title: NF-κB-dependent increase in tissue factor expression is responsible for hypoxic podocyte injury.
abstract: BACKGROUND
  Fibrin deposition within glomeruli is commonly seen in kidney biopsy specimens, suggesting enhanced coagulant activity. Tissue factor (TF) is a coagulation factor which is also related to various biological effects, and TF is upregulated by hypoxia in cancer cells. Recently, hypoxic podocyte injury has been proposed, therefore, we investigated TF expression in hypoxia.
  
  
  METHODS
  Conditionally immortalized human podocytes were differentiated and treated under hypoxic or normoxic conditions. mRNA expressions of TF and tissue factor pathway inhibitor (TFPI) were analyzed by quantitative RT-PCR. Protein levels of TF and TFPI were tested by enzyme-linked immunosorbent assay. We employed small interfering RNA (siRNA) to temporary knockdown early growth response protein 1 (Egr-1), hypoxia-inducible factor-1α (HIF-1α) and TF. The expression of CD2-associated protein (CD2AP) mRNA and phalloidin staining was examined to assess podocyte injury.
  
  
  RESULTS
  Hypoxia increased mRNA expression of TF (6 h: 2.3 ± 0.05 fold, p < 0.001, 24 h: 5.6 ± 2.4 fold, p < 0.05) and suppressed TFPI (6 h: 0.54 ± 0.04 fold, p < 0.05, 24 h: 0.24 ± 0.06 fold, p < 0.001) compared with normoxia. Similarly, protein levels of TF were increased and TFPI were decreased. Egr-1 siRNA did not change TF mRNA expression. Pyrrolidine dithiocarbamate (PDTC), a nuclear factor kappa B (NF-κB) inhibitor, significantly reduced hypoxia induced TF expression, and HIF-1α knockdown further increased TF. Hypoxia resulted in decreased CD2AP and actin reorganization in podocytes, and these changes were attenuated by TF siRNA.
  
  
  CONCLUSIONS
  Hypoxia increased the expression of TF in human podocytes NF-κB dependently. TF may have a critical role in the hypoxic podocyte injury.
authors.affiliation: Department of Cardiology and Nephrology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562, Japan.
authors.forename: Ikuyo
authors.initials: I
authors.lastname: Narita
authors.affiliation: Department of Cardiology and Nephrology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562, Japan. mshimada@hirosaki-u.ac.jp.
authors.forename: Michiko
authors.initials: M
authors.lastname: Shimada
authors.affiliation: Department of Cardiology and Nephrology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Yamabe
authors.affiliation: Department of Cardiology and Nephrology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562, Japan.
authors.forename: Takahiko
authors.initials: T
authors.lastname: Kinjo
authors.affiliation: Department of Cardiology and Nephrology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Tanno
authors.affiliation: Department of Cardiology and Nephrology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562, Japan.
authors.forename: Kimitaka
authors.initials: K
authors.lastname: Nishizaki
authors.affiliation: Department of Cardiology and Nephrology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562, Japan.
authors.forename: Misato
authors.initials: M
authors.lastname: Kawai
authors.affiliation: Department of Cardiology and Nephrology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Nakamura
authors.affiliation: Department of Cardiology and Nephrology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562, Japan.
authors.forename: Reiichi
authors.initials: R
authors.lastname: Murakami
authors.affiliation: Department of Cardiology and Nephrology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562, Japan.
authors.forename: Norio
authors.initials: N
authors.lastname: Nakamura
authors.affiliation: Department of Cardiology and Nephrology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Tomita
authors.affiliation: Bristol Renal, University of Bristol, Dorothy Hodgkin Building, Bristol, UK.
authors.forename: Moin A
authors.initials: MA
authors.lastname: Saleem
authors.affiliation: University of Hong Kong, Hong Kong, China.
authors.forename: Peter W
authors.initials: PW
authors.lastname: Mathieson
authors.affiliation: Department of Cardiology and Nephrology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Okumura
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D015687:Cell Hypoxia; D002460:Cell Line; D003035:Cobalt / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism; D051766:Early Growth Response Protein 1 / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D010590:Phalloidine / Q000378:metabolism; D050199:Podocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D011759:Pyrrolidines / Q000494:pharmacology; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013859:Thiocarbamates / Q000494:pharmacology; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C114545:CD2-associated protein; D003598:Cytoskeletal Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008074:Lipoproteins; D016328:NF-kappa B; D011759:Pyrrolidines; D012333:RNA, Messenger; D013859:Thiocarbamates; C051928:lipoprotein-associated coagulation inhibitor; D010590:Phalloidine; C020972:pyrrolidine dithiocarbamic acid; D003035:Cobalt; D013925:Thromboplastin; C018021:cobaltous chloride; D010100:Oxygen
keywords: Hypoxia; Podocytes; Tissue factor