FGF23 Fails to Inhibit Uremic Parathyroid Glands CANALEJO, Rocío; CANALEJO, Antonio; MARTINEZ-MORENO, Julio Manuel ...
Journal of the American Society of Nephrology,
07/2010, Letnik:
21, Številka:
7
Journal Article
Recenzirano
Odprti dostop
Fibroblast growth factor 23 (FGF23) modulates mineral metabolism by promoting phosphaturia and decreasing the production of 1,25-dihydroxyvitamin D(3). FGF23 decreases parathyroid hormone (PTH) mRNA ...and secretion, but despite a marked elevation in FGF23 in uremia, PTH production increases. Here, we investigated the effect of FGF23 on parathyroid function in normal and uremic hyperplastic parathyroid glands in rats. In normal parathyroid glands, FGF23 decreased PTH production, increased expression of both the parathyroid calcium-sensing receptor and the vitamin D receptor, and reduced cell proliferation. Furthermore, FGF23 induced phosphorylation of extracellular signal-regulated kinase 1/2, which mediates the action of FGF23. In contrast, in hyperplastic parathyroid glands, FGF23 did not reduce PTH production, did not affect expression of the calcium-sensing receptor or vitamin D receptor, and did not affect cell proliferation. In addition, FGF23 failed to activate the extracellular signal-regulated kinase 1/2-mitogen-activated protein kinase pathway in hyperplastic parathyroid glands. We observed very low expression of the FGF23 receptor 1 and the co-receptor Klotho in uremic hyperplastic parathyroid glands, which may explain the lack of response to FGF23 in this tissue. In conclusion, in hyperparathyroidism secondary to renal failure, the parathyroid cells resist the inhibitory effects of FGF23, perhaps as a result of the low expression of FGF23 receptor 1 and Klotho in this condition.
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.
Este trabajo de investigación se centrará en el estudio de las medidas que el profesorado toma en los centros para responder a la diversidad del alumnado, basándose en las adaptaciones curriculares. ...Para ello se ha entrevistado a un total de 10 profesores pertenecientes a distintos centros educativos del sur de Madrid (privados y públicos). Todos ellos tienen en sus aulas de primaria algún alumno con dificultades de aprendizaje que han precisado adaptaciones curriculares en una o varias áreas. En la entrevista realizada comentaron distintos aspectos relativos al tema de la atención a la diversidad en general y de los casos particulares que tratan en este momento.
Hyperphosphatemia is a key pathogenic factor in the development of secondary hyperparathyroidism and precludes its treatment with vitamin D. Calcimimetics are therapeutic drugs demonstrated to lower ...parathyroid hormone (PTH) levels through an increase in the intracellular calcium of parathyroid cells. The mechanism by which high phosphate levels stimulate PTH secretion is related to its ability to prevent the elevation of intracellular calcium. The aim of this study was to assess whether calcimimetics are able to normalize the phosphate-induced stimulation of PTH secretion.
In vivo experiments studied PTH-calcium curves, and were carried out by hypocalcemic or hypercalcemic clamp, in normal rats and those with hyperphosphatemic renal failure treated with the calcimimetic NPS R-568. For in vitro studies, parathyroid glands from normal rats were incubated in normal (1 mM) and high (4 mM) phosphate media with calcimimetic.
PTH-Ca curves showed that the calcimimetics produced a marked reduction in PTH secretion in both the hyperphosphatemic and control rats; maximal suppression of PTH was achieved with calcium of 0.9 mM vs. 0.7 mM, respectively. No effect was observed with calcium 0.6 mM. In vitro experiments showed that the addition of calcimimetic to medium with high phosphate concentration reduced PTH to values similar to those obtained from glands incubated in normal phosphate concentration.
Calcimimetics overcome the stimulatory effect of high phosphate on PTH secretion in vivo and in vitro. Thus, calcimimetics should be effective in patients with secondary hyperparathyroidism whose phosphorus levels would contraindicate vitamin D treatment alone.
