Titanium Dioxide Exacerbates Calcium Phosphate and Urate-Induced Oxidative Stress, Injury and Inflammatory Response...

The widespread use of titanium dioxide (TiO₂) nanoparticles (TNPs) in everyday products (ex; cosmetics, food additives) has growing concerns about their harmful effects on humans due to unavoidable exposure. We recently found that TNPs could stabilize both calcium phosphate (CaP) and uric acid (UA) crystals, implicating kidney stone formation. However, the impact of TNPs on the kidney, specifically on kidney tubular cells is unknown. Therefore, we investigated the effect of TiO₂ alone or in combination with UA/CaP microcrystals on Ca²⁺ dynamics by measuring Ca²⁺ transients, reactive oxygen species (ROS) production by H₂DCFDA staining and H₂O₂ release, oxidative stress and inflammatory response by gene expression analysis, cell death by DAPI and Annexin V/propidium iodide (PI) staining and cytotoxicity by lactate dehydrogenase (LDH) release in human kidney (HK-2) tubular cell line. The internalization of CaP/UA crystals with TiO₂ in HK-2 cells resulted in an enhanced store-operated Ca²⁺ entry (SOCE), which was blocked by Pyr6 (SOCE inhibitor). Indeed, the combination of CaP/UA crystals with TiO₂ -internalized HK-2 cells showed a pronounced increase in ROS production and LDH release compared to CaP and/or UA crystals groups, which were reduced by SOCE inhibition. Analyses of nuclear morphology via DAPI staining indicated a greater cell injury/death response in the presence of TiO₂ with CaP/UA crystals. Annexin V/PI staining revealed a greater percentage of apoptosis/necrosis after internalization of CaP/UA with TiO₂, which was decreased upon preapplication of Pyr6. Gene expression analyses show that internalization of CaP/UA+TiO₂ crystals evoked upregulation of oxidative stress (NOX4), inflammatory (MCP-1, TNF-α), fibrotic (FN-1) and apoptotic (BCL2) genes. Taken together, TNPs, when combined with CaP/UA, became the activator of SOCE to promote oxidative stress, inflammation, tubular injury and cytotoxicity, underscoring their implications in crystal-induced nephropathy and stone formation.