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.

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.

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.

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.

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.

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.

Correction of hypocalcemia allows optimal recruitment of FGF-23-dependent phosphaturic mechanisms in acute hyperphosphatemia post-phosphate enema.

Both parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) are phosphaturic hormones. These hormones should increase in response to phosphate excess. However, they also regulate serum calcium; PTH increases serum calcium concentration and FGF23 suppresses renal production of calcitriol, favoring hypocalcemia. We report the case of an 83-year-old woman with hyperphosphatemia and hypocalcemia resulting from phosphate-containing enemas. PTH and calcitriol increased in response to hypocalcemia, and FGF23 increased in response to hyperphosphatemia. Unexpectedly, peak FGF23 did not coincide with peak serum phosphate. Rather, peak FG23 was observed only after severe hypocalcemia was partially corrected with exogenous calcium administration, even though serum phosphate had been already decreasing for 32 h. Correction of severe hypocalcemia was thus associated with peak FGF23 values and with a precipitous decrease in PTH. Peak FGF23 was followed by an accelerated decrease in serum phosphate and significant phosphaturia. This clinical report is consistent with experimental data in rats showing a blunted FGF23 response to high phosphate in the presence of severe hypocalcemia. Thus, complementary experimental and clinical data suggest that partial correction of severe hypocalcemia is required for optimal FGF23-mediated phosphaturia, which takes place despite correction of PTH levels. We believe this the first human report suggesting blunting of the FGF23 response to high phosphate by severe hypocalcemia.

In vascular smooth muscle cells paricalcitol prevents phosphate-induced Wnt/ß-catenin activation.

The present study investigates the differential effect of two vitamin D receptor agonists, calcitriol and paricalcitol, on human aortic smooth muscle cells calcification in vitro. Human vascular smooth muscle cells were incubated in a high phosphate (HP) medium alone or supplemented with either calcitriol 10(-8)M (HP + CTR) or paricalcitol 3·10(-8) M (HP + PC). HP medium induced calcification, which was associated with the upregulation of mRNA expression of osteogenic factors such as bone morphogenetic protein 2 (BMP2), Runx2/Cbfa1, Msx2, and osteocalcin. In these cells, activation of Wnt/ß-catenin signaling was evidenced by the translocation of ß-catenin into the nucleus and the increase in the expression of direct target genes as cyclin D1, axin 2, and VCAN/versican. Addition of calcitriol to HP medium (HP + CTR) further increased calcification and also enhanced the expression of osteogenic factors together with a significant elevation of nuclear ß-catenin levels and the expression of cyclin D1, axin 2, and VCAN. By contrast, the addition of paricalcitol (HP + PC) not only reduced calcification but also downregulated the expression of BMP2 and other osteoblastic phenotype markers as well as the levels of nuclear ß-catenin and the expression of its target genes. The role of Wnt/ß-catenin on phosphate- and calcitriol-induced calcification was further demonstrated by the inhibition of calcification after addition of Dickkopf-related protein 1 (DKK-1), a specific natural antagonist of the Wnt/ß-catenin signaling pathway. In conclusion, the differential effect of calcitriol and paricalcitol on vascular calcification appears to be mediated by a distinct regulation of the BMP and Wnt/ß-catenin signaling pathways.

Direct and indirect effects of parathyroid hormone on circulating levels of fibroblast growth factor 23 in vivo.

Fibroblastic growth factor 23 (FGF23) is a bone-derived hormone that has a pivotal role in the pathogenesis of mineral disorders in chronic kidney disease. To study the effect of parathyroid hormone (PTH) on FGF23, rats were parathyroidectomized for a week and then implanted with constant-delivery infusion pumps to provide vehicle, a physiological, or a threefold supraphysiological dose of parathyroid hormone. Parathyroidectomy resulted in a significant decrease in blood ionized calcium, FGF23, and calcitriol along with an increase in phosphorus concentrations. PTH replacement produced a dose-dependent increase in ionized calcium and FGF23 with decreased phosphorus. Calcitriol was also increased but there was no dose effect of PTH treatment. To maintain normal plasma calcitriol levels, two additional groups of parathyroidectomized rats were given calcitriol and temporarily treated with vehicle or the supraphysiological dose of PTH. FGF23 was significantly increased by calcitriol in the vehicle-treated rats but was not further increased above that in rats given the supraphysiological dose of PTH in the absence of calcitriol. Klotho expression in the kidney decreased after parathyroidectomy but was restored by hormone supplementation. Hence, our results show a direct and an indirect effect of PTH on FGF23 secretion, the latter through changes in calcitriol concentrations.

