*Purpose: Ischemia induces altered bioenergetics with increased mitochondrial swelling and reactive oxygen species (ROS) and ultimately degradation of cellular function. Ischemia followed by reperfusion (IRI) induces mitochondrial fragmentation in proximal tubule (PT) cells. Therapeutic interventions that target improvement of mitochondrial health to repair, reprogram or replace mitochondria to restore respiratory functions may be beneficial for prevention of injury.

*Methods: Renal injury was assessed by plasma creatinine (PlCr; mg/dl). 8-wk old C57BL/6 mice were i.v. injected with exogenous mitochondria (Exo-Mito; 50 mcg protein equivalent) 1d before 26 mins/ 24 hours of IRI. Mitochondria was isolated from healthy mouse liver. For in vitro studies, mouse PT cells (TKPTS) were treated with 20 mcg of isolated mitochondria 1d prior to analysis that included measurement of ATP levels, mitochondrial functions (Seahorse flux analyzer), cytokines (PCR), immunofluorescence microscopy (uptake efficiency) and flow cytometry (mitotracker dyes [mitotracker or JC-1]).

*Results: In vivo studies demonstrated mitochondria (50mcg, mouse or human) treated mice are significantly protected compared to vehicle treated mice after IRI [PlCr (2.2±0.05 vs 0.62±0.2), p=0.01; using mouse liver mitochondria]. Mice treated with mitochondria had significantly higher kidney gene expression of PGC1α compared to vehicle treated mice. Structurally (sonicated) or functionally (Rotenone/Antimycin A) altered Exo-Mito no longer protected kidneys from IRI. Transfer of mitochondrial products (DNA, protein or ATP) do not protect kidneys from IRI. Labeled Exo-Mito signal was found in spleen (in macrophages), kidney (in PT, identified with anti-CD13 antibody [labels brush border]), liver and lungs. In kidneys the labeled Exo-Mito signal is found in close proximity to PT endogenous mitochondria. In in vitro studies, Exo-Mito are taken up by TKPTS in a dose dependent manner. Exo-Mito treated TKPTS had significantly higher levels of extra- and intracellular ATP, higher basal oxygen consumption rate and spare respiratory capacity measured by Seahorse analyzer and lower cytokines after LPS stimulation.

*Conclusions: Our data demonstrates that treatment with healthy mitochondria enhances recipient cells energy production to help replace damaged mitochondria by inducing PGC1α to rescue cellular functions. Our current study demonstrates that treatment with healthy mitochondria can be used as therapeutic modality for prevention IRI.

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