A new in vitro model to delay high phosphate-induced vascular calcification progression.
An increasing amount of patients affected by advanced chronic kidney disease suffer from vascular calcification (VC) that associates with cardiovascular morbidity and mortality. In this study, we created a new experimental in vitro model, trying to better elucidate high phosphate (Pi)-induced VC pathogenic mechanisms. Rat aortic vascular smooth muscle cells (VSMCs) were challenged for 7-10 days with high Pi with a repeated and short suspensions of high Pi treatment (intermittent suspension, IS) that was able to induce a significant inhibition of high Pi calcification, maximal at 5 h. Interestingly, the delay in calcification is a consequence of either the absence of free Pi or calcium-phosphate crystals being comparable to the total effect obtained during the 5 h-IS. The protective effect of IS was mediated by the reduction of apoptosis as demonstrated by the action of 20 μmol/L Z-VAD-FMK and by the preservation of the pro-survival receptor Axl expression. Furthermore, autophagy, during IS, was potentiated by increasing the autophagic flux, evaluated by LC3IIB western, while treating VSMCs with 1 mmol/L valproic acid did not affect VC. Finally, IS prevented VSMC osteoblastic differentiation by preserving smooth muscle lineage markers expression. Our data support the hypothesis that to delay significantly VC is necessary and sufficient the IS of high Pi challenge. The IS was able to prevent significantly apoptosis, to induce a potentiation in autophagy, and to prevent osteoblastic differentiation by preserving SM lineage markers.
Laboratory of Experimental Nephrology, Department of Health Sciences, University of Milan, Milan, Italy.
Paola
P
Ciceri
Laboratory of Experimental Nephrology, Department of Health Sciences, University of Milan, Milan, Italy.
Francesca
F
Elli
Laboratory of Experimental Nephrology, Department of Health Sciences, University of Milan, Milan, Italy.
Laura
L
Cappelletti
Laboratory of Experimental Nephrology, Department of Health Sciences, University of Milan, Milan, Italy.
Delfina
D
Tosi
Laboratory of Experimental Nephrology, Department of Health Sciences, University of Milan, Milan, Italy.
Paola
P
Braidotti
Laboratory of Experimental Nephrology, Department of Health Sciences, University of Milan, Milan, Italy.
Gaetano
G
Bulfamante
Renal Division and Laboratory of Experimental Nephrology, Dipartimento di Scienze della Salute, Università di Milano, S. Paolo Hospital, Milan, Via A. di Rudinì, 8, 20142, Milan, Italy. mario.cozzolino@unimi.it.
Mario
M
Cozzolino
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects; D002130:Calcium Phosphates / Q000378:metabolism; D019070:Cell Lineage; D054338:Cell Transdifferentiation / Q000187:drug effects; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D018450:Disease Progression; D008840:Microfilament Proteins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D010641:Phenotype; D010710:Phosphates / Q000494:pharmacology* / Q000633:toxicity; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D013997:Time Factors; D061205:Vascular Calcification / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D000094963:Axl Receptor Tyrosine Kinase
D002130:Calcium Phosphates; D050659:Core Binding Factor Alpha 1 Subunit; C477012:MAP1LC3A protein, human; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins; D009124:Muscle Proteins; D010710:Phosphates; D011518:Proto-Oncogene Proteins; C493709:RUNX2 protein, human; C051615:transgelin; D020794:Receptor Protein-Tyrosine Kinases; D000094963:Axl Receptor Tyrosine Kinase
Apoptosis; Phosphate; VSMC; Vascular calcification