The Mechanistic Study Of Crosstalk Between Renal Tubule And Podocyte Contributing To Podocyte Cytoskeleton Injury

BackgroundPatients with newly-diagnosed diabetes mellitus(DM)have significantly surged around the world in recent years,and China has become the country with the largest number of DM patients.DM has become a major social problem which threatens people's survival and health.Diabetic nephropathy(DN)is one of the common chronic complications of DM.Although many large clinical studies have shown that intensive hypoglycemic,hypotensive treatments and lowering lipid can significantly reduce urinary protein,but the residual risk of developing DN is still as high as 50%-86%.At present,DN has become the prominent pathogenic reason for end-stage renal disease(ESRD)in hospitalized patients in China.Guidelines recommend renin-angiotensin-aldosterone system(RAAS)treatment as the main treatment of DN.But there is still limitation in benefits in renal protection,and the evidence which reduces the occurrence of developing ESRD is still inadequate.In the past 30 years,despite the application of various treatments,the incidence of ESRD caused by DN has not decreased significantly,and its increasing incidence suggested that the potential mechanism of DN remains to be explored.Therefore,to further clarify the new pathogenesis of DN and to find more effective treatment methods and targets are the key issues that urgently need to be solved.The research on DN has always been a hot spot at home and abroad.At present,there are controversies regarding the theory of the initial changes of DN.In the past,most experts believed that DN was mainly caused by glomerular pathological changes,but since then,more and more evidence has shown that renal tubular lesions also play an important role in the occurrence and development of DN,especially in the early DN and renal tubulopathy appear earlier than glomerulopathy.Renal tubulopathy tightly correlated with the early renal function abnormality.Under physiological condition,about 3.3 g albumin filtrates from normal renal glomerulus per day.But only 3%(0.1 g/d)albumin discharges in the urine,and most of the proteins are absorbed by renal tubular epithelial cells,and 71%proteins are absorbed by proximal renal tubular epithelial cells(PTECs).In the progress of DN development,PTECs have been impaired and the reabsorption function is damaged when the glomerular filtration is normal.Glomerular filtration membrane is a highly differentiated hemofiltration barrier,which is responsible for the excretion of various metabolites in the kidney and plays an important role in maintaining water,electrolyte and acid-base balance in the body.Therefore,glomerular injury is regarded as a major reason for the occurrence and progression of DN proteinuria.Podocyte is the last barrier of glomerular filtration membrane and its injury plays a vital role in DN development.Podocyte is also one of the weakest parts in the renal homeostasis disorder of DN.Studies showed that podocyte injury was closely related to DN proteinuria.However,through the construction of rat DN model and in vitro functional experiments,we previously found that the apoptosis of PTECs in rats of early DN was significantly increased,while the apoptosis of glomerular cells was rare,which suggested that,in early DN,the apoptosis of renal tubules was earlier than that of glomerulus.Bim is a member of the Bcl-2 protein family and works as a pro-apoptotic protein.Only one Bcl-2 homology(BH3)domain is contained in Bim which mainly participates in the activation of the cell death pathway.And we further revealed that Bim was identified as the key factor mediating the early apoptosis of PTECs in early DN in vitro.Currently,most of the researches about the mechanism of DN are based on a single point and focus on the role of single cell such as podocyte and renal tubular epithelial cell in DN,and ignore the important role of communication between cells in the occurrence and development of DN as a whole tissue.However,as an organic whole,the intercellular communication of kidney promoting the occurrence and development of DN has attracted more and more attention.In recent years,targeting the interactions between different functional cells and exploring their roles in the mechanisms in DN have become a research focus.As the two most representative types of cells,glomerular podocytes and PTECs are undoubtedly the powerful executors of physiological functions.At present,the research on the interaction between the two cells is under the initial stage,and the mechanism of their interaction mediating DN is still unclear and needs to be further explored.The