The Role Of TRPC6 In Podocyte Injury

Partâ… Effects of puromycin aminonucleoside on the expression of TRPC6of murine podocytes and the significance of itObjective:To investigate the effect of PAN on the expression of TRPC6 of murinepodocytes and to further study the effect of overexpression of TRPC6 on Nephrin,α-actinin-4 andα-tubulin in murine podocytes in vitro.Methods:Conditionally immortalized murine podocyte cell line was cultured underpermissive condition at 33℃.After passage the cells were shifted to non-permissivecondition at 37℃and cultrued for 14 days.PAN was added to the medium at theconcentration of 30μg/ml.Immunofluorescent assay was used to observe the distribution ofTRPC6.The expression of TRPC6 mRNA was assessed by RT-PCR and the expression ofTRPC6 protein was measured by Western-blot at 6,12,24 and 48 hours.Mouse TRPC6cDNA eukaryotic expression vector pEGFP-N₁-mTRPC6 was transfected to podocytes byliposome.The fluorescent microscopy was used to examine the expression of EGFP after48 hours.The change of TRPC6 protein expression was observed by Western-blot after 48hours.Immunofluorescent assay was used to observe the distribution of Nephrin, α-actinin-4 andα-tubulin.RT-PCR and Western-blot were used to measure the expressionof mRNA and protein of Nephrin,α-actinin-4 andα-tubulin,respectively.Results:TRPC6 was expressed in podocytes,expecialy in the perinuclear and the cellmembrane.The expression of membrane TRPC6 increased after exposure to PAN for 24h.After treated with 50μg/ml PAN for 48h,more significant changes were observed.Podocytes treated with different concentrations of PAN resulted in the stimulation ofTRPC6 in a dose-and time-dependent manner.The addition of PAN to the media ofcultured mouse podocytes effectively stimulated the mRNA and protein production ofTRPC6.A noticeable increase in TRPC6 was detected at 30μg/ml PAN.A higherconcentration,50μg/ml,of PAN stimulated PAN production even more.At a fixed dose of50μg/ml PAN,the stimulation of TRPC6 production was evident in 12h.Additional time ofexposure to PAN appear to increase the quantity of TRPC6 mRNA and protein in the mousepodocytes further.About 35% of the cells expressed EGFP.A up-regulation of proteinexpression of TRPC6 was detected in podocytes when transfected with pEGFP-N₁-mTRPC6 (P＜0.01).The distribution changes of Nephrin andα-actinin-4 were found ascompared to control groups.The reductions of the mRNA and the protein expression inNephrin were apparently detected by about 42% and 26%,respectively.The mRNA andprotein expression levels ofα-actinin-4 decreased by about 22% and 25%,respectively.(P＜0.05).The overexpression of TRPC6 had no effect on the expression ofα-tubulin butchanged the distribution of it.Conclusion:The distribution of TRPC6 was changed after exposure toPAN.Exposureto PAN resulted in the stimulation of TRPC6 both in mRNA and protein level.Theoverexpression of TRPC6 interfere with the normal distribution and function of Nephrin,α-actinin-4 andα-tubulin. Partâ…¡Effect of overexpression of TRPC6 on angiotensinâ…¡-inducedapoptosis of mouse podocytesObjective:To study the effect of overexpression of TRPC6 on Angâ…¡-inducedapoptosis of mouse podocytes in vitro and to further explore the possible mechanisms.Methods:Conditionally immortalized murine podocyte cell line was cultured underpermissive condition at 33℃.After passage the cells were shifted to non-permissivecondition at 37℃and cultrued for 14 days.Podocytes were treated by differentconcentrations of Angâ…¡and the cell viability was dected by methyl thiazolyl tetrazoliummethod(MTT).Mouse TRPC6 cDNA eukaryotic expression vector pEGFP-N₁-mTRPC6was transfeced to podocytes by liposome.The fluorescent microscopy was used to examinethe expression of EGFP after 48 hours.The change of TRPC6 protein expression wasobserved by Western-blot.The podocyte intracellular calcium concentration was measuredwith laser-scanning confocal microscope.Podocytes were divided into groups and differentconcentrations of Angâ…¡were used.The expression of Bax and Bcl-2 