| Background:Glomerular disease is a urinary system disorder characterized by glomerular allergic inflammation.Podocyte injury is an important event in the early stage of glomerulonephritis,as morphological abnormality and extensive apoptosis of podocytes can seriously damage the filtering barrier function of the glomerulus,causing nephropathy syndrome with major clinical manifestations such as proteinuria,hypoproteinemia,and hyperlipidemia.Therefore,it is of great clinical significance and research merit to explore the pathogenesis and progression of nephrotic proteinuria,as well as to find out an effective therapeutic target.Plectin,a cytoskeleton connexin widely expressed in glomerular podocytes,has a dumbbell-shaped structure consisting of a 200-nm rod-like domain in the center and two large globular domains on both sides.Such multi-domain structure allows plectin to interact with many different proteins and significantly affect their functions.Since plectin plays an important role in maintaining the general morphology,skeletal stability,and structural completeness of the cells,its abnormal expression may direct compromise the morphology and survival of podocytes,thus undermining the filtering function of the glomerulus.In addition to functioning as a linker,plectin plays a crucial role in cellular processes involving actin cytoskeleton dynamics;moreover,plectin can function as a scaffolding platform for signaling molecules,such as integrinα6β4 and focal adhesion kinase(FAK),which is an intriguing new facet of its functional repertoire that has recently attracted attention from researchers.FAK is a key mediator of integrin signaling among different cellular functions in a variety of cells.Following activation by integrins,FAK undergoes autophosphorylation,forms a complex with other cellular proteins and triggers downstream signaling through its kinase activity or scaffolding function.FAK and downstream p38 MAPK signaling have been shown to play vital roles in ADR-induced podocyte apoptosis and F-actin cytoskeletal remodeling.Given that plectin is severely depleted in ADR-treated glomeruli,the present study aimed to evaluate its effects in ADR-induced podocyte apoptosis and F-actin cytoskeletal rearrangement and explore its relationships with integrin α6β4 and FAK.The pathological and physiological changes in the rat model of Adriamycin(ADR)-induced nephropathy are similar to those of the minimal change nephropathy in humans.Using the model ADR-induced podocyte injury is also likely to cause typical manifestations of podocyte injury,including obvious foot process fusion or absence,apoptosis,and protein expression changes.Both models are widely used in basic research.In this regard,the models of ADR-induced nephropathy and ADR-induced podocyte injury were adopted in this study,so as to observe and explore the mechanisms of plectin and integrin α6β4/FAK/p38 MAPK pathways activated in both podocyte injury and nephrotic proteinuria induced by ADR.By doing so,new theoretical basis and new ideas could be provided for early prevention and treatment of nephrotic proteinuria.Objectives:1.To identify the changes and effects of plectin in ADR-induced podocyte apoptosis and cytoskeletal disorder.2.To investigate the mechanisms of the plectin-related signaling pathways and the resulting podocyte injuries in ADR-induced renal podocyte apoptosis and cytoskeletal disorder.3.To observe the glomerular basal layer structural and podocyte morphological changes in the animal model of ADR-induced nephropathy,as well as in the express of the proteins in interest.Methods:1.Cell culture:Podocyte lines of immortalized mice were cultured in vitro.By using the culture dishes coated by type Ⅰ collagen,the podocytes were placed into RPMI 1640 medium containing 10%fetal bovine serum,along with 100 U/ml penicillin,100 mg/ml streptomycin,10U/ml recombinant mouse interferon-γ.The podocytes were culture at 33 ℃ for proliferation.Then the podocytes were transferred to conditions at 37℃ and the culture was continued in medium that did not contain recombinant mouse interferon-γ for 14 days until the podocytes were differentiated and mature.2.Establishing rat model of ADR-induced nephropathy:Twenty male Sprague-Dawley rats(200g±20g)were purchased and placed in a temperature-controlled room with natural light illumination,provided with free access to food and water.These rats were randomly divided into two groups(n = 10 in each group):a ADR group and a normal control group.In the ADR group,rat model of nephropathy was established by a single injection of 7.5mg/kg ADR via tail vein,while the control group was injected with the same dose of saline.No rat died throughout the trial.Four weeks after injection,the feeding was ended and all rats were weighted and then euthanized.Urine of each rat was collected from the metabolic cage to determine 24-hour urinary protein and 24-hour urine output.Blood was collected from the abdominal aorta and centrifuged to prepare serum samples,which were stored at-20 ℃.3.Detecting the changes in plectin expression and podocyte injury indicators after ADR stimulation:(1)Western blot and real-time PCR were used to detect the plectin expression at various ADR concentrations and intervention time points,and the measures were specified for subsequent cell-molecule experiments involving ADR.