Echinocystic acid ameliorates ischemic acute kidney injury in neonatal rats by attenuating ferroptosis via the Nrf2/GPX4 pathway.

Acute kidney injury (AKI) is the most common complication in neonates with hypoxic-ischemic encephalopathy (HIE), significantly contributing to both morbidity and mortality, and targeting key pathological processes, such as inflammation, ferroptosis and apoptosis, could be an effective approach to improving survival outcomes in these patients. In this context, echinocystic acid (EA), a pentacyclic triterpene, has shown promising anti-inflammatory, antioxidant, and anti-apoptotic effects in various disease models, suggesting its potential as a therapeutic agent for AKI in HIE. To evaluate the therapeutic potential and underlying mechanisms of EA in ameliorating ischemia/reperfusion (IR)-induced AKI in neonatal rats. Seven-day-old neonatal rat pups were subjected to an IR injury model to induce AKI and treated with EA via intraperitoneal injection. The effects of EA on renal injury were assessed using western blotting, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), immunofluorescence, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and hematoxylin and eosin (H&E) staining. Treatment with EA significantly reduced IR-induced renal pathology and injury scores, as well as serum levels of blood urea nitrogen (BUN) and creatinine (Cr). In addition, EA diminished the release of pro-inflammatory cytokines and reduced the levels of F4/80, a macrophage marker, in the IR-treated pups. EA also attenuated ferroptosis, as evidenced by decreased levels of iron (Fe²⁺), reactive oxygen species (ROS), myeloperoxidase (MPO), and malondialdehyde (MDA), while simultaneously increasing the activity of antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), and glutathione (GSH). Furthermore, EA reduced apoptosis, as demonstrated by lower levels of cleaved caspase 3 and cleaved poly(ADP-ribose) polymerase (PARP). Mechanistically, EA activated the Nrf2/GPX4 pathway, and inhibition of Nrf2 with ML385 reversed EA's beneficial effects on ferroptosis, inflammation, and renal injury. EA may relieve ischemic AKI in neonatal rats by modulating inflammation, ferroptosis and apoptosis, through the activation of the Nrf2/GPX4 pathway, indicating that it could be a promising therapeutic agent for AKI in neonates.