p53 negatively regulates the osteogenic differentiation of vascular smooth muscle cells in mice with chronic kidney disease

Abstract

Aim: To investigate the osteogenic differentiation of vascular smooth muscle cells (VSMCs) in mice with chronic kidney disease (CKD) and to evaluate the effects of p53 on the osteogenic differentiation of the VSMCs.

Methods: Experimental models of CKD-associated vascular calcification generated by five-sixth (5/6) nephrectomy (Nx) and a high-phosphate (HP) diet were used in p53+/+ and p53-/- mice. Following 5/6 Nx, aortic calcification, markers of osteogenic differentiation, VSMCs and p53 protein in aortic tissues were studied.

Results: Aortic calcification was observed after eight weeks following 5/6 Nx in mice of both genotypes, and expression of the markers of osteogenic differentiation in the VSMCs was increased. These changes were continuously observed up to 12 weeks after 57/6 Nx, and particularly after 5/6 Nx + HP. Compared with p53+/+ mice, aortic calcification in p53-/- mice was more severe (p < 0.001). Expression of the markers of osteogenic differentiation was noticeably increased (p < 0.001), while expression of the marker of VSMCs had decreased (p < 0.001). Statistical analysis demonstrated that the markers of osteogenic differentiation were negatively correlated with p53, and the marker of VSMCs was positively correlated with p53 (p < 0.001).

Conclusion: p53 has the potential to negatively regulate the osteogenic differentiation of VSMCs in CKD mice.

Fig. 1.

I: Histological appearance of the kidney in A and C: p53+/+ mice, and B and D: p53–/– mice at 12 weeks after 5/6 Nx or 5/6 Nx + HP diet. A and B: Nx mice, C and D: Nx + HP mice (HE staining, original magnification: × 200). II: Quantification of glomerular cell number, sclerosis index and tubulo-interstitial fibrosis in mice at 12 weeks after Nx or Nx + HP. Values are means ± SEM; n = 5 per group. NS = no significant difference vs p53–/– mice.

Fig. 2.

Ⅰ: Histopathological analysis of vascular calcification in the aorta at 12 weeks after Nx or Nx + HP (original magnification, × 200) with von Kossa staining. A and C: p53+/+ mice, and B and D: p53–/– mice. Ⅱ: Evaluation of the vascular calcification score in p53+/+ and p53–/– mice at 12 weeks after 5/6 Nx or 5/6 Nx + HP. Values are means ± SE; n = 5 per group. ^ap < 0.001 vs p53+/+ mice, ^bp < 0.01 vs Nx mice.

Fig. 3.

Indirect immunofluorescence staining analysis for BMP-2, RUNX2 and Osx in the aorta at 12 weeks after 5/6 Nx or 5/6 Nx + HP (original magnification, × 200). Positive staining of BMP-2, RUNX2 and Osx protein was detected in VSMCs, clumping or a green line demonstrated the expression of protein, but in p53+/+ or 5/6 Nx mice, a weaker signal was detected compared with those in p53–/– or 5/6 Nx + HP mice.

Fig. 4.

Immunohistochemistry analysis for p53, ALP and α-SMA in the aorta at 12 weeks after 5/6 Nx or 5/6 Nx + HP (original magnification, × 200). In p53–/– mice with 5/6 Nx + HP, ALP expression was high, but α-SMA was low, and no p53 was detected, while related heavy mineral deposition was also detected. On the other hand, a weak positive staining of ALP, and enhanced positive staining of p53 and α-SMA was detected in p53+/+ mice, while moderate mineral deposition could be detected. A brown colour indicated the presence of proteins and a black colour indicated mineral deposition.

Fig. 5.

Western blot analysis for p53, BMP-2 and RUNX2 in the aorta at 12 weeks after 5/6 Nx or 5/6 Nx + HP. A densitometry graph shows the expression levels of each calcification-related protein identified by Western blot analysis in the aorta. The graph indicates the relative band density when levels of GAPDH protein expression in each sample were calculated as 100%. Values are means ± SE; n = 5 per group. ^ap < 0.001 vs p53–/– mice, NS = no significant difference vs 5/6 Nx mice with the same genetype.

Fig. 6.

Western blot analysis for Osx, ALP and α-SMA in the aorta at 12 weeks after 5/6 Nx or 5/6 Nx + HP. A densitometry graph shows the expression levels of each calcification-related protein identified by Western blot analysis in the aorta. The graph indicates the relative band density when levels of GAPDH protein expression in each sample were calculated as 100%. Values are means ± SE; n = 5 per group. ^ap < 0.001 vs p53–/– mice, ^bp < 0.05, ^cp < 0.01 vs 5/6 Nx mice with the same genetype.