Ang-(1-7) Inhibited Mitochondrial Fission In High Glucose-induced Podocytes By Up-regulation Of Mir-30a And Down-regulation Of Drp1 And P53

BackgroundDiabetes is currently the most common disease in the world.Diabetic nephropathy(DN)is an important diabetic microangiopathy,which is one of the serious complications of diabetes and one of the leading causes of death.The incidence rate in China is also on the rise,and it has become the second leading cause of end-stage renal disease,second only to various glomerulinephritis.Diabetic nephropathy(DN)is one of the main causes of end-stage renal disease(ESRD),causing a huge social and economic burden that seriously affects the quality of life of patients.Renal podocytes injury is closely related to diabetic nephropathy.Mitochondria are the main sites of energy metabolism in eukaryotic cells,and play an important role in physiological and pathological activities such as free radical production,cell senescence,and apoptosis.Mitochondria fusion and division not only maintain normal mitochondria morphology in normal cells.And more importantly,The role is also involved in maintaining mitochondrial DNA stability,energy synthesis,mitochondrial division and fusion are synergistic,the process is highly conservative in evolution.Studies on mammalian cells have found that involved in the process of mitochondrial division Proteins can be divided into two classes:dynamin-related protein 1(Drp1)and receptor-like proteins.Drp1-mediated mitochondrial division is very similar to Dynamin-mediated endocytic vesicle division.Drpl is a large GTPase molecule with a GTPase domain at the N-terminus and a GTPase effector domain at the C-terminus(GED);in the middle is the Dynamin homology domain(also known as the middle zone)and the Insert B zone.Drpl is primarily localized in the cytoplasm and is present as a multimer(possibly a tetramer).The Dipl protein is a motor-associated protein 1,which belongs to the superficial family of dynamin.When Drpl is localized to mitochondria,it is driven by GTP to assemble into a helical structure,and this self-assemble can drive the membrane to shrink.Self-assemble can also promote GTP hydrolysis,this hydrolysis is likely to cause a change in the conformation within the Drpl helix,thereby completing the membrane splitting.P53 gene is a human tumor suppressorgene.The gene encodes a molecular weight of 53kD protein Named P53.P53 protein consists of 393Amino acidComposition with specific transcriptional activation.P53 captures in the cell cycle and plays an important role in DNA repair,cell senescence,differentiation,apoptosis,etc.It can repair damaged cells or remove severely damaged cells to avoid the harmful effects of these cells on the body.P53 mediated Cell signal transduction,The pathway plays an important role in regulating the normal life of cells,and its connection with other signal transduction pathways in the cell is very complicated.MicroRNAs(miRNAs)are non-coding small RNAs involved in posttranscrip-tional regulation of gene expression.Incomplete binding to the target mRNA results in instability or translational inhibition of the target gene.miRNAs can be used as tumor suppressor genes and oncogenes.It is involved in the regulation of gene expression in life activities such as growth,development,reproduction,senescence and death of animals and plants.MiRNAs are also associated with various kidney diseases such as IgA nephropathy,obstructive nephropathy,diabetic nephropathy,systemic lupus(systemic lupus).Erythematosus,SLE)related.MiRNA is involved in the pathophysiological process of the disease through differential expression.The expression of microRNA-30a in renal podocytes and collecting duct epithelial cells of normal mice was detected.At present,in the study of cardiovascular and nervous system diseases,it has been found that microRNA-30a can inhibit mitochondrial division by inhibiting Drp-1 pathway from upstream,but there are few studies on podocyte injury in diabetic nephropathy.Angiotensin-(1-7)(Ang-(1-7))is one of the important biologically active substances in the renin-angiotensin-aldosterone system(RAAS),which is regulated by the Mas receptor at the intracellular signal factor level.Improves endothelial cell function,inhibits vascular smooth muscle cell proliferation and migration,relaxes blood vessels,inhibits ventricular remodeling,etc.,thereby exerting functions of protecting cells,regulating blood pressure,protecting heart,kidney,etc.;and Ang-(1-7)through Mas receptor It can inhibit the formation of atherosclerotic plaque and enhance the stability of plaque.Ang-(1-7)is involved in angiogenesis