Estradiol is an important regulator of bone accumulation and maintenance. Circulating estrogens are primarily produced by the gonads. Aromatase, the enzyme responsible for the conversion of androgens to estrogen, is expressed by bone marrow cells (BMCs) of both hematopoietic and nonhematopoietic origin. While the significance of gonad-derived estradiol to bone health has been investigated, there is limited understanding regarding the relative contribution of BMC derived estrogens to bone metabolism. To elucidate the role of BMC derived estrogens in male bone, irradiated wild-type C57BL/6J mice received bone marrow cells transplanted from either WT (WT(WT)) or aromatase-deficient (WT(ArKO)) mice. MicroCT was acquired on lumbar vertebra to assess bone quantity and quality. WT(ArKO) animals had greater trabecular bone volume (BV/TV p = 0.002), with a higher trabecular number (p = 0.008), connectivity density (p = 0.017), and bone mineral content (p = 0.004). In cortical bone, WT(ArKO) animals exhibited smaller cortical pores and lower cortical porosity (p = 0.02). Static histomorphometry revealed fewer osteoclasts per bone surface (Oc.S/BS%), osteoclasts on the erosion surface (ES(Oc+)/BS, p = 0.04) and low number of osteoclasts per bone perimeter (N.Oc/B.Pm, p = 0.01) in WT(ArKO). Osteoblast-associated parameters in WT(ArKO) were lower but not statistically different from WT(WT). Dynamic histomorphometry suggested similar bone formation indices' patterns with lower mean values in mineral apposition rate, label separation, and BFR/BS in WT(ArKO) animals. Ex vivo bone cell differentiation assays demonstrated relative decreased osteoblast differentiation and ability to form mineralized nodules. This study demonstrates a role of local 17β-estradiol production by BMCs for regulating the quantity and quality of bone in male mice. Underlying in vivo cellular and molecular mechanisms require further study.

This cross-sectional study was performed to characterize the factors affecting bone mass in male hemodialysis subjects. We found that of all the factors analyzed, the strongest correlation was with body mass index. In fact, after adjusting for body weight, the correlations with bone turnover markers and sex hormones were no longer significant.

Estrogen regulation of the male skeleton was first clearly demonstrated in patients with aromatase deficiency or a mutation in the ERα gene. Estrogen action on the skeleton is thought to occur mainly through the action of the nuclear receptors ERα and ERβ. Recently, in vitro studies have shown that the G protein-coupled receptor GPR30 is a functional estrogen receptor (ER). GPR30-deficient mouse models have been generated to study the in vivo function of this protein; however, its in vivo role in the male skeleton remains underexplored. We have characterized size, body composition, and bone mass in adult male Gpr30 knockout (KO) mice and their wild-type (WT) littermates. Gpr30 KO mice weighed more and had greater nasal-anal length (p < .001). Both lean mass and percent body fat were increased in the KO mice. Femur length was greater in Gpr30 KO mice, as was whole-body, spine, and femoral areal bone mineral density (p < .01). Gpr30 KO mice showed increased trabecular bone volume (p < .01) and cortical thickness (p < .001). Mineralized surface was increased in Gpr30 KO mice (p < .05). Bromodeoxyuridine (BrdU) labeling showed greater proliferation in the growth plate of Gpr30 KO mice (p < .05). Under osteogenic culture conditions, Gpr30 KO femoral bone marrow cells produced fewer alkaline phosphatase-positive colonies in early differentiating osteoblast cultures but showed increased mineralized nodule deposition in mature osteoblast cultures. Serum insulin-like growth factor 1 (IGF-1) levels were not different. These data suggest that in male mice, GPR30 action contributes to regulation of bone mass, size, and microarchitecture by a mechanism that does not require changes in circulating IGF-1.

Wnt/β-catenin signaling is a critical regulator of skeletal physiology. However, previous studies have mainly focused on its roles in osteoblasts, while its specific function in osteoclasts is unknown. This is a clinically important question because neutralizing antibodies against Wnt antagonists are promising new drugs for bone diseases. Here, we show that in osteoclastogenesis, β-catenin is induced during the macrophage colony-stimulating factor (M-CSF)-mediated quiescence-to-proliferation switch but suppressed during the RANKL-mediated proliferation-to-differentiation switch. Genetically, β-catenin deletion blocks osteoclast precursor proliferation, while β-catenin constitutive activation sustains proliferation but prevents osteoclast differentiation, both causing osteopetrosis. In contrast, β-catenin heterozygosity enhances osteoclast differentiation, causing osteoporosis. Biochemically, Wnt activation attenuates whereas Wnt inhibition stimulates osteoclastogenesis. Mechanistically, β-catenin activation increases GATA2/Evi1 expression but abolishes RANKL-induced c-Jun phosphorylation. Therefore, β-catenin exerts a pivotal biphasic and dosage-dependent regulation of osteoclastogenesis. Importantly, these findings suggest that Wnt activation is a more effective treatment for skeletal fragility than previously recognized that confers dual anabolic and anti-catabolic benefits.

It has been nearly 90 years since the discovery of the antirachitic activity of vitamin D. During that period, vitamin D structure, metabolism, and mechanism of action at target tissues have been delineated. We now recognize that vitamin D acts as a steroid hormone to help maintain normal calcium and phosphate homeostasis. Many diseases characterized by deranged calcium and phosphate metabolism have been explained by dysregulated vitamin D production and/or action. Calcium-containing kidney stones are believed to result from excessive urinary calcium excretion due to increased intestinal absorption of calcium, increased bone resorption, and renal calcium loss. To try and explain the cause of this hypercalciuria, many studies have focused on the role of deranged vitamin D metabolism and action. Much of our understanding of how such derangements in vitamin D metabolism and action can contribute to the development of hypercalciuria and ultimately kidney stones has come from both clinical and basic approaches that are discussed in this chapter. However, some studies have failed to observe any alteration in vitamin D production or action, while others have implicated a role for increased 1,25(OH)(2)D production or increased tissue sensitivity in the face of normal circulating 1,25(OH)(2)D concentrations. Resolution of these discrepancies will require additional studies in hypercalciuric stone-forming patients that focus on the genetics of vitamin D metabolism and the cellular and molecular actions of vitamin D at its target tissues.

