Antonio Canalejo

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.

FGF23 Fails to Inhibit Uremic Parathyroid Glands

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.

Regulation of Parathyroid Vitamin D Receptor Expression by Extracellular Calcium

Low extracellular calcium (Ca) stimulates parathyroid hormone (PTH) secretion and also increases the renal synthesis of calcitriol (CTR), which is known to decrease PTH production. This study began with the hypothesis that the parathyroid cell response to CTR may be modulated by extracellular Ca concentration through an effect on parathyroid cell vitamin D receptor (VDR). In the present study, rat parathyroid glands were incubated in low (0.6 mM) and high (1.5 mM) Ca concentration. The parathyroid VDRmRNA was higher in 1.5 than 0.6 mM Ca. Furthermore, this effect was not observed in incubated slices of kidney cortex and medulla, tissues which also possess both Ca and vitamin D receptors. Experiments were also performed to evaluate the effect of Ca on VDR expression in vivo. Male Wistar rats received intraperitoneal injections of CaCl(2) or a single intramuscular injection of EDTA to obtain 6 h of hypercalcemic (ionized Ca, 1.4 to 1.6 mM) or hypocalcemic (ionized Ca, 0.85 to 0.95 mM) clamp; a third group of rats was used as control. A small dose of CTR was administered to hypercalcemic rats to match the serum CTR levels of hypocalcemic rats. Parathyroid gland VDRmRNA and VDR protein were increased in hypercalcemic rats as compared with hypocalcemic rats. Increasing doses of CTR upregulated VDRmRNA and VDR only in hypercalcemic rats. Additional experiments showed that the decrease in VDR in hypocalcemic rats prevented the inhibitory effect of CTR on PTHmRNA. In conclusion, our study shows that extracellular Ca regulates VDR expression by parathyroid cells independently of CTR and that by this mechanism hypocalcemia may prevent the feedback of CTR on the parathyroids.

99mTc-sestamibi Scintigraphy and Cell Cycle in Parathyroid Glands of Secondary Hyperparathyroidism

Double-phase parathyroid MIBI (99mTc-sestamibi) was performed in 27 patients with secondary hyperparathyroidism (SPT). Focal areas of increased uptake were scored for intensity on a three-point scale. All patients underwent subtotal parathyroidectomy (SPTx), and a total of 78 glands were removed at operation. Blood was obtained from the jugular vein before and after SPTx to measure the parathyroid hormone (PTH) levels. The volume and weight of the glands were calculated. The tissue was divided, with one aliquot being used for cell cycle analysis. The nuclei were acquired by flow cytometry and analyzed using CELLEIT software. Cell viability was assessed by flow cytometry and analyzed with LYSIS II software. Positive MIBI uptake was observed in 88.8% of patients. Focal MIBI uptake of one, two, or three glands was observed in 6, 11, and 8 patients, respectively. All patients experienced an 86% decrease in PTH blood level after SPTx compared to that before excision. A correlation was found between the volume of glands and the blood levels of intact PTH (iPTH) (r= 0.5, p < 0.05). A positive correlation was observed between MIBI uptake and the iPTH levels before SPTx (p < 0.01) and between the uptake of MIBI in the parathyroid glands and the cell cycle phases; low-grade uptake correlated with the G0 phase and higher uptake with G2+S phase (r= 7, p < 0.01). No correlation was observed between MIBI uptake and the weight of the glands. MIBI scintigraphy accurately reflects the functional status of the hyperplastic parathyroid glands: Higher uptake grades correlated with the active growth phase. MIBI uptake does not reveal parathyroid enlargement; rather, it identifies the presence of hyperfunctioning autonomous glands. SPTx and total parathyroidectomy with autografting (TPTx+A) are the most widely accepted surgical approaches for patients with SPT. Reoperation for recurrence is necessary in 6% to 15% of cases. MIBI is now considered to be the radionuclide of reference for parathyroid gland scanning, although it is widely accepted that it produces poor results when trying to detect hyperplastic glands.

Calcium-Sensing Receptor Expression and Parathyroid Hormone Secretion in Hyperplastic Parathyroid Glands from Humans

In uremic patients, severe parathyroid hyperplasia is associated with reduced parathyroid calcium-sensing receptor (CaR) expression. Thus, in these patients, a high serum Ca concentration may be required to inhibit parathyroid hormone (PTH) secretion. This study compares the magnitude of reduction in CaR expression and the degree of the abnormality in Ca-regulated PTH release in vitro. A total of 50 glands from 23 hemodialysis patients with refractory hyperparathyroidism were studied. Tissue slices were incubated in vitro to evaluate (1) the PTH secretory output in a normal Ca concentration (1.25 mM) and (2) the PTH secretory response to high (1.5 mM) and low (0.6 mM) Ca concentration. Tissue aliquots were processed for determination of CaRmRNA expression. The results showed that, corrected for DNA, parathyroid tissue with lowest CaR expression secreted more PTH than that with relatively high CaR expression (146 +/- 23 versus 60 +/- 2 pg/microg DNA; P < 0.01). Furthermore, glands with low CaR expression demonstrated a blunted PTH secretory response to both the inhibitory effect of high Ca and the stimulatory effect of low Ca. The study also showed that the larger the gland, the lower the CaRmRNA expression. Thus, large parathyroid glands produce a large amount of PTH not only as a result of the increased gland size but also because the parathyroid tissue secretory output is increased. These abnormalities in PTH regulation are related to low CaR expression.

Upregulation of parathyroid VDR expression by extracellular calcium is mediated by ERK1/2-MAPK signaling pathway

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.