24-25-dihydroxyvitamin-d-3 and Disease-Models--Animal

A rat model of postmenopausal osteoporosis was introduced, using ovariectomized rats on a low Ca diet. CT treatment of these animals for one month prevented the decrease in both mineral contents and physical properties of the femoral bone. Treatment of the animals with 1,25(OH)2D3 was effective in increasing bone mineral contents and maintaining positive mineral balance, but did not increase the physical tolerance of bones. In contrast, 24,25(OH)2D3 increased the breaking force of the femoral bone, with minimal effect on bone mineral contents and mineral balance. These results suggest that 1,25(OH)2D3 and 24,25(OH)2D3 act differently on the matrix phase and mineral phase of bones, but that they act together to maintain mineral balance and structural integrity of bones. The mechanism of how these vitamin D metabolites affect bone metabolism remain to be clarified.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Bone and Bones; Bone Development; Calcitonin; Calcitriol; Calcium; Dihydroxycholecalciferols; Disease Models, Animal; Female; Intestinal Absorption; Menopause; Minerals; Osteoporosis; Ovariectomy; Rats; Rats, Inbred Strains; Vitamin D

Osteoarthritis (OA) in humans is associated with low circulating 25-hydroxyvitamin D3 [25(OH)D3]. In vitamin D replete rats, radiolabeled 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] accumulates in articular cartilage following injection of [3H]-25(OH)D3. Previously, we showed that 24R,25(OH)2D3 blocks chondrocyte apoptosis via phospholipase D and p53, suggesting a role for 24R,25(OH)2D3 in maintaining cartilage health. We examined the ability of 24R,25(OH)2D3 to prevent degenerative changes in articular cartilage in an OA-like environment and the potential mechanisms involved. In vitro, rat articular chondrocytes were treated with IL-1β with and without 24R,25(OH)2D3 or 1α,25(OH)2D3. 24R,25(OH)2D3 but not 1α,25(OH)2D3 blocked the effects of IL-1β in a dose-dependent manner, and its effect was partially mediated through the TGF-β1 signaling pathway. In vivo, unilateral anterior cruciate ligament transections were performed in immunocompetent rats followed by intra-articular injections of 24R,25(OH)2D3 or vehicle (t = 0, 7, 14, 21 days). Tissues were harvested on day 28. Joints treated with vehicle had changes typical of OA whereas joints treated with 24R,25(OH)2D3 had less articular cartilage damage and levels of inflammatory mediators. These results indicate that 24R,25(OH)2D3 protects against OA, and suggest that it may be a therapeutic approach for preventing trauma-induced osteoarthritis.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Anterior Cruciate Ligament Injuries; Cartilage, Articular; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Gene Expression Regulation; Humans; Injections, Intra-Articular; Interleukin-1beta; Male; Osteoarthritis, Knee; Rats; Signal Transduction; Transforming Growth Factor beta1; Vitamins