BACKGROUND:Hyperoxaluria is a major risk factor for kidney stone formation. Although urinary oxalate measurement is part of all basic stone risk assessment, there is no standardized method for this measurement.METHODS:Urine samples from 24-h urine collection covering a broad range of oxalate concentrations were aliquoted and sent, in duplicates, to six blinded international laboratories for oxalate, sodium and creatinine measurement. In a second set of experiments, ten pairs of native urine and urine spiked with 10 mg/L of oxalate were sent for oxalate measurement. Three laboratories used a commercially available oxalate oxidase kit, two laboratories used a high-performance liquid chromatography (HPLC)-based method and one laboratory used both methods.RESULTS:Intra-laboratory reliability for oxalate measurement expressed as intraclass correlation coefficient (ICC) varied between 0.808 [95% confidence interval (CI): 0.427-0.948] and 0.998 (95% CI: 0.994-1.000), with lower values for HPLC-based methods. Acidification of urine samples prior to analysis led to significantly higher oxalate concentrations. ICC for inter-laboratory reliability varied between 0.745 (95% CI: 0.468-0.890) and 0.986 (95% CI: 0.967-0.995). Recovery of the 10 mg/L oxalate-spiked samples varied between 8.7 ± 2.3 and 10.7 ± 0.5 mg/L. Overall, HPLC-based methods showed more variability compared to the oxalate oxidase kit-based methods.CONCLUSIONS:Significant variability was noted in the quantification of urinary oxalate concentration by different laboratories, which may partially explain the differences of hyperoxaluria prevalence reported in the literature. Our data stress the need for a standardization of the method of oxalate measurement.

The classic definition of hypercalciuria, an upper normal limit of 200 mg/day, is based on a constant diet restricted in calcium, sodium, and animal protein; however, random diet data challenge this. Here our retrospective study determined the validity of the classic definition of hypercalciuria by comparing data from 39 publications analyzing urinary calcium excretion on a constant restricted diet and testing whether hypercalciuria could be defined when extraneous dietary influences were controlled. These papers encompassed 300 non-stone-forming patients, 208 patients with absorptive hypercalciuria type I (presumed due to high intestinal calcium absorption), and 234 stone formers without absorptive hypercalciuria; all evaluated on a constant restricted diet. In non-stone formers, the mean urinary calcium was well below 200 mg/day, and the mean for all patients was 127 +/- 46 mg/day with an upper limit of 219 mg/day. In absorptive hypercalciuria type I, the mean urinary calcium significantly exceeded 200mg/day in all studies with a combined mean of 259 +/- 55 mg/day. Receiver operating characteristic curve analysis showed the optimal cutoff point for urinary calcium excretion was 172 mg/day on a restricted diet, a value that approximates the traditional limit of 200 mg/day. Thus, on a restricted diet, a clear demarcation was seen between urinary calcium excretion of kidney stone formers with absorptive hypercalciuria type I and normal individuals. When dietary variables are controlled, the classic definition of hypercalciuria of nephrolithiasis appears valid.

The Westernized diet is acidogenic due to the high content of sulfur-containing amino acids and relative deficiency of potassium organic anions. Chronic acid loads result in hypercalciuria and negative calcium balance often associated with loss of bone mineral. Alkali therapy tends to reverse the hypercalciuria but little is known regarding its effect on bone as assessed by bone histomorphometry. The present study utilized dynamic bone histomorphometry to evaluate the effects of alkali therapy on acid-induced changes in bone turnover. Serum and urine analyses and bone histomorphometry were assessed in adult rats after 2 months of either a low casein (LC) or high casein (HC) diet supplemented with either potassium chloride (KCl) or potassium citrate (KCit). Compared to animals on LC-KCl diet, HC-KCl diet delivered a substantial acid load as shown by significant increases in urinary sulfate, ammonium, and net acid excretion, and a lower urinary pH and citrate excretion without detectable changes in serum parameters. The acid load also resulted in hypercalciuria. Dynamic and static bone histomorphometry disclosed a significant reduction in cancellous bone volume and trabecular number associated with a 2.5-fold increase in eroded and a 3.5-fold increase in osteoclastic surfaces. There was also a near 2-fold increase in bone formation rate in rats on the HC-KCl diet. When animals on the HC diet were given KCit instead of KCl, all of the aforementioned changes in urine biochemistry and bone turnover were significantly attenuated or entirely prevented. These findings underscore the deleterious effects of high animal protein intake in promoting hypercalciuria and increasing bone turnover. Co-administration of potassium alkali attenuates or prevents these changes. In this animal model of high dietary animal protein intake, the major skeletal effect of alkali therapy is to reduce bone resorption, with little or no effect on bone formation.

Background Bisphosphonates are the most commonly prescribed medications for the treatment of osteoporosis. Although existing evidence supports a good safety profile, there is concern that chronic administration of these agents could result in severe suppression of bone turnover with increased risk of nonvertebral fractures.Objective The objective of this study was to report the clinical presentation, selected bone histomorphometry and X-ray images of patients who developed mid-shaft long bone fractures during bisphosphonate therapy, six of whom had bone biopsy for histomorphometery.Results Of the 13 patients who sustained atraumatic mid-shaft fractures, 10 were on alendronate and three were on risedronate therapy before the fractures. In addition to bisphosphonates, three patients were on oestrogen and two on tamoxifen concomitantly. Four patients with glucocorticoid-induced osteoporosis were on alendronate for 3-11 years along with glucocorticoid therapy. Bone histomorphometry showed severe suppression of bone turnover in five patients and low bone turnover in one patient.Conclusion Long-term bisphosphonate therapy may increase the risk of unusual long bone mid-shaft fractures. This is probably due to prolonged suppression of bone turnover, which could lead to accumulation of microdamage and development of hypermineralized bone. At present, the scope of this complication in the larger context of patients receiving bisphosphonate therapy remains unknown, but appears to be small.

