Effect of down-regulation of TRPC6 on the puromycin aminonucleoside-induced apoptosis of mouse podocytes

Abstract

Eukaryotic expression vectors carrying the small hairpin RNA (shRNA) for TRPC6 mRNA were constructed, and the effects of knocking-down TRPC6 on puromycin aminonucleoside (PAN)-induced apoptosis of mouse podocytes were observed. Two eukaryotic expression vectors containing small hairpin structure targeting TRPC6 named pGCsi-TRPC6A and pGCsi-TRPC6B were designed and synthesized. The plasmids were transfected into conditionally immortalized murine podocyte cell line by liposome. The changes in the TRPC6 mRNA and protein expression were observed by RT-PCR and Western blot after 48 h. Cultured podocytes were divided into four groups: control group, PAN treatment group, PAN treatment+shRNA transfection group, and PAN treatment+ negative control group. The expression of Bax and Bcl-2 mRNA and proteins was detected by RT-PCR and Western-blot respectively. The apoptotic rate of podocytes was measured by flow cytometry. The results showed that the expression of TRPC6 mRNA and protein was decreased in the podocytes when transfected with pGCsi-TRPC6A, and pGCsi-TRPC6B. The expression of Bax was increased, and that of Bcl-2 was decreased at protein and mRNA levels in the podocytes after treated with PAN for 48 h. These changes was attenuated by knocking-down TRPC6. Knocking-down TRPC6 could effectively decrease the PAN-induced apoptosis of podocytes. It was concluded that TRPC6 may play an important role in the PAN-induced apoptosis of podocytes. Knocking-down TRPC6 gene could effectively prevent the podocytes from apoptosis induced by PAN.

Keywords

TRPC6 / RNA interference / puromycin aminonucleoside / podocyte / apoptosis

References

[1] ShanklandS.J.. Cell cycle regulatory proteins in glomerular disease. Kidney Int, 1999, 56(4): 1208-1215

[2] ZiyadehF.N., WolfG.. Pathogenesis of the podocytopathy and proteinuria in diabetic glomerulopathy. Curr Diabetes Rev, 2008, 4(1): 39-45

[3] HaraM., YanagiharaT., KiharaI., et al.. Apical cell membranes are shed into urine from injured podocytes: a novel phenomenon of podocyte injury. J Am Soc Nephrol, 2005, 16(2): 408-416

[4] KrizW., GretzN., LemleyK.V., et al.. Progression of glomerular diseases: Is the podocyte the culprit?. Kidney Int, 1998, 54(3): 687-697

[5] WinnM.P., ConlonP.J., LynnK.L., et al.. A mutation in the TRPC6 cation channel causes familial focal segmental glomerulosclerosis. Science, 2005, 308(5729): 1801-1804

[6] ReiserJ., PoluK.R., MollerC.C., et al.. TRPC6 is a glomerular slit diaphragm-associated channel required for normal renal function. Nat Genet, 2005, 37(7): 739-744

[7] MollerC.C., WeiC., AltintasM.M., et al.. Induction of TRPC6 channel in acquired forms of proteinuric kidney disease. J Am Soc Nephrol, 2007, 18(1): 29-36

[8] MundelP., ReiserJ., Zuniga Mejia BorjaA., et al.. Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines. Exp Cell Res, 1997, 236(1): 248-258

[9] JiangH.J., ChangY., ZhuZ.H., et al.. Role of CD2-associated protein in podocyte differentiation. Sheng Li Xue Bao (Chinese), 2008, 25(1): 135-142

[10] ZhangC., JiangH.J., ChangY., et al.. Downregulation of CD2-associated protein impaired the physiological functions of podocytes. Cell Biol Int, 2009, 33(6): 632-639

[11] KobayashiN., KrizW.. Introduction to the podocyte issue: more than eight enigmas. Microsc Res Tech, 2002, 57(4): 187-188

[12] SchlondorffJ.S., PollakM.R.. TRPC6 in glomerular health and disease: What we know and what we believe. Semin Cell Dev Biol, 2006, 17(6): 667-674

[13] MukerjiN., DamodaranT.V., WinnM.P.. TRPC6 and FSGS: the latest TRP channelopathy. Biochim Biophys Acta, 2007, 1772(8): 859-868

[14] BrummelkampT.R., BemardsR., AgamiR., et al.. A system for stable expression of short interfering RNAi in mammalian cells. Science, 2002, 296(5567): 550-553

[15] SharpP.A., ZamoreP.D.. RNA interference. Science, 2000, 287(5462): 2431-2433

[16] ElbashirS.M., HarborthJ., LendeckelW., et al.. Duplexes of 21 nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001, 411(6836): 494-498

[17] KimY.H., GoyalM., KurnitD., et al.. Podocyte depletion and glomerulosclerosis have a direct relationship in the PAN-treated rat. Kidney Int, 2002, 60(3): 957-968

[18] ZhengC.X., ChenZ.H., ZengC.H., et al.. Triptolide protects podocytes from puromycin aminonucleoside induced injury in vivo and in vitro. Kidney Int, 2008, 74(5): 596-612

[19] WadaT., PippinJ.W., MarshallC.B., et al.. Dexamethasone prevents podocyte apoptosis induced by puromycin aminonucleoside: Role of p53 and Bcl-2-related family proteins. J Am Soc Nephrol, 2005, 16(9): 2615-2625

[20] LiuS.F., DingJ., FanQ.F., et al.. Establishment of a podocyte cell injury model induced by puromycin aminonucleoside. Beijing Da Xue Xue Bao (Chinese), 2008, 40(6): 586-589

[21] SzegezdiE., MacdonaldD.C., Ní ChonghaileT., et al.. Bcl-2 family on guard at the ER. Am J Physiol Cell Physiol, 2009, 296(5): 941-953

[22] SchifferM., BitzerM., RobertsI.S., et al.. Apoptosis in podocytes induced by TGF-beta and Smad7. J Clin Invest, 2001, 108(6): 807-816

[23] WadaT., PippinJ.W., TeradaY., et al.. The cyclin-dependent kinase inhibitor p21 is required for TGF-beta1-induced podocyte apoptosis. Kidney Int, 2005, 68(4): 1618-1629

[24] DingG., ReddyK., KapasiA.A., et al.. Angiotensin II induces apoptosis in rat glomerular epithelial cells. Am J Physiol Renal Physiol, 2002, 283(1): 173-180