title: Development of bioartificial renal tubule devices with lifespan-extended human renal proximal tubular epithelial cells.
abstract: BACKGROUND
  The bioartificial renal tubule device is a cell therapy system for renal failure. The major obstacle in the development of the bioartificial renal tubule device is the obtainment of a large number of viable renal tubule cells to seed on the inner surface of hollow fibers. Although our previous studies had used a transformed cell line, they may be dangerous for clinical uses. Therefore, different approaches to amplify renal proximal tubular epithelial cells (RPTEC) in culture without oncogenes, vectors and carcinogens have been required.
  
  
  METHODS
  The limitation of the replicative lifespan of human RPTEC, which is ∼12 population doublings (PDs), was extended by invalidating messenger RNA of cell cycle-related genes with antisense oligonucleotide or small interfering RNA (siRNA).
  
  
  RESULTS
  Periodic transfection of siRNA to a tumor suppressor p53 or a cyclin-dependent kinase inhibitor p16(INK4a) extended the lifespan by 33 and 63 PDs, respectively, in 3 months of culture. The siRNA-mediated lifespan extension was controllable because cell division ceased within 2 weeks after the transfection was discontinued. Expressions of γ-glutamyltransferase 1 and glucose transporter 1 were recovered in siRNA-transfected RPTEC cultured on porous membranes. Bioartificial renal tubule devices (0.8 m(2)) constructed with these cells showed reabsorption of water (122.3 ± 4.2 mL/30 min), sodium (18.1 ± 0.7 mEq/30 min) and glucose (121.7 ± 4.4 mg/30 min) after 1 week of circulation. Furthermore, β2-microglobulin and pentosidine were metabolized by RPTEC in mini-devices (65 cm(2)) within 48 h of circulation.
  
  
  CONCLUSIONS
  These approaches enabled us to yield a high enough number of RPTEC for construction of bioartificial renal tubule devices repeatedly. Lifespan-extended RPTEC could recover their specific characteristics by culturing on porous membranes, and bioartificial renal tubule devices constructed with these cells showed good performances of reabsorption and metabolism.
  
  
  CONCLUSIONS
  A large number of human renal tubular cells required for construction of the bioartificial renal tubule device were prepared by extending the lifespan of the primary cells by invalidating mRNA of cell cycle-related genes. Constructed bioartificial renal tubule devices with lifespan-extended cells showed good performances of in vitro examination of reabsorption and metabolism. Requiring no oncogenes, vectors or cell cloning, the RNAi-mediated lifespan extension can help advance tissue-replacement therapy as well as basic research.
authors.affiliation: Division of Nephrology and Metabolism, Department of Medicine, Tokai University School of Medicine, Isehara, Japan.
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Sanechika
authors.forename: Kaichiro
authors.initials: K
authors.lastname: Sawada
authors.forename: Yukio
authors.initials: Y
authors.lastname: Usui
authors.forename: Kazuya
authors.initials: K
authors.lastname: Hanai
authors.forename: Takatoshi
authors.initials: T
authors.lastname: Kakuta
authors.forename: Hajime
authors.initials: H
authors.lastname: Suzuki
authors.forename: Genta
authors.initials: G
authors.lastname: Kanai
authors.forename: Satoshi
authors.initials: S
authors.lastname: Fujimura
authors.forename: Tun Aung
authors.initials: TA
authors.lastname: Yokoyama
authors.forename: Masafumi
authors.initials: M
authors.lastname: Fukagawa
authors.forename: Toshiro
authors.initials: T
authors.lastname: Terachi
authors.forename: Akira
authors.initials: A
authors.lastname: Saito
mesh_terms: D002478:Cells, Cultured; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D051246:Glucose Transport Proteins, Facilitative / Q000235:genetics / Q000378:metabolism; D006440:Hemofiltration / Q000295:instrumentation*; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000378:metabolism; D007688:Kidneys, Artificial; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D016062:Porosity; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005723:gamma-Glutamyltransferase / Q000235:genetics / Q000378:metabolism
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D051246:Glucose Transport Proteins, Facilitative; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D005723:gamma-Glutamyltransferase