Search

UnlabelledOsteoporosis causes an elevated fracture risk. We propose the continued use of T-scores as one means for diagnosis but recommend that, alternatively, hip fracture; osteopenia-associated vertebral, proximal humerus, pelvis, or some wrist fractures; or FRAX scores with ≥3% (hip) or 20% (major) 10-year fracture risk also confer an osteoporosis diagnosis.IntroductionOsteoporosis is a common disorder of reduced bone strength that predisposes to an increased risk for fractures in older individuals. In the USA, the standard criterion for the diagnosis of osteoporosis in postmenopausal women and older men is a T-score of  ≤ -2.5 at the lumbar spine, femur neck, or total hip by bone mineral density testing.MethodsUnder the direction of the National Bone Health Alliance, 17 clinicians and clinical scientists were appointed to a working group charged to determine the appropriate expansion of the criteria by which osteoporosis can be diagnosed.ResultsThe group recommends that postmenopausal women and men aged 50 years should be diagnosed with osteoporosis if they have a demonstrable elevated risk for future fractures. This includes having a T-score of less than or equal to -2.5 at the spine or hip as one method for diagnosis but also permits a diagnosis for individuals in this population who have experienced a hip fracture with or without bone mineral density (BMD) testing and for those who have osteopenia by BMD who sustain a vertebral, proximal humeral, pelvic, or, in some cases, distal forearm fracture. Finally, the term osteoporosis should be used to diagnose individuals with an elevated fracture risk based on the World Health Organization Fracture Risk Algorithm, FRAX.ConclusionsAs new ICD-10 codes become available, it is our hope that this new understanding of what osteoporosis represents will allow for an appropriate diagnosis when older individuals are recognized as being at an elevated risk for fracture.

Human idiopathic hypercalciuria (IH) is the most common cause of calcium oxalate nephrolithiasis with perturbed calcium metabolism with increased bone resorption and decreased renal calcium reabsorption, which can be phenotype-copied in the genetic hypercalciuric stone-forming (GHS) rat model. We previously demonstrated that high VDR expression plays important roles in the development of hypercalciuria in the GHS rats. However, the underlying mechanism through which VDR impact hypercalciuria development remains to be fully understood. Here, we sought to determine how VDR regulated its target genes that are implicated in calcium homeostasis and potentially hypercalciuria. We found that VDR expression in the GHS rats was elevated in the calcium transporting tissues, as well as in the thymus and prostate, but not in lung, brain, heart, liver and spleen, when compared with control SD rats. Snail expression in the GHS rats was significantly downregulated in kidney, intestine, thymus and testis. Intraperitoneal injection of 1,25(OH) 2 D 3 significantly upregulated the expression of renal calcium sensing receptor (CaSR), intestinal calcium transporters transient receptor potential vanilloid type 6 (TRPV6), and VDR in GHS rats, compared with that in control SD rats. ChIP assays revealed that VDR specifically bound to the proximal promoters of target genes, followed by histone H3 hyperacetylation or hypermethylation. Collectively, our results suggest that elevated VDR expression may contribute to the development of hypercalciuria by sensitizing VDR target genes to 1,25(OH) 2 D 3 through histone modifications at their promoter regions in a genetic hypercalciuric stone-forming (GHS) rat model., Competing Interests: There are no interests conflict., (© 2020 Chongqing Medical University. Production and hosting by Elsevier B.V.)

To study human idiopathic hypercalciuria (IH), we developed an animal model, genetic hypercalciuric stone-forming (GHS) rats, whose pathophysiology parallels that in IH. All GHS rats form kidney stones and have decreased BMD and bone quality compared with the founder Sprague-Dawley (SD) rats. To understand the bone defect, we characterized osteoclast and osteoblast activity in the GHS compared with SD rats. Bone marrow cells were isolated from femurs of GHS and SD rats and cultured to optimize differentiation into osteoclasts or osteoblasts. Osteoclasts were stained for TRAcP (tartrate resistant acid phosphatase), cultured to assess resorptive activity, and analyzed for specific gene expression. Marrow stromal cells or primary neonatal calvarial cells were differentiated to osteoblasts, and osteoblastic gene expression as well as mineralization was analyzed. There was increased osteoclastogenesis and increased resorption pit formation in GHS compared with SD cultures. Osteoclasts had increased expression of cathepsin K , Tracp , and MMP9 in cells from GHS compared with SD rats. Osteoblastic gene expression and mineralization was significantly decreased. Thus, alterations in baseline activity of both osteoclasts and osteoblasts in GHS rats, led to decreased BMD and bone quality, perhaps because of their known increase in vitamin D receptors. Better understanding of the role of GHS bone cells in decreased BMD and quality may provide new strategies to mitigate the low BMD and increased fracture risk found in patients with IH. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research., (© 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.)

Oleanolic acid (OA) and ursolic acid (UA) are the major chemical constituents in Fructus Ligustri Lucidi (FLL), a kidney-tonifying Chinese herb that is previously shown to improve bone properties and enhance calcium balance in aged female rats. The present study was designed to study if OA and UA act as the active ingredients in FLL to exert the positive effects on bone and mineral metabolism in aged rats. Aged (13-month-old) Sprague-Dawley female rats were randomly assigned to four groups with oral administration of drug or vehicle treatment for 12 weeks: medium calcium diet (MCD, 0.6% calcium), high calcium diet (HCD, 1.2% calcium), MCD + FLL (700 mg/kg/day), MCD + OA (23.6 mg/kg/day) + UA (8.6 mg/kg/day). A group of mature (3-month-old) female rats fed with MCD was included as positive control. The results demonstrated that FLL and OA+UA increased bone mineral density and improved microarchitectural properties of aged female rats. The osteoprotective effects of FLL and OA+UA might be, at least in part, associated with their actions on enhancing calcium balance and suppressing age-induced secondary hyperparathyroidism in aged female rats. FLL and OA+UA also significantly induced renal CYP27B1 protein expression and OA+UA treatment decreased CYP24A1 mRNA and protein expressions in aged female rats. In addition, FLL and OA+UA significantly increased the promoter activity, mRNA and protein expressions of renal CYP27B1 in vitro in human proximal tubule HKC-8 cells. The present findings suggest that OA+UA can be regarded as the active ingredients of FLL and might be a potential drug candidate for prevention and treatment of osteoporosis.