High ${\rm [HCO}_3^ - ]$ inhibits and low ${\rm [HCO}_3^ - ]$ stimulates bone resorption, which mediates part of the effect of chronic acidosis or acid feeding on bone. Soluble adenylyl cyclase (sAC) is a bicarbonate sensor that can potentially mediate the effect of bicarbonate on osteoclasts. Osteoclasts were incubated in 0, 12, and 24 mM ${\rm HCO}_3^ -$ at pH 7.4 for 7–8 days and assayed for tartrate‐resistant acid phosphatase (TRAP) and vacuolar‐ATPase expression, and H + accumulation. Total number and area of TRAP (+) multinucleated osteoclasts was decreased by ${\rm HCO}_3^ -$ in a dose‐dependent manner. V‐ATPase expression and H + accumulation normalized to cell cross‐sectional area or protein were not significantly changed. The ${\rm HCO}_3^ -$ ‐induced inhibition of osteoclast growth and differentiation was blocked by either 2‐hydroxyestradiol, an inhibitor of sAC or sAC knockdown by sAC specific siRNA. The model of ${\rm HCO}_3^ -$ inhibiting osteoclast via sAC was further supported by the fact that the ${\rm HCO}_3^ -$ dose‐response on osteoclasts is flat when cells were saturated with 8‐bromo‐cAMP, a permeant cAMP analog downstream from sAC thus simulating sAC activation. To confirm our in vitro findings in intact bone, we developed a 1‐week mouse calvaria culture system where osteoclasts were shown to be viable. Bone volume density (BV/TV) determined by micro‐computed tomography (µCT), was higher in 24 mM ${\rm HCO}_3^ -$ compared to 12 mM ${\rm HCO}_3^ -$ treated calvaria. This ${\rm HCO}_3^ -$ effect on BV/TV was blocked by 2‐hydroxyestradiol. In summary, sAC mediates the inhibition of osteoclast function by ${\rm HCO}_3^ -$ , by acting as a ${\rm HCO}_3^ -$ sensor. J. Cell. Physiol. 220: 332–340, 2009. © 2009 Wiley‐Liss, Inc.

Purpose: The Equil 2 computer program has been questioned by the new Joint Expert Speciation System program (Mayhem Unit Trust and Council for Scientific and Industrial Research, Pretoria, South Africa) for estimating the urinary saturation of stone forming salts to gauge the propensity for stone formation. To attempt resolution the supersaturation index according to the Joint Expert Speciation System and the relative saturation ratio according to Equil 2 were compared with the semi-empirically derived concentration-to-product ratio.Materials and Methods: Data were obtained from a recent article in The Journal of Urology (R), in which pH, calcium and citrate were varied over a wide range in 72 urine samples. We calculated the relative saturation ratio and the supersaturation index of brushite, and compared them with the available concentration-to-product ratio derived from the growth or dissolution of synthetic brushite.Results: The mean concentration-to-product ratio did not differ from the supersaturation index but the concentration-to-product ratio and the supersaturation index were significantly lower than the relative saturation ratio (p <0.004). On the saturation value and urinary variable plot the relative saturation ratio could be readily distinguished from the concentration-to-product ratio because it was consistently and significantly higher. While the supersaturation index pattern was similar to the concentration-to-product ratio, the supersaturation index was slightly lower at high urinary pH and calcium, and slightly higher at lower urinary pH and calcium (p <0.001). When the Ca(2)H(2)(PO(4))(2) complex was deleted from the Joint Expert Speciation System, the corrected supersaturation index was not significantly different from the relative saturation ratio determined by Equil 2.Conclusions: The relative saturation ratio overestimates brushite saturation by about 80%. The supersaturation index yields a good approximation of brushite saturation at modest degrees of saturation but it overestimates saturation at low pH or calcium (low saturation) and underestimates it at high pH or calcium (high saturation).

