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.
Abstract Background and Aims The physiological function of sclerostin (SOST) remains unknown. It is known that SOST is produced in osteocytes and functions as an inhibitor of the Wnt/β-catenin ...pathway. Similarly, it is well-established that low levels of SOST lead to bone alterations affecting cortical bone, resulting in osteopetrosis and reduced calciuria. High levels of SOST are associated with osteoporosis. In this study, we aimed to investigate the impact of three elevated doses of SOST on cortical, trabecular, and subchondral bone, as well as its relationship with other parameters of mineral metabolism. Method Bone histomorphometry, mCT, immunohistochemistry, and analysis of mineral metabolism parameters revealed that high doses of SOST over a 14-day period led to a reduction in trabecular bone volume due to a significant increase in bone resorption through the direct activation of osteoclastogenesis. Results Bone resorption, as measured by TRAP activity, was higher in trabecular, cortical, and subchondral bone. Similarly, high doses of SOST increased the number of hypertrophic chondrocytes, consequently expanding the growth plate area. Cortically, positive TRAP staining was observed, suggesting osteocytic osteolysis and trabecularization of cortical bone. The increased bone resorption resulted in a substantial rise in urinary excretion of phosphorus and calcium, accompanied by elevated levels of FGF23 and a significant decrease in PTH. Conclusion The findings suggest that elevated levels of SOST promote bone resorption through the activation of osteoclasts and the generation of osteocytic osteolysis. The increase in calcium and phosphorus levels led to changes in mineral metabolism, indicating a close relationship between SOST and other mineral metabolism parameters.
Abstract Background and Objectives Ongoing studies are shedding light on the potential connection between FGF23 and hypertension. In experimental settings, the administration of recombinant FGF23 in ...mice has been demonstrated to induce hypertension by elevating serum sodium, along with an increase in NCC receptors in the kidney. Clinical evidences further supports pro-hypertensive effects of high circulating levels of FGF23. Our group has previously demonstrated a direct influence of FGF23 on increased arterial stiffness by inducing a phenotypic transition in vascular smooth muscle cells (VSMCs) from a contractile to a synthetic phenotype. This study aimed comprehensively to evaluate, both in vivo and in vitro, whether FGF23 directly leads to hypertension through the stimulation of intracellular calcium entry in vascular cells. Methods To ascertain whether FGF23 contributes to hypertension through a direct impact on blood vessels, recombinant FGF23 (15 µg/day) was administered for 28 days via Alzet pumps. Blood pressure was meticulously measured in these animals, and we investigated alterations in vascular remodeling and calcium channels, as well as proteins associated with vascular contraction such as Orai1, STIM1, PKD1, SERCA2a, and AGTR1. In vitro experiments included the evaluation of the effect of elevated levels of recombinant FGF23 (2 ng/ml) over 9 days on intracellular calcium entry stimulated by Ang2 (100 nM) or Thapsigargin (2.5 µM). We used the FURA method to conduct these experiments. To elucidate the in vitro pro-hypertensive mechanisms of FGF23, studies were conducted with the administration of inhibitors targeting FGF Receptors 1-3 (AZD4547, 150 nM), FGF Receptor-4 (BLU9931, 10 nM), and Erk1/2 phosphorylation (PD98059, 10 μM). Additionally, changes in Orai1, STIM1, PKD1, SERCA2a, and AGTR1 were also evaluated in these VSMCs. Results After 28 days of recombinant FGF23 treatment, no significant differences were observed in serum sodium levels, yet there was a marked increase in urinary phosphorus excretion. Systolic and diastolic blood pressure values showed a progressive significant elevation in animals exposed to high levels of FGF23. The expression of AGTR1 and PKD1 levels significantly increased after FGF23 administration for 28 days (Figure). However, Orai1 and STIM1 in the thoracic aorta exhibited no significant differences following FGF23 treatment. In VSMCs, the exposure to recombinant FGF23 for 9 days resulted in increased calcium entry upon Ang2 stimulation (Figure). This effect was markedly diminished after inhibiting FGFR1-3 receptors and Erk1/2 phosphorylation (Figure). Notably, the inhibition of FGF23 receptor 4 (FGFR4) had no impact on the calcium entry induced by FGF23 (Figure). While Orai1, STIM1, IP3R, and TRPV5 in VSMCs remained unaffected by recombinant FGF23 administration, PKD1, SERCA2, and AGTR1 levels were significantly elevated compared to control cells (Figure). The addition of Thapsigargin in a Ca2+-free culture medium to VSMCs incubated with recombinant FGF23 for 9 days led to increased Ca2+ efflux from endoplasmic reticulum. Conclusion Elevated levels of FGF23 directly enhanced calcium entry in VSMCs. This effect is mediated by FGFR1-3 receptors, Erk1/2 phosphorylation, and the upregulation of related protein to cell contraction such as SERCA2a, PKD1 or AGTR1. These modifications could be responsible of FGF23-associated hypertension in the experimental model.
