Thyroparathyroidectomized Rats Research Articles

Preclinical development of EXT608, an investigational parathyroid hormone derivative with extended half-life for the treatment of hypoparathyroidism.

Hypoparathyroidism, a deficiency of parathyroid hormone (PTH), results in hypocalcemia, hyperphosphatemia, and hypercalciuria. The disease is poorly controlled by calcium and vitamin D supplements or native PTH(1-84) replacement therapy. A version of PTH is being developed using D-VITylation technology, whereby vitamin D is conjugated to a therapeutic peptide, which confers a long plasma half-life by virtue of binding to the abundant vitamin D binding protein (DBP). D-VITylation of PTH caused no reduction in activity at the PTHR1 receptor, and resulted in a plasma elimination half-life of 7-15h in rats and 24-32h in cynomolgus monkeys. Analysis of steady-state pharmacokinetics as a function of dose showed flat profiles with smaller peak:trough ratios at low doses, indicative of slower subcutaneous absorption. In thyroparathyroidectomized (TPTx) rats, PTH(1-34)-vitamin D conjugates restored serum calcium and phosphate levels into the normal range over the 24h dosing period, and increased bone turnover markers and reduced bone mineral density. Urinary calcium was initially elevated, but normalized by the end of treatment on day 27. In healthy monkeys, a single dose of PTH(1-34)-vitamin D conjugates elevated serum calcium levels above the normal range for a period of 24-48h while simultaneously reducing urinary calcium. Therefore, the lead compound, EXT608, is a promising candidate as a therapeutic that can truly mimic the endogenous activity of PTH and warrants further study in patients with hypoparathyroidism.

Synthesis and Biological Evaluations of Novel Human Parathyroid Hormone 1 Receptor (hPTHR1) Agonists Bearing Bicyclic Aromatic Moiety.

We have previously shown that the small molecule hPTHR1 agonist PCO371 (1) orally and dose-dependently induces PTH-like calcemic and hypophostemic activity in thyroparathyroidectomized rats. Compound 2a, bearing a bicyclic aromatic ring, was identified as a novel hPTHR1 agonist during hit to lead modification. It showed moderate PTHR1 agonistic activity with an EC20 value of 15 μM, and its metabolic stability in human liver microsome (hLM) as well as its solubility in phosphate buffer (PPb) and Fasted state simulated intestinal fluid (FaSSIF) were found to be poor. As results of the initial derivatization of 2a, we identified the indole derivatives as another scaffold. In this article, we report on the structure-activity relationship (SAR), structure-metabolism relationship (SMR), and structure-solubility relationship (SSR) of bicyclic aromatic derivatives, and the in vivo efficacy of 2j.

Dose-range analysis of the effects of the long-acting parathyroid hormone analog AZP-3601 versus PTH(1-34) delivered by daily injection or continuous infusion on blood calcium levels and bone metabolism in thyroparathyroidectomized (TPTX) rats

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Optimization of PTH/PTHrP Hybrid Peptides to Derive a Long-Acting PTH Analog (LA-PTH).

ABSTRACTProlonged signaling at the parathyroid hormone receptor 1 (PTHR1) correlates with the capacity of a ligand to bind to a G protein‐independent receptor conformation (R0). As long‐acting PTH (LA‐PTH) ligands hold interest as potential treatments for hypoparathyroidism (HP), we explored the structural basis in the ligand for stable R0 binding and prolonged cAMP signaling. A series of PTH/PTHrP hybrid analogs were synthesized and tested for actions in vitro and in vivo. Of the series, [Ala1,3,12,Gln10,Arg11,Trp14]‐PTH(1‐14)/PTHrP(15–36) (M‐PTH/PTHrP) bound with high affinity to R0, induced prolonged cAMP responses in UMR106 rat osteoblast‐derived cells, and induced the most prolonged increases in serum calcium (sCa) in normal rats. Daily s.c. injection of M‐PTH/PTHrP into thyroparathyroidectomized (TPTX) rats, a model of HP, normalized sCa without raising urine Ca. In contrast, oral alfacalcidol, a widely used treatment for HP, normalized sCa, but induced frank hypercalciuria. M‐PTH/PTHrP exhibited low solubility in aqueous solutions of neutral pH; however, replacement of Leu18, Phe22, and His26 with the less hydrophobic residues, Ala, Ala, and Lys, at those respective positions markedly improved solubility while maintaining bioactivity. Indeed, we recently showed that the resultant analog [Ala18,22,Lys26]‐M‐PTH/PTHrP or LA‐PTH, effectively normalizes sCa in TPTX rats and mediates prolonged actions in monkeys. These studies provide useful information for optimizing PTH and PTHrP ligand analogs for therapeutic development. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Lead Optimization and Avoidance of Reactive Metabolite Leading to PCO371, a Potent, Selective, and Orally Available Human Parathyroid Hormone Receptor 1 (hPTHR1) Agonist.

