Roxadustat ameliorates vascular calcification in CKD rats by regulating HIF-2/HIF-1

Vascular calcification (VC) is a common complication of chronic kidney disease (CKD). VC is a gene-regulated process similar to osteogenic differentiation. There are still no convincing schemes to prevent and reduce the development of VC. It has been reported that hypoxia-inducing factor 1 alpha (HIF-1 alpha) and endothelin-1(ET-1) are related to VC. In this study, we found that the expression of ET-1 and HIF-1 alpha was enhanced after VC, the interaction between HIF-1 alpha and ET-1 was confirmed by CO-IP and luciferase experiments. We found that ET-1 was an upregulated differential gene of calcified vascular smooth muscle cells (VSMCs) through gene sequencing. However, hypoxia-inducing factor 2 alpha (HIF-2 alpha) and HIF-1 alpha have antagonistic effects on each other. HIF-1 alpha is a pro-inflammatory cytokine, and HIF-2 alpha can improve inflammation and fibrosis. Roxadustat, as a selective PHD3 inhibitor, preferentially activates HIF-2 alpha. It is still unclear whether roxadustat improves VC in CKD by regulating the expression of HIF-2 alpha/HIF-1 alpha. Alizarin red staining and western blot as well as immunohistochemical results showed that roxadustat could significantly reduce the degree of vascular and VSMCs calcification in CKD rats. Serum HIF-1 alpha and ET-1 were significantly decreased after roxadustat treatment. In addition, western blot results showed that roxadustat could decrease the expression of HIF-1 alpha and ET-1 in vascular tissues and calcified VSMC, but HIF-2 alpha expression significantly increased. Interestingly, our study confirmed that activation of HIF-1 alpha or inhibition of HIF-2 alpha reversed the ameliorating effect of roxadustat on VC, proving that the effect mediated by roxadustat is HIF-2 alpha/HIF-1 alpha dependent. We have demonstrated for the first time that roxadustat improves VC in CKD rats by regulating HIF-2 alpha/HIF-1 alpha, thus providing a new idea for the application of roxadustat in VC of CKD.