Regulation of serum 1,25(OH)2 vitamin D3 levels by fibroblast growth factor 23 is mediated by FGF receptors 3 and 4

Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone implicated in the pathogenesis of several hypophosphatemic disorders. FGF23 causes hypophosphatemia by decreasing the expression of sodium phosphate cotransporters (NaPi-2a and NaPi-2c) and decreasing serum 1,25(OH)(2)Vitamin D(3) levels. We previously showed that FGFR1 is the predominant receptor for the hypophosphatemic actions of FGF23 by decreasing renal NaPi-2a and 2c expression while the receptors regulating 1,25(OH)(2)Vitamin D(3) levels remained elusive. To determine the FGFRs regulating 1,25(OH)(2)Vitamin D(3) levels, we studied FGFR3(-/-)FGFR4(-/-) mice as these mice have shortened life span and are growth retarded similar to FGF23(-/-) and Klotho(-/-) mice. Baseline serum 1,25(OH)(2)Vitamin D(3) levels were elevated in the FGFR3(-/-)FGFR4(-/-) mice compared with wild-type mice (102.2 ± 14.8 vs. 266.0 ± 34.0 pmol/l; P = 0.001) as were the serum levels of FGF23. Administration of recombinant FGF23 had no effect on serum 1,25(OH)(2)Vitamin D(3) in the FGFR3(-/-)FGFR4(-/-) mice (173.4 ± 32.7 vs. 219.7 ± 56.5 pmol/l; vehicle vs. FGF23) while it reduced serum 1,25(OH)(2)Vitamin D(3) levels in wild-type mice. Administration of FGF23 to FGFR3(-/-)FGFR4(-/-) mice resulted in a decrease in serum parathyroid hormone (PTH) levels and an increase in serum phosphorus levels mediated by increased renal phosphate reabsorption. These data indicate that FGFR3 and 4 are the receptors that regulate serum 1,25(OH)(2)Vitamin D(3) levels in response to FGF23. In addition, when 1,25(OH)(2)Vitamin D(3) levels are not affected by FGF23, as in FGFR3(-/-)FGFR4(-/-) mice, a reduction in PTH can override the effects of FGF23 on renal phosphate transport.     

Klotho ablation converts the biochemical and skeletal alterations in FGF23 (R176Q) transgenic mice to a Klotho-deficient phenotype

Transgenic mice overexpressing fibroblast growth factor (FGF23) (R176Q) (F(Tg)) exhibit biochemical {hypophosphatemia, phosphaturia, abnormal 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] metabolism} and skeletal (rickets and osteomalacia) abnormalities attributable to FGF23 action. In vitro studies now implicate the aging-related factor Klotho in the signaling mechanism of FGF23. In this study, we used a mouse genetic approach to validate in vivo the pivotal role of Klotho in the metabolic and skeletal derangements associated with FGF23 (R176Q) overexpression. To this end, we crossed mice heterozygous for the hypomorphic Klotho allele (Kl(+/-)) to F(Tg) mice and obtained F(Tg) transgenic mice homozygous for the Kl-hypomorphic allele (F(Tg)/Kl(-/-)). Mice were killed on postnatal day 50, and serum and tissues were procured for analysis and comparison with F(Tg), wild-type, and Kl(-/-) controls. From 4 wk onward, F(Tg)/Kl(-/-) mice were clearly distinguishable from F(Tg) mice and exhibited a striking phenotypic resemblance to the Kl(-/-) controls. Serum analysis for calcium, phosphorus, parathyroid hormone, 1,25(OH)(2)D(3), and alkaline phosphatase activity confirmed the biochemical similarity between the F(Tg)/Kl(-/-) and Kl(-/-) mice and their distinctness from the F(Tg) controls. The characteristic skeletal changes associated with FGF23 (R176Q) overexpression were also dramatically reversed by the absence of Klotho. Hence the wide, unmineralized growth plates and the osteomalacic abnormalities apparent in trabecular and cortical bone were completely reversed in the F(Tg)/Kl(-/-) mice. Nevertheless, independent actions of Klotho on bone were suggested as manifested by alterations in mineralized bone, and in cortical bone volume which were observed in both the Kl(-/-) and F(Tr)/Kl(-/-) mutants. In summary, our findings substantiate in vivo the essential role of Klotho in the mechanism of action of FGF23 in view of the fact that Klotho ablation converts the biochemical and skeletal manifestations resulting from FGF23 overexpression to a phenotype consistent with Klotho deficiency.     

