The mechanism of phosphorus as a cardiovascular risk factor in CKD

Abstract

Hyperphosphatemia and vascular calcification have emerged as cardiovascular risk factors among those with chronic kidney disease. This study examined the mechanism by which phosphorous stimulates vascular calcification, as well as how controlling hyperphosphatemia affects established calcification. In primary cultures of vascular smooth muscle cells derived from atherosclerotic human aortas, activation of osteoblastic events, including increased expression of bone morphogenetic protein 2 (BMP-2) and the transcription factor RUNX2, which normally play roles in skeletal morphogenesis, was observed. These changes, however, did not lead to matrix mineralization until the phosphorus concentration of the media was increased; phosphorus stimulated expression of osterix, a second critical osteoblast transcription factor. Knockdown of osterix with small interference RNA (siRNA) or antagonism of BMP-2 with noggin prevented matrix mineralization in vitro. Similarly, vascular BMP-2 and RUNX2 were upregulated in atherosclerotic mice, but significant mineralization occurred only after the induction of renal dysfunction, which led to hyperphosphatemia and increased aortic expression of osterix. Administration of oral phosphate binders or intraperitoneal BMP-7 decreased expression of osterix and aortic mineralization. It is concluded that, in chronic kidney disease, hyperphosphatemia stimulates an osteoblastic transcriptional program in the vasculature, which is mediated by osterix activation in cells of the vascular tunica media and neointima.

Figures

Figure 1.

Effects of BMP-7, 10 μg/kg intraperitoneally once weekly, on VC in CKD by BMP-7. (A) VC increased from 22 to 28 wk in vehicle-treated mice, but aortic Ca decreased in mice treated with BMP-7. (B) The serum phosphorus was unchanged between 22 and 28 wk but was reduced to normal levels by treatment with BMP-7. Data are group means ± SEM (n = 4 to 7).

Figure 2.

Effects of CaCO3 and LaCO3, both 3% mixed in the diet, on VC in CKD. Whereas VC significantly increased from 22 to 28 wk in vehicle-treated mice, aortic Ca was decreased below levels at 22 wk in mice treated with 3% CaCO3. there was no significant change from 22 wk in the 3% LaCO3-treated mice. Data are group means ± SEM (n = 4 to 5).

Figure 3.

Sections of the proximal aorta demonstrating large calcified atherosclerotic plaques in the LDLR−/− high fat–fed mice. (A) Large lipid-laden plaque (between arrows) in proximal aorta of a sham-operated high fat–fed LDLR−/− mouse. Thick arrows identify focal calcifications in the base of the plaque. (B) A large calcified plaque (between arrows) in the proximal aorta of a CKD high fat–fed LDLR−/− mouse. (C) A large lipid-laden plaque (between arrows) in the proximal aorta of a BMP-7–treated CKD high fat–fed LDLR−/− mouse. The stain is alizarin red. Magnification, ×400.

Figure 4.

Stimulation of matrix mineralization in cultures of hVSMC isolated from atherosclerotic donors. hVSMC from seven donors were grown in DMEM as described in the Concise Methods section (group A) or DMEM supplemented with 1 mM NaH2PO4/NaHPO4 (pH 7.4; group B). In groups C, D, and E, 0.1, 1, and 10 ng/ml BMP-7, respectively, was added to the NaH2PO4/NaHPO4-supplemented media. Cultures were ended at 14 (A) and 21 d (B). Data are means ± SEM (n = 7 donors).

Figure 5.