A 52-year-old white female with a history of profound hypophosphatemia, muscle weakness, and multiple debilitating atraumatic fractures was referred in March 2004 for evaluation at the Mineral Metabolism Clinic. Five years prior, she had sustained bilateral rib fractures which failed to heal. In May 2000, she was diagnosed with primary hyperparathyroidism based on serum calcium of 10.4 mg/dl (reference range 8.4–10.2 mg/dl), parathyroid hormone (PTH) of 97 pg/ml (reference 10–65 pg/ml), phosphorus of 2.0 mg/dl (reference 2.5–4.5 mg/dl), and alkaline phosphatase of 420 IU/l (reference 38–126 IU/l). Bone density measured by dual energy X-ray absorptiometry (DXA) revealed T-scores of -2.9 (lumbar spine) and -3.5 (femoral neck), indicative of osteoporosis. In July 2000, the patient underwent partial parathyroidectomy with removal of two parathyroid glands (histologic diagnosis: adenoma for left superior gland, normal for left inferior gland). Intraoperatively, serum PTH fell from 58 to 13 pg/ml at 24 min post-excision. In the ensuing months, serum calcium normalized, but PTH remained elevated and serum phosphorus remained low. One year after the first parathyroid surgery, the patient underwent a subtotal parathyroidectomy (histologic diagnosis: hyperplasia), leaving only about 20–30 mg of the right inferior gland, and was placed on low dose calcitriol (0.5 μg daily). Over the next three years, she progressively lost mobility due to muscle weakness, requiring assistance in ambulation. She developed multiple additional atraumatic fractures (bilateral superior and inferior pubic ramus, bilateral femoral head, radial neck, ulnar, and multiple vertebral) resulting in kyphosis. After repeated neurological and rheumatological evaluations, she was referred to our clinic. In June 2004, she complained of profound fatigue and severe pain in the low back and thoracic area and required a walker for ambulation. Physical examination revealed a well-nourished kyphotic woman, with a wide-based gait and generalized decreased muscle strength (4/5). An iliac crest bone biopsy and clinical biochemistry evaluation were performed. The bone biopsy (Figure 1) revealed a marked increase in osteoid parameters for both cortical and cancellous bone, including osteoid volume (11.9 and 56.1% of total bone volume, for cortical and cancellous bone respectively), osteoid surface (78.9 and 93.6% of total bone surface), and mean osteoid seam width (24 and 31 μm). Mineralized bone volume and mean thickness were reduced in both cortical and cancellous bone. Osteoblastic surfaces were present but reduced for the amount of osteoid present. Resorptive parameters were not increased and frankly low for cancellous bone. Tetracycline labeling was absent in all sections examined, indicating lack of new bone formation. Taken together, these findings are consistent with osteomalacia because of defective bone mineralization. The results of the initial clinical biochemistry evaluation, performed during a 4-day constant metabolic diet, are shown in Table 1. In the absence of a family history of hypophosphatemia, the combination of acquired severe hypophosphatemia, renal phosphate wasting, osteomalacia, and inappropriately low 1,25-dihydroxy vitamin D raised the suspicion of tumor-induced osteomalacia (TIO). Consistently, serum fibroblast growth factor 23 (FGF23) measured by enzyme immunosorbent assay directed against the C-terminal molecule (Alpco, Salem, NH) was elevated: 573 RU/ml (reference <230 RU/ml). A search for an underlying tumor was initiated. Magnetic resonance imaging of the chest and abdomen had been previously performed and were unremarkable. An 111In-octreotide scintigraphy (octreotide scan) was obtained in September 2004 and revealed an inappropriate focus of uptake slightly to the left of the midline of the head (Figure 2a). Single-photon emission computed tomography revealed an intense focus of radiotracer in the midline, inferior to the cranial vault. Magnetic resonance imaging of the orbit and face demonstrated a 1.5 × 1.4 × 1.4 cm rounded mass within the dorsal aspect of the left nasal cavity (Figure 2b). The mass abutted the dorsal aspect of the inferior nasal turbinate and nasal septum, without invasion of the pterygoid musculature.Table 1Initial metabolic evaluationSerum calcium8.5 mg/100 ml(reference 8.4–10.2 mg/100 ml)Serum phosphorus0.7 mg/100 ml(reference 2.5–4.5 mg/100 ml)Parathyroid hormone89 pg/ml(reference 10–65 pg/ml)Serum alkaline phosphatase168 IU/l(reference 38–126 IU/l)Serum creatinine0.7 mg/100 ml(reference 0.8–1.4 mg/100 ml)Serum 25-hydroxy vitamin D30 ng/ml(reference 5–60 ng/ml)Serum 1,25-dihydroxy vitamin D6 pg/ml(reference 10–65 pg/ml)Renal fractional excretion of phosphate40%Renal threshold phosphate concentration (TmP/GFR)0.25–0.30 mg/100 ml(reference 2.5–4.35 mg/100 ml)Intestinal calcium absorption (dual-isotope method)23%(mean normal 50%)Laboratory data were collected during a 4-day constant metabolic diet containing 400 mg calcium, 100 mEq sodium, and 800 mg phosphate daily. Open table in a new tab Laboratory data were collected during a 4-day constant metabolic diet containing 400 mg calcium, 100 mEq sodium, and 800 mg phosphate daily. While awaiting surgery, in October 2004 the patient was initiated on indomethacin 25 mg, p.o., t.i.d., both for bone pain control and to evaluate the putative effect of indomethacin on FGF23/phosphate metabolism (detailed in Discussion). No significant improvement of hypophosphatemia was noted and indomethacin was discontinued after 4 weeks. The patient was placed on replacement therapy with calcitriol (0.25 μg, p.o., t.i.d.) and phosphate (750 mg, p.o., t.i.d.) until a week before surgery. A transnasal, endoscopic excision of the nasal mass (Figure 2c) was performed in December 2004. Pathological examination showed diffuse hypercellular spindle cell proliferation arranged in an ill-defined fascicular growth pattern (Figure 3a). The neoplastic cells were mostly short spindled with round to oval nuclei showing mild to moderate cytological atypia (mild hyperchromasia and mild to moderate nuclear pleomorphism). The mitotic rate was low (1–2 per 10 high-power fields). The stroma exhibited prominent capillary-like, thick-walled, and hemangiopericytic-like vessels. Multiple osteoclast-like giant cells (Figure 3b) and focal extracellular mineralized chondroid matrix (Figure 3c) were noted. No necrosis was identified. The pathological diagnosis was phosphaturic mesenchymal tumor, mixed connective tissue variant. Immunohistochemical analysis of paraffin-fixed sections using a monoclonal anti-human FGF23 antibody showed expression of FGF23 protein in the cytoplasm of isolated cells in the excised tumor (Figure 3d–f). Figure 4 shows the evolution of markers of phosphate metabolism over 33 months starting from our initial evaluation, including 27 months post-surgery. Postoperative clinical improvement and normalization of biochemical abnormalities confirmed the initial diagnosis of TIO. Serum phosphorus normalized within 1 week after tumor removal. A rapid decrease in serum calcium (to 7.5 mg/100 ml within 24 h) prompted concern of hungry bone syndrome, and further hypocalcemia was prevented with temporary calcium supplementation. Serum 1,25-dihydroxy vitamin D levels normalized 24 h post-intervention, became elevated after 1 week (101 pg/ml), likely because of an abrupt increase in bone mineralization (Figure 4d), and returned to normal 3 months postoperatively. At that time, intestinal calcium absorption measured by dual isotope was increased (77%). Repeat octreotide scan 5 months post-intervention demonstrated no abnormal tracer uptake (Figure 2d). Bone density at the lumbar spine measured by dual-energy X-ray absorptiometry 6 months after surgery showed a 21% improvement. During 27 months of postoperative follow-up, the patient's clinical complaints of bone pain and muscle weakness progressively resolved, and self-ambulation was restored. TIO is a rare paraneoplastic syndrome characterized by renal phosphate wasting, hypophosphatemia, inappropriately normal or low serum 1,25-dihydroxy vitamin D, and inadequate bone mineralization.1.Drezner M.K. Tumor-induced osteomalacia.Rev Endocr Metab Disord. 2001; 2: 175-186Crossref PubMed Scopus (74) Google Scholar,2.Kumar R. Tumor-induced osteomalacia and the regulation of phosphate homeostasis.Bone. 2000; 27: 333-338Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar The clinical manifestations of TIO include fatigue, muscle weakness, bone pain (that can mimic joint pain), and insufficiency fractures (‘spontaneous’ fractures, caused by physiological levels of mechanical stress exerted upon weakened bone), in the absence of a family history of renal phosphate wasting and/or bone disorders. Disease progress can be slow and insidious, mimicking other rheumatological, neurological, or endocrine disorders, and thus correct diagnosis can be delayed for years or missed altogether. Without intervention, patients become progressively debilitated, experience a markedly decreased quality of life, and are at risk of life-threatening complications. Identification and surgical removal of the offending tumor followed by normalization of phosphate metabolism provides both definitive diagnosis and cure of TIO. Phosphorus homeostasis is maintained by the interplay between net intestinal absorption, tissue and bone turnover, and renal excretion, with net intestinal absorption matching renal excretion under balance conditions. The kidney assumes a pivotal role in maintaining total body phosphorus balance. Phosphorus circulating as unbound phosphate is freely filtered at the glomerulus and reabsorbed primarily across the proximal tubule. Renal reabsorption of phosphate is regulated by multiple humoral factors, including a class of incompletely understood circulating phosphaturic factors collectively called phosphatonins.3.Schiavi S.C. Moe O.W. Phosphatonins: a new class of phosphate-regulating proteins.Curr Opin Nephrol Hypertens. 2002; 11: 423-430Crossref PubMed Scopus (54) Google Scholar, 4.Quarles L.D. FGF23, PHEX, and MEPE regulation of phosphate homeostasis and skeletal mineralization.Am J Physiol Endocrinol Metab. 2003; 285: E1-E9Crossref PubMed Scopus (268) Google Scholar, 5.Schiavi S.C. Kumar R. The phosphatonin pathway: new insights in phosphate homeostasis.Kidney Int. 2004; 65: 1-14Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 6.Berndt T.J. Schiavi S. Kumar R. ‘Phosphatonins’ and the regulation of phosphorus homeostasis.Am J Physiol Renal Physiol. 