change of cytoskeleton is the main cause of early podocyte lesions in DN,and is also an important factor to induce early DN proteinuria.Therefore,to elucidate the pathogenesis of podocyte cytoskeleton dysfunction and further delay and prevent the damage of glomerular filtration barrier is still an important link in the prevention and treatment of DN.Studies have found that Bim can not only promote apoptosis,but also lead to the induction of cytokine secretion and play other roles other than pro-apoptosis.Currently,it has not been reported whether the Bim protein that promotes PTECs apoptosis can play a role in inducing cytoskeleton injury of glomerular podocytes through certain factor,thus leading to DN development.Our study was designed to investigate the exact mechanism of Bim-mediated tubule-podocyte communication in DN podocyte injury.Objectives1.Through the establishment of an in vitro PTECs-podocytes co-culture model,the effect of PTECs on the damage of podocytes under high glucose was clarified,that is,the existence of tubule-podocyte communication was confirmed.2.To clarify the molecular mechanism of PTECs mediating podocyte cytoskeleton injury under high glucose.3.Exploring the effects of Bim in renal tubule on the podocyte cytoskeleton injury by constructing in vivo DN mice model.Materials and methods1.Screening of optimal high glucose concentration and treatment time:Human podocytes were treated with different glucose concentration gradients for various time gradients.And the expression of synaptopodin in podocytes was detected to determine the optimal high glucose concentration and the optimal treatment time of high glucose.2.RT-PCR and Western blot:To clarify the mRNA and protein expression levels of Bim in PTECs under high glucose treatment for 48 h.3.Down-regulation of Bim by lentivirus and construction of stable PTECs which were transfected with lentivirus:.(1)Transfection with lentivirus which down-regulates Bim expression and construction of stable PTECs which were transfected with lentivirus:1)Preliminary experiment of lentivirus infection was conducted to confirm the optimal time of infection and multiplicity of infection(MOI)value:Set up different lentivirus concentrations and PTECs(HK-2 cell lines)with the confluence was 20-30%was prepared to be transfected with lentivirus.When infection efficiency reached about 80%and HK-2 cells grew well,the corresponding infection time and MOI value could be used as the basis for subsequent infection experiments.2)Formal experiment of lentivirus infection:According to the infection conditions obtained from the preliminary experiment and the optimal MOI,the lentivirus was infected to HK-2 cells.After infection for about 72 hours,the infection efficiency was observed with a fluorescence microscope.(2)Stable PTECs which were transfected with lentivirus were selected by puromycin:Different concentrations of puromycin were set up for screening of optimal concentration of puromycin.Puromycin was added in HK-2 cells after lentivirus infection for 3-5 days,and the puromycin selection period was one week.After one week,the fluorescence intensity was observed.The cultured cells which were resistant to puromycin could be used for subsequent verification and functional experiments.4.Co-culture model construction:Human podocytes and HK-2 cells were digested when the density reached 80-90%.The resuspended podocytes and HK-2 cells were seeded into six-well plates and Transwell chamber which contained normal glucose medium at the density of 5×104,respectively and then continued to be cultured in an incubator containing 5%CO2.After cells were completely adherent,the medium which was contained with 40 mM high glucose was added in the Transwell chamber and six-well plate.Using tweezers to put the Transwell chamber which contained HK-2 on the six-well plate which contained podocytes and constructing an in vitro co-culture system.The co-culture system was placed in an incubator containing 5%CO2 for 48 hours for further observation and treatment.5.Western blot:Western blot was performed to detect the expression level of Bim protein in HK-2 cells of different groups to verify the intervention effect after lentivirus transfection.The expression of synaptopodin protein in podocytes was detected,and the influence of co-culture with different PTECs on the expression of synaptopodin in podocytes was determined.6.Immunofluorescence was used to detect the positive expression of synaptopodin and the arrangement of cytoskeleton filaments.(1)Positive expression of cytoskeleton-related protein synaptopodin in podocytes was detected by immunofluorescence:Podocytes in various groups were stained by immunofluorescence and then the slides were placed under a fluorescence microscope for observation and pictures were collected.