Bone health in children with severe cerebral palsy.

Aim: To describe bone health and associated factors in children with severe cerebral palsy. Method: In a retrospective, single-centre study, we performed a comprehensive bone evaluation (including clinical, densitometric and bone biomarker assessments) of children with severe cerebral palsy. Results: None of the 19 included children had a normal BMCTBLH Z score, and only one had a BMDTBLH Z score greater than -2. Six children had a BMDLS Z score greater than -2. The bone biomarker data were suggestive of excessive bone remodelling. Levels of bone remodelling markers factors and densitometric variables were not significantly related. Age, weight and pubertal stage were significantly related to bone mass. Discussion: Our results highlights the insufficient increase in bone mass with age (probably due to excessive bone remodelling) and confirms the high prevalence of low bone mineral density in children with severe cerebral palsy. Possible preventive measures might include calcium + vitamin D supplementation and the systematic management of underweight and delayed puberty. Bone remodelling markers might be of value for follow-up.

Relationship between bone marrow adipose tissue and kidney function in postmenopausal women.

Introduction: Bone marrow adipose tissue (BMAT) is associated with aging, osteoporosis, and chronic kidney disease (CKD). To date, the association between BMAT and kidney function in postmenopausal women has not been thoroughly investigated. The main purpose of this study was to determine whether a relationship exists between proton density fat fraction (PDFF) and kidney function in postmenopausal women. Methods: We investigated the cross-sectional association between estimated glomerular filtration rate (eGFR) - calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation - and PDFF - measured at the lumbar spine and proximal femur using Water Fat Imaging (WFI) MRI - in 199 postmenopausal women from the ADIMOS cohort study. We also performed DXA scans and laboratory measurements of sclerostin and c-terminal Fibroblast Growth Factor 23 (cFGF23). Results: Participants' mean age was 67.5 (standard deviation, SD 10.0) years. Their median eGFR was 85.0 (interquartile range, IQR 72.2-95.0) ml/min/1.73 cm2, and their mean lumbar spine PDFF was 57.9 % (SD 9.6). When classified by eGFR-based CKD stages, 41.7 % of the cohort had an eGFR ≥ 90 (n = 83), 47.2 % had an eGFR of 60-89.9 (n = 94), and 11.1 % had an eGFR of 30-59.9 (n = 22). Participants with eGFR ≥ 90 had a lower lumbar spine PDFF than those with eGFR 60-89.9 (mean 55.8 % (9.8) vs. 58.9 % (9.0), p = 0.031) and those with eGFR 30-59.9 (55.8 % (9.8) vs. 60.8 % (9.8), p = 0.043). However, the differences did not remain significant after adjusting for predetermined confounders, including age, diabetes, Charlson comorbidity index, recent history of fragility fracture, appendicular lean mass, and lumbar spine BMD. The inclusion of sclerostin and/or cFGF23 as suspected mediators did not alter the findings. When proximal hip imaging-based PDFF was considered, no significant differences were found between the eGFR categories in the unadjusted and adjusted analyses. Conclusion: No evidence of an association between kidney function and bone marrow adiposity was found either in the lumbar spine or proximal femur in a cohort of postmenopausal women.

Questions about Using the Induced Membrane Technique to Manage Cases of Congenital Tibial Pseudarthrosis.

The induced membrane technique is an innovative approach for repairing critical bone defects and has been applied recently in patients with congenital pseudarthrosis of the tibia (CPT). CPT is frequently associated with neurofibromatosis type 1 (NF1). Here, we briefly describe the clinical results of the induced membrane technique in NF1-deficient patients with CPT and in an animal model of CPT. Furthermore, we discuss the hypotheses used to explain inconsistent outcomes for the induced membrane technique in CPT-especially when associated with NF1.

Chronic kidney disease is a key risk factor for aortic stenosis progression.