2005; 289: F1170-F1182Crossref PubMed Scopus (169) Google Scholar These were initially discovered as humoral factors secreted by the tumors of patients with TIO, and include FGF23, FGF7, matrix extracellular phosphoglycoprotein, and secreted frizzled-related protein 4. Phosphatonins downregulate renal phosphate reabsorption at least in part by decreasing the abundance of apical sodium/phosphate co-transporters (NaPi-IIa) in the proximal tubule.6.Berndt T.J. Schiavi S. Kumar R. ‘Phosphatonins’ and the regulation of phosphorus homeostasis.Am J Physiol Renal Physiol. 2005; 289: F1170-F1182Crossref PubMed Scopus (169) Google Scholar FGF23 is the most studied phosphatonin, and has a causative role in the pathogenesis of hereditary hypophosphatemias—X-linked hypophosphatemic rickets (XLHR) and autosomal dominant hypophosphatemic rickets—as well as in some cases of acquired hypophosphatemia from TIO.7.Shimada T. Mizutani S. Muto T. et al.Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia.Proc Natl Acad Sci. 2001; 98: 6500-6505Crossref PubMed Scopus (1120) Google Scholar,8.Stubbs J. Liu S. Quarles L.D. Role of fibroblast growth factor 23 in phosphate homeostasis and pathogenesis of disordered mineral metabolism in chronic kidney disease.Semi Dial. 2007; 20: 302-308Crossref PubMed Scopus (95) Google Scholar Hypophosphatemia can be caused by inadequate dietary intake, decreased intestinal absorption, excessive urinary excretion, or the shift of phosphate from serum into cells and/or bone. Inappropriately high excretion (renal wasting) of phosphate occurs in various genetic and acquired disorders. One common cause is hyperparathyroidism, which is usually associated with moderate hypophosphatemia. Parathyroid hormone (PTH)-independent renal phosphate wasting is usually more severe and may occur because of intrinsic proximal tubular defects (Fanconi syndrome), or because of increased production and/or decreased degradation of phosphatonins. Phosphatonin-related disorders may in turn be genetic (XLHR, autosomal dominant hypophosphatemic rickets, fibrous dysplasia), or acquired (TIO). Of note, secondary hyperparathyroidism and tertiary hyperparathyroidism have been observed in patients with XLHR and TIO9.Huang Q.L. Feig D.S. Blackstein M.E. Development of tertiary hyperparathyroidism after phosphate supplementation in oncogenic osteomalacia.J Endocrinol Invest. 2000; 23: 263-267Crossref PubMed Scopus (26) Google Scholar,10.Savio R.M. Gosnell J.E. Posen S. et al.Parathyroidectomy for tertiary hyperparathyroidism associated with X-linked dominant hypophosphatemic rickets.Arch Surg. 2004; 139: 218-222Crossref PubMed Scopus (27) Google Scholar and may further contribute to phosphate loss in these patients. The mechanism for this is uncertain, but may be related to reduced 1,25-dihydroxy vitamin D levels leading to decreased intestinal absorption of calcium, hypocalcemia, and consequent upregulation of PTH secretion. Hyperparathyroidism in our patient was initially diagnosed as primary, but in retrospect it is reasonable to suggest that it was actually secondary or tertiary. Understanding the mechanism of hypophosphatemia is critical for correct diagnosis. Renal phosphate wasting, a hallmark of TIO, should be investigated in any patient with persistent hypophosphatemia. Determination of phosphate clearance and fractional excretion of phosphate do not distinguish true renal phosphate wasting (inappropriate reabsorption from the glomerular filtrate) from other causes of abnormally elevated phosphate excretion (increased net renal inflow of phosphate and/or increased glomerular filtration rate (GFR), exceeding a normal reabsorptive capacity). Renal wasting can be confirmed by using the nomogram of Walton and Bijvoet11.Walton R.J. Bijvoet O.L.M. Nomogram for derivation of renal threshold phosphate concentration.Lancet. 1975; 306: 309-310Abstract Scopus (618) Google Scholar to derive renal threshold phosphate concentration (TmP/GFR) from serum phosphorus and tubular reabsorption of phosphate (TRP=1–phosphate clearance/creatinine clearance, using fasting urine and serum phosphate and creatinine). A low TmP/GFR in spite of hypophosphatemia is indicative of renal phosphate wasting. If FGF23 is markedly increased in the absence of a family history of hypophosphatemia, TIO is likely—although approximately 20% of XLHR cases are because of sporadic mutations and thus have a similar presentation.12.Dixon P.H. Christie P.T. Wooding C. et al.Mutational analysis of PHEX gene in X-linked hypophosphatemia.J Clin Endocrinol Metab. 1998; 83: 3615-3623Crossref PubMed Scopus (103) Google Scholar However, a normal FGF23 level does not exclude TIO, as other tumor-secreted phosphatonins may be involved and are not measured. FGF23 was likely the offending phosphatonin in our case, as FGF23 protein was expressed in the excised tumor (Figure 3d–f), and serum FGF23 normalized after surgery (Figure 4b). A search for an underlying tumor should be initiated as soon as the clinical and biochemical findings indicate that TIO is likely. Various tumor locations have been described, including distal extremities, and, most commonly, the nasopharynx and sinuses. Plain radiographs, computed tomography, magnetic resonance imaging, and even positron-emission tomography scanning have been successfully used to localize TIO tumors, and computed-tomography-guided fine-needle biopsy of the tumor has been used to aid in diagnosis. When other imaging techniques fail to identify the tumor (as was the case in our patient), octreotide scan should be considered.13.Jan de Beur S.M. Streeten E.A. Civelek A.C. et al.Localisation of mesenchymal tumours by somatostatin receptor imaging.Lancet. 2002; 359: 761-763Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar,14.Nguyen B.D. Wang E.A. Indium-111 pentetreotide scintigraphy of mesenchymal tumor with oncogenic osteomalacia.Clin Nucl Med. 1999; 24: 130-131Crossref PubMed Scopus (51) Google Scholar This technique has high specificity but a negative octreotide scan does not exclude TIO, as some but not all TIO tumors have surface somatostatin receptors. Selective venous sampling for FGF23 has also been successfully used to localize FGF23-secreting tumors.15.Takeuchi Y. Suzuki H. Ogura S. et al.Venous sampling for fibroblast growth factor-23 confirms preoperative diagnosis of tumor-induced osteomalacia.J Clin Endocrinol Metab. 2004; 89: 3979-3982Crossref PubMed Scopus (131) Google Scholar,16.van Boekel G. Ruinemans-Koerts J. Joosten F. et al.Tumor producing fibroblast growth factor 23 localized by two-staged venous sampling.Eur J Endocrinol. 2008; 158: 431-437Crossref PubMed Scopus (42) Google Scholar Most tumors associated with TIO are slow-growing, benign, polymorphous neoplasms of mesenchymal origin, classified as osteoblastoma-like, ossifying fibrous-like, non-ossifying fibrous-like, and phosphaturic mesenchymal tumor, mixed connective tissue variant.17.Jan de Beur S.M. Tumor-induced osteomalacia.JAMA. 2005; 294: 1260-1267Crossref PubMed Scopus (166) Google Scholar Of these, phosphaturic mesenchymal tumor, mixed connective tissue variant is the most common, and contains characteristic spindle-shaped neoplastic cells with low or absent mitotic activity in a matrix with calcifications, osteoclast-like giant cells, and rich microvasculature.18.Folpe A.L. Fanburg-Smith J.C. Billings S.D. et al.Most osteomalacia-associated mesenchymal tumors are a single histopathologic entity: an analysis of 32 cases and a comprehensive review of the literature.Am J Surg Pathol. 2004; 28: 1-30Crossref PubMed Scopus (436) Google Scholar Rarely, TIO can result from neurofibromatosis, fibrous dysplasia of bone, carcinomas, or metastases.17.Jan de Beur S.M. Tumor-induced osteomalacia.JAMA. 2005; 294: 1260-1267Crossref PubMed Scopus (166) Google Scholar,18.Folpe A.L. Fanburg-Smith J.C. Billings S.D. et al.Most osteomalacia-associated mesenchymal tumors are a single histopathologic entity: an analysis of 32 cases and a comprehensive review of the literature.Am J Surg Pathol. 2004; 28: 1-30Crossref PubMed Scopus (436) Google Scholar Complete removal of the underlying tumor (and thus removal of the source of excess circulating phosphatonins) is to date the only definitive treatment. In most cases this involves surgical resection, but successful computed-tomography-guided radiofrequency ablation of a TIO tumor has also been reported.19.Hesse E. Rosenthal H. Bastian L. Radiofrequency ablation of a tumor causing oncogenic osteomalacia.N Engl J Med. 2007; 357: 422-424Crossref PubMed Scopus (54) Google Scholar Before surgery or in nonexcisable tumors (failed localization or surgical contraindication), aggressive phosphate and calcitriol replacement can improve the symptoms, normalize serum phosphorus, but does not improve renal phosphate wasting. Treatment with unlabeled octreotide was attempted with mixed results in patients with somatostatin receptor-positive tumors (as established by radiolabeled octreotide scan).20.Paglia F. Dionisi S. Minisola S. Octreotide for tumor-induced osteomalacia.N Engl J Med. 2002; 346: 1748-1749Crossref PubMed Scopus (1) Google Scholar,21.Seufert J. Ebert K. Muller J. et al.Octreotide therapy for tumor-induced osteomalacia.N Engl J Med. 2001; 345: 1883-1888Crossref PubMed Scopus (189) Google Scholar The therapeutic trial with indomethacin in our patient was prompted by previous findings in hyp mice. The hyp mouse is a murine model of XLHR with decreased FGF23 degradation and increased urinary prostaglandin E2 excretion. Treatment with indomethacin led to amelioration of hypophosphatemia and fractional excretion of phosphate in these mice, without changes in GFR.22.Baum M. Loleh S. Saini N. et al.Correction of proximal tubule phosphate transport defect in Hyp mice in vivo and in vitro with indomethacin.Proc Natl Acad Sci USA. 2003; 100: 11098-11103Crossref PubMed Scopus (20) Google Scholar To our knowledge, this intervention has not been attempted in humans with renal phosphate wasting. The lack of improvement in our patient may be because of insufficient dosage (a higher dose is however not recommended), or because of inherent differences between human TIO and the hyp mouse model. In our patient, TIO remained undiagnosed for several years. Careful metabolic evaluation and extensive investigations eventually led to the diagnosis, and TIO was cured by surgical removal of the offending tumor. We emphasize the importance of determining serum phosphorus and TmP/GFR and considering TIO early in the differential diagnosis of unexplained persistent bone pain, apparent joint pain, muscle weakness, and spontaneous fractures. This work was supported by the National Institutes of Health grants P01-DK20543 and M01-RR00633.

