Effect Of Bim On Partial Epithelial To Mesenchymal Transition In Proximal Renal Tubular Cell In Diabetic Kidney Disease

Effect of Bim on partial epithelial to mesenchymal transition in proximal renal tubular cell in Diabetic kidney disease[Background]The proximal tubular center theory is a research focus in recent years for diabetic kidney disease(DKD).Proximal renal tubular epithelial cell(PTEC)apoptosis was found earlier than that of glomerular cells in the kidneys of early DKD rats,our previous studies also confirmed that Bim is a key target of PTEC apoptosis.In addition to apoptosis,the injury mechanism of PTEC in DKD also includes epithelial to mesenchymal transition(EMT),which has been confirmed by previous studies.Early targeted inhibition of proximal tubular EMT can reverse tubular interstitial fibrosis(TIF)to protect PTEC.Traditionally,the EMT process has been thought to cause the transformation of PTEC into myofibroblasts,thus leading to TIF.However,whether the myofibroblast is the source of PTEC is controversial.Some scholars believe that renal tubular epithelial cells do not break through the basement membrane in the process of EMT,but present a state of "partial epithelial to mesenchymal transition(PEMT)".In other words,renal tubular epithelial cells acquire the characteristics and motor phenotype of interstitial cells,but do not completely break through the basement membrane and transform into myoblasts.Both apoptosis and PEMT are the signature events of proximal renal tubular cell injury in early DKD.We have been committed to studying the molecular mechanism of PTEC damage in DKD,and the results show that Bim is a key factor mediating the apoptosis of proximal renal tubular epithelial cells,and it is clear that Bim/NFAT2-mediated tubuleball communication plays an important role in DKD.Therefore,on the basis of previous studies,this paper explores whether Bim is involved in affecting the occurrence of PEMT in addition to the direction of PTEC apoptosis mediated by Bim,so as to further improve the regulatory network of BIM-mediated PTEC damage and provide new ideas and directions for the prevention and treatment of DKD.[Aims]1.The Bim knockout mice with PTEC were used to construct DKD model,and the mechanism of Bim mediated anti-diabetic nephropathy PEMT in vivo was studied.2.In vitro studies were conducted to determine the effect of interfering Bim expression in HK-2 cells under high glucose conditions on PEMT.[Methods and materials]Animal experiments:A PTEC conditional bim gene knock out mouse model(Bim CKO)was constructed,and comparison was observed with wild-type mice(normal strain mice without specific Bim knock out,wild type,WT).The genes of each group were identified by agarose gel electrophoresis.The DKD model was established on the basis of the above mice,and the influence of Bim expression in proximal renal tubules on diabetic nephropathy PEMT was determined in vivo.Male mice aged 6-8 weeks were selected for unilateral right nephrectomy,and 50 mg/kg streptozotocin(STZ)was injected intrabitoneally one week after surgery for 5 consecutive days.The blood glucose was measured 72h after injection,and the random blood glucose≥16.7mmol/L at the same time for two consecutive times was regarded as the successful modeling of diabetes,and the continuous increase of albuminuria at 24h was monitored,which was regarded as the successful modeling of DKD.The Bim knockout mice and wild-type mice were divided into the following four groups:A:WT+Con group;B:WT+DKD group;C:Bim CKO+Con group;D:Bim CKO+DKD group,the body weight,blood glucose and 24h urinary protein of mice were monitored before,4,8 and 12 weeks after modeling,respectively.At the end of the 12th week,the mice were sacrificed and sampled to detect the levels of blood urea nitrogen and creatinine.The localization and expression of Bim in kidney of mice in each group were observed by immunohistochemistry.Hematoxylin-Eosin(HE)staining was used to observe the pathological changes of kidney tissue,and Masson staining was used to observe the degree of renal fibrosis.The expression of transforming growth factor 1(TGF-β1),mesenchymal cell marker Vimentin and Epithelial cell marker E-cadherin(e-cadherin)in mouse kidney was observed by immunofluorescence staining.Cell experiment:Human proximal renal tubular epithelial cell line(HK-2)was cultured in vitro.Lentivirus and purinomycin were used to construct a silencing Bim stable cell line for subsequent experiments.PEMT was induced in HK-2 cells by high glucose(40 mmol/L)intervention.Cell experiment groups were as follows:A:positive glucose group(5.5 mmol/L.NG);B:high glucose group(40 mmol/L,HG);C:high glucose+negative control virus group(HG+Bim shNC);D:High sugar+Bim lentivirus group(HG+Bim shRNA).Total proteins were extracted and Bim silencing efficiency was verified by Western blot.The expression of intermediate cell marker Vimentin and epithelial cell marker cytokeratin(CK-18)and nuclear translocation of nuclear transcription factor(NFATc1)were observed by immunofluorescence staining in the above cells after 72h intervention with different glucose concentrations,respectively.The expressions of transforming growth factor 1(TGF-β1),Vimentin and cytokeratin(CK-18)in HK-2 cells were detected by Western blot.The transverse and longitudinal migration ability of HK-2 cells were observed by scratch test and Transwell migration test respectively.The observation time of scratch experiment was 24h,48h and 72h in order to observe the migration of HK-2 cells at different time points.