Effects Of Autophagy On Apoptosis Induced By Extracellular Histones In Human Renal Tubular Epithelial Cells

BackgroundAcute kidney injury(AKI)is one of the common diseases in intensive care units and the morbidity of AKI is increasing.A lot of studies have shown that renal tubular cell apoptosis is one of the important pathogenesis.Histones is the main structure proteins of eukaryotes chromatin.However,it has been demonstrated that extracellular histones is also a kind of endogenous damage-associated molecular patterns and they play an important role in the pathogenesis of AKI.Researchers find that histones can induce leukocyte recruitment,microvascular vascular leakage,renal inflammation,and structural features of AKI after they direct inject histones into the renal arteries of mice.Their experiments in vivo and in vitro have shown extracellular histones can induce renal tubular epithelial cells apoptotic cell death.Autophagy is a dynamic process involving the rearrangement of subcellular membranes to sequester cytoplasm and organelles for degradation,which has the function of cellular homeostasis maintained and organelle turnover.Autophagy is also a kind of defense mechanism and it has been proved that autophagy is upregulated in many disease models of AKI.Many researchs suggest that autophagy can protect cell from apoptosis.For example,autophagy is a pro-survival mechanism under nutrition deficiency or the lack of growth factors.A study reports that autophagy is upregulated in the mice model of renal ischemia reperfusion injury.However,with autophagy inhibitor 3-Methyladenine(3-MA)or chloroquine pretreatment,the number of renal cells apoptosis become more.It has been confirmed that excessive reactive oxygen species(ROS)can lead to cell apoptosis under pathological conditions.It's not clear whether autophagy can protect human renal proximal tubular epithelial cells(HK-2)from apoptosis induced by extracellular histones through a decrease in excessive ROS production.Our study will explore the above question.Objectives1.To investigate whether extracellular histones can induce HK-2 apoptosis or autophagy.2.To explore whether autophagy can protect HK-2 from apoptosis induced by extracellular histones through a decrease production of ROS.Methods1.To primarily realize the effect of histones on the autophagy and apoptosis,HK-2 cells were treated with rising concentrations of histones.The rate of apoptosis and the expressions of autophagy-related protiens LC3II and Beclinl of HK-2 cells were detected separately using flow cytometry and immunoblotting.2.To definite the effect of autophagy on apoptosis of HK-2 cells,cells were incubated with histones after one hour pretreatment with 3-MA,a pharmacological inhibitor.The rate of apoptosis and the activation of caspase3 of HK-2 cells were detected separately using flow cytometry and immunoblotting.3.To further definite the effect of autophagy on apoptosis of HK-2 cells through a decrease production of ROS,cells were incubated with histones after one hour pretreatment with 3-MA,the production of ROS was detected indirectly using flow cytometry.Statistical analysisThe datas were analyzed in IBM SPSS 21 statistical software.Datas were presented as mean±standard deviation and analyzed with one-way analysis of wariance(ANOVA)followed by LSD test or Dunnett's T3 test.P values of less than 0.05.were considered statistically significant.Results1.Effects of different concentrations of histones on HK-2 apoptosis There were statistic differences in the effects of different concentrations of histones on HK-2 apoptosis(F=57.497,P<0.01).As compared to control group,histones significantly enhanced apoptosis of HK-2 cells in a dose-idependent manner.(P<0.05,P<0.01,P<0.01)?2.Effects of different concentrations of histones on the expressions of LC3? and Beclinl of HK-2There were statistic differences in the effects of different concentrations of histones on the expressions of LC3? and Beclinl of HK-2.As compared to control group,histones significantly enhanced the expressions of LC3II and Beclinl of HK-2.3.Effects of 3-MA on apoptosis and the activation of caspase3 of HK-2 induced by histonesThe 3-MA group didn't have a significant increase in apoptosis and the activation of caspase3 of HK-2 as compared to control group.However,the 3-MA+histones group had a significant increase in apoptosis and the activation of caspase3 of HK-2 as compared to 3-MA group.4.Effects of 3-MA on intracellular ROS accumulation of HK-2 induced by histones The 3-MA group didn't have a significant increase in ROS intensity as compared to control group.However,the 3-MA+histones group had a significant increase in ROS intensity of HK-2 as compared to 3-MA group.ConclusionAutophagy can protect human renal proximal tubular epithelial cells from apoptosis induced by extracellular histones through a decrease in excessive ROS production.We suggest the stimulation of autophagy pathways in AKI has potential application value.