title: VEGF regulates TRPC6 channels in podocytes.
abstract: BACKGROUND
  Both, increased plasma concentrations of vascular endothelial growth factor (VEGF) and increased expression of transient receptor potential canonical type 6 (TRPC6) channels in podocytes have been associated with proteinuric kidney diseases. Now, we investigated the hypothesis that VEGF regulates TRPC6 in podocytes.
  
  
  METHODS
  TRPC6 messenger RNA (mRNA) and TRPC6 protein expression were analyzed in cultured podocytes after administration of VEGF165 using quantitative real-time reverse transcription-polymerase chain reaction and immunoblotting, respectively. YFP-tagged TRPC6 in podocytes was analyzed using confocal laser scanning microscopy. TRPC6-associated calcium influx was measured fluorometrically. Both, immunofluorescence and immunohistochemistry were performed in renal tissue from patients with diabetes mellitus and controls.
  
  
  RESULTS
  Administration of VEGF165 to podocytes significantly increased TRPC6 mRNA expression and TRPC6 protein levels. The effects of VEGF165 were dose dependent and could be blocked by phosphoinositide-3-kinase inhibitors. In the presence of cycloheximide, an inhibitor of protein biosynthesis, we did not observe an effect of VEGF on TRPC6 protein levels, indicating the requirement of de novo protein synthesis. VEGF165 significantly increased TRPC6-mediated calcium influx in podocytes. Calcium influx was significantly lower in podocytes after gene knockdown using siRNA against TRPC6. Immunohistochemistry showed both increased TRPC6 channel protein and VEGF receptor type 2 (VEGFR-2) protein in podocytes from patients with diabetic nephropathy compared to control subjects. There was a significant association between VEGFR-2 mRNA and TRPC6 mRNA (n = 48; r(2) = 0.585; P < 0.0001) in human renal cortex.
  
  
  CONCLUSIONS
  VEGF regulates TRPC6 in podocytes.
authors.affiliation: Medizinische Klinik, Nephrologie, Charité Campus Benjamin Franklin, Berlin, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Thilo
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Christoph
authors.initials: C
authors.lastname: Loddenkemper
authors.forename: Ralf
authors.initials: R
authors.lastname: Schuelein
authors.forename: Antje
authors.initials: A
authors.lastname: Schmidt
authors.forename: Zengcheng
authors.initials: Z
authors.lastname: Yan
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Zhu
authors.forename: Andreas
authors.initials: A
authors.lastname: Zakrzewicz
authors.forename: Maik
authors.initials: M
authors.lastname: Gollasch
authors.forename: Martin
authors.initials: M
authors.lastname: Tepel
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003922:Diabetes Mellitus, Type 1 / Q000378:metabolism* / Q000473:pathology; D003924:Diabetes Mellitus, Type 2 / Q000378:metabolism* / Q000473:pathology; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008875:Middle Aged; D050199:Podocytes / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D050052:TRPC Cation Channels / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000074287:TRPC6 Cation Channel; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; C492146:Trpc6 protein, mouse; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2; D002118:Calcium