Beta-Glycerophosphate-Induced ORAI1 Expression and Store Operated Ca²⁺ Entry in Megakaryocytes.

Area of Science:

• Nephrology
• Cell Biology
• Molecular Biology

Background:

• Impaired renal phosphate elimination in CKD elevates plasma phosphate.
• Elevated phosphate upregulates NFAT5 and SGK1, which in turn increases ORAI1 calcium channels.
• Store-operated calcium entry (SOCE) mediated by ORAI1 is crucial for platelet and megakaryocyte function.

Purpose of the Study:

• To investigate if phosphate-donor ß-glycerophosphate augments NFAT5, ORAI1,2,3, and STIM1,2 expressions and SOCE in megakaryocytes.
• To compare NFAT5, SGK1, ORAI1/2/3, and STIM1/2 expression and NFAT5/ORAI1 protein levels in platelets from CKD patients versus healthy controls.

Main Methods:

• Human megakaryocytic Meg01 cells were treated with ß-glycerophosphate.
• Platelets were isolated from CKD patients and healthy volunteers.
• Gene and protein expression (NFAT5, ORAI1/2/3, SGK1, STIM1/2) were analyzed using q-RT-PCR and Western blots.
• Cytosolic calcium concentration and SOCE were measured using Fura-2 fluorescence.

Main Results:

• ß-glycerophosphate increased NFAT5, ORAI1/2/3, STIM1/2 transcript levels, and SOCE in Meg01 cells.
• Platelets from CKD patients showed significantly higher transcript levels of NFAT5, SGK1, ORAI1/2/3, and STIM1/2.
• NFAT5 and ORAI1 protein abundance was also elevated in platelets from CKD patients.

Conclusions:

• Phosphate-donor ß-glycerophosphate activates a signaling cascade involving NFAT5/SGK1/ORAI/STIM.
• This cascade leads to the upregulation of store-operated calcium entry (SOCE).
• The findings highlight a molecular mechanism linking phosphate dysregulation in CKD to altered platelet function via SOCE.