Abstract
Vascular calcification is a life-threatening clinical condition in chronic kidney disease (CKD) and is associated with reduced zinc serum levels. Anemia is another frequent complication of CKD. Hypoxia-inducible factor (HIF) stabilizers, also known as HIF prolyl hydroxylase inhibitors (PHI), are promising candidates to treat CKD-associated anemia by increasing erythropoietin synthesis. Recent evidence suggests that HIFs play a pivotal role in vascular calcification. Our study explored feasible impacts of HIF PHI on phosphate (Pi)-induced calcification of vascular smooth muscle cells (VSMCs) and tested whether zinc might inhibit this mineralization process. Treatment of VSMCs with PHI aggravated Pi-induced calcium deposition and Pi uptake. PHI promoted Pi-induced loss of smooth muscle cell markers (ACTA-2, MYH11, SM22α) and enhanced osteochondrogenic gene expression (Msx-2, BMP-2, Sp7) triggering osteochondrogenic phenotypic switch of VSMCs. These effects of PHI paralleled with increased pyruvate dehydrogenase kinase 4 (PDK4) expression, decreased Runx2 Ser451 phosphorylation, and reduced cell viability. Zinc inhibited Pi-induced mineralization of VSMCs in a dose-dependent manner and also attenuated the pro-calcification effect of PHI in Pi-induced mineralization. Zinc inhibited osteochondrogenic phenotypic switch of VSMCs reflected by lowering Pi uptake, decreasing the expressions of Msx-2, BMP-2, and Sp7 as well as the loss of smooth muscle cell-specific markers. Zinc preserved phosphorylation state of Runx2 Ser451, decreased PDK4 level, and restored cell viability. PHI alone reduced the expression of smooth muscle markers without inducing mineralization, which was also inhibited by zinc. In addition, we observed a significantly lower serum zinc level in CKD as well as in patients undergoing carotid endarterectomy compared to healthy individuals. Conclusion - PHI promoted the loss of smooth muscle markers and augmented Pi-induced osteochondrogenic phenotypic switch leading to VSMCs calcification. This mineralization process was attenuated by zinc. Enhanced vascular calcification is a potential risk factor during PHI therapy in CKD which necessitates the strict follow up of vascular calcification and zinc supplementation.
Highlights
Anemia is a clinical hallmark of chronic kidney disease (CKD) representing a worldwide burden on public health (Levey et al, 2007; Webster et al, 2017) by reducing the quality of life and increasing cardiovascular disease and mortality (KDOQI and National Kidney Foundation, 2006)
We showed that neither high Pi nor PHD inhibitors (PHI) and high Pi + PHI induced BMP-2 and Msx-2 expression as early as 3 or 6 days (Supplementary Figures 2A–D), and only Sp7 gene expression was elevated at 6 days in response to Pi + PHI (20 μmol/L), which was ameliorated by zinc
Since reactive oxygen species (ROS) and oxidative stress are linked to vascular calcification (Scholze et al, 2016), we examined whether zinc inhibits Pi provoked mineralization of VSMC in an MT-dependent fashion
Summary
Anemia is a clinical hallmark of chronic kidney disease (CKD) representing a worldwide burden on public health (Levey et al, 2007; Webster et al, 2017) by reducing the quality of life and increasing cardiovascular disease and mortality (KDOQI and National Kidney Foundation, 2006). A promising approach to correct CKD-associated anemia is the pharmacologic inhibition of hypoxia-inducible factor (HIF) prolyl-4-hydroxylase domain (PHD) proteins. Clinical trials have demonstrated that PHI FG4592 effectively corrects anemia in CKD patients (Besarab et al, 2015, 2016; Provenzano et al, 2016; Chen et al, 2017), together with other HIF PHIs, has gained attention as a promising alternative for erythropoiesis-stimulating agents (Gupta and Wish, 2017). Recent evidence suggests that HIFs play a pivotal role in vascular calcification (Mokas et al, 2016; Zhu et al, 2018). It is uncovered whether HIF stabilizer PHIs have any effect on the mineralization of VSMC. We provide evidence that PHI FG4592 facilitates phosphate induced calcification in VSMC, which is attenuated by zinc supplementations
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