Dietary Mg Supplementation Decreases Oxidative Stress, Inflammation, and Vascular Dysfunction in an Experimental Model of Metabolic Syndrome with Renal Failure.

BACKGROUND: Metabolic syndrome (MetS) and chronic kidney disease (CKD) are commonly associated with cardiovascular disease (CVD) and in these patients Mg concentration is usually decreased. This study evaluated whether a dietary Mg supplementation might attenuate vascular dysfunction through the modulation of oxidative stress and inflammation in concurrent MetS and CKD. METHODS: A rat model of MetS (Zucker strain) with CKD (5/6 nephrectomy, Nx) was used. Nephrectomized animals were fed a normal 0.1%Mg (MetS+Nx+Mg0.1%) or a supplemented 0.6%Mg (MetS+Nx+Mg0.6%) diet; Sham-operated rats with MetS receiving 0.1%Mg were used as controls. RESULTS: As compared to controls, the MetS+Nx-Mg0.1% group showed a significant increase in oxidative stress and inflammation biomarkers (lipid peroxidation and aortic interleukin-1b and -6 expression) and Endothelin-1 levels, a decrease in nitric oxide and a worsening in uremia and MetS associated pathology as hypertension, and abnormal glucose and lipid profile. Moreover, proteomic evaluation revealed changes mainly related to lipid metabolism and CVD markers. By contrast, in the MetS+Nx+Mg0.6% group, these parameters remained largely similar to controls. CONCLUSION: In concurrent MetS and CKD, dietary Mg supplementation reduced inflammation and oxidative stress and improved vascular function.

The direct effect of fibroblast growth factor 23 on vascular smooth muscle cell phenotype and function.

BACKGROUND: In chronic kidney disease (CKD) patients, increased levels of fibroblast growth factor 23 (FGF23) are associated with cardiovascular mortality. The relationship between FGF23 and heart hypertrophy has been documented, however, it is not known whether FGF23 has an effect on vasculature. Vascular smooth muscle cells VSMCs may exhibit different phenotypes; our hypothesis is that FGF23 favours a switch from a contractile to synthetic phenotype that may cause vascular dysfunction. Our objective was to determine whether FGF23 may directly control a change in VSMC phenotype. METHODS: This study includes in vitro, in vivo and ex vivo experiments and evaluation of patients with CKD stages 2-3 studying a relationship between FGF23 and vascular dysfunction. RESULTS: In vitro studies show that high levels of FGF23, by acting on its specific receptor FGFR1 and Erk1/2, causes a change in the phenotype of VSMCs from contractile to synthetic. This change is mediated by a downregulation of miR-221/222, which augments the expression of MAP3K2 and PAK1. miR-221/222 transfections recovered the contractile phenotype of VSMCs. Infusion of recombinant FGF23 to rats increased vascular wall thickness, with VSMCs showing a synthetic phenotype with a reduction of miR-221 expression. Ex-vivo studies on aortic rings demonstrate also that high FGF23 increases arterial stiffening. In CKD 2-3 patients, elevation of FGF23 was associated with increased pulse wave velocity and reduced plasma levels of miR-221/222. CONCLUSION: In VSMCs, high levels of FGF23, through the downregulation of miR-221/222, causes a change to a synthetic phenotype. This change in VSMCs increases arterial stiffening and impairs vascular function, which might ultimately worsen cardiovascular disease.

Silver jubilee: 25 years of the first demonstration of the direct effect of phosphate on the parathyroid cell.

Although phosphorus is an essential element for life, it is not found in nature in its native state but rather combined in the form of inorganic phosphates (PO43-), with tightly regulated plasma levels that are associated with deleterious effects and mortality when these are out of bounds. The growing interest in the accumulation of PO43- in human pathophysiology originated in its attributed role in the pathogenesis of secondary hyperparathyroidism (SHPT) in chronic kidney disease. In this article, we review the mechanisms by which this effect was justified and we commemorate the important contribution of a Spanish group led by Dr. M. Rodríguez, just 25 years ago, when they first demonstrated the direct effect of PO43- on the regulation of the synthesis and secretion of parathyroid hormone by maintaining the structural integrity of the parathyroid glands in their original experimental model. In addition to demonstrating the importance of arachidonic acid (AA) and the phospholipase A2-AA pathway as a mediator of parathyroid gland response, these findings were predecessors of the recent description of the important role of PO43- on the activity of the calcium sensor-receptor, and also fueled various lines of research on the importance of PO43- overload not only for the pathophysiology of SHPT but also in its systemic pathogenic role.

