BackgroundVascular calcification (VC) is prevalent in patients suffering from chronic kidney disease. Factors promoting calcification include abnormalities in mineral metabolism, particularly high phosphate levels. Inorganic phosphate (Pi) is a classical inducer of in vitro VC. Recently, an inverse relationship between serum magnesium concentrations and VC has been reported. The present study aimed to investigate the effects of magnesium on Pi-induced VC at the cellular level using primary HAVSMC.MethodsAlive and fixed HAVSMC were assessed during 14 days in the presence of Pi with increasing concentrations of magnesium (Mg2+) chloride. Mineralization was measured using quantification of calcium, von Kossa and alizarin red stainings. Cell viability and secretion of classical VC markers were also assessed using adequate tests. Involvement of transient receptor potential melastatin (TRPM) 7 was assessed using 2-aminoethoxy-diphenylborate (2-APB) inhibitor.ResultsCo-incubation with Mg2+ significantly decreased Pi-induced VC in live HAVSMC, no effect was found in fixed cells. At potent concentrations in Pi-induced HAVSMC, Mg2+ significantly improved cell viability and restored to basal level increased secretions of osteocalcin and matrix gla protein, whereas a decrease in osteopontin secretion was partially restored. The block of TRPM7 with 2-APB at 10−4 M led to the inefficiency of Mg2+ to prevent VC.ConclusionsIncreasing Mg2+ concentrations significantly reduced VC, improved cell viability and modulated secretion of VC markers during cell-mediated matrix mineralization clearly pointing to a cellular role for Mg2+ and 2-APB further involved TRPM7 and a potential Mg2+ entry to exert its effects. Further investigations are needed to shed light on additional cellular mechanism(s) by which Mg2+ is able to prevent VC.

No abstract

Chronic kidney disease (CKD) is associated with vascular calcifications and atherosclerosis. There is a need for novel predictors to allow earlier diagnosis of these disorders, predict disease progression, and improve assessment of treatment response. We focused on microRNAs since they are implicated in a variety of cellular functions in cardiovascular pathology. We examined changes of microRNA expression in aortas of CKD and non-CKD wild type mice and apolipoprotein E knock-out mice, respectively. Both vascular smooth muscle-specific miR-143 and miR-145 expressions were decreased in states of atherosclerosis and/or CKD or both, and the expression level of protein target Myocardin was increased. The inflammatory miR-223 was increased in more advanced stages of CKD, and specific protein targets NFI-A and GLUT-4 were dramatically decreased. Expression of miR-126 was markedly increased and expression of protein targets VCAM-1 and SDF-1 was altered during the course of CKD. The drug sevelamer, commonly used in CKD, corrected partially these changes in microRNA expression, suggesting a direct link between the observed microRNA alterations and uremic vascular toxicity. Finally, miR-126, -143 and -223 expression levels were deregulated in murine serum during the course of experimental CKD. In conclusion, these miRNAs could have role(s) in CKD vascular remodeling and may therefore represent useful targets to prevent or treat complications of CKD.

Rationale: Vascular calcification is a process similar to bone formation leading to an inappropriate deposition of calcium phosphate minerals in advanced atherosclerotic plaques. Monocyte-derived macrophages, located in atherosclerotic lesions and presenting heterogeneous phenotypes, from classical proinflammatory M1 to alternative anti-inflammatory M2 macrophages, could potentially display osteoclast-like functions.Objective: To characterize the phenotype of macrophages located in areas surrounding the calcium deposits in human atherosclerotic plaques. Methods and Results: Macrophages near calcium deposits display an alternative phenotype being both CD68and mannose receptor-positive, expressing carbonic anhydrase type II, but relatively low levels of cathepsin K. In vitro interleukin-4-polarization of human primary monocytes into macrophages results in lower expression and activity of cathepsin K compared with resting unpolarized macrophages. Moreover, interleukin-4 polarization lowers expression levels of the osteoclast transcriptional activator nuclear factor of activated T cells type c-1, associated with increased gene promoter levels of the transcriptional repression mark H3K27me3 (histone 3 lysine 27 trimethylation). Despite higher expression of the receptor activator of nuclear factor κB receptor, receptor activator of nuclear factor κB ligand/macrophage colony-stimulating factor induction of nuclear factor of activated T cells type c-1 and cathepsin K expression is defective in these macrophages because of reduced Erk/cfos-mediated downstream signaling resulting in impaired bone resorption capacity. Conclusions:

