title: Lentiviral-mediated RNA interference against TGF-beta receptor type II in renal epithelial and fibroblast cell populations in vitro demonstrates regulated renal fibrogenesis that is more efficient than a nonlentiviral vector.
abstract: BACKGROUND
  Lentiviral constructs reportedly can integrate into the genome of non-dividing, terminally differentiated cells and dividing cells, for long-term gene expression. This investigation tested whether a third generation lentiviral-mediated small interfering RNA (siRNA) delivered into renal epithelial and fibroblast cells against type II transforming growth factor-beta receptor (siRNA-TBRII) could better attenuate renal fibrogenesis in comparison with a non-lentiviral construct.
  
  
  METHODS
  HIV-derived lentiviral and non-lentiviral constructs were used to transfect cells with siRNA-TBRII or siRNA-EGFP control. Human embryonic kidney (HEK-293T), renal epithelial cells (NRK-52E) and renal fibroblasts (NRK-49F) were transfected and gene silencing quantified (fluorescence microscopy, Western blotting, fluorescence-activated cell sorting). Renal fibrogenesis was assessed using extracellular matrix protein synthesis (fibronectin and collagen-III; Western immunoblot), and α-smooth muscle actin (α-SMA) was analysed as a marker of fibroblast activation and epithelial-to-mesenchymal transdifferentiation (EMT).
  
  
  RESULTS
  Lentiviral-mediated siRNA-TBRII significantly suppressed TBRII expression in all cell lines, and also significantly suppressed renal fibrogenesis. In comparison with the non-lentiviral construct, lentiviral-mediated siRNA-TBRII produced stronger and more persistent inhibition of collagen-III in NRK-49F cells, fibronectin in all renal cell lines, and α-SMA in renal epithelial cells.
  
  
  CONCLUSIONS
  Lentiviral vector systems against TBRII can be delivered into renal cells to efficiently limit renal fibrogenesis by sequence-specific gene silencing.
authors.affiliation: Centre for Kidney Disease Research, School of Medicine, The University of Queensland at Princess Alexandra Hospital, Brisbane, QLD 4102, Australia.
authors.forename: Tao
authors.initials: T
authors.lastname: Yang
authors.forename: Bing
authors.initials: B
authors.lastname: Zhang
authors.forename: Betty K
authors.initials: BK
authors.lastname: Pat
authors.forename: Ming Q
authors.initials: MQ
authors.lastname: Wei
authors.forename: Glenda C
authors.initials: GC
authors.lastname: Gobe
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D004847:Epithelial Cells / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism*; D005355:Fibrosis; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007668:Kidney / Q000473:pathology*; D016086:Lentivirus / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D051381:Rats; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism; D013489:Suppression, Genetic; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D000199:Actins; D018125:Receptors, Transforming Growth Factor beta; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II