The Mediating Role Of Rab7 In Renal Tubular Epithelial Cells Injury Induced By Proteinuria And Its Correlation With Extracellular Matrix Metabolism

Part 1: The correlation study of Rab7 and matrix metalloproteinase-2(MMP-2)of renal tubular epithelial cells in proteinuric nephropathyObjective: Evaluating the correlation between Rab7 and matrix metalloproteinase-2(MMP-2)in renal tubular epithelial cells from the renal biopsy specimens with different kinds of kidney diseases,to provide new ideas for the mechanism research of proteinuric nephrology.Methods: Seventy-seven renal biopsy specimens with five kinds of kidney diseases were collected,including Ig A nephropathy(Ig A N),diabetic nephropathy(DN),membranous nephropathy(MN),focal segmental glomerulosclerosis(FSGS)and lupus nephritis(LN).The expressions of Rab7 and MMP-2 were detected by immunohistochemistry.Based on this,the renal tubular basement membrane was observed by PAS staining.Statistical software SPSS 19.0 was used to analyze the relationship between MMP-2 and Rab7 expression.Results: The positive expressions of MMP-2 and Rab7,as brown-yellow granules,mainly locate in the cytoplasm of renal tubular epithelial cells.Our data demonstrated that Rab7 expression level presented a positive linear correlation with MMP-2.The linear correlation coefficient was 0.6036(p<0.0001).The expression of Rab7 was obviously aweak in tubular epithelial cells with marked fibrosis and thickened basement membrane.Conclusion: There was significant correlation between endocytosis,autophagy and MMP-2 in renal tubular epithelial cells of patients with proteinuria.The expression of Rab7 differed in tubular epithelial cells along with the degree of injuries.The biopsy specimens were so limited that MMP-2 activity couldn't be detected,because the tissues were almost used up for the required staining examination.The thickening of tubular basement membrane suggested that MMP-2 activity was inadequate.Thus the underlying mechanism need to be explored.Part 2: Construction of rat model with renal fibrosis induced by albumin and analyses of Rab7 expression and MMP-2 activityObjective: In order to further understand the relationship of Rab7 expression and MMP-2 activity in albumin-exposed renal tubular epithelial cells,corresponding rat model was constructed.And assess the morphological changes,endocytosis,autophagy,MMP-2 activity and renal fibrosis in renal tubular epithelial cells.Methods: Twenty Wistar female rats were randomly divided into two groups of ten,the control group(saline group)and the model group(albumin group).The rats in the model group and control group were intraperitoneally administrated with 1000 mg BSA solution or saline daily,respectively.Five rats from each group were sacrificed at the end of the first week and other rats were continuously injected until the 10 th week.24 h urine protein,NAG and serum creatinine were measured.a-SMA and Vimentin were detected by HE staining,sirius red staining and immunohistochemical staining to evaluate the degree of renal injury and fibrosis.The ultrastructural structures of renal tubules,endocytosis and autophagy of renal tubular epithelial cells were observed by electron microscopy.MMP-2 activity was detected by gelatin zymography.Western Blot was used to examine the expression of collagen IV,MMP-2,LC3(47),P62 and Rab7.Results: Increased expression of Rab7 was observed in the kidneys of BSA-treated rats for one week.The 24 h urine protein and NAG in the model group were significantly higher than those in the control group,while there was no marked difference in serum creatinine.Inflammatory cells infiltration,fibroblast proliferation,protein casts and edema of renal tubular epithelial cells were found with HE staining.There was abundant collagen in the kidneys of model rats.Enhanced a-SMA and Vimentin and decreased autophagic levels of renal tubular epithelial cellswere detected in the model rats.Simultaneously,elevated collagen IV and MMP-2 protein levels were noticed in the model rats,while there was an inverse effect forLC3(47),Rab7,and MMP-2 activity.Conclusion: The rats with renal fibrosis were successfully constructed.In the early stage of albumin stimulation,the expression of Rab7 was up-regulated in the renal tubular epithelial cells,while in the advanced stage,the decreased expression of Rab7 and MMP-2 activity,more collagen deposit and weakened autophagic level were observed.Part 3: Role of Rab7 in albumin induced acute injury in renal tubular epithelial cellsObjective: To clarify the effects of Rab7 and autophy in the regulation of MMP-2 activity in the renal tubular epithelial cells acute injury induced by albumin.Methods: The renal tubular epithelial cells(TECs)were insulted by overloaded albumin nanoparticles.Rab7 was transfected into TECs(HK-2 cellline)using lenti-virus.The endosomes and autophagosomes were examined by laser-scanning confocal microscope and TEM.The expressions of LC3(47),P62 and MMP-2 regulatory moleculeswere were detected by WB and immunofluorescence staining.MMP-2 activity was analyzed by zymography.The expression and activity of Cathepsin B and the activity of acid phosphatase were measured to clarify the autophagy