Hiporrespuesta o resistencia a la acción de la hormona paratiroidea en la enfermedad renal crónica / Hyporesponsiveness or resistance to the action of parathyroid hormone in chronic kidney disease

El hiperparatiroidismo secundario es uno de los componentes integrales de las alteraciones del metabolismo óseo-mineral en la enfermedad renal crónica (ERC) o complejo chronic kidney disease-mineral bone disorder. Se ha demostrado que en el desarrollo y progresión del hiperparatiroidismo secundario intervienen muchos factores, estrechamente interrelacionados, pero la presencia e importancia de la hiporrespuesta (o resistencia) a la acción de la hormona paratiroidea (PTH) es poco comprendida. En esta revisión analizaremos sus antecedentes, factores que intervienen, así como alguno de los mecanismos moleculares que podrían explicarla. La presencia de resistencia a la acción biológica de la PTH no es única en la ERC, ya que también se presenta para otras hormonas, habiéndose incluso usado el término de «uremia como una enfermedad de receptores». Esta hiporrespuesta a la PTH tiene importantes implicaciones clínicas, dado que no solo permite explicar parte de la patogenia progresiva de la hipersecreción de PTH e hiperplasia paratiroidea, sino también la creciente prevalencia de enfermedad ósea adinámica en la población con ERC. De este modo, subrayamos la importancia de controlar, sin normalizar completamente, los niveles de PTH en los distintos estadios de ERC, dado que un cierto incremento de sus niveles supone inicialmente una adaptación clínica. Futuros estudios a nivel molecular sobre la uremia, o la reciente descripción del efecto directo del fosfato sobre la actividad del receptor sensor de calcio como sensor de fosfato, podrían resultar valiosos incluso más allá de explicar la hiporrespuesta a la PTH en la ERC. (AU)

Secondary hyperparathyroidism (SHPT) is an integral component of the chronic kidney disease–mineral and bone disorder (CKD–MBD). Many factors have been associated with the development and progression of SHPT but the presence of skeletal or calcemic resistance to the action of PTH in CKD has often gone unnoticed. The term hyporesponsiveness to PTH is currently preferred and, in this chapter, we will not only review the scientific timeline but also some of the molecular mechanisms behind. Moreover, the presence of resistance to the biological action of PTH is not unique in CKD since resistance to other hormones has also been described (“uremia as a receptor disease”). This hyporesponsiveness carries out important clinical implications since it explains, at least partially, not only the progressive nature of the pathogenesis of CKD-related PTH hypersecretion and parathyroid hyperplasia but also the increasing prevalence of adynamic bone disease in the CKD population. Therefore, we underline the importance of PTH control in all CKD stages, but not aiming to completely normalize PTH levels since a certain degree of SHPT may represent an adaptive clinical response. Future studies at the molecular level, i.e. on uremia, or the recent description of the calcium-sensing receptor as a phosphate sensor, may become of great value beyond their significance to explain just the hyporesponsiveness to PTH in CKD. (AU)

Magnesium supplementation reduces inflammation in rats with induced chronic kidney disease.

