Loïc Louvet

Magnesium prevents phosphate-induced calcification in human aortic vascular smooth muscle cells

Background Vascular 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. Methods Alive 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. Results Co-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. Conclusions Increasing 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.

High extracellular inorganic phosphate concentration inhibits RANK–RANKL signaling in osteoclast-like cells†

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‐κB 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‐κB. 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‐κB. The effects of Pi on osteoclast differentiation and DNA‐binding activity of NF‐κB 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. J. Cell. Physiol. 215: 47–54, 2008.

Daily peritoneal administration of sodium pyrophosphate in a dialysis solution prevents the development of vascular calcification in a mouse model of uraemia

BACKGROUND The high rate of cardiovascular mortality in patients with end-stage renal disease (ESRD) is a significant barrier to improved life expectancy. Unique in this population is the marked development and aggressive worsening of vascular calcification (VC). Pyrophosphate (PPi), an endogenous molecule, appears to naturally inhibit soft tissue calcification, but may be depressed in chronic kidney disease (CKD) and ESRD. Although once thought to be a promising therapeutic, PPis very short half-life in circulation curtailed earlier studies. We tested the possibility that a slow, continuous entry of PPi into the circulation and prevention of VC might be achieved by daily peritoneal dialysis (PD). METHODS Pharmacokinetic studies were first carried out in rats with renal impairment resulting from a 5/6 nephrectomy. Efficacy studies were then performed in the apolipoprotein E gene knockout mouse model overlaid with CKD. PPi was delivered by means of a permanent peritoneal catheter in a solution simulating PD, but without the timed removal of spent dialysate. von Kossas staining followed by semiquantitative morphological image processing, with separation of inside (intimal) and outside (presumed medial) lesions, was used to determine aortic root calcification. RESULTS In comparison to an intravenous bolus, delivery of PPi in a PD solution resulted in a slower, extended delivery over >4 h. Next, the efficacy studies showed that a 6-day/week PD-simulated administration of PPi resulted in a dose-dependent inhibition of aortic calcification in both intimal and medial lesions. A dose-response effect on total aortic calcification was also documented, with a full inhibition seen at the highest dose. A limited peritoneal catheter-related inflammation was observed, as expected, and included the placebo-treated control groups. This inflammatory response could have masked a lower level PPi-specific adverse effect, but none was observed. CONCLUSIONS Our findings suggest potential for PPi, administered during PD, to prevent the development of VC and to potentially extend the life of ESRD patients.

Characterisation of Calcium Phosphate Crystals on Calcified Human Aortic Vascular Smooth Muscle Cells and Potential Role of Magnesium

Background Cardiovascular 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 Findings In 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/Significance For 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.

Guanidino compounds as cause of cardiovascular damage in chronic kidney disease: an in vitro evaluation

Chronic kidney disease is considered a major cause of cardiovascular risk and non-traditional risk factors remain largely unknown. The in vitro toxicity of 10 guanidino compounds (GCs) was evaluated via a standardized approach on different cell systems of relevance in cardiovascular disease. The parameters evaluated were production of reactive oxygen species, expression of surface molecules, cell proliferation, cytotoxicity and calcification. Several GCs had a stimulatory effect on monocytes and granulocytes (SDMA, creatine and guanidinobutyric acid (GBA)). Some GCs (guandine (G), guanidinosuccinic acid (GSA) and SDMA) inhibited endothelial cell proliferation or reduced calcification in osteoblast-like human VSMC (ADMA, GSA and SDMA). Stimulation of osteoclastogenesis could be demonstrated for ADMA, G, guanidinoacetic acid and GBA in a RAW264.7 cell line. No compounds were cytotoxic to AoSMC or endothelial cells, nor influenced their viability. GCs, especially SDMA, likely contribute to cardiovascular complications in uremia, mainly those related to microinflammation and leukocyte activation.

Oxidized low density lipoprotein decreases Rankl‐induced differentiation of osteoclasts by inhibition of Rankl signaling

The role of OxLDL in the generation and progression of atherosclerosis is well admitted. In addition, it is well known that atherosclerosis is often accompanied by perturbations in bone remodeling, resulting in osteoporosis. In the current studies, the effect of Cu2+‐oxidized LDL (OxLDL) on RANKL‐induced RAW264.7 mouse monocytes‐macrophages differentiation to osteoclasts and on RANKL signaling pathway was investigated. OxLDL, within the range of 10–50 µg protein/ml, prevented RANKL‐induced generation of multinucleated osteoclast‐like cells and RANKL‐induced tartrate resistant acid phosphatase (TRAP) activity. OxLDL also prevented the RANKL‐induced phosphorylation of ERK, p38 and JNK kinases, together with the RANKL‐induced DNA binding activities of NFkappaB and NFAT transcription factors. Concomitantly, OxLDL enhanced RANKL‐induced generation of reactive oxygen species in a dose‐dependent manner. The antioxidant glutathione (GSH) prevented whereas the prooxidant compound buthionine‐sulfoximine (BSO) enhanced the effect of OxLDL on RANKL‐induced oxidative stress and RANKL‐induced differentiation. Finally, OxLDL also prevented RANKL‐induced TRAP activity and RANKL‐induced bone resorbing activity of human peripheral blood mononuclear cells. These results demonstrate that OxLDL, by generation of an intracellular oxidative stress, prevents the differentiation of osteoclasts by inhibition of RANKL signaling pathway. This might be related to the fact that atherosclerosis is accompanied by perturbations in bone and vascular remodeling, leading to osteoporosis and vascular calcification. J. Cell. Physiol. 221: 572–578, 2009.

