| BackgroudDiabetes mellitus(DM)is a kind of chronic disease that has emerged as a serious problem of public health throughout the world.China has become the country with the highest number of diabetes worldwide.Diabetic kidney disease is one of the most important cause of death in patients with diabetic nephropathy,However,the pathogenesis of diabetic kidney disease is still unclear.Injury of podocytes is a major determinant of proteinuria as the clinical hallmark in the early stage of diabetic kidney disease and loss of podocytes because of apoptosis has been reported to be the vital role in the pathogenesis.Podocytes are terminally differentiated epithelial visceral cells which form crucial component of the glomerular filtration barrier which also include the other two major components:the fenestrated endothelial cell and the glomerular basement membrane.The podocytes wrap around the capillaries and leave slits between them for blood filtration,known as filtration slits which is composed of several proteins including nephrin,podocin,and NEPH-1,etc.It was reported that under conditions of high glucose,oxidative stress,activation of the renin angiotensin aldosterone system(RAAS),activation of Notch and P38MARK signal pathways contribute to apoptosis of podocytes.But the underlying mechanism is still unclear.In recent years,studies have shown that hypoxia is an important factor in the apoptosis of podocytes,and it is also considered as a key pathway for progressive renal damage in diabetic nephropathy.The hypoxia inducible transcription factor a(HIF-1?)is a critical regulator of cellular responses to hypoxia and continuous activation of HIF-1? is hallmark for hypoxia.Vascular endothelial growth factor(VEGF)is one of the fator upregulated by HIF-1? and is the primary cytokine related to angiogenesis.In glomeruli;VEGF is predominantly produced by podocytes and ensure adequate supply of oxygen for solute transport through activating VEGF receptor on endothelial cells.Mitochondria plays a central role in the energy metabolism and act as sensor and target in hypoxia.Mitochondrial dysfunction of fusion and fission can lead to apoptosis.Mitochondrial dynamin related protein 1(Drp-1),a member of the dynamin family of GTPases,plays an important role in mitochondrial dynamics.Drp-1 translocates from the cytosol to the mitochondria and mediates mitochondrial fission and apoptosis.Considering the significance of miRNA in the pathogenesis of various kidney diseases,it has been attracting more and more attention currently.Some studies showed that miRNA is crucial for differentiation,foot process function of podocytes,and the integrity of the glomerular filtration barrier,and abnormal regulation of their target genes may induce progressive proteinuria and glomerulosclerosis.MiR-30a was detected in collecting tubular epithelial cells and podocytes in kidney and its expression reduced in the Dicer knockout mouse which showed serious defects in kidney including destruction of foot process effacement,irregular and split areas of the glomerular basement membrane,apoptosis and depletion of podocytes,mesangial expansion,capillary dilation,and glomerulosclerosis.Studies of cardiovascular and neurological disorders have shown that miR-30a could prevent mitochondria fission and apoptosis through inhibiting Drp-1 pathway.But its role in diabetic nephropathy is still unclear.Angiotensin receptor antagonist(ARB)is commonly used for the treatment of diabetic kidney disease,Losartan is widely used in the clinical treatment.Losartan works as an angiotensin receptor antagonist to exert protective effects against kidney injury of diabetic nephropathy.The Hemodynamic effects of Losaran includes the amelioration in glomerular hyperperfusion,high filtration and high glomerular capillary pressure.Losartan also improves non-hemodynamics of diabetic nephropathy including oxidative stress,apoptosis,hyperplasia of extracellular matrix and permeability of glomerular filtration membrane,etc.Recently the non-hemodynamic improvements attracted more researchers ' interest and a lot of studies showed that ARB may protect podocytes and reduce proteinuria by reduce the shedding,depletion,foot processes of podocytes.However,the mechanism is still unclear.Proteinuria is a hallmark of diabetic nephropathy,and studies have highlighted that the reduction of podocytes may cause proteinuria and glomerulosclerosis directly.Losaran can improve proteinuria through reducing injury of podocytes to exert its protective effects in diabetic nephropathy,but its non-hemodynamic mechanism remains unclear.Hypoxia is deeply involved in the pathogenesis of diabetic nephropathy.HIF-1 a and its target VEGF are activated in hypoxia.Hypoxia affects mitochondrial dynamics and Drp-1 regulates cell apoptosis through mediating mitochondria