The Effect Of MiR-21/Dnm1l Signal Pathway In PAN-induced Potocyte Injury And Mitochondrial Dysfunction

The common pathological types of renal glomerular diseases include focal segmental glomerulosclerosis,minimal change nephropathy and membranous nephropathy.The injury of the structure and function of podocytes is a major mechanism of glomerular disease.Our previous studies have proved that mitochondrial dysfunction is an early event of podocyte injury and in the pathophysiology of renal diseases.MiRNAs play essential roles in the pathophysiology of many kidney diseases.The first report of miR-21 in fibrosis is firstly shown in heart failure by Thum et al.Subsequently,there are a number of researches of the relationship between miR-21 and acute kidney injury,diabetic nephropathy,chronic kidney disease and renal fibrosis.But the effect and mechanism of miR-21 on potocyte is still unclear,which needs further study.Part1 The effect of miR-21 in PAN-induced potocyte injury and mitochondrial dysfunctionObjective:To explore the effect of miR-21 in PAN induced podocyte injury and mitochondrial dysfunction.Methods:1.20 male Spague-Dwley(SD)rats were randomly divited innto the following groups(n=10 for each group):the control group(sham)and the PAN group(PAN).One single dose--150mg/kg of puromycin aminonucleoside was injected into rats of the PAN group by tail-vein injection while the rats in the control group were given intravenous injection of saline.2.MPC5 were treated with different dose(25,50,75,100 mg/ml)of PAN and the controls were given saline.3.We divided the cultured MPC5 into the control group(vehi)and the overexpression of miR-21 group(miR-21 OE).The overexpression of miR-21 group was treated with transfection reagent siRNA-mate and of miR-21 mimicss,with siRNA mate at a concentration of 2μl/ml and miR-21 mimicss concentration of 20nm.The control group was given equal transfection reagent and negative control sequence.24 hours urinary protein excretion was detected by the Bradford prptein assay.Potocyte injury was observed by the transmission electron microscope.Nephrin,podocin,mitochondrial DNA(mtDNA)copy numbers and miR-21 expression levels were measured by real-time PCR.Nephrin and podocin expression level was detected by western blot.Cellular ROS production was determined by DCFDA fluorescence.The mitochondrial membrane potential was examined by JC-1 staining.MPC5 apoptosis was assessed using Hoechst 33258 staining and annexin V/flow cytometry detection.Results:1-In vivo,PAN induced SD rats appear proteinuria and podocyte injury,and PAN induced the miR-21 expression.24 hour urine albumin was increased significantly in the presence of PAN,but not in sham group.Examination of kidney ultrastructure morphology showed foot-process effacement after 14 days of PAN treatment,which appeared as sheets covering the glomerular basement membrane,with disappearance of the slit diaphragm gap.Examination of kidney ultrastructure morphology showed numerous electrondense granulae appeared in the cytoplasm of the podocytes,and the cell configuration changed,with appearance of microvilli,vacuoles,and large plasma membrane extensions.The expressions of nephrin and podocin decreased in PAN group.The mtDNA copy number in the kidney cortex at 14 day after PAN infusion was significantly decreased.Consistent with the proteinuria,mitochondial dysfunction and podocyte damage,the expressions of miR-21 increased several times after 14 days in PAN-infused SD rats.2.In vitro,podocytes were exposed to 0-100 nmol/L of PAN,which is dose dependently,reduced nephrin and podocin protein expression with a noticeable effect at 50μg/mL and a maximal effect at 100μg/mL.Then we treated MPC5 with PAN at a concentration of 50μg/ml,after the treatment,ROS increased while JC-1 and mtDNA copy numbers decreased.3.Transient transfection of MPC5 with miR-21 mimicss can increase the expression of miR-21 by 4 times.The apoptosis of podocytes was induced by miR-21.Overexpression of miR-21 also decreased the expression of nephrin and podocin.MiR-21 reduced mtDNA copy number and JC-1,and induced mitochondrial superoxide anion production.Conclusion:MiR-21 is involved in PAN induced podocyte injury and mitochondrial dysfunction。Part2 Dnm1l is a direct target gene of miR-21Objective:To validate Dnm1l is a direct target gene of miR-21.Methods:1.Computational tools were used to predict the potential targets genes of miR-21.2.A dual luciferase reporter assay was used to validate Dnm1l is a direct target gene of miR-21.3.MPC5 cultured in vitro and divided into the over expression of miR-21 group(miR-21 OE)and control group(vehi).4.Transient transfect of plasmid Dnm1l coding sequence to MPC5 by transfection reagent lip2000 with a concentration of 3 μl/ml and a concentration of s 1 μg/ml of plasmid,and then over express miR-21.MPC5 were divided into control group(vehi),miR-21 over expression group(miR-21 OE)and miR-21 and Dnm1l co-expression group(miR-210E+Dnm1l).The expression of Dnm1l was detected by real-time PCR and western blot.MPC5 apoptosis was assessed using Hoechst 33258 staining and annexin V/flow cytometry detection.Cellular ROS production was determined by DCFDA fluorescence.The mitochondrial membrane potential was examined by JC-1 staining.Nephrin and podocin expression level was detected by western blot.Results:1.We used computational tools(TargetScan,PicTar Genome and RNA Hybrid)to predict potential targets of miR-21,and found that Dnm1l is a potential target gene for miR-21 as there is a seven-nucleotide complementary sequence between miR-21 and the Dnm1l 3’UTR.2.Luciferase reporter assay showed that miR-21 upregulation significantly decreased the luciferase activity of wild-type Dnm1l but not the mutant Dnm1l.3.MiR-21 overexpression significantly inhibited Dnm1l protein and mRNA expression in podocytes.4.Both mRNA level and protein level of Dnm1l were markedly increased compared with vehi group.5.MiR-21 could remarkably induce JC-1,mtDNA copy numbes and nephrin expression and enhanced ROS production,whereas Dnm1l transfection could almost block such effects.Conclusion:Dnm1l is a direct target gene of miR-21.Part3 The effect of miR-21/Dnm1l signaling pathway in PAN-induced potocyte injury and mitochondrial dysfunctionObjective:To investigate whether miR-21 induces PAN-induced podocyte injury and mitochondrial dysfunction by downregulating Dnm1l.Methods:We cultured MPC5 and divided it into control group(vehi),PAN stimulation group(vehi+PAN)and miR-21 low expression plus PAN stimulation group(miR-21 KD+PAN).Transfected reagents siRNA mate and miR-21 inhibitor were co-transfected into cells of the low expression plus PAN stimulation group witn the concentration of siRNA mate for 2 μl/ml and miR-21 inhibitor concentration for 20nM while the other two groups received equal transfection reagent and negative control sequence.Nephrin and Dnm1l expression level was detected by western blot.MPC5 apoptosis was assessed using Hoechst 33258 staining and annexin V/flow cytometry detection.Cellular ROS production was determined by DCFDA fluorescence.The mitochondrial membrane potential was examined by JC-1 staining.MtDNA copy numbers expressing was determined by real-time PCR.Results:The protein level of nephrin and Dnm1l was decreased in podocytes after PAN stimulation,and knockdown of miR-21 can attenuates the decrease.The miR-21 konckdown podocytes were treated with PAN,and we found that downregulation of miR-21 attenuated the podocytes apoptosis induced by PAN.Consistently,miR-21 knockdwn ameliorated mtDNA copy number and JC-1 and decreased ROS induced by PAN.Conclusion:MiR-21 induces PAN-induced podocyte injury and mitochondrial dysfunction by downregulating Dnm1l.