Magnesium Attenuates Phosphate-Induced Deregulation of a MicroRNA Signature and Prevents Modulation of Smad1 and Osterix during the Course of Vascular Calcification

Vascular calcification (VC) is prevalent in patients suffering from chronic kidney disease (CKD). High phosphate levels promote VC by inducing abnormalities in mineral and bone metabolism. Previously, we demonstrated that magnesium (Mg2+) prevents inorganic phosphate- (Pi-) induced VC in human aortic vascular smooth muscle cells (HAVSMC). As microRNAs (miR) modulate gene expression, we investigated the role of miR-29b, -30b, -125b, -133a, -143, and -204 in the protective effect of Mg2+ on VC. HAVSMC were cultured in the presence of 3 mM Pi with or without 2 mM Mg2+ chloride. Total RNA was extracted after 4 h, 24 h, day 3, day 7, and day 10. miR-30b, -133a, and -143 were downregulated during the time course of Pi-induced VC, whereas the addition of Mg2+ restored (miR-30b) or improved (miR-133a, miR-143) their expression. The expression of specific targets Smad1 and Osterix was significantly increased in the presence of Pi and restored by coincubation with Mg2+. As miR-30b, miR-133a, and miR-143 are negatively regulated by Pi and restored by Mg2+ with a congruent modulation of their known targets Runx2, Smad1, and Osterix, our results provide a potential mechanistic explanation of the observed upregulation of these master switches of osteogenesis during the course of VC.

High inorganic phosphate concentration inhibits osteoclastogenesis by modulating miR-223

Chronic kidney disease-mineral and bone disorder (CKD-MBD) is a common complication of CKD, and uremic toxins have been shown to be instrumental in this process. We have previously shown that miR-223 is increased in smooth muscle cells subjected to the uremic toxin inorganic phosphate (Pi). In the present study we investigated the influence of this miRNA in osteoclastogenesis in order to elucidate its role in the course of CKD-MBD. RT-qPCR demonstrated that high Pi concentration decreased miR-223 expression in differentiated RAW 264.7 cells. Up- and down-regulation of miR-223 was performed using specific pre-miR and anti-miR-223. Differentiation of monocyte/macrophage precursors was assessed by using RAW 264.7 cells and peripheral blood mononuclear cells (PBMC). TRAP activity and bone resorption were used to measure osteoclast activity. Pi induced a marked decrease in osteoclastogenesis in RAW cells and miR-223 levels were concomitantly decreased. Anti-miR-223 treatment inhibited osteoclastogenesis in the same way as Pi. In contrast, overexpression of miR-223 triggered differentiation, as reflected by TRAP activity. We showed that miR-223 affected the expression of its target genes NFIA and RhoB, but also osteoclast marker genes and the Akt signalling pathway, which induces osteoclastogenesis. These results were confirmed by measuring bone resorption activity of human PBMC differentiated into osteoclasts. We thus demonstrate a role of miR-223 in osteoclast differentiation, with rational grounds to use deregulation of this miRNA to selectively increase osteoclast-like activity in calcified vessels of CKD-MBD. This approach could alleviate vascular calcification without altering bone structure.

Uremic toxin indoxyl sulfate inhibits human vascular smooth muscle cell proliferation.

Uremic syndrome is attributed to the progressive retention of a large number of compounds, such as indoxyl sulfate, which under physiological conditions are excreted by the kidneys. Previous in vitro studies have demonstrated that uremic indoxyl sulfate concentrations induce a weak increase in the proliferation of both rat and human vascular aortic smooth muscle cells (hVASMC) after short term exposition to the toxin (i.e. 24 h). In the present study, we evaluated indoxyl sulfate effects on the proliferation of hVASMC at three different concentrations after long‐term exposure (seven days). In contrast to previously published studies, we observed a dose‐dependent and significant inhibitory effect of this toxin on hVASMC proliferation. We also demonstrated that indoxyl sulfate inhibits epidermal growth factor‐induced hVASMC proliferation after long‐term exposure. Indoxyl sulfate effects were associated with a dose‐dependent induction of intracellular reactive oxygen species and up‐regulation of p21 and p27 protein expression. Chronic exposure to indoxyl sulfate produces a significant inhibitory effect on hVASMC proliferation. The relevance of these findings must be evaluated by further studies, particularly in an in vivo setting.

N-methyl-2-pyridone-5-carboxamide (2PY)—Major Metabolite of Nicotinamide: An Update on an Old Uremic Toxin

N-methyl-2-pyridone-5-carboxamide (2PY, a major metabolite of nicotinamide, NAM) was recently identified as a uremic toxin. Recent interventional trials using NAM to treat high levels of phosphorus in end-stage renal disease have highlighted new potential uremic toxicities of 2PY. In the context of uremia, the accumulation of 2PY could be harmful—perhaps by inhibiting poly (ADP-ribose) polymerase-1 activity. Here, we review recently published data on 2PY’s metabolism and toxicological profile.