(2)Detection of cytoskeleton arrangement by staining TRITC phalloidin:Podocytes in various groups were stained with TRITC phalloidin and then the slides were placed under a fluorescence microscope for observation and pictures were collected.7.RT-PCR:For examining the mRNA expression levels of various NFAT subtypes in different PTECs groups.8.Immunofluorescence staining for NFAT2 nuclear translocation in PTECs in treatment group.9.The expression of NFAT2 in PTECs was up-regulated and down-regulated by transfection of plasmids and siRNA,respectively.The intervention effect was detected by RT-PCR and Western blot,respectively:(1)HK-2 cells with stable down-regulation of Bim were transfected with NFAT2 plasmids:The mixture of NFAT2 plasmid and LipofectaminetM3000 was transfected into HK-2 cells which were transfected with Lenti-Bim-shRNA.(2)Transfection of small interfering RNA which inhibited NFAT2 mRNA expression(NFAT2-siRNA)into HK-2 cells:The mixture of NFAT2-siRNA and LipofectaminetM2000 was transfected into HK-2 cells.(3)After successful transfection of NFAT2 plasmid and siRNA for 48 h,RT-PCR was applied to examine NFAT2 mRNA expression and Western blot was conducted to examine NFAT2 protein expression in different groups.10.After the expression of NFAT2 in various PTECs was regulated,the co-culture model was constructed again,Western blot was used to detect the expression of podocyte synaptopodin in different groups.The positive expression of synaptopodin and the arrangement of cytoskeleton filaments in different groups were detected by immunofluorescence.11.Feeding of mice:We selected healthy and strong C57BL/6J male mice in good condition which were aged 6-8 weeks for subsequent animal experiment.Mice experienced adaptive feeding in our lab which longed for one week,and they were randomly grouping,including unilateral nephrectomy group(UNX),diabetic nephropathy group(DN)and sham operation group(Sham)for follow-up treatment and operation.12.DN mice model construction:Unilateral nephrectomy and small dose of streptozotocin(STZ)injection were used to construct DN mice model.13.Mice's body weight,blood glucose,urine volume,urinary protein content,and biochemical indexes including serum creatinine and blood urea nitrogen levels were examined:Blood samples were collected from the tail vein of mice before surgery,3 days after STZ injection,and in 2,4,6,8,10 and 12 weeks after DM model was established.Body weight and blood glucose level were measured at each time point.The next day,the mice were fasting but fed with water and moved into metabolic cages.Then 24 h urine was collected and urine volume was examined.We determined the concentration of urinary protein of mice by applying for Coomassie brilliant blue method.At the end of the 12th week,the heart apical blood of mice was collected.The serum of mice was collected,and the serum creatinine and urea nitrogen levels of mice in each group were detected.14.Renal glycogen staining:Mice were sacrificed at the 12th week of the experiment,and left kidney was taken.The kidney of mice was prepared into tissue slices which were thick about 3 ?m.The renal glycogen staining(PAS)was conducted.The pathological changes of kidney tissues were observed under light microscopy.15.Immunohistochemical staining for Bim location and expression in the kidney tissue of mice in each group.16.The protein expression levels of Bim and synaptopodin in kidney tissues were detected by Western blot.17.The localization and expression of cytoskeleton-related protein synaptopodin in podocytes in different groups of mice were detected by immunofluorescence.18.Data processing and statistical analysis:We repeated all experiments and samples more than 3 times for data processing and statistical analysis.Data were statistically analyzed by SPSS 22.0 software.Statistical significance assessment:T test was used for comparison between the two groups and data were shown with mean± S.E.M.One-way analysis of variance(One-way ANOVA)was used for more than three groups.P<0.05(bilateral)was considered statistically significant.The bar graph was drawn using GraphPad Prism 5.0 software.Results1.40 mM high glucose treatment for 48 h significantly induced the down-regulation of synaptopodin in podocytes and up-regulation of Bim in PTECs.(1)In order to determine the optimal high glucose concentration and the optimal treatment time of high glucose,podocytes were treated with various high glucose for different time.Western blot results showed that compared with 5.5 mM,high glucose concentration of 40 mM for 48 h significantly induced a decrease in the expression of synaptopodin in podocytes(P<0.05).