Mycophenolic acid (MPA) is an important immunosuppressant broadly used in renal transplantation. However, the large inter-patient variability in mycophenolic acid (MPA) pharmacokinetics (PK) limits its use. We hypothesize that extrahepatic metabolism of MPA may have significant impact on MPA PK variability. Two intestinal UDP-glucuronosyltransferases 1A8 and 1A10 plays critical role in MPA metabolism. Both in silico and previous genome-wide analyses suggested that vitamin D (VD) may regulate intestinal UGT1A expression. We validated the VD response elements (VDREs) across the UGT1A locus with chromatin immunoprecipitation (ChIP) and luciferase reporter assays. The impact of 1-alpha,25-dihydroxyvitamin D3 (D3) on UGT1A8 and UGT1A10 transcription and on MPA glucuronidation was tested in human intestinal cell lines LS180, Caco-2 and HCT-116. The correlation between transcription levels of VD receptor (VDR) and the two UGT genes were examined in human normal colorectal tissue samples (n = 73). PK alterations of MPA following the parent drug, mycophenolate mofetil (MMF), and D3 treatment was assessed among renal transplant recipients (n = 10). Our ChIP assay validate three VDREs which were further demonstrated as transcriptional enhancers with the luciferase assays. D3 treatment significantly increased transcription of both UGT genes as well as MPA glucuronidation in cells. The VDR mRNA level was highly correlated with that of both UGT1A8 and UGT1A10 in human colorectal tissue. D3 treatment in patients led to about 40% reduction in both AUC 0-12 and Cmax while over 70% elevation of total clearance of MPA. Our study suggested a significant regulatory role of VD on MPA metabolism and PK via modulating extrahepatic UGT activity., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ronacaleret is an orally-active calcium-sensing receptor (CaSR) antagonist that has the potential for therapeutic utility in the stimulation of PTH release, notably as a bone anabolic agent comparable to recombinant human PTH(1-34) (rhPTH(1-34)). A recent study has shown that, despite the ability to increase circulating PTH levels in postmenopausal women in a dose-dependent manner, minimal effects of ronacaleret on bone mineral density have been observed. Therefore, the purpose of this study was to characterize the PTH profile as well as calcium metabolism parameters as a marker of PTH biological activity following the administration of ronacaleret or rhPTH(1-34). Administration of ronacaleret led to lower peak levels of PTH than were observed with rhPTH(1-34), however, greater total PTH exposure was observed. Further, chronic administration of either agent was associated with increases in urinary calcium excretion and serum calcium levels, with the magnitude of the changes following ronacaleret significantly greater than that for rhPTH(1-34). The greater magnitude of effects observed with ronacaleret is likely due to the greater total PTH exposure, and is potentially reflective of a state comparable to mild hyperparathyroidism. It is not clear whether the administration of all calcilytics would lead to a similar result, or is due to characteristics specific to ronacaleret., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Genetic hypercalciuric stone-forming (GHS) rats have increased intestinal Ca absorption, decreased renal tubule Ca reabsorption and low bone mass, all of which are mediated at least in part by elevated tissue levels of the vitamin D receptor (VDR). Both 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and bone morphogenetic protein 2 (BMP2) are critical for normal maintenance of bone metabolism and bone formation, respectively. The complex nature of bone cell regulation suggests a potential interaction of these two important regulators in GHS rats. In the present study, BMP2 expression is suppressed by the VDR-1,25(OH)2D3 complex in Bone Marrow Stromal Cells (BMSCs) from GHS and SD rat and in UMR-106 cell line. We used chromatin immunoprecipitation (ChIP) assays to identify VDR binding to only one of several potential binding sites within the BMP2 promoter regions. This negative region also mediates suppressor reporter gene activity. The molecular mechanisms underlying the down-regulation of BMP2 by 1,25(OH)2D3 were studied in vitro in BMSCs and UMR-106 cells using the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC) and the histone deacetylase inhibitor trichostatin A (TSA). Both DAC and TSA activate BMP2 expression in combination with 1,25(OH)2D3. Bisulfite DNA pyrosequencing reveals 1,25(OH)2D3 to completely hypermethylate a single CpG site in the same BMP2 promoter region identified by the ChIP and reporter gene assays. ChIP assays also show that 1,25(OH)2D3 can increase the repressive histone mark H3K9me2 and reduce the acetylation of histone H3 at the same BMP2 promoter region. Taken together, our results indicate that 1,25(OH)2D3 binding to VDR down-regulates BMP2 gene expression in BMSCs and osteoblast-like UMR-106 cells by binding to the BMP2 promoter region. The mechanism of this 1,25(OH)2D3-induced transcriptional repression of BMP2 involves DNA methylation and histone modification. The study provides novel evidence that 1,25(OH)2D3 represses bone formation through down-regulating BMP2 expression both in vivo and in vitro.

Genetic hypercalciuric stone-forming (GHS) rats, bred to maximize urine (U) calcium (Ca) excretion, have increased intestinal Ca absorption and bone Ca resorption and reduced renal Ca reabsorption, leading to increased UCa compared with the Sprague-Dawley (SD) rats. GHS rats have increased vitamin D receptors (VDR) at each of these sites, with normal levels of 1,25(OH)(2)D(3) (1,25D), indicating that their VDR is undersaturated with 1,25D. We tested the hypothesis that 1,25D would induce a greater increase in UCa in GHS rats by feeding both strains ample Ca and injecting 1,25D (25 ng · 100 g body wt(-1) · day(-1)) or vehicle for 16 days. With 1,25D, UCa in SD increased from 1.7 ± 0.3 mg/day to 24.4 ± 1.2 (Δ = 22.4 ± 1.5) and increased more in GHS from 10.5 ± 0.7 to 41.9 ± 0.7 (Δ = 29.8 ± 1.8; P = 0.003). To determine the mechanism of the greater increase in UCa in GHS rats, we measured kidney RNA expression of components of renal Ca transport. Expression of transient receptor potential vanilloid (TRPV)5 and calbindin D(28K) were increased similarly in SD + 1,25D and GHS + 1,25D. The Na(+)/Ca(2+) exchanger (NCX1) was increased in GHS + 1,25D. Klotho was decreased in SD + 1,25D and GHS + 1,25D. TRPV6 was increased in SD + 1,25D and increased further in GHS + 1,25D. Claudin 14, 16, and 19, Na/K/2Cl transporter (NKCC2), and secretory K channel (ROMK) did not differ between SD + 1,25D and GHS + 1,25D. Increased UCa with 1,25D in GHS exceeded that of SD, indicating that the increased VDR in GHS induces a greater biological response. This increase in UCa, which must come from the intestine and/or bone, must exceed any effect of 1,25D on TRPV6 or NCX1-mediated renal Ca reabsorption.

Sarcoidosis affects the bone directly in only a minority of patients. Nonetheless, bone health should be considered in the management of all patients with sarcoidosis. Deficiency in vitamin D, an important contributor to bone health, has been linked to autoimmune disease incidence. Studies have shown that patients with sarcoidosis frequently have low levels of vitamin D-25 but may have normal or increased levels of vitamin D-1,25. In addition, granuloma formation has been linked to a failure of the innate immune system, which could be related to a deficiency in vitamin D, although this relationship has not been fully characterized. Furthermore, many patients with sarcoidosis are treated with corticosteroids, which are known to induce osteoporosis. Therefore, bone health may be impacted in several ways in sarcoidosis--by direct involvement with granulomas, vitamin D deficiency, or corticosteroid therapy.

Patients with idiopathic hypercalciuria (IH) and genetic hypercalciuric stone-forming (GHS) rats, an animal model of IH, are both characterized by normal serum Ca, hypercalciuria, Ca nephrolithiasis, reduced renal Ca reabsorption, and increased bone resorption. Serum 1,25-dihydroxyvitamin D [1,25(OH)(2)D] levels are elevated or normal in IH and are normal in GHS rats. In GHS rats, vitamin D receptor (VDR) protein levels are elevated in intestinal, kidney, and bone cells, and in IH, peripheral blood monocyte VDR levels are high. The high VDR is thought to amplify the target-tissue actions of normal circulating 1,25(OH)(2)D levels to increase Ca transport. The aim of this study was to elucidate the molecular mechanisms whereby Snail may contribute to the high VDR levels in GHS rats. In the study, Snail gene expression and protein levels were lower in GHS rat tissues and inversely correlated with VDR gene expression and protein levels in intestine and kidney cells. In human kidney and colon cell lines, ChIP assays revealed endogenous Snail binding close to specific E-box sequences within the human VDR promoter region, whereas only one E-box specifically bound Snail in the rat promoter. Snail binding to rat VDR promoter E-box regions was reduced in GHS compared with normal control intestine and was accompanied by hyperacetylation of histone H(3). These results provide evidence that elevated VDR in GHS rats likely occurs because of derepression resulting from reduced Snail binding to the VDR promoter and hyperacetylation of histone H(3)., (Copyright 2010 American Society for Bone and Mineral Research.)