BACKGROUND: Inflammation is a common feature in chronic kidney disease (CKD) that appears specifically associated with cardiovascular derangements in CKD patients. Observational studies have revealed a link between low Mg levels and inflammation. In this study, we hypothesize that Mg might have a modulatory effect on the inflammation induced under the uraemic milieu. METHODS: In vivo studies were performed in a 5/6 nephrectomized rat model of CKD. Furthermore, a possible direct effect of Mg was addressed through in vitro studies with vascular smooth muscle cells (VSMCs). RESULTS: Uraemic rats fed a normal (0.1%) Mg diet showed a systemic inflammatory response evidenced by the elevation in plasma of the pro-inflammatory cytokines TNF-α, IL-1ß and IL-6, and GPx activity, a marker of oxidative stress. Importantly, an increased expression of these cytokines in the aortic tissue was also observed. In contrast, a dietary Mg supplementation (0.6%) greatly prevented the oxidative stress and the pro-inflammatory response. In vitro, in VSMCs cultured in a pro-inflammatory high phosphate medium, incubation with Mg 1.6 mM inhibited the increase in the production of ROS, the rise in the expression of TNF-α, IL-1ß, IL-6 and IL-8 and the activation of NF-κB signalling that was observed in cells incubated with a normal (0.8 mM) Mg. CONCLUSION: Mg supplementation reduced inflammation associated with CKD, exerting a direct effect on vascular cells. These findings support a possible beneficial effect of Mg supplementation along the clinical management of CKD patients.

Hyporesponsiveness or resistance to the action of parathyroid hormone in chronic kidney disease.

Secondary hyperparathyroidism (SHPT) is an integral component of the chronic kidney disease-mineral and bone disorder (CKD-MBD). Many factors have been associated with the development and progression of SHPT but the presence of skeletal or calcemic resistance to the action of PTH in CKD has often gone unnoticed. The term hyporesponsiveness to PTH is currently preferred and, in this chapter, we will not only review the scientific timeline but also some of the molecular mechanisms behind. Moreover, the presence of resistance to the biological action of PTH is not unique in CKD since resistance to other hormones has also been described ("uremia as a receptor disease"). This hyporesponsiveness carries out important clinical implications since it explains, at least partially, not only the progressive nature of the pathogenesis of CKD-related PTH hypersecretion and parathyroid hyperplasia but also the increasing prevalence of adynamic bone disease in the CKD population. Therefore, we underline the importance of PTH control in all CKD stages, but not aiming to completely normalize PTH levels since a certain degree of SHPT may represent an adaptive clinical response. Future studies at the molecular level, i.e. on uremia or the recent description of the calcium-sensing receptor as a phosphate sensor, may become of great value beyond their significance to explain just the hyporesponsiveness to PTH in CKD.

Fibroblast growth factor 23 predicts carotid atherosclerosis in individuals without kidney disease. The CORDIOPREV study.

BACKGROUND: Fibroblast growth factor 23 (FGF23) is a major determinant of mineral metabolism derangements and emerges as a possible risk factor underlying the negative cardiovascular outcome in CKD patients. However, its contribution in non-CKD individuals is less clear. This cross-sectional study investigated the associations between FGF23 and mineral metabolism parameters and with carotid atherosclerosis in a population at high cardiovascular risk with preserved renal function. METHODS: We employed 939 subjects with coronary heart disease enrolled in the CORDIOPREV study (mean eGFR=93.0 ±â€¯0.7 ml/min/1.73 m2 and median FGF23=44.9 (IQR=13.1) pg/ml), in which intima-media thickness of both common carotid arteries (IMT-CC) was measured. RESULTS: Adjusted for anthropometric factors, FGF23 associated positively with creatinine, phosphate, calcium and 25(OH)-vitaminD and negatively with eGFR and calcitriol. In multivariable-adjusted models all of them were independent contributors to FGF23 levels. FGF23 showed a positive relationship with IMT-CC; both the higher third and fourth quartiles associated significantly with IMT-CC (Beta= 0.135 and 0.187, respectively) and after additional adjustment for established cardiovascular risk factors and morbidities FGF23 remained as a significant contributor to IMT-CC. Logistic regression analysis confirmed its predictive ability to differentiate patients at higher atherosclerotic risk defined by an IMT-CC≥0.7 mm (OR for FGF23 quartiles 3 and 4 vs. 1: 1.860; 95%CI 1.209-2.862 and 2.114; 95%CI 1.339-3.337, respectively). CONCLUSION: Even in the setting of a normally functioning phosphate-FGF23-calcitriol system, FGF23 independently associated with IMT-CC, a surrogate of atherosclerotic vascular dysfunction. This supports the notion of FGF23 as a predictor of cardiovascular risk independent of renal failure.