Klotho lacks a vitamin D independent physiological role in glucose homeostasis, bone turnover, and steady-state PTH secretion in vivo

Apart from its function as co-receptor for fibroblast growth factor-23 (FGF23), Klotho is thought to regulate insulin signaling, intracellular oxidative stress, and parathyroid hormone (PTH) secretion in an FGF23 independent fashion. Here, we crossed Klotho deficient (Kl^−/−) mice with vitamin D receptor (VDR) mutant mice to examine further vitamin D independent functions of Klotho. All mice were fed a rescue diet enriched with calcium, phosphorus, and lactose to prevent hyperparathyroidism in VDR mutants, and were killed at 4 weeks of age after double fluorochrome labeling. Kl^−/− mice displayed hypercalcemia, hyperphosphatemia, dwarfism, organ atrophy, azotemia, pulmonary emphysema, and osteomalacia. In addition, glucose and insulin tolerance tests revealed hypoglycemia and profoundly increased peripheral insulin sensitivity in Kl^−/− mice. Compound mutants were normocalcemic and normophosphatemic, did not show premature aging or organ atrophy, and were phenocopies of VDR mutant mice in terms of body weight, bone mineral density, bone metabolism, serum calcium, serum phosphate, serum PTH, gene expression in parathyroid glands, as well as urinary calcium and phosphate excretion. Furthermore, ablation of vitamin D signaling in double mutants completely normalized glucose and insulin tolerance, indicating that Klotho has no vitamin D independent effects on insulin signaling. Histomorphometry of pancreas islets showed similar beta cell volume per body weight in all groups of animals. In conclusion, our findings cast doubt on a physiologically relevant vitamin D and Fgf23 independent function of Klotho in the regulation of glucose metabolism, bone turnover, and steady-state PTH secretion in vivo.     

The autosomal dominant hypophosphatemic rickets R176Q mutation in fibroblast growth factor 23 resists proteolytic cleavage and enhances in vivo biological potency

Missense mutations in fibroblast growth factor 23 (FGF23) are the cause of autosomal dominant hypophosphatemic rickets (ADHR). The mutations (R176Q, R179W, and R179Q) replace Arg residues within a subtilisin-like proprotein convertase (SPC) cleavage site (RXXR motif), leading to protease resistance of FGF23. The goals of this study were to examine in vivo the biological potency of the R176Q mutant FGF23 form and to characterize alterations in homeostatic mechanisms that give rise to the phenotypic presentation of this disorder. For this, wild type and R176Q mutant FGF23 were overexpressed in the intact animals using a tumor-bearing nude mouse system. At comparable circulating levels, the mutant form was more potent in inducing hypophosphatemia, in decreasing circulating concentrations of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and in causing rickets and osteomalacia in these animals compared with wild type FGF23. Parameters of calcium homeostasis were also altered, leading to secondary hyperparathyroidism and parathyroid gland hyperplasia. However, the raised circulating levels of parathyroid hormone were ineffective in normalizing the reduced 1,25(OH)(2)D(3) levels by increasing renal expression of 25(OH)D(3)-1alpha-hydroxylase (Cyp40) to promote its synthesis and by decreasing that of 25(OH)D(3)-24-hydroxylase (Cyp24) to prevent its catabolism. The findings provide direct in vivo evidence that missense mutations from ADHR kindreds are gain-of-function mutations that retain and increase the protein's biological potency. Moreover, for the first time, they define a potential role for FGF23 in dissociating parathyroid hormone actions on mineral fluxes and on vitamin D metabolism at the level of the kidney.     

