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

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Abstract
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 Ca 2+ 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.

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