Regulation of Mitochondrial Respiration in Diabetic Kidney Disease

University Of Tx Md Anderson Can Ctr, Houston TX

Investigators

Abstract

PROJECT SUMMARY Current treatments of diabetic kidney disease (DKD) mainly focus on managing the symptoms of diabetes and do not address the root causes of DKD. Among causal mechanisms associated with the progression of DKD, there is growing recognition of the central role of mitochondrial dysfunction. Importantly, various aspects of mitochondrial function in kidney cells are known to be impaired in experimental models and in individuals with DKD, including enhanced generation of reactive oxygen species (ROS), substantial alterations in mitochondrial dynamics and structure, and mitochondrial electron transport chain dysfunction. Dysregulated mitochondrial electron transport chain (ETC) has recently emerged as a leading cause of mitochondrial dysfunction in DKD, contributing to the progression of DKD and other micro/macrovascular complications of diabetes. However, the nature or ETC dysfunction in the development of DKD remains unknown. This proposal is based on our recent observations (Mise K. et al. Nature Commu 2024), indicating that: (1) the expression of Ndufs4 (NADH: ubiquinone oxidoreductase iron-sulfur protein 4), a subunit of the complex I of the ETC, is consistently reduced in kidney podocytes of diabetic mice; (2) conditional overexpression of Ndufs4 in the podocytes of diabetic mice (Ndufs4podTg) restored ETC function and improved mitochondrial dynamics and structure in podocytes; and (3) diabetic mice with Ndufs4podTg exhibit significant improvement in albuminuria and kidney morphology compared to control DKD mice. Overall, these discoveries highlight the role of ETC remodeling under diabetic conditions. However, several key questions remain to be carefully addressed, including, a) is mitochondrial ETC regulated in DKD? and if so, how? b) can we unbiasedly identify novel regulators of ETC remodeling in DKD? and c) what are the molecular mechanisms and physiological consequences of altered ETC regulators in DKD? In this application, we propose to address these questions by designing an unbiased genome-wide single-cell CRISPR (clustered regularly interspaced short palindromic repeats) platform to identify and validate regulatory genes that affect mitochondrial ETC fitness in DKD. As a proof of principle, we recently conducted a pooled genome-wide CRISPR interference (CRISPRi) screen using a mouse sgRNA library and a mitochondrial ROS sensitive reporter (mito-roGFP2) in Ndufs4 deficient (Ndufs4+/-) vs. Ndufs4+/+ wild type cultured podocytes. We identified a subset of genes that are involved in mROS generation in a Ndufs4-dependent manner in podocytes. We now propose under three well- defined Specific Aims to identify and dissect the molecular mechanisms of action of putative regulators of Ndufs4- mediated ETC dysfunction. We believe that the successful completion of this application will not only fill the knowledge gap on regulators of ETC but will also provide foundational insights into the pathogenesis of DKD. Developing novel strategies based on restoring ETC activity in DKD could be a promising approach toward correcting kidney cells energetics which represents a paradigm shift in the management of DKD patients.