We have previously shown that the oral administration of the small molecule hPTHR1 agonist PCO371 and its lead compound, 1 (CH5447240) results in PTH-like calcemic and hypophostemic activity in thyroparathyroidectomized rats. However, 1 was converted to a reactive metabolite in a human liver microsome assay. In this article, we report on the modification path that led to an enhancement of PTHR1 agonistic activity and reduction in the formation of a reactive metabolite to result in a potent, selective, and orally active PTHR1 agonist 1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione (PCO371, 16c). This compound is currently being evaluated in a phase 1 clinical study for the treatment of hypoparathyroidism.

Pharmacodynamic Actions of a Long-Acting PTH Analog (LA-PTH) in Thyroparathyroidectomized (TPTX) Rats and Normal Monkeys.

Hypoparathyroidism is a disease of chronic hypocalcemia and hyperphosphatemia due to a deficiency of parathyroid hormone (PTH). PTH and analogs of the hormone are of interest as potential therapies. Accordingly, we examined the pharmacological properties of a long-acting PTH analog, [Ala(1,3,12,18,22) , Gln(10) ,Arg(11) ,Trp(14) ,Lys(26) ]-PTH(1-14)/PTHrP(15-36) (LA-PTH) in thyroparathyroidectomized (TPTX) rats, a model of HP, as well as in normal monkeys. In TPTX rats, a single intravenous administration of LA-PTH at a dose of 0.9 nmol/kg increased serum calcium (sCa) and decreased serum phosphate (sPi) to near-normal levels for longer than 48 hours, whereas PTH(1-34) and PTH(1-84), each injected at a dose 80-fold higher than that used for LA-PTH, increased sCa and decreased sPi only modestly and transiently (<6 hours). LA-PTH also exhibited enhanced and prolonged efficacy versus PTH(1-34) and PTH(1-84) for elevating sCa when administered subcutaneously (s.c.) into monkeys. Daily s.c. administration of LA-PTH (1.8 nmol/kg) into TPTX rats for 28 days elevated sCa to near normal levels without causing hypercalciuria or increasing bone resorption markers, a desirable goal in the treatment of hypoparathyroidism. The results are supportive of further study of long-acting PTH analogs as potential therapies for patients with hypoparathyroidism. © 2016 American Society for Bone and Mineral Research.

Effect of Sodium Fluoride Administration to Rats on Bone Phosphorous Content and Phosphatemia

Fluoride (F) has a known mitogenic effect on bone cells. The daily administration of 40 micromol NaF per 100 g of body weight (bw) increases bone mass in rats. Nevertheless, the quality and composition of bone formed under F stimulus is still matter of study. The objective of this work was to investigate the effect of sodium fluoride (NaF, CAS 7681-49-4) administration on phosphate metabolism and its impact on bone. Experiments were carried out in female fasted 50-day-old rats. Unless otherwise stated, NaF dose was 40 micromol NaF/day . 100 g bw, administered by gastric tube. Four groups of 4 rats each were given the following daily NaF doses: Group A: 0, B: 20, C: 40 and D: 60 micromol NaF/day x 100 g bw. After 30 days rats were killed saving their femora and plasma. Bone phosphorous contents (BPC) and phosphatemia (mg/dl) were measured. BPC decreased significantly as a function of NaF dose. A: 93.3+/-14.1; B: 78.7+/-15.5; C: 61.1+/-14.7**; D: 59.6+/-8.6 **mg P/g dry bone (** significant difference to group A, p < 0.01). Phosphatemia (mg/dl) increased significantly with a peak at 90 min after NaF dose (basal: 5.34+/-0.06; 90 min: 8.15 +/-0.43, p < 0.001). Phosphaturia (microg/min) increased though differences were not significant (basal: 46.7+/-42.8; 4 h: 1275 +/-757, p > 0.05). In thyroparathyroidectomized rats, plasma phosphate increased continuously for at least 240 min. Renal plasmatic flow, glomerular filtration rate and renal blood flow were not affected by NaF treatment. In isolated perfused rat kidneys, urinary phosphate excretion remained unaffected after NaF administration. Phosphate concentration was measured in the plasma and erythrocytes of rats after one dose of NaF (n = 8). Phosphate content of erythrocyte was not affected by NaF, in spite of the concurrent increase in phosphatemia. It is concluded that the treatment with NaF causes a transitory increase in plasma phosphate levels. Neither renal hemodynamic factors nor the inhibitory effect on parathormone actions appear to be the causes of hyperphosphatemia. Efflux of phosphate from cells might not be the cause of the increase in phosphatemia. The loss of phosphorous from bone appears as the most probable determinant of hyperphosphatemia after fluoride administration.

