title: Iron citrate reduces high phosphate-induced vascular calcification by inhibiting apoptosis.
abstract: 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. Pi + Fe3+, p < 0.01). We found that 50 μM Fe3+completely prevents high Pi-induced apoptosis measured as DNA fragmentation (1.51 ± 0.08 vs. 1.03 ± 0.06, Pi vs. Pi + Fe3+; p < 0.01), 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, high Pi-induced osteoblastic differentiation is partially affected by Fe3+, since BMP2 increase is prevented and OPN is enhanced, but RUNX2 increase and α-actin and SM22α decrease are not modified. Interestingly, the addition of Fe3+at different time points after Pi challenge completely blocks 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.
authors.affiliation: Laboratory of Experimental Nephrology, Department of Health Sciences, University of Milan, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Ciceri
authors.affiliation: Laboratory of Experimental Nephrology, Department of Health Sciences, University of Milan, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Elli
authors.affiliation: Division of Pathology, Department of Health Sciences, University of Milan, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Braidotti
authors.affiliation: Division of Pathology, Department of Health Sciences, University of Milan, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Falleni
authors.affiliation: Division of Pathology, Department of Health Sciences, University of Milan, Italy.
authors.forename: Delfina
authors.initials: D
authors.lastname: Tosi
authors.affiliation: Division of Pathology, Department of Health Sciences, University of Milan, Italy.
authors.forename: Gaetano
authors.initials: G
authors.lastname: Bulfamante
authors.affiliation: Nephrology, Denver Nephrologists PC, Denver, CO, USA.
authors.forename: Geoffrey A
authors.initials: GA
authors.lastname: Block
authors.affiliation: Laboratory of Experimental Nephrology, Department of Health Sciences, University of Milan, Italy. Electronic address: mario.cozzolino@unimi.it.
authors.forename: Mario
authors.initials: M
authors.lastname: Cozzolino
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D001343:Autophagy; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism; D002118:Calcium / Q000737:chemistry; D002454:Cell Differentiation; D002478:Cells, Cultured; D019343:Citric Acid / Q000494:pharmacology*; D018450:Disease Progression; D007501:Iron / Q000737:chemistry*; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D010006:Osteoblasts / Q000166:cytology; D010710:Phosphates / Q000009:adverse effects*; D051381:Rats; D061205:Vascular Calcification / Q000139:chemically induced / Q000188:drug therapy*
chemical_list: D000199:Actins; C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D010710:Phosphates; D019343:Citric Acid; D007501:Iron; D002118:Calcium
keywords: Apoptosis; Iron; Phosphate; VSMC; Vascular calcification