in human atherosclerotic plaques.Recent studies in China have shown that Ang-(1-7)can protect podocyte injury induced by serum in preeclampsia patients in vitro,and its effect may be related to the effect of Ang-(1-7)and Mas receptor binding,but specific molecules of the biological mechanism is not clear.Based on the currently recognized role of Ang-(1-7),the intracellular signaling pathway of Ang-(1-7)via Mas receptor is closely related to mitochondrial function,in which miR-30a is involved in the regulation of mitochondrial dynamics.Expression of P53 and dynamin-related protein 1(Drpl),and analysis of whether Ang-(1-7)regulates mitochondrial function through receptors,and what is the expression of apoptotic proteins P53 and Drpl,The effect of in-depth study of the intracellular signaling pathway of Ang-(1-7)through the receptor provides a theoretical basis for revealing the molecular biological mechanism of Ang-(1-7).In conclusion,podocyte Injury may play a key role in the development of diabetic nephropathy.Ang-(1-7)can play a protective role in diabetic nephropathy by reducing podocyte damage.The molecular biological mechanism of Ang-(1-7)protecting podocytes cultured in high glucose environment is explored by using Ang-(1-7)to interfere with podocytes cultured in high glucose environment.Three experiments are designed in this study:(1)To observe the effect of Ang-(1-7)on the morphology of podocytes under high glucose environment;(2)to make the podocyte mitochondria morphological changes.Western blot and immunocytochemical techniques were used to observe the effects of Ang-(1-7)on the expression of Drpl and P53 in podocytes under high glucose environment;(3)qRT-PCR was used to study the effects of Ang-(1-7)on the expression of microRNA30a in podocytes under high glucose environment.MicroRNA30a mimics and inhibitors were used in podocytes induced by high glucose,andDrpl and P53 were identified as target genes of microRNA 30A by qRT-PCR,Western blot and immunocytochemistry.Part I The study of the effect of Ang-(1-7)on mitochondrialmorphology of podocytes in high glucose environmentObjectivesThe effects of Ang-(1-7)on mitochondrial morphology of podocytes in high glucose environment were studied.The characteristics of mitochondrial division and fusion of podocytes were observed by transmission electron microscopy.Morphologically,Ang-(1-7)was clarified the protective effect of podocytes in high glucose environment.MethodsThe well-grown mouse podocytes were inoculated into 6-well cell culture plates,and 1.5×105cells were seeded per well,and 1.5 ml of RPMI-1640 medium containing 10%FBS was added to each well to grow podocytes.After overnight,the cell density reached 60-70%.the medium was discarded.and fresh 10%FBS RPMI-1640 medium was added to the wells of the culture plate according to the experiment:(1)Normal RPMI-1640 medium group(2)normal RPMI-1640 medium + Ang-(1-7)(10nmol/L)group;(3)high glucose(33 mmol/L)medium group;(4)high glucose(33 mmol/L)culture Base +Ang-(1-7)(lOnmol/L)group;After 48 hours of continuous culture,the experiment was carried out.Under the inverted phase contrast microscope,the state and quantity of the cells were observed.After observation under an inverted phase contrast microscope,the podocytes were fixed with 2.5%glutaraldehyde,then coated with agar,ultrathin sections,and examined by transmission electron microscopy.Focus on the morphology and quantity of mitochondria in podocytes,whether there are swelling and rupture,and whether there is vacuolization,etc.Each group selects multiple fields of view for photographing.ResultsAfter 48 hours of treatment of each group of cells,the culture plate was taken out from the incubator,and the color of the medium in each well was visually observed by the naked eye,and the medium of the high glucose group was dark.The nonnal group and the high glucose+ Ang-(1-7)group were lighter in color.The culture plate was placed under an inverted phase contrast microscope.The normal control group and normal RPMI-1640 medium + Ang-(1-7)(10nmol/L)were observed,podocytes have no morphological changes,podocytes are evenly distributed at the bottom of the plate,cells are dendritic,with multiple pseudopods,which grow close to the bottom of the culture plate;2.High sugar(33mmol/L)In the RPMI-1640 medium,the podocyte volume increased,the cell density increased,and the high glucose(33 mmol/L)RPMI-1640 medium + Ang-(1-7)(10 nmol/L)group.The cells are still growing more evenly and the morphology is near normal(Figure 1).After the podocytes of each group were fixed and coated with agar,ultrathin sections were taken.The mitochondrial