily subcutaneous injection. This dose was based on previous in vivo experiments using ZK191784 (3)
. Mice were treated for 28 days, after which the animals were housed in metabolic cages enabling ration feeding and collection of 24 h urine samples under mineral oil, preventing evaporation. At the end of the experiment the animals were killed, blood samples were taken, and duodenum, kidney, and femur were sampled. The animal ethics board of the Radboud University Nijmegen approved all animal studies.
Analytical procedures
Serum and urine Ca2+ concentrations were determined using a colorimetric assay as described previously (21
, 22)
. Mouse serum PTH was measured using an immunoradiometric assay (Immutopics, San Clemente, CA). Na+ and Li+ concentrations were determined flame-spectrophotometrically (Eppendorf FCM 6343, Hamburg, Germany). Urine pH was measured using an electronic ion analyzer (Hanna Instruments, Szeged, Hungary).
In vivo 45Ca2+ absorption assay
Intestinal Ca2+ absorption was assessed by measuring serum 45Ca2+ at early time points after oral gavage. Mice were treated for 28 days with 50 µg/kg/day ZK191784 or vehicle as described above and were fasted 12 h before the experiment. Animals were hemodynamically stable under anesthesia during the assay. The 45CaCl2 was administrated by oral gavage as described previously (19)
. Blood samples were obtained at indicated time intervals, and serum was analyzed by liquid scintillation counting. The change in the plasma Ca2+ concentration (
µM) was calculated from the 45Ca2+ content of the serum samples and the specific activity of the administered Ca2+.
Real-time quantitative polymerase chain reaction analysis
Total RNA was extracted from duodenum, kidney, and bone using TriZol Total RNA Isolation Reagent (GIBCO, Breda, the Netherlands). Femurs, from which the bone marrow was removed by flushing with PBS, were first homogenized using a Mikro Dismembrator S (Sartorius, Goettingen, Germany). The obtained RNA was subjected to DNase treatment and reverse transcribed (22)
. Subsequently, the cDNA was used to determine TRPV6 and calbindin-D9K mRNA levels in duodenum, TRPV5 and calbindin-D28K mRNA expression in kidney, and TRPV5 and TRPV6 mRNA in bone by real-time quantitative polymerase chain reaction (PCR) as described previously (22
, 23)
. In addition, mRNA expression of the housekeeping gene hypoxanthine-guanine phosphoribosyl transferase (HPRT) was determined as an endogenous control, which enabled calculation of specific mRNA expression levels as a ratio of HPRT. To determine the mRNA expression level of TRPV6 in human osteoblast cultures, species-specific primers and probes were used. For TRPV6, these were the forward primer, 5'-GCTTTGCTTCAGCCTTCTATATCAT-3'; reverse primer, 5'-TGGTAAGGAACAGCTCGAAGGT-3' and 5'-AGGAGCTAGGCCACTTCTACGACTAC CCCA-3' as probe. In the case of the housekeeping gene GAPDH, these were 5'-ATGGGGAAGGTGAAGGTCG-3', 5'-TAAAAGCAGCCCTGGTGACC-3', and 5'-CGCC CAATACGACCAAATCCGTTGAC-3'.
Immunohistochemistry
Staining of kidney sections for TRPV5 and calbindin-D28K was performed on cryosections of periodate-lysine-paraformaldehyde fixed kidney samples as described previously (22
, 23)
. For semiquantitative determination of protein abundance, images were made using a Zeiss fluorescence microscope equipped with a digital camera (Nikon DXM1200). Images were analyzed with the Image Pro Plus 4.1 image analysis software (Media Cybernetics, Silver Spring, MD), resulting in quantification of the protein levels as the mean of integrated optical density (IOD).