flux.EMT(epithelial-mesenchymal transition)and apoptosis were analyzed with immunofluorescence staining and flow cytometry.Finally,CQ(an autophagic inhibitor)was used to determine the role of autophagy and Rab7.Results: In vitro,albumin stimulation resulted in over-activation of MMP-2,obstructed autophagic flux due to lysosomal dysfunction.Furthermore,over-activation of MMP-2 was mediated by its related regulatory molecules(MT1-MMP,TIMP-2 and RECK)on the membrane of TECs.Over-activation of MMP-2 was alleviated by Rab7 overexpression,which was reversed by chloroquine(CQ).In addition,our data indicated that up-regulation of Rab7 suppressed EMT andapoptosis in the albumin-treated TECs.Conclusions: The expression of Rab7 was up-regulated in the early stage of albumin-induced renal tubular epithelial cells and Rab7 played a protective role by inhibiting MMP-2 activity.And autophagy was confirmed to be involved in the regulation of MMP-2 activity.Part 4: Role of Fe3O4 magnetic albumin nanomaterials in renal tubular epithelial cells injury and tubulointerstitial fibrosis in the mice exposed to albuminObjective: Based on the previous data-Fe3O4 magnetic nanoparticles enhance the activity of MMP-2.The Fe3O4 magnetic albumin nanoparticles were used to wild-type mice and Rab7 over-expressed transgenic mice with albumin overload.The effect of Fe3O4 magnetic nanoparticles on the regulation of MMP-2 activity,renal tubular injury and interstitial fibrosis were investigated,and to further explore the Rab7-related mechanisms.Methods: The Fe3O4 magnetic albumin nanoparticles were prepared by the chemical precipitation method.The surface of Fe3O4 magnetic nanoparticles was modified by polyethyleneimine(PEI).Fe3O4 magnetic nanoparticles were encapsulated with bovine serum albumin(BSA)using glutaraldehyde crosslinking.Transmission electron microscopy(TEM)and dynamic light scattering analyzer(DLS)were used to characterize the magnetic albumin nanoparticles.Cationic albumin was intravenously injected into the mice to construct animal model with albumin overload.Then,Fe3O4 magnetic albumin nanoparticles were intravenously injected into the mice.Subsequently,24 h urine protein,urinary NAG,serum albumin,creatinine and urea nitrogen were detected.The HE staining was conducted to measure the degree of renal tubular injury,the collagen deposit was measured by sirius red staining,and the expression of a-SMA was examined by immunohistochemical staining.The renal ultrastructure,endocytosis and autophagy were observed by electron microscopy.The activity of MMP-2 was detected by gelatin zymography in the kidney tissues.Western Blot was used to analyze the expression of Beclin1 and LC3(47).Results: Fe3O4 magnetic albumin nanoparticles presented sphere-like shape,of uniform size with the diameter around 100 nm.and showed good dispersibility.The 24 h urine protein and urinary NAG presented a high level in the albumin treated wild type mice and Rab7 over-expressed transgenic mice compared to the control rats.This phenomenon was inhibited by Fe3O4 magnetic albumin nanoparticles.However,the results of Rab7 over-expressed transgenic mice were higher than those of wild mice.And the data were confirmed by the serum biochemical exmaination.Inflammatory cell infiltration,tubular edema,protein casts and fibroblast proliferation were observed in tubulointerstitium of Wild Type(WT)mice and Rab7 over-expressed transgenic mice with albumin overload,which was alleviated after application of magnetic albumin nanoparticles.However,the pathological changes were more serious in Rab7 over-expressed transgenic mice.The results from sirius red staining and a-SMA immunohistochemical staining demonstrated that strong expression was observed in both groups of the mice with albumin overload compared with that in control group,and a higher expression level was noticed in the Rab7-overexpressed transgenic mice than that in the WT mice,but MMP-2 activity presented a opposite trened.The effects were reversed after application of nanomaterials,and the activity of MMP-2 was weaker in the Rab7 over-expressed transgenic mice than that of wild type mice.The WB results showed that Beclin1 and LC3(47)expressions were lessened in the renal tissues of the WT mice and Rab7 over-expressed transgenic mice with albumin overload,which was increased after treated by the nanomaterials.In addition,the protein levels of Beclin1 and LC3(47)were higher in the Rab7 over-expressed transgenic mice than those of WT mice.Conclusion: The Fe3O4 magnetic albumin nanoparticles were successfully prepared and the WT mice and Rab7 over-expressed transgenic mice with tubulointerstitial fibrosis were successfully constructed by induction of albumin.At the advanced stage of albumin stimulation,the decreased MMP-2 activity and exacerbated renal tubular injury were noted in the renal tubular epithelial cells.The application of Fe3O4 magnetic albumin nanoparticles enhanced the activity of MMP-2 and alleviated renal tubular injury induced by albumin.At the advanced stage of proteinuria combined with over-expression of Rab7,could futher decrease the MMP-2 activity,exacerbate renal tubular injury and promote the progress of tubulointerstitial fibrosis.