Effect of calcium phosphate crystals induced by uremic serum on calcification of human aortic smooth muscle cells / 中华肾脏病杂志

ABSTRACT

Objective To investigate the impact of calcium phosphate crystals induced by uremic serum on calcification of human aortic smooth muscle cells (HASMCs).Methods Uremic serum was incubated at 37℃ for 3 days.Calcium phosphate crystals and uremic supernatant were isolated from uremic serum by uhracentrifugation.Scanning electron microscope (SEM) and energy dispersive X-ray spectroscopy (EDX) were performed for analysis of morphological and chemical characteristics of the crystals.HASMCs were treated in vitro with control medium,uremic serummedium,calcium phosphate crystals-medium and uremic supernatant-medium.Calcification was visualizcd by Alizarin red staining.Calcium loads in cells were quantified by o-cresolphthalein complexone method.Gene expression of bone morphogenetic protein-2 (BMP-2),osteopontin (OPN) and core-binding factor α1 (Cbfα1),alkaline phosphate (ALP) and matrix gamma carboxyglutamic acid protein (MGP) were quantified by real-time PCR.Cbfα1,OPN and BMP-2 protein levels were determined by Western blotting or ELISA.Results Calcium phosphate crystals which induced by uremic serum displayed laminated shapes containing crystallized needle-like projections and ranged from 30-500 nm,with Ca/P ratios of 1.41 ±0.05.Compared with the cells in control group,uremic serum induced HASMCs calcification,increased calcium loads (P ＜ 0.05),up-regulated BMP-2,OPN,Cbfα1 mRNA and protein expression (all P＜ 0.01).Similar to uremic serum,calcium phosphate crystals also induced HASMCs calcification,increased calcium loads (P＜0.05),and up-regulated BMP-2,OPN,Cbfα1 mRNA and protein expression (all P ＜ 0.01).However,there was no significant difference between HASMCs growing in uremic supernatant and control medium in calcium loads or the expression levels of these osteogenic proteins (P ＞ 0.05).Conclusions Calcium phosphate crystals induced by uremic serum promote HASMCs calcification,which might be one of the mechanisms of uremic vascular calcification.