Inflammation both increases and causes resistance to FGF23 in normal and uremic rats.

Fibroblast growth factor 23 (FGF23) increases phosphorus excretion and decreases calcitriol (1,25(OH)2D) levels. FGF23 increases from early stages of renal failure. We evaluated whether strict control of phosphorus intake in renal failure prevents the increase in FGF23 and to what extent inflammation impairs regulation of FGF23. The study was performed in 5/6 nephrectomized (Nx) Wistar rats fed diets containing 0.2-1.2% phosphorus for 3 or 15 days. FGF23 levels significantly increased in all Nx groups in the short-term (3-day) experiment. However, at 15 days, FGF23 increased in all Nx rats except in those fed 0.2% phosphorus. In a second experiment, Nx rats fed low phosphorus diets (0.2 and 0.4%) for 15 days received daily intraperitoneal lipopolysaccharide (LPS) injections to induce inflammation. In these rats, FGF23 increased despite the low phosphorus diets. Thus, higher FGF23 levels were needed to maintain phosphaturia and normal serum phosphorus values. Renal Klotho expression was preserved in Nx rats on a 0.2% phosphorus diet, reduced on a 0.4% phosphorus diet, and markedly reduced in Nx rats receiving LPS. In ex vivo experiments, high phosphorus and LPS increased nuclear ß-catenin and p65-NFκB and decreased Klotho. Inhibition of inflammation and Wnt signaling activation resulted in decreased FGF23 levels and increased renal Klotho. In conclusion, strict control of phosphorus intake prevented the increase in FGF23 in renal failure, whereas inflammation independently increased FGF23 values. Decreased Klotho may explain the renal resistance to FGF23 in inflammation. These effects are likely mediated by the activation of NFkB and Wnt/ß-catenin signaling.

Serum Magnesium is associated with Carotid Atherosclerosis in patients with high cardiovascular risk (CORDIOPREV Study).

This study aimed to ascertain whether there is an independent association between serum magnesium (Mg) and the Carotid Intima-Media Thickness (IMT-CC), a well-accepted atherosclerotic-biomarker surrogate of cardiovascular disease (CVD), in a population with high cardiovascular risk. Serum Mg and traditional atherosclerotic risk factors were recorded in 939 patients (mean age, 59.6 ± 0.3 years, 83.2% men) with coronary heart disease (CHD) enrolled in the CORDIOPREV trial. Serum Mg strongly associated with IMT-CC. Before adjusting for potential confounding factors, IMT-CC decreased by 0.111 ± 0.011 mm per mg/dl increase in serum Mg (p < 0.001). After adjustment, the effect of Mg did not appear mediated through factors related to glucose metabolism, the lipid profile or the mineral metabolism and renal function. Multivariate models showed the lower Mg levels (quartile 1) as a strong independent factor contributing to IMT-CC along with age, sex, SBP, HDL-C, and diuretic use. Logistic regression analysis confirmed the predictive ability of serum Mg to differentiate patients at higher atherosclerotic risk as defined by an IMT-CC ≥ 1.0 mm, yielding a OR for the lower quartile of 10.623 (95%CI 2.311-48.845; P = 0.002) and a ROC-derived cutoff of 1.61 mg/dl. Therefore, our findings outline low serum magnesium as a possible independent risk factor for carotid atherosclerosis.

Calcimimetics maintain bone turnover in uremic rats despite the concomitant decrease in parathyroid hormone concentration.

