Wulingsan alleviates cisplatin-induced acute kidney injury and inhibits renal tubular epithelial cell apoptosis in association with the CaSR/CaMKKβ/AMPK pathway.

INTRODUCTION

The calcium-sensing receptor (CaSR), a G protein-coupled receptor (GPCR), plays a critical role in kidney injury by promoting inflammation and apoptosis when activated. Whether Wulingsan (WLS), a traditional Chinese herbal formula used for kidney disorders, exerts its protective effects against acute kidney injury (AKI) via modulation of the CaSR pathway remains unclear.

METHODS

WLS's chemical profile was characterized by ultra-high performance liquid chromatography coupled with Q-Exactive mass spectrometry (UHPLC-QE-MS). C57BL/6J mice were divided into Control, cisplatin (CP)-induced AKI, WLS (2.34/4.68/9.36 g/kg), and CaSR inhibitor NPS2143 (4.5 mg/kg) groups. Mice were pretreated with WLS/NPS2143 before CP injection (20 mg/kg, i. p.). Renal function, histopathology, inflammation, oxidative stress, and apoptosis were evaluated. Cisplatin-induced mouse renal tubular epithelial cells (mRTECs) were treated with WLS-containing serum (5%-10%) for in vitro validation. Molecular docking predicted the binding affinity of WLS components to CaSR.

RESULTS

UHPLC-QE-MS identified 15 major WLS components, with 13 binding to CaSR (Polyporusterone E: binding energy of -10.4 kcal/mol). WLS pretreatment significantly improved renal function (reduced serum creatinine (SCr)/blood urea nitrogen (BUN)), attenuated tubular injury, suppressed inflammation (downregulated monocyte chemoattractant protein-1 (MCP-1)/tumor necrosis factor-α (TNF-α)/interleukin-1β (IL-1β)), alleviated oxidative stress (restored glutathione (GSH)/superoxide dismutase (SOD), reduced malondialdehyde (MDA)), and inhibited apoptosis (decreased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells, modulated Bcl-2-associated X protein (Bax)/B-cell lymphoma-2 (Bcl-2) ratio, downregulated cleaved Caspase 3). Mechanistically, WLS inhibited renal CaSR/calmodulin-dependent protein kinase β (CaMKKβ)/adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway activation (reduced CaSR/CaMKKβ/p-AMPK expression). In vitro, WLS-containing serum reduced cisplatin-induced reactive oxygen species (ROS) production, intracellular Ca2+ overload, and apoptosis in mRTECs, effects reversed by the CaSR agonist cinacalcet.

CONCLUSION

This study demonstrates that WLS protects against CP-induced kidney injury and inhibits apoptosis in renal tubular epithelial cells in association with the CaSR/CaMKKβ/AMPK pathway.