(2)PTECs were treated with 40 mM high glucose and normal glucose,respectively.We found that in contrast with 5.5 mM normal glucose group,the expression of Bim protein in PTECs was significantly promoted after treatment with 40 mM high glucose by conducting Western blot(P<0.05).2.Stable PTEC cell lines which were transfected with lentivirus inhibiting Bim expression were successfully constructed.(1)The preliminary results showed that when the MOI of the lentivirus diluted by enhance solution was 20,the transfection efficiency of lentivirus down-regulating Bim was the highest and the fluorescence expression intensity was the strongest.(2)Lentivirus with MOI of 20 was transfected into PTECs,and 1 ?g/ml puromycin was applied for selection of stable PTEC cell lines for 1 week.RT-PCR and Western blot were used to verify the efficacy of the intervention.The results showed that compared with the PTECs transfected with negative control lentivirus group,the expressions of Bim mRNA and protein in PTECs transfected with lentivirus which can intervene Bim expression were significantly down-regulated(P<0.05),which confirmed that the stable PTECs transfected with lentivirus which can down-regulate Bim expression were successfully constructed and they were cultured for subsequent experiments.3.In vitro co-culture model was established successfully:Co-culture model was established and the groups included co-culture with control PTECs under high glucose,co-culture with PTECs transfected with lentivirus inhibiting Bim expression under high glucose,and co-culture with PTECs transfected with negative control lentivirus under high glucose.4.Up-regulation of Bim in PTECs induced by high glucose caused podocyte cytoskeleton injury and disordered arrangement.Co-culture with PTECs with down-regulation of Bim significantly alleviated the cytoskeleton injury of podocytes.(1)Immunofluorescence staining showed that after co-culture with PTECs for 48 hours under high glucose,the arrangement of F-actin in podocytes was disordered.However,after the intervention of Bim in PTECs for 48 hours,the arrangement of F-actin in podocytes returned to polarity.(2)Immunofluorescence staining showed the expression of synaptopodin in podocytes was significantly down-regulated after co-culture with control PTECs under high glucose for 48 hours.However,the expression of synaptopodin in podocytes was restored and up-regulated after co-culture with PTECs that transfected with lentivirus which downregulated Bim for 48 hours.(3)Western blot was conducted to detect the synaptopodin expression in podocytes in different co-culture groups and the results were consistent with immunofluorescence staining results.The results showed that the expression of synaptopodin in podocytes was also significantly down-regulated after co-culture with control PTECs under high glucose for 48 hours,but the expression of synaptopodin in podocytes was restored and up-regulated after co-culture with PTECs with Bim intervention for 48 hours(P<0.05).5.Further screening the downstream molecules that were regulated by Bim in PTECs to induce the injury of podocyte cytoskeleton,and the results revealed that the expression of NFAT2 and nuclear translocation were regulated by Bim.(1)The subtypes in the NFAT family which were regulated by Bim were screened by RT-PCR,and Western blot further confirmed that NFAT2 protein expression also were regulated by Bim.(2)NFAT family works as a class of transcription factor,and exerts gene regulation function by nuclear localization.Therefore,we detected the nuclear translocation changes of NFAT2 in PTECs after the regulation of Bim by immunofluorescence confocal microscopy.The results showed that after Bim intervention in PTECs,the nuclear translocation of NFAT2 was significantly reduced.6.After transfection of NFAT2 plasmid and siRNA for 48 h,RT-PCR was applied to examine NFAT2 mRNA expression and Western blot was conducted to examine NFAT2 protein expression in different groups.Results showed that NFAT2 mRNA and protein expression in PTECs were significantly down-regulated after transfection of NFAT2-siRNA compared with negative control siRNA(P<0.05).In contrast with the empty plasmid which could not carry the NFAT2 gene,both of NFAT2 mRNA expression and protein expression in PTECs were obviously up-regulated after transfection of NFAT2 plasmid(P<0.05).7.Bim in PTECs induced podocyte cytoskeleton dysfunction by activating NFAT2 under high glucose.In order to further determine the effects of Bim in PTECs on podocyte cytoskeleton by regulation of NFAT2,siRNA interfering with NFAT2 expression was transfected into PTECs,and plasmid upregulating NFAT2 expression was transfected into PTECs cell lines stably transfected with Lenti-Bim-shRNA.And then,synaptopodin expression and cytoskeleton filament arrangement in podocytes were detected.