BACKGROUND: Rapid progression of aortic stenosis (AS) has been observed in patients undergoing dialysis, but existing cross-sectional evidence is contradictory in non-dialysis-dependent chronic kidney disease (CKD). The present study sought to evaluate whether CKD is associated with the progression of AS over time in a large cohort of patients with AS. METHODS: We retrospectively studied all consecutive patients diagnosed with AS [peak aortic jet velocity (Vmax) ≥2.5 m/s] and left ventricular ejection fraction ≥50% in the echocardiography laboratories of two tertiary centers between 2000 and 2018. The estimated glomerular filtration rate (eGFR) (mL/min/1.73 m2) was calculated from serum creatinine values. Patients were divided into five CKD stages according to the baseline eGFR. Annual rates of change in the aortic valve area (AVA) were determined by a linear mixed-effects model. RESULTS: Among the 647 patients included, 261 (40%) had CKD. After a median follow-up of 2.9 (interquartile range 1.8-4.8) years, the mean overall rate of change in AVA was -0.077 (95% confidence interval -0.082; -0.073) cm2/year. There was an inverse relationship between the progression rate and kidney function. The more severe the CKD stage, the greater the AVA narrowing (P < .001). By multivariable linear regression analysis, the eGFR was also negatively associated (P < .001) with AS progression. An eGFR strata below 45 mL/min/1.73 m2 was associated with higher odds of rapid progression of AS than normal kidney function. During the clinical follow-up, event-free survival (patients free of aortic valve replacement or death) decreased as CKD progressed. Rapid progression of AS in patients with kidney dysfunction was associated with worse outcomes. CONCLUSIONS: Patients with CKD exhibit more rapid progression of AS over time and require close monitoring. The link between kidney dysfunction and rapid progression of AS is still unknown and requires further research.

Vasorin plays a critical role in vascular smooth muscle cells and arterial functions.

Within the cardiovascular system, the protein vasorin (Vasn) is predominantly expressed by vascular smooth muscle cells (VSMCs) in the coronary arteries and the aorta. Vasn knockout (Vasn-/- ) mice die within 3 weeks of birth. In the present study, we investigated the role of vascular Vasn expression on vascular function. We used inducible Vasn knockout mice (VasnCRE-ERT KO and VasnSMMHC-CRE-ERT2 KO , in which respectively all cells or SMCs only are targeted) to analyze the consequences of total or selective Vasn loss on vascular function. Furthermore, in vivo effects were investigated in vitro using human VSMCs. The death of VasnCRE-ERT KO mice 21 days after tamoxifen injection was concomitant with decreases in blood pressure, angiotensin II levels, and vessel contractibility to phenylephrine. The VasnSMMHC-CRE-ERT2 KO mice displayed concomitant changes in vessel contractibility in response to phenylephrine and angiotensin II levels. In vitro, VASN deficiency was associated with a shift toward the SMC contractile phenotype, an increase in basal intracellular Ca2+ levels, and a decrease in the SMCs' ability to generate a calcium signal in response to carbachol or phenylephrine. Additionally, impaired endothelium-dependent relaxation (due to changes in nitric oxide signaling) was observed in all Vasn knockout mice models. Our present findings highlight the role played by Vasn SMC expression in the maintenance of vascular functions. The mechanistic experiments suggested that these effects are mediated by SMC phenotype switching and changes in intracellular calcium homeostasis, angiotensin II levels, and NO signaling.

The Endothelium and COVID-19: An Increasingly Clear Link Brief Title: Endotheliopathy in COVID-19.

The endothelium has a fundamental role in the cardiovascular complications of coronavirus disease 2019 (COVID-19). Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particularly affects endothelial cells. The virus binds to the angiotensin-converting enzyme 2 (ACE-2) receptor (present on type 2 alveolar cells, bronchial epithelial cells, and endothelial cells), and induces a cytokine storm. The cytokines tumor necrosis factor alpha, interleukin-1 beta, and interleukin-6 have particular effects on endothelial cells-leading to endothelial dysfunction, endothelial cell death, changes in tight junctions, and vascular hyperpermeability. Under normal conditions, apoptotic endothelial cells are removed into the bloodstream. During COVID-19, however, endothelial cells are detached more rapidly, and do not regenerate as effectively as usual. The loss of the endothelium on the luminal surface abolishes all of the vascular responses mediated by the endothelium and nitric oxide production in particular, which results in greater contractility. Moreover, circulating endothelial cells infected with SARS-CoV-2 act as vectors for viral dissemination by forming clusters that migrate into the circulation and reach distant organs. The cell clusters and the endothelial dysfunction might contribute to the various thromboembolic pathologies observed in COVID-19 by inducing the formation of intravascular microthrombi, as well as by triggering disseminated intravascular coagulation. Here, we review the contributions of endotheliopathy and endothelial-cell-derived extracellular vesicles to the pathogenesis of COVID-19, and discuss therapeutic strategies that target the endothelium in patients with COVID-19.

