1. ¹Department of Nephrology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
2. ²State Key Laboratory of Cancer Biology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
3. ³Department of Geriatrics, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China

Correspondence: Daiming Fan, State Key Laboratory of Cancer Biology, Xijing Hospital, Fourth Military Medical University, No 15 Chang le West Road, Xi'an, Shaanxi Province 710032, China. E-mail: fandaim@fmmu.edu.cn

⁴These authors contributed equally to this work.

Received 12 June 2008; Revised 11 December 2008; Accepted 13 January 2009; Published online 11 March 2009.

Abstract

Epithelial-to-mesenchymal transition (EMT) induced by chronic hypoxia is one of the critical causes of renal fibrosis. Twist, a basic helix-loop-helix transcription factor, is believed to be important in promoting EMT. We found that the expression of Twist was increased in human tubule cell lines (HK-2 and HKC) grown under hypoxic conditions. This was accompanied by reduced expression of the epithelial markers E-cadherin and ZO-1 and enhanced expression of the mesenchymal markers vimentin and -smooth muscle actin. When Twist was overexpressed in these cells it induced a mesenchymal phenotype, whereas its knockdown by short interfering RNA (siRNA) effectively reversed hypoxia-induced EMT. We showed that transfection with siRNA to hypoxia-inducible factor-1 (HIF-1), another basic helix-loop-helix transcription factor, reduced Twist expression. Twist promoters contain HIF1--binding sites and transfection of reporter constructs using the promoter showed increased transcription in cells subjected to hypoxia. Electrophoretic mobility shift and chromatin immunoprecipitation assays identified the presence of a functional HIF-1-binding site within the proximal Twist gene promoter. In an in vivo assay using the rat remnant kidney we found that both Twist and HIF-1 were overexpressed in tubular epithelial cells showing EMT. These studies suggest that HIF-1 induces Twist expression in hypoxic tubular cells and that this plays a role in EMT during renal fibrogenesis.