(1)Immunofluorescence staining showed that on the basis of the intervention of Bim in PTECs,upregulation of NFAT2 expression further induced the disordered cytoskeleton filament and reduced synaptopodin expression in podocytes,which indicated that upregulation of Bim in PTECs under high glucose promoted NFAT2 expression and then contributed to the cytoskeleton injury of podocytes.(2)Western blot was conducted to identify the changes of synaptopodin expression in podocytes after various co-culture treatment and the findings showed consistence with the results of synaptopodin expression by immunofluorescence staining.8.The morphology and appearance of mice:During the whole experiment,mice in UNX and Sham groups were generally in good condition,with smooth and shiny hair and sensitive response.Mice in DN group had disorganized and yellow hair,with emaciation,listlessness,lethargy and slow reaction after model construction.9.Body weight,blood glucose and urine volume:Distinct with mice in Sham and UNX groups,body weight gain of mice in DN group slowed down,and some mice even lost weight.The blood glucose of mice in DN group was significantly increased,and no spontaneous blood glucose recovery in DN group was found during the experiment.Additionally,the drinking intake and urine volume of mice in DN group was also markedly increased.10.Changes of urinary protein,kidney weight/body weight ratio,serum creatinine,and blood urea nitrogen in mice:Mice in DN group possessed obviously incremental 24 h urinary protein content,kidney weight/body weight ratio,serum creatinine and urea nitrogen levels when compared to mice in Sham and UNX groups.11.Renal glycogen staining:The glomerular structure of mice in UNX and Sham group was clear.The structure of renal tubules was normal and tubules arranged orderly.The mice in DN group had renal hypertrophy and mild hyperplasia of mesangial cells.Obvious glycogen accumulation was observed in the glomerulus of mice in DN group.The renal tissues in DN mice manifested as typical pathological changes which included disarrangement of renal tubules and thickening of basal membrane of renal tubules commonly presented in DN mice.12.Immunohistochemical staining showed that compared with UNX and Sham groups,Bim which was positively stained in proximal renal tubules of mice in DN group was significantly increased.13.Western blot results showed that compared with mice in UNX and Sham groups,obviously up-regulated expression of Bim protein in kidney tissue of mice in DN group was found(P<0.05),and significantly down-regulated expression of cytoskeleton-related protein synaptopodin in glomerular podocytes was also found(P<0.05).14.Immunofluorescence results showed that the expression of synaptopodin in glomerular podocytes of DN group was significantly down-regulated compared with UNX and Sham groups.Conclusions1.Through the establishment of an in vitro PTECs-podoctyes co-culture model,we found that the up-regulation of Bim protein in PTECs can promote the disorder and loss of polarity of podocytes cytoskeleton arrangement,that is,tubule-podocyte crosstalk existed.2.The mechanism of tubule-podocyte crosstalk was preliminarily explored:under high glucose,up-regulation of Bim in PTECs induced the disorder of cytoskeleton arrangement in podocytes by activating the downstream factor,NFAT2,that is,Bim/NFAT2-mediated tubule-podocyte communication played a potential regulatory role in the occurrence and development of DN.3.Bim protein expression in renal tubules was increased,and the expression of podocyte synaptopodin was reduced in DN mice,which may contribute to the occurrence of proteinuria in DN.BackgroundThe mechanisms of diabetic nephropathy(DN)involve a variety of factors,including genetic factors and signal transduction pathways which often regulate the expression of DN-related gene phenotypes.However,the increasing incidence of DN indicates that a deeper understanding of the underlying molecular mechanisms is still needed to develop better therapies of DN.More and more evidence showed that genes related to DN development were not only regulated by classical signaling pathways,but also controlled by epigenetic mechanisms such as histone chromatin modification,DNA methylation and non-coding RNA.The identification of the epigenetic mechanism of DN through the Genome Wide Association Study(GWAS)may also