title: Inactivation of AMPK mediates high phosphate-induced extracellular matrix accumulation via NOX4/TGFß-1 signaling in human mesangial cells.
abstract: OBJECTIVE
  High phosphate (Pi) levels and extracellular matrix (ECM) accumulation are associated with chronic kidney disease progression. However, how high Pi levels contribute to ECM accumulation in mesangial cells is unknown. The present study investigated the role and mechanism of high Pi levels in ECM accumulation in immortalized human mesangial cells (iHMCs).
  
  
  METHODS
  iHMCs were exposed to normal (0.9 mM) or increasing Pi concentrations (2.5 and 5 mM) with or without diferent blockers or activators. NOX4, phosphorylated AMPK (p-AMPK), phosphorylated SMAD3 (p-SMAD3), fibronectin (F/N), collagen IV (C-IV) and alpha-smooth muscle actin (α-SMA) expression was assessed via western blot and immunofluorescence. Lucigenin-enhanced chemiluminescence, and dihydroethidium (DHE) assessed NADPH oxidase activity and superoxide (SO), respectively.
  
  
  RESULTS
  In iHMCs, a Pi transporter blocker (PFA) abrogated high Pi-induced AMPK inactivation, increase in NADPH oxidase-induced reactive oxygen species (ROS) levels, NOX4, p-SMAD3, α-SMA and C-IV expression. AMPK activation by AICAR, NOX4 silencing or NADPH oxidase blocker prevented high Pi-induced DHE levels, p-SMAD3, F/N, C-IV and α-SMA expression.
  
  
  CONCLUSIONS
  AMPK inactivation with NOX4-induced ROS formation and transforming growth factor ß-1 (TGFß-1) signaling activation mediates high Pi-induced ECM accumulation in iHMCs. Maneuvers increasing AMPK or reducing NOX4 activity may contribute to renal protection under hyperphosphatemia.
authors.affiliation: Renal Pathophysiology Laboratory, Investigation on Diabetes Complications, Faculty of Medical Sciences, State University of Campinas (Unicamp), Campinas, São Paulo, Brazil.
authors.forename: Alexandros
authors.initials: A
authors.lastname: Papadimitriou
authors.forename: Elisa B M I
authors.initials: EB
authors.lastname: Peixoto
authors.forename: Kamila C
authors.initials: KC
authors.lastname: Silva
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Lopes de Faria
authors.forename: José B
authors.initials: JB
authors.lastname: Lopes de Faria
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D002478:Cells, Cultured; D024141:Collagen Type IV / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D050527:Mesangial Cells / Q000378:metabolism*; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D051900:Smad3 Protein / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D024141:Collagen Type IV; D005353:Fibronectins; D010710:Phosphates; D017382:Reactive Oxygen Species; C497790:SMAD3 protein, human; D051900:Smad3 Protein; C508960:TGFB1 protein, human; D053773:Transforming Growth Factor beta1; D013481:Superoxides; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; D055372:AMP-Activated Protein Kinases