Mirjam E. Peter

Magnesium reduces calcification in bovine vascular smooth muscle cells in a dose-dependent manner

Background. Vascular calcification (VC), mainly due to elevated phosphate levels, is one major problem in patients suffering from chronic kidney disease. In clinical studies, an inverse relationship between serum magnesium and VC has been reported. However, there is only few information about the influence of magnesium on calcification on a cellular level available. Therefore, we investigated the effect of magnesium on calcification induced by β-glycerophosphate (BGP) in bovine vascular smooth muscle cells (BVSMCs). Methods. BVSMCs were incubated with calcification media for 14 days while simultaneously increasing the magnesium concentration. Calcium deposition, transdifferentiation of cells and apoptosis were measured applying quantification of calcium, von Kossa and Alizarin red staining, real-time reverse transcription–polymerase chain reaction and annexin V staining, respectively. Results. Calcium deposition in the cells dramatically increased with addition of BGP and could be mostly prevented by co-incubation with magnesium. Higher magnesium levels led to inhibition of BGP-induced alkaline phosphatase activity as well as to a decreased expression of genes associated with the process of transdifferentiation of BVSMCs into osteoblast-like cells. Furthermore, estimated calcium entry into the cells decreased with increasing magnesium concentrations in the media. In addition, higher magnesium concentrations prevented cell damage (apoptosis) induced by BGP as well as progression of already established calcification. Conclusions. Higher magnesium levels prevented BVSMC calcification, inhibited expression of osteogenic proteins, apoptosis and further progression of already established calcification. Thus, magnesium is influencing molecular processes associated with VC and may have the potential to play a role for VC also in clinical situations.

Relationship between Magnesium and Clinical Biomarkers on Inhibition of Vascular Calcification

Background: Arteriosclerosis and cardiovascular disease are strongly associated with vascular calcification. Hyperphosphatemia is an essential risk factor for increased vascular calcification. End-stage renal disease (ESRD) patients could serve as an in vivo model for accelerated calcification. This study focuses on the most likely protective effects of magnesium ion (Mg2+) on phosphate-induced vascular calcification ex vivo/in vitro. Furthermore, plasma Mg2+ concentrations of ESRD and healthy controls were investigated for association with surrogate parameters of vascular calcification in vivo. Methods: Aortic segments of male Wistar-Kyoto rats were incubated and the phosphate concentration of the medium was elevated. The aortic segments were incubated in the absence and presence of MgCl2; tissue calcification was quantified by different methods. Serum Mg2+ concentrations of patients with chronic kidney disease (CKD stage 5; ESRD) and patients without CKD (controls) were associated with carotid intima media thickness (IMT) and aortic pulse wave velocity (PWV) as surrogate parameter for arteriosclerosis and arterial stiffening. Results: Incubation of aortic segments in the presence of β-glycerophosphate and NaH2PO4 caused an increased tissue Ca2+ deposition compared to control conditions. This increased amount of Ca2+ in the aortic rings was significantly decreased in the presence of Mg2+. In CKD patients, but not in controls, magnesium serum concentration was associated with the IMT of the carotid arteries. In addition, CKD patients with higher magnesium serum concentration had a significantly lower PWV. Discussion and Conclusion: Elevated phosphate concentrations in the culture media induce ex vivo/in vitro medial calcification in intact rat aortic rings in the presence of alkaline phosphatase. Mg2+ ions reduced ex vivo/in vitro vascular calcification despite increased phosphate concentration. This hypothesis is additionally based on the fact that CKD patients with high Mg2 serum levels had significantly lower IMT and PWV values, which may result in a lower risk for cardiovascular events and mortality in these patients. Therefore, Mg2+ supplementation may be an option for treatment and prevention of vascular calcification resulting in a reduction of cardiovascular events in CKD patients.

