Oxidative stress by Ca²⁺ overload is critical for phosphate-induced vascular calcification

Hyperphosphatemia is the primary risk factor for vascular calcification, which is closely associated with cardiovascular morbidity and mortality. Recent evidence showed that oxidative stress by high inorganic phosphate (Pi) mediates calcific changes in vascular smooth muscle cells (VSMCs). However, intracellular signaling responsible for Pi-induced oxidative stress remains unclear. Here, we investigated molecular mechanisms of Pi-induced oxidative stress related with intracellular Ca²⁺ ([Ca²⁺]_i) disturbance, which is critical for calcification of VSMCs. VSMCs isolated from rat thoracic aorta or A7r5 cells were incubated with high Pi-containing medium. Extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin were activated by high Pi that was required for vascular calcification. High Pi upregulated expressions of type III sodium-phosphate cotransporters PiT-1 and -2 and stimulated their trafficking to the plasma membrane. Interestingly, high Pi increased [Ca²⁺]_i exclusively dependent on extracellular Na⁺ and Ca²⁺ as well as PiT-1/2 abundance. Furthermore, high-Pi induced plasma membrane depolarization mediated by PiT-1/2. Pretreatment with verapamil, as a voltage-gated Ca²⁺ channel (VGCC) blocker, inhibited Pi-induced [Ca²⁺]_i elevation, oxidative stress, ERK activation, and osteogenic differentiation. These protective effects were reiterated by extracellular Ca²⁺-free condition, intracellular Ca²⁺ chelation, or suppression of oxidative stress. Mitochondrial superoxide scavenger also effectively abrogated ERK activation and osteogenic differentiation of VSMCs by high Pi. Taking all these together, we suggest that high Pi activates depolarization-triggered Ca²⁺ influx via VGCC, and subsequent [Ca²⁺]_i increase elicits oxidative stress and osteogenic differentiation. PiT-1/2 mediates Pi-induced [Ca²⁺]_i overload and oxidative stress but in turn, PiT-1/2 is upregulated by consequences of these alterations. NEW & NOTEWORTHY The novel findings of this study are type III sodium-phosphate cotransporters PiT-1 and -2-dependent depolarization by high Pi, leading to Ca²⁺ entry via voltage-gated Ca²⁺ channels in vascular smooth muscle cells. Cytosolic Ca²⁺ increase and subsequent oxidative stress are indispensable for osteogenic differentiation and calcification. In addition, plasmalemmal abundance of PiT-1/2 relies on Ca²⁺ overload and oxidative stress, establishing a positive feedback loop. Identification of mechanistic components of a vicious cycle could provide novel therapeutic strategies against vascular calcification in hyperphosphatemic patients.