| Lead(Pb)is a common environmental pollutant.Once absorbed,it accumulates in the body and can cause serious damage to human health.It exerts multisystem and multiorgan toxicity.Kidney is one of the most sensitive target organs for Pb toxicity,while the proximal tubule is the major site of Pb-induced renal injury.Autophagy is an evolutionarily conserved biological process which maintains cellular homeostasis via the degradation and recycling of damaged organelles,misfolded proteins,and long-lived macromolecules in lysosomes.It has been demonstrated that autophagy plays a protective role in toxicants-induced acute renal injury,while the underlying molecular mechanism of Pb-mediated primary rat proximal tubular(rPT)cytotoxicity remains to be elucidated.Lysosomes play a vital role in effective degradation of autophagic cargo.Various death stimuli can induce lysosomal membrane permeabilization(LMP)which is characterized by the release of cathepsin B and cathepsin D from the lysosomal lumen to the cytosol.Once released into the cytosol,these two cathespsins can activate the apoptotic pathway and play its cytotoxic effect.We have previously demonstrated that the apoptotic death was the chief mechanism in low-dose(0-1.0 μM)Pb-induced cytotoxicity in rPT cells.However,the role of LMP in Pb-induced apoptosis in rPT cells is still unknown.Due to the dual role of lysosomes in autophagy and apoptosis,this study will offer further evidences to elucidate the possible interaction mechanism between autophagy inhibition,impairment of lysosomal function and apoptosis in Pb-exposed rPT cells.These findings will shed new light on understanding the molecular mechanism of Pb-induced nephrotoxicity.First,the reason of Pb-induced accumulation of autophagosomes was studied.Protein levels of LC3-II in rPT cells treated with Pb for 3 h,6 h and 12 h was detected by western blotting to investigate the effect of Pb exposure on autophagy,respectively.Then we selected the most obvious exposure dose and time in subsequent experiments.The results show that Pb promoted the accumulation of autophagosomes in rPT cells and 0.5 μM Pb exposure for 12 hled to the most significant accumulation of autophagosomes.Two autophagy inhibitors(3-MA,blocks the upstream steps of autophagy and CQ,blocks the downstream steps of autophagy)were used to detect its effects on Pb-induced accumualtion of autophagosomes in rPT cells transiently transfected with GFP-LC3 plasmids.Effects of Pb in the presence or absence of autophagy modulators on the levels of autophagic markers LC3-II,p62/SQSTM1 was monitored by western blotting,respectively.To further determine the effect of Pb on autophagy,the delivery of RFP-GFP-LC3 to the lysosome was detected by confocal microscopy.The results showed that Pb-induced increased numbers of autophagosomes is the result of impaired autophagic flux.Next,we investigated the detailed mechanism of Pb-mediated impairment of autophagic flux.We used confocal microscopy to check for autolysosome formation(a complex structure of autophagosome fused with lysosome)during Pb exposure by double labeling rPT cells with endogeneous LC3,as a marker of autophagosome,and LAMP-1,as a marker of lysosome.Two sensitive lysosomotropic pH probes,Lyso-Tracker Red and Acridine Orange,were applied to label lysosomes and the effect of Pb on lysosomal pH in rPT cells was detected by confocal microscopy.Changes in protein expression of three V-ATPase subunits which regulated lysosomal acidification during Pb treatment were assessed by western blotting.The results showed that Pb-mediated suppression of two V-ATPase subunits might result in lysosomal alkalinization.Then fluorescence intensity of DQ-BSA-Green and protein levels of total cathepsin B and cathepsin D during Pb treatment was detected by confocal microscopy and western blotting,respectively.These results indicated that Pb-induced autophagy blockade is not the result of defective autophagosome-lysosome fusion,but the result of impaired lysosomal function(including lysosomal alkalinization and decreased lysosomal degradation capacity).Finally,we investigated the relationship between LMP and apoptosis.The subcellular distribution of cathepsin B and cathepsin D during Pb treatment was monitored by immunofluorescence and western blotting,respectively.Meanwhile,rPT cells were pre-incubated with CA 074(cathepsin B inhibitor)or Pep A(cathepsin D inhibitor)following Pb treatment.Then the effect of cathepsins inhibitor on Pb-induced protein expression of cleaved caspase-3,morphological changes and changes of apoptosis rate was detected bywestern blotting,confocal microscopy and flow cytometry,respectively.These data supported the notion that Pb can cause LMP,leading to the release of cathepsin B and cathepsin D from lysosomes into the cytoplasm,and then induced apoptosis.In conclusion,lysosomal function was impaired during Pb exposure as evidenced by lysosomal alkalinization and decreased lysosomal degradation,which subsequently led to autophagy blockade.Inhibition of autophagic flux may lead to nephrotoxicity.Moreover,Pb-mediated lysosomal membrabe permeabilization with the translocation of cathepsin B and cathepsin D from lysosomes to the cytosol induced downstream apoptosis,which increased nephrotoxicity. |
