High phosphate-induced vascular calcification (VC) and iron deficiency-induced anemia are two major contributors of cardiovascular morbidity and mortality in patients affected by chronic kidney disease (CKD). Since phosphate (Pi) control and iron replacement are common therapies in CKD, the aim of our study was to investigate the effect of iron on high-Pi induced VC in rat vascular smooth muscle cells (VSMCs).
We treated VSMCs with 5 mM Pi and iron citrate (Fe3+) to evaluate Ca deposition by Alizarin Red destaining, DNA fragmentation by ELISA, gene expression by RT-PCR and protein expression by Western Blot.
Pretreatment with Fe3+ prevents high Pi-induced calcium (Ca) deposition concentration-dependently with 90.1% inhibition at 50 µM (0.716±0.04 vs 0.071±0.01, OD/mg protein; Pi vs Fe3+, p<0.01). We found that 50 µM Fe3+ completely prevents apoptosis measured as DNA fragmentation (1.51±0.08 vs 1.03±0.06, Pi vs Fe3+; p<0.01), and that this action is through the prevention of the downregulation of the pro-survival pathway GAS6/AXL. Moreover, Fe3+ stimulates autophagy, a protective phenomenon in VC, as demonstrated by electron microscopy and by autophagy flux detected by LC3IIβ protein expression. Finally, osteoblastic differentiation is partially affected by Fe3+, since increased BMP2 is prevented, but increased RUNX2 and decreased α-actin and SM22α are not modified. Interestingly, the addition of Fe3+ at different time points after Pi challenge blocks completely the progression of Ca deposition.
In conclusion, iron citrate inhibits high Pi-induced Ca deposition by prevention of apoptosis, induction of autophagy, and partially affecting osteoblastic differentiation.