Effects of ONO-5334, a novel orally-active inhibitor of cathepsin K, on bone metabolism

In the present study, we examined the in vitro and in vivo pharmacological effects of ONO-5334, a novel inhibitor of cathepsin K, on bone metabolism. In vitro experiments indicated that ONO-5334 is a potent inhibitor of cathepsin K with Ki value of 0.1nM. Although this compound inhibited other cysteine proteases, such as cathepsin S, L and B, its inhibitory activity for these enzymes was 8 to 320 fold lower than that for cathepsin K. ONO-5334 also inhibited human osteoclasts bone resorption in vitro at a concentration more than 100 fold lower than that of alendronate, a bisphosphonate. While alendronate disrupted actin ring and induced pyknotic nuclei in osteoclasts, ONO-5334 did not have such effects, suggesting that this compound does not affect osteoclasts viability. In in vivo experiments, oral administration of ONO-5334 dose-dependently reduced plasma calcium level increased by parathyroid hormone related peptide in thyroparathyroidectomized rats. Furthermore, in vivo experiment using normal monkeys demonstrated that ONO-5334 decreases serum and urine C-telopeptide of type I collagen level, a bone resorption marker, soon after oral dosing. These levels were consistently decreased below pre-dose levels by repeated oral dosing with ONO-5334 for 7days. ONO-5334 on the other hand did not affect bone formation markers, serum osteocalcin and bone specific alkaline phosphatase. These findings indicate that ONO-5334 is a specific inhibitor for cathepsin K and thus may be a novel therapeutic agent for metabolic bone diseases.

Evidence for a signaling axis by which intestinal phosphate rapidly modulates renal phosphate reabsorption

The mechanisms by which phosphorus homeostasis is preserved in mammals are not completely understood. We demonstrate the presence of a mechanism by which the intestine detects the presence of increased dietary phosphate and rapidly increases renal phosphate excretion. The mechanism is of physiological relevance because it maintains plasma phosphate concentrations in the normal range after ingestion of a phosphate-containing meal. When inorganic phosphate is infused into the duodenum, there is a rapid increase in the renal fractional excretion of phosphate (FE Pi). The phosphaturic effect of intestinal phosphate is specific for phosphate because administration of sodium chloride does not elicit a similar response. Phosphaturia after intestinal phosphate administration occurs in thyro-parathyroidectomized rats, demonstrating that parathyroid hormone is not essential for this effect. The increase in renal FE Pi in response to the intestinal administration of phosphate occurs without changes in plasma concentrations of phosphate (filtered load), parathyroid hormone, FGF-23, or secreted frizzled related protein-4. Denervation of the kidney does not attenuate phosphaturia elicited after intestinal phosphate administration. Phosphaturia is not elicited when phosphate is instilled in other parts of the gastrointestinal tract such as the stomach. Infusion of homogenates of the duodenal mucosa increases FE Pi, which demonstrates the presence of one or more substances within the intestinal mucosa that directly modulate renal phosphate reabsorption. Our experiments demonstrate the presence of a previously unrecognized phosphate gut-renal axis that rapidly modulates renal phosphate excretion after the intestinal administration of phosphate.

