Abstract 4365476: The Klotho Protein Reduces Vascular Calcification via Suppressing GPX4-mediated Ferroptosis in Vascular Smooth Muscle Cells

Objectives: Chronic kidney disease (CKD) patients exhibit elevated cardiovascular risk linked to vascular calcification—pathological calcium-phosphate deposition impairing vascular compliance. Previous studies identified reduced α-Klotho expression in CKD, potentially exacerbating phosphate dyshomeostasis. Hyperphosphatemia promotes vascular calcification by inducing ferroptosis in vascular smooth muscle cells (VSMCs) via GSH/GPX4 pathway suppression. This study aimed to elucidate α-Klotho's role in regulating ferroptosis-dependent calcification. Methods: VSMCs were treated with high-phosphate (2.6 mM) medium to mimic CKD calcific stress, with/without recombinant α-Klotho (100 ng/ml). Osteogenic markers (Runx2, OST, ALP) were assessed by western blot. Ferroptosis markers (Ptgs2/COX2, GSH, GPX4) and lipid peroxidation (Liperfluo) were analyzed via RT-qPCR/western blot. Intracellular Fe 2+ (FerroOrange) and viability (CCK-8) were quantified. Ferroptosis inducer RSL3 was used for validation. Results: (1) High-phosphate-induced calcification&ferroptosis were attenuated by α-Klotho: (i) High-phosphate significantly increased osteogenic markers (Runx2, ALP, OCN; P<0.05 vs. control), suppressed by α-Klotho (Fig 1A). (ii) α-Klotho inhibited RSL3-induced VSMC calcification (Fig 1B). (2) α-Klotho restored redox homeostasis: (i) High-phosphate upregulated COX2 (P<0.05) and downregulated GSH/GPX4; α-Klotho reversed these effects (Fig 2A-B). (ii) High-phosphate increased lipid ROS and Fe 2+ accumulation while reducing viability; α-Klotho suppressed lipid ROS/Fe 2+ and restored viability (Fig 2C-E). (iii) α-Klotho blocked RSL3-triggered calcification (Fig 2F-H). Conclusion: α-Klotho inhibits High-phosphate-driven calcification via dual mechanisms: 1) Reactivating GSH/GPX4 to suppress ferroptosis; 2) Inhibiting VSMC osteogenic transdifferentiation. Conclusions: α-Klotho inhibits High-phosphate-induced VSMC ferroptosis by activating GSH/GPX4, reducing lipid ROS/Fe 2+ accumulation, and restoring viability. Concurrently, it downregulates osteogenic markers (Runx2/ALP/OCN), preventing phenotypic transition. These findings reveal α-Klotho's dual protection against vascular calcification via coordinated suppression of ferroptosis and osteogenic differentiation (Fig 1-2).