Maria E. Rodríguez-Ortiz, Antonio Canalejo, Carmen Herencia, Julio M. Martínez-Moreno, Alan Peralta-Ramírez, Pablo Perez-Martinez, Juan F. Navarro-González, Mariano Rodríguez, Mirjam Peter, Kristina Gundlach, Sonja Steppan, Jutta Passlick-Deetjen, Juan R. Muñoz-Castañeda, Yolanda Almaden, Magnesium modulates parathyroid hormone secretion and upregulates parathyroid receptor expression at reasonably low calcium focus, Nephrology Dialysis Transplantation, Quantity 29, Challenge 2, February 2014, Pages 282–289, https://doi.org/10.1093/ndt/gft400
Summary
INTRODUCTION
The manufacturing of parathyroid hormone (PTH) is important for calcium (Ca) homeostasis and the upkeep of regular mineral metabolism. Parathyroid cells have cell-surface calcium-sensing receptors (CaRs), which allow them to reply to modifications in extracellular Ca [1, 2]; thus, even small modifications in extracellular Ca induce fast modifications in PTH secretion. Calcitriol and FGF23 additionally lower parathyroid operate by their particular parathyroid cell receptors: the vitamin D receptor (VDR) and the FGFR-Klotho receptor system, respectively. In vivo and in vitro research have additionally proven a direct impact of phosphate on PTH synthesis and secretion, though a selected cell sensor for phosphate has not been recognized, but.
The activation of the CaR leads to Gq/11-dependent activation of phosphatidylinositol-specific phospholipase C, cytosolic Ca improve, protein kinase C (PKC) activation [3] and triggering of the PLA2-AA-MAPKase pathway [4, 5] that regulates PTH synthesis and different important proteins similar to CaR, VDR, Klotho and FGFR1 [6–9].
Though Ca is the principle CaR agonist, different divalent and trivalent cations are additionally capable of activate the CaR [2]. For instance, a rise in extracellular magnesium (Mg) inhibits PTH secretion and reduces agonist-stimulated cAMP accumulation from parathyroid cells [10–13]. In dialysis sufferers, excessive serum Mg concentrations have been related to low serum PTH ranges. This inverse relationship between extracellular ranges of Mg and PTH led to the notion of an inhibitory impact of Mg on PTH secretion in these sufferers [2, 14–17]. Nonetheless, the PTH secretory response to modifications in extracellular Mg focus has not been formally evaluated in sufferers.
The curiosity on Mg has grown since medical research have proven the efficacy of Mg-containing phosphate binders [18], and a few concern has arisen associated to the potential impact of a reasonable improve in serum Mg on PTH secretion. In sufferers with regular serum Ca focus and managed PTH ranges, it’s not clear whether or not a rise in Mg could elicit an over suppression of PTH. Due to this fact, one particular query that needs to be addressed is whether or not modifications in serum Mg have the identical influence on PTH secretion when parathyroid cells are uncovered to low, regular or excessive serum Ca concentrations.
The goal of this in vitro examine was to guage the direct impact of Mg on the regulation of the parathyroid operate utilizing intact rat parathyroid glands. Particularly, the current work investigates the impact of Mg at totally different Ca concentrations on PTH secretion and on the expression of the parathyroid cell receptors CaR, VDR and FGFR1/Klotho.
MATERIALS AND METHODS
Animals
Wistar rats (250 g physique wt) had been fed a food plan containing 1.1% Ca and 0.8% phosphorus and vitamin D (1 IU/g). All animals acquired optimum care in compliance with the ‘Principles of Laboratory Animal Care,’ formulated by the Spanish Nationwide Society for Medical Analysis and the ‘Guide for the Care and Use of Laboratory Animals’, ready by the Nationwide Academy of Sciences. The experimental protocols had been reviewed and authorised by the Ethics Committee for Animal Analysis of the Universidad de Cordoba.
Parathyroid gland tradition
Rat parathyroid glands had been obtained as beforehand described [19]. Briefly, rats had been anaesthetized with thiopental sodium (50 mg/kg ip). Blood was drained by aortic puncture, and inside 2 min the parathyroid glands had been dissected freed from the thyroid gland and eliminated.
