| Objectives: Idiopathic nephrotic syndrome(INS)is one of the most common glomerular disorders in childhood.About 10% patients with nephrotic syndrome in childhood are steroid resistant,30% patients with steroid sensitive nephrotic syndrome are steroid dependent or frequent relapsed.It's difficult to treat these patients and alternative treatments are needed for them to relieve the proteinuria and prevent the severe side effects of steroids.Some overseas patients with INS were remitted by vincristine(VCR)treatment,which suggested the possibility of vincristine application in nephrotic syndrome.We found that vincristine can induce complete remission in INS patients with relapsing,and more important,it can decrease relapse frequency without obvious side effects.Minimal change disease characterized with foot process effacement is the most common pathological type of INS in childhood.Adriamycin(ADR)infusion by caudal vein in mouse and rat can induce foot process fusion and mass proteinuria,which is a typical nephropathy model presented with foot process lesion.In this experiment,we aimed to explore the protect effect of vincristine on podocyte cytoskeleton,the therapeutic effect of vincristine in nephrotic syndrome model and the effect of vincristine on expression of foot process related proteins based on podocyte and ADR-induced nephrotic syndrome.Finally,we aimed to clarify the therapeutic effect of vincristine in nephrotic syndrome and related mechanism by in vivo and in vitro experiment.Methods:(1)Podocyte culture and establishment of podocyte injury model and VCR treatment.Immortalized mouse podocyte cell line was gifted from Professor Jianghua Chen.Podocytes were proliferated under condition of 33°C and 5% CO2,then differentiated under condition of 37°C.The optimum concentration of ADR and VCR on podocyte were obtained by analysis of podocyte morphology and F-actin filament distribution based on phalloidin immuno-fluorescence staining.VCR was administered to podocytes after 24 and 48 hours of ADR induction,and Dexamethasone(DXM)was used as control in treatment groups.Podocyte morphology and F-actin filament distribution were observed subsequently and RNA was extracted for further experiment.(2)Establishment of ADR induced nephrotic syndrome model in rat and VCR treatment.Dose of 7.5mg/kg ADR was administered by caudal vein infusion to establish nephrotic syndrome model.After 4 weeks,VCR was given with dose of 0.2mg/kg twice in one week,meanwhile,DXM with dose of 1.8mg/kg was given daily by intraperitoneal injection as control compared to VCR group.After treatment for 4 weeks,24 hours urine proteinuria,serum albumin,serum cholesterol were quantified.Rats were sacrificed and kidneys were prepared for light microscopy and electron microscopy analysis.RNA and protein of renal cortex were extracted for further analysis.(3)Establishment of ADR induced nephrotic syndrome model in mouse and VCR treatment.Dose of 10mg/kg ADR was administered by caudal vein infusion to establish nephrotic syndrome model.After 4 weeks,VCR was given with dose of 0.45mg/kg twice in one week,meanwhile,DXM with dose of 1.35mg/kg was given daily by intraperitoneal injection as control compared to VCR group.After treatment for 4 weeks,24 hours urine proteinuria was quantified.Mice were sacrificed and renal cortex RNA was extracted for further analysis.(4)Podocyte related protein expression.The expression of podocyte related protein were analysed by RT-PCR in VCR treated podocyte and nephrotic syndrome model induced by ADR.These proteins included focal adhension kinase and ?3?1-integrin distributed close to glomerular basement membrane,nephrin and podocin distributed on slit diaphragm,and podocalyxin distributed on the apical surface of podocyte.And expression of actin-associated protein such as ?-actinin-4 and synaptopodin were also analysed.Phospho-FAK expression was analysed by western blot in VCR treated nephrotic syndrome model induced by ADR.Results:(1)VCR treatment can rescue podocyte injury caused by ADR.By phalloidin immuno-fluorescence staining,VCR with dose of 5nmol/L had no cytotoxicity on podocyte after 24 hours or 48 hours,and it will repair the podocyte injury caused by 0.5umol/L ADR as effectively as 1umol/L DXM.Injured podocyte morphology and F-actin filament distribution can be recovered to normal status under treatment of VCR with dose of 5nmol/L.(2)VCR had therapeutical effect on ADR induced nephrotic syndrome in rat.After 1 week of ADR infusion in rat,proteinuria were increased obviously after 2 weeks,reached climax at 6 weeks and lasted until 8 weeks.Proteinuria were started to decrease after 2 week-VCR treatment and lasted for 4 weeks,while serum albumin and cholesterol level were recovered to normal range.As to DXM treatment,proteinuria were started to decrease after 4 week-DXM administration,although serum albumin and cholesterol level were recovered to normal range at same time.Glomerular mesangial matrix proliferation and infiltrated inflammatory cells in interstitium can be observed in ADR induced rat by HE stain.While proliferation and infiltration were disappeared after VCR of DXM treatment.Foot process showed focal fusion by electron microscopy after 4-week ADR infusion,and deteriorated to diffuse foot process fusion another 4 weeks later.But the fusional foot processes were recovered to normal after 4 week treatment of VCR or DXM.(3)Proteinuria can be spontaneous remitted in ADR induced mice.In mice,proteinuria were increased obviously after 2 weeks of ADR infusion,reached climax at 3 weeks,reversed to decrease after 6 weeks and subsequently to normal range.While serum albumin level was still low compared to normal level.(4)VCR can inhibit FAK and ?3?1-integrin expression.VCR administration can inhibit the m RNA expression of FAK to baseline,which were upregulated in ADR induced podocyte and nephrotic syndrome model in mice and rat.The same results can be aquired of ?3?1-integrin m RNA expression.VCR administration had almost no effect on nephrin m RNA expression,which was upregulated in ADR induced podocyte and nephrotic syndrome model in mice.But VCR administration can inhibit the m RNA expression of podocin to baseline,which was upregulated in ADR induced nephrotic syndrome model in rat.Podocalyxin m RNA expression was upregulated in ADR induced nephrotic syndrome model in mice and rat.While VCR or DXM can inhibit the upregulated expression.And VCR administration can inhibit the m RNA expression of podocalyxin to baseline,which was upregulated expressed in ADR induced rat.VCR or DXM can inhibit the m RNA expression of synaptopodin to baseline,which was upregulated expressed in ADR induced podocyte.But this was not observed in ADR induced mice and rat.VCR administration had almost no effect on ?-actinin-4 m RNA expression in ADR induced podocyte,mice and rat.Pho-FAK in ADR induced podocyte was increased by western blot analysis.VCR or DXM administration can inhibit the pho-FAK expression to baseline.FAK expression was increased by western blot in rat after 8 weeks of ADR infusion,while it can be reversed to baseline under VCR treatment.Conclusions 1.VCR administeration can rescue podocyte injury caused by ADR via keeping normal podocyte morphology and F-actin filament expression and distribution.2.VCR had therapeutic effect in ADR induced nephrotic syndrome in rat.VCR administration can reduce proteinuria,recover serum albumin and cholestrol level to normal range,and repair the fusional foot precess to normal morphology.What's more,the therapeutic effect of VCR in ADR induced rat can be showed earlier compared to the effect of DXM treatment.3.Proteinuria can be spontaneous remitted in ADR induced mice.4.The mechanism of therapeutic effect of VCR in ADR induced nephrotic syndrome in rat maybe as following: VCR administration can stabilize podocyte cytoskeleton and prevent foot process infusion via inhibiting the expression of FAK and ?3?1-integrin,which were upregulated caused by ADR induction. |
PDF Full Text Request