The Effect And Molecular Mechanism Of TRPC6 In Podocyte Injury Induced By TGF-?1 In Vitro

Part? Effect of TGF-?1 on the Expression of TRPC6 on Podocyte Injury and the Effect of Overexpression of TRPC6 on Nephrin,DesminObjective:The purpose of this study was to investigate the effect of TRPC6(Transient Receptor Potential Cation Channel 6)on transforming growth factor-?1(TGF-?1)induced podocyte injury.Methods: Adding different concentrations of TGF-?1(ng/ml ?4ng/ml?8ng/ml?12ng/ml)to the medium.Then morphology of podocytes was observed under inverted microscope.MTT method was used to test cell inhibition rates Immunofluorescence assay was used to observer the distibution of TRPC6,while TRPC6 m RNA and protein expression levels were detected by real time PCR and Western blot after 72 h.Cultuerd podocytes were divided into four groups again,TGF-?1 was add to the medium at the concentration of 12ng/ml.Immunofluorescence assay was used to observer the distibution of TRPC6 and TRPC6 m RNA and protein expression levels were detected by real time PCR and Western blot at 0,24,48 and 72 h.Mouse TRPC6 c DNA eukaryitic expression vector p EX-3-TRPC6 wsa transfected to podocytes by liposome.TRPC6 protein expression levels were detected by Western blot after 48 h in order to evaluate its transmissi on efficiency.Immunofluorescence,real time PCR and Western blot were used to observer the changes on the distibution and expression of TRPC6 on podocytes.Results: Compared with normal group,shrinking of podocytes structure and foot processes fusion,even disappearing observed under inverted microscope in the TGF-?1 12ng/ml group.TGF-?1 could inhibits podocytes proliferation,the proliferation inhibition rate was close to 50% when concentration of TGF-?1 was increased to 12ng/ml for 72 h.TGF-?1 could also induce the change of TRPC6 on podocytes.TRPC6 m RNA and protein expression were increased after exposue to 12ng/ml TGF-? for 72 h,but there was not statistical difference between model group and blank control group(P>0.05),after treated with 8?12ng/ml TGF-?1 for 72 h,more significant changes were observed(P<0.05).The TRPC6 m RNA and protein expression were increased immediately when treated with 12ng/ml TGF-?1 for 24 h,but there was not statistical difference between model group and blank control group(P>0.05),more significant changes were observed after treated with 12ng/ml TGF-?1 for 48 and 72 h,(P<0.05).Podocytes treated with different concentrations of TGF-?1 resulted in the stimulation of TRPC6 in a dose-and time-dependent manner.A up–regulation of protein expression of TRPC6 was detected on podocytes when tranfected with p EX-3-TRPC6 for 48(P<0.05),the distibution of nephrin had no changed,but the reductions of the m RNA and the protein expression were apparently detected by about 25% and 42% respectively(P <0.05).The distribution changes of desmin was found as compared to control groups.The m RNA and the protein expression leves of desmin increased by about 132% and 116%(P<0.05).Conclusions:TGF-?1 could change the distribution and increase the expression of TRPC6 on podocyte.The overexpression of TRPC6 could interfere with the expression of nephrin and desmin,interfere with the normal distribution of desmin,which may be one of the mechanism of podocyte damage with TRPC6.Part?The effect of TRPC6 on the podocyte injury induced by TGF-?1Objective: To investigate the effect of TRPC6 on the expression of nephrin,desmin,caspase9 and apoptosis on podocyte injury induled by TGF-?1.Methods: Murine podocyte cell line were cultured in vitro,two DNA sequences containing small hairpin structure targeting TRPC6 plasmid vector PGPU6/GFP/Neo-TRPC6-mus-581 and negative control plasmid vector PGPU6/GFP/Neo-NC were transfeced to mouse podocytes by liposome.Using fluorescent microscopy to examine the expression of GFP after 24 and 48 hours.The changes of TRPC6 protein expression was observed by Western-blot after 48 hours.Conditionally immortalized podocytes were cultured in vitro and they were divided into four groups: control;TGF-?1 treatment;TGF-?1 with TRPC6 knockdown and TGF-?1 without TRPC6 knockdown.Real time RT-PCRand Western blot analysis were employed to determine the m RNA and protein of expression of nephrin,desmin and caspase-9,respectively.Flow cytometry was used to examine the apoptotic rate of podocytes and DAPI fluorescent staining was used to determine apoptotic morphology.Results: After transfecting 48 h the expression of GFP on podocytes were more stronger than 24 hours.A down-regulation of protein expression of TRPC6 was detected in mouse podocytes when transfected with PGPU6/GFP/Neo-TRPC6-mus-581(P < 0.05)while compared with the control group and negative control group.The m RNA and protein expression of desmin and caspase-9 were increased in cultured TGF-?1 treated podocytes for 48 hours,whereas nephrin was declined as compared with the control group.Importantly,TRPC6 knockdown significantly attenuated the upregulated desmin and caspase-9,and alleviated impairment of nephrin induced by TGF-?1.After exposure to TGF-?1 for 48 hours,apoptosis cells were increased and presented some morphologic features of apoptosis.The podocyte apoptosis rate in TGF-?1 treatment group was 14%+2.1%,while koncing-down TRPC6 the podocyte apoptosis rate down-regulated to 10.9%+0.56%(P<0.05).Compared with TGF-?1 treatment group the podocyte apoptosis rate was increased in negative control group,but no statistically significant(P>0.05).Conclusions:TGF-?1 could induce apoptosis of podocyte,decrease the expression of nephrin and increase the expression of caspase9 and desmin,which may via TRPC6 signal pathway.Inhibition of TRPC6 alleviates these changes in podocytes-treated with TGF-?1,which had a protective effect on podocyte lesion.