Unlabelled: A United States-specific cost-effectiveness analysis, which incorporated the cost and health consequences of clinical fractures of the hip, spine, forearm, shoulder, rib, pelvis and lower leg, was undertaken to identify the 10-year hip fracture probability required for osteoporosis treatment to be cost-effective for cohorts defined by age, sex, and race/ethnicity. A 3% 10-year risk of hip fracture was generally required for osteoporosis treatment to cost less than $60,000 per QALY gained., Introduction: Rapid growth of the elderly United States population will result in so many at risk of osteoporosis that economically efficient approaches to osteoporosis care warrant consideration., Methods: A Markov-cohort model of annual United States age-specific incidence of clinical hip, spine, forearm, shoulder, rib, pelvis and lower leg fractures, costs (2005 US dollars), and quality-adjusted life years (QALYs) was used to assess the cost-effectiveness of osteoporosis treatment ($600/yr drug cost for 5 years with 35% fracture reduction) by gender and race/ethnicity groups. To determine the 10-year hip fracture probability at which treatment became cost-effective, average annual age-specific probabilities for all fractures were multiplied by a relative risk (RR) that was systematically varied from 0 to 10 until a cost of $60,000 per QALY gained was observed for treatment relative to no intervention., Results: Osteoporosis treatment was cost-effective when the 10-year hip fracture probability reached approximately 3%. Although the RR at which treatment became cost-effective varied markedly between genders and by race/ethnicity, the absolute 10-year hip fracture probability at which intervention became cost-effective was similar across race/ethnicity groups, but tended to be slightly higher for men than for women., Conclusions: Application of the WHO risk prediction algorithm to identify individuals with a 3% 10-year hip fracture probability may facilitate efficient osteoporosis treatment.

Unlabelled: Application of the WHO fracture prediction algorithm in conjunction with an updated US economic analysis indicates that osteoporosis treatment is cost-effective in patients with fragility fractures or osteoporosis, in older individuals at average risk and in younger persons with additional clinical risk factors for fracture, supporting existing practice recommendations., Introduction: The new WHO fracture prediction algorithm was combined with an updated economic analysis to evaluate existing NOF guidance for osteoporosis prevention and treatment., Methods: The WHO fracture prediction algorithm was calibrated to the US population using national age-, sex- and race-specific death rates and age- and sex-specific hip fracture incidence rates from the largely white population of Olmsted County, MN. Fracture incidence for other races was estimated by ratios to white women and men. The WHO algorithm estimated the probability (%) of a hip fracture (or a major osteoporotic fracture) over 10 years, given specific age, gender, race and clinical profiles. The updated economic model suggested that osteoporosis treatment was cost-effective when the 10-year probability of hip fracture reached 3%., Results: It is cost-effective to treat patients with a fragility fracture and those with osteoporosis by WHO criteria, as well as older individuals at average risk and osteopenic patients with additional risk factors. However, the estimated 10-year fracture probability was lower in men and nonwhite women compared to postmenopausal white women., Conclusions: This analysis generally endorsed existing clinical practice recommendations, but specific treatment decisions must be individualized. An estimate of the patient's 10-year fracture risk should facilitate shared decision-making.

Estrogen deficiency impairs intestinal Ca absorption and induces bone loss, but its effects on the vitamin D-endocrine system are unclear. In the present study, calciotropic hormones levels, renal vitamin D metabolism, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-dependent intestinal calcium absorption, and bone properties in 3-mo-old sham-operated (sham) or ovariectomized (OVX) rats fed either a normal-Ca (NCD; 0.6% Ca, 0.65% P) or a low-Ca (LCD; 0.1% Ca, 0.65% P) diet for 2 wk were determined. LCD increased serum 1,25(OH)2D3 levels in both sham and OVX rats. Serum parathyroid hormone [PTH(1-84)] levels were highest in OVX rats fed LCD. Renal 25-hydroxyvitamin D1alpha-hydroxylase (1-OHase) protein expression was induced in both sham and OVX rats during LCD, while renal 1-OHase mRNA expression was highest in OVX rats fed LCD. Renal vitamin D receptor (VDR) and mRNA expressions in rats were induced by ovariectomy in rats fed NCD but suppressed by ovariectomy in rats fed LCD. The induction of intestinal calcium transporter-1 and calbindin-D9k mRNA expressions by LCD were not altered by ovariectomy. As expected, bone Ca content, cancellous bone mineral density, and bone strength index in proximal metaphysis of rat tibia were reduced by both ovariectomy and LCD (P<0.05) as analyzed by two-way ANOVA. Taken together, the data demonstrate that ovariectomy alters the responses of circulating PTH levels, renal 1-OHase mRNA expression, and renal VDR expression to LCD. These results suggest that estrogen is necessary for the full adaptive response to LCD mediated by both PTH and 1,25(OH)2D3.

Introduction: Bone fragility is determined by bone mass, measured as bone mineral density (BMD), and by trabecular structure, which cannot be easily measured using currently available noninvasive methods. In previous studies, radiographic texture analysis (RTA) performed on the radiographic images of the spine, proximal femur, and os calcis differentiated subjects with and without osteoporotic fractures. The present cross-sectional study was undertaken to determine whether such differentiation could also be made using high-resolution os calcis images obtained on a peripheral densitometer., Methods: In 170 postmenopausal women (42 with and 128 without prevalent vertebral fractures) who had no secondary causes of osteoporosis and were not receiving treatment for osteoporosis, BMD of the lumbar spine, proximal femur, and os calcis was measured using dual energy x-ray absorptiometry. Vertebral fractures were diagnosed on densitometric spine images. RTA, including Fourier-based and fractal analyses, was performed on densitometric images of os calcis., Results: BMD at all three sites and all texture features was significantly different in subjects with and without fractures, with the most significant differences observed for the femoral neck and total hip measurements and for the RTA feature Minkowski fractal (p<0.001). In univariate logistic regression analysis, Minkowski fractal predicted the presence of vertebral fractures as well as femoral neck BMD (p<0.001). In multivariate logistic regression analysis, both femoral neck BMD and Minkowski fractal yielded significant predictive effects (p=0.001), and when age was added to the model, the effect of RTA remained significant (p=0.002), suggesting that RTA reflects an aspect of bone fragility that is not captured by age or BMD. Finally, when RTA was compared in 42 fracture patients and 42 nonfracture patients matched for age and BMD, the RTA features were significantly different between the groups (p=0.003 to p=0.04), although BMD and age were not., Conclusion: This study suggests that RTA of densitometer-generated calcaneus images provides an estimate of bone fragility independent of and complementary to BMD measurement and age.

It is not clear how bone mineral density (BMD) measurements from several regions of lumbar spine and proximal femur should be utilized in assessing fracture risk. We examined how well the newest ISCD recommendations differentiate subjects with and without prevalent vertebral fractures in 187 postmenopausal women presenting for routine bone densitometry. The association between T-scores from proximal femur and lumbar spine sites and the probability of having a vertebral fracture was modeled via logistic regression with adjustment for age. The lowest T-score of any hip or spine sites (the current ISCD recommendation) and the proximal femur measurements, particularly the femoral neck and total hip, displayed the strongest association with the probability of vertebral fractures.Subjects with a T-score < -2.5 at multiple hip sites had a higher probability of having a vertebral fracture. The sensitivity and specificity associated with particular T-score cutoff values varied greatly depending on the site of measurement.Consequently, T-score values from different sites that had comparable sensitivity/specificity for detecting the presence of vertebral fractures differed by as much as 1.5 T-score units. This finding implies that a single cutoff value, such as -2.5, might not be clinically acceptable when applied to T-scores from different sites.

