The effects of alpha pinene on methotrexate induced renal damage in rats

THE EFFECTS OF ALPHA-PINENE ON METHOTREXATE-INDUCED KIDNEY INJURY IN RATS Objective: Alpha-pinene (AP) is a natural monoterpene that exhibits anti-inflammatory, antioxidant, anti-tumour and anti-apoptotic properties. In this study, the possible protective effects of AP against renal damage induced by methotrexate (MTX), a chemotherapeutic agent widely used in the clinic, were investigated. Materials and Methods: Male Sprague-Dawley rats (2.5-3 months old) were used in the study and animals were divided into 5 groups (n=7) as Control, Vehicle, MTX, AP and AP+MTX. AP administration was performed at a dose of 50 mg/kg for 14 days, while MTX was administered as a single 20 mg/kg dose on the 12th day of the study. On the 14th day of the study, animals were placed in metabolic cages and urine samples were collected, and on the 15th day, animals were sacrificed under anaesthesia and blood and tissue samples were collected. Blood urea nitrogen (BUN) and creatinine levels were measured spectrophotometrically. Serum and urine creatinine values were used in glomerular filtration rate (GFR) calculation. Serum cystatin C, NGAL (Neutrophil gelatinase-associated lipocalin), and Kim-1 (Kidney injury molecule-1) levels were measured using ELISA, while MDA levels were determined spectrophotometrically. Histological evaluation was performed with hematoxylin-eosin staining. Statistical analysis was conducted using one-way ANOVA followed by Tukey post hoc test. Results: Compared with the Vehicle group, statistically significant increases in BUN (p<0.001), serum creatinine (p < 0.001) and cystatin C (p < 0.001) levels and a decrease in GFR (p<0.001) were found in the MTX group. Statistically significant increases were found in urinary levels of NGAL (p<0.001) and Kim-1 (p<0.001), as well as in renal concentrations of Kim-1 (p<0.001) and MDA (p<0.001). AP pretreatment caused a significant improvement in BUN (p<0.05), creatinine (p<0.05), cystatin C (p<0.05), GFR (p<0.05) and urinary NGAL (p<0.01) levels of AP+MTX group, but these values did not completely return to the levels observed in the Vehicle group. There was a more significant improvement in tissue and urine Kim-1 concentration and renal MDA levels, and no statistically significant difference was found among the Control, Vehicle and AP+MTX groups in terms of these parameters. Histopathological evaluation revealed that tubular and glomerular damage caused by MTX was significantly improved with AP pretreatment. Conclusion: In our study, the protective effect of AP against MTX-induced nephrotoxicity was confirmed by biochemical and histopathological findings. While this protective effect, arising from AP's antioxidant properties, cannot entirely eliminate the adverse effects of MTX, it suggests that AP could be a therapeutic candidate for alleviating renal damage. Further experimental studies are required to elucidate the molecular targets and signalling pathways of AP, and to establish the most effective dosage and treatment duration. Keywords: Alpha-pinene, KIM-1, Methotrexate, NGAL, Cystatin C, GFR