Intact rat parathyroid glands had been positioned inside a nylon basket in particular person wells containing 2 mL of buffered (pH 7.4) incubation medium (125 mM NaCl, 5.9 mM KCl, 1 mM sodium pyruvate, 4 mM glutamine, 12 mM glucose and 25 mM HEPES with 0.1 IU/mL human insulin, 0.1% bovine serum albumin, 100 IU/mL penicillin G, and 100 mg/mL streptomycin). A phosphate focus of 1 mM was achieved by addition of 1:2 NaH2PO4–Na2HPO4. Calcium concentrations starting from 0.8 to 1.5 mM (measured by a selective electrode Spotlyte Ca2+/pH Analyzer, Menarini Diagnostics, Barcelona, Spain) was achieved by addition of CaCl2. Mg was added as MgCl2·6H20 to realize Mg concentrations starting from 0.5 to five.0 mM. The glands had been maintained at 37°C with fixed rocking and shaking (mannequin AOS-0, SBS Devices, Badalona, Spain). Cell viability after the experiment was >80%. All chemical merchandise had been obtained from Sigma (St Louis, MO).
PTH secretion
After extraction, rat parathyroid glands had been stabilized in incubation medium at 1.25 mM Ca for six h. Then, the mixed impact of modifications in Mg and Ca concentrations on PTH secretion was decided. The parathyroid glands had been consecutively incubated for a 1 h interval in Ca concentrations of 0.8, 1.0, 1.2 and 1.5 mM. The identical experiment was repeated utilizing Mg concentrations of 0.5, 1.0, 2.0 and 5.0 mM. On the finish of the incubation interval, an aliquot of the medium was frozen at −20°C for PTH measurement. Intact rat PTH ranges had been quantified utilizing an Elisa package (Immutopics, San Clemente, CA). Set factors of the PTH-Ca curve had been calculated because the focus of Ca akin to the midrange between the values of maximal and minimal PTH secretion.
mRNA expression
Parathyroid glands had been incubated for six h at 0.8, 1.0 and 1.5 mM Ca in presence of physiological (0.5 mM) or elevated (2 mM) Mg stage. On the finish of the incubation interval, the glands had been freeze-dried and saved at −80°C for later PTH mRNA, VDR mRNA, CaR mRNA, FGFR mRNA and klotho mRNA measurements. For RNA isolation, 1 mL of phenol-guanidine isothiocyanate answer (Tri-Reagent, Sigma) was added to the glands.
The glands had been uncovered to ultrasound for five min at 4°C to permit for full cell rupture. Thereafter, complete RNA was extracted following a modification of the earlier protocol [20]. Extracted complete RNA was dissolved in nuclease-free water (Sigma, St. Louis, MO) and heated for 10 min at 60°C. Complete RNA was quantified by spectrophotometry (NanoDrop, Wilmington, DE). CaR, VDR, FGFR and Klotho mRNA expression had been decided by real-time RT-PCR, and beta-actin mRNA ranges had been assessed as inner controls (SensiMix SYBR No-ROX One-Step Package, Bioline, London, UK). Quantitative real-time reverse transcriptase-PCR (Mild cycler; Roche Diagnostics, Basel, Switzerland) was carried out utilizing specific primers and 50 ng of complete RNA/pattern. Desk 1 exhibits the sequences of the primers.