morphology of the podocytes was observed by transmission electron microscopy.We observed the normal mitochondrial morphology of the control group.The arrow in Figure2 indicates that the mitochondrial membrane is intact and the outer membrane is more Smooth,mitochondrial cristae is regular,no bubble change.Compared with the control group,the high glucose group increased the mitochondrial division of the podocytes,mitochondrial cristae is irregular,mitochondria swollen,vacuolated,as indicated by the arrow.The actual number of mitochondria increased,but the volume became smaller and a large number of vacuolar fusions were seen(Fig.2).On the other hand,compared with the HG group,the mitochondrial division of podocytes in the Ang-(1-7)+ high glucose group was reduced,and the mitochondrial structure was close to the normal control group.Compared with the HG group,the swelling and vacuolar change were significantly higher.The sugar group is reduced as indicated by the arrow.ConclusionsAng-(12-7)can inhibit the division of mitochondria in podocytes under high glucose environment.It can prevent mitochondrial swelling and vacuolar degeneration,and protect podocytes in high glucose environment.Part Ⅱ Research on the effect of Ang-(1-7)on the expression ofDrpl and P53 in podocytes in high glucose environmentObjectivesTo clarify the influence of Ang-(1-7)on the expression of Drpl and P53 in podocyte in high glucose environment,To determine the effect of high glucose on the expression of P53 and Drpl in renal podocytes,it is predicted that Ang-(1-7)can act as a protective factor for podocytes in high glucose environment,inhibit the expression of P53 and Drpl in podocytes,and thus protect podocytes.MethodsThe well-grown mouse podocytes were inoculated into 6-well cell culture plates,and 1.5×105 cells were seeded per well,and 1.5 ml of RPMI-1640 medium containing 10%FBS was added to each well to grow podocytes.After overnight,the cell density reached 60-70%,the medium was discarded,and fresh 10%FBS RPMI-1640 medium was added to the wells of the culture plate according to the experiment:(1)Normal RPMI-1640 medium group(2)normal RPMI-1640 medium + Ang-(1-7)(10nmol/L)group;(3)high glucose(33 mmol/L)medium group;(4)high glucose(33 mmol/L)culture Base +Ang-(1-7)(IOnmol/L)group;the experiment was carried out after 48 hours of continuous culture.The cells of each group were collected and protein were extracted.The expression levels of Drpl and P53 were detected by Western blotting.The images of podocytes in each group were observed by laser confocal scanning microscopy.The expression of Drpl was more intuitive.ResultsThe podocytes of each group were cultured for 48 hours,and the cells were collected and protein were extracted.The expression levels of Drpl and P53 were detected by Western blotting.The expression of P53 was increased in high glucose environment,while Ang-(1-7)could reduce the expression of P53 in high glucose environment;similarly,the expression of Drpl was significantly increased in high glucose environment,while Ang-(1-7)could reduce the expression of Drp1 in high glucose environment.ConclusionsIn this part of the experiment,we found that Ang-(1-7)can inhibit the expression of P53 and Drpl in podocytes in high glucose environment at a certain concentration,and conclude that Ang-(1-7)may regulatethe expression of P53 and Drpl in podocytes and serve to protect podocytes through Mas receptor.PartⅢ Effect of Ang-(1-7)on the expression of miR30a in podocytes in high glucose environmentObjectivesIt is speculated that miR-30a is the upstream target gene of P53 and Drpl.In high glucose environment,podocytes should have low expression of miR-30a.After application of Ang-(1-7),the level of miR-30a in podocytes can be up-regulated.Methods1.The well-grown mouse podocyte strains were inoculated into 6-well cell culture plates,and 1.5×105 cells were seeded per well,and 1.5 ml of RPMI-1640 medium containing 10%FBS was added to each well to grow podocytes.After overnight,the cell density reached 60-70%,the medium was discarded,and fresh 10%FBS RPMI-1640 medium was added to the wells of the culture plate according to the experiment:(1)Normal RPMI-1640 medium group(2)normal RPMI-1640 medium +Ang-(1-7)(10nmol/L)group;(3)high glucose(33 mmol/L)medium group;(4)high glucose(33 mmol/L)culture Base +Ang-(1-7)(10nmol/L)group;the experiment was carried out after 48 hours of continuous culture.Total RNA was extracted from podocytes with Trizol reagent.RNA was extracted from the four groups according to the instructions.After RNA purity detection,the remaining RNA was further subjected to tailing and reverse