Effect and mechanism of acidification on vascular calcification in chronic renal failure rats

Abstract

Objective To explore the effect and mechanism of acidification on calcification in chronic renal failure rats. Methods In vivo, male SD rats(n=22) were randomly divided into sham operated group(n=6), 5/6 nephrectomy(Nx,n=5), 5/6 Nx+ calcitriol group(n=5), 5/6 Nx+ calcitriol + acidosis group(n=6). Vascular calcification was determined by von Kossa stain and quantification of calcium. L-type calcium channels(LTCC)β3 subunit and runt-related transcription factor 2(runx2) in aortic were measured by immunohistochemistry. In vitro, vascular smooth muscle cells(VSMCs) were primarily cultured and calcification was induced by β-glycerophosphate(β-GP). They were then randomly divided into control +pH7.4 group, high phosphorus +pH7.4 group(10 mmol/L β-GP), high phosphorus +pH7.1 group(10 mmol/L β-GP) and verapamil intervention group(10 mmol/L β-GP+20 mmol/L verapamil). Calcium deposition was measured by Alizarin red staining and quantification of calcium; and alkaline phosphatase(ALP) activity was measured by enzyme linked immunosorbent assay. RT-PCR and Western blotting were used to observe the expression of VSMCs LTCC β3 subunit and runx2 mRNA and protein respectively. Results In vivo, compared to that in 5/6 Nx+calcitriol group, calcification was significantly reduced in 5/6Nx + calcitriol + acidosis group(P <0.05); the expressions of LTCC β3 subunit and runx2 were obviously down-regulated by acidification(P <0.05). In vitro, compared to that in high phosphorus +pH7.4 group, calcification was significantly reduced in high phosphorus+pH7.1 group(P <0.05), as well as ALP activity(P <0.05); the expressions of runx2 and LTCCβ3 subunit were down-regulated in high phosphorus +pH7.1 group(P <0.05). Calcium influx in VSMCs was partially blocked during acidification(P <0.05). The calcification level and expression of runx2 in verapamil intervention group were lower than that in high phosphorus + pH7.4 group(P < 0.05). Conclusion One of the possible mechanisms of the inhibitory effect of acidification on VSMCs calcification is that acidification prevents LTCCβ3 subunit expression and calcium influx, leading to the degradation of VSMCs phenotype transforming. Key words: Kidney failure, chronic; Calcinosis; Myocytes, smooth muscle