Pathogenic role of Fgf23 in Dmp1-null mice

Autosomal recessive hypophosphatemic rickets (ARHR), which is characterized by renal phosphate wasting, aberrant regulation of 1alpha-hydroxylase activity, and rickets/osteomalacia, is caused by inactivating mutations of dentin matrix protein 1 (DMP1). ARHR resembles autosomal dominant hypophosphatemic rickets (ADHR) and X-linked hypophosphatemia (XLH), hereditary disorders respectively caused by cleavage-resistant mutations of the phosphaturic factor FGF23 and inactivating mutations of PHEX that lead to increased production of FGF23 by osteocytes in bone. Circulating levels of FGF23 are increased in ARHR and its Dmp1-null mouse homologue. To determine the causal role of FGF23 in ARHR, we transferred Fgf23 deficient/enhanced green fluorescent protein (eGFP) reporter mice onto Dmp1-null mice to create mice lacking both Fgf23 and Dmp1. Dmp1(-/-) mice displayed decreased serum phosphate concentrations, inappropriately normal 1,25(OH)(2)D levels, severe rickets, and a diffuse form of osteomalacia in association with elevated Fgf23 serum levels and expression in osteocytes. In contrast, Fgf23(-/-) mice had undetectable serum Fgf23 and elevated serum phosphate and 1,25(OH)(2)D levels along with severe growth retardation and focal form of osteomalacia. In combined Dmp1(-/-)/Fgf23(-/-), circulating Fgf23 levels were also undetectable, and the serum levels of phosphate and 1,25(OH)(2)D levels were identical to Fgf23(-/-) mice. Rickets and diffuse osteomalacia in Dmp1-null mice were transformed to severe growth retardation and focal osteomalacia characteristic of Fgf23-null mice. These data suggest that the regulation of extracellular matrix mineralization by DMP1 is coupled to renal phosphate handling and vitamin D metabolism through a DMP1-dependent regulation of FGF23 production by osteocytes.     

1,25-(OH)2-16ene-23yne-D3 reduces secondary hyperparathyroidism in uremic rats with little calcemic effect

OBJECTIVES: To compare the effects of vitamin D analogs versus calcitriol on serum levels of Ca, P and parathyroid hormone (PTH). A compound better than calcitriol should increase the Ca x P product less than calcitriol for an equivalent decrease in PTH levels. METHODS: Biological activity of 4 vitamin D analogs, 1,25-(OH)(2)-16ene- D(3) (RO(1)), 1,25-(OH)(2)-16ene-23yne-D(3) (RO(2)), 1,25-(OH)(2)-26,27-hexafluoro-16ene-23yne-D(3) (RO(3)) and 1,25-(OH)(2)-16ene-23yne-26,27-hexafluoro-19nor-D(3) (RO(4)) was tested vs. calcitriol in parathyroidectomized rats. In a second set of experiments, the effects of RO(2), RO(4) and calcitriol were studied in 5/6 nephrectomized rats with secondary hyperparathyroidism. RESULTS: In parathyroidectomized rats, all analogs (250 pmol/day) led calcemia to rise after 7 days. In uremic rats, all treatments reduced PTH levels. RO(4) revealed toxicity. RO(2) was as effective as calcitriol in suppressing PTH in a dose dependent manner. Mean plasma ionized calcium did not change from baseline to day 14 and day 28 on RO(2) (250 or 500 pmol/day) whereas it increased significantly on RO(2) (1,000 pmol/day) and calcitriol (125 or 250 pmol/day). Increasing the dose of calcitriol led Ca x P to rise more dramatically than increasing the dose of RO(2), which appears to have a wider therapeutic window than calcitriol. CONCLUSION: 1,25-(OH)(2)-16ene-23yne-D(3) (RO(2)) may represent a novel candidate for the treatment of renal osteodystrophy in humans.     

Direct,Acute Effects of Klotho and FGF23 on Vascular Smooth Muscle and Endothelium

Chronic kidney disease (CKD) is regarded as a state of Klotho deficiency and FGF23 excess. In patients with CKD a strong association has been found between increased serum FGF23 and mortality risk, possibly via enhanced atherosclerosis, vascular stiffness, and vascular calcification. The aim of this study was to examine the hypothesis that soluble Klotho and FGF23 exert direct, rapid effects on the vessel wall. We used three in vitro models: mouse aorta rings, human umbilical vein endothelial cells, and human vascular smooth muscle cells (HVSMC). Increasing medium concentrations of soluble Klotho and FGF23 both stimulated aorta contractions and increased ROS production in HVSMC. Klotho partially reverted FGF23 induced vasoconstriction, induced relaxation on phosphate preconstricted aorta and enhanced endothelial NO production in HUVEC. Thus Klotho increased both ROS production in HVSMC and NO production in endothelium. FGF23 induced contraction in phosphate preconstricted vessels and increased ROS production. Phosphate, Klotho and FGF23 together induced no change in vascular tone despite increased ROS production. Moreover, the three compounds combined inhibited relaxation despite increased NO production, probably owing to the concomitant increase in ROS production. In conclusion, although phosphate, soluble Klotho and FGF23 separately stimulate aorta contraction, Klotho mitigates the effects of phosphate and FGF23 on contractility via increased NO production, thereby protecting the vessel to some extent against potentially noxious effects of high phosphate or FGF23 concentrations. This novel observation is in line with the theory that Klotho deficiency is deleterious whereas Klotho sufficiency is protective against the negative effects of phosphate and FGF23 which are additive.     

