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Journal of Biomedicine and Biotechnology
Volume 2010, Article ID 859240, 12 pages
http://dx.doi.org/10.1155/2010/859240
Research Article

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

1Centre for Kidney Disease Research, School of Medicine, The University of Queensland at Princess Alexandra Hospital, Brisbane, QLD 4102, Australia
2Discipline of Pathology, School of Medicine, University of Western Sydney, Sydney, NSW 2751, Australia
3Department of Employment, Economic Development and Innovation, Brisbane, QLD 4003, Australia
4Department of Medicine, University of Alabama, Birmingham, AL 35487, USA
5Division of Molecular and Gene Therapies, School of Medical Science, Griffith University, Gold Coast, QLD 4222, Australia

Received 29 March 2010; Revised 5 July 2010; Accepted 18 August 2010

Academic Editor: G. S. Stein

Copyright © 2010 Tao Yang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.