provide information for precision medicine research and have more benefits for diagnosis and treatment to prevent the development of end-stage renal disease.We previously clarified that tubular injury occurred much earlier than glomerular dysfunction in early DN and found that Bim upregulation of renal tubules caused podocyte cytoskeleton disorder by establishing in vitro proximal renal tubular epithelial cells(PTECs)-podocytes co-culture model,that is,tubular injury induced podocyte dysfunction in early occurrence of DN.At present,the research on the communication between renal tubules and podocytes has just started,and the exact mechanism of their interaction mediating DN remains to be explored.And in clinical work,we found that due to the-inconspicuous onset of DN,many patients often missed the opportunity to intervene early renal tubular lesions when treated,and the lesions have spread to the glomeruli.Therefore,preventing the progression of glomerular lesions has become the focus of research.In order to find the "prime culprit" that induces podocyte injury after tubular change in DN,we intend to investigate the initial mechanism of podocyte dysfunction after tubule-podocyte crosstalk in this part.Long noncoding RNAs(IncRNAs)are a class of non-coding RNAs whose transcripts are more than 200 nucleotides and do not show the ability of coding protein.Recent researches have shown that lncRNAs could participate in many physiological processes,and regulate cells' growth,development,senescence and death.Several evidences revealed that IncRNAs were involved in gene control,including regulating gene transcription,splicing,mRNA stability,epigenetic regulation,cell cycle control,differentiation,immune response and other biological mechanisms and processes.LncRNAs can also act as host RNAs for miRNAs and function as regulatory molecules with independent modules that enable them to bind specifically to DNA,RNA,and/or proteins,thereby affecting gene expression.Therefore,IncRNA often serve as an important transcriptional regulator to participate in the initiation and development of diseases.After many lncRNA expressions were found to be abnormally regulated in human diseases,the role of IncRNA in disease development was gradually revealed.Recent studies have shown that IncRNA also plays an important role in mediating the occurrence of DN.LncRNA PVT1 was involved in fibrosis and DN pathogenesis.Another study found that 21 IncRNAs were up-regulated in two renal fibrosis models and IncRNAs were down-regulated in Smad3 knockout mice models,suggesting that the TGF?/Smad3 signaling pathway may mediate renal inflammation and fibrosis through regulating these IncRNAs expression.A study in 2016 found that IncRNA MGC was the host IncRNA of nearly 40 miRNAs in miR-379 clusters and promoted the deposition of extracellular matrix(ECM)and induced mesangial cells hypertrophy in early DN mouse models.In addition,some studies have shown that overexpression of lncRNA CYP4B1-PS 1-001 inhibited the proliferation and fibrosis of mesangial cells in DN.Although there have been many studies on the mechanism of the role of lncRNAs in the occurrence and development of DN,these studies have only explored the mechanism from one perspective,and the role of lncRNAs in the communication between cells mediating DN has not been thoroughly reported.For clarifying the initial mechanism in the early lesions of podocytes after co-culture and achieving better treatment,we conducted IncRNA and mRNA array analyses to reveal the changes of lncRNA and mRNA expression in podocytes which were co-cultured with various PTECs under high glucose,and the potential pathological mechanistic study of tubule-podocyte communication inducing abnormal podocyte cytoskeleton arrangement was further carried out for providing interpretation of DN pathogenesis.ObjectivesOur previous studies have confirmed that Bim in PTECs mediated podocyte cytoskeleton injury.In order to further clarify the initial mechanism of podocyte cytoskeleton disorder caused by the communication of tubules and podocytes after the establishment of the co-culture model in vitro,the following explorations are conducted.Materials and methods1.High-throughput analysis of Sino human lncRNA array version 3.0.High-throughput analysis of Sino human lncRNA array version 3.0 was carried out.The contents included lncRNA and mRNA array detection.2.RT-PCR was used to detect the expression of lncRNA to verify whether the expression levels of lncRNA were consistent with the results of array analysis.3.The expression of lncRNA NONHSAT179542.1 in podocytes was regulated,and the effect of regulation was detected by RT-PCR:(1)Small interfering RNA of lncRNA NONHSAT179542.1 and negative control small interfering RNA were transfected into podocytes.