Calcimimetics decrease parathyroid hormone (PTH) secretion in patients with secondary hyperparathyroidism. The decrease in PTH should cause a reduction in bone turnover; however, the direct effect of calcimimetics on bone cells, which express the calcium-sensing receptor (CaSR), has not been defined. In this study, we evaluated the direct bone effects of CaSR activation by a calcimimetic (AMG 641) in vitro and in vivo. To create a PTH "clamp," total parathyroidectomy was performed in rats with and without uremia induced by 5/6 nephrectomy, followed by a continuous subcutaneous infusion of PTH. Animals were then treated with either the calcimimetic or vehicle. Calcimimetic administration increased osteoblast number and osteoid volume in normal rats under a PTH clamp. In uremic rats, the elevated PTH concentration led to reduced bone volume and increased bone turnover, and calcimimetic administration decreased plasma PTH. In uremic rats exposed to PTH at 6-fold the usual replacement dose, calcimimetic administration increased osteoblast number, osteoid surface, and bone formation. A 9-fold higher dose of PTH caused an increase in bone turnover that was not altered by the administration of calcimimetic. In an osteosarcoma cell line, the calcimimetic induced Erk1/2 phosphorylation and the expression of osteoblast genes. The addition of a calcilytic resulted in the opposite effect. Moreover, the calcimimetic promoted the osteogenic differentiation and mineralization of human bone marrow mesenchymal stem cells in vitro. Thus, calcimimetic administration has a direct anabolic effect on bone that counteracts the decrease in PTH levels.

Dietary magnesium supplementation prevents and reverses vascular and soft tissue calcifications in uremic rats.

Although magnesium has been shown to prevent vascular calcification in vitro, controlled in vivo studies in uremic animal models are limited. To determine whether dietary magnesium supplementation protects against the development of vascular calcification, 5/6 nephrectomized Wistar rats were fed diets with different magnesium content increasing from 0.1 to 1.1%. In one study we analyzed bone specimens from rats fed 0.1%, 0.3%, and 0.6% magnesium diets, and in another study we evaluated the effect of intraperitoneal magnesium on vascular calcification in 5/6 nephrectomized rats. The effects of magnesium on established vascular calcification were also evaluated in uremic rats fed on diets with either normal (0.1%) or moderately increased magnesium (0.6%) content. The increase in dietary magnesium resulted in a marked reduction in vascular calcification, together with improved mineral metabolism and renal function. Moderately elevated dietary magnesium (0.3%), but not high dietary magnesium (0.6%), improved bone homeostasis as compared to basal dietary magnesium (0.1%). Results of our study also suggested that the protective effect of magnesium on vascular calcification was not limited to its action as an intestinal phosphate binder since magnesium administered intraperitoneally also decreased vascular calcification. Oral magnesium supplementation also reduced blood pressure in uremic rats, and in vitro medium magnesium decreased BMP-2 and p65-NF-κB in TNF-α-treated human umbilical vein endothelial cells. Finally, in uremic rats with established vascular calcification, increasing dietary magnesium from 0.1% magnesium to 0.6% reduced the mortality rate from 52% to 28%, which was associated with reduced vascular calcification. Thus, increasing dietary magnesium reduced both vascular calcification and mortality in uremic rats.

Magnesium Chloride promotes Osteogenesis through Notch signaling activation and expansion of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSC) are osteoblasts progenitors and a variety of studies suggest that they may play an important role for the health in the field of bone regeneration. Magnesium supplementation is gaining importance as adjuvant treatment to improve osteogenesis, although the mechanisms involving this process are not well understood. The objective of this study was to investigate the effects of magnesium on MSC differentiation. Here we show that in rat bone marrow MSC, magnesium chloride increases MSC proliferation in a dose-dependent manner promoting osteogenic differentiation and mineralization. These effects are reduced by 2-APB administration, an inhibitor of magnesium channel TRPM7. Of note, magnesium supplementation did not increase the canonical Wnt/ß-catenin pathway, although it promoted the activation of Notch1 signaling, which was also decreased by addition of 2-APB. Electron microscopy showed higher proliferation, organization and maturation of osteoblasts in bone decellularized scaffolds after magnesium addition. In summary, our results demonstrate that magnesium chloride enhances MSC proliferation by Notch1 signaling activation and induces osteogenic differentiation, shedding light on the understanding of the role of magnesium during bone regeneration.

Differential regulation of renal Klotho and FGFR1 in normal and uremic rats.