Bone analysis
To evaluate the effects of ZK191784 on bone and the possible correlation between epithelial Ca2+ channel expression and bone homeostasis, femurs from control and ZK191784-treated TRPV5+/+ and TRPV5–/– mice were scanned using the SkyScan 1072 microtomograph (SkyScan, Antwerp, Belgium; ref 19
). Scans were processed, and a three-dimensional morphometric analysis of the bone was performed, using the 3D-Calculator project free software (http://www.eur.nl/fgg/orthopaedics/Downloads.html). Measured parameters were expressed according to bone histomorphometry nomenclature (24)
.
45Ca2+ uptake assay in the intestinal Caco-2 cell line
We performed a 45Ca2+ uptake assay in the human colon cancer Caco-2 cell line, which displays duodenal characteristics, to determine the effect of ZK191784 in an established intestinal cell model. The 45Ca2+ uptake assay was performed as described previously (15)
. In short, confluent monolayers of Caco-2 cells were incubated for 48 h in normal Dulbecco’s modified Eagle’s medium (DMEM) culture medium (15)
or culture medium supplemented with 1 · 10–7 M 1,25(OH)2D3, 1 · 10–7 M ZK191784, or 1 · 10–7 M 1,25(OH)2D3 together with 1 · 10–7 M ZK191784, respectively. Cells were washed with Krebs-Henseleit buffer (KHB) and, subsequently, were preincubated for 8 min in KHB or KHB supplemented with 10 µM of the TRPV6 blocker ruthenium red (Fluka, St. Louis, MO; ref 1
). Thereafter, the preincubation buffer was exchanged for 45Ca2+ uptake buffer, containing 0.1 mM CaCl2, 2 mM NaH2PO4, 10 µM felodipine, 10 µM verapamil, and 1 µCi 45CaCl2. After incubation for 8 min, cells were washed three times with ice-cold stop buffer consisting of KHB supplemented with 0.5 mM CaCl2 and 1.5 mM LaCl3. Cells were lysed in 0.1% (wt/v) SDS, and radioactivity of the lysate was measured using a liquid scintillation counter.
Transcellular Ca2+ transport in rabbit kidney CNT and CCD primary cell cultures
Rabbit kidney CNT and CCD tubules were immunodissected from the kidney cortex of New Zealand White rabbits and grown on permeable filters (Costar 0.33 cm2), as described in detail previously (25)
. Filters containing confluent monolayers of CNT and CCD cells were incubated with normal DMEM culture medium (25)
or culture medium supplemented with 1 · 10–7 M 1,25(OH)2D3, 1 · 10–7 M ZK191784, or 1 · 10–7 M 1,25(OH)2D3 together with 1 · 10–7 M ZK191784, respectively. After 48 h of incubation, transepithelial Ca2+ transport was measured during 90 min as described previously.
Osteocalcin secretion in rat osteosarcoma cells
Osteocalcin is produced by mature postproliferative osteoblasts at the onset of extracellular matrix (ECM) production. Ligand-induced osteocalcin production by reactive oxygen species (ROS) 17/2.8 cells was used to assess the bone formation potential of ZK191784. ROS 17/2.8 cells were cultured in DMEM culture medium containing 5% (v/v) charcoal treated fetal calf serum. Increasing concentrations of 1,25(OH)2D3 and ZK191784 were applied for 72 h to obtain a dose-response curve. Subsequently, the amount of osteocalcin produced was measured.
Statistical analysis
Data are mean ± SE. Statistical comparisons were analyzed by one-way ANOVA and Fisher’s multiple comparison. P values <0.05 were considered statistically significant. All analyses were performed using the StatView Statistical Package software (Power PC version 4.51, Berkely, CA) on an Apple iMac computer.