Immunohistochemistry
After 6 h of incubation with regular (0.5 mM) or excessive (2 mM) Mg and 0.8, 1.0 or 1.5 mM Ca, the parathyroid glands had been fixed in 4% formalin. Three-micrometre sections had been deparaffinized and incubated in 0.3% H2O2 in methanol for 30 min. Then, sections had been microwave-treated in 0.01 mmol/L citrate buffer (pH 6) for 20 min. Sections had been blocked with goat serum 10% for 40 min, then the sections had been incubated in a single day at 4°C in a humidified chamber with main affinity-purified rat anti-VDR mAb (Chemicon Int., Temecula, CA; 1:100 dilution), mouse anti-FGFR1 mAb (GeneTex, Irvine, CA; 1:200 dilution), mouse anti-CaR mAb (Abcam, Cambridge, UK; 1:500 dilution) or rabbit anti-Klotho antibody (Alpha Diagnostic, Int., San Antonio, TX; 1:50 dilution). After rinsing, the sections had been incubated for 40 min at room temperature with a peroxidase-labelled polymer conjugated to goat antimouse/rabbit Igs and handled with 3,3′ diaminobenzidine-tetrachloride chromogen answer for 10 to twenty min (En-Imaginative and prescient + System HRP [DAB]; DakoCytomation, Glostrup, Denmark; 1:100 dilution). Each step was adopted by three washes with phosphate-buffered saline for five min. Sections had been counterstained with haematoxylin (Dako). Immunoreactivity was assessed utilizing NIH picture freeware 1.62. Distinct optimistic staining was quantified in randomly chosen areas on every specimen, in a blinded method, over a minimal of 5 fields in additional than three sections. The expression of CaR, VDR, Klotho and FGR1 was measured by calculating the typical optical density (OD) per part of tissue by dividing the sum built-in OD by the sum space (background staining was subtracted from this worth). To keep away from the interference of nonspecific optimistic staining, plots of <30 pixels had been excluded. Statistical evaluation Outcomes are expressed because the imply ± SEM (n = 9 in every group: three totally different experiments with three repetitions). The distinction between imply values for 3 or extra teams was assessed by one-way evaluation of variance (ANOVA) adopted by submit hoc Duncan evaluation. The distinction between means for 2 totally different teams was decided by t check. A P worth of <0.05 was thought of vital. These analyses had been carried out with the help of a pc program (SPSS 15.0, Chicago, IL).
RESULTS
The impact of magnesium on PTH secretion in vitro
Outcomes from in vitro testing of PTH secretory response to modifications in Ca and Mg are offered in Determine 1. Determine 1A exhibits the PTH leads to pg/mL. Moreover, so as to standardize the change in PTH and keep away from small variations in PTH values attributable to minimal variations within the complete quantity of parathyroid tissue within the 10 glands of every nicely, the outcomes are additionally offered as % versus basal calcium in Determine 1B. Incubation of parathyroid glands with growing Ca concentrations produced a concentration-dependent lower in PTH values. Rising Mg concentrations from 0.5 to 1 and a pair of mM produced a left shift of the PTH-Ca curves. Thus, the set factors of the PTH curves had been 1.12; 1.02 and 0.91 mM Ca for Mg concentrations of 0.5; 1 and a pair of, respectively. With the intense Mg focus of 5 mM, the secretory response was virtually abolished in any respect Ca concentrations.
The addition of Mg to the medium diminished PTH secretion, however the diploma of inhibition various in accordance with the Ca focus. That is proven in Determine 1C, which depicts the info another way in order that variations turn out to be extra clearly seen. On the lowest Ca focus (0.8 mM) with regular 0.5 mM Mg, maximal PTH secretion was noticed; PTH elevated to greater than twice as with regular 1.25 mM Ca and 0.5 mM Mg. Mg concentrations of 1.0 and a pair of.0 mM had no vital impact on PTH secretion. Solely a particularly excessive Mg focus of 5.0 mM produced a major 62% lower in PTH secretion to values barely decrease than the PTH noticed with regular Ca and Mg of 0.5 mM. At a Ca focus of 1.0 mM with 0.5 mM Mg, PTH secretion was virtually twice as excessive as with regular Ca. Right here, the addition of each Mg 2.0 and 5.0 mM considerably decreased PTH secretion by 38 and 68%, respectively, to succeed in ranges that had been 120 and 60% of the PTH noticed with regular Ca and Mg of 0.5 mM (Determine 1C); Mg 1.0 mM didn’t considerably scale back PTH secretion on this setting. At a traditional 1.2 mM Ca focus, Mg concentrations of 1.0 mM and a pair of.0 mM didn’t scale back PTH secretion considerably. Solely a particularly excessive Mg focus (5.0 mM) produced a major 54% discount in PTH secretion to ranges that had been 56% of that with regular Ca and Mg of 0.5 mM. Lastly, when parathyroid glands had been incubated with excessive Ca (1.5 mM), solely the focus of 5.0 mM Mg was capable of lower PTH secretion (by 37%) to ranges that had been 43% of regular Ca and 0.5 mM Mg, whereas no impact was detected with Mg 1.0 and a pair of.0 mM (Determine 1C).
