Melatonin Attenuates Triple Whammy-Induced Acute Kidney Injury: In Vivo, In Vitro, and In Silico Findings

Abstract

This study assessed the nephroprotective potential of melatonin in a triple whammy-induced acute kidney injury (AKI) model using in vivo, in vitro, and in silico approaches. Thirty male rats were randomly assigned to 6 groups: Normal control, negative control, N-acetylcysteine (NAC) as a positive control, and 3 melatonin-treated groups (0.5, 1.0 and 1.5 mg/kg). Treatments were administered orally for 5 days, followed by AKI induction via the triple whammy regimen for 2 days. Renal function was evaluated using serum and urine biomarkers, and kidney tissues were examined histologically. In vitro antioxidant assays and molecular docking studies were performed to assess melatonin and NAC interactions with NF-κB and caspase-3. The triple whammy regimen significantly increased renal injury markers and caused structural kidney damage. High-dose melatonin (1.5 mg/kg) significantly improved serum creatinine, urea, and urinary protein levels (p < 0.05) and preserved glomerular and tubular architecture. In vitro, melatonin demonstrated strong antioxidant activity (IC₅₀ = 3.19 µg/mL), comparable to NAC (IC₅₀ = 3.10 µg/mL). Molecular docking revealed moderate to strong binding affinities of melatonin to caspase-3 (−6.0 kcal/mol) and NF-κB (−7.5 kcal/mol). In conclusion, melatonin confers nephroprotection in triple whammy-induced AKI potentially through antioxidant effects and interaction with apoptotic and inflammatory protein targets. Further studies are warranted to evaluate the translational potential of these findings in clinical settings. HIGHLIGHTS This is the first study showing melatonin’s nephroprotective effect against triple whammy-induced acute kidney injury (AKI) using integrated in vivo, in vitro, and in silico High-dose melatonin markedly improved renal function, preserved kidney histology, and showed potent activity comparable to N-Acetylcysteine (NAC) Molecular docking revealed melatonin had stronger binding affinities to caspase-3 and NF-κB than NAC, supporting its potential as a promising therapeutic candidate for drug-induced AKI. GRAPHICAL ABSTRACT

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