Project Summary Chronic kidney disease (CKD) is a common condition with limited treatment options. Because late-stage CKD is progressive and irreversible, molecular research to better understand the etiology of CKD progression and identify novel therapeutic targets is critically needed to improve clinical outcomes. Mitochondria play an important role in regulating kidney function, supporting the high energy demands of this end-organ. Mitochondrial DNA copy number (mtDNA-CN), a quantitative indicator of mitochondrial function, has been associated with the incidence of CKD. Our recently published data (CJASN 2022; 17: 966-975) further showed that lower baseline mtDNA-CN is associated with risk of CKD progression. However, longitudinal studies, with repeated measures of mtDNA-CN, have never been conducted in a CKD setting. Such research is needed to 1) evaluate whether mtDNA-CN decline predicts kidney function decline and 2) delineate its upstream determinants and downstream mechanisms. Kidney function may also be influenced by mtDNA quality, which can be assessed as mtDNA heteroplasmy (mtDNA-Het). mtDNA-Het reflects the presence of both mutant and wild-type mtDNA copies. In the only study to examine the association of mtDNA-Het with CKD progression, our pilot work targeting mutations at 25 random mtDNA markers showed that CKD patients with mtDNA-Het had a significant 2.4-fold higher hazard of CKD progression than those without mtDNA-Het. Sequencing of the entire mtDNA genome (16,569 markers) is needed to fully characterize the role of mtDNA-Het in CKD progression. Overall, our preliminary findings provide strong premise for our main hypothesis: mtDNA quality and quantity associate with CKD progression through known and novel CKD related risk factors. To characterize the role of mtDNA quality and quantity in CKD progression, we propose to measure mtDNA-Het at baseline and mtDNA-CN at baseline and 3- and 6-years of follow-up among 5,499 participants of the Chronic Renal Insufficiency Cohort (CRIC). The stringently ascertained CKD progression events and annually assessed data on clinical and molecular biomarkers in CRIC will allow us to assess the contribution of baseline mtDNA-Het (Aim 1) and mtDNA-CN changes (Aim 2) to CKD progression; delineate downstream mechanisms of mtDNA- Het and mtDNA-CN (Aim 3); and discover unique upstream determinants of mtDNA-CN decline (Aim 4) in a CKD setting. We will replicate CRIC findings among 3,498 CKD patients of the Trans-Omics for Precision Medicine program. The proposed work represents the first study to examine the role of mtDNA-Het in CKD progression and the first longitudinal study of mtDNA-CN decline in a CKD setting. Innovative methods, including state-of-the-art mtDNA variant calling pipeline to account for the circular nature of mtDNA and cross- lagged panel modeling to delineate causal directions, will be employed. Since mtDNA-CN and mtDNA-Het are modifiable, our findings have potential to not only revea...