Hypercalciuria in inbred genetic hypercalciuric stone-forming (GHS) rats is due, in part, to a decrease in renal tubule Ca reabsorption. Activation of the renal Ca receptor (CaR) may decrease renal tubule Ca reabsorption and cause hypercalciuria through suppression of Ca-sensitive potassium channel activity. Because the rat renal CaR gene is regulated by extracellular calcium and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and GHS rats have increased renal vitamin D receptor content, the current study was undertaken to determine the level of CaR gene expression in GHS rat kidney and whether CaR gene expression is regulated by 1,25(OH)2D3. Male GHS and normal control (NC) rats were fed a Ca-sufficient diet (0.6% Ca). Western blotting revealed a four-fold increase in CaR protein in GHS rat renal tissue, and 1,25(OH)2D3 administration increased renal CaR in both GHS and NC rats. Northern blot analysis of extracts of renal cortical tissue from GHS and NC rats revealed a major 7-kb transcript of CaR and a more modest 4-kb transcript, both of which were readily detectable. Both Northern blotting and real-time reverse transcription-PCR revealed increased basal CaR mRNA expression levels in GHS rat kidney. 1,25(OH)2D3 administration increased renal CaR mRNA levels 2.0- and 3.3-fold in GHS and NC rats, respectively. Despite the greater incremental increase by 1,25(OH)2D3 in NC rats, CaR mRNA levels remained higher in GHS rat kidney, and the elevation was more sustained. 1,25(OH)2D3 increased CaR mRNA through both elevated CaR gene expression and prolonged tissue half-life. These results demonstrate that GHS rats have high levels of CaR gene expression and CaR protein that may contribute to the hypercalciuria and calcium nephrolithiasis.

Unlabelled: The functional status and mechanism of increased VDR in GHS rats were investigated. Basal VDR and calbindins were increased in GHS rats. 1,25(OH)(2)D(3) increased VDR and calbindins in controls but not GHS rats. VDR half-life was prolonged in GHS rats. This study supports the mechanism and functional status of elevated VDR in GHS rats., Introduction: Genetic hypercalciuric stone-forming (GHS) rats form calcium kidney stones from hypercalciuria arising from increased intestinal calcium absorption and bone resorption and decreased renal calcium reabsorption. Normal serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels and increased vitamin D receptor (VDR) protein suggest that high rates of expression of vitamin D-responsive genes may mediate the hypercalciuria. The mechanism of elevated VDR and state of receptor function are not known., Materials and Methods: GHS and non-stone-forming control (NC) male rats (mean, 249 g), fed a normal calcium diet, were injected intraperitoneally with 1,25(OH)2D3 (30 ng/100 g BW) or vehicle 24 h before cycloheximide (6 mg/100 g, IP) and were killed 0-8 h afterward. Duodenal VDR was measured by ELISA and Western blot, and duodenal and kidney calbindins (9 and 28 kDa) were measured by Western blots., Results and Conclusions: Duodenal VDR protein by Western blot was increased 2-fold in GHS versus NC rats (633 +/- 62 versus 388 +/- 48 fmol/mg protein, n = 4, p < 0.02), and 1,25(OH)2D3 increased VDR and calbindins (9 and 28 kDa) further in NC but not GHS rats. Duodenal VDR half-life was prolonged in GHS rats (2.59 +/- 0.2 versus 1.81 +/- 0.2 h, p < 0.001). 1,25(OH)2D3 prolonged duodenal VDR half-life in NC rats to that of untreated GHS rats (2.59 +/- 0.2 versus 2.83 +/- 0.3 h, not significant). This study supports the hypothesis that prolongation of VDR half-life increases VDR tissue levels and mediates increased VDR-regulated genes that result in hypercalciuria through actions on vitamin D-regulated calcium transport in intestine, bone, and kidney.

The responses of renal vitamin D metabolism to its major stimuli alter with age. Previous studies showed that the increase in circulating 1,25-dihydroxyvitamin D (1,25(OH)2D3) as well as renal 25-hydroxyvitamin D3 1-alpha hydroxylase (1-OHase) activity in response to dietary Ca or P restriction reduced with age in rats. We hypothesized that the mechanism involved in increasing circulating 1,25(OH)2D3 in response to mineral deficiency alters with age. In the present study, we tested the hypothesis by studying the expression of genes involved in renal vitamin D metabolism (renal 1-OHase, 25-hydroxyvitamin D 24-hydroxylase (24-OHase) and vitamin D receptor (VDR)) in young (1-month-old) and adult (6-month-old) rats in response to low-phosphate diet (LPD). As expected, serum 1,25(OH)2D3 increased in both young and adult rats upon LPD treatment and the increase was much higher in younger rats. In young rats, LPD treatment decreased renal 24-OHase (days 1-7, P<0.01) and increased renal 1-OHase mRNA expression (days 1-5, P<0.01). LPD treatment failed to increase renal 1-OHase but did suppress 24-OHase mRNA expression (P<0.01) within 7 d of LPD treatment in adult rats. Renal expression of VDR mRNA decreased with age (P<0.001) and was suppressed by LPD treatment in both age groups (P<0.05). Feeding of adult rats with 10 d of LPD increased 1-OHase (P<0.05) and suppressed 24-OHase (P<0.001) as well as VDR (P<0.05) mRNA expression. These results indicate that the increase in serum 1,25(OH)2D3 level in adult rats during short-term LPD treatment is likely to be mediated by a decrease in metabolic clearance via the down-regulation of both renal 24-OHase and VDR expression. The induction of renal 1-OHase mRNA expression in adult rats requires longer duration of LPD treatment than in younger rats.

Vitamin D3, an important seco-steroid hormone for the regulation of body calcium homeostasis, promotes immature myeloid precursor cells to differentiate into monocytes/macrophages. Vitamin D receptor (VDR) belongs to a nuclear receptor super-family that mediates the genomic actions of vitamin D3 and regulates gene expression by binding with vitamin D response elements in the promoter region of the cognate gene. Thus by regulating gene expression, VDR plays an important role in modulating cellular events such as differentiation, apoptosis, and growth. Here we report lipopolysaccharide (LPS), a bacterial toxin; decreases VDR protein levels and thus inhibits VDR functions in the human blood monocytic cell line, THP-1. The biologically active form of vitamin D3, 1alpha,25-dihydroxy vitamin D3 [1,25(OH)2D3], induced VDR in THP-1 cells after 24 h treatment, and LPS inhibited 1,25(OH)2D3-mediated VDR induction. However, LPS and 1,25(OH)2D3 both increased VDR mRNA levels in THP-1 cells 20 h after treatment, as observed by real time RT-PCR. Moreover, LPS plus 1,25(OH)2D3 action on VDR mRNA level was additive and synergistic. A time course experiment up to 60 h showed an increase in VDR mRNA that was not preceded with an increase in VDR protein levels. Although the proteasome pathway plays an important role in VDR degradation, the proteasome inhibitor lactacystin had no effect on the LPS-mediated down-regulation of 1,25(OH)2D3 induced VDR levels. Reduced VDR levels by LPS were accompanied by decreased 1,25(OH)2D3/VDR function determined by VDR responsive 24-hydroxylase (CYP24) gene expression. The above results suggest that LPS impairs 1,25(OH)2D3/VDR functions, which may negatively affect the ability of 1,25(OH)2D3 to induce myeloid differentiation into monocytes/macrophages.