mRNA was assessedby RT-PCR and the expression of Bax and Bcl-2 protein was measured by Western-blot.The apoptotic ratio of podocytes was monitored by flow cytometry and Hoechst33258staining.Results:The cell viability han no change after treated with low-dose (10^-10mol/L) ofAngâ…¡but decreased 50% by using high-dose (10^-6mol/L)of Angâ…¡for 24 hours.About35% of the cells expressed EGFP.A up-regulation of protein expression of TRPC6 wasdetected in podocytes when transfected with pEGFP-N₁-mTRPC6(P＜0.01).Theoverexpression of TRPC6 promoted the Angâ…¡-induced influx of extracellular calcium andelevated the expression of Bax but decreased the expression of Bcl-2(P＜0.01,P＜0.05).The apoptotic ratio of podocyte was (2.50±0.72)% on the effect of low-doseAngâ…¡(10^-10mol/L),it was increased to (4.33±0.45)% when transfected with pEGFP-N₁ -mTRPC6(P＜0.05).Transfection with pEGFP-N₁-mTRPC6 increased apoptosis ratefrom (15.46±1.40)% to (18.33±0.87)% (P＜0.01) on the high-dose (10^-6mol/L) of Angâ…¡.Conclusion:TRPC6 plays an important role in the Angâ…¡-induced apoptosis ofpodocytes.This effect can be attributed to its ability of promoting the influx of extracellularcalcium and initiating the apoptosis cascade.Partâ…¢Effect of Down-regulation of TRPC6 on the Mouse Podocyte injuryinduced by Puromycin AminonucleosideObjective:To construct eukaryotic expression vectors carrying the small hairpinRNA(shRNA) for TRPC6 mRNA and observe the effect of knocking-down TRPC6 onPAN-induced injury of mouse podocytes.Methods:Two DNA sequences containing small hairpin structure targeting TRPC6were designed and synthesized and were then inserted into the plasmid pGC containinggreen fluorescence protein (GFP) to form plasmid pGCsi-TRPC6A and pGCsi-TRPC6B.The inserted sequences were verified by enzyme digestion and DNA sequeneing.Plasmidexpressing irrelevant shRNA was used as negative control named PGCsi-NC.The plasmidswere transfeced to conditionally immortalized murine podocyte cell line by liposome.Thefluorescent microscopy was used to examine the expression of GFP after 24 and 48 hours.The changes of TRPC6 mRNA and protein expression were observed by RT-PCR andWestern-blot after 48 hours.Cultured podocytes were divided into four groups:controlgroup;PAN treatment group;PAN treatment+shRNA transfection group and PANtreatment+negative control group.The expression of Nephrin and CD2AP protein wasmeasured by Western-blot.The expression of Bax and Bcl-2 mRNA was assessed byRT-PCR and the expression of Bax and Bcl-2 protein was measured by Western-blot.The apoptotic ratio ofpodocytes was monitored by flow cytometry.Results:The sequence of recombinant plasmids pGCsi-TRPC6A and pGCsi-TRPC6Bwas confirmed by further sequencing after extracted using plasmid extraction kit.Theresults showed that the inserted sequence of the pGCsi-U6-Neo-GFP-shRNA was the sameas the designed sequence.About 45% of the cells expressed GFP.A down-regulation ofmRNA and protein expression of TRPC6 was detected in mouse podocytes whentransfected with pGCsi-TRPC6A and pGCsi-TRPC6B(P＜0.05).Plasmid pGCsi-TRPC6Bwas used for subsequent study because of its higher efficiency of knocking-down theTRPC6 gene compared with plasmid pGCsi-TRPC6A.Compaired with the control group,dreased protein of Nephrin and CD2AP was detected in the cultured podocytes treated withPAN for 48 hours (P＜0.05).This effect was interfered by transfecting the plasmidpGCsi-TRPC6B into the podocytes(P＜0.05).The expression of Bax increased and theexpression of Bcl-2 decreased at protein and mRNA levels after treated with PAN for 48hours but knocking-down TRPC6 could prevent these changes.Knocking-down TRPC6could effectively decreased the PAN-induced apoptosis of podocytes.Conclusion:The eukaryotic expression vectors carrying the small hairpin RNA(shRNA) for TRPC6 mRNA was successfully constructed.The reeombinant plasmid couldexpress stably and eode small interfering RNA in cells to make target genes silence.Knocking-down TRPC6 gene could effectively prevent the podocytes from injury inducedby PAN.