1alpha,25(OH)(2)D(3) regulates rat growth plate chondrocytes via nuclear vitamin D receptor (1,25-nVDR) and membrane VDR (1,25-mVDR) mechanisms. To assess the relationship between the receptors, we examined the membrane response to 1alpha,25(OH)(2)D(3) in costochondral cartilage cells from wild type VDR(+/+) and VDR(-/-) mice, the latter lacking the 1,25-nVDR and exhibiting type II rickets and alopecia. Methods were developed for isolation and culture of cells from the resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) of the costochondral cartilages from wild type and homozygous knockout mice. 1alpha,25(OH)(2)D(3) had no effect on [(3)H]-thymidine incorporation in VDR(-/-) GC cells, but it increased [(3)H]-thymidine incorporation in VDR(+/+) cells. Proteoglycan production was increased in cultures of both VDR(-/-) and VDR(+/+) cells, based on [(35)S]-sulfate incorporation. These effects were partially blocked by chelerythrine, which is a specific inhibitor of protein kinase C (PKC), indicating that PKC-signaling was involved. 1alpha,25(OH)(2)D(3) caused a 10-fold increase in PKC specific activity in VDR(-/-), and VDR(+/+) GC cells as early as 1 min, supporting this hypothesis. In contrast, 1alpha,25(OH)(2)D(3) had no effect on PKC activity in RC cells isolated from VDR(-/-) or VDR(+/+) mice and neither 1beta,25(OH)(2)D(3) nor 24R,25(OH)(2)D(3) affected PKC in GC cells from these mice. Phospholipase C (PLC) activity was also increased within 1 min in GC chondrocyte cultures treated with 1alpha,25(OH)(2)D(3). As noted previously for rat growth plate chondrocytes, 1alpha,25(OH)(2)D(3) mediated its increases in PKC and PLC activities in the VDR(-/-) GC cells through activation of phospholipase A(2) (PLA(2)). These responses to 1alpha,25(OH)(2)D(3) were blocked by antibodies to 1,25-MARRS, which is a [(3)H]-1,25(OH)(2)D(3) binding protein identified in chick enterocytes. 24R,25(OH)(2)D(3) regulated PKC in VDR(-/-) and VDR(+/+) RC cells. Wild type RC cells responded to 24R,25(OH)(2)D(3) with an increase in PKC, whereas treatment of RC cells from mice lacking a functional 1,25-nVDR caused a time-dependent decrease in PKC between 6 and 9 min. 24R,25(OH)(2)D(3) dependent PKC was mediated by phospholipase D, but not by PLC, as noted previously for rat RC cells treated with 24R,25(OH)(2)D(3). These results provide definitive evidence that there are two distinct receptors to 1alpha,25(OH)(2)D(3). 1alpha,25(OH)(2)D(3)-dependent regul

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Cell Division; Cell Membrane; Cells, Cultured; Chondrocytes; Disease Models, Animal; Growth Plate; Mice; Mice, Knockout; Phospholipase D; Phospholipases A; Protein Kinase C; Proteoglycans; Rats; Receptors, Calcitriol; Rickets; Signal Transduction; Sulfates; Type C Phospholipases; Vitamin D

The aim of this work was to study the regulation by calcium of the synthesis of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) in Walker 256 carcinosarcoma (W256) cells compared to renal phenotype LLC-PK1 cells. We have detected synthesis of 1,25(OH)2D3 and 24,25(OH)2D3 in W256 cells. Levels of 1,25(OH)2D3 in W256 were similar to those of LLC-PK1 cells. Levels of 24,25(OH)2D3 were higher in W256 than in LLC-PK1 cells. High levels of calcium inhibited 1,25(OH)2D3 synthesis in LLC-PK1 cells but not in W256 cells.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Calcitriol; Calcium; Carcinoma 256, Walker; Disease Models, Animal; Female; LLC-PK1 Cells; Phenotype; Rats; Rats, Wistar; Swine

We have previously established an uremic rat model which is suitable for investigating the effect of various treatment modalities on the progression of renal osteodystrophy [1]. Four months subsequent to 5/6 nephrectomy, animals were treated three times a week for 3 months with either vehicle, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], 1,25(OH)2D3 + 24,25-dihydroxyvitamin D3 [24,25(OH)2D3], 1,25(OH)2D3 + calcitonin (CT), or 1,25(OH)2D3 + 24,25(OH)2D3 + CT. At termination of the study, clinical chemistry, chemical composition, and mechanical properties of femurs, calvarial parathyroid hormone (PTH)-elicited adenylate cyclase (AC), and phospholipase C (PL-C) activities, femoral cross-sectional area, and bone histomorphometry were analyzed. The main findings were that 1,25(OH)2D3 +/- 24,25(OH)2D3 treatment enhanced elasticity as well as time to fracture at the femoral metaphysis. CT potentiated the increase in elasticity obtained by 1,25(OH)2D3 +/- 24,25(OH)2D3 treatment. Only 24,25(OH)2D3 administration rectified the supernormal PTH-stimulated uremic bone AC, and only 1,25(OH)2D3 medication normalized the diminished CT-elicited AC. The obliterated uremic bone PTH-sensitive PL-C was fully normalized by all drug regimens. Femoral shaft inner zone diameter was enhanced by uremia, however, all drug treatments normalized it. Ditto effect was registered with either drug treatment on the subnormal outer and inner zone widths. Histomorphometrical analyses showed that 1,25(OH)2D3 administration reduced both eroded and osteoid surfaces. Most prominently, adjuvant 24,25(OH)2D3 or CT administration potentiated the beneficial effect of 1,25(OH)2D3 on fibrosis and osteomalacia. We assert that vitamin D3 treatment markedly reverses the development of renal osteodystrophy, and CT potentiates the effect of vitamin D3.

