INDOLE-3-PROPIONIC ACID-MEDIATED NEPHROPROTECTION AGAINST GENTAMICIN INDUCED OXIDATIVE STRESS AND FERROPTOSIS IN HK-2 RENAL CELLS: AN IN VITRO STUDY
Abstract
Objective: Gentamicin remains an effective aminoglycoside antibiotic; however, nephrotoxicity limits its therapeutic use. Proximal tubular epithelial injury induced by gentamicin involves oxidative stress, mitochondrial dysfunction, lipid peroxidation and ferroptosis. Indole-3-propionic acid (IPA), a gut microbiota-derived tryptophan metabolite, has antioxidant and cytoprotective potential. This study evaluated whether IPA protects HK-2 human renal proximal tubular epithelial cells against gentamicin-induced oxidative stress and ferroptosis. Materials and Methods: HK-2 cells were exposed to gentamicin 3 mM for 24 h. Cells were pretreated with IPA at 25, 50 and 100 µM for 2 h before gentamicin exposure. Ferrostatin-1 was used as a ferroptosis-inhibitor control. Cell viability, LDH release, intracellular ROS, malondialdehyde, glutathione, SOD, catalase, ferrous iron, lipid ROS and 4-HNE were evaluated. qRT-PCR assessed GPX4, SLC7A11, ACSL4, PTGS2, NRF2 and HO-1 mRNA expression. Western blotting assessed GPX4, SLC7A11, ACSL4, NRF2, HO-1 and β-actin. Results: Gentamicin reduced cell viability and increased LDH release, ROS generation, MDA, lipid ROS, ferrous iron and 4-HNE. IPA pretreatment improved viability and restored antioxidant defense in a concentration-dependent manner. Gentamicin downregulated GPX4, SLC7A11 and NRF2 and upregulated ACSL4 and PTGS2, while IPA reversed these alterations and enhanced NRF2/HO-1 signaling. IPA 100 µM produced the strongest protective response and showed a pattern comparable to ferrostatin-1 for key ferroptosis markers. Conclusion: IPA protected HK-2 renal tubular cells against gentamicin-induced oxidative stress and ferroptosis by reducing lipid peroxidation and iron-associated injury while restoring the SLC7A11/GPX4 axis and activating NRF2/HO-1 signaling. IPA may represent a promising nephroprotective candidate for further preclinical validation.