dynamics.The study of miRNA in the pathogenesis of kidney diseases has become a hot research in recent years.MiR-30a,highly expressed in renal tissues,is a promising miRNA for diabetic nephropathy.We speculate that miR-30a is involved in injury of podocytes in condition of high glucose.This study includes two sections:First,Establishing mouse model of diabetic nephropathy,and detect effects of Losaran on proteinuria and expression of nephrin,podocin,HIF-1?,VEGF,miR-30a and Drp-1 through immunohistochemistry,western blot and qRT-PCR;Sencond,Determining the effects of Losaran on apoptosis of podocytes,mitochondria,expression of HIF-1?,VEGF,miR-30a,Drp-1 throughn flow cytometry,electron microscopy,qRT-PCR,Western blot and immunofluorescence techniques in podocytes induced by high glucose,and then we transfect mimic and inhibitor of miR-30a in podocytes induced by high glucose and detect effects of miR-30a on expression of HIF-la and Drp-1.The experiment will provide a new evidence-based experimental basis for the pathogenesis of diabetic nephropathy and the therapeutic effect of ARB,and will contribute to provide new methods for the treatment of diabetic nephropathy.1.To investigate the effect of Part ? Effects of Losaran on HIF-1?,VEGF,Drp-1 and miR-30a in kidney of mice with Diabetic NephropathyObjectivesLosaran on urinary protein and injury of podocytes in mice with diabetic kidney disease.2.To investigate the effects of Losaran on HIF-1?,VEGF,Drp-1 and miR-30a expression in renal tissue of mice with diabetic kidney diseaseMethods1.Animal grouping:30 male Kunming mice,approximately 4 weeks of age,weighing 18-20 g,were randomly divided into three groups of 10 mice,namely group of normal control(group C),group of diabetic models(group DN)and group of treatment with Losartan(group DN+Los).Group C was given the same amount of normal feed,and DN group and DN+Los group were fed with high sugar and high fat diets to induce diabetes with abnormal glucose and lipid levels.At the end of the tenth week,Streptozotocin(STZ)with a concentration of 35 mg/Kg was intraperitoneally injected,and high-sugar and high-fat diets were continued to be fed for 12th week.Randomized blood glucose>16.7 mmol/L was detected for successful modeling.C and DN groups were intragastrically administered with normal saline(10 mg/Kg/d),and DN+Los group were givenLosaran(10 mg/Kg/d)by gavage.2.Blood glucose,serum creatinine,serum urea nitrogen,urinary ACR test:Measure mouse body weight weekly,take urine randomly at the 16th,20th,and 24th weeks and detect microalbumin and creatinine in urine,then calculate ACR.Blood were taken from the tail vein to measure blood glucose,and blood was taken from internal hemorrhoids to measure creatinine and urea nitrogen in serum.3.The mice were sacrificed by cervical dislocation at 24th week and the kidneys were isolated and fixed the kidneys in the 10%neutral buffered formalin.HE staining,PAS staining and PASM-Masson staining were used to observe the pathological changes in each group.4.Western blot was used to detect the expression of nephrin and podocin in renal tissue at 24th week.5.Immunohistochemical staining was used to detect the expression of HIF-1?,VEGF and Drp-1 in renal tissue at 24th week.6.Real-time fluorescence quantitative RT-PCR was used to detect the expression of miR-30a in renal tissue at 24th week.7.Statistical methods:Measurement data were expressed as mean±standard error.According to the homogeneity test of variance,the independent sample t-test was used for comparison between the two groups,and multiple-group comparisons were performed using one-way analysis of variance.All data were completed by statistical software SPSS 19.0.The difference was statistically significant at P<0.05.Results1.General condition:In group C,the mice were in good mental state,responsiveness and smooth coat;in group DN,the mice were apathetic,sluggish,dull and thirsty,and unresponsive,with dull hair,and the symptoms increased with weeks.Obviously;the statu of mice in the group DN+Los was between the group C and group DN.2.Blood glucose,serum creatinine,serum urea nitrogen,and ACR:At 16th,20th,and 24th week,blood glucose was higher significantly in group DN and group DN+Los than in the group C(P<0.01).There was no significant difference in blood glucose between group DN and group DN+Los(P>0.05);Compared with group C,ACR in group DN and group DN+Los treatment group were significantly higher(P<0.01);Compared with group DN,urinary ACR in group DN+Los were lower(P<0.05).There was no significant difference in creatinine and urea nitrogen in serum between the three groups(P>0.05).3.The pathological changes of renal tissue in mice of each group:under light microscope,glomerular structure was clear in group C,the basement membrane was regular;in group DN glomerular