Calcium‐sensing receptor and recovery from hypocalcaemia in thyroparathyroidectomized rats

Plasma ionized calcium (p-Ca(2+)) is kept within a very narrow range and deviations are rapidly corrected by flux of Ca(2+) between extracellular fluid and the labile calcium pool at the quiescent bone surface. The calcium sensing at the bone surface represents a physiological interesting model for the rapid minute-to-minute regulation of p-Ca(2+). Our aim was to study whether the calcium-sensing receptor (CaR) has a role in the rapid recovery of p-Ca(2+) from acute induced hypocalcaemia. Male Wistar rats were thyroparathyroidectomized (TPTX). Acute hypocalcaemia in the animals was induced by infusion of EGTA (40-50 mM EGTA, 3.0 mL h(-1) for 30 min). Thereafter the recovery of p-Ca(2+) was followed. Vehicle or the CaR activators, R-568 (2 mg as a bolus twice) or gentamycin were administrated intravenously. EGTA infusion resulted in significantly lower nadir of hypocalcaemia in R-568- or gentamycin-treated rats compared to vehicle-treated rats (P < 0.01). During recovery phase p-Ca(2+) remained significantly lower in R-568 rats (P < 0.001). As such p-Ca(2+) levels recovered to basal levels in the vehicle group within 70 min after stopping EGTA, while R-568 or gentamycin rats remained significantly hypocalcaemic. The CaR activators R-568 and gentamycin, both significantly delayed the recovery of p-Ca(2+) from acute EGTA-induced hypocalcaemia in TPTX rats. This novel finding suggests the existence of calcium sensing by bone of importance for the rapid minute-to-minute regulation of p-Ca(2+).

Bone Health in Chronic Kidney Disease–Mineral and Bone Disease

Chronic kidney disease (CKD) is accompanied by disturbances in calcium, phosphate, vitamin D, and parathyroid hormone (PTH) homeostasis that play an important role in the pathophysiology of renal bone disease. The increased cardiovascular morbidity and mortality observed among patients with CKD has recently been recognized to be associated with these disturbances in mineral metabolism. Thus, disturbances in mineral metabolism observed in renal failure results in a multisystem disorder, making the development of a standardized definition of these disorders a top priority. Therefore, the Board of Directors of Kidney Disease: Improving Global Outcomes proposed to define the broader category of mineral disorders associated with CKD as CKD-mineral and bone disorder (CKD-MBD). This newly proposed definition will include the disorders of mineral metabolism, bone histology (renal osteodystrophy), and the extraskeletal manifestations such as vascular calcification. This new definition and stratification of disease should result in improvement not only in the clinical management of patients but also will facilitate the interpretation and translation of clinical research. Renal osteodystrophy is now considered as 1 component of this disorder and will be defined as a morphologic alteration only, based on unification of the histomorphometric definitions that will include parameters of turnover, mineralization, and volume. An internationally accepted classification system will enable the consensus for bone biopsy evaluation as well as for the role of biomarkers. This article will focus on the newly proposed definitions of bone disease as part of CKD-MBD, based on the complex pathophysiologic process underlying bone disease in CKD stages 2 to 5.

Parathyroid hormone-dependent endocytosis of renal type IIc Na-Picotransporter

Hereditary hypophosphatemic rickets with hypercalciuria results from mutations of the renal type IIc Na-P(i) cotransporter gene, suggesting that the type IIc transporter plays a prominent role in renal phosphate handling. The goal of the present study was to investigate the regulation of the type IIc Na-P(i) cotransporter by parathyroid hormone (PTH). Type IIc Na-P(i) cotransporter levels were markedly increased in thyroparathyroidectomized (TPTX) rats. Four hours after administration of PTH, type IIc transporter protein levels were markedly decreased in the apical membrane fraction but recovered to baseline levels at 24 h. Immunohistochemical analyses demonstrated the presence of the type IIc transporter in the apical membrane and subapical compartments in the proximal tubular cells in TPTX animals. After administration of PTH, the intensity of immunoreactive signals in apical and subapical type IIc transporter decreased in the renal proximal tubular cells in TPTX rats. Colchicine completely blocked the internalization of the type IIc transporter. In addition, leupeptin prevented the PTH-mediated degradation of the type IIa transporter in lysosomes but had no effect on PTH-mediated degradation of the lysosomal type IIc transporter. In PTH-treated TPTX rats, the internalization of the type IIc transporter occurred after administration of PTH(1-34) (PKA and PKC activator) or PTH(3-34) (PKC activator). Thus the present study demonstrated that PTH is a major hormonal regulator of the type IIc Na-P(i) cotransporter in renal proximal tubules.

