NRF2-suppressed vascular calcification by regulating the antioxidant pathway in chronic kidney disease

Vascular calcification (VC), in which vascular smooth muscle cells (VSMCs) undergo differentiation and osteogenic transition, is a common complication of chronic kidney disease (CKD). Recent findings show that nuclear factor-erythroid-2-related factor 2 (NRF2) is an evolutionarily conserved antioxidant and beneficial in preventing vascular senescence and calcification. The roles of NRF2 in the initiation and progression of VC in CKD still need further investigation. CKD-associated VC model rats exhibited significant upregulation of NRF2, NAD(P)H: quinone oxidoreductase-1 (NQO1), osteogenic markers such as alkaline phosphatase (ALP), runt-related transcription factor-2 (RUNX2) and osteopontin (OPN), and β-catenin compared to CKD rats. Immunohistochemistry further verified these results. In addition, rat aortic VSMCs were isolated and subjected to four treatments: normal control, phosphorus-induced (Pi), Pi + NRF2 activator DMF, and Pi + NRF2 inhibitor ML385. The reactive oxygen species (ROS) generation, malondialdehyde (MDA) content, and calcium deposition of the four treatments were determined. The mRNA and protein expression levels of NRF2, NQO1, and haem oxygenase 1 (HO1) and the osteogenic markers ALP, Runx1, OPN, bone morphogenetic protein 2 (BMP2), and β-catenin were quantified by RT-PCR and western blotting. VSMC apoptosis was calculated by flow cytometry. The in vitro results suggested that intracellular oxidative stress and calcification were closely associated with NRF2 activity and that the activation of NRF2 could significantly suppress osteogenic transition and apoptosis in VSMCs. Thus, this study indicated that the NRF2-related antioxidant pathway can positively respond to and protect against the initiation and progression of VC in CKD by reducing oxidative stress. This study may contribute insights facilitating the application of the NRF2 antioxidative system as a therapeutic treatment for vascular diseases such as CKD.