RESULTS
Metabolic studies in ZK191784-treated WT and TRPV5–/– mice
WT and TRPV5–/– mice were treated for 28 days with 50 µg/kg/day ZK191784 or vehicle by daily subcutaneous injection. The obtained metabolic data are shown in Fig. 2
and Table 1
. Genetic ablation of TRPV5 resulted in a 10-fold increase in Ca2+ excretion compared with WT mice (Fig. 2A
). The in vivo 45Ca2+ absorption measurements showed a profound enhancement of intestinal Ca2+ absorption in these TRPV5–/– mice (Fig. 2B
). This was accompanied by a minor but significant increase in the serum Ca2+ concentration (Table 1)
. ZK191784 treatment in TRPV5–/– mice normalized the intestinal Ca2+ hyperabsorption as well as the serum Ca2+ concentration. In addition, Ca2+ excretion was decreased by ZK191784 administration in TRPV5–/– mice but remained significantly elevated compared with WT mice (Fig. 2A
). Furthermore, ZK191784 treatment significantly diminished intestinal Ca2+ absorption and decreased Ca2+ excretion in WT mice, without altering serum Ca2+ levels (Fig. 2B
; Table 1
). Urine volume, Na+ excretion, and Li+ clearance were not affected by ZK191784 in both TRPV5–/– and TRPV5+/+ mice (Table 1)
. Furthermore, serum PTH levels did not significantly differ in the treated groups.
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Duodenal mRNA expression of Ca2+ transport proteins
To study the effect of ZK191784 on the abundance of Ca2+ transporters in the intestine, TRPV6 and calbindin-D9K mRNA expression was determined by real-time quantitative PCR analysis. TRPV5–/– mice showed profoundly increased TRPV6 (Fig. 3
A) and calbindin-D9K (Fig. 3B
) mRNA levels in duodenum compared with WT mice. Administration of ZK191784 to TRPV5–/– mice significantly reduced the TRPV6 and calbindin-D9K mRNA abundance, resulting in a complete normalization of the expression of the intestinal Ca2+ transporters. ZK191784 treatment did not significantly alter TRPV6 and calbindin-D9K mRNA levels in WT mice.
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Renal expression of Ca2+ transport proteins
To evaluate the effect of ZK191784 on the expression of the Ca2+ transport proteins in the kidney, TRPV5 and calbindin-D28K mRNA, as well as protein abundance, was determined by real-time quantitative PCR (Fig. 4
) and semiquantitative immunohistochemistry, respectively (Fig. 5
). ZK191784 significantly increased TRPV5 and calbindin-D28K mRNA levels and enhanced protein abundance of these Ca2+ transporters in DCT and CNT of WT mice. In contrast, ZK191784 did not significantly alter the reduced calbindin-D28K mRNA and protein expression in TRPV5–/– mice.
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Expression of TRPV5 and TRPV6 mRNA in bone
Besides duodenum and kidney, bone is an important tissue in Ca2+ homeostasis and it was previously shown that the epithelial Ca2+ channels TRPV5 and TRPV6 are expressed in this tissue (18)
. Bone TRPV5 mRNA levels as determined in femur were not affected by ZK191784 in WT mice (Fig. 6
A). TRPV5 gene ablation did not alter the TRPV6 mRNA levels in femur (Fig. 6B
). However, TRPV6 mRNA expression was significantly enhanced in ZK191784-treated WT and TRPV5–/– mice.
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Bone analysis
To evaluate the effects of ZK191784 on bone morphology, femurs were scanned using microcomputed tomography (Fig. 7
). Detailed three-dimensional morphometric analysis demonstrated that trabecular thickness (Tb.Th) is reduced in the femoral head of TRPV5–/– mice (Table 2
). ZK191784 did not significantly affect the bone morphometric parameters in the femoral head of both mice strains. Analysis of the metaphysis and diaphysis showed that cortical thickness (Ct.Th) is also significantly reduced in TRPV5–/– mice compared with WT mice but is unaffected by ZK191784 treatment. There were no differences observed in the other trabecular and cortical bone parameters between the treated groups.
