Although methotrexate is widely used in clinical practice, its therapeutic application is frequently restricted by adverse effects, particularly nephrotoxicity. Oxidative stress is considered a principal mechanism underlying methotrexate-induced renal injury. Harmine, a plant-derived alkaloid, exhibits notable antioxidant and anti-inflammatory properties. This study aimed to investigate the protective effects of harmine against methotrexate-induced nephrotoxicity. Mice were randomly allocated into six experimental groups: control (saline), methotrexate (20 mg/kg), harmine (20 mg/kg), and methotrexate (20 mg/kg) combined with harmine at doses of 5, 10, or 20 mg/kg. All treatments were administered intraperitoneally for 14 days. At the end of the treatment period, blood and kidney tissues were collected for biochemical, molecular, and histopathological analyses. Renal tissues were evaluated using hematoxylin–eosin (H&E) staining, quantitative real-time PCR (qRT-PCR), and oxidative stress–related biochemical assays. Methotrexate administration resulted in a significant elevation of serum creatinine and blood urea nitrogen levels, whereas treatment with harmine at doses of 10 and 20 mg/kg significantly attenuated these changes. Harmine also improved the number and diameter of glomeruli in methotrexate-treated mice. In addition, methotrexate markedly increased renal malondialdehyde and nitric oxide levels while reducing total antioxidant capacity and superoxide dismutase activity. Harmine treatment significantly reduced oxidative stress markers and restored antioxidant defense parameters. Furthermore, harmine suppressed methotrexate-induced oxidative stress by downregulating the mRNA expression of Nqo1, Ho-1, Trx1, and Nrf2. Histopathological alterations caused by methotrexate were also markedly ameliorated by harmine administration. These findings indicate that harmine exerts a protective effect against methotrexate-induced nephrotoxicity, primarily through the modulation of oxidative stress and enhancement of antioxidant defenses.