(2)RT-PCR was used to detect the expression of lncRNA NONHSAT179542.1 mRNA in podocytes after regulation of lncRNA NONHSAT 179542.1.4.The expression of MICAL2 in podocytes was regulated,and the regulatory effect was detected by RT-PCR:(1)Plasmid of overexpressed MICAL2 and negative control plasmid were transfected into podocytes.(2)RT-PCR was used to detect the expression of MICAL2 mRNA after regulation of MICAL2 in podocytes.5.Measurement of cytoskeleton arrangement in podocytes after co-culture by immunofluorescence:(1)Further constructing the co-culture model in vitro,and regulating the expression level of lncRNA NONHSAT179542.1 in podocytes on the basis of inhibiting Bim expression in PTECs,and observing the cytoskeleton arrangement in podocytes.(2)After constructing an in vitro co-culture model,regulating the expression level of MICAL2 in podocytes on the basis of interfering expression of Bim in PTECs,and observing the cytoskeleton arrangement in podocytes.6.The expression of cytoskeleton-related protein synaptopodin in podocytes in different groups was detected by immunofluorescence:(1)The co-culture model in vitro was constructed,and the expression level of lncRNA NONHSAT179542.1 in podocytes was regulated on the basis of interfering Bim expression in PTECs.And the expression of synaptopodin in podocytes was observed.(2)After constructing an in vitro co-culture model,regulating the expression level of MICAL2 in podocytes and observing the expression of synaptopodin in podocytes.7.KEGG enrichment analysis and cis/trans-based target gene prediction:(1)The cytoskeleton-related pathways were enriched by KEGG enrichment;(2)The downstream target genes of lncRNA NONHSAT179542.1 were predicted by using cis/trans target gene prediction.8.Western blot was used to detect the changes of protein expression in podocytes in different co-culture groups:(1)The expression of synaptopodin protein in podocytes in different co-culture groups was detected:1)Co-culture model was constructed.On the basis of interfering Bim expression in PTECs,the expression level of lncRNA NONHSAT179542.1 in podocytes was further regulated,and the expression of synaptopodin protein in podocytes was observed.2)After constructing a co-culture model,regulating the expression level of MICAL2 in podocytes on the basis of interfering Bim expression in renal tubules,and observing the expression of synaptopodin in podocytes.(2)Detecting the expression of MICAL2 protein in podocytes in different co-culture groups:1)Constructing a co-culture model to detect the expression of MICAL2 in podocytes co-cultured with PTECs and Lenti-Bim-shRNA.2)Constructing a co-culture model to detect the expression of MICAL2 in podocytes co-cultured with Lenti-B im-shRN A,podocytes transfected with siRNA down-regulating lncRNA NONHSAT179542.1 on the basis of down-regulating Bim in PTECs,and podocytes transfected with negative control RNA on the basis of down-regulating Bim in PTECs.3)Constructing a co-culture model to detect the expression of MICAL2 in podocytes co-cultured with Lenti-Bim-shRNA,podocytes transfected with MICAL2 overexpression plasmid on the basis of co-culture with Lenti-Bim-shRNA,podocytes transfected with MICAL2 negative control plasmid on the basis of co-culture with Lenti-B im-shRNA.9.Immunohistochemical staining for MICAL2 location and expression in the kidney tissue of mice in each group.10.Statistical analysis:All samples were repeated more than 3 times for statistical analysis.The obtained data were statistically processed and analyzed by SPSS 22.0 software.Statistical significance assessment:T test was used for comparison between the two groups,with mean± S.E.M.One-way analysis of variance(One-way ANOVA)was used for more than three groups.P<0.05(bilateral)was considered statistically significant.The bar graph was drawn using GraphPad Prism5.0 software.Results1.Selection of differentially expressed lncRNAs:(1)We set the fold change(FC)to 1.5 times and P<0.05 as the selection criteria.Between the podocytes co-cultured with control PTECs under high glucose and podocytes co-cultured with Bim-knocked down PTECs under high glucose,35 differentially expressed lncRNAs were obtained.Seventeen lncRNAs were up-regulated in podocytes co-cultured with Lenti-Bim-shRNA,while 18 lncRNAs were down-regulated in podocytes co-cultured with Lenti-B im-shRNA.We selected the top ten differentially expressed lncRNAs with the largest fold change.(2)Between the podocytes co-cultured with PTECs transfected with negative control lentivirus under high gl...