HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Nijenhuis, T. Articles by Bindels, R. J. M. Search for Related Content PubMed PubMed Citation Articles by Nijenhuis, T. Articles by Bindels, R. J. M.

The novel vitamin D analog ZK191784 as an intestine-specific vitamin D antagonist

Tom Nijenhuis^*, Bram C. J. van der Eerden, Ulrich Zügel, Andreas Steinmeyer, Harrie Weinans, Joost G. J. Hoenderop^*, Johannes P. T. M. van Leeuwen and René J. M. Bindels^*,1

^* 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

SPECIFIC AIMS

The main physiological function of vitamin D₃ [1,25(OH)₂D₃] is to stimulate intestinal and renal Ca²⁺ (re)absorption and regulate bone Ca²⁺ turnover. In addition, 1,25(OH)₂D₃ has potent antiproliferative, immunosuppressive, and immunomodulatory activity. However, therapeutic administration of 1,25(OH)₂D₃ has dose-limiting hypercalcemic side effects. Therefore, there has been great effort in identifying new 1,25(OH)₂D₃ analogs that retain a beneficial therapeutic profile with minimal calcemic action. The 1,25(OH)₂D₃ analog ZK191784 was developed in an effort to dissociate the immunomodulatory and hypercalcemic actions of 1,25(OH)₂D₃. However, the in vivo effects of ZK191784 regarding Ca²⁺ homeostasis have not been evaluated in detail.

1,25(OH)₂D₃-stimulated transcellular Ca²⁺ (re)absorption involves Ca²⁺ entry across the luminal membrane via the epithelial Ca²⁺ channels TRPV5 and TRPV6. 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 duodenum. TRPV5 knockout (TRPV5^–/–) mice display profound renal Ca²⁺ wasting due to impaired active Ca²⁺ reabsorption in DCT and CNT. Furthermore, these mice show hypervitaminosis D leading to intestinal Ca²⁺ hyperabsorption and display reduced bone thickness.

The aim of this study was, therefore, to evaluate the effect of ZK191784 on Ca²⁺ absorption, Ca²⁺ excretion and expression of the Ca²⁺ transport proteins in intestine and kidney in wild-type (WT) and TRPV5^–/– mice. Furthermore, the actions of ZK191784 on intestinal, renal, and osteosarcoma cell lines were characterized to reveal the biological profile of this novel 1,25(OH)₂D₃ analog.

PRINCIPAL FINDINGS

1. 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. Genetic ablation of TRPV5 resulted in a 10-fold increase in Ca²⁺ excretion compared with WT mice (Fig. 1 A) and enhancement of intestinal Ca²⁺ absorption as determined by in vivo ⁴⁵Ca²⁺ absorption measurements (Fig. 1B ), accompanied by a minor increase in the serum Ca²⁺ concentration. 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. 1A ). Furthermore, ZK191784 treatment significantly diminished intestinal Ca²⁺ absorption and decreased Ca²⁺ excretion in WT mice, (Fig. 1B ) without altering serum Ca²⁺ levels.

View larger version (23K): [in this window] [in a new window]   Figure 1. Renal Ca2+ excretion and intestinal Ca2+ absorption during treatment with ZK191784 in TRPV5+/+ and TRPV5–/– mice. Effect of ZK191784 on renal Ca2+ excretion was determined in metabolic cages (A). Intestinal Ca2+ absorption was determined in an in vivo 45Ca2+ absorption assay, measuring serum 45Ca2+ at early time points after oral gavage (B). Controls, mice treated with vehicle only; ZK191784, mice treated for 28 days with the 1,25(OH)2D3 analog ZK191784 (50 µg/kg/day). = 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.

2. ZK191784 inhibits 1,25(OH)₂D₃-stimulated Ca²⁺ absorption and intestinal Ca²⁺ transporter expression
To study the in vivo effect of ZK191784 on the abundance of Ca²⁺ transporters in the intestine, TRPV6 and calbindin-D_9K mRNA expression was determined by real-time quantitative polymerase chain reaction (PCR) analysis. TRPV5^–/– mice showed profoundly increased TRPV6 and calbindin-D_9K mRNA levels in duodenum compared with WT mice. ZK191784 significantly reduced the TRPV6 and calbindin-D_9K mRNA abundance in TRPV5^–/– mice, resulting in a complete normalization of the expression of the intestinal Ca²⁺ transporters.

To determine the effect of ZK191784 in an intestinal cell model, ⁴⁵Ca²⁺ uptake was determined in the human Caco-2 cell line. 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. 2 A). In contrast, incubation with 1 · 10^–7 M ZK191784 did not stimulate ⁴⁵Ca²⁺ uptake. Importantly, concomitant application of 1 · 10^–7 M ZK191784 and 1 · 10^–7 M 1,25(OH)₂D₃ significantly inhibited the 1,25(OH)₂D₃-dependent ⁴⁵Ca²⁺ uptake by Caco-2 cells.

View larger version (17K): [in this window] [in a new window]   Figure 2. Differential effect of 1,25(OH)2D3 and ZK191784 on 45Ca2+ uptake in the intestinal Caco-2 cell line and on transepithelial Ca2+ transport in rabbit kidney CNT/CCD primary cell cultures. 45Ca2+ uptake was determined in Caco-2 cells incubated for 48 h in normal culture medium (Control) 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 (A). Data are depicted as ruthenium red (RR)-sensitive uptake. Transcellular Ca2+ transport was determined in immunodissected rabbit CNT/CCD cultures incubated for 48 h in normal culture medium (Control), 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 (B). Data are mean ± SE. *P < 0.05 vs. untreated cells (Control); #P < 0.05 vs. 1,25(OH)2D3-treated cells.

