Introduction: Renal oncocytoma (RO) is the most commonly resected benign renal neoplasm. ROs are characterized by a dense accumulation of dysfunctional mitochondria possibly resulting by increased mitochondrial biogenesis and defective mitophagy. Although it is well known that ROs harbor recurrent inactivating mutations in mitochondrial genes encoding Complex I (NADH dehydrogenase) in the electron transport chain, the mechanism of defective mitophagy remains unclear. Here, we hypothesize that mitophagy inhibition in RO is regulated by Complex I loss, leading to increased glutathione that oxidizes key players in the mitophagy pathway. Methods: RO and normal kidney (NK) tissues were obtained from patients undergoing surgical resection. DNA was isolated from frozen RO samples (n=18) and matched whole blood. Long-range PCR was performed to sequence mitochondrial DNA (mtDNA). Protein expression was analyzed using Western Blot. Complex I-IV activity, as measured by oxygen consumption rate, was analyzed by the Seahorse XF96 analyzer. Gene expression changes were evaluated using the Nanostring nCounter human metabolic pathway panel and analyzed by Rosalind. Metabolic profiles were measured using 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 I loss-of-function (LoF) mutations with 8 (44.4%) having high allele frequency (>50%). Compared to NK, nearly all ROs had reduced Complex I activity with a compensatory increase of Complex IV activity, irrespective of identified mtDNA mutation. On the protein level, there was a universal loss of Complex I with resultant compensatory increase of other complexes regardless of mtDNA status. Gene expression profiling revealed increased activation of AMPK pathway in ROs as well as altered glutathione metabolism, including increased biosynthesis and decreased breakdown. Metabolic profiling confirmed that both reduced and oxidized forms of glutathione were significantly enriched in ROs compared to NKs. The mechanism of glutathione inhibition of mitophagy is currently being investigated with possible candidates having dysregulated oxidation. Conclusions: The universal loss of Complex I activity in ROs is independent of mtDNA mutations. Complex I loss in RO is associated with increased glutathione, which in turn may result in impaired mitophagy. We continue to develop models to interrogate key drivers in this process. SOURCE OF Funding: AUA Urology Care Foundation Residency Research Award AUA Research Scholar Award
