title: The mTORC2/Akt/NFκB Pathway-Mediated Activation of TRPC6 Participates in Adriamycin-Induced Podocyte Apoptosis.
abstract: OBJECTIVE
  Although increased expression and gain function of transient receptor potential cation channel 6 (TRPC6) has been associated with the pathogenesis of some proteinuric glomerular diseases, it remains elusive how TRPC6 participates in the process of podocyte damage.
  
  
  METHODS
  The potential signaling responsible for TRPC6 activation was investigated using immunoblot assays in an in vitro podocyte injury model induced by Adriamycin (ADR). Podocyte apoptosis was measured using FITC-conjugated Annexin V and Propidium Iodide staining. The channel activity of TRPC6 was assessed using the Ca2+ influx assay.
  
  
  RESULTS
  Increase of TRPC6 expression was detected in ADR-treated podocytes, and TRPC6 knockdown significantly decreased ADR-induced podocytes apoptosis. Following ADR treatment, phospho-mTORSer2481 and phospho-AktSer473 was significantly increased in a time-dependent manner, whereas phospho-mTORSer2448 and phospho-p70S6KThr389 showed no change. ADR-induced apoptosis was prevented by ku0063794 (a dual mTOR complexes inhibitor), not by rapamycin (a specific mTORC1 inhibitor). Furthermore, nuclear translocation of NFκB/p65 was detected in ADR-treated podocytes, which was prevented by an Akt inhibitor triciribine. Of note, NFκB inhibitor PDTC prevented ADR-induced increase of TRPC6, and decreased ADR-induced apoptosis. We found that Akt activation and NFκB nuclear translocation was significantly inhibited by knockdown of mTORC2 protein Rictor, not by mTORC1 protein Raptor. In comparison with control, the Ca2+ influx was significantly increased in ADR-treated podocytes, which was remarkably prevented by TRPC6 knockdown. ADR-induced increase of TRPC6 channel activity was dramatically prevented by ku0063794, but not by rapamycin. Additionally, knockdown of Rictor, not Raptor, prevented ADR-induced increase of the Ca2+ influx. Moreover, the application of NFκB inhibitor PDTC also prevented the Ca2+ influx in ADR-treated podocytes.
  
  
  CONCLUSIONS
  Our findings revealed that the mTORC2/Akt/NFκB pathway-mediated activation of TRPC6 participates in ADR-induced podocyte apoptosis.
authors.affiliation: Department of Pediatrics, Harbin Medical University Second Hospital, Harbin, Heilongjiang, PR China.
authors.forename: Hai-Tao
authors.initials: HT
authors.lastname: Zhang
authors.forename: Wei-Wei
authors.initials: WW
authors.lastname: Wang
authors.forename: Li-Hong
authors.initials: LH
authors.lastname: Ren
authors.forename: Xia-Xia
authors.initials: XX
authors.lastname: Zhao
authors.forename: Zhi-Hui
authors.initials: ZH
authors.lastname: Wang
authors.forename: De-Li
authors.initials: DL
authors.lastname: Zhuang
authors.forename: Yun-Nuo
authors.initials: YN
authors.lastname: Bai
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology*; D000076225:Mechanistic Target of Rapamycin Complex 2; D051379:Mice; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D050199:Podocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D050052:TRPC Cation Channels / Q000378:metabolism*; D000074287:TRPC6 Cation Channel; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D046912:Multiprotein Complexes; D016328:NF-kappa B; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C492146:Trpc6 protein, mouse; D004317:Doxorubicin; D000076225:Mechanistic Target of Rapamycin Complex 2; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D002118:Calcium