Ginsenoside Ro alleviated cuproptosis induced by high oxalate via inhibiting zinc transporter ZnT1 in renal tubular epithelial cells.

BACKGROUND: Kidney stone disease (KSD), notably characterized by heightened oxidative stress and cell death within renal tubular epithelial cells due to high oxalate. Ginsenoside Ro (Ro), a scarce oleanane-type saponin isolated from Panax ginseng Mey. and Achyranthes bidentata Bl., has garnered attention for the anti-tumor, anti-oxidant, and anti-inflammatory properties. This study aims to investigate the protective effects and mechanisms of Ro on crystal-induced renal injury in vivo and in vitro. MATERIALS AND METHODS: In vitro, we assessed renal injury, renal crystal deposition, and inflammatory infiltration in a glyoxylic acid (Gly)-induced stone formation mouse model, with administration of Ro. The protective effects of Ro on proximal tubular epithelial cell line HK-2 damaged by hyperoxaluria were assessed via MTS assay and live/dead cell staining in vivo. Additionally, we examined reactive oxygen species (ROS) levels and crystal adhesion-related proteins. Subsequently, we identified ZnT1 as the target of Ro through RNA-seq, immunohistochemical (IHC) staining, Western blotting (WB), molecular docking, molecular dynamics simulations, bio-layer interferometry (BLI) and cellular thermal shift assay (CETSA). Further, we constructed HK-2 cells over-expressing and knocking out of ZnT1 to evaluated cellular damage and crystal adhesion effects. Finally, the role of Ro in high oxalate-induced cuproptosis in HK-2 was examined via cuproptosis-related events, including DLAT oligomerization, cuproptosis-associated proteins FDX1 and HSP70, mitochondrial ROS levels, JC-1 staining, and GSH levels. RESULTS: Ro effectively mitigated renal injury induced by Gly in mice. Moreover, it notably ameliorated renal crystal deposition and infiltration of F4/80-positive macrophages observed in Gly mice. Additionally, in vitro studies demonstrated that Ro alleviated oxidative damage and crystal adhesion induced by high oxalate in HK-2 cells. Mechanistically, Ro significantly suppressed ZnT1 expression, and notably, over-expression of ZnT1 reversed the inhibitory of Ro on NaOx-induced proliferation suppression, crystal adhesion, and augmented ROS generation in HK-2 cells. Additionally, NaOx elevated cuproptosis in HK-2 cells, and this elevation was blocked by ZnT1 over-expression, which in turn was reversed by TTM, a cuproptosis inhibitor. CONCLUSION: This study provided evidence that Ro mitigated cuproptosis HK-2 cells induced by high oxalate through inhibiting ZnT1, thus effectively suppressing oxidative stress and crystal deposition triggered by high oxalate.