In renal failure, hyperphosphatemia occurs despite a marked elevation in serum fibroblast growth factor (FGF)-23. Abnormal regulation of the FGFR1-Klotho receptor complex may cause a resistance to the phosphaturic action of FGF23. The purpose of the present study was to investigate the regulation of renal Klotho and FGF receptor (FEFR)-1 in healthy and uremic rats induced by 5/6 nephrectomy. In normal rats, the infusion of rat recombinant FGF23 enhanced phosphaturia and increased renal FGFR1 expression; however, Klotho expression was reduced. Uremic rats on a high-phosphate (HP) diet presented hyperphosphatemia with marked elevation of FGF23 and an increased fractional excretion of phosphate (P) that was associated with a marked reduction of Klotho expression and an increase in FGFR1. After neutralization of FGF23 by anti-FGF23 administration, phosphaturia was still abundant, Klotho expression remained low, and the FGFR1 level was reduced. These results suggest that the expression of renal Klotho is modulated by phosphaturia, whereas the FGFR1 expression is regulated by FGF23. Calcitriol (CTR) administration prevented a decrease in renal Klotho expression. In HEK293 cells HP produced nuclear translocation of ß-catenin, together with a reduction in Klotho. Wnt/ß-catenin inhibition with Dkk-1 prevented the P-induced down-regulation of Klotho. The addition of CTR to HP medium was able to recover Klotho expression. In summary, high FGF23 levels increase FGFR1, whereas phosphaturia decreases Klotho expression through the activation of Wnt/ß-catenin pathway.-Muñoz-Castañeda, J. R., Herencia, C., Pendón-Ruiz de Mier, M. V., Rodriguez-Ortiz, M. E., Diaz-Tocados, J. M., Vergara, N., Martínez-Moreno, J. M., Salmerón, M. D., Richards, W. G., Felsenfeld, A., Kuro-O, M., Almadén, Y., Rodríguez, M. Differential regulation of renal Klotho and FGFR1 in normal and uremic rats.

High phosphate induces a pro-inflammatory response by vascular smooth muscle cells and modulation by vitamin D derivatives.

In chronic kidney disease patients, high phosphate (HP) levels are associated with cardiovascular disease, the major cause of morbidity and mortality. Since serum phosphate has been independently correlated with inflammation, the present study aimed to investigate an independent direct effect of HP as a pro-inflammatory factor in VSMCs. A possible modulatory effect of vitamin D (VitD) was also investigated. The study was performed in an in vitro model of human aortic smooth muscle cells (HASMCs). Incubation of cells in an HP (3.3 mM) medium caused an increased expression of the pro-inflammatory mediators intercellular adhesion molecule 1 (ICAM-1), interleukins (ILs) IL-1ß, IL-6, IL-8 and tumour necrosis factor α (TNF-α) (not corroborated at the protein levels for ICAM-1), as well as an increase in reactive oxygen/nitrogen species (ROS/RNS) production. This was accompanied by the activation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signalling as demonstrated by the increase in the nuclear translocation of nuclear factor κ-light-chain-enhancer of activated B cells protein 65 (p65-NF-κΒ) assessed by Western blotting and confocal microscopy. Since all these events were attenuated by an antioxidant pre-incubation with the radical scavenger Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), it is suggested that the inflammatory response is upstream mediated by the ROS/RNS-induced activation of NF-κΒ. Addition of paricalcitol (PC) 3·10-8 M to cells in HP prevented the phosphate induced ROS/RNS increase, the activation of NF-κΒ and the cytokine up-regulation. A bimodal effect was observed, however, for different calcitriol (CTR) concentrations, 10-10 and 10-12 M attenuated but 10-8 M stimulated this phosphate induced pro-oxidative and pro-inflammatory response. Therefore, these findings provide novel mechanisms whereby HP may directly favour vascular dysfunctions and new insights into the protective effects exerted by VitD derivatives.

Energy-dense diets increase FGF23, lead to phosphorus retention and promote vascular calcifications in rats.