Increased PTH and 1.25(OH)(2)D levels associated with increased markers of bone turnover following bariatric surgery

The objective of this study was to characterize changes in metabolic bone parameters following bariatric surgery. Seventy-three obese adult patients who underwent either gastric banding (GB), Roux-en-Y gastric bypass (RYGB), or biliopancreatic diversion with duodenal switch (BPD/DS) were followed prospectively for 18 months postoperatively. Changes in the calcium-vitamin D axis (25-hydroxyvitamin D (25OHD), 1,25-dihydroxyvitamin D (1,25(OH)(2)D), calcium, parathyroid hormone (PTH)), markers of bone formation (osteocalcin, bone-specific alkaline phosphatase) and resorption (urinary N-telopeptide (NTx)), as well as bone mineral density (BMD) were assessed at 3-month intervals during this time period. Bariatric surgery resulted in significant and progressive weight loss over 18 months. With supplementation, 25OHD levels increased 65.3% (P < 0.0001) by 3 months, but leveled off and decreased <30 ng/ml by 18 months. PTH initially decreased 21.4% (P = 0.01) at 3 months, but later approached presurgery levels. 1,25(OH)(2)D increased significantly starting at month 12 (50.3% increase from baseline, P = 0.008), and was positively associated with PTH (r = 0.82, P = 0.0001). When stratified by surgery type, median PTH and 1,25(OH)(2)D levels were higher following combined restrictive and malabsorptive operations (RYGB and BPD/DS) compared to GB. Bone formation/resorption markers were increased by 3 months (P < 0.05) and remained elevated through 18 months. Radial BMD decreased 3.5% by month 18, but this change was not significant (P = 0.23). Our findings show that after transient improvement, preoperative vitamin D insufficiency and secondary hyperparathyroidism persisted following surgery despite supplementation. Postoperative secondary hyperparathyroidism was associated with increased 1,25(OH)(2)D levels and increased bone turnover markers.     

In vivo administration of 1,25-dihydroxyvitamin D3 suppresses the expression of RANKL mRNA in bone of thyroparathyroidectomized rats constantly infused with PTH

It is known that pharmacological or toxic doses of vitamin D induce bone resorption both in vivo and in vitro, whereas physiological doses of the vitamin have a protective effect on bone in vivo. To investigate the discrepancies of the dose-dependent effect of vitamin D on bone resorption, we examined the in vivo effect of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] on the expression of the receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) and osteoprotegerin (OPG) mRNAs in bone of thyroparathyroidectomized (TPTX) rats infused with or without parathyroid hormone (PTH). Continuous infusion of 50 ng/h of PTH greatly increased the expression of RANKL mRNA in bone of TPTX rats. Expression of OPG mRNA was not altered by PTH infusion. When graded doses of 1,25(OH)(2)D(3) was daily administered orally for 14 days to normocalcemic TPTX rats constantly infused with PTH, 0.01 and 0.1 microg/kg of 1,25(OH)(2)D(3) inhibited the PTH-induced RANKL mRNA expression, but 0.5 microg/kg of the vitamin did not inhibit it. Regulator of G protein signaling-2 (RGS-2) gene expression was suppressed by 1,25(OH)(2)D(3) dose-dependently, but PTH/PTHrP receptor mRNA expression was not altered. Bone morphometric analyses revealed that 1,25(OH)(2)D(3) suppressed PTH-induced osteoclast number in vivo. These results suggest that pharmacological or toxic doses of 1,25(OH)(2)D(3) stimulate bone resorption by inducing RANKL, but a certain range of physiological doses of the vitamin inhibit PTH-induced bone resorption, the latter mechanism appeared to be mediated, at least in part, by the suppression of the PTH/PTHrP receptor-mediated signaling.     

Synthesis and biological evaluation of a 3-positon epimer of 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D3 (ED-71)