Abstract
Background and Aims
Iron (Fe) deficiency significantly contributes to anemia in chronic kidney disease (CKD). High FGF23 levels, commonly found in CKD, are related to adverse outcomes in ...patients. In the normal setting, an association between Fe deficiency and FGF23 has been observed. However, it is unknown whether Fe deficiency-induced anemia might contribute to the excessive FGF23 levels in CKD. Thus, this study was intended to evaluate the interplay between Fe deficiency, CKD, and FGF23.
Method
Male Wistar rats with normal renal function received either standard (40 ppm Fe) or Fe-deficient (4 ppm Fe) diets for 6 weeks to induce anemia. Then, several normal and all the anemic rats were switched to an adenine-enriched (0.6%) diet to induce renal damage. Groups of anemic-uremic animals were treated with oral (ferric citrate, FC and ferrous sulfate, FS) or intravenous (ferric carboxymaltose, FCx) Fe at a dose of 9 mg/kg for 5 weeks. Hematological and mineral metabolism parameters were determined at sacrifice. All experimental protocols were approved by the Ethics Committee for Animal Research of the University of Córdoba.
Results
Plasma and serum biochemistries are shown in Table 1. CKD per se was associated with derangements in hematological parameters. The presence of Fe deficiency-induced anemia further decreased these parameters, being statistically significant in the case of serum Fe (P = 0.003). Iron therapy improved hematocrit, hemoglobin, and Fe levels, although statistical significance for all of them was only reached when FC was administered (P = 0.012, P = 0.038, and P = 0.002, respectively). As expected, CKD rats exhibited disturbances in mineral metabolism. Interestingly, uremic and anemic rats showed higher intact (P = 0.039) and c-terminal (P = 0.048) FGF23 levels when compared with CKD group. Despite normalization of serum Fe, intact FGF23 further increased following administration of FS (P = 0.022), and same trend was observed with all the compounds. However, cFGF23 levels remained unchanged in all the Fe-treated groups.
Conclusion
The presence of Fe-induced anemia in an experimental model of CKD is associated with further increases in the levels of both intact and c-terminal FGF23. Thus, other factors distinct than serum Fe might influence FGF23 levels. Our results support the need for investigating the precise mechanisms underlying the relationship between Fe and FGF23 to optimize the clinical management of anemia, particularly in the context of renal dysfunction.
Abstract
Background and Aims
In chronic kidney disease (CKD) patients, high serum phosphate (HP) levels are associated with the development of a cardiorenal renal syndrome type 4 (CRS4). In this ...syndrome, the heart is damaged by oxidative stress, inflammation, and fibrosis in renal tissue. In this study we have evaluated if dietary Mg supplementation could reduce CRS4.
Method
The in vivo studies were performed in 5/6Nx rats fed a high (0.9%) phosphate diet with high or low dietary content of Mg to evaluate: 1) The effect of dietary Mg supplementation (0.3%) on oxidative stress, inflammation and fibrosis of kidney and heart. 2) The contribution of hypomagnesemia (using a low Mg diet (0,03%) to the progression of CRS4 in Nx rats and normal rats as controls. 3) Whether dietary Mg supplementation (8 weeks) reduces renal and cardiac fibrosis in Nx rats with established CRS4. In vitro, we evaluated the effects of Mg (Mg2Cl) on mesangial and tubular cells and also cardiomyocytes cells expose to TGF-β.
Results
Dietary Mg supplementation (0,3%) improved renal function, decreased oxidative stress, FGF23 levels, hypertension, renal and cardiac fibrosis and recovered renal expression of Klotho. A low Mg diet increased FGF23 and renal fibrosis but a subsequent switch to dietary Mg supplementation did not significantly improve CRS4 parameters although it reduced the values of blood pressure. In HK2 and rat mesangial cells treated with TGFβ (100 ng/ml), high Mg levels (1,4- and 2,8-times basal levels in the medium) reduced the amount of pro-fibrotic proteins such as α-smooth muscle actin, fibronectin, or renin, recovering Klotho expression and decreasing Smad3 phosphorylation.
Conclusion
Dietary Mg supplementation is a useful tool to improve renal function and prevent CRS4, by reduction the progression of renal and cardiac fibrosis.
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