Mineral Composition of Phosphate-Induced Calcification in a Rat Aortic Tissue Culture Model

Aim: High phosphorus conditions promote vascular calcification (VC) in both chronic kidney disease (CKD) patients and experimental models. However, the composition of medial calcification has not been accurately determined, so the objective of this study was to evaluate the mineral composition of calcification in a tissue culture model, not a cell culture system. Methods: Aortic rings obtained from male Sprague-Dawley rats were incubated in serum-supplemented medium for 10 days. The inorganic phosphate (Pi) concentration of the medium was increased to induce VC, which was assessed by histology, imaging, and spectroscopy. The mineral composition of the calcification was analyzed using Fourier transform infrared (FTIR) spectroscopic imaging, scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDX) mapping. Results: The calcium content significantly increased only in aortic rings cultured for 10 days in the high-Pi medium (HiP: 3.8 mmol/L). The concentration of the phosphate transporter Pit-1 in the aortic tissue exposed to HiP was higher than that in the control incubated sections. The FTIR images and spectra indicated that PO43－ was mostly distributed as hydroxyapatite in the medial calcification of aortic rings cultured in HiP. A small quantity of carbonate was identified. The SEM-EDX overlay map demonstrated that phosphorus and calcium simultaneously accumulated and localized in the area of medial calcification induced by exposure to HiP. Conclusion: This is the first report of accurate determination of the chemical composition of aortic medial calcification. Exposure to high Pi concentration augments aortic calcification via an increase in Pit-1, which mainly contains calcium phosphate.