Topics: 24,25-Dihydroxyvitamin D 3; Adenylyl Cyclases; Animals; Bone and Bones; Bone Density; Calcitonin; Calcitriol; Calcium; Chronic Kidney Disease-Mineral and Bone Disorder; Creatinine; Disease Models, Animal; Drug Administration Schedule; Female; Femur; Rats; Rats, Wistar; Type C Phospholipases; Uremia

To examine changes in mechanical competence of bone caused by ovariectomy, and to assess the effect of 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) administration on mass and structure, we conducted mechanical tests on canine lumbar vertebrae and femur 31 months after surgery. Beagles weighing 9-10 kg were ovariectomized (OVX) or sham operated (n = 3, group 1). OVX dogs were divided into three groups. Group 2 (n = 3) received only the agent vehicle, groups 3 (n = 4) and 4 (n = 4) received daily 24R,25(OH)2D3 doses of 2 and 10 mcg/kg, respectively from 1 month after surgery. In group 4, the dose level was increased up to 100 mcg/kg by the 17 month. Then, L3 and L4 vertebrae and left femur were excised from each animal. Torsional tests at the femoral diaphysis were conducted. On the L3 specimen, the circumferential shell was removed to obtain a cancellous core specimen. The shell was left intact on the L4 specimen. In compression tests, the loading was stopped just after maximal strength was reached for minimum specimen collapse, from which 7-mcm thick, undecalcified, midcross sections parallel to the base of the specimen were obtained. Neither femoral morphology, bone mineral contents (BMCs) nor structural stiffness indicated a significant difference among groups. Though L3 and L4 BMCs were reduced in group 2, in group 3 and 4 they were significantly larger than in group 2. Compression tests on lumbar vertebral specimens showed a significant decrease in mechanical parameters in group 2. On the cancellous core specimen of L3, the mean structural stiffness in group 2 was 31.8% of that in group 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 24,25-Dihydroxyvitamin D 3; Analysis of Variance; Animals; Biomechanical Phenomena; Bone and Bones; Bone Density; Disease Models, Animal; Dogs; Female; Femur; Humans; Lumbar Vertebrae; Osteoporosis, Postmenopausal; Ovariectomy; Stress, Mechanical

In order to determine whether the administration of 24R,25(OH)2D3 had any beneficial effect on the regulation of bone turnover and the prevention of bone atrophy, we examined beagles for 31 months after ovariectomy (OVX). Fourteen beagle dogs (8.54 +/- 1.22 kg body wt-b.w.) were divided into four groups. Group 1 (n = 3) was the sham, and Group 2 (n = 3) served as the OVX control. In Group 3 (n = 4) and Group 4 (n = 4), 24,25-dihydroxyvitamin D3(24R,25(OH)2D3) was given daily at dose levels of 2 and 10 mcg/kg B.W., respectively. In Group 4, the dose level was increased to 100 mcg/kg by 17 months. During the experiments, urinary hydroxyproline (U-HPr), serum chemistry, serum bone gla-protein (BGP), and vitamin D metabolite levels were monitored. At the end of the experiment, bone mineral content (BMC) in the 6th and 7th lumbar vertebrae and right femur was determined by single photon absorptiometry. The left iliac bone sample was obtained after tetracycline labeling, and undecalcified sections were observed. In Group 2, excretion of U-HPr increased after OVX and had reached a level of approximately twice the baseline values by 10 months; then it gradually came down to the original level. In Group 3, however, U-HPr excretion remained at the same level as the baseline value, as it did in Group 1. In Group 4, it was remarkably reduced down to 50-60% of the baseline values. Serum BGP level was markedly reduced in Group 4. Serum 24,25(OH)2D levels were markedly increased in Groups 3 and 4.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Bone and Bones; Bone Resorption; Disease Models, Animal; Dogs; Drug Evaluation, Preclinical; Female; Humans; Hydroxyproline; Ilium; Osteocalcin; Osteoclasts; Osteoporosis, Postmenopausal; Ovariectomy

