2.4. Analysis of complex I and complex II mediated respiration in kidney mitochondria

In order to define specific alterations in the mitochondrial respiration in LPS model of sepsis and test the potential protection by mito2HOBA we have adopted the Seahorse protocol for mitochondrial studies [32] in the presence of mitochondrial substrates Glutamate + Malate (GM) or succinate. To define the specific role of complex I mediated respiration we performed measurements in the presence of complex II inhibitor malonate (5 mM). We have measured basal respiration in the presence of mitochondria plus substrates, coupled respiration after addition of ADP (2 mM), proton leak following addition of oligomycin A (2.5 μM), uncoupled respiration after supplementation of CCCP (1 μM), and non-mitochondrial respiration with mixture of antimycin A and rotenone (1 μM) [32]. Mitochondrial studies where independently verified in two labs using Oroboros O2k high-resolution respirometry and Fluorescence Lifetime Micro Oxygen Monitoring System (Instech Laboratories, Inc). Kidney mitochondria were isolated from control sham mice, LPS-injected mice (25 μg/g, 16 h post-injection), mito2HOBA supplemented mice (0.1 g/Liter drinking water, 4 days), or mito2HOBA plus LPS (0.1 g/Liter mito2HOBA for 3 days plus LPS injection). One kidney was used for mitochondrial studies and second kidney was used for histopathological studies.