3. ZK191784 up-regulates renal Ca²⁺ transport proteins and stimulates transcellular Ca²⁺ reabsorption
To evaluate the effect of ZK191784 on the expression of the Ca²⁺ transport proteins in the kidney, TRPV5 and calbindin-D_28K mRNA as well as protein abundance were determined by real-time quantitative PCR and semiquantitative immunohistochemistry, respectively. ZK191784 significantly increased TRPV5 and calbindin-D_28K mRNA levels and enhanced protein abundance of these Ca²⁺ transporters in DCT and CNT of WT mice.

Transcellular Ca²⁺ transport was measured in primary cultures of immunodissected rabbit kidney CNT and cortical collecting duct (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. 2B ). 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.

4. Effects of ZK191784 on bone
Bone TRPV5 mRNA levels were not affected by ZK191784 in WT mice, but TRPV6 mRNA expression was significantly enhanced in ZK191784-treated WT and TRPV5^–/– mice. Ligand-induced osteocalcin production by reactive oxygen species (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 extracellular matrix production. Both 1,25(OH)₂D₃ and ZK191784 induced osteocalcin production in a dose-dependent manner.

To evaluate the in vivo effects of ZK191784 on bone morphology, femurs from control and ZK191784-treated mice were scanned using microcomputed tomography. Detailed three-dimensional morphometric analysis demonstrated that both trabecular and cortical thickness are reduced in TRPV5^–/– mice. ZK191784 did not significantly affect bone morphometric parameters in both mice strains nor were there differences in the other trabecular and cortical bone parameters between the treated groups.

CONCLUSIONS AND SIGNIFICANCE

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, ZK191784 exerted partial agonistic actions on Ca²⁺ handling in kidney and bone. This tissue-specific partial 1,25(OH)₂D₃ agonism/antagonism reflects a biological profile unlike any other 1,25(OH)₂D₃ analog used so far (Fig. 3 ).

View larger version (49K): [in this window] [in a new window]   Figure 3. Schematic representation of the differential tissue-specific effects of ZK191784 compared with 1,25(OH)2D3 regarding Ca2+ homeostasis and expression of Ca2+ transporters. ZK191784 inhibits 1,25(OH)2D3-stimulated Ca2+ absorption in vivo and in the intestinal Caco-2 cell line and down-regulates 1,25(OH)2D3-stimulated intestinal Ca2+ transporter expression. ZK191784 and 1,25(OH)2D3 both display stimulatory effects in vivo on Ca2+ reabsorption and renal Ca2+ transporter expression as well as in primary cultures of immunodissected rabbit kidney CNT and CCD cells. Furthermore, ZK191784 and 1,25(OH)2D3 stimulate osteocalcin secretion by ROS17/2.8 cells.

ZK191784 normalized the increased expression of the intestinal Ca²⁺ transporters and, thereby, antagonized the Ca²⁺ hyperabsorption in TRPV5^–/– mice. Previous studies from our laboratory demonstrated that 1,25(OH)₂D₃ and several analogs enhance Ca²⁺ transporter expression and intestinal Ca²⁺ absorption. Furthermore, ZK191784 diminished Ca²⁺ absorption in WT mice. These 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 expresses TRPV6 and calbindin-D_9K. In the absence of 1,25(OH)₂D₃, ZK191784 did 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, unlike 1,25(OH)₂D₃, ZK191784 does not stimulate Ca²⁺ uptake by the intestine and exerts a unique antagonistic effect on 1,25(OH)₂D₃-stimulated active Ca²⁺ absorption.

Renal Ca²⁺ transporter expression is tightly regulated by 1,25(OH)₂D₃. The concomitantly increased renal TRPV5 and calbindin-D_28K expression accompanied by 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 the in vivo findings. Interestingly, ZK191784 did not increase calbindin-D_28K abundance in TRPV5^–/– mice. Previous studies from our group demonstrated that calbindin-D_28K expression is highly dependent on the TRPV5-mediated Ca²⁺ influx in DCT and CNT cells. This could explain the significantly reduced calbindin-D_28K expression in TRPV5^–/– mice, despite elevated 1,25(OH)₂D₃ levels, and the absence of a stimulatory effect of ZK191784 in these mice. ZK191784 still resulted in a Ca²⁺-sparing action in TRPV5^–/– mice that, obviously, cannot be explained by stimulation of active Ca²⁺ reabsorption. However, abolishment of Ca²⁺ hyperabsorption and reduced serum Ca²⁺ results in a decreased filtered Ca²⁺ load and, therefore, diminished Ca²⁺ excretion.

The functional role of TRPV5 and TRPV6 in bone remains largely elusive. Recently, it was demonstrated that TRPV5 is exclusively expressed in the ruffled border of osteoclasts and that cultured osteoclasts from TRPV5^–/– mice display reduced bone resorptive capacity. ZK191784 did not alter TRPV5 expression but increased bone TRPV6 expression. 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 and ZK191784, the efficacy of the latter was rather weak. Bone morphometry did not suggest altered bone turnover in ZK191784-treated mice.

In conclusion, this study demonstrated that ZK191784 is an intestine-specific 1,25(OH)₂D₃ antagonist, being one of few synthetic 1,25(OH)₂D₃ ligand displaying tissue-specific effects in vivo. These unique properties might be of benefit in clinical practice where complete inhibition, or stimulation, of the 1,25(OH)₂D₃ endocrine system is mostly undesirable. Our results indicate this compound will display reduced hypercalcemic potential compared with 1,25(OH)₂D₃ and its analogs currently used in clinical practice.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5155fje

This Article Abstract Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Nijenhuis, T. Articles by Bindels, R. J. M. Search for Related Content PubMed PubMed Citation Articles by Nijenhuis, T. Articles by Bindels, R. J. M.