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‐1β 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‐1β, 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.
Periodontitis is a complex pathology characterized by the loss of alveolar bone. The causes and the mechanisms that promote this bone resorption still remain unknown. The knowledge of the critical ...regulators involved in the alteration of alveolar bone homeostasis is of great importance for developing molecular therapies. Procaine is an anesthetic drug with demethylant properties, mainly used by dentists in oral surgeries. The inhibitor role of Wnt signaling of procaine was described in vitro in colon cancer cells.
In this work we evaluated the role of procaine (1 uM) in osteo/odontogenesis of rat bone marrow mesenchymal stem cells. Similarly, the mechanisms whereby procaine achieves these effects were also studied.
Procaine administration led to a drastic decrease of calcium content, alkaline phosphatase activity, alizarin red staining and an increase in the expression of Matrix Gla Protein. With respect to osteo/odontogenic markers, procaine decreased early and mature osteo/odontogenic markers. In parallel, procaine inhibited canonical Wnt/β-catenin pathway, observing a loss of nuclear β-catenin, a decrease in Lrp5 and Frizzled 3, a significant increase of sclerostin and Gsk3β and an increase of phosphorylated β-catenin. The combination of osteo/odontogenic stimuli and Lithium Chloride decreased mRNA expression of Gsk3β, recovered by Procaine. Furthermore it was proved that Procaine alone dose dependently increases the expression of Gsk3β and β-catenin phosphorylation. These effects of procaine were also observed on mature osteoblast. Interestingly, at this concentration of procaine no demethylant effects were observed.
Our results demonstrated that procaine administration drastically reduced the mineralization and osteo/odontogenesis of bone marrow mesenchymal stem cells inhibiting Wnt/β-catenin pathway through the increase of Gsk3β expression and β-catenin phosphorylation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Transforming growth factor-β (TGF-β) is a key cytokine during differentiation of mesenchymal stem cells (MSC) into vascular smooth muscle cells (VSMC). High phosphate induces a phenotypic ...transformation of vascular smooth muscle cells (VSMC) into osteogenic-like cells. This study was aimed to evaluate signaling pathways involved during VSMC differentiation of MSC in presence or not of high phosphate.
Our results showed that TGF-β induced nuclear translocation of Smad3 as well as the expression of vascular smooth muscle markers, such as smooth muscle alpha actin, SM22α, myocardin, and smooth muscle-myosin heavy chain. The addition of high phosphate to MSC promoted nuclear translocation of Smad1/5/8 and the activation of canonical Wnt/β-catenin in addition to an increase in BMP-2 expression, calcium deposition and alkaline phosphatase activity. The administration of TGF-β to MSC treated with high phosphate abolished all these effects by inhibiting canonical Wnt, BMP and TGF-β pathways. A similar outcome was observed in high phosphate-treated cells after the inhibition of canonical Wnt signaling with Dkk-1. Conversely, addition of both Wnt/β-catenin activators CHIR98014 and lithium chloride enhanced the effect of high phosphate on BMP-2, calcium deposition and alkaline phosphatase activity.
