Bindels, R.J.M.

Klotho prevents renal calcium loss (Article)

2009-11-01T00:00:00Z

Disturbed calcium (Ca2+) homeostasis, which is implicit to the aging phenotype of klotho-deficient mice, has been attributed to altered vitamin D metabolism, but alternative possibilities exist. We hypothesized that failed tubular Ca2+absorption is primary, which causes increased urinary Ca2+excretion, leading to elevated 1,25-dihydroxyvitamin D3[1,25(OH)2D3] and its sequelae. Here, we assessed intestinal Ca2+absorption, bone densitometry, renal Ca2+excretion, and renal morphology via energy-dispersive x-ray microanalysis in wild-type and klotho-/-mice. We observed elevated serum Ca2+and fractional excretion of Ca2+(FECa) in klotho-/-mice. Klotho-/-mice also showed intestinal Ca2+hyperabsorption, osteopenia, and renal precipitation of calcium-phosphate. Duodenal mRNA levels of transient receptor potential vanilloid 6 (TRPV6) and calbindin-D9Kincreased. In the kidney, klotho-/-mice exhibited increased expression of TRPV5 and decreased expression of the sodium/calcium exchanger (NCX1) and calbindin-D28K, implying a failure to absorb Ca2+through the distal convoluted tubule/connecting tubule (DCT/CNT) via TRPV5. Gene and protein expression of the vitamin D receptor (VDR), 25-hydroxyvitamin D-1-α-hydroxylase (1αOHase), and calbindin-D9Kexcluded renal vitamin D resistance. By modulating the diet, we showed that the renal Ca2+wasting was not secondary to hypercalcemia and/or hypervitaminosis D. In summary, these findings illustrate a primary defect in tubular Ca2+handling that contributes to the precipitation of calcium-phosphate in DCT/CNT. This highlights the importance of klotho to the prevention of renal Ca2+loss, secondary hypervitaminosis D, osteopenia, and nephrocalcinosis. Copyright

Bone resorption inhibitor alendronate normalizes the reduced bone thickness of TRPV5-/- mice (Article)

2008-11-01T00:00:00Z

TRPV5 is a Ca2+-selective channel involved in transcellular Ca2+absorption expressed in kidney and in the ruffled border of osteoclasts. Studies in hypercalciuric TRPV5 knockout (TRPV5-/-) mice, which display significantly increased vitamin D levels, showed that TRPV5 ablation increases number and size of osteoclasts but impairs osteoclast-mediated bone resorption. The latter is not in line with the observed decreased bone thickness in TRPV5-/-mice. Bisphosphonates also inhibit osteoclast-mediated bone resorption. The aim of this study was to evaluate the effect of alendronate on the expression of the Ca2+transporters in bone, kidney, and duodenum and, importantly, the bone phenotype in TRPV5-/-mice. Wildtype (TRPV5+/+) and TRPV5-/-mice were treated during 10 wk with 2 mg/kg alendronate or vehicle weekly and housed in metabolic cages at the end of treatment. Urine and blood samples were taken for biochemical analysis, and duodenum, kidney, and femur were sampled. Expression of Ca2+transporters and osteoclast ruffled border transporters in bone and cultured osteoclasts was determined by QPCR analysis. Femurs were scanned using μCT, and resorption pit assays were performed in bone marrow cultures isolated from TRPV5+/+and TRPV5-/-mice. Alendronate treatment enhanced bone thickness in TRPV5+/+mice but also normalized the disturbed bone morphometry parameters in TRPV5-/-mice. Bone TRPV5 expression was specifically enhanced by alendronate, whereas the expression of Ca2+transporters in kidney and intestine was not altered. The expression of the osteoclast ruffled border membrane proteins chloride channel 7 (CLC-7) and the vacuolar H+-ATPase did not differ between both genotypes, but alendronate significantly enhanced the expression and PTH levels in TRPV5-/-mice. The expression of TRPV5, CLC-7, and H+-ATPase in osteoclast cultures was not affected by alendronate. The number of resorption pits was reduced in TRPV5-/-bone marrow cultures, but the response to vitamin D was similar to that in TRPV5+/+cultures. The alendronate-induced upregulation of TRPV5 in bone together with the decreased resorptive capacity of TRPV5-/-osteoclasts in vitro suggests that TRPV5 has an important role in osteoclast function. However, our data indicate that significant bone resorption still occurs in TRPV5-/-mice, because alendronate treatment normalized bone thickness in these mice. Thus, TRPV5-/-mice are able to rescue the resulting defect in osteoclast-mediated bone resorption, possibly mediated by the long-term hypervitaminosis D or other (non)hormonal compensatory mechanisms.

Murine TNFΔARE Crohn's disease model displays diminished expression of intestinal Ca2+ tansporters (Article)

