Auotophagy Modulated By Advanced Oxidation Protein Products(AOPPs) Mediates The HK-2 Cells Injury

ObjectivesIn this study,we cultured HK-2 cells(a human proximal tubular epithelial cell line)in vitro.We first investigated the effects of AOPPs on HK-2 cells autophagy.Furthermore,we investigated whether the PI3K/Akt/mTOR signaling pathway is involved in AOPP-modulated activity of autophagy in HK-2 cells.Lastly,we examined whether autophagy participated in AOPPs induced HK-2 cells injury.Methods1.AOPPs preparation and content determination2.Cell culture of HK-23.The effects of AOPPs on HK-2 cells autophagy:HK-2 cells were cultured with 200 ig/mL of AOPPs for(0,3,6,12,24 h)or control medium,native BSA(100 ig/mL),or AOPPs(100,200,400 ig/mL)for 12 h.RT-qPCR and western blotting were used to analyse the mRNA and protein expression of autophagy related protein Beclinl and p62 and the conversion of LC3-? to LC3-?.HK-2 cells were cultured with control medium or 200 ig/mL of AOPPs for 12 h.Immunostaing and TEM were used to observe the LC3B positive dots and the formation of autophagosome(AP)and autolysosome(AL).4.The mechanism of AOPPs modulating HK-2 cells autophagy.HK-2 cells were cultured with 200 ig/mL of AOPPs for(0,3,6,12,24 h)or control medium,native BSA(100 ig/mL),or AOPPs(100,200,400 ig/mL)for 12 h.Western blotting assay was applied to detect the phosphorylation of PI3K,AKT and mTOR.HK-2 cells were treated with control medium or 200 ig/mL of AOPPs for 12 h in the presence or absence of LY294002(10 iM).RT-qPCR and western blotting were used to analyse the mRNA and protein change of autophagy related protein Beclinl and p62 and the conversion of LC3-? to LC3-?.Immuno-staining and TEM were also used to observe the LC3B positive dots and the formation of AP and AL.5.The effects of autophagy on AOPPs induced HK-2 cells injury.HK-2 cells were cultured with 200 ig/mL of AOPPs for(0,6,12,24,48 h)or control medium,native BSA(100 ig/mL),or AOPPs(100,200,400 ig/mL)for 24 h.RT-qPCR and ELISA were used to determine the mRNA and protein change of KIM-1 and NGAL.HK-2 cells were treated with control medium or 200 ig/mL of AOPPs for 24 h in the presence or absence of CQ(1 mM)or Rapamycin(1 uM).RT-qPCR and ELISA were used to determine the mRNA and protein change of KIM-1 and NGAL.Results1.AOPPs inhibited autophagy activity in HK-2 cells.Compared to control group,AOPPs treatment up-regulated the gene and protein levels of p62 but down-regulated that of Beclinl and conversion of LC3-?to LC3-?.Immunofluorescent technology verified that the LC3-? positive dots were decreased significantly in HK-2 cells treated with AOPPs.TEM showed a marked decrease in the number of typical AP and AL.However,these effects were not observed in the unmodified-BSA group.2.PI3K/AKT/mTOR signaling pathway mediated AOPPs inhibited HK-2 cells autophagy.Treatment with AOPPs induced phosphorylation of PI3K,AKT and mTOR as compared with the levels in control medium cells.However,the phosphorylation of PI3K,AKT,mTOR was not observed in unmodified BSA-treated cells.After addition of PI3K inhibitor LY294002,AOPP-decreased expression of Beclinl and conversion of LC3-? to LC3-? and AOPP-increased expression of p62 was significantly reversed Furthermore,the decreased LC3B positive dots induced by AOPPs were also partly reversed by LY294002.And transmission electron microscopy analysis demonstrated that LY294002 could also partly reverse AOPP-decreased number of AP and AL.3.Autophagy mediated AOPP-induced injury of HK-2 cells through activation of the PI3K/AKT/mTOR signaling pathway.AOPPs treatment increased the mRNA and protein level of KIM-1 and NGAL.However,it was not changed in control cells and in unmodified BSA-treated cells,which indicated that the over-expression of KIM-1 and NGAL were associated with the advanced oxidation of BSA.AOPP-induced over-expression of KIM-1 and NGAL in HK-2 cells was also partly suppressed after treatment with rapamycin,the autophagy enhancer.AOPP-induced over-expression of KIM-1 and NGAL in HK-2 cells was further aggravated after treatment with CQ,the autophagy inhibitor.ConclusionOur results indicated that AOPPs inhibited autophagy in RTEC through the activation of PI3K/AKT/mTOR pathway,and autophagy inhibition was associated with the RTEC injury induced by AOPPs.AOPPs have been shown to be associated with the pathogenesis of CKD.Our results provide a framework for developing novel nephroprotective therapies by targeting autophagy.