Cholecalciferol-1-hydroxylase activities were estimated in renal cortex homogenates and mitochondrial preparations from three groups of 6- to 12-week-old pigs. Five animals suffering from an inherited form of vitamin D-deficiency rickets showed symptoms of florid rickets when used for this study. Six pigs were normocalcemic heterozygous litter mates of the rachitic strain and three pigs were normal controls (German land-race and wild pigs). The renal cortex homogenates and mitochondrial preparations were incubated for 5-30 min at 37 degrees C with 25-(26-27-methyl-3H) OHD3 as substrate. 1,25(OH)2D3 was subsequently identified in normal phase (Zorbax-Sil) and reversed phase (Zorbax-ODS) HPLC eluates. 1-hydroxylase activities were demonstrated in both normal controls and heterozygote offspring and were ten times above minimum detectability of the assay. The km was 255 +/- 74 (SD) and 278 +/- 85 nmol X 1(-1) in controls and heterozygote offspring, respectively. The Vmax in the two groups was between 0.11 and 0.794 and decreased with age of the animals. Km and Vmax did not differ between the two groups. In homozygous, hypocalcemic rachitic animals no 1-hydroxylase activity was detectable in either homogenates or mitochondrial preparations. Addition of kidney homogenate from a rachitic animal to a homogenate from a normal pig did not specifically depress 1-hydroxylase activity in the mixture. Treatment of rachitic pigs with 1.0 microgram/day of 1,25(OH)2D3 for 4 weeks also had no effect on 1-hydroxylase activity. It is concluded that the rachitic pigs suffer from an inborn error of renal 1,25(OH)2D3 production.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 24,25-Dihydroxyvitamin D 3; 25-Hydroxyvitamin D3 1-alpha-Hydroxylase; Animals; Calcitriol; Dihydroxycholecalciferols; Disease Models, Animal; In Vitro Techniques; Kidney; Kidney Cortex; Kinetics; Rickets; Steroid Hydroxylases; Swine

The common marmoset, a New World monkey, requires a large amount of vitamin D3 to maintain its normal growth. This monkey is reported to have an end-organ resistance to 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3). In this study, the bone morphology of marmosets fed a high vitamin D3 diet (intake of vitamin D3, 110 IU/day/100 g of body weight) was compared by X-ray and histological examinations with that of rhesus monkeys (Old World monkey) fed a normal diet (intake of vitamin D3, 5 IU/day/100 g of body weight). Three of 20 marmosets were found by X-ray examination to have osteomalacic changes in their bones despite the high daily intake of vitamin D3, whereas none of the 5 rhesus monkeys showed any signs of osteomalacia. Osteomalacic marmosets had distinct increases in osteoid surface, relative osteoid volume, and active osteoclastic bone resorption, whereas non-osteomalacic marmosets had no increase in osteoid tissues in their bones. None of the marmosets, either osteomalacic or non-osteomalacic, was hypercalcemic despite the extremely high circulating levels of 1 alpha,25(OH)2D3. However, the serum 25-hydroxyvitamin D3 (25OHD3) and 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) levels were significantly lower in the osteomalacic than in the non-osteomalacic marmosets. These results suggest that the marmoset is likely to exhibit osteomalacic bone changes despite the high daily intake of vitamin D3. These changes resemble those in vitamin D-dependent rickets, type II.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Bone and Bones; Calcifediol; Calcitriol; Callitrichinae; Diet; Dihydroxycholecalciferols; Disease Models, Animal; Female; Macaca mulatta; Male; Osteomalacia; Rickets; Species Specificity

