Study Of Lead-inducing Apoptosis And Epithelial-mesenchymal Transdifferentiation In Proximal Renal Tubular Cells HK-2 And The Isolation And Identification Of Lead-binding Proteins

Since the lead pollution of environment is common and the kindney is sensitive to lead,the disservice of nephrotoxicity of lead is universal both at home and abroad at present.The toxic effect of Lead on kidney is an insidious and progressive pathological process,and the early effct is subclinical and reversible,but it will turn out inreversible if devolops to a certain stage.Investigations show that the main target site in kidney of lead toxic effect is proximal tubules.The early pathological changes of lead nephrosis(LN) are mainly the formation of inclusion bodies in renal tubular epithelial cells and the damage of renal tubular epithelial cells, then renal interstitial fibrosis,renal tubule atrophy and loss of nephron gradually develop,finally lead to renal dysfunction.However,only a little has been known about the damage mechanism of renal tubular epithelial cells,which are the main target renal cells of lead,also there is a lack of systematic researchs on molecular toxicological mechanisms of LN.in tthis study,We observe apoptosis and epithelial-mesenchymal transdifferentiation(EMT) of human proximal tubular epithelial cell line(HK-2) by lead acetate inducing and the dose-response relationship of cytotoxic effects of lead acetate(Pb) to human proximal tubular epithelial cell.as well as the effect of lead acetate on the molecules that related to apoptosis and EMT signaling pathway,and the antagonistic effects of potassium iodide(KI).And then,we employ the functional proteomics methods to isolate and identify the high affinity lead-binding proteins,try to explore the real mechanism of lead-inducing apoptosis and EMT of HK-2.Previous investigations had shown that the intracellular lead could interact with proteins of specific and high affinity and form the lead-binding proteins(PbBPs).PbBPs are defined as the intracellular lead-receptors and target proteins with high affinity capacity of banding lead concerned with reactiveness and adaptability of cells.Are there PbBPs to being involved in the processes of HK-2 apoptosis and EMT by lead? What's the role of PbBPs in these processes? For this reason,we had to isolate and identify PbBPs in the human renal tubular epithelial cell apoptosis and EMT model.In this way we could realize the real mechanism of lead-inducing apoptosis and phenotype transformation and identify the intracellular molecular targets,and might be able to identify a new high affinity PbBPs and provide evidence for exploration of protein chelating agent.lead affinity column-metal chelate affinity chromatography, one-dimensional SDS-PAGE electrophoresis and ESI-MS/MS liquid chromatography tandem mass spectrometry were used to isolate and identify In HK-2 showing obviously apoptosis and EMT,in this study.and immunoblotting was used to verify the PbBPs.Then bioinformatics was used to analyze the potential role of PbBPs in apoptosis and EMT of HK-2.The study is the first time observation EMT in HK-2 by lead acetate inducing in vitro;six high affinity lead-binding proteins involved in lead-inducing apoptosis and EMT in HK-2 were isolated and identified; A new high-throughout technical method to isolate and identify PbBPs had be set up.The study was divided into three parts:Partâ… The apoptosis in HK-2 mediated by lead and the antagonistic effect of potassium iodideObjective:To investigate the dose-response relationship of cytotoxic effects of lead acetate(Pb) to human proximal tubular epithelial cell and the antagonistic effects of potassium iodide(KI).And to explore the molecular mechanism involved in Pb inducing apoptosis. Methods:human proximal tubular epithelial cell line(HK-2) were divided in two groups.(1)treated with Pb(0,2.5,5,10,20,40μmol/L).(2)pretreated with Pb(0,2.5,5,10,20,40μmol/L) puls KI of final concentration 40mg/L.The cells viability was measured with MTT assay and the cell morphology changes was observed with phase-contrast microscopy.The apoptosis was monitored by using flow cytometry and Hoechst 33258 staining with fluorescence microscopy.NF-κB,bcl-2 protein expression quantity was detected by flow cytometry.Results:(1) The minimum concentration of lead acetate which decreased The viability of HK-2 cells was 10μmol/L.The viability of HK-2 cells was negatively correlated with dosages of Pb(r= -0.878, P＜0.01) after exposure of Pb for 72h..(2) The concentrations of Pb that inhibited 50%(IC₅₀) of HK-2 cells at 72h was 15.68μmol/L.(3) HK-2 cells cultured in high dosages of Pb showed obvious morphologic changes,including elongated,contracted and rounded even agglomerated (4) apoptosis ratio was elevated significantly(p＜0.05).(5) chromatin was condensed and cell nuclei exhibited the dense fluorescence in every dosages of Pb.(6) Pb also decreased NF-κB and Bcl-2 protein expression,Significant differences of NF-κB protein expression was found between 0μmol/L and 10μmoI/L,and of Bcl-2,were 0μmol/L and 5μmol/L and 10μmoI/L.(7) Correlation analysis showed that the rates of apoptosis were inversely correlated with the expression of NF-κB and Bcl-2(r= -0.625,p＜0.01;r=-0.709.