hypertrophy,basement membrane thickening,and mesangial membrane were observed,mesangial matrix was accumulated;The statue of change in kidney of group DN+Los was between the group C and group DN.4.Losaran can increase the expression of nephrin and podocin in renal tissues of mice with diabetic kidney disease:The results of the Western blot showed that the expression of nephrin and podocin protein decreased significantly in the group DN and group DN+Los compared with the group C(P<0.01).Compared with the group DN,the expression of nephrin and podocin protein increased in the group DN+Los(P<0.05).5.Losaran can reduce the levels of HIF-1?,VEGF and Drp-1 protein in renal tissue of mice with diabetic kidney disease.The results of immunohistochemical staining showed that HIF-1? was mainly expressed in the nucleus.the expression of HIF-1? was rare in group C.compared with group C,HIF-1? expression in group DN and group DN+Los increased significantly(P<0.01),compared with group DN,HIF-1? expression in group DN+Los decreased(P<0.05).VEGF and Drp-1 were mainly expressed in cytoplasm.VEGF and Drp-1 expression were rare in group C.Compared with group C,VEGF and Drp-1 expression were increased significantly in group DN and group DN+Los(P<0.01,P<0.05).compared with group DN,the expression of VEGF and Drp-1 was decreased in group DN+Los(P<0.05).6.Losaran can increase the expression of miR-30a in kidney of mice with diabetic kidney disease:Immunofluorescence result showed hat,Compared with group C,the level of miR-30a in group DN and group DN+Los were significantly lower(P<0.01,P<0.05),compared with group DN,the level of miR-30a in group DN+Los was higher than group DN(P<0.05).Conclusion1.Losaran can reduce the urinary ACR in mice with diabetic kidney disease.2.Losartan can upregulate the expression of nephrin and podocin proteins in podocytes of mice with diabetic kidney disease3.Losartan may improve injury of podocytes in mice with diabetic kidney disease through upregulating the expression of miR-30a and downregulating the expression of HIF-1?,VEGF and Drp-1.Part II Effects of Losaran on HIF-la,VEGF,Drp-1 and miR-30a of Podocytes Induced by High GlucoseObjective1.To investigate the effect of Losaran on apoptosis and mitochondrial injury of podocytes induced by high glucose.2.To investigate the effect of Losaran on the expression of HIF-1?±,VEGF,Drp-1 and miR-30a of podocytes induced by high glucose.3.To investigate the effect of miR-30a on the expression of HIF-1? and Drp-1 of podocytes induced by high glucose.Methods1.Culture and grouping of podocytes:the podocytes were cultured in RPMI 1640 medium containing 15%heat-inactivated fetal bovine serum and 100 U/ml penicillin and 100 mg/mL streptomycin.The podocytes were cultured in RPMI 1640 medium containing 10 U/ml of mouse recombinanty-IFN at 33?,and then the podocytes were differentiated and matured at 37? in a medium free of y-interferon for about 14 days.Differentiated podocytes were cultured in RPMI 1640 medium for 24 hours.Podocytes were divided into the following three groups:(1)group of normal control(group C):incubated in RPMI 1640 containing 5.6 mM glucose;(2)group of high glucose(group HG):incubated in RPMI 1640 containing 30 mM glucose;(3)group of Losaran treatment(group HG+Los):Incubated in RPMI 1640 containingLosaran 10-5M and 30 mM glucose.2.Effects of Losaran on apoptosis of podocytes induced by high glucose:Flow cytometry was used to detect the podocyte apoptosis at 0h,12h,24h and 48h.3.Electron microscopy was used to observe the change of mitochondria in podocytes at 24h.4.Real-time fluorescence quantitative RT-PCR was used to detect the expression of HIF-1?,VEGF,Drp-1 mRNA and miR-30a in podocytes induced by high glucose at 0h,12h,24h and 48h.5.Immuno:fluorescence and Western blot were used to detect the expression of HIF-1?,VEGF and Drp-1 in high glucose induced podocytes at Oh,12h,24h and 48h.6.Transfection was performed on podocytes induced by high glucose by miR-30a mimic and inhibitor to observe the change of miR-30a,Drp-1,and HIF-la mRNA and protein.Podocytes were divided into the following five groups:high glucose group(group HG),high glucose with miR-30a mimic group(group HG+mimic),high glucose with miR-30a mimic negative control group(group HG+ mimic-NC),high glucose with miR-30a inhibitor group(group HG+ inhibitor)and high-glucose with miR-30a inhibitor negative control group(group HG+ inhibitor-NC).Flow cytometry was used to detect the apoptosis of podocytes at Oh,24h in each group.Immunofluorescence was used to detect the expression of miR-30a,HIF-1?,and Drp-1 mRNA of each group at at Oh,24h and 48h.Western blot was used to detect the expression of HIF-1 a and Drp-lprotein of each group at 24h.7.Statistical methods:Measurement data are expressed as mean ± standard error.According to the test results