Project Summary/Abstract Mitochondrial dysfunction is a hallmark of normative aging and of kidney disease and mitochondrial DNA (mtDNA) damage and mutation accumulation has been proposed as one underlying cause. A clear understanding of the functional role of somatic mtDNA mutation in age-related mitochondrial dysfunction has been impeded, however, by the limited accuracy of modern mutation detection techniques and the complexities of experimental approaches to isolate specific cells and their components. Furthermore, many studies have underestimated the importance of tissue-specific analysis of mtDNA mutation by broadly applying single organ studies to make assumptions of organismal-level mechanisms. By implementing Duplex Sequencing, an ultra- accurate sequencing method designed to detect mutations with a frequency as low as 1x10-7, we have been able to characterize the tissue-specific patterns of somatic mtDNA mutation across 10 tissues from young and aged mice. In doing so, we identified unique aging mutation patterns between organs, with kidney cortex showing the highest frequency of somatic mtDNA mutations. Even within the kidney we found regional differences by comparing mutation rates in the tubule-rich kidney cortex to isolated renal glomeruli, thus revealing that the glomerulus has a significantly lower point mutation frequency, a lower frequency of oxidative mtDNA mutations and differential accumulation of mutations in mtDNA genes, as compared to the whole cortex. These results demonstrate that mtDNA somatic mutation accumulation is cell-specific within the kidney. Based on the premise that age-associated somatic mtDNA mutation in the kidney is determined by cell-specific differences in the ability to respond to mutation accumulation, we will utilize advanced technological approaches, including Duplex Sequencing, to address two Aims. In Aim 1, mitochondria from unique renal cell populations will be accurately isolated and analyzed by taking advantage of a Cre-Lox mitochondrial reporter mouse (MITO-Tag) crossed with mice expressing either a glomerular podocyte (podocin) or tubule epithelia (KSP) Cre. Mutation burden, mitochondrial energetics and mitophagy will be analyzed from single cell-type populations in the context of somatic mutation accumulation through natural aging. In Aim 2, kidney-specific mitochondrial dysfunction will be generated through uni-nephrectomy and by introducing a high fat/high sucrose diet as a model of premature kidney aging; this will allow us to elucidate the molecular mechanisms involved in somatic mutagenesis of renal mtDNA under oxidative stress and in response to interventions aimed at protecting the mitochondria; specifically, SS-31, a rejuvenating peptide with potential translational applications. This project will develop novel tools to clarify the role of cell-type and age-associated somatic mtDNA mutation in the kidney and provide a new perspective on the contribution of DNA mutation and aging to kid...