Idiopathic hypercalciuria (IH) is the most common cause of calcium oxalate nephrolithiasis. Increased intestinal calcium absorption and bone resorption and decreased tubule calcium reabsorption may be caused by elevated serum 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] in some patients but not in those with normal serum 1,25(OH)(2)D(3) levels. Because 1,25(OH)(2)D(3) exerts its biological actions through binding to the cellular vitamin D receptor (VDR), the present study was undertaken to test the hypothesis that VDR levels are elevated in IH patients. Ten male IH calcium oxalate stone-formers were paired with controls matched in age within 5 yr and lacking a history of stones or family history of stones. Blood was obtained for serum, peripheral blood monocytes (PBMs) were separated from lymphocytes and other mononuclear cells, and PBM VDR content was measured by Western blotting. The PBM VDR level was 2-fold greater in IH men at 49 +/- 21 vs. 20 +/- 15 fmol/mg protein, mean +/- sd; P < 0.008. Serum 1,25(OH)(2)D(3) levels were not higher than controls (48 +/- 14 vs. 39 +/- 11 pg/ml; P < 0.068). In conclusion, PBM VDR levels are elevated in IH calcium oxalate stone-formers. The elevation could not be ascribed to increased serum 1,25(OH)(2)D(3) levels. These results suggest that the molecular basis for IH involves a pathological elevation of tissue VDR level, which may elevate intestinal calcium absorption and bone resorption and decrease renal tubule calcium reabsorption. The mechanism for increased VDR in IH patients with normal serum 1,25(OH)(2)D(3) levels is unknown.

The current study was undertaken to evaluate the clinical utility of DVA, a system for imaging the lateral spine on the Lunar Prodigy densitometer. DVA images were obtained and bone density of the lumbar spine and proximal femur measured in 297 subjects (272 women), aged 64+/-13 years. The images were classified as: normal (N) if no fractures were detected and all vertebrae between T6 and L4 were visualized, fracture (F) if any vertebra had a fracture (defined as 25% or more reduction in the vertebral height) even if some of the other vertebrae could not be visualized, and un-interpretable (U) if at least one of the vertebra between T6 and L4 could not be classified and no fractures were detected in the visualized vertebrae. A subset of 66 patients also had standard radiographs of the thoracic and lumbar spine. Compared to radiographs, DVA had a 95% sensitivity to detect fractures and 82% specificity (to exclude them). Among all 297 subjects studied, DVAs were interpretable in 87%. They were classified as N in 204 (68%), F in 55 (19%) and U in 38 (13%). The reasons for un-interpretability were: scoliosis, scapular or rib shadow, severe arthritic changes and multiple vertebral compression fracture with severe spinal deformities. Only 11% of F subjects gave a history of a vertebral fracture, and only 56% of F subjects met the BMD criteria for osteoporosis (T score <-2.5). These results indicate that adding DVA, a low radiation and relatively low cost "point of service" procedure, to BMD measurement provides the clinician with a more comprehensive fracture risk assessment than that afforded by clinical evaluation and BMD measurement alone.

Regulation of vitamin D metabolism alters with age. The present study is undertaken to investigate if the loss of renal 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) production in response to dietary phosphate (P) restriction in adult rats is due to an alteration in the renal expression of 25-hydroxyvitamin D(3) 1-alpha hydroxylase (1-OHase). Young (4-6 weeks old) and adult (12-14 weeks old) male Sprague Dawley rats were fed either normal P (NPD) or low P diet (LPD) for 0-5 days. Basal expression of 1-OHase protein was higher in adult rats. Young rats, but not adult rats, significantly increased 1-OHase protein and mRNA expressions in response to LPD in a time-dependent manner. To determine if the stability of renal 1-OHase protein changes with LPD feeding, young and adult rats fed either NPD or LPD for 5 days were injected intravenously with cycloheximide (CHX), a protein synthesis inhibitor. CHX decreased 1-OHase protein expression in young rats fed NPD. However, CHX did not alter 1-OHase protein expression in young rats fed LPD nor in adult rats fed either diet. The results indicate that the stability of renal 1-OHase protein increased with age and that LPD increased its stability only in young rats.

Bone fragility is determined by bone mass and trabecular structure. While bone mass can be readily measured as bone density, bone trabecular structure cannot be easily assessed by currently available methods. The realization of the importance of bone structure in determining fracture risk has led to the development of several imaging modalities aimed at evaluating the contribution of bone quality to its biomechanical strength and fragility. High-resolution magnetic resonance imaging and computed tomography have limited spatial resolution and high cost but have a potential to generate true three-dimensional images of trabecular structure in vivo. Bone radiographs subjected to various forms of texture analysis have higher resolution and lower cost but provide only a two-dimensional representation of bone structure. Both two- and three-dimensional methods have been shown to predict biomechanical strength in vitro and to differentiate between subjects with and without fractures in vivo. Therefore, all of these methods deserve closer evaluation and also need further technical improvements before they can be considered for use in clinical practice.

Concern that people who form kidney stones may have reduced bone mineral density (BMD) and increased fracture risk has motivated clinical and population-based studies, but findings are inconsistent. In this cross-sectional study, we use the Third National Health and Nutrition Examination Survey (NHANES III) to determine whether a history of kidney stones (n = 793) is associated with lower femoral neck BMD and whether the association is similar for men and women. We further ask whether dietary calcium modifies the association between kidney stone history and BMD and whether there is an association between kidney stone history and prevalent spine or wrist fracture. We find that men with kidney stone history have lower femoral neck BMD than men without kidney stone history after adjusting for age, body mass index (BMI), race/ethnicity, and other potential confounders. The effect of kidney stone history on BMD is weaker for women. Men with kidney stone history also are more likely to report prevalent wrist and spine fractures. Dietary calcium, represented by usual milk consumption, is associated positively with BMD for both men and women and modifies the effect of kidney stone history on BMD for men. For men who form kidney stones, milk consumption is associated more strongly with femoral neck BMD than for men without such a history. The effect modification is such that the difference in BMD between men with and without kidney stone history is observed only at lower levels of milk consumption.

This study compared the effects of oral alendronate and intranasal calcitonin for treatment of osteoporosis in postmenopausal women. Women at least 5 yr postmenopause (n = 299) were randomized to either 10 mg alendronate, matching alendronate placebo, or open-label intranasal calcitonin 200 IU daily for 12 months. Hip and spine bone mineral density (BMD) and markers of bone turnover were measured, and safety and tolerability were assessed. Alendronate produced greater increases in BMD than calcitonin at 12 months at the lumbar spine (5.16% vs. 1.18%; P < 0.001), trochanter (4.73% vs. 0.47%; P < 0.001), and femoral neck (2.78% vs. 0.58%; P < 0.001). Changes in BMD with calcitonin were greater than with placebo at the femoral neck, but were not different from placebo at either the trochanter or lumbar spine. Greater decreases in bone turnover were seen with alendronate than with calcitonin (serum bone-specific alkaline phosphatase, 43% vs. 9%, P < 0.001; urinary N-telopeptide, 62% vs. 11%, P < 0.001). Similar percentages of patients in each group reported an adverse experience during the study. We conclude that, in postmenopausal women with osteoporosis, 12 months of therapy with alendronate produced significantly greater increases in BMD of the hip and spine and greater decreases in bone turnover than intranasal calcitonin.

