Abstract
In chronic kidney disease, hyperphosphatemia upregulates the Ca2+ channel ORAI and its activating Ca2+ sensor STIM in megakaryocytes and platelets. ORAI1 and STIM1 accomplish store-operated Ca2+ entry (SOCE) and play a key role in platelet activation. Signaling linking phosphate to upregulation of ORAI1 and STIM1 includes transcription factor NFAT5 and serum and glucocorticoid-inducible kinase SGK1. In vascular smooth muscle cells, the effect of hyperphosphatemia on ORAI1/STIM1 expression and SOCE is suppressed by Mg2+ and the calcium-sensing receptor (CaSR) agonist Gd3+. The present study explored whether sustained exposure to Mg2+ or Gd3+ interferes with the phosphate-induced upregulation of NFAT5, SGK1, ORAI1,2,3, STIM1,2 and SOCE in megakaryocytes. To this end, human megakaryocytic Meg-01 cells were treated with 2 mM ß-glycerophosphate for 24 h in the absence and presence of either 1.5 mM MgCl2 or 50 µM GdCl3. Transcript levels were estimated utilizing q-RT-PCR, protein abundance by Western blotting, cytosolic Ca2+ concentration ([Ca2+]i) by Fura-2 fluorescence and SOCE from the increase in [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). As a result, Mg2+ and Gd3+ upregulated CaSR and blunted or virtually abolished the phosphate-induced upregulation of NFAT5, SGK1, ORAI1,2,3, STIM1,2 and SOCE in megakaryocytes. In conclusion, Mg2+ and the CaSR agonist Gd3+ interfere with phosphate-induced dysregulation of [Ca2+]i in megakaryocytes.
Highlights
The impairment of renal phosphate excretion in chronic kidney disease (CKD) increases the plasma phosphate concentration with subsequent osteogenic reprogramming of vascular smooth muscle cells (VSMCs) [1,2,3,4] and vascular calcification [5,6,7,8]
U1.prUegpurleagtiuonlaotfioCnaSoRftrCaanSscRriptrtiaonnsbcyriMptgiColn2 abnyd rMevgeCrsal2l oafnßd-grlyecveerorpsahlosopfhßa-teg-liyndceurcoedphosphate-induced NNFFAATT5,5S,GSKG1,KO1R, AOI1R,AOIR1A,IO2,ROARAI2I3,OSTRIAMI13a,nSdTSITMIM12atnradnsScTriIpMtio2ntinramnesgcarkipartyioocnytiens mbyeMggaCkla2.ryocytes by MgCl2. (A–H) Arithmetic means (±SEM, n = 6) of calcium-sensing receptor (CaSR) (A), nuclear factor of activated T cells 5 (NFAT5) (B), serum and glucocorticoid-inducible kinase 1 (SGK1) (C), ORAI1 (D), ORAI2 (E), ORAI3 (F), STIM1 (G) and STIM2 (H) transcript levels in megakaryocytes without (CTR) and with prior 24-h exposure to 1.5 mM MgCl2 alone (MgCl2), 2 mM ß-glycerophosphate alone (Pi) or 2 mM ß-glycerophosphate and 1.5 mM MgCl2 (Pi+MgCl2). * (p < 0.05), ** (p < 0.01), *** (p < 0.001) indicate statistically significant differences compared to CTR; # (p < 0.05), ## (p < 0.01) indicate statistically significant differences compared to Pi alone (ANOVA)
Sci. 2021, 22, 3292 (A–H) Arithmetic means (± SEM, n = 6) of CaSR (A), NFAT5 (B), SGK1 (C), ORAI1 (D), ORAI2 (E), ORAI3 (F), STIM1 (G) and STIM2 (H) transcript levels in megakaryocytes without (CTR) and with prior 24-h exposure to 1.5 mM MgCl2 alone (MgCl2), 2 mM ß-glycerophosphate alone (Pi) or 2 mM ß-glycerophosphate and 1.5 mM MgCl2 (Pi+MgCl2). * (p < 0.05), ** (p < 0.01), *** (p < 0.001) indicate statistically significant differences compared to CTR; # (p < 0.05), ## (p < 0.01) indicate statistically significant differences compared to Pi alone (ANOVA)
Summary
The impairment of renal phosphate excretion in chronic kidney disease (CKD) increases the plasma phosphate concentration with subsequent osteogenic reprogramming of vascular smooth muscle cells (VSMCs) [1,2,3,4] and vascular calcification [5,6,7,8]. (A–H) Arithmetic means (± SEM, n = 6) of CaSR (A), NFAT5 (B), SGK1 (C), ORAI1 (D), ORAI2 (E), ORAI3 (F), STIM1 (G) and STIM2 (H) transcript levels in megakaryocytes without (CTR) and with prior 24-h exposure to 1.5 mM MgCl2 alone (MgCl2), 2 mM ß-glycerophosphate alone (Pi) or 2 mM ß-glycerophosphate and 1.5 mM MgCl2 (Pi+MgCl2). The upregulation of NFAT5, SGK1, ORAI1, ORAI2, ORAI3, STIM1 and STIM2 transcript levels by 2 mM ß-glycerophosphate was significantly blunted by additional exposure to 50 μM GdCl3. In the absence of ß-glycerophosphate, 50 μM GdCl3 did not significantly modify the transcript levels of NFAT5, SGK1, ORAI1, ORAI2, ORAI3, STIM1 and STIM2 (Figure 4). In the absence of ß-glycerophosphate, 50 μM GdCl3 did not significantly modify SOCE
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