Although methotrexate is widely used in clinical practice, its therapeutic application is frequently restricted due to adverse effects, particularly nephrotoxicity. Oxidative stress is recognized as a primary mechanism underlying methotrexate-induced renal injury. Harmine, a naturally occurring plant-derived compound, exhibits antioxidant and anti-inflammatory activities. This study aimed to investigate the protective effects of harmine against methotrexate-induced kidney toxicity. Mice were randomly assigned to 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 over a 14-day period. At the end of the experiment, blood and kidney tissues were collected for further analysis. Histopathological evaluation was performed using hematoxylin–eosin (H&E) staining, while molecular and biochemical assessments were conducted using qRT-PCR and standard biochemical assays. Administration of methotrexate 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 both the number and diameter of glomeruli in methotrexate-treated mice. In addition, methotrexate markedly increased renal malondialdehyde and nitric oxide levels and reduced total antioxidant capacity and superoxide dismutase activity. Harmine treatment effectively reduced oxidative stress markers and restored antioxidant defense systems. Furthermore, harmine downregulated the methotrexate-induced upregulation of Ho-1, Nqo1, Trx1, and Nrf2 mRNA expression. Histological abnormalities caused by methotrexate were also substantially alleviated by harmine administration. These findings indicate that harmine exerts a protective effect against methotrexate-induced nephrotoxicity, likely through the suppression of oxidative stress and enhancement of antioxidant mechanisms.