Full VSMC differentiation induced by TGF-β may not be achieved when extracellular phosphate levels are high. Moreover, TGF-β prevents high phosphate-induced osteogenesis by decreasing the nuclear translocation of Smad 1/5/8 and avoiding the activation of Wnt/β-catenin pathway.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Introduction Periodontitis is a complex pathology characterized by the loss of alveolar bone. The causes and the mechanisms that promote this bone resorption still remain unknown. The knowledge of ...the critical regulators involved in the alteration of alveolar bone homeostasis is of great importance for developing molecular therapies. Procaine is an anesthetic drug with demethylant properties, mainly used by dentists in oral surgeries. The inhibitor role of Wnt signaling of procaine was described in vitro in colon cancer cells. Methods In this work we evaluated the role of procaine (1 uM) in osteo/odontogenesis of rat bone marrow mesenchymal stem cells. Similarly, the mechanisms whereby procaine achieves these effects were also studied. Results Procaine administration led to a drastic decrease of calcium content, alkaline phosphatase activity, alizarin red staining and an increase in the expression of Matrix Gla Protein. With respect to osteo/odontogenic markers, procaine decreased early and mature osteo/odontogenic markers. In parallel, procaine inhibited canonical Wnt/beta-catenin pathway, observing a loss of nuclear beta-catenin, a decrease in Lrp5 and Frizzled 3, a significant increase of sclerostin and Gsk3beta and an increase of phosphorylated beta-catenin. The combination of osteo/odontogenic stimuli and Lithium Chloride decreased mRNA expression of Gsk3beta, recovered by Procaine. Furthermore it was proved that Procaine alone dose dependently increases the expression of Gsk3beta and beta-catenin phosphorylation. These effects of procaine were also observed on mature osteoblast. Interestingly, at this concentration of procaine no demethylant effects were observed. Conclusions Our results demonstrated that procaine administration drastically reduced the mineralization and osteo/odontogenesis of bone marrow mesenchymal stem cells inhibiting Wnt/beta-catenin pathway through the increase of Gsk3beta expression and beta-catenin phosphorylation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
ABSTRACT
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 highphosphate (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 β‐cateniv together with a reduction in Klotho. Wnt/β‐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/β‐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. FASEB J. 31, 3858–3867 (2017). www.fasebj.org—Muñoz‐Castañeda, Juan R., Herencia, Carmen, Pendón‐Ruiz de Mier, Maria Victoria, Rodriguez‐Ortiz, Maria Encarnación, Diaz‐Tocados, Juan M., Vergara, Noemi, Martínez‐Moreno, Julio M., Salmerón, Maria Dolores, Richards, William G., Felsenfeld, Arnold, Kuro‐O, Makoto, Almadén, Yolanda, Rodríguez, Mariano Differential regulation of renal Klotho and FGFR1 in normal and uremic rats. FASEB J. 31, 3858–3867 (2017)
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 β‐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.
In vivo studies used 5/6 nephrectomy with parathyroidectomy and replacement of exogenous PTH to eliminate the osteogenic effect of PTH comparing the bone effects of calcitriol vs calcimimetic. The osteogenesis of mesenchymal stem cells in presence of calcitriol was also evaluated. Results show calcimimetic increases bone formation, while calcitriol reduces it. In vitro, calcitriol presence reduces mineralization and osteogenesis of stem cells into osteoblasts. In this experimental study and independently of PTH, we demonstrate that calcitriol administration reduces bone formation.
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.
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.
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.
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.
The progression of chronic kidney disease (CKD) involves the development of alterations in mineral metabolism that are closely related to cardiovascular outcomes and bone disease. Hypomagnesemia is ...associated with more rapid progression of CKD and other comorbidities. Our objective was to analyze in CKD patients stages 3–4 the impact of the administration of magnesium (Mg) carbonate on bone mineral density (BMD) and hemodynamic changes associated with by vascular calcification (VC).
Patients with CKD stages 3–4 were randomized into controls (n=12) or intervention (n=7) group receiving 360mg of Mg carbonate daily during a 15-month period. Parameters related to mineral metabolism, BMD, VC, and pulse wave velocity (PWV) were evaluated.
Supplementation with Mg produced an increase in the urinary excretion of Mg while serum Mg levels remained stable and no episodes of hypermagnesemia were reported. In addition, no significant changes were found in the degree of VC assessed by Adragao index, however, both serum and urine Mg were significantly associated with a decrease in PWV, suggesting an increase in vascular compliance. Likewise, BMD did not change following treatment, but serum Mg significantly correlated with the levels of N-terminal propeptide of collagen alpha-1(I) chain (PINP), a marker of bone synthesis.
In sum, these results suggest a possible beneficial effect of Mg on vascular compliance with no detrimental effects on bone status. In addition, our results highlight the need to consider monitorization of urinary Mg status in CKD patients.