The calcimimetic AMG 641 accelerates regression of extraosseous calcification in uremic rats.

The purpose of the present study was to test the hypothesis that extraskeletal calcification regresses in uremic rats after reduction in phosphorus intake and treatment with calcimimetics. Extraosseous calcification was induced in five to six nephrectomized rats fed a high-phosphorus (1.2%) diet who received calcitriol (80 ng/kg ip) every other day for a period of 14 days. Next, dietary phosphorus was reduced to 0.6%, and rats were treated with vehicle (n = 20), calcitriol [80 ng/kg ip/48 h (n = 20)], or the calcimimetic AMG 641 [1.5 mg/kg sc/48 h (n = 20)]. Aortic and soft-tissue calcium and phosphorus content was evaluated after 14 and 28 days. At 28 days, reduction of phosphorus intake resulted in a significant decrease in tissue mineral content in vehicle- and AMG 641-treated rats but not in rats receiving calcitriol. Aortic calcium and phosphorus was lower in rats treated with AMG 641 (96.7 +/- 26.4 mg/g) than in rats receiving vehicle (178.3 +/- 38.6 mg/g). An infiltrate of phagocytic cells expressing the calcium-sensing receptor was identified in areas surrounding foci of calcification. Additional studies in parathyroidectomized rats demonstrated that AMG 641 increased the urinary excretion of calcium (6.2 +/- 0.6 vs. 3.1 +/- 0.5 mg/day, vehicle) (P < 0.001). In conclusion, experimentally induced extraosseous calcification in uremic rats can be partially resolved by reducing phosphorus intake; the addition of calcimimetics may accelerate the regression process through mechanisms potentially involving a direct stimulatory effect on mineral phagocytic cells plus an increase in urinary calcium excretion.

Dynamics of PTH secretion in hemodialysis patients as determined by the intact and whole PTH assays.

BACKGROUND: Renal hyperparathyroidism is assessed by measurement of parathyroid hormone (PTH) levels. The intact PTH assay (I-PTH) not only reacts with 1-84 PTH but also with large, truncated fragments of non-1-84 PTH. Because the whole PTH assay (W-PTH) is specific for 1-84 PTH, non-1-84 PTH is determined by subtracting W-PTH from I-PTH values. These large circulating PTH fragments may exert a hypocalcemic effect by contributing to skeletal resistance to 1-84 PTH. METHODS: The dynamic secretion of both 1-84 PTH and non-1-84 PTH was evaluated during the induction of hypo- and hypercalcemia in eight hemodialysis patients. RESULTS: The basal ionized calcium concentration was 1.23 +/- 0.03 mmol/L at which time I-PTH, W-PTH, and non-1-84 PTH values were 276 +/- 78 pg/mL, 164 +/- 48 pg/mL, and 102 +/- 28 pg/mL, respectively. The induction of hypo- and hypercalcemic changes resulted in a sigmoidal response for all three PTH moieties, I-PTH, W-PTH, and non-1-84 PTH. During hypocalcemia, maximal values of W-PTH were greater than those of non-1-84 PTH. But during hypercalcemia, minimal values of W-PTH and non-1-84 PTH were similar. Neither the set points nor the basal/maximal ratios for W-PTH, I-PTH, and non-1-84 PTH were different. At the baseline ionized calcium concentration, the W-PTH (1-84 PTH)/non-1-84 PTH ratio was 1.53 +/- 0.15. Changes in ionized calcium resulted in a sigmoidal relationship with hypocalcemia, increasing this ratio to a maximum of 2.01 +/- 0.30 and hypercalcemia decreasing this ratio to a minimum of 1.18 +/- 0.15 (P < 0.01 vs baseline for both hypo- and hypercalcemia). CONCLUSION: Although acute changes in serum calcium produce similar secretory responses in 1-84 PTH and non-1-84 PTH, the secretory responses are not proportional for these PTH moieties. Changes in the serum calcium concentration modulate the ratio of 1-84 PTH/non-1-84 PTH in a sigmoidal pattern with hypocalcemia maximizing this ratio. Whether changes in the 1-84 PTH/non-1-84 PTH ratio specifically modulate the calcemic action and other biologic effects of 1-84 PTH remain to be determined.