transcription reaction.U6 was used as the internal reference for miRNA quantification.RNA was quantified by qRT-PCR using SYBR Green PCR Master Mix.Thus,the expression changes of miR-30a in the cells were verified.2.Podocytes induced by high glucose were transfected with miRNA30a(mir-30a-mimic),which mimics the endogenous microRNA 30a of organisms,and mir-30a-inhibitor,a specific target of miRNA 30a.According to the experimental requirements,podocytes were divided into five groups:(1)High sugar group(HG group);(2)High glucose + microRNA-30a mimic group(HG + mimic group);(3)High glucose + mimic-30a mimic negative control group(HG+ mimic-NC group);(4)HG + inhibitor group;(5)High glucose + inhibitor-30a negative control group(HG + inhibitor-NC group);Experiments were carried out after 24 hours of continuous culture.(1)Real-time fluorescence quantitative RT-PCR was used to detect the changes of podocyte microRNA-30a,P53 and Drp-1 in each group.(2)Western blot was used to detect the changes of Drp-1 and P53 protein levels in podocrtes of each group.(3)Immunofluorescence was used to observe and detect the expression of Drp-1 in the cytoplasm of podocytes.Results1.After 48 hours of podocyte culture,podocyte cells were collected and RNA was extracted.The expression of microRNA-30a in podocytes was detected by RNA purity detection and real-time quantitative PCR.The results showed that the expression of microRNA-30a in podocytes in high glucose environment decreased compared with the control group.The expression of microRNA-30a in podocytes increased significantly after Ang-(1-7)intervention.2.Quantitative fluorescence RT-PCR showed that the expression of P53 and Drp-1 in podocytes was affected by mimic and inhibitor of microRNA-30a for 24 hours.Compared with HG group,the expression of microRNA-30a in HG+mimic group was significantly increased(P<0.01),and the expression of microRNA-30a in HG + inhibitor group was significantly decreased(P<0.05).Compared with HG group,the expression of P53 and Drp-1 in HG+inhibitor group was significantly decreased(P<0.05).Compared with HG+mimic group,P53 and Drp-1 in HG-inhibitor group increased significantly(P<0.01).3.Western blot results showed that compared with HG group,the expression levels of P53 and Drp-1 protein in HG+mimic group decreased(P<0.05),while the expression levels of P53 and Drp-1 protein in HG+inhibitor group increased(P<0.05);compared with HG+mimic group,the expression levels of P53 and Drp-1 protein in HG + inhibitor group increased significantly(P<0.01).4.The expression of Drp-1 in podocytes was determined by immunofluorescence.The expression of Drpl was determined by laser scanning confocal microscopy.Podocyte Drpl was stained with Drpl antibody(green)and nucleus was stained with DAPI(blue).Immunofluorescence results showed that after 24 hours of intervention,the expression of Drp-1 protein in HG-mimic group decreased and that in HG-inhibitor group increased.Image-ProPlus software was used to automatically quantify the average fluorescence intensity of Drp-1 in the whole image,which was expressed by fluorescence unit(FU).Average.Compared with the control group,the expression of Drp-1 in podocytes of HG+mimic group decreased,P<0.01;the level of Drp-1 in HG+inhibitor group increased,P<0.01.conclusion1.In this part of the experiment,we found that Ang-(1-7)can increase the expression of microRNA-30a in podocytes under high glucose environment,which may be one of the mechanisms of Ang-(1-7)inhibiting mitochondrial division and fragmentation of podocytes under high glucose environment,thus protecting podocytes.2.The expression of Drp-1 and P53 can be negatively regulated by microRNA-30a to improve podocyte injury induced by high glucose.Overall Conclusions:1.Mitochondrial division and vacuolation of podocytes were observed in high glucose environment.At the same time,the expression of P53 and Drpl in podocytes increased,and the expression of microRNA-30a in podocytes decreased.2.The expression of Drp-1 and P53 can be negatively regulated by microRNA-30a to improve podocyte injury induced by high glucose.3.Ang-(1-7)could significantly down-regulate the expression of P53 and Drp 1 in podocytes under high glucose environment,and increase the expression of microRNA-30a in podocytes under high glucose environment.4.It is presumed that Ang-(1-7)is regulated by the gene of microRNA30a through Mas receptor,which affects the expression of downstream transcription regulators Drpl and P53,and then inhibits the mitochondrial division and fragmentation of podocytes under high glucose environment,thus realizing the role of podocyte protection.