Decreased monocyte calcium sensing receptor expression in patients with chronic kidney disease is associated with impaired monocyte ability to reduce vascular calcification.

In chronic kidney disease (CKD), calcium-sensing receptor (CaSR) expression and function have been extensively studied in parathyroid tissue and vascular tissues. To examine whether similar changes occurred in other tissues, we measured total and surface CaSR expression in monocytes of patients with various stages of CKD and healthy volunteers respectively in cross-sectional studies. We further explored in vitro the impact of uremic serum on CaSR expression in monocytes (U937 and THP-1 cell lines), and whether human peripheral blood mononuclear cells or U937 and THP-1 monocytes might modify vascular calcium deposition in rat carotid arteries in vitro. CKD was associated with a decrease in peripheral blood mononuclear cell CaSR expression both in total and at the monocyte surface alone (43% and 34%, respectively in CKD stages 4-5). This decrease was associated with a reduction in the ability of monocytes to inhibit vascular calcification in vitro. Pretreatment with the calcimimetic NPSR568 of peripheral blood mononuclear cells isolated from patients with CKD significantly improved monocyte capacity to reduce carotid calcification in vitro. The fewer peripheral blood mononuclear cells expressing cell surface CaSR, the more calcimimetic treatment enhanced the decrease of carotid calcium content. Thus, we demonstrate that monocyte CaSR expression is decreased in patients with CKD and provide in vitro evidence for a potential role of this decrease in the promotion of vascular calcification. Hence, targeting this alteration or following monocyte CaSR expression as an accessible marker might represent a promising therapeutic strategy in CKD-associated arterial calcification.

Early steroid withdrawal has a positive effect on bone in kidney transplant recipients: a propensity score study with inverse probability-of-treatment weighting.

BACKGROUND: Long-term corticosteroid use after kidney transplantation is associated with a decrease in bone mineral density (BMD) and a high fracture risk. We hypothesized that patients with early steroid withdrawal (ESW) would display a gain in BMD in the year following kidney transplantation, when compared with patients on long-term corticosteroid therapy. METHODS: In a cohort of kidney transplant recipients, 356 patients were included between 2012 and 2019. Dual-energy X-ray absorptiometry was performed 1 and 12 months after transplantation. The data were analyzed using linear regression with inverse probability-of-treatment weighting (based on a propensity score). RESULTS: At 1 year after transplantation, the gain in BMD was significantly greater in recipients with ESW than in recipients on long-term corticosteroid therapy for the lumbar spine (+0.036 g/cm2, p < 0.001) and the femoral neck (+0.020 g/cm2, p = 0.035). Among patients with ESW, (i) none had osteoporosis, (ii) the percentage with normal BMD increased from 33.3% at month 1 to 54.4% at month 12, and (iii) the percentage with osteopenia fell from 56.2% to 45.6%. In patients undergoing long-term corticosteroid therapy, the fracture incidence was 13.5 per 1000 person-years. None of the patients in the ESW group experienced a fracture. CONCLUSION: ESW has a positive effect on bone in kidney transplant recipients.

Circulating Receptor Activator of Nuclear Factor kB Ligand and triglycerides are associated with progression of lower limb arterial calcification in type 2 diabetes: a prospective, observational cohort study.

BACKGROUND: Lower limb arterial calcification is a frequent, underestimated but serious complication of diabetes. The DIACART study is a prospective cohort study designed to evaluate the determinants of the progression of lower limb arterial calcification in 198 patients with type 2 diabetes. METHODS: Lower limb arterial calcification scores were determined by computed tomography at baseline and after a mean follow up of 31.20 ± 3.86 months. Serum RANKL (Receptor Activator of Nuclear factor kB Ligand) and bone remodeling, inflammatory and metabolic parameters were measured at baseline. The predictive effect of these markers on calcification progression was analyzed by a multivariate linear regression model. RESULTS: At baseline, mean ± SD and median lower limb arterial calcification scores were, 2364 ± 5613 and 527 respectively and at the end of the study, 3739 ± 6886 and 1355 respectively. Using multivariate analysis, the progression of lower limb arterial log calcification score was found to be associated with (ß coefficient [slope], 95% CI, p-value) baseline log(calcification score) (1.02, 1.00-1.04, p < 0.001), triglycerides (0.11, 0.03-0.20, p = 0.007), log(RANKL) (0.07, 0.02-0.11, p = 0.016), previous ischemic cardiomyopathy (0.36, 0.15-0.57, p = 0.001), statin use (0.39, 0.06-0.72, p = 0.023) and duration of follow up (0.04, 0.01-0.06, p = 0.004). CONCLUSION: In patients with type 2 diabetes, lower limb arterial calcification is frequent and can progress rapidly. Circulating RANKL and triglycerides are independently associated with this progression. These results open new therapeutic perspectives in peripheral diabetic calcifying arteriopathy. Trial registration NCT02431234.