The impact of magnesium on parathyroid CaR, VDR, FGFR1 and Klotho expression
From earlier experiments, it grew to become obvious that the inhibition of PTH by Mg was noticed primarily when the serum Ca was 1.0 mM. The following set of experiments evaluating the impact of Mg on parathyroid CaR, VDR, FGFR1 and Klotho expression had been carried out utilizing a Ca focus of 1.0 mM and a Mg focus of 0.5 or 2.0 mM.
The mRNA ranges of CaR, VDR, FGFR1 and Klotho exhibited a 2.2, 1.6, 3.9 and 4.6-fold improve, respectively, at a Mg focus of two.0 mM in comparison with 0.5 mM Mg (P < 0.05 in all of the comparisons) (Determine 2). Because the expression of the totally different mRNAs (CaR, VDR, FGFR1 and Klotho) modified in the identical path, we evaluated the expression of PTH mRNA to show that the management gene efficiency was steady on this in vitro setting. PTH mRNA ranges lowered to a 73 ± 5% at Mg 2.0 mM in comparison with Mg 0.5 mM, P < 0.05 (Determine 2E). The expression of those receptors at protein stage was additionally evaluated by immunohistochemistry; protein content material paralleled mRNA expression ranges. Parathyroid CaR staining confirmed a 1.4-fold improve at 2.0 mM Mg in comparison with 0.5 mM Mg (P < 0.05) (Determine 3A). Parathyroid VDR protein expression was additionally upregulated at 2.0 mM Mg by 1.2-fold with respect to the degrees discovered at 0.5 mM Mg (P < 0.05) (Determine 3B). Lastly, immunohistochemical evaluation revealed that each FGFR1 and Klotho staining had been 1.8- and 1.4-fold higher, respectively, at 2.0 mM than at 0.5 mM Mg (P < 0.05).
DISCUSSION – “magnesium and parathyroid disease”
The current examine evaluates the mixed impact of modifications in Ca and Mg on parathyroid gland operate. The work was carried out by incubating complete parathyroid glands in Ca concentrations starting from 0.8 to 1.5 mM; the experiments had been repeated at totally different Mg concentrations. Our outcomes present that Mg reduces PTH secretion primarily if serum Ca is considerably under regular. As well as, Mg upregulates pivotal parathyroid CaR, VDR and FGFRI/Klotho at mRNA and protein ranges.
In vivo animal and medical research [2, 14–17, 21–23] have proven that prime Mg concentrations inhibit PTH secretion. Medical research on sufferers on haemodialysis and peritoneal dialysis confirmed that serum Mg was inversely and independently related to PTH concentrations, even after adjusting for Ca and phosphorus ranges [16, 17]. In an try and isolate the particular impact of Mg on PTH secretion, in vitro research had been additionally carried out a few years in the past [10–13]. Nonetheless, these had been carried out in remoted parathyroid cells that quickly lose CaR [24]. Thus, it appeared worthy to guage this impact in an in vitro mannequin of intact parathyroid glands as this permits the elucidation of a direct impact of phosphorus on PTH secretion [19], which had been elusive in remoted parathyroid cells.