We investigated the effects of a multi‐load exercise program and KMgCit supplementation on the preservation of bone quality following 5 weeks of bedrest as measured by ultrasound critical‐angle reflectometry (UCR). The bone elasticity of 35 subjects (26 M, 9 F, 35 ± 10 yrs) was studied before and after bedrest. The subjects were divided into 4 groups: sedentary with no drug (n=12); sedentary with KMgCit (n=11); exercise (n=11); and exercise with KMgCit (n=4). The exercise group underwent intense multi‐joint rowing ergometry (30–45 min/d, 6 d/wk) and resistive strength training (2 d/wk). Subjects receiving the study drug were given 42:21:63 mEq KMgCit a day. Bone elasticity, a correlate of bone strength, was assessed by UCR on the tibia and calcaneus at multiple angles. In the sedentary group, prolonged bedrest resulted in a small decrease in bone elasticity, most notable in the more responsive trabecular bone (2.5%, 2779±140, 2709±92 m/s, p=0.06). KMgCit increased trabecular elasticity (4.2%, 2705±125, 2814±140 m/s, p<0.05), while exercise showed a similar trend (3.5%, 2701±121, 2798 ±100 m/s, p=0.08). Treatment with both exercise and KMgCit demonstrated a 1.9% increase, but included only 4 subjects. These findings suggest that KMgCit and exercise training are effective countermeasures against trabecular bone quality loss during prolonged periods in microgravity. Supported by NSBRI.

