Universal Loss of Complex I in Oncocytoma is Associated with Defective Mitophagy Via Altered Glutathione Metabolism

NTRODUCTION AND OBJECTIVE: Renal oncocytoma (RO)is the most commonly resected benign renal neoplasm. ROs arecharacterized by a dense accumulation of dysfunctional mitochondriapossibly resulting by increased mitochondrial biogenesis and defectivemitophagy. Although it is well known that ROs harbor recurrent inacti-vating mutations in mitochondrial genes encoding Complex I (NADHdehydrogenase) in the electron transport chain, the mechanism ofdefective mitophagy remains unclear. Here, we hypothesize thatmitophagy inhibition in RO is regulated by Complex I loss, leading toincreased glutathione that oxidizes key players in the mitophagypathway.METHODS: RO and normal kidney (NK) tissues were obtainedfrom patients undergoing surgical resection. DNA was isolated fromfrozen RO samples (n[18) and matched whole blood. Long-rangePCR was performed to sequence mitochondrial DNA (mtDNA).Protein expression was analyzed using Western Blot. Complex I-IVactivity, as measured by oxygen consumption rate, was analyzed bythe Seahorse XF96 analyzer. Gene expression changes wereevaluated using the Nanostring nCounter human metabolic pathwaypanel and analyzed by Rosalind. Metabolic profiles were measuredusing liquid chromatography-tandem mass spectrometry (LC-MS).Statistical calculations were performed using Graphpad Prism.RESULTS: A total of 13 ROs (72.2%) had mtDNA Complex Iloss-of-function (LoF) mutations with 8 (44.4%) having high allelefrequency (>50%). Compared to NK, nearly all ROs had reducedComplex I activity with a compensatory increase of Complex IVactivity, irrespective of identified mtDNA mutation. On the proteinlevel, there was a universal loss of Complex I with resultantcompensatory increase of other complexes regardless of mtDNAstatus. Gene expression profiling revealed increased activation ofAMPK pathway in ROs as well as altered glutathione metabolism,including increased biosynthesis and decreased breakdown.Metabolic profiling confirmed that both reduced and oxidized forms ofglutathione were significantly enriched in ROs compared to NKs. Themechanism of glutathione inhibition of mitophagy is currently beinginvestigated with possible candidates having dysregulated oxidation.CONCLUSIONS: The universal loss of Complex I activity inROs is independent of mtDNA mutations. Complex I loss in RO isassociated with increased glutathione, which in turn may result inimpaired mitophagy. We continue to develop models to interrogate keydrivers in this process