Targeting mitochondrial dysfunction to mitigate renal ischemia-reperfusion injury: mitochondrial transplantation and oxygen-loaded nanocarriers

Renal ischemia, caused by a reduction or interruption of blood supply to the kidney, severely compromises proximal tubular cells, which rely on oxidative phosphorylation. During reperfusion, the restoration of blood flow triggers the release of reactive oxygen species and other inflammatory mediators into circulation, favouring the activation of the immune response and cell death pathways. As a consequence, the process of ischemia-reperfusion causes severe histological and functional damage to the kidney. Aim of the present thesis was to test emerging nanotherapeutic strategies: mitochondrial transplantation, oxygen-loaded nanobubbles (OLNBs), and oxygen-loaded nanodroplets (OLNDs) to counteract hypoxia and restore mitochondrial function. We first set up a model of mitochondrial isolation from Human Kidney-2 (HK-2) cells using four different protocols. Once the best method was identified – based on flow cytometry results, protein yield and ATP production – isolated mitochondria were tested in an in vitro model recapitulating ischemia-reperfusion renal damage, either immediately after isolation or after storage at different temperatures in the presence or absence of DMSO. Their effects on tricarboxylic acid (TCA) cycle enzyme activities, electron transport chain (ETC) function, and overall ATP levels were determined. Additionally, the molecular mechanism responsible for the cellular uptake of fluorescently-labelled isolated mitochondria was investigated through flow cytometry. In parallel, oxygen-loaded nanocarriers were also studied. OLNBs and OLNDs, both nanosized bubbles consisting of an inner core surrounded by an outer shell, differ in their chemical composition. OLNBs and OLNDs were quantified through Nanoparticle Tracking Analysis and tested in our in vitro model of renal chemical anoxia. Cytotoxicity of both OLNBs and OLNDs was determined using MTT assays and the effects of OLNDs on TCA cycle enzymes, ETC function and overall ATP levels were assessed. Overall, our results demonstrate that both isolated mitochondria (either freshly isolated or stored) and OLNDs exert significant beneficial effects on metabolically damaged renal cells. These approaches represent promising therapeutic strategies in the regenerative medicine field for the treatment of renal ischemia-reperfusion injury