The role of proteotoxicity and cross-organelle stress response in drug-induced acute kidney injury
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Nephrotoxicity is a dose-limiting side effect of gentamicin that accounts for a significant portion of clinical acute kidney injury (AKI). The mechanism of gentamicin-induced nephrotoxicity is uncertain and effective therapy for gentamicin-induced renal cell injury is limited by incomplete mechanistic insight. To address this challenge, we hypothesized that RNAi signal pathway screening can identify both a unifying mechanism of gentamicin-induced cell injury and a therapeutic that ameliorates its toxicity. Dual shRNA screens of 5,000 individually barcoded signal pathway genes were performed in gentamicin-exposed human proximal tubule cell lines and differentially expressed shRNAs were analyzed by Ingenuity Pathways Analysis (IPA) software. Computational analysis of RNAi signal screens identified the Cross-Organelle Stress Response (CORE), the Unfolded Protein Response (UPR), and cell chaperones as key injury targets of gentamicin-induced proteotoxicity. To validate these injury mechanisms, we assessed the effect of gentamicin on the CORE, UPR and cell chaperone function, and tested the therapeutic efficacy of enhancing cell chaperone content. Early gentamicin exposure disrupted the CORE, evidenced by a rise in the ATP:ADP ratio, mitochondrial-specific H2O2 accumulation, Drp-1 associated mitochondrial fragmentation, and endoplasmic reticulum-mitochondrial dissociation. CORE disruption preceded measurable increases in whole cell oxidative stress, misfolded protein content, transcriptional UPR activation and its untoward downstream effects: CHOP expression, PARP cleavage and cell death. Geranylgeranylacetone, a therapeutic that increases cell chaperone content, prevented mitochondrial H2O2 accumulation, preserved the CORE, reduced the burden of misfolded proteins and CHOP expression, and significantly improved survival in gentamicin-exposed cells. We identify CORE disruption as an early and remediable cause of gentamicin proteotoxicity that precedes downstream cytosolic UPR activation and cell death. Preserving the CORE is associated with improved renal cell survival likely by reducing organelle-specific proteotoxicity during gentamicin exposure.
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