The Masquelet technique: Current concepts, animal models, and perspectives.

Bone reconstruction within a critical-sized defect remains a real challenge in orthopedic surgery. The Masquelet technique is an innovative, two-step therapeutic approach for bone reconstruction in which the placement of a poly (methylmethacrylate) spacer into the bone defect induces the neo-formation of a tissue called "induced membrane." This surgical technique has many advantages and is often preferred to a vascularized bone flap or Ilizarov's technique. Although the Masquelet technique has achieved high clinical success rates since its development by Alain-Charles Masquelet in the early 2000s, very little is known about how the process works, and few animal models of membrane induction have been developed. Our successful use of this technique in the clinic and our interest in the mechanisms of tissue regeneration (notably bone regeneration) prompted us to develop a surgical model of the Masquelet technique in rats. Here, we provide a comprehensive review of the literature on animal models of membrane induction, encompassing the defect site, the surgical procedure, and the histologic and osteogenic properties of the induced membrane. We also discuss the advantages and disadvantages of those models to facilitate efforts in characterizing the complex biological mechanisms that underlie membrane induction.

The Metabolism of Epoxyeicosatrienoic Acids by Soluble Epoxide Hydrolase Is Protective against the Development of Vascular Calcification.

This study addressed the hypothesis that soluble epoxide hydrolase (sEH), which metabolizes endothelium-derived epoxyeicosatrienoic acids, plays a role in vascular calcification. The sEH inhibitor trans-4-(4-(3-adamantan-1-yl-ureido)-cyclohexyloxy)-benzoic acid (t-AUCB) potentiated the increase in calcium deposition of rat aortic rings cultured in high-phosphate conditions. This was associated with increased tissue-nonspecific alkaline phosphatase activity and mRNA expression level of the osteochondrogenic marker Runx2. The procalcifying effect of t-AUCB was prevented by mechanical aortic deendothelialization or inhibition of the production and action of epoxyeicosatrienoic acids using the cytochrome P450 inhibitor fluconazole and the antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE), respectively. Similarly, exogenous epoxyeicosatrienoic acids potentiated the calcification of rat aortic rings through a protein kinase A (PKA)-dependent mechanism and of human aortic vascular smooth muscle cells when sEH was inhibited by t-AUCB. Finally, a global gene expression profiling analysis revealed that the mRNA expression level of sEH was decreased in human carotid calcified plaques compared to adjacent lesion-free sites and was inversely correlated with Runx2 expression. These results show that sEH hydrolase plays a protective role against vascular calcification by reducing the bioavailability of epoxyeicosatrienoic acids.

The CaSR in Pathogenesis of Breast Cancer: A New Target for Early Stage Bone Metastases.

The Ca2+-sensing receptor (CaSR) is a class-C G protein-coupled receptor which plays a pivotal role in calciotropic processes, primarily in regulating parathyroid hormone secretion to maintain systemic calcium homeostasis. Among its non-calciotropic roles, where the CaSR sits at the intersection of myriad processes, it has steadily garnered attention as an oncogene or tumor suppressor in different organs. In maternal breast tissues the CaSR promotes lactation but in breast cancer it acts as an oncoprotein and has been shown to drive the pathogenesis of skeletal metastases from breast cancer. Even though research has made great strides in treating primary breast cancer, there is an unmet need when it comes to treatment of metastatic breast cancer. This review focuses on how the CaSR leads to the pathogenesis of breast cancer by contrasting its role in healthy tissues and tumorigenesis, and by drawing brief parallels with the tissues where it has been implicated as an oncogene. A class of compounds called calcilytics, which are CaSR antagonists, have also been surveyed in the instances where they have been used to target the receptor in cancerous tissues and constitute a proof of principle for repurposing them. Current clinical therapies for treating bone metastases from breast cancer are limited to targeting osteoclasts and a deeper understanding of the CaSR signaling nexus in this context can bolster them or lead to novel therapeutic interventions.