A model of low-phosphate, vitamin D-deficient rachitic rats was used to compare the effects of 1 alpha(OH)D3, 1,25(OH)2D3, and 24,25(OH)2D3 on cartilage and bone. The rats were maintained for 3 weeks on a high-calcium, low-phosphate, vitamin D-deficient diet, during which period they developed severe rickets. The rachitic rats were injected for 2 or 3 consecutive days with a physiologic dose of either metabolite. Other littermates were given a single dose of 50,000 IU of cholecalciferol in combination with a normal diet. Samples of cartilage fluid (Cfl) and of blood were removed prior to sacrifice for biochemical studies of some parameters of calcification. These parameters were correlated with the results of light and electron microscopic studies of the growth plate cartilage and bone. Treatment with 1 alpha (OH)D3 or with 1,25(OH)2D3, in spite of increasing Ca and P levels in the Cfl, induced only partial healing of the rickets. In contrast, 24,25(OH)2D3 or vitamin D with a normal diet resulted in complete morphologic and biochemical healing of the rickets. Transmission electron microscopic (TEM) studies have shown partial mineralization of the wide hypertrophic zone of the growth plate following treatment with 1 alpha(OH)D3 or with 1,25(OH)2D3. Mineralization was more complete with 24,25(OH)2D3 treatment. The results of this study emphasize the importance of 24,25(OH)2D3 for normal endochondral bone formation and mineralization.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Calcitriol; Calcium; Dihydroxycholecalciferols; Disease Models, Animal; Growth Plate; Male; Phosphates; Rats; Rats, Inbred Strains; Rickets

The hypophosphatemic male mouse (Hyp/y), the proposed model for human vitamin D-resistant rickets (VDRR), is characterized by chronic hypophosphatemia, dwarfism, and rachitic and osteomalacic bone lesions. We have reported that treatment of Hyp/y mice with phosphate salts (Pi) heals rickets but does not correct the defective endosteal bone mineralization. In an attempt to cure osteomalacia, mutant male animals were treated with Pi combined with 25-hydroxyvitamin D3 (25OHD3, 1 microgram/kg/day), 24,25-dihydroxyvitamin D3 [24,25(OH)2D3, 0.5 microgram/kg/day], or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3, 0.05--0.25 microgram/kg/day] infused constantly for 3 weeks. The biochemical and skeletal effects of treatment were assessed by analytical methods and bone histomorphometry. The results show that only 1,25(OH)2D3 produced a dose-dependent elevation of serum calcium and phosphorus, and greatly improved bone mineralization at doses high enough to increase serum calcium and phosphorus concentrations within or above the normal range. Better improvement of bone mineralization was obtained when Pi was combined to 1,25(OH)2D3. In conjunction with the correction of hypocalcemia, Pi + 1,25(OH)2D3 suppressed the stimulation of bone turnover induced by Pi supplementation. The results show that, as in VDRR children, 1,25(OH)2D3 produces beneficial effects on bone lesions in Hyp/y mice, mainly through enhancement of mineral availability. However, the persistence of osteomalacia despite correction of serum mineral concentrations suggests that there is a specific bone cell resistance to mineral and/or hormonal influences in Hyp/y mice.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Bone and Bones; Calcitriol; Calcium; Dihydroxycholecalciferols; Disease Models, Animal; Dose-Response Relationship, Drug; Hydroxycholecalciferols; Hypophosphatemia, Familial; Male; Mice; Mice, Inbred C57BL; Osteomalacia; Phosphates; Phosphorus; Rats; Vitamin D