Ambrisentan ameliorates methotrexate nephrotoxicity through modulation of RhoA/ROCK and PI3K/MDM2-associated signaling: network pharmacology-guided discovery and in vivo confirmation

Introduction Methotrexate (MTX) is a potent antineoplastic agent; however, its clinical application is frequently limited by nephrotoxicity, which may result in treatment interruption. Previous evidence indicates that MTX can promote endothelin-1 (ET-1)-associated renal injury. Therefore, this study investigated whether blocking ET-1 receptors with Ambrisentan (AMB) could alleviate MTX-induced acute kidney injury (AKI). Methods Network pharmacology analysis was performed to identify potential targets and pathways associated with AMB and MTX-induced AKI. For In-vivo validation, twenty-four rats were divided into four groups: control, AMB, MTX, and MTX+AMB. Results and Discussion Network pharmacology identified 102 overlapping targets between AMB and MTX-induced AKI, with 20 hub genes identified through protein–protein interaction network analysis, highlighting PPAR-γ as a key target. KEGG pathway enrichment revealed the involvement of RhoA/ROCK and PI3K-associated signaling pathways, in correlation with inflammatory and apoptotic markers. Experimentally, AMB co-treatment improved renal function parameters and histopathological alterations, restored oxidative balance, and attenuated MTX-induced inflammatory and apoptotic responses, including ICAM-1, NF-κB, RhoA/ROCK, Bax, Caspase-3 and p53. Furthermore, AMB restored the expression of PPAR-γ, PI3K, MDM2, and Bcl-2, suggesting the involvement of PI3K/MDM2-associated signaling alterations in AMB-mediated nephroprotection. In conclusion, AMB may mitigate MTX-induced nephrotoxicity through modulation of oxidative stress, inflammation, and apoptosis. Further studies are required to evaluate its effects on MTX pharmacokinetics, renal elimination, antineoplastic efficacy, and therapeutic potential in established MTX-induced AKI.