Generation of an anti-fibrotic therapeutic gene using a tubular epithelium-specific expression gene casette and iKB cDNA

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To identify tubular epithelium-specific, positive regulatory elements in the promoter of osteopontin (OPN) gene, we generated luciferase reporter gene constructs bearing various fragments of the OPN promoter and performed transient transfection experiments using murine tubular epithelial cells (mPTEC). In addition to mPTEC, fibroblasts (3T3) and monocytes (RAW264) were also employed for the transfection experiments. The -600 to -825 bp fragment of the OPN promoter constitutively showed positive transcription activity in mPTEC, but not in 3T3 and RAW264. In contrast, the -115 to -189 bp fragment showed positive transcription activity in mPTEC as well as 3T3 and RAW264 in response to recombinant transforming growth factor-b1 treatment. Thus, the -600 to -825 bp fragment is suggested to contain tubular epithelium-specific, positive regulatory elements. We are now generating tubular epithelium-specific expression gene casette using this fragment, and testing whether tubular epithelium-specific expression of iKB can attenuate fibrosis through down-regulation of NF-kB activity in vitro and in vivo.