Fig. 2: The LCN2 expression level was increased in two CKD-VC model animals and in HP-stimulated VSMCs.

A Scheme of the 5/6 nephrectomy and HP diet-induced CKD-VC rat model and AP diet-induced CKD-VC mouse model. B Representative Von Kossa staining and IHC images of LCN2 in the aortic smooth muscle layer from CKD-VC model rats and mice. Scale bar: 200 μm for Von Kossa staining, 100 μm for IHC images. C Quantification of the LCN2-positive area in CKD-VC model rats (n = 6 for the CTL group; n = 8 for the CKD-VC model group). D Quantification of the LCN2-positive area in CKD-VC model mice (n = 5 for the CTL group; n = 7 for the CKD-VC model group). E The expression of LCN2 mRNA in the aortas of CTL and CKD-VC rats was analysed by qRT-PCR (n = 5 per group). Mouse VSMCs were treated with growth medium (CTL) or calcifying medium containing 3.0 mM HP sodium for 5 days. Calcium deposition in VSMCs was assessed by a calcium concentration assay normalised to the protein concentration (n = 3 per group) (F) and alizarin red staining (G) (positive staining: red; scale bar: 200 μm). H–K The protein levels of osteogenic phenotype markers (RUNX2, BMP2) and LCN2 were determined via Western blot (n = 3 per group). L LCN2 mRNA expression in VSMCs exposed to HP (3.0 mM) (n = 6 per group). Data are presented as mean ± SD. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 and ^****p < 0.0001. CKD-VC chronic kidney disease-related vascular calcification, HP high phosphate, VSMCs vascular smooth muscle cells, IHC immunohistochemical, AP adenine and phosphate, CTL control, qRT-PCR quantitative real-time polymerase chain reaction, RUNX2 runt-related transcription factor 2, BMP2 bone morphogenetic protein 2, SD standard deviation.