Indoxyl Sulfate Drives Vascular Calcification by the mTORC1-ER Stress- Integrated Stress Response Axis

Medial vascular calcification is a major contributor to cardiovascular morbidity in chronic kidney disease (CKD) and is exacerbated by the accumulation of uremic toxins such as indoxyl sulfate (IS), a gut microbiota-derived metabolite. However, the molecular mechanisms by which IS promotes vascular smooth muscle cell (VSMC) calcification remain incompletely defined. In this study, we investigated the role of the mechanistic target of rapamycin complex 1 (mTORC1)–endoplasmic reticulum (ER) stress–integrated stress response (ISR) signaling axis in IS-induced calcification of human aortic smooth muscle cells (hASMCs). Exposure of cultured hASMCs to IS led to dose-dependent increases in reactive oxygen species (ROS) production, osteochondrogenic transdifferentiation, cellular senescence, and calcium deposition. These pathological responses were further amplified under high-phosphate conditions. Mechanistically, IS activated MEK–ERK1/2 and PI3K–AKT signaling, resulting in increased mTORC1 activity, suppressed autophagy, ER stress, and ISR activation, as evidenced by elevated levels of phosphorylated eIF2α, ATF4, and CHOP. Importantly, pharmacologic interventions that raised intracellular NAD⁺ (via nicotinamide mononucleotide, NMN) or reduced ER stress (via tauroursodeoxycholic acid, TUDCA) attenuated IS-induced ISR activation, osteogenic transdifferentiation, and senescence in hASMCs. These findings demonstrate that IS promotes hASMC calcification through ROS-mediated activation of the mTORC1–ER stress–ISR axis. Targeting this pathway may offer novel therapeutic opportunities to prevent or reduce vascular calcification and cardiovascular complications in patients with CKD.