(2)Phalloidin-fluorescein staining was used to observe the tissue structure of podocyte F-actin cytoskeleton and to detect podocyte injury.(3)Flow cytometry assay was used to detect the ADR-induced apoptosis of podocytes.(4)Western blot was used to detect the expression levels of injury marker protein WT1,synaptopodin,and desmin in the podocytes intervened by ADR.4.Determining the role of plectin in ADR-induced podocyte injury:(1)Western blot,real time PCR,and flow cytometry were used to determine the plectin expression and ROS production intervened by a specific concentration of ADR at various time points.(2)Western blot and flow cytometry were used to determine the changes of plectin and ROS production in ADR-induced podocyte injury after the mitochondrial protective agent was administered.(3)Western blot,flow cytometry,and cell immunofluorescence were used to determine the plectin expression,changes in apoptosis rate,changes in podocyte F-actin cytoskeleton,and changes in podocyte-specific marker proteins,including WT1,synaptopodin,and desmin,in the podocytes intervened by ADR and transfected by plectin cDNA plasmid.(4)Flow cytometry and immunofluorescent staining were used to determine the podocyte apoptosis and F-actin cytoskeleton changes after the plectin-specific small interfering RNA(siPlectin)was administered.5.Determining the plectin-regulated signaling pathways and their mechanisms in podocyte injury:(1)Western blot was used to determine the expressions of integrinα6β4,FAK,and p3 8,as well as the phosphorylation levels of FAK AMD p3 8,in ADR-induced podocyte injury.After siPlectin was administered,the expression and phosphorylation levels of integrin α6β4,FAK,and p38 in the podocytes were determined.(2)Western blot was used to determine the changes in the expression and phosphorylation levels of integrin a6p4,FAK,and p38 in the ADR-induced podocytes,after plectin expression was restored by plasmid transfection.(3)Western blot and flow cytometry were used to determine the expression and phosphorylation levels of integrin α6β4,FAK,and p38,as well as the time-course effect of apoptosis rate,in the podocytes,after siPlectin was administered.(4)Western blot was used to determine the expression and phosphorylation levels of FAK and p38,apoptosis rate of podocytes,and changes in cytoskeleton,as well as changes in the expressions of injury marker proteins WT1,synaptopodin,and desmin,after the integrin α6β4 mutate at site Y1494 was transfected and siPlectin was given.(5)Western blot,flow cytometry,and immunofluorescent staining were used to determine the expression and phosphorylation levels of integrin α6β4 and p38,apoptosis rate of podocytes,changes in cytoskeleton,and expressions of injury marker proteins WT1,synaptopodin,and desmin,after the inhibitor of FAK’s site Y397 was administered and siPlectin was given.(6)Western blot,flow cytometry,and immunofluorescent staining were used to determine the expression and phosphorylation levels of integrinα6β4 and FAK,expression levels of apoptosis regulatory proteins Bax and caspase-3,expression level of cytoskeleton regulator synaptopodin,and changes in apoptosis rate and cytoskeleton,after p3 8-specific inhibitor was administered and siPlectin was given.6.Determining the expressions of plectin and related signaling pathway proteins and renal injury indicator in rat model of ADR-induced nephropathy:(1)After successful modeling by vein injection of ADR at the rat tail,the bodyweight,24-hour total urine protein,blood urea nitrogen,serum creatinine,and 24-hour total urine output of the rats in both groups were recorded.(2)The histological changes of the glomerulus and tubular-interstitial regions of the rats’ kidney in the model group were observed under an optical microscope.(3)The morphological changes of the podocytes in the model group were observed under an electron microscope.(4)Western blot was used to determine the expression levels of podocyte marker protein WT1,synaptopodin,and desmin in both groups.(5)Western blot and immunohistochemistry were used to determine the amount and location of plectin in renal cortex.