The adaptive increase in renal proximal tubule 25-hydroxyvitamin D-alpha-hydroxylase activity (1-OHase) during dietary calcium restriction is mediated by an increase in parathyroid hormone (PTH) and is inhibited by aging. Recent studies in mature (3-4 mo) rats demonstrated that insulin-like growth factor-I (IGF-I) restored stimulation of renal 1,25-dihydroxycholecalciferol [1,25(OH)(2)D(3)] production by low phosphorus diet (LPD), another major stimulus of 1-OHase. These studies were designed to determine whether IGF-I stimulates 1-OHase during low calcium intake in old rats. Male rats were fed a normal calcium diet (NCD, 6 g Ca/kg diet) or low calcium diet (LCD, 0.2 g Ca/kg diet) for 14 d, and recombinant human IGF-I [rhIGF-I, 1.4 mg/(24h 160 kg body wt)] or vehicle was administrated via miniosmotic pump for 72 h before killing. In 4-mo-old male Sprague-Dawley rats, LCD increased in vitro renal 1-OHase activity in the presence but not in the absence of rhIGF-I. LCD increased in vitro1-OHase activity in young (1-mo-old) but not old (24-mo-old) male Fischer 344 rats. RhIGF-I increased 1-OHase activity in 24 mo-old rats fed LCD to levels that were not different from those in 1-mo-old rats fed LCD. The results indicate that the adaptive increase in 1-OHase activity due to a LCD is lost by 4 mo in rats and can be restored by pharmacologic doses of rhIGF-I.

Rationale and Objectives: In the noninvasive evaluation of bone quality, bone mineral density (BMD) has been shown to be the single most important predictor of bone strength and osteoporosis-related fracture. Among the methods of measuring BMD, dual x-ray absorptiometry (DXA) has widespread acceptance due to its low radiation, low cost, and high precision. However, DXA measures area BMD instead of true volumetric density; thus, a larger bone will tend to have a high BMD than will a smaller bone. Therefore, the comparison of BMDs of bones of different sizes can be misleading. In this study, the authors tried to compensate for the size effect by normalizing the area BMD with bone size as measured from a standard pelvic radiograph., Materials and Methods: The overall method for calculation of normalized BMD included conventional area-based BMD from DXA and the extraction of geometric measures from pelvic radiographs. The database for analysis included 34 femoral neck specimens. Regression analysis was performed between the normalized volumetric BMD, measured from femoral neck region, and the mechanical properties obtained from trabecular bone cubes machined from the same region., Results: After normalization of the area BMD, the coefficient of determination increased from 0.30 to 0.43 for the Young modulus and from 0.27 to 0.37 for bone compressive strength., Conclusion: A noninvasive method of normalizing BMD can improve the prediction of bone mechanical properties and has potential in monitoring changes in growing skeletons and in the clinical evaluation of bone quality.

We are developing computerized methods for characterizing the bone texture pattern from digitized skeletal radiographs. For this method to be useful clinically, it must be able to distinguish between weak and strong bone under the range of exposure conditions potentially encountered in the clinical setting. In this study, we examined the effect of exposure conditions on Fourier-based texture features. Thirty-four femoral specimens from total hip arthroplasties were radiographed multiple times under different exposure conditions. The specimens underwent mechanical strength testing from which load to failure values were obtained. The performance of the texture features were investigated in the task of distinguishing between strong and weak bone as characterized by the load to failure values. The texture features showed no dependence upon focal spot size of the x-ray tube or magnification. The texture features did show a dependence with relative exposure, peak kilovoltage, and amount of scattering material.

We are investigating computerized methods to ultimately characterize bone trabecular pattern from clinical skeletal radiographs. In this paper, we present a "phantom" for potential use in the development and evaluation of computerized methods for characterizing radiographic trabecular patterns and ultimately bone strength. Femoral neck specimens were excised during total hip arthroplasties from subjects exhibiting a range of diseases. To mimic the femoral neck in vivo, a "simulated clinical" setup was implemented in which specimens were exposed under conditions that yielded radiographs similar in appearance to standard pelvis radiographs. Fourier-based and fractal-based texture measures were used in the computer analysis; including RMS variation, first moment of the power spectrum, angular-dependent forms of these measures, and fractal dimension. The texture measures obtained from the "simulated clinical" specimen films correlated modestly with those from direct exposure "verification" films of the specimens (r= 0.59-0.69; p<0.0001). From our study, we conclude that the femoral neck specimen "phantoms" may be useful in the development and evaluation of computerized methods for analyzing bone trabecular patterns from skeletal radiographs. The use of a phantom that simulates the clinical radiographic examination allows for repeat exposures without the concern of excessive radiation exposure to a patient.

Both bone mineral density (BMD) and trabecular structure are important determinates of bone mechanical properties. However, neither BMD or trabecular structural features can completely explain the variations in bone mechanical properties. In this study, we combine BMD and bone structural features to characterize bone mechanical behavior. Radiographs were obtained from 34 femoral neck specimens excised during total hip arthroplasties. Each neck radiograph was digitized and a region of interest (ROI) was selected from the medial side of the femoral neck. Textural features, the global Minkoswski dimension and trabecular orientation, were extracted from each ROI image using Minkowski dimension analysis. The BMD of each specimen was measured using dual-energy x-ray absorptiometry (DXA) and subsequently normalized by bone size as measured from a standard pelvis radiograph. Mechanical testing was performed on the trabecular bone cubes machined from each femoral neck to yield bone mechanical properties. Multiple regression was performed to select the best features to predict bone mechanical properties. The results suggest that, using multiple predictors including normalized BMD structural features, and patient age, the coefficients of determination (R2) improved over the use of BMD alone. For bone strength, the R2 was improved from 0.24 using conventional BMD to 0.48 using a four-predictor model. Similar results were obtained in the prediction of Young's modulus, i.e., the R2 was improved from 0.25 to 0.55 in going from the model using conventional BMD to a four-predictor model. This study demonstrates the contributions of normalized BMD, structural features, and age to bone mechanical properties, and suggests a potential method for the noninvasive evaluation of bone mechanical properties.

Hypercalciuria in genetic hypercalciuric stone-forming (GHS) rats is accompanied by intestinal Ca hyperabsorption with normal serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels, elevation of intestinal, kidney, and bone vitamin D receptor (VDR) content, and greater 1,25(OH)2D3-induced bone resorption in vitro. To test the hypothesis that hyperresponsiveness of VDR gene expression to 1,25(OH)2D3 may mediate these observations, male GHS and wild-type Sprague- Dawley normocalciuric control rats were fed a normal Ca diet (0.6% Ca) and received a single intraperitoneal injection of either 1,25(OH)2D3 (10-200 ng/100 g body wt) or vehicle. Total RNAs were isolated from both duodenum and kidney cortex, and the VDR and calbindin mRNA levels were determined by Northern blot hybridization using specific cDNA probes. Under basal conditions, VDR mRNA levels in GHS rats were lower in duodenum and higher in kidney compared with wild-type controls. Administration of 1,25(OH)2D3 increased VDR gene expression significantly in GHS but not normocalciuric animals, in a time- and dose-dependent manner. In vivo half-life of VDR mRNA was similar in GHS and control rats in both duodenum and kidney, and was prolonged significantly (from 4-5 to > 8 h) by 1,25(OH)2D3 administration. Neither inhibition of gene transcription by actinomycin D nor inhibition of de novo protein synthesis with cycloheximide blocked the upregulation of VDR gene expression stimulated by 1,25(OH)2D3 administration. No alteration or mutation was detected in the sequence of duodenal VDR mRNA from GHS rats compared with wild-type animals. Furthermore, 1,25(OH)2D3 administration also led to an increase in duodenal and renal calbindin mRNA levels in GHS rats, whereas they were either suppressed or unchanged in wild-type animals. The results suggest that GHS rats hyperrespond to minimal doses of 1,25(OH)2D3 by an upregulation of VDR gene expression. This hyperresponsiveness of GHS rats to 1,25(OH)2D3 (a) occurs through an increase in VDR mRNA stability without involving alteration in gene transcription, de novo protein synthesis, or mRNA sequence; and (b) is likely of functional significance, and affects VDR-responsive genes in 1, 25(OH)2D3 target tissues. This unique characteristic suggests that GHS rats may be susceptible to minimal fluctuations in serum 1, 25(OH)2D3, resulting in increased VDR and VDR-responsive events, which in turn may pathologically amplify the actions of 1,25(OH)2D3 on Ca metabolism that thus contribute to the hypercalciuria and stone formation.

