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Background: Mesenchymal stem cells (MSCs) repair injured tissue in a paracrine manner. To enhance their therapeutic properties, preconditioning with various factors has been researched. We have previously showed that MSCs cultured in serum-free medium (SF-MSCs) promote their immunosuppressive ability, thereby enhancing their anti-fibrotic effect. Here, we examined whether serum-free medium and hypoxic preconditioning synergistically enhance the therapeutic effects of MSCs on renal fibrosis in rats with ischemia-reperfusion injury (IRI). Methods: SF-MSCs were incubated under 1% O2 conditions (hypo-SF-MSCs) or 21% O2 conditions (normo-SF-MSCs) for 24 h before collection. After IRI procedure, hypo-SF-MSCs or normo-SF-MSCs were injected through the abdominal aorta. At 7 or 21 days post-injection, the rats were killed and their kidneys were collected to evaluate inflammation and fibrosis. In in vitro experiments, we investigated whether hypo-SF-MSCs enhanced secretion of anti-fibrotic humoral factors using transforming growth factor (TGF)-β1-stimulated HK-2 cells incubated with conditioned medium from hypo-SF-MSCs or normo-SF-MSCs. Results: Normo-SF-MSCs showed attenuation of senescence, which increased their proliferative capacity. Although no significant difference in cellular senescence was found between normo-SF-MSCs and hypo-SF-MSCs, hypo-SF-MSCs further increased their proliferative capacity compared with normo-SF-MSCs. Additionally, administration of hypo-SF-MSCs more strongly ameliorated renal fibrosis than that of normo-SF-MSCs. Moreover, although hypo-SF-MSCs strongly attenuated infiltration of inflammatory cells compared with the control rats, which were treated with PBS, this attenuation was almost equal between normo-SF-MSCs and hypo-SF-MSCs. In vitro experiments revealed that hypo-SF-MSCs more significantly inhibited transforming growth factor (TGF)-β/Smad signaling compared with normo-SF-MSCs. Moreover, hypoxic preconditioning increased hepatocyte growth factor (HGF) secretion even under serum-free conditions, whereas knockdown of HGF in hypo-SF-MSCs attenuated inhibition of TGF-β/Smad signaling. Conclusions: These results indicate that administration of ex vivo-expanded, hypoxia-preconditioned SF-MSCs may be a useful cell therapy to prevent renal fibrosis. [ABSTRACT FROM AUTHOR]

Organophosphorus (OP) compounds such as sarin are toxic agents that irreversibly inhibit the enzyme acetylcholinesterase. A recent study showed that OP compounds also have multiple toxicity mechanisms, and another suggested that endoplasmic reticulum (ER) dysfunction contributes to OP toxicity. However, the signaling pathway and mechanisms involved are poorly understood. We examined whether the sarin-like OP agent bis(isopropyl methyl)phosphonate (BIMP), which exhibits toxicity similar to that of sarin, induced ER stress in human astrocytoma CCF-STTG1 cells. Our results demonstrate that BIMP exposure reduced cell viability. Moreover, it induced changes in mitochondrial membrane potential and increased cleavage of caspase 3. Treatment with BIMP increased the mRNA levels of the ER stress marker genes binding immunoglobulin protein (BiP) and the transcription factor C/EBP homologous protein (CHOP). Furthermore, BIMP increased the protein expressions and phosphorylation of BiP, CHOP, and protein kinase RNA-like ER kinase and the phosphorylation of eukaryotic translation initiation factor 2. Compared to BIMP treatment alone, pretreatment with the CHOP siRNA, siCHOP, decreased BIMP-dependent CHOP expression and improved CCF-STTG1 cell viability. Our findings suggest that BIMP induced mitochondrial dysfunction and apoptotic cell death event mediated by ER stress in CCF-STTG1 cells and that treatment targeted at managing ER stress has the potential to attenuate the toxicity of OP nerve agents. [ABSTRACT FROM AUTHOR]