Magnesium Inhibits Wnt/β-Catenin Activity and Reverses the Osteogenic Transformation of Vascular Smooth Muscle Cells

Magnesium reduces vascular smooth muscle cell (VSMC) calcification in vitro but the mechanism has not been revealed so far. This work used only slightly increased magnesium levels and aimed at determining: a) whether inhibition of magnesium transport into the cell influences VSMC calcification, b) whether Wnt/β-catenin signaling, a key mediator of osteogenic differentiation, is modified by magnesium and c) whether magnesium can influence already established vascular calcification. Human VSMC incubated with high phosphate (3.3 mM) and moderately elevated magnesium (1.4 mM) significantly reduced VSMC calcification and expression of the osteogenic transcription factors Cbfa-1 and osterix, and up-regulated expression of the natural calcification inhibitors matrix Gla protein (MGP) and osteoprotegerin (OPG). The protective effects of magnesium on calcification and expression of osteogenic markers were no longer observed in VSMC cultured with an inhibitor of cellular magnesium transport (2-aminoethoxy-diphenylborate [2-APB]). High phosphate induced activation of Wnt/β-catenin pathway as demonstrated by the translocation of β-catenin into the nucleus, increased expression of the frizzled-3 gene, and downregulation of Dkk-1 gene, a specific antagonist of the Wnt/β-catenin signaling pathway. The addition of magnesium however inhibited phosphate-induced activation of Wnt/β-catenin signaling pathway. Furthermore, TRPM7 silencing using siRNA resulted in activation of Wnt/β-catenin signaling pathway. Additional experiments were performed to test the ability of magnesium to halt the progression of already established VSMC calcification in vitro. The delayed addition of magnesium decreased calcium content, down-regulated Cbfa-1 and osterix and up-regulated MGP and OPG, when compared with a control group. This effect was not observed when 2-APB was added. In conclusion, magnesium transport through the cell membrane is important to inhibit VSMC calcification in vitro. Inhibition of Wnt/β-catenin by magnesium is one potential intracellular mechanism by which this anti-calcifying effect is achieved.

Magnesium modulates parathyroid hormone secretion and upregulates parathyroid receptor expression at moderately low calcium concentration

Background The interest on magnesium (Mg) has grown since clinical studies have shown the efficacy of Mg-containing phosphate binders. However, some concern has arisen for the potential effect of increased serum Mg on parathyroid hormone (PTH) secretion. Our objective was to evaluate the direct effect of Mg in the regulation of the parathyroid function; specifically, PTH secretion and the expression of parathyroid cell receptors: CaR, the vitamin D receptor (VDR) and FGFR1/Klotho. Methods The work was performed in vitro by incubating intact rat parathyroid glands in different calcium (Ca) and Mg concentrations. Results Increasing Mg concentrations from 0.5 to 2 mM produced a left shift of PTH–Ca curves. With Mg 5 mM, the secretory response was practically abolished. Mg was able to reduce PTH only if parathyroid glands were exposed to moderately low Ca concentrations; with normal–high Ca concentrations, the effect of Mg on PTH inhibition was minor or absent. After 6-h incubation at a Ca concentration of 1.0 mM, the expression of parathyroid CaR, VDR, FGFR1 and Klotho (at mRNA and protein levels) was increased with a Mg concentration of 2.0 when compared with 0.5 mM. Conclusions Mg reduces PTH secretion mainly when a moderate low calcium concentration is present; Mg also modulates parathyroid glands function through upregulation of the key cellular receptors CaR, VDR and FGF23/Klotho system.

Effect of a magnesium-based phosphate binder on medial calcification in a rat model of uremia

Calcium-based phosphate binders are used to control hyperphosphatemia; however, they promote hypercalcemia and may accelerate aortic calcification. Here we compared the effect of a phosphate binder containing calcium acetate and magnesium carbonate (CaMg) to that of sevelamer carbonate on the development of medial calcification in rats with chronic renal failure induced by an adenine diet for 4 weeks. After 1 week, rats with chronic renal failure were treated with vehicle, 375 or 750 mg/kg CaMg, or 750 mg/kg sevelamer by daily gavage for 5 weeks. Renal function was significantly impaired in all groups. Vehicle-treated rats with chronic renal failure developed severe hyperphosphatemia, but this was controlled in treated groups, particularly by CaMg. Neither CaMg nor sevelamer increased serum calcium ion levels. Induction of chronic renal failure significantly increased serum PTH, dose-dependently prevented by CaMg but not sevelamer. The aortic calcium content was significantly reduced by CaMg but not by sevelamer. The percent calcified area of the aorta was significantly lower than vehicle-treated animals for all three groups. The presence of aortic calcification was associated with increased sox9, bmp-2, and matrix gla protein expression, but this did not differ in the treatment groups. Calcium content in the carotid artery was lower with sevelamer than with CaMg but that in the femoral artery did not differ between groups. Thus, treatment with either CaMg or sevelamer effectively controlled serum phosphate levels in CRF rats and reduced aortic calcification.