Within the current examine, we discovered that Mg diminished PTH secretion in an in vitro mannequin of intact rat parathyroid glands. That is in accordance with earlier research in dispersed human and bovine parathyroid cells [10–13, 25]. Nonetheless, we discovered that prime Mg was capable of scale back PTH solely when parathyroid glands had been uncovered to reasonably low Ca concentrations. The PTH–Ca curves obtained with progressively growing Mg concentrations (from 0.5 to five mM) had been constantly shifted to the left; thus, there was a lower within the set level of Ca for PTH secretion. With regular to excessive Ca concentrations, solely a particularly excessive Mg focus of 5.0 mM was capable of lower PTH secretion; the impact of decrease Mg concentrations was minor or absent. Nonetheless, at 1 mM Ca (8 mg/dL complete Ca), a serum focus on the decrease finish in dialysis sufferers, the inhibitory impact of a Mg focus of two.0 mM was clearly evidenced. Curiously, at 0.8 mM Ca the inhibitory impact of Mg was not noticed anymore. It seems that within the setting of marked low calcium focus, the parathyroid glands are strongly stimulated, in order that solely very excessive doses of Mg are capable of lower PTH secretion. Within the basic dialysis inhabitants, low Ca focus will not be the key concern; fairly excessive Ca concentrations have been described to be related to detrimental outcomes. However at excessive Ca concentrations, excessive Mg concentrations don’t appear to pose an issue in accordance with our experiments.
The relative potencies of Ca and Mg in inhibiting PTH secretion had been beforehand addressed by Habener and Potts [10] and Brown’s group [12, 13], who reported that Ca was about thrice stronger than Mg in decreasing PTH secretion in vitro. Our outcomes permit the next conclusion: at a Mg focus of 0.5 mM a rise in Ca from 1 to 1.5 mM (delta of 0.5 mM) reduces PTH by 57%; alternatively, if Ca is maintained at 1 mM and the Mg focus is elevated by 0.5 mM (from 0.5 to 1 mM), the PTH secretion decreases by 14%. The ratio 57/14% is 4.0, indicating that Ca is 4 occasions stronger than Mg in suppressing PTH. Nonetheless, the flexibility of Mg to lower PTH is fixed in any respect calcium concentrations. At 1 mM Ca, a rise in Mg focus from 0.5 to 2.0 mM (delta of 1.5 mM) reduces PTH by 38%. An equal 38% lower in PTH secretion is obtained with solely a small improve in Ca from 1.0 to 1.2 mM (see Determine 1). Thus, the relative efficiency of Ca and Mg in decreasing PTH secretion varies in accordance with the basal serum Ca focus and the diploma of change in each Mg and Ca. Nonetheless, the efficiency of Ca in suppressing PTH is a minimum of 4 occasions higher than that of Mg, which is near what has been printed earlier than.
It’s recognized that, like Ca, Mg is ready to activate the CaR; although binding websites for Ca and Mg appear to be totally different. Nonetheless, variations between Ca and Mg-elicited actions could also be additional associated to mechanisms apart from how Mg interacts with the CaR [2]. Mg and Ca could activate totally different intracellular signalling pathways. The paradoxical lower in PTH secretion noticed at any given Ca focus below Mg deficiency in vivo, in vitro and in sufferers with extreme hypomagnesaemia [26–28] was defined by a mechanism involving an intracellular improve within the exercise of G alpha subunits of heterotrimeric G-proteins [29]. Moreover, as deduced from a examine by the group of Fitzpatrick et al. [30], addition of extracellular Mg to single remoted bovine parathyroid cells was capable of have an effect on PTH secretion by modifications in intracellular Ca concentrations by way of two totally different CaR-independent mechanisms.
Ca additionally regulates parathyroid operate by modulating the expression of key inhibitory receptors such because the CaR, VDR and FGFR/Klotho. Within the current examine we addressed whether or not Mg can be capable of regulate the CaR expression. We did this by incubating parathyroid glands at a Ca focus of 1.0 mM and Mg concentrations of 0.5 or 2.0 mM, that are contained in the physiologically doable vary. The expression of CaR, measured each at mRNA and protein ranges, was upregulated by excessive Mg focus. Thus, this distinct impact of Mg on the CaR expression represents a further contribution of this cation to scale back PTH secretion. From earlier personal experiments [9, 31], we all know that the minimal incubation interval required for seeing a response in gene expression is 6 h. Due to this fact, the inhibitory impact of Mg on PTH secretion noticed in our in vitro experiments can’t be attributed to modifications in parathyroid CaR expression as a result of the PTH secretion was evaluated after a 1 h of incubation, which isn’t sufficient time to permit for modifications in CaR expression.