Rats with normal renal function (Experiment 1, n = 12) and uninephrectomized (1/2Nx) rats (Experiment 2, n = 12) were fed diets with normal P (NP) and either normal (NF) or high fat (HF). Rats with intact renal function (Experiment 3, n = 12) were also fed NF or HF diets with high P (HP). Additionally, uremic (5/6Nx) rats (n = 16) were fed HP diets with NF or HF. Feeding the HF diets resulted in significant elevation of plasma FGF23 vs rats fed NF diets: Experiment 1, 593 ± 126 vs 157 ± 28 pg/ml (p < 0.01); Experiment 2, 538 ± 105 vs 250 ± 18 pg/ml (p < 0.05); Experiment 3, 971 ± 118 vs 534 ± 40 pg/ml (p < 0.01). Rats fed HF diets showed P retention and decreased renal klotho (ratio klotho/actin) vs rats fed NF diets: Experiment 1, 0.75 ± 0.06 vs 0.97 ± 0.02 (p < 0.01); Experiment 2, 0.69 ± 0.07 vs 1.12 ± 0.08 (p < 0.01); Experiment 3, 0.57 ± 0.19 vs 1.16 ± 0.15 (p < 0.05). Uremic rats fed HF diet showed more severe vascular calcification (VC) than rats fed NF diet (aortic Ca = 6.3 ± 1.4 vs 1.4 ± 0.1 mg/g tissue, p < 0.001). In conclusion, energy-rich diets increased plasma levels of FGF23, a known risk factor of cardiovascular morbidity and mortality. Even though FGF23 has major phosphaturic actions, feeding HF diets resulted in P retention, likely secondary to decreased renal klotho, and aggravated uremic VC.

Procaine Inhibits Osteo/Odontogenesis through Wnt/ß-Catenin Inactivation.

INTRODUCTION: Periodontitis is a complex pathology characterized by the loss of alveolar bone. The causes and the mechanisms that promote this bone resorption still remain unknown. The knowledge of the critical regulators involved in the alteration of alveolar bone homeostasis is of great importance for developing molecular therapies. Procaine is an anesthetic drug with demethylant properties, mainly used by dentists in oral surgeries. The inhibitor role of Wnt signaling of procaine was described in vitro in colon cancer cells. METHODS: In this work we evaluated the role of procaine (1 uM) in osteo/odontogenesis of rat bone marrow mesenchymal stem cells. Similarly, the mechanisms whereby procaine achieves these effects were also studied. RESULTS: Procaine administration led to a drastic decrease of calcium content, alkaline phosphatase activity, alizarin red staining and an increase in the expression of Matrix Gla Protein. With respect to osteo/odontogenic markers, procaine decreased early and mature osteo/odontogenic markers. In parallel, procaine inhibited canonical Wnt/ß-catenin pathway, observing a loss of nuclear ß-catenin, a decrease in Lrp5 and Frizzled 3, a significant increase of sclerostin and Gsk3ß and an increase of phosphorylated ß-catenin. The combination of osteo/odontogenic stimuli and Lithium Chloride decreased mRNA expression of Gsk3ß, recovered by Procaine. Furthermore it was proved that Procaine alone dose dependently increases the expression of Gsk3ß and ß-catenin phosphorylation. These effects of procaine were also observed on mature osteoblast. Interestingly, at this concentration of procaine no demethylant effects were observed. CONCLUSIONS: Our results demonstrated that procaine administration drastically reduced the mineralization and osteo/odontogenesis of bone marrow mesenchymal stem cells inhibiting Wnt/ß-catenin pathway through the increase of Gsk3ß expression and ß-catenin phosphorylation.

Vitamin D modulates tissue factor and protease-activated receptor 2 expression in vascular smooth muscle cells.

Clinical and epidemiologic studies reveal an association between vitamin D deficiency and increased risk of cardiovascular disease. Because vascular smooth muscle cell (VSMC)-derived tissue factor (TF) is suggested to be critical for arterial thrombosis, we investigated whether the vitamin D molecules calcitriol and paricalcitol could reduce the expression of TF induced by the proinflammatory cytokine TNF-α in human aortic VSMCs. We found that, compared with controls, incubation with TNF-α increased TF expression and procoagulant activity in a NF-κB-dependent manner, as deduced from the increased nuclear translocation of nuclear factor κ-light-chain-enhancer of activated B cells protein 65 (p65-NF-κB) and direct interaction of NF-κB to the TF promoter. This was accompanied by the up-regulation of TF signaling mediator protease-activated receptor 2 (PAR-2) expression and by the down-regulation of vitamin D receptor expression in a miR-346-dependent way. However, addition of calcitriol or paricalcitol blunted the TNF-α-induced TF expression and activity (2.01 ± 0.24 and 1.32 ± 0.14 vs. 3.02 ± 0.39 pmol/mg protein, P < 0.05), which was associated with down-regulation of NF-κB signaling and PAR-2 expression, as well as with restored levels of vitamin D receptor and enhanced expression of TF pathway inhibitor. Our data suggest that inflammation promotes a prothrombotic state through the up-regulation of TF function in VSMCs and that the beneficial cardiovascular effects of vitamin D may be partially due to decreases in TF expression and its activity in VSMCs.