Background & Aims: Children with inflammatory bowel disease (IBD) are at risk for osteoporosis because of undernutrition, delayed puberty, and prolonged corticosteroid use. The aim of this study was to compare bone mineral density (BMD) in children with IBD with that in normal children and to assess the effects of nutritional and hormonal factors and corticosteroid dosages on BMD., Methods: One hundred sixty-two subjects (99 with IBD and 63 healthy sibling controls) were enrolled. Patients underwent anthropometric assessment, pubertal staging, bone age radiography, and BMD assessment by dual energy x-ray absorptiometry of the lumbar spine, femoral neck, and radius. Laboratory evaluations included serum calcium, phosphate, alkaline phosphatase, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, parathyroid hormone, osteocalcin, urinary N-telopeptides, albumin, insulin-like growth factor I, and testosterone or estradiol. Cumulative corticosteroid doses were calculated., Results: BMD Z scores at the lumbar spine and femoral neck were lower in patients with IBD, and lower in those with Crohn's disease compared with those with ulcerative colitis. Low BMD persisted after correction for bone age in girls with Crohn's disease (lumbar spine, P = 0.004; femoral neck, P = 0.002). Cumulative corticosteroid dose was a significant predictor of reduced BMD. BMD did not correlate with measures of calcium homeostasis, except elevated serum phosphate and urine calcium levels in girls., Conclusions: Low BMD occurs in children with IBD (more in Crohn's disease than in ulcerative colitis), especially pubertal and postpubertal girls. Cumulative corticosteroid dose is a predictor of low BMD, but other factors in Crohn's disease remain undetermined.

Dietary P restriction increases renal 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] biosynthesis through stimulation of proximal tubule 25-hydroxyvitamin D3-1 alpha-hydroxylase (1-OHase). Because insulin-like growth factor I (IGF-I) is required for 1-OHase stimulation by low-P diet (LPD) and because 1-OHase stimulation by low-Ca diet and parathyroid hormone is lost with aging, studies were undertaken to determine whether 1-OHase activity during LPD is impaired with age and whether IGF-1 can increase 1-OHase activity in adult rats. Five days of LPD increased in vitro 1-OHase activity in young (97.3 +/- 13.5 vs. 49.7 +/- 6.8 pg.mg protein-1.5 min-1, P < 0.005) but not adult (42.3 +/- 5.37 vs. 41.2 +/- 8.9) rats. In LPD-fed adult rats, recombinant human IGF-I (rhIGF-I, 1.4 mg.kg body wt-1.day-1) for 72 h increased 1-OHase (65.2 +/- 5.88 vs. 95.1 +/- 7.26 pg.mg protein-1.5 min-1, P < 0.005). The results show that the rise in 1-OHase activity during LPD is lost in adult rats and that rhIGF-I can overcome the inhibition and stimulate renal 1-OHase activity to levels observed in young animals. The studies indicate that the age-related loss of 1-OHase activity is reversible.

Studies of the relationship between PTH structure and function in the activation of protein kinases have revealed that different regions within the biologically active PTH-(1-34) peptide are responsible for different functions. The first two N-terminal amino acids are required for plasma membrane adenylyl cyclase stimulation, and the C-terminal region 29-32 is necessary for the translocating activity of protein kinase C. In the present study, we explored the structure-function relationship of human (h) PTH in the regulation of the vitamin D receptor (VDR) in osteoblast-like cells (ROS 17/2.8). VDR-rich cytosol extract was prepared after the confluent cells were incubated with different hPTH fragments for 16 h. hPTH-(1-34) at concentrations of 10(-9)-10(-7) M caused a dose-dependent decrease in VDR content from a control level of 70.2 +/- 2.2 fmol/mg protein to 62.1 +/- 3.3 (-16%) at 10(-9) M, 52.3 +/- 5.3 (-25.5%; P < 0.02) at 10(-8) M, and 45.5 +/- 3.5 fmol/mg protein (-35.3%; P = 0.001) at 10(-7) M (n = 6). hPTH-(1-31) also decreased VDR content from 65.5 +/- 3.6 to 55.2 +/- 7.9 (-19.5%) at 10(-9) M, 44.3 +/- 5.8 (-32.4%; P < 0.05) at 10(-8) M, and 40.6 +/- 3.2 fmol/mg protein (-38.9%; P < 0.05) at 10(-7) M (n = 6). Incubation of ROS 17/2.8 cells with 0.5 nM 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] led to up-regulation of VDR content by 340-370% of the control value. hPTH-(1-34) decreased the VDR up-regulatory effect of 1,25-(OH)2D3 from 340% to 230% of the control value at 10(-8) M (P < 0.0001) and 170% of the control value (P < 0.0001) at 10(-7) M, respectively (n = 6). hPTH-(1-31) also decreased the receptor up-regulatory effect of 1,25-(OH)2D3 from 370% to 286% (P < 0.02) at 10(-8) M and 220% (P < 0.002) at 10(-7) M, respectively (n = 6). hPTH-(3-34) and -(13-34) at concentrations of 10(-9)-10(-7) M did not decrease VDR content in either the absence or presence of 1,25-(OH)2D3. Quantitation of VDR messenger RNA by reverse transcription-polymerase chain reaction showed that PTH-(1-34) and -(1-31) at 10(-7) M, but not PTH-(3-34) and -(13-34), inhibited ROS 17/2.8 cell VDR gene expression in both the absence and presence of 1,25-(OH)2D3.(ABSTRACT TRUNCATED AT 400 WORDS)