La progresión de la enfermedad renal crónica (ERC) supone el desarrollo de alteraciones del metabolismo mineral que están estrechamente relacionadas con eventos cardiovasculares y enfermedad ósea. La hipomagnesemia se asocia con una progresión más rápida de la ERC, así como con otras comorbilidades. Nuestro objetivo fue analizar en pacientes con ERC estadios 3-4 el impacto de la administración de carbonato de magnesio (Mg) sobre la densidad mineral ósea (DMO) y los cambios hemodinámicos asociados a la calcificación vascular (CV).
Los pacientes con enfermedad renal crónica estadios 3-4 se distribuyeron aleatoriamente en los grupos control (n=12) o intervención (n=7), que recibió 360 mg de carbonato de Mg diariamente durante un periodo de 15 meses. Se evaluaron parámetros relacionados con el metabolismo mineral además de la DMO, CV, y velocidad de onda de pulso (VOP).
La suplementación con Mg produjo un aumento en la excreción urinaria de Mg, mientras que los niveles séricos de Mg permanecieron estables y no se reportaron episodios de hipermagnesemia. Además, no se observaron cambios significativos en cuanto al grado de CV valorado en base al índice de Adragao. No obstante, tanto los niveles séricos como urinarios de Mg se asociaron significativamente con un descenso en la VOP, lo que sugiere un aumento en la distensibilidad vascular. De manera similar, la DMO no se modificó con la administración del tratamiento, pero los niveles séricos de Mg correlacionaron significativamente con los del propétido N-terminal del colágeno tipo I (PINP), un marcador de la síntesis ósea.
En conjunto, estos resultados sugieren un posible efecto beneficioso del Mg sobre la distensibilidad vascular sin efectos negativos a nivel óseo. Además, nuestros resultados subrayan la necesidad de considerar la monitorización del nivel de Mg urinario en pacientes con ERC.
We have previously demonstrated that the activation of rat parathyroid calcium-sensing receptor (CaSR) upregulates VDR expression in vivo (Garfia B, Cañadillas S, Luque F, Siendones E, Quesada M, ...Almadén Y, Aguilera-Tejero E, Rodríguez M. J Am Soc Nephrol 13: 2945-2952, 2002; Rodriguez ME, Almaden Y, Cañadillas S, Canalejo A, Siendones E, Lopez I, Aguilera-Tejero E, Martin D, Rodriguez M. Am J Physiol Renal Physiol 292: F1390-F1395, 2007). The present study was designed to characterize the signaling system that mediates the stimulation of parathyroid VDR gene expression by extracellular calcium. Experiments were performed in vitro by the incubation of rat parathyroid glands and in vivo with normal and uremic (Nx) rats receiving injections of CaCl(2) or EDTA to obtain hypercalcemic or hypocalcemic clamps. A high calcium concentration increased VDR expression. The addition of arachidonic acid (AA) to the low-calcium medium produced an increase in VDR mRNA of the same magnitude as that observed with high calcium. The addition of ionophore to the low-calcium medium also increased VDR mRNA expression. High calcium or the addition of AA to the low-calcium medium induced the activation (phosphorylation) of ERK1/2-MAPK. The specific inhibition of the ERK1/2-MAPK activity prevented the stimulation of VDR expression by high calcium or AA. These results suggest that AA regulates parathyroid VDR gene expression through the activation of the ERK1/2-MAPK. CaSR activation induced the activation of transcription factor Sp1, but not of NF-κB p50 or p65 or activator protein-1. The addition of AA to the low-calcium medium increased specific DNA-binding activity of Sp1 to almost the same level as high calcium, which was prevented by the inhibition of ERK1/2. Furthermore, mithramycin A (a Sp1 inhibitor) prevented the upregulation of VDR mRNA by high calcium. Finally, both sham and Nx hypercalcemic rats showed similar increased levels of VDR mRNA compared with sham and Nx hypocalcemic rats. Our results demonstrate that extracellular calcium stimulates VDR expression in parathyroid glands through the elevation of the cytosolic calcium level and the stimulation of the PLA(2)-AA-dependent ERK1/2-pathway. Furthermore, the transcription factor Sp1 mediates this effect.
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