1alpha,25-Dihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3) (ED-71), an analog of active vitamin D(3), 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], possesses a hydroxypropoxy substituent at the 2beta-position of 1,25(OH)(2)D(3). ED-71 has potent biological effects on bone and is currently under phase III clinical studies for bone fracture prevention. It is well-known that the synthesis and secretion of parathyroid hormone (PTH) is regulated by 1,25(OH)(2)D(3). Interestingly, during clinical development of ED-71, serum intact PTH in osteoporotic patients did not change significantly upon treatment with ED-71. The reason remains unclear, however. Brown et al. reported that 3-epi-1,25(OH)(2)D(3), an epimer of 1,25(OH)(2)D(3) at the 3-position, shows equipotent and prolonged activity compared to 1,25(OH)(2)D(3) at suppressing PTH secretion. Since ED-71 has a bulky hydroxypropoxy substituent at the 2-position, epimerization at the adjacent and sterically hindered 3-position might be prevented, which may account for its weak potency in PTH suppression observed in clinical studies. We have significant interest in ED-71 epimerization at the 3-position and the biological potency of 3-epi-ED-71 in suppressing PTH secretion. In the present studies, synthesis of 3-epi-ED-71 and investigations of in vitro suppression of PTH using bovine parathyroid cells are described. The inhibitory potency of vitamin D(3) analogs were found to be 1,25(OH)(2)D(3)>ED-71> or =3-epi-1,25(OH)(2)D(3)>3-epi-ED-71. ED-71 and 3-epi-ED-71 showed weak activity towards PTH suppression in our assays.     

2-methylene-19-nor-20(S)-1alpha-hydroxy-bishomopregnacalciferol [20(S)-2MbisP], an analog of vitamin D3 [1,25(OH)2D3], does not stimulate intestinal phosphate absorption at levels previously shown to suppress parathyroid hormone

Chronic kidney disease results in a reduction in 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) synthesis and an accumulation of phosphorus in the blood, leading to secondary hyperparathyroidism and renal osteodystrophy. Vitamin D analogs that retain the ability to suppress PTH but that are less calcemic and phosphatemic than the native hormone are preferred therapies for secondary hyperparathyroidism. However, even the most favored analog currently approved for the treatment of chronic kidney disease patients, i.e. 1,25-dihydroxy-19-nor-vitamin D2 (19-nor-D2, Zemplar), still retains some ability to stimulate intestinal absorption of calcium and phosphate. A recently described analog of vitamin D3, 2-methylene-19-nor-20(S)-1alpha-hydroxy-bishomopregnacalciferol [20(S)-2MbisP], suppresses PTH levels, but is unable to stimulate intestinal calcium absorption or bone resorption in rats. The present study shows that 20(S)-2MbisP is unable to stimulate intestinal phosphate absorption at levels known to suppress PTH secretion. Further, 19-nor-vitamin D2 under the same circumstances does stimulate phosphate absorption. Thus, 2MbisP has significant potential in the management of secondary hyperparathyroidism of renal failure.     

Compound deletion of Fgfr3 and Fgfr4 partially rescues the Hyp mouse phenotype

Uncertainty exists regarding the physiologically relevant fibroblast growth factor (FGF) receptor (FGFR) for FGF23 in the kidney and the precise tubular segments that are targeted by FGF23. Current data suggest that FGF23 targets the FGFR1c-Klotho complex to coordinately regulate phosphate transport and 1,25-dihydroxyvitamin D [1,25(OH)(2)D] production in the proximal tubule. In studies using the Hyp mouse model, which displays FGF23-mediated hypophosphatemia and aberrant vitamin D, deletion of Fgfr3 or Fgfr4 alone failed to correct the Hyp phenotype. To determine whether FGFR1 is sufficient to mediate the renal effects of FGF23, we deleted Fgfr3 and Fgfr4 in Hyp mice, leaving intact the FGFR1 pathway by transferring compound Fgfr3/Fgfr4-null mice on the Hyp background to create wild-type (WT), Hyp, Fgfr3(-/-)/Fgfr4(-/-), and Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice. We found that deletion of Fgfr3 and Fgfr4 in Fgfr3(-/-)/Fgfr4(-/-) and Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice induced an increase in 1,25(OH)(2)D. In Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice, it partially corrected the hypophosphatemia (P(i) = 9.4 ± 0.9, 6.1 ± 0.2, 9.1 ± 0.4, and 8.0 ± 0.5 mg/dl in WT, Hyp, Fgfr3(-/-)/Fgfr4(-/-), and Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice, respectively), increased Na-phosphate cotransporter Napi2a and Napi2c and Klotho mRNA expression in the kidney, and markedly increased serum FGF23 levels (107 ± 20, 3,680 ± 284, 167 ± 22, and 18,492 ± 1,547 pg/ml in WT, Hyp, Fgfr3(-/-)/Fgfr4(-/-), and Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice, respectively), consistent with a compensatory response to the induction of end-organ resistance. Fgfr1 expression was unchanged in Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice and was not sufficient to transduce the full effects of FGF23 in Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice. These studies suggest that FGFR1, FGFR3, and FGFR4 act in concert to mediate FGF23 effects on the kidney and that loss of FGFR function leads to feedback stimulation of Fgf23 expression in bone.     

