MiR-181c-5p-SIRT1 axis-driven Pink1/Parkin-mediated mitophagy prevents ferroptosis and vascular calcification in chronic kidney disease

Background: Vascular calcification (VC) is a severe cardiovascular complication of chronic kidney disease (CKD), driven by vascular smooth muscle cell (VSMC) osteogenic trans-differentiation and exacerbated by oxidative stress and cellular dysfunction. Despite its clinical relevance, the molecular mechanisms underlying CKD-associated VC remain incompletely understood. This study investigates the role of Sirtuin 1 (SIRT1) in modulating VC through Ferroptosis inhibition and Mitophagy activation and examines whether microRNA-181c-5p (miR-181c-5p) contributes to SIRT1 dysregulation in this context.

Methods: A CKD-associated VC model was induced in rats by 5/6 nephrectomy followed by high calcium/phosphate and calcitriol loading, and an in vitro calcification model was established in primary rat VSMCs. SIRT1 was manipulated using AAV9-mediated overexpression in vivo and plasmid overexpression or inhibition in vitro. Upstream regulation of SIRT1 by miR-181c-5p was predicted bioinformatically and validated by RNA pull-down and dual-luciferase assays. Ferroptosis was assessed by redox and Fe²⁺ indices, and Mitophagy by Pink1/Parkin, LC3-II and p62 expression. Rescue experiments employed erastin, Mdivi-1 and Parkin knockdown.

Results: SIRT1 expression was markedly reduced in calcified aortic tissues and VSMCs. SIRT1 overexpression suppressed VC by reducing calcium deposition, downregulating osteogenic markers, and increasing fetuin-A levels. SIRT1 also suppressed Ferroptosis by restoring the GSH/GPX4/SLC7A11 axis and limiting ROS and lipid peroxidation, whereas erastin abolished these effects. Mechanistically, miR-181c-5p was found to directly target SIRT1 and promote VSMC calcification by repressing SIRT1. Moreover, SIRT1 promoted Mitophagy via the Pink1/Parkin pathway activation. Furthermore, inhibition of Mitophagy reversed the anti-ferroptotic effects of SIRT1, confirming their functional interplay.

Conclusions: SIRT1, negatively regulated by miR-181c-5p, mitigates CKD-associated VC by suppressing Ferroptosis and activating Pink1/Parkin-dependent Mitophagy in VSMCs, highlighting a potential therapeutic axis for vascular protection in CKD.

Clinical trial registration number: Not applicable.

Chronic kidney disease; Ferroptosis; Mitophagy; Pink1/Parkin; SIRT1; Vascular calcification.