Mechanism of phosphate-induced calcification in rat aortic tissue culture: possible involvement of Pit-1 and apoptosis.
BACKGROUND
Hyperphosphataemia is a known contributing factor in the progression of vascular calcification in dialysis patients. The cellular mechanisms underlying phosphate-induced calcification are still unclear despite intense study, so in this study, we investigated the possible involvement of the type III sodium-dependent phosphate cotransporter, Pit-1, in an aortic tissue culture model.


METHODS
Aortic segments from 9-week-old male Sprague-Dawley rats were incubated in serum-supplemented medium for 10 days. The phosphate concentration of the medium was elevated to induce calcification, which was assessed by histology and calcium content. Phosphonoformic acid (PFA) was used to inhibit phosphate uptake. The involvement of apoptosis was examined using the terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate (dUTP) nick-end labelling (TUNEL) assay, caspase 3 activation, and inhibition of apoptosis using a general caspase inhibitor. Phenotypic changes in vascular smooth muscle cells (VSMC) were assessed using expression of osteochondrogenic differentiation markers.


RESULTS
Medial vascular calcification was induced in aortas cultured in a high phosphate medium. PFA decreased the rates of calcification and apoptosis of VSMC in the media, concomitant with calcification. Caspase inhibitor reduced calcification. No phenotypic transition of VSMC was seen in this model.


CONCLUSIONS
These results indicate that phosphate uptake through the type III sodium-dependent phosphate cotransporter, Pit-1, leads to induction of apoptosis and subsequent calcification of VSMC.
Mune
Sachiko
S
Department of Nephrology and Blood Purification Medicine, Wakayama Medical University, Kimiidera, Wakayama, Japan. smune@wakayama-med.ac.jp
Shibata
Maki
M
Hatamura
Ikuji
I
Saji
Fumie
F
Okada
Tadashi
T
Maeda
Yuka
Y
Sakaguchi
Toshifumi
T
Negi
Shigeo
S
Shigematsu
Takashi
T
D000818:Animals; D001011:Aorta / Q000473:pathology / Q000502:physiology; D017209:Apoptosis; D002114:Calcinosis / Q000139:chemically induced*; D053148:Caspase 3 / Q000378:metabolism; D017245:Foscarnet / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000473:pathology / Q000502:physiology; D010710:Phosphates / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D050611:Sodium-Phosphate Cotransporter Proteins, Type III / Q000502:physiology*; D046509:Tissue Culture Techniques
D010710:Phosphates; C493588:Slc20a1 protein, rat; D050611:Sodium-Phosphate Cotransporter Proteins, Type III; D017245:Foscarnet; D053148:Caspase 3