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45Ca2+ uptake assay in the intestinal Caco-2 cell line
To determine the effect of ZK191784 in an isolated intestinal cell model, 45Ca2+ uptake was determined in the human Caco-2 cell line, which has duodenal characteristics and expresses TRPV6 and calbindin-D9K (1
, 26)
. Application of 1 · 10–7 M 1,25(OH)2D3 for 48 h enhanced the ruthenium red-sensitive 45Ca2+ uptake, substantiating the presence of 1,25(OH)2D3-responsive and TRPV6-mediated Ca2+ absorption in these polarized epithelial intestinal cells (Fig. 8
A). In contrast, incubation with 1 · 10–7 M ZK191784 did not stimulate 45Ca2+ uptake. Importantly, concomitant application of 1 · 10–7 M ZK191784 with 1 · 10–7 M 1,25(OH)2D3 significantly inhibited the 1,25(OH)2D3-dependent 45Ca2+ uptake by Caco-2 cells.
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Transcellular Ca2+ transport in rabbit kidney CNT and CCD primary cell cultures
Transcellular Ca2+ transport was measured in primary cultures of immunodissected rabbit kidney CNT and CCD cells grown to confluency on permeable filter supports. Application of 1 · 10–7 M 1,25(OH)2D3 for 48 h enhanced transcellular Ca2+ absorption by the confluent monolayers (Fig. 8B
). Importantly, 1 · 10–7 M ZK191784 also stimulated Ca2+ transport. Addition of 1 · 10–7 M 1,25(OH)2D3 in the presence of 1 · 10–7 M ZK191784 did not result in a further enhancement of transepithelial Ca2+ transport.
Osteocalcin secretion in rat osteosarcoma cells
Ligand-induced osteocalcin production by ROS 17/2.8 cells was used to assess the potential of ZK191784 to induce bone formation. Osteocalcin is produced by mature osteoblasts at the onset of ECM production. Both 1,25(OH)2D3 and ZK191784 induced osteocalcin production in a dose-dependent manner (Fig. 9
). The concentration for half maximal increase (EC50) was 2.3 x 10–10 M for 1,25(OH)2D3 and 5.3 x 10–8 M for ZK191784. The efficacy of ZK191784 compared with 1,25(OH)2D3 was 41%.
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DISCUSSION
The present study demonstrated that ZK191784 acts as an intestinal 1,25(OH)2D3 antagonist by diminishing 1,25(OH)2D3-stimulated Ca2+ absorption. Studies in TRPV5–/– mice indicated that this action was achieved by directly down-regulating intestinal Ca2+ transport protein expression. In contrast, in vivo and in vitro experiments indicated that ZK191784 exerts partial agonistic actions on Ca2+ handling in kidney. This tissue-specific partial 1,25(OH)2D3 agonism/antagonism reflects a biological profile unlike any other 1,25(OH)2D3 analog tested so far. Because ZK191784 does not stimulate intestinal Ca2+ absorption, this compound will be associated with less hypercalcemic side effects compared with1,25(OH)2D3 and its analogs currently used in clinical practice.