(6)Western blot was used to determine the expression and phosphorylation levels of integrin α6β4,FAK,and p38,as well as the expression levels of the podocyte apoptosis regulators Bax and caspase-3,in both groups.Results:1.Twelve hours after the podocytes were intervened with 0.5ug/ml ADR,the structure of F-actin cytoskeleton of the podocytes was disordered and collapsed to the periphery.The rate of podocyte apoptosis was also significantly higher than that of the control group(P<0.01).In addition,compared with the control group,the typical marker proteins of podocyte,WT1 and synaptopodin,were significantly decreased(P<0.01)and the podocyte injury-sensitive marker protein,desmin,was significantly increased(P<0.01).2.After the podocytes were intervened by 0.5ug/ml ADR for one hour,two hours,four hours,six hours,and twelve hours,ROS generated by mitochondrial oxidative stress were always significantly increased(P<0.01).However,the expression levels of plectin and the apoptosis rate of podocytes did not significantly increase until 4 to 6 hours later(P<0.01).After mitochondrial protective agent was administered,the expression levels of plectin recovered much better than the group intervened by ADR alone(P<0.01).The group intervened by ADR and transfected by plectin cDNA plasmid was compared with the group intervened by ADR alone,and it was found that the apoptosis rate of podocytes decreased drastically(P<0.01),F-actin cytoskeletal disorder was alleviated,the expression levels of WT1 and synaptopodin increased significantly(P<0.01),and the expression level of desmin decreased significantly(P<0.01).However,in the in the normal siPlectin-transfected podocytes,serious apoptosis and cytoskeletal disorders were found,the expression levels of WT1 and synaptopodin dropped significantly(P<0.01),and the expression level of desmin rose significantly(P<0.01).3.ADR intervention of podocytes could cause a significant increase in the intracellular phosphorylation levels of integrin α6β4,FAK,and p38(P<0.01).In contrast,transfecting the ADR-intervened podocytes with plectin cDNA plasmid could inhibit the increase in the phosphorylation levels of integrin α6β4,FAK,and p38,while alleviating cell injuries.Transfection of normal podocytes with siPlectin could simulate the increased phosphorylation levels of integrin α6β4,FAK,and p38 induced by ADR(P<0.01).The time-course effects of podocyte transfection with siPlectin showed that the phosphorylation levels of integrin α6β4,FAK and p38 were successively increased,and the apoptosis rate of podocytes also increased accordingly.Plasmids transfected with Y1494 site mutations in integrin β4 could prevent the increase in the phosphorylation levels of FAK and p38 induced by siPlectin.Administration of FAK inhibitors prevented the increase of p38 phosphorylation induced by siPlectin but did not affect the increasing phosphorylation of integrin α6β4.Administration of the p38 inhibitor did not prevent the increasing phosphorylation of integrin α6β4 and FAK induced by siPlectin but inhibited the expression of proteins Bax and caspase-3 induced by siPlectin.Both FAK and p38 inhibitors could improve the abnormal expressions of WT1,synaptopodin,and desmin induced by siPlectin.4.After ADR was injected via tail vein of the rats,several changes could b observed,including glomerular atrophy and loss,proliferation of mesangial cells and increased deposition of mesangial matrix,and foot process fusion.Compared with the podocytes in the control group,the expressions of specific marker proteins WT1 and synaptopodin dropped(P<0.01),the expression of injury marker protein desmin rose(P<0.01),the expressions of apoptosis marker proteins Bax and caspase-3 rose(P<0.01),and the phosphorylation levels of signaling pathway proteins integrin α6β4,FAK,and p38 rose(P<0.01).Conclusions:1.ADR could inhibit the expression of plectin through mitochondrial oxidative stress.The reduced plectin played a key role in the increased apoptosis rate of podocytes and cytoskeletal disorders induced by ADR.2.The decrease of plectin activated the signaling pathways integrin α6β4/FAK/p38 MAPK.As a result,the phosphorylation levels of the signaling pathway proteins increased,and the expression of apoptosis regulatory proteins Bax and caspase-3 in the pathway target proteins were increased,with increased apoptosis.The expression of the cytoskeleton regulator synaptopodin in the pathway target proteins decreased and cytoskeletal disorders could be observed.3.In the rat model of ADR-induced nephropathy,there were pathological changes in the glomerular basal layer and morphological changes of the podocytes,decreased plectin,increased Bax and caspase-3,and higher phosphorylation levels of integrinα6β4,FAK,and p38MAPK.These results were consistent with the findings of experiments in vitro. |
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