New Insights into the Roles of Monocytes/Macrophages in Cardiovascular Calcification Associated with Chronic Kidney Disease.

Cardiovascular disease (CVD) is an important cause of death in patients with chronic kidney disease (CKD), and cardiovascular calcification (CVC) is one of the strongest predictors of CVD in this population. Cardiovascular calcification results from complex cellular interactions involving the endothelium, vascular/valvular cells (i.e., vascular smooth muscle cells, valvular interstitial cells and resident fibroblasts), and monocyte-derived macrophages. Indeed, the production of pro-inflammatory cytokines and oxidative stress by monocyte-derived macrophages is responsible for the osteogenic transformation and mineralization of vascular/valvular cells. However, monocytes/macrophages show the ability to modify their phenotype, and consequently their functions, when facing environmental modifications. This plasticity complicates efforts to understand the pathogenesis of CVC-particularly in a CKD setting, where both uraemic toxins and CKD treatment may affect monocyte/macrophage functions and thereby influence CVC. Here, we review (i) the mechanisms by which each monocyte/macrophage subset either promotes or prevents CVC, and (ii) how both uraemic toxins and CKD therapies might affect these monocyte/macrophage functions.

Pharmacokinetics of a New Pharmaceutical Form of Vitamin D3 100,000 IU in Soft Capsule.

Vitamin D deficiency is frequent in the general population and both subjects and health professionals could benefit from a broader range of vitamin D3 formulations. We conducted a single-dose, open-label, parallel-group, randomized bioequivalence study to compare a single dose of a newly developed vitamin D3 100,000 IU in a soft capsule (Group 1) with the reference drug vitamin D3 100,000 IU oral solution in ampoule (Group 2) in healthy volunteers over a four-month period. The primary endpoint was the area under the curve (AUC) of serum 25-hydroxyvitamin-D (25(OH)D) concentrations on Day 112. This study was conducted in France from February to June 2014 in 53 young adults with a mean age of 26.9 years. At baseline, low mean serum 25(OH)D levels were observed in both groups (10.6 ng/mL in Group 1 and 9.0 ng/mL in Group 2). On Day 112, the AUC of serum 25(OH)D concentration was 2499.4 ± 463.8 nmol/mL (7.8 ± 0.2 for LogAUC) for Group 1 and 2152.3 ± 479.8 nmol/mL (7.6 ± 0.2 for LogAUC) for Group 2. Bioequivalence of the two treatments was not demonstrated. Superiority of vitamin D3 100,000 IU soft capsule was observed with p = 0.029 for AUC and p = 0.03 for LogAUC using a non-parametric Wilcoxon test. The profile of the serum 25(OH)D concentration showed a significant difference in favor of Group 1 on Days 1, 3, 7, 14 and 90. Mean serum 25(OH)D concentrations in Group 1 were between 20 and 30 ng/mL during the four-month period and under 20 ng/mL throughout the study in Group 2, except on Day 112. Mean Cmax for Group 1 was significantly higher (p = 0.002). Fourteen days were needed to reach Tmax by more than half the subjects in Group 1 compared to 45 days in Group 2. Both treatments were well tolerated, with no severe or related adverse events reported. In conclusion, the pharmacokinetic profile of the new formulation of vitamin D3 100,000 IU soft capsule is superior to that of the oral solution in ampoule. The new formulation increased serum 25(OH)D levels to above 20 ng/mL and maintained levels from 20 ng/mL to 30 ng/mL for four months in late winter and spring.

Activation of the calcium-sensing receptor in human valvular interstitial cells promotes calcification.