To investigate whether the effect of the calcimimetic AMG 641 and calcitriol on CaSR and VDR expression could be separated from their ability to reduce parathyroid cell proliferation, five-sixth ...nephrectomized (5/6 Nx) rats received vehicle, AMG 641, calcitriol, or AMG 641+calcitriol either daily for 13 days (long-term protocol) or in a single dose (short-term protocol). In the long-term protocol, AMG 641, calcitriol, and their combination significantly reduced the percentage of proliferating parathyroid cells. Proliferation was uncontrolled in the short-term protocol. A significant increase in CaSR mRNA (% vs. beta-actin) was detected in rats treated with both calcitriol (1.60 +/- 0.30) and AMG 641 (1.66 +/- 0.25) for 13 days (P = 0.01 vs. 5/6 Nx+vehicle, 0.89 +/- 0.09); and there was a further increase when both drugs were administered simultaneously (2.46 +/- 0.33). In the short-term protocol, only rats receiving AMG 641 alone (2.01 +/- 0.33, P < 0.001) showed increased expression of CaSR mRNA, whereas the combination (1.81 +/- 0.20) produced no additional benefit. AMG 641 also increased CaSR mRNA expression in vitro. Changes in VDR mRNA paralleled those of CaSR mRNA. In the long-term treatment, both AMG 641 (0.87 +/- 0.14) and calcitriol (0.99 +/- 0.12) increased VDR mRNA (P < 0.05 vs. 5/6 Nx+vehicle, 0.49 +/- 0.10), and the increase was more accentuated when the drugs were combined (1.49 +/- 0.45). In the short-term protocol, only treatment with AMG 641, alone (1.52 +/- 0.41) or combined with calcitriol (1.86 +/- 0.24), increased VDR mRNA. In conclusion, our results demonstrate an acute increase in CaSR mRNA and VDR mRNA in the parathyroid glands of uremic rats treated with AMG 641, in which cell proliferation was uncontrolled, thus supporting a direct effect of calcimimetics on CaSR and VDR expression by hyperplastic parathyroid cells.
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.
Este trabajo de investigación se centrará en el estudio de las medidas que el profesorado toma en los centros para responder a la diversidad del alumnado, basándose en las adaptaciones curriculares. ...Para ello se ha entrevistado a un total de 10 profesores pertenecientes a distintos centros educativos del sur de Madrid (privados y públicos). Todos ellos tienen en sus aulas de primaria algún alumno con dificultades de aprendizaje que han precisado adaptaciones curriculares en una o varias áreas. En la entrevista realizada comentaron distintos aspectos relativos al tema de la atención a la diversidad en general y de los casos particulares que tratan en este momento.
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/β-catenin signaling was evidenced by the translocation of β-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 β-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 β-catenin and the expression of its target genes. The role of Wnt/β-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/β-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/β-catenin signaling pathways.
Background. Many experimental studies have demonstrated that parathyroid cell proliferation is induced by uremia and further aggravated by hypocalcemia, phosphorus retention and vitamin D deficiency. ...However, these factors may also promote parathyroid growth without uremia. In the present study, we examined the onset and progression of parathyroid hyperplasia regardless of the uremic setting, a situation that might occur soon during the early renal disease. Thus, the novelty of this work resides in the close examination of the time course for the expected changes in proliferation rates and their association with parathyroid hormone (PTH) release in normal rats under the physiological demands of a high-phosphate diet (HPD) or a low-calcium diet (LCD). Methods. We evaluated the functional response of the parathyroid glands in normal rats to different physiological demands an HPD 0.6% Ca, 1.2% P) and LCD 0.2% Ca, 0.6% P) and compared it with that of uremic rats. Furthermore, we also evaluated the time course for the reversal of high-P and low-Ca-induced parathyroid cell growth and PTH upon normalization of dietary Ca and P intake (0.6% Ca, 0.6% P). Proliferation was measured by flow cytometry and calcium receptor (CaR) and vitamin D receptor (VDR) expression were assessed by qRT–PCR. Results. The pattern in the development of parathyroid hyperplasia by the two dietary models was different. The HPD produced a stronger stimulus than the number of proliferating cells doubled after only 1 day, while the LCD required 5 days to induce an increase; the elevated calcitriol might be a mitigating factor. The increase in cell proliferation was accompanied by a transient down-regulation of VDR expression (higher in the HPD); the expression of CaR was not affected by either diet. Cell proliferation and VDR mRNA levels were restored to control values by Day 15; it is as though the gland had attained a sufficient level of hyperplasia to respond to the PTH challenge. Compared to normal rats, the response of uremic rats to the HPD showed sustained and much higher rates of PTH secretion and cell proliferation and sustained down-regulation of both VDR mRNA and CaR mRNA. Finally, the recovery from the HPD or LCD to a control diet resulted in a rapid restoration of PTH values (1 to 2 days), but the reduction in cell proliferation was delayed (3 to 5 days). Conclusions. Regardless of uremia, a physiological demand to increase the PTH secretion driven either by a high P or a low Ca intake is able to induce a different pattern of parathyroid hyperplasia, which might be aggravated by the down-regulation of VDR expression. The recovery from the HPD or LCD to a control diet results in a more rapid reduction in PTH than in cell proliferation.
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^sup -8^M (HP + CTR) or paricalcitol 3.10^sup -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/β-catenin signaling was evidenced by the translocation of β-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 β-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 β-catenin and the expression of its target genes. The role of Wnt/β-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/β-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/β-catenin signaling pathways. PUBLICATION ABSTRACT
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