Stimulation of a BMP-2/MSX2-directed osteoblastic differentiation program by NaH2PO4/NaHPO4 through induction of osterix. Basal levels of gene expression in hVSMC cultured in DMEM as described in the Concise Methods section and detected by RT-PCR (insets) were set as a reference value of 1 (□, group A). Fold induction by high phosphorus [2 mM NaH2PO4/(Na)2HPO4] culture media (▪, group B) and 2 mM phosphorus plus 10 ng/ml BMP-7 (□, group C) was determined. An osteoblastic transcriptional program directed by BMP-2/MSX2 was present in hVSMC cultures as demonstrated by the presence of osteocalcin (A), BMP-2 (B), MSX2 (C), and RUNX2 (D) gene transcription. (E) Osterix was only weakly expressed in basal culture conditions. Phosphorus stimulated transcription of osterix. BMP-7 inhibited osterix and osteocalcin expression. Data were normalized to the expression level in basal culture conditions and expressed as fold induction (mean ± SEM; n = 3).

Figure 6.

Effects of a BMP-specific inhibitor, noggin, on VSMC mineralization and gene expression. (A) Noggin (10 to 100 ng/ml), added to high-phosphorus media (□), inhibited matrix mineralization stimulated by phosphorus. (B) Noggin, 100 ng/ml, blocked phosphorus-induced osterix expression.

Figure 7.

Effects of osterix knockdown on phosphorus-stimulated mineralization by osteoblast-like cells. SAOS cell lines expressing siRNA to osterix were developed as described in the Concise Methods section. (A) The levels of mRNA to osterix were decreased by >50% in two separate cell lines (osx siRNA #1 and osx siRNA #2). (B) Protein levels of osterix were greatly diminished by Western analysis in osx siRNA #1 and osx siRNA #2. Histone H3 levels were determined as a loading control on the Western blots. (C through G) Von Kossa (C through E) and alizarin red (F and G) stains of cultures stimulated by high-phosphorus conditions. (C) Mineralized nodules in the periphery of wells containing cells expressing the scrambled siRNA at 7 d in the high-phosphorus mineralizing conditions. (D) Mineralized nodules in the center of wells containing cells expressing the scrambled siRNA at 7 d in the high-phosphorus mineralizing conditions. (E) Absence of mineralization detected by von Kossa staining in periphery of wells expressing osx siRNA #1 at 7 d in the high-phosphorus mineralizing conditions. (F) Alizarin red–stained nodules in the periphery of wells containing cells expressing the scrambled siRNA at 7 d in the high-phosphorus mineralizing conditions. (G) Absence of mineralization detected by alizarin red staining in periphery of wells expressing osx siRNA #1 at 7 d in the high-phosphorus mineralizing conditions. The orange is the background stain in alizarin red–negative stains. (H) Ca levels in the matrices of cultures of cell lines expressing siRNA to osterix.

Figure 8.

Expression of contractile VSMC markers in hVSMC. (A through C) Calponin (A), α-smooth muscle actin (α-SMA) (B), heavy-chain myosin (MHSCM) (C), and SM22 α (D) message levels detected by RT-PCR were low in hVSMC in basal conditions (□, group A) and in the presence of high-phosphorus media (▪, group B). BMP-7 (□, group C) induced expression of each of the marker proteins. Data were normalized to the expression level in basal culture conditions and expressed as fold induction (mean ± SEM, n = 3).

Figure 9.

Gene expression in aortas of various groups of LDLR−/− high fat–fed mice measured by real-time RT-PCR. The various groups of mice were as follows: sham operated (s), CKD vehicle treated (C), CKD BMP-7 treated (B7), CKD 3% CaCO3 treated (CC), and CKD 3% LaCO3 treated (LC). (A) MSX2 expression was low, not induced by CKD, and suppressed with each of the three treatments. Inset shows data for scale of the ratio between 0.0 and 0.5. (B) CBFA1 expression was not stimulated by CKD, but it was inhibited by BMP-7, 3% CaCO3, and 3% LaCO3 treatment. (C) Osterix expression was stimulated by CKD and was inhibited by BMP-7, CaCO3, or LaCO3 treatment. (D) Osteocalcin expression was strongly induced by CKD (note different y axis scale) and inhibited by BMP-7, CaCO3, or LaCO3. The number of aortas/mice in each group was four. Data are means of the transcript levels relative to GAPDH expression in each aorta ± SEM.