In the past quarter century, more than 50 metabolites of vitamin D have been described. To date, only a few of these have been quantified in blood, but this has widened our understanding of the pathologic role that altered vitamin D metabolism plays in the development of diseases of calcium homeostasis. Currently, awareness is growing of the prevalence of vitamin D insufficiency in the general population in association with an increased risk of several diseases. However, for many researchers, it is not clear which vitamin D metabolites should be quantified and what the information gained from such an analysis tells us. Only 2 metabolites, namely, 25-hydroxyvitamin D [25(OH)D] and 1,25-dihydroxyvitamin D [1,25(OH)2D], have received the greatest attention. Of these, the need for measuring serum 1,25(OH)2D is limited, and this metabolite should therefore not be considered as part of the standard vitamin D testing regimen. On the other hand, serum 25(OH)D provides the single best assessment of vitamin D status and thus should be the only vitamin D assay typically performed. Currently, numerous formats exist for measuring serum 25(OH)D concentrations, each with its own advantages and disadvantages. This article reviews the currently available methods for serum 25(OH)D quantitation and considers important issues such as whether both the D2 and the D3 forms of the vitamin should be assayed, whether total or free concentrations are most important, and what measures should be taken to ensure the fidelity of the measurements.

There is sufficient epidemiological and clinical data demonstrating an association between reduced bone mineral density and idiopathic hypercalciuria (IH). There have been relatively few studies that have addressed the underlying defect in bone remodeling. The limited studies to date suggest that increased bone turnover occurs in some forms of IH such as fasting hypercalciuria or renal calcium leak and explains the bone loss observed in these forms of IH. On the other hand, defective bone formation is the major defect observed in patients with IH resulting from intestinal hyperabsorption of calcium. These alterations in bone remodeling have been ascribed to genetic, metabolic, and nutritional causes. Although there are several therapeutic options available for treating such patients and preventing stone recurrence, prevention of future bone loss should also be considered to prevent the increased risk of osteoporotic fracture in patients with IH.

s 0.73, 95% CI 0.56 -0.96) or acute renal failure (OR 0.69, 95% CI 0.55 -0.88) with no difference in the frequency of hepatic encephalopathy.Despite this favorable profile, both uninsured (OR 1.36, 95% CI 1.02 -1.77) and Medicaid (OR 1.54, 95% CI 1.16 -2.04) patients had higher in-hospital mortality on multivariate analysis.This disparity was stronger for uninsured patients who developed acute renal failure (OR 1.75).After adjusting for comorbidity, there was no difference in inter-hospital transfers between uninsured, Medicaid and privately insured patients.However, both Medicaid (OR 0.73, 95% CI 0.56 -0.95) and uninsured patients were less likely to undergo liver biopsy (OR 0.53, 95% CI 0.38 -0.73) or OLT (OR 0.62, 95% CI 0.39 -0.97 for Medicaid and OR 0.04, 95% CI 0.01 -0.17 for uninsured respectively).Conclusion: Uninsured and Medicaid patients have higher mortality in ALF and are less likely to undergo OLT.

Hypercalciuria of intestinal origin has been linked with bone loss in calcium nephrolithiasis and idiopathic osteoporosis. This retrospective data analysis was performed to explore potential pathogenetic link between intestinal hyperabsorption of calcium and postmenopausal osteoporosis. Data were retrieved from postmenopausal women who were evaluated for osteoporosis or osteopenia at the Mineral Metabolism Clinic of UT Southwestern Medical Center. A total of 319 patients underwent the test of calciuric response to oral calcium load to obtain an indirect measure of intestinal calcium absorption. Serum and urinary biochemistry and L2–L4 bone mineral density (BMD) were compared between five quintiles of calciuric response. There was a statistically significant trend toward a rise in 24-h urinary calcium and a decrease in urinary deoxypyridinoline (DPD) and BMD, with increasing order of quintiles. The presentation of those in the 1st quintile was consistent with vitamin D insufficiency or deficiency, with impaired calcium absorption, secondary hyperparathyroidism, and stimulated bone turnover (high normal urinary DPD). In contrast, patients in the 5th quintile displayed a picture of absorptive hypercalciuria of stone disease, with intestinal hyperabsorption of calcium, high or high normal urinary calcium and suppressed bone turnover (low or low normal urinary DPD). Thus, the assessment of intestinal calcium absorption in a seemingly homogeneous group of postmenopausal women with osteoporosis or osteopenia revealed a spectrum of calciuric response whose extremes may represent two physiologically distinct subtypes that have important diagnostic and therapeutic implications.