An Orally Active Cathepsin K Inhibitor, Furan-2-Carboxylic Acid, 1-{1-[4-Fluoro-2-(2-oxo-pyrrolidin-1-yl)-phenyl]-3-oxo-piperidin-4-ylcarbamoyl}-cyclohexyl)-amide (OST-4077), Inhibits Osteoclast Activity in Vitro and Bone Loss in Ovariectomized Rats

Human cathepsin K, a cysteine proteinase of the papain family, has been recognized as a potential drug target for the treatment of osteoporosis. The predominant expression of cathepsin K in osteoclasts has rendered the enzyme into a major target for the development of novel antiresorptive drugs. Now, we report the pharmacological properties of OST-4077 [furan-2-carboxylic acid (1-{1-[4-fluoro-2-(2-oxo-pyrrolidin-1-yl)-phenyl]-3-oxo-piperidin-4-ylcarbamoyl}-cyclohexyl)-amide] as a novel selective cathepsin K inhibitor. Human and rat cathepsin K were inhibited in vitro by OST-4077 with the IC50 values of 11 and 427 nM, respectively. OST-4077 suppressed bone resorption induced by rabbit osteoclasts (IC50, 37 nM) but did not affect bone mineralization or cellular alkaline phosphatase activity in MC3T3-E1 cells. Parathyroid hormone-induced bone resorption was inhibited in a dose-dependent manner in thyroparathyroidectomized rats gavaged with a single dose of OST-4077 (ED50, 69 mg/kg). When given orally twice daily for 4 weeks to 3-month-old ovariectomized (OVX) rats, OST-4077 dose-dependently prevented bone loss, as monitored by bone densitometry, ash content, and urinary excretion of deoxypyridinoline. No change in serum osteocalcin in the OVX rats by OST-4077 suggested that bone formation might not be affected by the agent. In summary, OST-4077 selectively inhibited bone resorbing activities of osteoclasts and prevented bone loss induced by estrogen deficiency but did not affect bone formation. OST-4077, an orally active selective human cathepsin K inhibitor, may have the therapeutic potential for the treatment of diseases characterized by excessive bone loss including osteoporosis.

Action of Calciotropic Hormones on Bone Metabolism – Role of Vitamin D3 in Bone Remodeling Events

Vitamin D3 is known to have immunosuppressive effects that can be beneficial for treatment of immune disorders and transplant rejection, however therapeutic application is limited due to hypercalcemia and hypercalcuria. The goal of our studies was to explore both the acute and steady state effects of vitamin D3 on bone remodeling as potential limiting factors to broader use of vitamin D3 in the clinic. Vitamin D3 was evaluated for its skeletal effects in both thyroparathyroidectomized (TPTx) and intact rat models. In TPTx rats, deprivation of thyroid and parathyroid hormones and calcitonin creates a low state of bone modeling and remodeling ideal for evaluation of changes imposed by drug intervention. The use of both models allowed for discrimination of individual (TPTx) versus combined (intact) effects of calciotropic hormones on bone and calcium metabolism. Our studies have confirmed the limitations of using vitamin D3 for treatment/co- treatment of immune disease in humans due to the intrinsic hypercalcemic properties of the hormone, and also highlighted the potential of vitamin D3 to negatively impact skeletal integrity due to excessive bone remodeling driven by bone resorption. Taken together our data emphasize the importance of including biomarkers of bone remodeling as an integral part of clinical and preclinical studies using vitamin D3 to treat immune disorders and suggest the need for co-treatment with an antiresorptive agent to counteract hypercalcemia and deterioration of bone.

