Exosomes Derived From Human Umbilical Cord Mesenchymal Stem Cells Reduce Injury Of HK-2 Cells Induced By Oxalate And Calcium Oxalate Monohydrate

Objective:To investigate whether the exosome derived from human umbilical cord mesenchymal stem cells（hucMSCs）can alleviate epithelial-mesenchymal transition（EMT）and oxidative stress injury induced by oxalate and calcium oxalate monohydrate（COM）crystals in renal tubular epithelial cellsMethods:（1）The HK-2 cells were induced by oxalate and COM crystals（2）Human umbilical cord mesenchymal stem cells were cultured with the exo-free FBS medium.Exosomes from conditioned medium of human umbilical cord mesenchymal stem cells（hucMSC-Ex）was isolated and purified by ultracentrifugation,the morphology was verified by transmission electron microscopy（TEM）,and specific biomarkers TSG101,CD63 and Alix was detected by western blot.Nanoparticle tracking analysis（NTA）was used to characterize the size.（3）The HK-2 cells were divided into control group,exosome group,oxalate+COM group,exosome+oxalate+COM group.The cell viability was detected by MTT assay at 24h,48h and 72h.The oxidative stress level was measured by LDH、H₂O₂、MDA and ROS assays.The expression of N-cadherin,TGF-βand ZO-1 was detected by immunofluorescence.The expression of epithelial markers E-cadherin,ZO-1,mesenchymal markers N-cadherin,α-SMA and related signaling pathway protein TGF-βand Smad2 was detected by Western blot.Results:（1）After 48h incubation of oxalate and COM crystals,the cells lose cellular adhesion and present long,spindle-shaped and scattered morphology,and the number of cells was significantly decreased.And the expression of elements in TGF-β/Smad signaling pathway,including TGF-βand Smad2,mesenchymal markers,including N-cadherin andα-SMA was up-regulated and epithelial markers,including ZO-1and E-cadherin were down-regulated.And the LDH、H₂O₂、MDA and ROS were increased.（2）The morphology of the exosome showed a double-layer membrane,hollow,circular or elliptical,and the diameter was distributed in the 30-100nm.TSG101,CD63,Alix protein were positively expressed,their diameters were distributed in 80-300nm.（3）The cell viability of HK-2 cells after 48h and 72h after treatment with Oxalate and COM crystals decreased significantly,and the cell activity increased after the treatment of hucMSC-Ex.Under transmission electron microscope,ZO-1 expression of oxalate+COM group was enhanced,and the expression of TGF-βand N-cadherin was decreased after the treatment of hucMSC-Ex.Under the white light,the cells in control and hucMSC-Ex group were closely connected and showed a paving stone-like morphology.The cells of oxalate+COM group lose cellular adhesion and present long,spindle-shaped and scattered morphology,and the number of cells was significantly decreased,while the morphology of cells in oxalate+COM+exosome group had less changes.Western blot results found that,in oxalate+COM+exosome group,the expression of N-cadherin,α-SMA and TGF-β,Smad2 in HK-2 cells was reduced,and the expression of E-cadherin and ZO-1 was up-regulated.Conclusions:（1）HucMSC-Ex can be successfully isolated by ultracentrifugation.（2）Oxalate and COM crystals can induce EMT and oxidative injury in HK-2 cells.（3）Exosomes derived from human umbilical cord mesenchymal stem cells reduce the oxidative injury and alleviate epithelial-mesenchymal transition induced by oxalate and COM crystals in HK-2 cells...