Vitamin D analogs: therapeutic applications and mechanisms for selectivity

The vitamin D endocrine system plays a central role in mineral ion homeostasis through the actions of the vitamin D hormone, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], on the intestine, bone, parathyroid gland, and kidney. The main function of 1,25(OH)(2)D(3) is to promote the dietary absorption of calcium and phosphate, but effects on bone, kidney and the parathyroids fine-tune the mineral levels. In addition to these classical actions, 1,25(OH)(2)D(3) exerts pleiotropic effects in a wide variety of target tissues and cell types, often in an autocrine/paracrine fashion. These biological activities of 1,25(OH)(2)D(3) have suggested a multitude of potential therapeutic applications of the vitamin D hormone for the treatment of hyperproliferative disorders (e.g. cancer and psoriasis), immune dysfunction (autoimmune diseases), and endocrine disorders (e.g. hyperparathyroidism). Unfortunately, the effective therapeutic doses required to treat these disorders can produce substantial hypercalcemia. This limitation of 1,25(OH)(2)D(3) therapy has spurred the development of vitamin D analogs that retain the therapeutically important properties of 1,25(OH)(2)D(3), but with reduced calcemic activity. Analogs with improved therapeutic indices are now available for treatment of psoriasis and secondary hyperparathyroidism in chronic kidney disease, and research on newer analogs for these indications continues. Other analogs are under development and in clinical trials for treatment of various types of cancer, autoimmune disorders, and many other diseases. Although many new analogs show tremendous promise in cell-based models, this article will limit it focus on the development of analogs currently in use and those that have demonstrated efficacy in animal models or in clinical trials.     

1,25(OH)2D3 alters growth plate maturation and bone architecture in young rats with normal renal function

Whereas detrimental effects of vitamin D deficiency are known over century, the effects of vitamin D receptor activation by 1,25(OH)(2)D(3), the principal hormonal form of vitamin D, on the growing bone and its growth plate are less clear. Currently, 1,25(OH)(2)D(3) is used in pediatric patients with chronic kidney disease and mineral and bone disorder (CKD-MBD) and is strongly associated with growth retardation. Here, we investigate the effect of 1,25(OH)(2)D(3) treatment on bone development in normal young rats, unrelated to renal insufficiency. Young rats received daily i.p. injections of 1 μg/kg 1,25(OH)(2)D(3) for one week, or intermittent 3 μg/kg 1,25(OH)(2)D(3) for one month. Histological analysis revealed narrower tibial growth plates, predominantly in the hypertrophic zone of 1,25(OH)(2)D(3)-treated animals in both experimental protocols. This phenotype was supported by narrower distribution of aggrecan, collagens II and X mRNA, shown by in situ hybridization. Concomitant with altered chondrocyte maturation, 1,25(OH)(2)D(3) increased chondrocyte proliferation and apoptosis in terminal hypertrophic cells. In vitro treatment of the chondrocytic cell line ATDC5 with 1,25(OH)(2)D(3) lowered differentiation and increased proliferation dose and time-dependently. Micro-CT analysis of femurs from 1-week 1,25(OH)(2)D(3)-treated group revealed reduced cortical thickness, elevated cortical porosity, and higher trabecular number and thickness. 1-month administration resulted in a similar cortical phenotype but without effect on trabecular bone. Evaluation of fluorochrome binding with confocal microscopy revealed inhibiting effects of 1,25(OH)(2)D(3) on intracortical bone formation. This study shows negative effects of 1,25(OH)(2)D(3) on growth plate and bone which may contribute to the exacerbation of MBD in the CKD pediatric patients.     

Humoral hypercalcemia of malignancy: Some enigmas on the clinical features

Humoral hypercalcemia of malignancy (HHM) is a common paraneoplastic syndrome mediated by tumor-derived parathyroid hormone-related peptide (PTHRP), which bears structural and functional similarities to PTH. Thus the clinical features of HHM are very similar to those of primary hyperparathyroidism (1° HPT), a prototype of humoral hypercalcemia caused by PTH. On the other hand, HHM syndrome differs from 1° HPT in several aspects, including serum 1,25(OH)₂D levels, acid-base balance, and bone remodeling process, the reason of which remains largely unknown. We approached these questions using a unique animal model of HHM, nude rats implanted with PTHRP-overproducing human carcinomas. In this review we will summarize the results and discuss the implications in understanding the disease mechanism.