Changes in amelogenesis in the rat incisor following short-term hypocalcaemia

There is a relationship between hypocalcaemia and the enamel hypoplasia. Earlier studies in rats have reported a severe hypocalcaemia and enamel hypoplasia a month after thyro-parathyroidectomy (TPTX). The aims of this study were to look at earlier stages and to attempt to correlate morphological changes with alterations in the distribution of amelogenin. Twenty-five Wistar rats were, under anaesthesia, thyro-parathyroidectomized. Sham operated rats were included as controls. After 14, 30 or 57 days, the animals were reanesthatized and the tissues fixed by intracardiac perfusion of fixative. The lower incisors were processed for light microscopy and immunogold electron microscopy. After 14 days the thyro-parathyroidectomised rats were severely hypocalcaemic but amelogenesis was morphologically similar to controls. After 30 and 57 days, enamel defects were observed in the late secretory and early maturation stages in the thyro-parathyroidectomised rats. The immunocytochemical study revealed a concentration of stippled material immunolabelled for amelogenin at the secretory pole of the ameloblasts in the hypocalcaemic rats. The absence of enamel defects after 14 days suggests that this was an insufficient hypocalcaemic period to induce morphological alterations. The concentration of stippled material containing amelogenin suggests that alterations in matrix formation may be the basis of the morphological changes.

Circulating FGF-23 Is Regulated by 1α,25-Dihydroxyvitamin D3 and Phosphorus in Vivo

Fibroblast growth factor-23 (FGF-23), a novel phosphate-regulating factor, was elevated in hypophosphatemic patients with X-linked hypophosphatemic rickets/osteomalacia and also in patients with chronic kidney disease. These observations suggested the pathophysiological importance of FGF-23 on phosphate homeostasis. However, regulation of FGF-23 production is still unclear. We investigated effects of both dietary phosphorus and 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) on circulating FGF-23 in vivo Administration of. 1alpha,25(OH)(2)D(3) dose-dependently increased serum FGF-23 in thyroparathyroidectomized rats without correlating with serum inorganic phosphorus or serum parathyroid hormone. On the other hand, vitamin D receptor null mice had very low serum FGF-23 and did not respond to the 1alpha,25(OH)(2)D(3) administration. These observations suggested 1alpha,25(OH)(2)D(3) directly or indirectly regulates circulating FGF-23. Serum FGF-23 had a strong correlation with serum inorganic phosphorus controlled by dietary phosphorus in 5/6 nephrectomized rats. High phosphate diet elicited a 5-fold increase in serum FGF-23 compared with sham-operated rats, whereas serum FGF-23 did not correlate with serum calcium or serum creatinine in 5/6 nephrectomized rats. Administration of 1alpha,25-dihydroxyvitamin D(3) also elicited a severalfold increase in serum FGF-23 in the uremic rats. Taken together, this shows that both serum phosphorus and 1alpha,25(OH)(2)D(3) regulate circulating FGF-23 independent of each other. Therefore, we proposed there was a feedback loop existing among serum phosphorus, 1alpha,25(OH)(2)D(3), and FGF-23, in which the novel phosphate-regulating bone-kidney axis integrated with the parathyroid hormone-vitamin D(3) axis in regulating phosphate homeostasis.

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.

Secreted frizzled-related protein 4 is a potent tumor-derived phosphaturic agent

Tumors associated with osteomalacia elaborate the novel factor(s), phosphatonin(s), which causes phosphaturia and hypophosphatemia by cAMP-independent pathways. We show that secreted frizzled-related protein-4 (sFRP-4), a protein highly expressed in such tumors, is a circulating phosphaturic factor that antagonizes renal Wnt-signaling. In cultured opossum renal epithelial cells, sFRP-4 specifically inhibited sodium-dependent phosphate transport. Infusions of sFRP-4 in normal rats over 2 hours specifically increased renal fractional excretion of inorganic phosphate (FEPi) from 14% ± 2% to 34% ± 5% (mean ± SEM, P < 0.01). Urinary cAMP and calcium excretion were unchanged. In thyro-parathyroidectomized rats, sFRP-4 increased FEPi from 0.7% ± 0.2% to 3.8% ± 1.2% (P < 0.05), demonstrating that sFRP-4 inhibits renal inorganic phosphate reabsorption by PTH-independent mechanisms. Administration of sFRP-4 to intact rats over 8 hours increased FEPi, decreased serum phosphate (1.95 ± 0.1 to 1.53 ± 0.09 mmol/l, P < 0.05) but did not alter serum 1α, 25-dihydroxyvitamin D, renal 25-hydroxyvitamin D 1α-hydroxylase cytochrome P450, and sodium-phosphate cotransporter mRNA concentrations. Infusion of sFRP-4 antagonizes Wnt action as demonstrated by reduced renal β-catenin and increased phosphorylated β-catenin concentrations. The sFRP-4 is detectable in normal human serum and in the serum of a patient with tumor-induced osteomalacia. Thus, sFRP-4 displays phosphatonin-like properties, because it is a circulating protein that promotes phosphaturia and hypophosphatemia and blunts compensatory increases in 1α, 25-dihydroxyvitamin D.