(8) Compared with same dosages of Pb group,after pretreatment with KI,cell viability was increased,IC₅₀ being enhanced 3.33 times.Morphologic changes was significantly improved and apoptosis ratio was lowered.Conclusions:(1) There was a dramatically dose-response relationship between dosages of Pb and the toxic effects to HK-2 cells,. The low-dose lead acetate(2.5μmol/L) could cause significant apoptosis and cell morphology changes,while the high dose(10μmol/L) caused high degree of cells necrosis,and LC₅₀ of lead acetate in the HK-2 cells is 15.68μmol/L;(2) Pb induces apoptosis by inhibiting NF-κB and Bcl-2 expression in human proximal tubular epithelial cell line HK-2.(3) 40 mg./L KI can protect Pb-induced cytotoxicity in part.Partâ…¡Changes in expression of fibrotic markers and phenotype transformation in HK-2 exposed to lead acetate and potassium iodideObjective:To investigate whether lead acetate could induce human renal proximal tubule epithelial cells phenotype transformatation,and the possible mechanism,as well as the impacts of potassium iodide.Methods:human renal proximal tubule epithelial lines(HK-2) were cultured for 72 hours in different conditions as(1) treated with lead acetate(0,2.5,5,10μmol/L).(2)treated with lead acetate(0,2.5,5,10μmol/L) puls KI(40mg/L).The cell morphology changes was observed with phase-contrast microscopy,the expression of a-SMA,FN,TGF-β1and CTGF were assessed by immunofluorescence-flow cytometry.RT-PCR. was performed to test the expression of TGF-β1and CTGF mRNA.Results:(1) HK-2 cells in the control group were approximately of square or oval shape in a cobblestone-like arrangement way,and the cells connected to each other closely integrating into a monolayer.Some cells in the 5μmol/l lead group became longer and fusiform and grew separately.Besides the changes similar to 5μmol/l lead group,in 10μmol/L lead group,some cells' borderlines were not clear,and a few cells shrunk and turned rounder.(2) Compared with the control group,the rate of the tubular epithelial cells expressing a-SMA significantly increased in 5,10μmol/L lead group(p＜0.01).(3) The marked proteins FN in extracellular matrix(ECM) increased as lead acetate dose increased,and compared with the control group,the quantity of 5,10μmol/l lead groups increased to 8.3,8.4 times respectively(P＜0.01).(4)Compared with the control group,the TGF-β1,CTGFmRNA and protein expression of HK-2 cells increased significantly(5,10μmol/L lead groups,P＜0.05).(5) treating with lead acetate(0,2.5,5,10μmol/L) puls KI(40mg/L),the changes described above can be inhibited to some extent. Conclusions:lead acetate can induce tubular EMT.TGF-β1/CTGF. Signaling pathway mediates the lead-induced EMT.Part Three:The isolation and identification of the lead-binding proteins in human renal tubular epithelial cells HK-2Objective:To screen the lead-binding protein in the lead-exposed human renal tubular epithelia cell HK-2,analyze the potential effects of lead-binding protein in the processes of lead-inducing cell apoptosis and EMT,and explore a new high-throughout technical method to isolation and identification of the lead-binding proteins.Method:(1) human renal proximal tubule epithelial lines(HK-2) were incubated with lead acetate for 72 h in twe conditions as treated with 0μmol/L and 5μmol/L lead acetate.(2) the cells were lysed with nondenaturing lysis buffer,then they were desalted and got rid of EDTA with 3 kDa ultrafiltration.(3) Prepared lead affinity column.(4) Isolated lead-binding proteins with lead affinity column metal chelate affinity chromatography.(5) Separated proteins with 1D-SDS-PAGE.(6) Enzymaticly hydrolyzed the proteins in gel.(7) identited the mixed protein peptides with ESI-Q-TOF,and the pkl document were outputted by the equipment.(8) searched protein database(NCBInr) with Mascot software.(9) proteins bioinformatics Analysis(10) confirmed proteins with Westein-bloting.Results:(1) There were 5 clear bands in treated with 5μmol/L lead acetate HK-2 protein sample on SDS-PAGE gel,while the control group protein sample,only one clear band.(2) 6 lead-binding proteins were identited from the HK-2 treated with 5μmol/L lead acetate.â‘¡gi|306891 90kDa heat shock protein, HSP90;â‘¡gi|1082886 tumor necrosis factor type 1 receptor associated protein,TRAP-1;â‘¢gi|13540513 oxysterol binding protein 2 isoform a, OSBP;â‘£gi|3820484 ataxin-2-like protein,A2LP;⑤gi|119573321 G patch domain containing 4,isoform CRA_b,GPATC4;â‘¥gi|60594178 Trex2 3' Exonuclease Structure,Trex2.And two lead-binding protein were identited from the control group,ie HSP90,TRAP-1.(3) structure and function of Proteins:HSP90,TRAP-1,Trex2 belong to metal-binding proteins.OSBP contains a negative-charge concentrating domain in space conformation,and no specific lead-binding sites were found in GPATC4,A2LP.Subcellular Localization:There were three cytoplasmic proteins,one mitochondrial protein,one nuclear protein, one external membrane protein.HSP90,TRAP-1 are the main molecular chaperones in the anti-apoptotic signaling pathway.and Trex2,A2LP, GPATC4 are involved in damaged-DNA repair.and OSBP is related to oxysteroid transport,detoxification,and the aggregation of the vimentin.then HSP90 might be a collaborative chaperone of the TGF-β/Smads signaling pathway.(4) westein-bloting showed that the protein expression of HSP90 and TRAP-1 in lead-treated group was higher compared with the control group.Conclusions:(1) Six high affinity lead-binding proteins were identited from HK-2 cell.(2) lead-inducing apoptosis and transdifferentiation in HK-2 may concerned with lead-bind proteins interfering the processes of anti-apoptosis,damaged-DNA repair and transdifferenation signal pathway.(3) The method of lead affinity column metal chelate affinity chromatography combining with 1D -SDS-PAGE and liquid chromatography-tandem mass spectrometry can isolate and identity multitude specific lead-binding proteins.