Treating kidney injury using soundwaves combined with mesenchymal stem cells and their extracellular vesicles - PROJECT SUMMARY In the present proposal, we aim to validate novel translational approaches (i.e. targeted local delivery into the kidney and microenvironment modulation of the kidney) and novel regenerative therapies (i.e. pulsed focused ultrasound (pFUS) primed mesenchymal stem cells (MSCs) and their secreted extracellular vesicles (MSC-EVs)) to treat acute kidney injury (AKI) resulting from Ischemia-Reperfusion Injury (IRI). IRI-AKI is a significant cause of morbidity following major cardiac and vascular surgery, kidney transplantation, sepsis or hemorrhagic shock, and has a complex and dynamic pathophysiology as it progresses through 3 phases: ischemia (which causes metabolic shifts due to mitochondrial dysfunction and ATP depletion), reperfusion (which causes reactive oxygen species (ROS) production) and inflammation (which can activate the immune system and, if left unchecked, can trigger fibrotic pathways). We have developed and characterized a reproducible IRI-AKI preclinical mouse model, that has molecular changes closely matching those seen in human patients. Given the translational potential of this model, we now propose to evaluate bone marrow derived MSCs (BM-MSCs) and EVs (BM-EVs) as therapies for IRI-AKI. Based on our characterization studies, these therapies have the optimal functional phenotype to address the activated pathways in IRI-AKI compared to other MSC sources, given their high expression of pro- angiogenic, metabolic, growth, immunomodulatory and anti-inflammatory factors. Interestingly, we have developed a novel approach to reproducibly stimulate BM-MSCs to produce primed therapies (i.e. pBM-MSCs and pBM-EVs) using pFUS, with therapies having an enhanced therapeutic phenotype, enriched metabolic cargo, reduced expression of factors that can activate the coagulation system, and increased expression of connexin-43 (Cx43) that can form gap junctions (GJs) with corresponding Cx43 proteins whose expression are also increased on cells in the injured kidney. Hence, we will fully optimize the generation pBM-MSCs (Aim 1) and pBM-EVs (Aim 2) and characterize their phenotype to ensure reproducibility among different donors from different sexes. Next, we will validate their therapeutic capabilities in vitro in a hypoxia-normoxia injury model with renal organoids, and then in vivo in an IRI-AKI animal model especially after intra-arterial (IA) delivery of these therapies directly into the injured kidney. To confirm that our primed therapies are working to restore metabolic shifts by transferring their cargo into injured cells via Cx43-GJs, we will next test these therapies in which Cx43 has been silenced. To determine how pFUS modulates the injured kidney microenvironment in IRI- AKI, we will assess how different sonication protocols can stimulate regenerative pathways, correct metabolic shifts and even help pBM-MSCs and pBM-EVs retention, thus facilitating their spatial co-localization next to injured cells for cargo transfer (Aim 3). Finally, we will validate our optimized therapeutic candidate and approach (i.e. dose, delivery route, timing, and pFUS priming protocol) following IRI-AKI -/- KO mouse.