Angiotensin II prevents calcification in vascular smooth muscle cells by enhancing magnesium influx.

BACKGROUND: Vascular calcification (VC) is highly prevalent in patients with chronic kidney disease (CKD). Low magnesium levels are associated with VC, and recent in vitro studies confirm a protective role of magnesium, which is mediated by its entry into the VSMCs through the Transient Receptor Potential Melastatin 7 (TRPM7) channel. The role of Angiotensin II (Ang II) on VC is still unclear. As Ang II is able to stimulate TRPM7 activity, we hypothesize that it might prevent VC. Thus, the aim of this study was to dissect the direct effect of Ang II on VC. MATERIALS AND METHODS: We worked with a model of high phosphate (HP)-induced calcification in human aortic smooth muscle cells, which resembles the CKD-related VC. RESULTS: Addition of Ang II to cells growing in HP decreased calcification, which was associated with the upregulation of the osteogenic factors BMP2, Runx2/Cbfa1, Osterix and ALP. A reduction of magnesium entry into the HP-calcifying cells was found. The treatment with Ang II avoided this reduction, which was reversed by the cotreatment with the TRPM7-inhibitor 2-APB. The protective effect of Ang II was related to AT1R-induced ERK1/2 MAPKinase activation. HP-induced calcification was also associated with the upregulation of the canonical Wnt/beta-catenin pathway, while its downregulation was related to attenuation of calcification by Ang II. CONCLUSION: As hypothesized, Ang II prevented phosphate-induced calcification in VSMCs, which appears mediated by the increase of magnesium influx and by the activation of the ERK1/2 and the inhibition of the canonical Wnt/beta-catenin signalling pathways.

Tumor-induced rickets in a child with a central giant cell granuloma: a case report.

Tumor-induced osteomalacia/rickets is a rare paraneoplastic disorder associated with a tumor-producing fibroblast growth factor 23 (FGF23). We present a child with symptoms of rickets as the first clinical sign of a central giant cell granuloma (CGCG) with high serum levels of FGF23, a hormone associated with decreased phosphate resorption. A 3-year-old boy presented with a limp and 6 months later with painless growth of the jaw. On examination gingival hypertrophy and genu varum were observed. Investigations revealed hypophosphatemia, normal 1,25 and 25 (OH) vitamin D, and high alkaline phosphatase. An MRI showed an osteolytic lesion of the maxilla. Radiographs revealed typical rachitic findings. Incisional biopsy of the tumor revealed a CGCG with mesenchymal matrix. The CGCG was initially treated with calcitonin, but the lesions continued to grow, making it necessary to perform tracheostomy and gastrostomy. One year after onset the hyperphosphaturia worsened, necessitating increasing oral phosphate supplements up to 100 mg/kg per day of elemental phosphorus. FGF23 levels were extremely high. Total removal of the tumor was impossible, and partial reduction was achieved after percutaneous computed tomography-guided radiofrequency, local instillation of triamcinolone, and oral propranolol. Compassionate use of cinacalcet was unsuccessful in preventing phosphaturia. The tumor slowly regressed after the third year of disease; phosphaturia improved, allowing the tapering of phosphate supplements, and FGF23 levels normalized. Tumor-induced osteomalacia/rickets is uncommon in children and is challenging for physicians to diagnose. It should be suspected in patients with intractable osteomalacia or rickets. A tumor should be ruled out if FGF23 levels are high.