Influence of Bicarbonate/Low-GDP Peritoneal Dialysis Fluid (Bicavera) on In Vitro and Ex Vivo Epithelial-to-Mesenchymal Transition of Mesothelial Cells

♦ Background: Peritoneal membrane damage induced by peritoneal dialysis (PD) is largely associated with epithelial-to-mesenchymal transition (EMT) of mesothelial cells (MCs), which is believed to be a result mainly of the glucose degradation products (GDPs) present in PD solutions. ♦ Objectives: This study investigated the impact of bicarbonate-buffered, low-GDP PD solution (BicaVera: Fresenius Medical Care, Bad Homburg, Germany) on EMT of MCs in vitro and ex vivo. ♦ Methods: In vitro studies: Omentum-derived MCs were incubated with lactate-buffered standard PD fluid or BicaVera fluid diluted 1:1 with culture medium. Ex vivo studies: From 31 patients randomly distributed to either standard or BicaVera solution and followed for 24 months, effluents were collected every 6 months for determination of EMT markers in effluent MCs. ♦ Results: Culturing of MCs with standard fluid in vitro resulted in morphology change to a non-epithelioid shape, with downregulation of E-cadherin (indicative of EMT) and strong induction of vascular endothelial growth factor (VEGF) expression. By contrast, in vitro exposure of MCs to bicarbonate/low-GDP solution had less impact on both EMT parameters. Ex vivo studies partially confirmed the foregoing results. The BicaVera group, with a higher prevalence of the non-epithelioid MC phenotype at baseline (for unknown reasons), showed a clear and significant trend to gain and maintain an epithelioid phenotype at medium- and longer-term and to show fewer fibrogenic characteristics. By contrast, the standard solution group demonstrated a progressive and significantly higher presence of the non-epithelioid phenotype. Compared with effluent MCs having an epithelioid phenotype, MCs with non-epithelioid morphology showed significantly lower levels of E-cadherin and greater levels of fibronectin and VEGF. In comparing the BicaVera and standard solution groups, MCs from the standard solution group showed significantly higher secretion of interleukin 8 and lower secretion of collagen I, but no differences in the levels of other EMT-associated molecules, including fibronectin, VEGF, E-cadherin, and transforming growth factor β1. Peritonitis incidence was similar in both groups. Functionally, the use of BicaVera fluid was associated with higher transport of small molecules and lower ultrafiltration capacity. ♦ Conclusions: Effluent MCs grown ex vivo from patients treated with bicarbonate/low-GDP BicaVera fluid showed a trend to acquire an epithelial phenotype, with lower production of proinflammatory cytokines and chemokines (such as interleukin 8) than was seen with MCs from patients treated with a lactate-buffered standard PD solution.

Removal of protein-bound, hydrophobic uremic toxins by a combined fractionated plasma separation and adsorption technique.

Protein-bound uremic toxins, such as phenylacetic acid, indoxyl sulfate, and p-cresyl sulfate, contribute substantially to the progression of chronic kidney disease (CKD) and cardiovascular disease (CVD). However, based on their protein binding, these hydrophobic uremic toxins are poorly cleared during conventional dialysis and thus accumulate in CKD-5D patients. Therefore, we investigated whether hydrophobic and cationic adsorbers are more effective for removal of protein-bound, hydrophobic uremic toxins than conventional high-flux hemodialyzer. Five CKD-5D patients were treated using the fractionated plasma separation, adsorption, and dialysis (FPAD) system for 5 h. A control group of five CKD patients was treated with conventional high-flux hemodialysis. Plasma concentrations of phenylacetic acid, indoxyl sulfate, and p-cresyl sulfate were measured. Removal rates of FPAD treatment in comparison to conventional high-flux hemodialysis were increased by 130% for phenylacetic acid, 187% for indoxyl sulfate, and 127% for p-cresol. FPAD treatment was tolerated well in terms of clinically relevant biochemical parameters. However, patients suffered from mild nausea 2 h after the start of the treatment, which persisted until the end of treatment. Due to the high impact of protein-bound, hydrophobic uremic toxins on progression of CKD and CVD in CKD-5D patients, the use of an adsorber in combination with dialysis membranes may be a new therapeutic option to increase the removal rate of these uremic toxins. However, larger, long-term prospective clinical trials are needed to demonstrate the impact on clinical outcome.