Acute kidney injury (AKI) is often caused by ischemia-reperfusion injury (IRI). IRI significantly affects kidney metabolism, which elicits pro-inflammatory responses and kidney injury. The ischemia/reperfusion of the kidney is associated with transient high mitochondrial-derived reactive oxygen species (ROS) production rates. Excessive mitochondrial-derived ROS damages cellular components and, together with other pathogenic mechanisms, elicits a range of acute injury mechanisms that impair kidney function. Mitochondrial-derived ROS production also stimulates epithelial cell secretion of extracellular vesicles (EVs) containing RNAs, lipids, and proteins, suggesting that EVs are involved in AKI pathogenesis. This literature review focuses on how EV secretion is stimulated during ischemia/reperfusion and how cell-specific EVs and their molecular cargo may modify the IRI process. Moreover, critical pitfalls in the analysis of kidney epithelial-derived EVs are described. In particular, we will focus on how the release of kidney epithelial EVs is affected during tissue analyses and how this may confound data on cell-to-cell signaling. By increasing awareness of methodological pitfalls in renal EV research, the risk of false negatives can be mitigated. This will improve future EV data interpretation regarding EVs contribution to AKI pathogenesis and their potential as biomarkers or treatments for AKI.

Extracellular vesicle (EV) secretion rate is stimulated by hypoxia that causes increased reactive oxygen species (ROS) production by the mitochondrial electron transport chain (ETC) and hypoxia‐induced factor (HIF)‐1 signaling; however, their contribution to the increased EV secretion rate is unknown. We found that the EV marker secretion rate in our EV reporter cell line CD9truc‐EGFP was unaffected by the HIF‐1α stabilizer roxadustat; yet, ETC stimulation by dichloroacetic acid (DCA) significantly increased EV secretion. The DCA‐induced EV secretion was blocked by the antioxidant TEMPO and rotenone, an inhibitor of the ETC's Complex I. Under hypoxic conditions, the limited oxygen reduction impedes the ETC's Complex III. To mimic this, we inhibited Complex III with antimycin A, which increased ROS‐dependent EV secretion. The electron transport between Complex I and III is accomplished by coenzyme Q created by the mevalonate pathway and tyrosine metabolites. Blocking an early step in the mevalonate pathway using pitavastatin augmented the DCA‐induced EV secretion, and 4‐nitrobenzoate—an inhibitor of the condensation of the mevalonate pathway with tyrosine metabolites—increased ROS‐dependent EV secretion. Our findings indicate that hypoxia‐mimetics targeting the ETC modify EV secretion and that ROS produced by the ETC is a potent stimulus for EV secretion.

Human aging is linked to bone loss, resulting in bone fragility and an increased risk of fractures. This is primarily due to an age-related decline in the function of bone-forming osteoblastic cells and accelerated cellular senescence within the bone microenvironment. Here, we provide a detailed discussion of the hypothesis that age-related defective bone formation is caused by senescence of skeletal stem cells, as they are the main source of bone forming osteoblastic cells and influence the composition of bone microenvironment. Furthermore, this review discusses potential strategies to target cellular senescence as an emerging approach to treat age-related bone loss.

Phenformin and metformin are antihyperglycemic drugs that belong to the class of biguanides. Previously, we demonstrated that metformin elicits renoprotective effects in unilateral ureteral obstructed mice by reducing the infiltration of immune cells into the kidney. Since phenformin is a more potent drug as compared to metformin, we investigated the renoprotective properties of phenformin. We studied the efficacy of both drugs using mice that underwent unilateral ureteral obstruction. Renal damage was evaluated on RNA and protein level by qPCR, Western blotting, and immunohistochemistry. Moreover, we studied immune cell infiltration using flow cytometry. Both biguanides significantly reduced UUO-induced kidney injury, as illustrated by a reduction in KIM-1 protein expression. In addition, both metformin and phenformin impacted the gene expression of several inflammatory markers but to a different extent. Moreover, in contrast to metformin, phenformin did not impact immune cell infiltration into UUO kidneys. In conclusion, we demonstrated that phenformin has similar renoprotective effects as metformin, but the mechanism of action differs, and phenformin is more potent. The beneficial effects of phenformin are probably due to inhibition of the STAT3 pathway and mitochondrial complex I. Further research is needed to unveil the therapeutic potential of phenformin for the treatment of renal injury, either at low, non-toxic concentrations or as part of a combination therapy.

Human bone marrow-derived stromal cells (also termed mesenchymal stem cell -MSCs) are progenitors capable of differentiating into bone forming osteoblasts and fat storing adipocytes. Due to the loss of bone mass being associated with increased marrow fat, trans-differentiation of osteoblasts into adipocytes been hypothesized as a contributor to osteoporotic bone loss and fragility. Reprogramming of transcriptional networks is a prerequisite for cellular differentiation, however, to which extent cell-type specific transcriptional networks modulate cellular plasticity within stromal cells remains unknown. In this study we performed gene expression analysis at bulk and single cell level in stromal cells being repeatedly exposed to osteogenic and adipogenic inducers in vitro . Surprisingly, cell type specific gene networks are not suppressive but instead promoting to obtain an opposing phenotype, e.g., enhanced osteoblast differentiation of adipogenic pre-stimulated stromal cells compared to undifferentiated ones. Mechanistically, lineage-selective genes with enhanced response upon interconversion are primed in the stem cell state and obtain modest activity levels during exposure to opposing lineage conditions. Finally, the presence of cells simultaneously showing an osteogenic and adipogenic phenotype highlighting a strong molecular plasticity of transcriptional networks in stromal cells. These observations provide a strong molecular support for the notion of not only progenitor specification but also the plasticity of differentiated cells contributing to the balance of bone mass and marrow fat content. Summary: We provide mechanistic evidence, that cells with established lineage-selective gene programs are more vulnerable to obtain gene signatures and phenotypes of opposing lineages, building ground for trans-differentiation between osteoblasts and adipocytes being a coordinated process rather than random and misdirected.

Human bone marrow-derived stromal cells (also termed mesenchymal stem cell) are progenitors capable of differentiating into bone-forming osteoblasts and fat-storing adipocytes. Due to the loss of bone mass being associated with increased marrow fat, trans-differentiation of osteoblasts into adipocytes has been hypothesized as a contributor to osteoporotic bone loss and fragility. Reprogramming of transcriptional networks is a prerequisite for cellular differentiation; however, to which extent cell type-specific transcriptional networks modulate cellular plasticity within stromal cells remains unknown. In this study, we performed gene expression analysis at bulk and single cell level in stromal cells being repeatedly exposed to osteogenic and adipogenic inducers in vitro. Surprisingly, cell type-specific gene networks are not suppressive but instead promoting to obtain an opposing phenotype, for example, enhanced osteoblast differentiation of adipogenic prestimulated stromal cells compared to undifferentiated ones. Mechanistically, lineage-selective genes with enhanced response upon interconversion are primed in the stem cell state and obtain modest activity levels during exposure to opposing lineage conditions. Finally, the presence of cells simultaneously shows an osteogenic and adipogenic phenotype highlighting a strong molecular plasticity of transcriptional networks in stromal cells. These observations provide a strong molecular support for the notion of not only progenitor specification but also the plasticity of differentiated cells contributing to the balance of bone mass and marrow fat content.