[Result]1、Bim knockout with conditional PTEC alleviates early renal injury with DKD.(1)The general indexes and renal function of mice were observed.The results showed that compared with the control group(WT+Con,Bim CKO+Con),the weight gain of mice in the two groups(WT+DKD,Bim CKO+DKD)slowed down,and some mice lost weight;The blood glucose level of diabetic nephropathy group was significantly higher than that of non-diabetic nephropathy group,and the difference was statistically significant(P<0.05);Compared with WT+Con group,the blood urea nitrogen and urine protein levels of WT+DKD group were significantly increased.After Bim gene knockout in mice,the blood urea nitrogen and urine protein levels of mice in Bim CKO+DKD group were reduced,but there was no significant difference in body weight and blood glucose compared with that in WT+DKD group,indicating that Bim gene knockout in mice can improve renal function injury to a certain extent.At the end of week 12,we found no significant difference in serum creatinine levels among the four groups,indicating that renal impairment was still in its early stages.(2)Immunohistochemical results showed that Bim positive staining increased in the proximal renal tubules of diabetic kidney disease mice(WT+DKD group),but no positive staining was observed in the glomeruli.No significant expression of Bim was found in non-diabetic kidney disease group(WT+Con).In addition,no Bim expression was found in the kidney tissues of the two groups of mice(Bim CKO+Con and Bim CKO+DKD)conditionally knocked out Bim gene by PTEC,which confirmed the success of Bim knockout.(3)HE staining showed no obvious pathological changes in the kidney tissue of mice in the two groups of non-diabetic kidney disease;The glomeruli and renal tubules of diabetic kidney disease group showed obvious lesions,which were relieved in diabetic gene knockout mice.In addition,Masson staining showed that obvious blue collagen fiber deposition was observed in the kidney tissue of mice in WT+DKD group,and the kidney damage caused by DKD could be improved in mice after Bim knockout.2、Bim knockout by conditional PTEC inhibited diabetic nephropathy PEMT.Immunofluorescence staining of animal tissues showed that compared with WT+Con group,the expression of vimentin and TGF-β1 in kidney tissues of WT+DKD group was increased,while the expression of E-cadherin was decreased,suggesting the occurrence of PEMT in diabetic nephrotic group.However,the expression of vimentin and TGF-β1 decreased and E-cadherin increased in Bim CKO+DKD kidney tissue after Bim knockout,confirming that Bim gene knockout with conditional PTEC can inhibit diabetic nephrotic PEMT in animal kidney tissue.3、Down-regulating Bim expression in HK-2 cells inhibited hyperglycemic induced PEMT,inhibited NFATcl nuclear translocation,and reduced cell migrationFirstly,Western blot results confirmed that the Bim protein expression level in Bim shRNA transfected lentivirus cells was significantly decreased(P<0.05)compared with the negative transfected control virus group(Bim shNC),which confirmed the success of lentivirus transfection.The transfected cell lines were used for follow-up in vitro experiments.The results of immunofluorescence staining showed that the expression level of vimentin in HK-2 cells was significantly increased after the intervention of high glucose,while the expression level of CK-18 was significantly decreased.At the same time,it was also observed that NFATc1 nuclear translocation increased in proximal renal tubule cells under high glucose environment,and interfering Bim expression could inhibit the nuclear translocation of NFATc1 in HK-2 cells under the intervention of high glucose.In addition,Western blot results showed that compared with HG group,the protein levels of TGF-β1 and vimentin were decreased in HG+Bim shRNA group,while the protein levels of CK-18 were increased,which was consistent with the results of cellular immunofluorescence.It was confirmed that down-regulating Bim expression in HK-2 cells could inhibit high glucose induced cell PEMT.Finally,the results of scratch and migration experiments showed that the migration ability of HK-2 cells in HG+Bim shRNA group was significantly decreased compared with that in HG group.Inhibition of Bim expression could reduce the migration ability of HK-2 cells under high glucose condition.[Conclusion]1.Bim knockout by conditional PTEC in vivo can reduce the blood urea nitrogen and urine protein levels of mice,alleviate early renal function injury of DKD,and inhibit the level of PEMT.2.In vitro down-regulation of Bim expression in HK-2 cells inhibits high glucoseinduced cell PEMT and suppresses cell migration ability.Inhibition of Bim expression in HK-2 cells inhibits NFATc1 nuclear translocation.