In this work, we investigated the effect of inorganic phosphate (Pi) on the differentiation of monocyte/macrophage precursors into an "osteoclastic" phenotype, and we delineated the molecular mechanisms which could be involved in this phenomenon. This was achieved by stimulating human peripheral blood monocytic cells and RAW 264.7 monocyte-macrophage precursor cells to differentiate into osteoclast-like cells in the presence of receptor activator of NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). RANKL has been previously reported to stimulate the signaling kinases ERK 1/2, p38, Akt, JNK, and the DNA-binding activity of the transcription factors AP-1 and NF-kappaB. Increase in extracellular Pi concentration (1.5-4.5 mM) dose-dependently inhibits both osteoclastic differentiation and bone resorption activity induced by RANKL and M-CSF. Pi was found to specifically inhibit the RANKL-induced JNK and Akt activation, while RANKL-induced p38 and ERK 1/2 phosphorylation were not significantly affected. Moreover, we found that Pi significantly reduced the RANKL-stimulated DNA-binding activity of NF-kappaB. The effects of Pi on osteoclast differentiation and DNA-binding activity of NF-kappaB were prevented by Foscarnet, a sodium-phosphate cotransport inhibitor, suggesting that the effects of Pi occur subsequently to its intake. These results demonstrate that Pi downregulates the differentiation of osteoclasts via a negative feedback exerted on RANK-RANKL signaling.

Our findings strongly suggest that IS not only inhibits osteoblast function but also has an inhibitory effect on OCL function and thus could affect bone remodelling in CKD patients.

Vascular dysfunction is an essential element found in many cardiovascular pathologies and in pathologies that have a cardiovascular impact such as chronic kidney disease (CKD). Alteration of vasomotricity is due to an imbalance between the production of relaxing and contracting factors. In addition to becoming a determining factor in pathophysiological alterations, vascular dysfunction constitutes the first step in the development of atherosclerosis plaques or vascular calcifications. In patients with CKD, alteration of vasomotricity tends to emerge as being a new, less conventional, risk factor. CKD is characterized by the accumulation of uremic toxins (UTs) such as phosphate, para-cresyl sulfate, indoxyl sulfate, and FGF23 and, consequently, the deleterious role of UTs on vascular dysfunction has been explored. This accumulation of UTs is associated with systemic alterations including inflammation, oxidative stress, and the decrease of nitric oxide production. The present review proposes to summarize our current knowledge of the mechanisms by which UTs induce vascular dysfunction.

BackgroundCardiovascular disease including vascular calcification (VC) remains the leading cause of death in patients suffering from chronic kidney disease (CKD). The process of VC seems likely to be a tightly regulated process where vascular smooth muscle cells are playing a key role rather than just a mere passive precipitation of calcium phosphate. Characterisation of the chemical and crystalline structure of VC was mainly led in patients or animal models with CKD. Likewise, Mg2+ was found to be protective in living cells although a potential role for Mg2+ could not be excluded on crystal formation and precipitation. In this study, the crystal formation and the role of Mg2+ were investigated in an in vitro model of primary human aortic vascular smooth muscle cells (HAVSMC) with physical techniques.Methodology/Principal FindingsIn HAVSMC incubated with increased Ca x Pi medium, only calcium phosphate apatite crystals (CPA) were detected by Micro-Fourier Transform InfraRed spectroscopy (µFTIR) and Field Effect Scanning Electron Microscope (FE — SEM) and Energy Dispersive X-ray spectrometry (EDX) at the cell layer level. Supplementation with Mg2+ did not alter the crystal composition or structure. The crystal deposition was preferentially positioned near or directly on cells as pictured by FE — SEM observations and EDX measurements. Large µFTIR maps revealed spots of CPA crystals that were associated to the cellular layout. This qualitative analysis suggests a potential beneficial effect of Mg2+ at 5 mM in noticeably reducing the number and intensities of CPA µFTIR spots.Conclusions/SignificanceFor the first time in a model of HAVSMC, induced calcification led to the formation of the sole CPA crystals. Our data seems to exclude a physicochemical role of Mg2+ in altering the CPA crystal growth, composition or structure. Furthermore, Mg2+ beneficial role in attenuating VC should be linked to an active cellular role.