INTRODUCTION AND AIMS: Calcific aortic valve disease (CAVD) is the most common heart valve disease in western countries. It has been reported that activation of the calcium-sensing receptor(CaSR) expressed by vascular smooth muscle cells prevents vascular calcification. However, to date, the CaSR's expression and function in cardiac valves have not been studied. The present study sought to evaluate the presence of the CaSR within human valvular interstitial cells (hVICs), assess the CaSR's functionality, and ascertain its involvement in hVIC calcification. METHODS AND RESULTS: Data from Western blot, flow cytometry and immunocytochemistry experiments demonstrated that primary hVICs express the CaSR. The receptor was functional, since the incubation of hVICs with the calcimimetic R-568 significantly increased Ca2+-induced ERK1/2 phosphorylation, and exposure to the calcilytic NPS2143 reduced ERK1/2 activation. A reduction in endogenous CaSR expression by hVICs (using siRNA) was associated with significantly lower levels of Ca2+-induced mineralization (quantified using Alizarin Red staining). Similar data were obtained after the pharmacological inhibition of CaSR activity by the calcilytic NPS2143. In contrast, overexpression of a functional CaSR amplified Ca2+-induced calcification. Pharmacological activation of the CaSR with the calcimimetic R-568 showed similar effects. CaSR's procalcific properties are associated with increased osteogenic transition (as characterized by elevated mRNA expression of bone morphogenetic protein 2 and osterix), and reduced the expression of the calcification inhibitor osteopontin. Histological analysis of 12 human aortic tricuspid valves showed that CaSR expression was greater in calcified areas than in non-calcified areas. These data were confirmed by Western blots. CONCLUSIONS: To the best of our knowledge, this study is the first to have demonstrated that hVICs express a functional CaSR. Taken as a whole, our data suggest that activation of the CaSR expressed by hVICs might be a key promoter of CAVD progression.

Differential Determinants of Tubular Phosphate Reabsorption: Insights on Renal Excretion of Phosphates in Kidney Disease.

We assessed the tubular reabsorption of phosphate (TRP) and maximal renal threshold for phosphate reabsorption to glomerular filtration rate (TmPi/GFR) and their determinants in 64 stages 2-4 chronic kidney disease (CKD) patients in order to define the early changes in phosphate metabolism in CKD. In multivariable analysis, TmPi/GFR correlates were estimated GFR (eGFR), intact parathyroid hormone (iPTH), and hemoglobin (R2 = 0.417), while TRP correlates were eGFR, iPTH, 24-h phosphaturia, and calcitriol (R2 = 0.72). This suggests that TmPi/GFR and TRP, respectively, assess hemoglobin-phosphate and bowel-kidney phosphate regulation axis. Iron supplementation based on TmPi/GFR or earlier phosphate restriction based on TRP should be investigated in view of modifying clinical outcomes in CKD.

Randomized Clinical Trial of Sevelamer Carbonate on Serum Klotho and Fibroblast Growth Factor 23 in CKD.

BACKGROUND AND OBJECTIVES: Epidemiologic studies suggest that higher serum phosphaturic hormone fibroblast growth factor 23 levels are associated with increase morbidity and mortality. The aim of the FGF23 Reduction Efficacy of a New Phosphate Binder in CKD Trial was to evaluate the effect of sevelamer carbonate on serum C-terminal fibroblast growth factor 23 levels in normophosphatemic patients with CKD stage 3b/4. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Patients with CKD, eGFR between 45 and 15 ml/min per 1.73 m2, fasting serum phosphate concentration >3.1 mg/dl, and serum C-terminal fibroblast growth factor 23 >80 relative units/ml were included in our double-blind, placebo-controlled, randomized multicenter study. All patients received 100,000 IU cholecalciferol at time of randomization. Participants received either placebo or sevelamer carbonate 4.8 g daily during a 12-week period. Biologic parameters, including serum C-terminal fibroblast growth factor 23, intact fibroblast growth factor 23, and α-klotho, were evaluated at baseline and 12 weeks after inclusion. RESULTS: Of 96 screened patients, 78 (mean±SD age: 63±13 years old; 70% men; mean eGFR: 27±9 ml/min per 1.73 m2) met the inclusion criteria. At baseline, mean eGFR was 27±9 ml/min per 1.73 m2, mean serum phosphate level was 3.8±0.5 mg/dl, and median (interquartile range) serum C-terminal fibroblast growth factor 23 level was 157 (120-241) relative units/ml. After 12 weeks of treatment, urinary phosphate-to-creatinine ratio fell significantly in the sevelamer group. The sevelamer and placebo groups did not differ significantly in terms of median change in serum C-terminal fibroblast growth factor 23 levels: the median (interquartile range) change was 38 (-13-114) relative units/ml in the placebo group and 37 (-1-101) relative units/ml in the sevelamer group (P=0.77). There was no significant difference in serum intact fibroblast growth factor 23, α-klotho, or phosphate levels changes between the two groups. Serum total and LDL cholesterol levels fell significantly in the sevelamer group. CONCLUSIONS: In our double-blind, placebo-controlled, randomized study performed in normophosphatemic patients with CKD, a 12-week course of sevelamer carbonate significantly reduced phosphaturia without changing serum phosphorus but did not significantly modify serum C-terminal fibroblast growth factor 23 and intact fibroblast growth factor 23 or α-klotho levels.