Secreted frizzled-related protein 4 is a potent tumor-derived phosphaturic agent.

Tumors associated with osteomalacia elaborate the novel factor(s), phosphatonin(s), which causes phosphaturia and hypophosphatemia by cAMP-independent pathways. We show that secreted frizzled-related protein-4 (sFRP-4), a protein highly expressed in such tumors, is a circulating phosphaturic factor that antagonizes renal Wnt-signaling. In cultured opossum renal epithelial cells, sFRP-4 specifically inhibited sodium-dependent phosphate transport. Infusions of sFRP-4 in normal rats over 2 hours specifically increased renal fractional excretion of inorganic phosphate (FEPi) from 14% +/- 2% to 34% +/- 5% (mean +/- SEM, P < 0.01). Urinary cAMP and calcium excretion were unchanged. In thyro-parathyroidectomized rats, sFRP-4 increased FEPi from 0.7% +/- 0.2% to 3.8% +/- 1.2% (P < 0.05), demonstrating that sFRP-4 inhibits renal inorganic phosphate reabsorption by PTH-independent mechanisms. Administration of sFRP-4 to intact rats over 8 hours increased FEPi, decreased serum phosphate (1.95 +/- 0.1 to 1.53 +/- 0.09 mmol/l, P < 0.05) but did not alter serum 1alpha, 25-dihydroxyvitamin D, renal 25-hydroxyvitamin D 1alpha-hydroxylase cytochrome P450, and sodium-phosphate cotransporter mRNA concentrations. Infusion of sFRP-4 antagonizes Wnt action as demonstrated by reduced renal beta-catenin and increased phosphorylated beta-catenin concentrations. The sFRP-4 is detectable in normal human serum and in the serum of a patient with tumor-induced osteomalacia. Thus, sFRP-4 displays phosphatonin-like properties, because it is a circulating protein that promotes phosphaturia and hypophosphatemia and blunts compensatory increases in 1alpha, 25-dihydroxyvitamin D.

Vitamin D and bone.

It is now well established that supraphysiological doses of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] stimulate bone resorption. Recent studies have established that osteoblasts/stromal cells express receptor activator of NF-kappaB ligand (RANKL) in response to several bone-resorbing factors including 1alpha,25(OH)(2)D(3) to support osteoclast differentiation from their precursors. Osteoclast precursors which express receptor activator of NF-kappaB (RANK) recognize RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into osteoclasts in the presence of macrophage-colony stimulating factor (M-CSF). Osteoprotegerin (OPG) acts as a decoy receptor for RANKL. We also found that daily oral administration of 1alpha,25(OH)(2)D(3) for 14 days to normocalcemic thyroparathyroidectomized (TPTX) rats constantly infused with parathyroid hormone (PTH) inhibited the PTH-induced expression of RANKL and cathepsin K mRNA in bone. The inhibitory effect of 1alpha,25(OH)(2)D(3) on the PTH-induced expression of RANKL mRNA occurred only with physiological doses of the vitamin. Supraphysiological doses of 1alpha,25(OH)(2)D(3) increased serum Ca and expression of RANKL in vivo in the presence of PTH. These results suggest that the bone-resorbing activity of vitamin D does not occur at physiological dose levels in vivo. A certain range of physiological doses of 1alpha,25(OH)(2)D(3) rather suppress the PTH-induced bone resorption in vivo, supporting the concept that 1alpha,25(OH)(2)D(3) or its derivatives are useful for the treatment of various metabolic bone diseases such as osteoporosis and secondary hyperparathyroidism.