title: Hyperphosphatemia induces protective autophagy in endothelial cells through the inhibition of Akt/mTOR signaling.
abstract: OBJECTIVE
  Hyperphosphatemia-induced endothelial dysfunction has been shown to play a pathogenic role in the development of atherosclerosis in chronic kidney disease (CKD) through unclear mechanisms. Emerging evidence indicates that autophagy is involved in the maintenance of normal cardiovascular function. However, it is unclear whether autophagy participates in the molecular mechanism underlying high phosphate (Pi)-induced endothelial dysfunction.
  
  
  METHODS
  The autophagy activity was determined by the immunofluorescence staining of the expression of endothelial microtubule-associated protein 1 light chain 3 (LC3) in the 5/6 nephrectomy rat model of CKD and sham-operated control rats. The LC3-II/LC3-I ratio and the activation of the Akt/mammalian target of rapamycin (mTOR) signaling pathway were determined in cultured human microvascular endothelial cell (HMEC-1) endothelial cells that were exposed to a high concentration of Pi with or without the Pi influx blocker phosphonoformic acid, the autophagy inhibitor 3-methyladenine, and the autophagy inducer rapamycin. The impacts of autophagy on Pi-induced apoptotic damage were assessed by flow cytometry.
  
  
  RESULTS
  The in vivo rat model of CKD revealed that hyperphosphatemia is associated with increased endothelial LC3 staining. The exposure of HMEC-1 cells to high Pi induced both dose-dependent and time-dependent increases in the LC3-II/LC3-I expression ratio accompanied by the inhibition of the Akt/mTOR signaling pathway. In HMEC-1 cells, high Pi-induced autophagy and the inhibition of Akt/mTOR signaling were reversed by phosphonoformic acid through the blockage of Pi influx. Apoptosis, characterized by the levels of cleaved caspase 3 and poly(ADP-ribose) polymerase, along with autophagy was induced by high Pi, and the inhibition of autophagy by 3-methyladenine significantly aggravated high Pi-induced apoptosis. The flow cytometry results confirmed that the blockage of autophagy promoted the apoptosis of endothelial cells.
  
  
  CONCLUSIONS
  Hyperphosphatemia induces endothelial autophagy, possibly through the inhibition of the Akt/mTOR signaling pathway, which may play a protective role against high Pi-induced apoptosis.
authors.affiliation: Division of Nephrology, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.
authors.forename: Yu-Juei
authors.initials: YJ
authors.lastname: Hsu
authors.affiliation: Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan.
authors.forename: Shih-Che
authors.initials: SC
authors.lastname: Hsu
authors.affiliation: Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan; Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan.
authors.forename: Shih-Ming
authors.initials: SM
authors.lastname: Huang
authors.affiliation: Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.
authors.forename: Herng-Sheng
authors.initials: HS
authors.lastname: Lee
authors.affiliation: Division of Nephrology, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.
authors.forename: Shih-Hua
authors.initials: SH
authors.lastname: Lin
authors.affiliation: Division of Cardiovascular Surgery, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.
authors.forename: Chien-Sung
authors.initials: CS
authors.lastname: Tsai
authors.affiliation: Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan.
authors.forename: Chun-Che
authors.initials: CC
authors.lastname: Shih
authors.affiliation: Division of Cardiovascular Surgery, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan; Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan. Electronic address: linrock@ms26.hinet.net.
authors.forename: Chih-Yuan
authors.initials: CY
authors.lastname: Lin
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001343:Autophagy / Q000187:drug effects; D002460:Cell Line; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D004789:Enzyme Activation; D017245:Foscarnet / Q000494:pharmacology; D006801:Humans; D054559:Hyperphosphatemia / Q000201:enzymology* / Q000209:etiology / Q000473:pathology; D008297:Male; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D017208:Rats, Wistar; D051436:Renal Insufficiency, Chronic / Q000150:complications; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
chemical_list: D000225:Adenine; D017245:Foscarnet; D008869:Microtubule-Associated Proteins; D010710:Phosphates; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; C526109:Anxa3 protein, rat; C477012:MAP1LC3A protein, human; C025946:3-methyladenine; C546842:MTOR protein, human; C546845:mTOR protein, rat