The impact of Mg on the expression of the parathyroid VDR, one other key receptor within the regulation of PTH secretion, was additionally evaluated. The expression of VDR is upregulated by Ca [6] and calcimimetics [31]. Right here, we present that Mg upregulates the VDR expression at mRNA and protein ranges. This impact of Mg would favour the inhibition of PTH secretion by vitamin D.
Latest research have proven that FGF23 regulates parathyroid operate. FGF23 exerts an inhibitory impact on PTH secretion and parathyroid cell proliferation by binding to the receptor FGFR1 within the presence of its co-receptor Klotho [9, 32]. Klotho and FGFR are abundantly expressed in regular parathyroid cells, however in hyperplastic parathyroid glands the expression of FGFR1 and Klotho is markedly diminished, which doubtless makes the tissue proof against the motion of FGF23 [9]. Each FGFR1 and Klotho gene expression are upregulated by Ca [9]. Thus, we assessed whether or not Mg can be capable of modulate the expression of those receptors. The outcomes of this examine present increased ranges of each receptors within the parathyroid glands incubated with excessive (2.0 mM) Mg focus in comparison with these noticed with regular (0.5 mM) Mg focus. Due to this fact, the elevated ranges of expression of FGFR1 and Klotho could also be thought of as a further mechanism underlying the downregulation of PTH secretion by Mg.
The capability of Mg to modulate the expression of the CaR and, subsequently, of the VDR and the FGFR1/Klotho at long run means that this cation could possibly be thought of as a wide-range inhibitor of the entire parathyroid operate. Moreover, the inhibitory results of Mg on parathyroid operate, primarily when Ca is reasonably low, may assist to regulate secondary hyperparathyroidism. Curiously, Mg additionally would favour the effectivity of therapeutic molecules focusing on the parathyroid CaR, VDR or FGF23/Klotho. Then again, additionally it is necessary to emphasise that the lower in PTH secretion didn’t end in an oversuppression of PTH at reasonably elevated Mg concentrations. Our information definitely counsel that hypomagnesaemia needs to be averted. In these circumstances of suppression of PTH by excessive Ca, the elevated ranges of Mg had restricted further suppressive impact on PTH. Within the current examine, carried out with parathyroid glands from rats with regular renal operate, we noticed that Mg induced up-regulation of CaR, VDR and FGFR/Klotho. Nonetheless, because the impact of Mg is thru the activation of the CaR, and this receptor is normally down-regulated in CKD, outcomes could possibly be totally different in hyperplastic parathyroid glands from uraemic rats. Though this certainly deserves to be evaluated in future research, it could possibly be assumed that increased ranges of Mg needs to be wanted to take care of comparable ranges of CaR exercise in addition to the corresponding upregulation of the opposite receptors. Thus, avoiding hypomagnesaemia (and hypocalcaemia) in CKD prevents downregulation of key parathyroid receptors (CaR, VDR, Klotho FGFR1).
In conclusion, the outcomes of this examine present that parathyroid glands are delicate to an inhibitory impact of Mg solely when a reasonable low calcium focus is current. Mg additionally modulates the operate of parathyroid glands by upregulation of the important thing mobile receptors CaR, VDR and the FGF23/Klotho system.
CONFLICT OF INTEREST STATEMENT
M.E.R.-O., A.C., C.H., J.M.-M.A.Z., A.P.-R., P.P.-M., J.F.N.-G., J.R.M.-C. and Y.A. don’t have anything to declare. M.R. has acquired analysis grants from Amgen and Fresenius and lecture charges from Amgen, Abbott, Shire and Fresenius. M.P., Ok.G. and S.S. are staff of Fresenius Medical Care Deutschland GmbH. JP-D is a guide of Fresenius Medical Care Deutschland GmbH.
CONFLICT OF INTEREST STATEMENT
The outcomes offered on this paper haven’t been printed beforehand in complete or half, besides in summary format.