1,25(OH)2D3 is an important stimulatory hormone of intestinal Ca2+ absorption and is known to enhance the expression of the duodenal Ca2+ transporters (1)
. TRPV5–/– mice were previously shown to display hypervitaminosis D due to the profound renal Ca2+ wasting caused by abolishment of active Ca2+ transport in DCT and CNT (19)
. Indeed, these mice demonstrated significantly enhanced duodenal TRPV6 and calbindin-D9K expression and Ca2+ hyperabsorption. Importantly, the 1,25(OH)2D3 analog ZK191784 normalized the increased expression of the intestinal Ca2+ transporters and, thereby, antagonized intestinal Ca2+ hyperabsorption in TRPV5–/– mice. Furthermore, ZK191784 diminished Ca2+ absorption in WT mice. In contrast, previous studies from our laboratory demonstrated that 1,25(OH)2D3 and several of its analogs enhance Ca2+ transporter expression and intestinal Ca2+ absorption in these mice (1
, 27
, 28)
. The fact that duodenal Ca2+ transporter expression was not significantly altered in WT mice suggests that ZK191784 might also antagonize nongenomic effects of 1,25(OH)2D3 in the intestine. This finding is in line with previous studies that suggested that when dietary Ca2+ content is sufficient, there is only limited genomic 1,25(OH)2D3-dependent stimulation of active Ca2+ absorption and, therefore, competitive binding of ZK191784 to the nuclear VDR does not significantly affect Ca2+ transporter expression in these mice (28)
. Taken together, these in vivo data indicated that ZK191784 specifically inhibits 1,25(OH)2D3-stimulated intestinal Ca2+ absorption. To test this hypothesis in vitro, we used the intestine-derived Caco-2 cell line, which was previously shown to express both TRPV6 and calbindin-D9K (26)
. These experiments showed that in the absence of 1,25(OH)2D3, ZK191784 does not affect Ca2+ uptake by these cells. However, applied in combination with 1,25(OH)2D3, ZK191784 was able to significantly diminish 1,25(OH)2D3-stimulated Ca2+ uptake. Thus, the present data demonstrated that ZK191784, unlike 1,25(OH)2D3, does not stimulate Ca2+ uptake by the intestine and exerts a unique antagonistic effect on 1,25(OH)2D3-stimulated active Ca2+ absorption. This can explain why ZK191784 possesses reduced hypercalcemic potency, when administered in a dose known to exert immunosuppressive effects in vivo (3)
.
ZK191784 reduced renal Ca2+ excretion and significantly enhanced the expression levels of TRPV5 and calbindin-D28K in WT mice. These proteins are tightly regulated by 1,25(OH)2D3 and are crucial for renal Ca2+ reabsorption, which is exemplified by the robust hypercalciuria in TRPV5–/– mice (1
, 19)
. Therefore, the concomitantly increased Ca2+ transporter expression and reduced Ca2+ excretion in WT mice suggested that ZK191784 exerts a 1,25(OH)2D3-agonistic action on renal active Ca2+ reabsorption. The stimulatory effect of ZK191784 on transcellular Ca2+ transport in primary cultures of rabbit CNT and CCD substantiated these findings. Interestingly, ZK191784 did not increase calbindin-D28K abundance in TRPV5–/– mice. However, previous studies from our group demonstrated that blockade of TRPV5-mediated Ca2+ influx in DCT and CNT cells down-regulates calbindin-D28K expression (29)
. This indicated that regulation of the latter protein is highly dependent on the magnitude of the Ca2+ influx through TRPV5. This could explain the significantly reduced calbindin-D28K expression in TRPV5–/– mice, despite elevated 1,25(OH)2D3 levels (19)
, as well as the absence of a stimulatory effect of ZK191784 in these mice. Interestingly, ZK191784 still resulted in a Ca2+-sparing action in TRPV5–/– mice that, obviously, cannot be explained by stimulation of active Ca2+ reabsorption. The unaffected Li+ clearance compared with controls, as an inverse measure of proximal tubular Na+ reabsorption to which passive Ca2+ reabsorption is functionally coupled, does also not support enhanced proximal tubular Ca2+ reabsorption. However, abolishment of the compensatory intestinal Ca2+ hyperabsorption and reduced serum Ca2+ levels will likely result in a decreased filtered Ca2+ load and, therefore, would be in line with the decreased Ca2+ excretion. Likewise, the fact that ZK191784 had only a small effect on Ca2+ excretion in TRPV5–/– mice demonstrated that the 1,25(OH)2D3-mediated Ca2+ hyperabsorption does not contribute largely to the hypercalciuria. Together, these findings underline the presence of a primary renal Ca2+ leak in TRPV5–/– mice.
