Comparative effects of calcitriol and calcimimetic on bone health in renal insufficiency.

Calcitriol and calcimimetics are used to treat hyperparathyroidism secondary to chronic kidney disease (CKD). Calcitriol administration and the subsequent increase in serum calcium concentration decrease parathyroid hormone (PTH) levels, which should reduce bone remodeling. We have previously reported that, when maintaining a given concentration of PTH, the addition of calcimimetics is associated with an increased bone cell activity. Whether calcitriol administration affects bone cell activity while PTH is maintained constant should be evaluated in an animal model of renal osteodystrophy. The aim of the present study was to compare in CKD PTH-clamped rats the bone effects of calcitriol and calcimimetic administration. The results show that the administration of calcitriol and calcimimetic at doses that induced a similar reduction in PTH secretion produced dissimilar effects on osteoblast activity in 5/6 nephrectomized (Nx) rats with secondary hyperparathyroidism and in Nx rats with clamped PTH. Remarkably, in both rat models, the administration of calcitriol decreased osteoblastic activity, whereas calcimimetic increased bone cell activity. In vitro, calcitriol supplementation inhibited nuclear translocation of ß-catenin and reduced proliferation, osteogenesis, and mineralization in mesenchymal stem cells differentiated into osteoblasts. In conclusion, besides the action of calcitriol and calcimimetics at parathyroid level, these treatments have specific effects on bone cells that are independent of the PTH level.

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.

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.

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.

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.

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.

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.

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.

Atherosclerosis and cardiovascular disease in systemic lupus erythematosus: effects of in vivo statin treatment.

OBJECTIVE: Statins may have beneficial vascular effects in systemic lupus erythematosus (SLE) beyond their cholesterol-lowering action, although the mechanisms involved are not completely understood. We investigated potential mechanisms involved in the efficacy of fluvastatin in preventing atherothrombosis in SLE. METHODS: Eighty-five patients with SLE and 62 healthy donors were included in the study. Selected patients (n=27) received 20 mg/day fluvastatin for 1 month. Blood samples were obtained before the start and at the end of treatment. Monocytes from five patients were treated in vitro with fluvastatin. RESULTS: Increased prothrombotic and inflammatory variables were found in patients with SLE. SLE monocytes displayed altered mitochondrial membrane potential and increased oxidative stress. Correlation and association analyses demonstrated a complex interplay among autoimmunity, oxidative stress, inflammation and increased risk of atherothrombosis in SLE. Fluvastatin treatment of patients for 1 month reduced the SLE Disease Activity Index and lipid levels, oxidative status and vascular inflammation. Array studies on monocytes demonstrated differential expression in 799 genes after fluvastatin treatment. Novel target genes and pathways modulated by fluvastatin were uncovered, including gene networks involved in cholesterol and lipid metabolism, inflammation, oxidative stress and mitochondrial activity. Electron microscopy analysis showed increased density volume of mitochondria in monocytes from fluvastatin-treated patients, who also displayed higher expression of genes involved in mitochondrial biogenesis. In vitro treatment of SLE monocytes confirmed the results obtained in the in vivo study. CONCLUSIONS: Our overall data suggest that fluvastatin improves the impairment of a redox-sensitive pathway involved in processes that collectively orchestrate the pathophysiology of atherothrombosis in SLE.

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.

Cardiotrophin-1 decreases liver apoptosis through calpastatin induction.

BACKGROUND: Cardiotrophin-1 (CT1) has been used to prevent cell death in different models of liver injury in rats. D-galactosamine induces cell death in culture rat and human hepatocytes. The present study evaluated the cytoprotective effects of CT1 in an experimental model of apoptosis induced by D-galactosamine in hepatocytes. METHODS: DNA fragmentation, calpain activity and Western blots of caspase-3, calpastatin and Stat3, and Akt phosphorylation were measured. Stat3 and Akt inhibitors were used to analyze the mechanisms of action of CT1. RESULTS: CT1 caused an increase in Stat3 and Akt phosphorylation and a decrease of DNA fragmentation, calpain activity, and caspase-3 induced by D-galactosamine. The reduction of calpain activity by CT1 was associated with an increase of calpastatin (its endogenous inhibitor). The effects of CT1 were also dependent on the activation of Sta3 or Akt. CONCLUSIONS: CT1 decreases cell death through a mechanism related to Stat3 and Akt phosphorylation and activation of calpastatin in D-galactosamine-treated hepatocytes.

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.

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.

The effect of phosphate binders, calcium and lanthanum carbonate on FGF23 levels in chronic kidney disease patients.

BACKGROUND: Recent publications show that elevation of FGF23 is independently associated with progression or renal disease, left ventricular hypertrophy and cardiovascular mortality. Dietary restriction of phosphate and phosphate binders are used for control phosphate balance and elevation of serum FGF23 levels. The aim of this study is to compare the effectiveness of calcium carbonate vs. lanthanum carbonate in reducing serum FGF23 levels in Chronic Kidney Disease (CKD) patients. METHODS: 32 patients from the Nephrology outpatient clinic with CKD 4 - 5 non-dialysis were included. Patients receive a 4-month treatment period of calcium carbonate or lanthanum carbonate. Patients had normal serum calcium concentration, 25 (OH) levels >30 ng/ml and they were not on VDR activators or cinacalcet. RESULTS: As compared with calcium carbonate, patients on lanthanum carbonate had lower serum levels of FGF23 (226 ± 11 vs. 158 ± 9 pg/ml) and less urinary excretion of phosphate. No significant changes in serum calcium and PTH levels were observed in both groups. CONCLUSIONS: In conclusion, in CKD 4 - 5 patients lanthanum carbonate is effective in reducing phosphate load and FGF23 levels; this effect was not observed with calcium carbonate.

Calcium deficiency reduces circulating levels of FGF23.

Fibroblast growth factor (FGF) 23 inhibits calcitriol production, which could exacerbate calcium deficiency or hypocalcemia unless calcium itself modulates FGF23 in this setting. In Wistar rats with normal renal function fed a diet low in both calcium and vitamin D, the resulting hypocalcemia was associated with low FGF23 despite high parathyroid hormone (PTH) and high calcitriol levels. FGF23 correlated positively with calcium and negatively with PTH. Addition of high dietary phosphorus to this diet increased FGF23 except in rats with hypocalcemia despite high PTH levels. In parathyroidectomized rats, an increase in dietary calcium for 10 days increased serum calcium, with an associated increase in FGF23, decrease in calcitriol, and no change in phosphorus. Also in parathyroidectomized rats, FGF23 increased significantly 6 hours after administration of calcium gluconate. Taken together, these results suggest that hypocalcemia reduces the circulating concentrations of FGF23. This decrease in FGF23 could be a response to avoid a subsequent reduction in calcitriol, which could exacerbate hypocalcemia.

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.

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.

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.

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.