AKI can progress to AKD and CKD despite apparent recovery. We tested whether persistent mitochondrial dysfunction after gentamicin-induced AKI promotes this transition and whether rapamycin-induced mitophagy is protective.

NRK-52E renal tubular epithelial cells were exposed to gentamicin and allowed to recover in normal culture medium before undergoing hypoxia/reoxygenation (H/R) injury with or without rapamycin treatment. Mitochondrial respiration, mitochondrial dynamics, oxidative stress, apoptosis, and fibrosis-associated markers were assessed. For in vivo study, a sequential “two-hit” mouse model was established using gentamicin-induced AKI followed two weeks later by unilateral ischemia–reperfusion injury (IRI). Renal function, tubular apoptosis, and interstitial fibrosis were examined to characterize progression toward AKD and CKD.

Despite apparent recovery, gentamicin-pre-exposed NRK-52E cells exhibited persistent mitochondrial dysfunction characterized by increased reactive oxygen species generation, impaired mitochondrial respiration, and enhanced susceptibility to subsequent H/R injury. Apoptosis was significantly higher in gentamicin-pre-exposed cells than in cells subjected to H/R alone (37.8% vs. 22.4%). Secondary injury further increased cleaved caspase-3, fibronectin, and α-smooth muscle actin expression, indicating activation of profibrotic pathways. Rapamycin restored basal and maximal oxygen consumption rates, improved mitochondrial morphology, enhanced expression of mitophagy-related proteins (PINK1, Beclin-1, and LC3), increased MFN1 and ATP synthase β levels, and reduced expression of the mitochondrial fission protein Drp1. These protective effects were abolished by the autophagy inhibitor 3-methyladenine, supporting a mitophagy-dependent mechanism. In vivo, serum creatinine and NGAL levels normalized following gentamicin-induced AKI; however, kidneys previously exposed to gentamicin developed significantly greater tubular apoptosis, renal dysfunction, collagen deposition, and interstitial fibrosis after secondary IRI compared with controls. Rapamycin administration before IRI attenuated these pathological changes and preserved renal function.

Gentamicin-induced AKI leaves persistent mitochondrial injury that increases vulnerability to later insults and promotes AKD/CKD progression. Enhancing mitophagy with rapamycin restores mitochondrial homeostasis and reduces fibrosis, suggesting a potential strategy to prevent CKD after AKI.