Dietary phosphorus restriction up-regulates intestinal vitamin D receptor (VDR), but the tissue specificity of the up-regulation and the mechanism of receptor accumulation remain unknown. Therefore, the effects of low phosphorus diet (LPD) on VDR content in intestine, kidney, and splenic monocytes/macrophages were examined. Male Sprague-Dawley rats weighing 50-100 g were fed a normal diet (NPD; 0.6% Ca, 0.65% P) as controls followed by an LPD (0.6% Ca, 0.1% P) for 1-10 days (D1-D10). LPD rapidly decreased serum P levels by D1 from 11.11 +/- 0.19 mg/dl (mean +/- SE) to 4.98 +/- 0.37 mg/dl (n = 9). LPD increased total serum Ca from 10.54 +/- 0.09 mg/dl to 11.63 +/- 0.15, 12.17 +/- 0.15, and 12.39 +/- 0.18 mg/dl by D1, D2, and D3, respectively, and then remained stable. Serum 1,25-(OH)2D3 rapidly increased from 123 +/- 5.4 pg/ml to 304 +/- 35 pg/ml by D1, reached a plateau through D5, and then gradually increased to 464.9 +/- 27.7 pg/ml by D10. Intestinal VDR quantitated by ligand binding assay increased 3.5-fold from 169.6 +/- 13.7 fmol/mg of cytosol protein in rats fed NPD (n = 12) to a peak of 588.3 +/- 141.88 fmol/mg of protein by D3 (n = 6; p < 0.001) and then decreased to a plateau level of 2.5-fold greater than NPD (p < 0.05) during D5 to D10. In contrast, LPD did not up-regulate kidney or splenic monocyte/macrophage VDR. Northern blot analysis showed that intestinal VDR mRNA increased 2-fold by D2 (n = 3) of LPD and then gradually decreased to control levels after D5. In contrast, kidney VDR mRNA levels did not change during the first 5 days of P restriction and then subsequently decreased to 50% of NPD controls. The results of these studies indicate that VDR up-regulation during dietary phosphorus restriction is tissue-specific and that the mechanism of the up-regulation is time-dependent. Acutely (D1-D5), phosphorus restriction up-regulates intestinal VDR through increased VDR gene expression, whereas chronic (D5-D10) phosphorus restriction appears to alter VDR metabolism through nongenomic mechanisms that are consistent with prolongation of the half-life of the receptor. The nature of the tissue-specific regulation of VDR during phosphorus restriction remains to be determined.

X-linked recessive nephrolithiasis (XRN) was described in a large kindred in which nephrolithiasis; proximal tubular dysfunction, proteinuria, nephrocalcinosis, and renal failure occur only in males. Carrier females are asymptomatic, but formal studies of them have not been done. The gene for XRN has been mapped to the pericentromeric region of the X chromosome, close to the loci for several eye disease genes. We studied six affected males, 13 carrier females, and 25 normal members of this family including 7 females whose genetic haplotype predicted them to be carriers. Studies were done in the Clinical Research Unit on a diet containing 400 mg of calcium and 2 g of sodium, and by an additional outpatient urine collection was obtained on a 1-g calcium intake. Hypercalciuria occurred in five of six affected males, 4 of 12 carrier females, and three of seven predicted carriers. Significant proteinuria was present in all affected males and in no other subjects. Low-molecular-weight proteinuria was present in all affected males: the excretion of alpha 1-microglobulin exceeded normal by 3- to 14-fold, of beta 2-microglobulin exceeded normal by 100- to 400-fold, and of retinol-binding protein exceeded normal by 1,000- to 3,000-fold. The excretion of these proteins was less strikingly elevated in carrier females, but the excretion of alpha 1-microglobulin was abnormal in 9 of 15 carriers, beta 2-microglobulin was abnormal in 12 of 15, and retinolbinding protein in was abnormal 12 of 13, and this pattern was similar in predicted carriers. The urinary concentrating ability was abnormal in four affected males with renal insufficiency but normal in all other subjects. Urinary wasting of potassium, phosphorous, and glucose occurred infrequently, and no subject was hypouricemic. Formal ophthalmologic studies were normal in five affected males. Thus, the most consistent urinary abnormalities in XRN are hypercalciuria and low-molecular-weight proteinuria, the latter of which appears to be a marker for the carrier state.

Calcium oxalate nephrolithiasis is a common syndrome that recurs and may be complicated by infection, obstruction, bleeding, and rarely, impairment in renal function. The formation of Ca oxalate stones depends on the state of urinary supersaturation with respect to Ca and oxalate and the action of urinary inhibitors of crystal nucleation, aggregation, and growth. Idiopathic hypercalciuria is the most common cause of Ca oxalate stones and is characterized by hypercalciuria, normocalcemia, and intestinal Ca hyperabsorption with or without elevated serum 1,25(OH)2D3 levels in the absence of other known causes of hypercalciuria. Current diagnostic evaluation of recurrent Ca oxalate nephrolithiasis should be conducted while the patients follow their usual diets and includes the following: 1. Analysis of stone composition by polarization microscopy. 2. Measurement of serum Ca, phosphate, uric acid, 1,25(OH)2D3, and creatinine. 3. Twenty-four-hour urine collection for an analysis of volume, pH, and excretion of Ca, phosphorus, magnesium, uric acid, citrate, sodium, oxalate, and creatinine. Therapy to prevent stone recurrence is designed to reduce urinary supersaturation of Ca oxalate by increasing urine volume, reducing urine Ca to below 200 mg/24 hr with thiazide, maintaining dietary Ca intake at 600 to 800 mg/day, and adding potassium citrate if urine citrate levels are reduced. If elevated, urine oxalate excretion can be reduced by dietary oxalate restriction. Stones less than 2 cm in diameter located in the renal parenchyma or upper urinary tract can be fragmented with ESWL, whereas larger stones or those in the lower urinary tract should be removed by either percutaneous nephrolithotomy or ureteroscopic procedures.

Osteoporosis is a clinical syndrome of bone fragility resulting in fractures with minimal or no trauma. Bone strength is directly related to bone mass, therefore factors that influence bone mass will determine the subsequent risk of osteoporotic fracture. Estrogen loss at the menopause is the most common cause of osteoporosis. Factors that limit the process of bone formation during adolescence and early adulthood can interfere with reaching peak adult bone mass and thereby predispose to osteoporosis in later adult years. Bone remodeling (bone resorption followed by formation) during the early and mid-adult years may be altered by many diseases, medications and lifestyle (alcohol, inadequate exercise, low calcium intake), resulting in reduced bone mass and increased risk for osteoporotic fracture. Extremes of physical training can also cause bone loss in women athletes, and can be reversed by estrogen and adequate calcium intake. More research is needed to determine which forms of training optimize the effects of exercise on bone mass.

Human idiopathic hypercalciuria (IH) is a common cause of hypercalciuria that contributes to calcium oxalate nephrolithiasis. The disorder is characterized by normocalcemia, increased intestinal Ca absorption, and normal or elevated circulating 1,25(OH)2D3. Intestinal Ca hyperabsorption, which is a source of excess urine Ca excretion, may result from either a primary increase in renal 1,25(OH)2D3 production; a primary, vitamin D-independent defect in enterocyte regulation of Ca transport; or a secondary increase in 1,25(OH)2D3 production in response to a defect in renal tubular Ca reabsorption. Breeding male and female Sprague Dawley rats with spontaneous hypercalciuria has resulted in offspring with hypercalciuria, increased intestinal Ca absorption, and normal serum 1,25(OH)2D3. In male IH rats, vitamin D receptor (VDR) content measured by saturation binding and western blotting revealed a twofold increase in VDR number in the duodenum, kidney cortex, and splenic monocytes. The molecular basis for the increase in VDR appears not to be due to increased VDR gene expression, but may result from increased efficiency of translation of the VDR message or prolongation of the half-life of VDR. Comparable migration of normal and IH intestinal VDR on western blots and of intestinal VDR mRNA on northern blots suggests that the abundant VDR in IH rat intestine is not a mutation of the wild-type VDR. These observations strongly suggest that, in IH rats, normal serum 1,25(OH)2D3 and increased VDR results in increased VDR-1,25(OH)2D3 complexes and enhanced biologic actions of 1,25(OH)2D3, including increased intestinal Ca transport. IH in rats may be the first genetic disorder due to a pathologic increase in the VDR.