Absorptive hypercalciuria (AH), a common stone-forming condition characterized biochemically by intestinal hyperabsorption of calcium and hypercalciuria may be associated with bone loss. In AH type I (AH-1), hypercalciuria persists despite restriction in dietary calcium intake. We therefore hypothesized that the skeleton may contribute to the hypercalciuria in this subgroup of patients. Histomorphometric analysis of iliac crest biopsies were performed on nine stone-formers with AH-1 and on nine matched normal subjects. After stabilization on a stone-prevention diet, calcium homeostasis in the stone formers was then evaluated on inpatient constant metabolic diet before and after short-term blockade of bone resorption by alendronate (10 mg daily, 17 days total). Compared with controls, the stone-formers had lower indices of bone formation (osteoblast surface/bone surface 1.8+/-2.1 vs 3.0+/-1.5%, P=0.04; wall thickness 35.8+/-6.9 vs 47.2+/-7.6%, P=0.001) and relatively higher bone resorption (osteoclast surface/bone surface 0.4+/-0.2 vs 0.2+/-0.2%, P=0.05). In the stone-formers, a short-term course of alendronate treatment corrected fasting urinary calcium (0.14+/-0.06 to 0.06+/-0.04 mg Ca/mg Cr, P=0.001) and marginally reduced 24-h urinary calcium by 48 mg/day (P=0.06). Increased intestinal calcium absorption and hypercalciuria persisted, but estimated calcium balance improved (P=0.007). Our results suggest that the hypercalciuria of AH-1 originates primarily from intestinal hyperabsorption of calcium, but bone resorption in excess of bone formation may contribute.

PURPOSE:Exposure to the microgravity environment of space increases the risk of kidney stone formation, particularly for calcium oxalate and uric acid stones. This study was performed to evaluate the efficacy of potassium alkali as potassium-magnesium citrate in reducing renal stone risk and bone turnover. MATERIALS AND METHODS:This study was performed as a double-blind, placebo controlled trial. We studied 20 normocalciuric subjects randomized to either placebo or potassium-magnesium citrate (42 mEq potassium, 21 mEq magnesium, 63 mEq citrate per day) before and during 5 weeks of strict bed rest. The study was performed in the General Clinical Research Center and under a controlled dietary regimen composed of 100 mEq of sodium, 800 mg of calcium, 0.8 gm/kg animal protein and 2,200 kcal per day. Two 24-hour urine collections were obtained under oil each week for assessment of stone risk parameters and relative saturation of calcium oxalate, brushite and undissociated uric acid. Blood was also collected for determination of serum immunoreactive parathyroid hormone and vitamin D metabolites. RESULTS:Bed rest promoted a rapid increase in urinary calcium excretion of approximately 50 mg per day in both groups. Despite this increase subjects treated with potassium-magnesium citrate demonstrated significant decreases in the relative saturation of calcium oxalate and in the concentration of undissociated uric acid compared to placebo. Immunoreactive parathyroid hormone, serum 1,25-dihydroxyvitamin D and intestinal calcium absorption all decreased in both groups with no difference in response between the 2 treatment arms. CONCLUSIONS:Provision of alkali as potassium-magnesium citrate is an effective countermeasure for the increased risk of renal stone disease associated with immobilization. Despite an increase in urine calcium concentration, the relative saturation of calcium oxalate decreased due to citrate chelation of calcium and the concentration of undissociated uric acid decreased due to the significant increase in urine pH.

Kidney stones increase after menopause, suggesting a role for estrogen deficiency. ArKO mice have hypercalciuria and lower levels of calcium transport proteins, whereas levels of the klotho protein are elevated. Thus, estrogen deficiency is sufficient to cause altered renal calcium handling.Introduction: The incidence of renal stones increases in women after menopause, implicating a possible role for estrogen deficiency. We used the aromatase deficient (ArKO) mouse, a model of estrogen deficiency, to test the hypothesis that estrogen deficiency would increase urinary calcium excretion and alter the expression of molecular regulators of renal calcium reabsorption.Materials and Methods: Adult female wildtype (WT), ArKO, and estradiol-treated ArKO mice (n = 5-12/ group) were used to measure urinary calcium in the fed and fasting states, relative expression level of some genes involved in calcium reabsorption in the distal convoluted tubule by real-time PCR, and protein expression by Western blotting or immunohistochemistry. Plasma membrane calcium ATPase (PMCA) activity was measured in kidney membrane preparations. ANOVA was used to test for differences between groups followed by posthoc analysis with Dunnett's test.Results: Compared with WT, urinary Ca:Cr ratios were elevated in ArKO mice, renal mRNA levels of transient receptor potential cation channel vallinoid subfamily member 5 (TRPV5), TRPV6, calbindin-D-281, the Na+/Ca+ exchanger (NCXI), and the PMCA1b were significantly decreased, and klotho mRNA and protein levels were elevated. Estradiol treatment of ArKO mice normalized urinary calcium excretion, renal mRNA levels of TRPV5, calbindin-D-28k, PMCA1b, and klotho, as well as protein levels of calbindin-D-28k and Klotho. ArKO mice treated with estradiol had significantly greater PMCA activity than either untreated ArKO mice or WT mice.Conclusions: Estrogen deficiency caused by aromatase inactivation is sufficient for renal calcium loss. Changes in estradiol levels are associated with coordinated changes in expression of many proteins involved in distal tubule calcium reabsorption. Estradiol seems to act at the genomic level by increasing or decreasing (klotho) protein expression and nongenomically by increasing PMCA activity. PMCA, not NCX1, is likely responsible for extruding calcium in response to in vivo estradiol hormonal challenge. These data provide potential mechanisms for regulation of renal calcium handling in response to changes in serum estrogen levels.