Overexpression of a functional calcium-sensing receptor dramatically increases osteolytic potential of MDA-MB-231 cells in a mouse model of bone metastasis through epiregulin-mediated osteoprotegerin downregulation.

INTRODUCTION AND AIMS: Osteolytic bone metastases are observed in advanced cases of breast cancer. In vitro data suggest that the activity of the calcium-sensing receptor (CaSR) expressed by metastatic cells could potentiate their osteolytic potential. This study aimed to demonstrate in vivo the involvement of the CaSR in breast cancer cells osteolytic potential and to identify potential targets linked to CaSR activity. METHODS AND RESULTS: MDA-MB-231 stably transfected with plasmids containing either a full-length wild-type CaSR (CaSR-WT), or a functionally inactive dominant negative mutant (CaSR-DN) or an empty vector (EV) were intratibially injected into Balb/c-Nude mice. X-ray analysis performed 19 days after injection showed a dramatic increase of osteolytic lesions in mice injected with CaSR-WT-transfected cells as compared to mice injected with EV- or CaSR-DN-transfected cells. This was associated with decreased BV/TV ratio and increased tumor burden. Epiregulin, an EGF-like ligand, was identified by a DNA microarray as a possible candidate involved in CaSR-mediated osteolysis. Indeed, in vitro, CaSR overexpression increased both epiregulin expression and secretion as compared to EV- or CaSR-DN-transfected cells. Increased epiregulin expression was also detected in osteolytic bone lesions from mice injected with CaSR-WT-transfected MDA-MB-231. In vitro, exposure of osteoblastic cells (HOB and SaOS2) to exogenous epiregulin significantly decreased OPG mRNA expression. Exposure of osteoblastic cells to conditioned media prepared from CaSR-WT-transfected cells also decreased OPG expression. This effect was partially blocked after addition of an anti-epiregulin antibody. CONCLUSIONS: Overexpression of a functional CaSR in metastatic breast cancer cells dramatically amplifies their osteolytic potential through epiregulin-mediated OPG downregulation.

Association between metformin use and below-the-knee arterial calcification score in type 2 diabetic patients.

BACKGROUND: Vascular calcification (VC) is common in type 2 diabetes, and is associated with cardiovascular complications. Recent preclinical data suggest that metformin inhibits VC both in vitro and in animal models. However, metformin's effects in patients with diabetic VC have not previously been characterized. The present study investigated the association between metformin use and lower-limb arterial calcification in patients with type 2 diabetes and high cardiovascular risk. METHODS: The DIACART cross-sectional cohort study included 198 patients with type 2 diabetes but without severe chronic kidney disease. Below-the-knee calcification scores were assessed by computed tomography and supplemented by colour duplex ultrasonography. Data on anti-diabetic drugs were carefully collected from the patients' medical records and during patient interviews. Biochemical and clinical data were studied as potential confounding factors. RESULTS: Metformin-treated patients had a significantly lower calcification score than metformin-free patients (mean ± standard deviation: 2033 ± 4514 and 4684 ± 9291, respectively; p = 0.01). A univariate analysis showed that metformin was associated with a significantly lower prevalence of severe below-the-knee arterial calcification (p = 0.02). VC was not significantly associated with the use of other antidiabetic drugs, including sulfonylureas, insulin, gliptin, and glucagon like peptide-1 analogues. A multivariate logistic regression analysis indicated that the association between metformin use and calcification score (odds ratio [95% confidence interval] = 0.33 [0.11-0.98]; p = 0.045) was independent of age, gender, tobacco use, renal function, previous cardiovascular disease, diabetes duration, neuropathy, retinopathy, HbA1c levels, and inflammation. CONCLUSIONS: In patients with type 2 diabetes, metformin use was independently associated with a lower below-the-knee arterial calcification score. This association may contribute to metformin's well-known vascular protective effect. Further prospective investigations of metformin's potential ability to inhibit VC in patients with and without type 2 diabetes are now needed to confirm these results.