Rac1 Promotes Proximal Tubule Kidney Repair by Coupling the Actin Cytoskeleton to Mitochondrial Function

Background: The kidney proximal tubule (PT) is a specialized polarized epithelium that functions as a high capacity resorptive machine. PT cells are exquisitely sensitive to ischemia due to their high mitochondrial content and high metabolic rate. The small GTPase Rac1 regulates epithelial function by promoting polarity through its effects on the actin cytoskeleton. While Rac1 classically associates with the plasma membrane, it can also tightly control the homeostasis of other organelles including mitochondria via unknown mechanisms. We assessed the hypothesis that Rac1 plays a role in the repair of the proximal tubule. Methods: We deleted Rac1 in the PT by crossing γGT-Cre with Rac1-floxed mice and performed ischemia-reperfusion injury (AKI-IRI). We assessed metabolism by Seahorse and mitochondrial structure by super resolution imaging and electron microscopy. We used CRISPR to generate Rac1 Knockout PT cells in vitro and subjected them to Seahorse and confocal live imaging of actin cytoskeleton and mitochondrial dynamics using various reporters. Results: γGT:Rac1f/f (mutant) mice showed decreased proximal tubular reabsorption function and cortical ATP levels after AKI followed by near complete tubular atrophy 3 months after injury. Injured mutant kidneys demonstrated reduced mitochondrial oxygen consumption, loss of normal mitochondrial morphology, and lipid droplet accumulation, all indicative of decreased mitochondrial function. This was confirmed by ultrastructural assessment which revealed extensive persistent mitochondrial damage and a drastic decrease of mito-autolysosomes suggesting a defect in mitochondrial clearance. Rac1 null PT cells in vitro were unable to regulate mitochondrial dynamics and metabolic recovery due to a defect in actin cytoskeleton-driven mitophagy after depolarizing injury. Induction of mitophagy using UMI-77 after AKI-IRI in mutant mice restored mitochondrial structure, metabolism, and tubular morphology. Conclusion: In summary, we propose that Rac1 promotes PT metabolism after injury by actin-dependently clearing damaged mitochondria which is required to restore mitochondrial integrity providing the energy to maintain tubular cell shape and function after injury. Funding: NIDDK Support, Veterans Affairs Support