of homogeneity of variance,two groups were compared using independent sample t-test,and multiple groups were compared using one-way analysis of variance.All data were analyzed by statistical software SPSS 19.0.Completed,P<0.05 was considered statistically significant.Results1.Losaran can inhibit the apoptosis of podocytes induced by high glucose:The results of Flow Cytometry showed that,at 12h,24h and 48h,compared with group C,the apoptosis rate of group HG and group HG+Los increased significantly(P<0.01),and apoptosis rate gradually increased with time;Compared with group HG,the apoptosis rate of group HG+Los was decreased(P<0.05).2.Losaran can improve the mitochondrial impairment of podocytes induced by high glucose:Electron microscope results showed that,at 24h,In group C,the mitochondria were normal in shape which was rod-shaped or oval-shaped.The adventitia was smooth and complete,and the enamel morphology was intact.The electron density in the endometrium was even.Compared with group C,mitochondria of podocytes in HG group were swollen,outer membrane was thickened or vacuolization,the intima and tendon were ruptured or disappeared,outer membrane was ruptured,and the number of mitochondria decreased.Compared with HG group,mitochondria injury of group HG+Los was less.3.Losaran can increase miR-30a expression and decrease HIF-1?,VEGF and Drp-1 mRNA of podocytes induced by high glucose:Real-time fluorescence quantitative RT-PCR results show that,at 12h,24h,and 48h,compared with group C,miR-30a in group HG and group HG +Los were significantly lower than that in group C significantly(P<0.01,P<0.05),compared with group HG,miR-30a in group HG+Los was significantly higher than that in HG group(P<0.05,P<0.01,/P<0.01).Compared with group C,at 12h,24h,and 48h,the levels of HIF-1?,VEGF,and Drp-1 mRNA in group HG and group HG +Los were lower significantly(P<0.01).Compared with HG,the levels of HIF-1?,VEGF,and Drp-1 mRNA in group HG+Los decreased(P<0.05).4.Losaran can reduce expression of HIF-1?,VEGF and Drp-1 in podocytes induced by high glucose:Immunofluorescence results showed that,at 12h,24h,and 48h,HIF-la was expressed mainly in the nucleus of podocytes,VEGF and Drp-1 are mainly expressed in the cytoplasm of podocytes.Compared with group C,the levels of HIF-la,VEGF and Drp-1 protein in group HG and group HG+Los increased.Compared with HG,the levels of HIF-1?,VEGF,and Drp-1 protein in group HG+Los decreased at 12h,24h,48h.Western blot results showed that the levels of HIF-1?,VEGF and Drp-1 protein in group HG and group HG+Los increased significantly(P<0.01).Compared with HG,the levels of HIF-la,VEGF,and Drp-1 protein in group HG+Los decreased at 12h,24h,48h(P<0.05).5.Effects of miR-30a mimic and inhibitor on apoptosis of podocytes induced by high glucose:At 24h,Compared with group HG,the apoptosis of podocytes in group HG+mimic was lower(P<0.05),the apoptosis of podocytes in group HG+inhibitor was higher(P<0.05),Compared with group HG+mimic,the apoptosis of podocytes in group HG+inhibitor was significantly higher(P<0.01).6.Effects of miR-30a mimic and inhibitor on HIF-la and Drp-1mRNA:Real-time fluorescence quantitative RT-PCR results showed that,Compared with group HG,the level of miR-30a in group HG+mimic was significantly higher(P<0.01),the level of miR-30a in group HG+inhibior was significantly lower(P<0.01),Compared with group HG,the levels of HIF-1? and Drp-1 in group HG+mimic were lower(P<0.05),the levels of HIF-1? and Drp-1 in group HG+inhibitor were higher significantly(P<0.05).Compared with group HG+mimic,the levels of HIF-l?and Drp-1 in group HG+inhibitor were all higher significantly(P<0.01).7.Effects of miR-30a mimic and inhibitor on the levels of HIF-la and Drp-1 protein level:Immunofluorescence results showed that,at 24h,expression of HIF-la and Drp-1 protein in group HG+mimic were lower,expression of HIF-1? and Drp-1 protein in group HG+inhibitor were higher.Western blot results showed that,at 24h,Compared with group HG,the levels of HIF-1? and Drp-1 protein in group HG+mimic were higher(P<0.05);Compared with group HG,the levels of HIF-1?and Drp-1 protein group HG+inhibitor was higher significantly(P<0.01).Conclusion1.Losaran can inhibit apoptosis and mitochondrial impairment of podocytes induced by high glucose.2.Losaran may improve injury of podocytes induced by high glucose through upregulating miR-30a and downregulating Dip-I,HIF-1? and VEGF.3.MiR-30a may improve injury of podocytes induced by high glucose through regulate Drp-1 and HIF-la negatively.Conclusion of full textLosartan can protect podocytes and reduce urinary protein in diabetic kidney disease,because it may upregulate miR-30a and then downregulate HIF-1?,VEGF and Drp-1,improve mitochondrial impairment and apoptosis of podocytes. |
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