2008-06-01T00:00:00Z

Background: Patients suffering from Crohn's disease (CD) show increased incidence of low bone mineral density. Investigating this complication is difficult because the exact etiology of CD remains elusive. Mice carrying a deletion in the tumor necrosis factor (TNF) AU-rich elements (ARE) are reported as a model for human CD and are characterized by elevated TNF-α levels and inflammations in the terminal ileum. To evaluate whether these mice have a Ca2+handling problem, this study analyzed the Ca2+homeostasis in heterozygous TNFΔAREmice (TNFΔARE/+) in comparison to wildtype littermates. Methods: Beside serum Ca2+and vitamin D levels, the expression of Ca2+transporters was analyzed in intestine, kidney and bone using quantitative real-time PCR, Western blot and immunohistochemistry. Bone scans were performed to measure bone parameters. Results: Ca2+transporters in duodenum (TRPV6, calbindin-D9K, PMCA1b) and kidney (TRPV5, calbindin-D28K, NCX1) showed significantly reduced mRNA expression levels in TNPΔARE/+mice, except for renal TRPV5. In bone, only calbindin-D9KmRNA displayed a significant down-regulation. These findings were supported by declined duodenal calbindin-D9Kand renal calbindin-D28Kprotein values. Likely, this down-regulation of Ca2+transporters in TNPΔARE/+mice is mediated by the 58 ± 9% reduction in serum 1,25(OH)2D3levels. Diminished expression of Ca2+transporters combined with unchanged serum Ca2+levels assumes Ca2+loss from bone to compensate for the body's overall Ca2+shortage. Indeed, microcomputed tomography scanning demonstrated reduced trabecular and corticol bone thickness and volume in TNFΔARE/+mice. This finding is further supported by increased total deoxypyridinoline in serum. Conclusions: Our results imply that TNFΔARE/+mice have a disturbed Ca2+homeostasis characterized by reduced duodenal and renal Ca2+transporters, diminished 1,25(OH)2D3levels, and increased bone resorption associated with profound bone abnormalities. Copyright

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

2006-10-01T00:00:00Z

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-D(9K), 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-D(28K) 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.

The epithelial Ca2+ channel TRPV5 is essential for proper osteoclastic bone resorption. (Article)

2005-11-29T00:00:00Z

Bone remodeling involves the interplay of bone resorption and formation and is accurately controlled to maintain bone mass. Both processes require transcellular Ca(2+) transport, but the molecular mechanisms engaged remain largely elusive. The epithelial Ca(2+) channel TRPV5 is one of the most Ca(2+)-selective transient receptor potential (TRP) channels. In this study, the functional role of TRPV5 in bone was investigated. TRPV5 mRNA was expressed in human and murine bone samples and in osteoclasts along with other genes involved in transcellular Ca(2+) transport, including calbindin-D(9K) and calbindin-D(28K), Na(+)/Ca(2+) exchanger 1, and plasma membrane Ca(2+)-ATPase 1b. TRPV5 expression in murine osteoclasts was confirmed by immunostaining and showed predominant localization to the ruffled border membrane. However, TRPV5 was absent in osteoblasts. Analyses of femoral bone sections from TRPV5 knockout (TRPV5(-/-)) mice revealed increased osteoclast numbers and osteoclast area, whereas the urinary bone resorption marker deoxypyridinoline was reduced compared with WT (TRPV5(+/+)) mice. In an in vitro bone marrow culture system, the amount of osteoclasts and number of nuclei per osteoclast were significantly elevated in TRPV5(-/-) compared with TRPV5(+/+) mice. However, using a functional resorption pit assay, we found that bone resorption was nearly absent in osteoclast cultures from TRPV5(-/-) mice, supporting the impaired resorption observed in vivo. In conclusion, TRPV5 deficiency leads to an increase in osteoclast size and number, in which Ca(2+) resorption is nonfunctional. This report identifies TRPV5 as an epithelial Ca(2+) channel that is essential for osteoclastic bone resorption and demonstrates the significance of transcellular Ca(2+) transport in osteoclastic function.

Hypervitaminosis D mediates compensatory Ca2+ hyperabsorption in TRPV5 knockout mice (Article)

2005-11-01T00:00:00Z

Vitamin D plays an important role in Ca(2+) homeostasis by controlling Ca(2+) (re)absorption in intestine, kidney, and bone. The epithelial Ca(2+) channel TRPV5 mediates the Ca(2+) entry step in active Ca(2+) reabsorption. TRPV5 knockout (TRPV5(-/-)) mice show impaired Ca(2+) reabsorption, hypercalciuria, hypervitaminosis D, and intestinal hyperabsorption of Ca(2+). Moreover, these mice demonstrate upregulation of intestinal TRPV6 and calbindin-D(9K) expression compared with wild-type mice. For addressing the role of the observed hypervitaminosis D in the maintenance of Ca(2+) homeostasis and the regulation of expression levels of the Ca(2+) transport proteins in kidney and intestine, TRPV5/25-hydroxyvitamin-D(3)-1alpha-hydroxylase double knockout (TRPV5(-/-)/1alpha-OHase(-/-)) mice, which show undetectable serum 1,25(OH)(2)D(3) levels, were generated. TRPV5(-/-)/1alpha-OHase(-/-) mice displayed a significant hypocalcemia compared with wild-type mice (1.10 +/- 0.02 and 2.54 +/- 0.01 mM, respectively; P < 0.05). mRNA levels of renal calbindin-D(28K) (7 +/- 2%), calbindin-D(9K) (32 +/- 4%), Na(+)/Ca(2+) exchanger (12 +/- 2%), and intestinal TRPV6 (40 +/- 8%) and calbindin-D(9K) (26 +/- 4%) expression levels were decreased compared with wild-type mice. Hyperparathyroidism and rickets were present in TRPV5(-/-)/1alpha-OHase(-/-) mice, more pronounced than observed in single TRPV5 or 1alpha-OHase knockout mice. It is interesting that a renal Ca(2+) leak, as demonstrated in TRPV5(-/-) mice, persisted in TRPV5(-/-)/1alpha-OHase(-/-) mice, but a compensatory upregulation of intestinal Ca(2+) transporters was abolished. In conclusion, the elevation of serum 1,25(OH)(2)D(3) levels in TRPV5(-/-) mice is responsible for the upregulation of intestinal Ca(2+) transporters and Ca(2+) hyperabsorption. Hypervitaminosis D, therefore, is of crucial importance to maintain normocalcemia in impaired Ca(2+) reabsorption in TRPV5(-/-) mice.