The expression of TRPV5 and TRPV6 in bone was previously demonstrated, but the functional role of these epithelial Ca2+ channels in this tissue remained unknown (18)
. In the present study, we showed that TRPV5–/– mice display unaltered bone TRPV6 expression, suggesting that TRPV6 does not compensate for the absence of TRPV5. This argues against redundancy of epithelial Ca2+ channels in bone and indicates that both channels play distinct roles in bone Ca2+ homeostasis. Recently, the exclusive expression of TRPV5 in the ruffled border of osteoclasts in bone was demonstrated (17)
. Furthermore, cultured osteoclasts from TRPV5–/– mice displayed reduced bone resorptive capacity, suggesting that this channel is involved in osteoclastic bone resorption. However, ZK191784 did not alter TRPV5 expression in WT mice but increased bone TRPV6 expression in both mice strains. Ligand-induced osteocalcin production by ROS 17/2.8 cells was used to assess the bone formation properties of ZK191784. Although rat osteosarcoma cells secreted osteocalcin on treatment with 1,25(OH)2D3 as well as ZK191784, the efficacy of the latter was rather weak. Accordingly, detailed microcomputed tomography of bone did not suggest a significantly altered bone turnover in ZK191784-treated mice. On the other hand, we cannot exclude that prolonged ZK191784 administration will affect bone mineral density. In addition, morphometric analysis showed that trabecular and cortical thickness was reduced in TRPV5–/– mice. The exact explanation for this bone phenotype remains elusive but in addition to a primary defect due to TRPV5 ablation could be a consequence of the negative Ca2+ balance or a direct result of the hypervitaminosis D. When administered chronically in supraphysiological concentrations, 1,25(OH)2D3 was previously shown to reduce cortical bone thickness (30)
.
In summary, this study demonstrated that ZK191784 acts as an intestine-specific 1,25(OH)2D3 antagonist. Similar tissue-specific effects have been described for e.g., the partial estrogen receptor agonist tamoxifen, which was shown to exert a breast-selective, bone-sparing antagonistic action (31
32
33
34)
. Furthermore, our results in TRPV5–/– mice suggest that ZK191784 may ameliorate the clinical picture in disorders that are characterized by high 1,25(OH)2D3 levels or intestinal Ca2+ hyperabsorption. Taken together, the unique properties of this new 1,25(OH)2D3 analog might be of great benefit in clinical practice, where complete inhibition or stimulation, of the 1,25(OH)2D3 endocrine system is mostly undesirable.
ACKNOWLEDGMENTS
The work was financially supported by the Dutch Kidney Foundation (C10.1881, C03.6017) and the Dutch Organization of Scientific Research (Zon-Mw 016.006.001). The authors thank the staff of the Central Animal Facility, Radboud University Nijmegen, for technical support.
Received for publication November 25, 2005. Accepted for publication May 15, 2006.
REFERENCES
,25-dihydroxyvitamin D3 analog with reduced hypercalcemic activity. J. Invest. Dermatol. 119,1434-1442[CrossRef][Medline],25(OH)2-vitamin D3. Steroids 67,457-466[CrossRef][Medline],25-dihydroxyvitamin D3-26, 23-lactone analogs on differentiation of human leukemia cells (HL-60) induced by 1,25-dihydroxyvitamin D3. J. Biol. Chem. 274,16392-16399,25-dihydroxyvitamin D3 analogue toward vitamin D receptor function. J. Biol. Chem. 274,32376-32381-methyl introduction on antagonistic activity. Bioorg. Med. Chem. 11,3621-3631[CrossRef][Medline]-hydroxylase knockout mice. Kidney Int. 66,1082-1089[CrossRef][Medline]
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= TRPV5+/+ controls, treated with vehicle only; 
= ZK191784-treated TRPV5+/+; 
= TRPV5–/– controls, treated with vehicle only; 
= ZK191784-treated TRPV5–/–. Data are mean ± SE. *P < 0.05 vs. TRPV5+/+ controls; #P < 0.05 vs. TRPV5–/– controls; n = 9 animals per group.
