Hypoxia promotes fibrogenesis in vivo via HIF-1 stimulation of epithelial-to-mesenchymal transition
Debra F. Higgins, … , Masayuki Iwano, Volker H. Haase
Debra F. Higgins, … , Masayuki Iwano, Volker H. Haase
Published November 21, 2007
Citation Information: J Clin Invest. 2007;117(12):3810-3820. https://doi.org/10.1172/JCI30487.
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Research Article Nephrology

Hypoxia promotes fibrogenesis in vivo via HIF-1 stimulation of epithelial-to-mesenchymal transition

Abstract

Hypoxia has been proposed as an important microenvironmental factor in the development of tissue fibrosis; however, the underlying mechanisms are not well defined. To examine the role of hypoxia-inducible factor–1 (HIF-1), a key mediator of cellular adaptation to hypoxia, in the development of fibrosis in mice, we inactivated Hif-1α in primary renal epithelial cells and in proximal tubules of kidneys subjected to unilateral ureteral obstruction (UUO) using Cre-loxP–mediated gene targeting. We found that Hif-1α enhanced epithelial-to-mesenchymal transition (EMT) in vitro and induced epithelial cell migration through upregulation of lysyl oxidase genes. Genetic ablation of epithelial Hif-1α inhibited the development of tubulointerstitial fibrosis in UUO kidneys, which was associated with decreased interstitial collagen deposition, decreased inflammatory cell infiltration, and a reduction in the number of fibroblast-specific protein–1–expressing (FSP-1–expressing) interstitial cells. Furthermore, we demonstrate that increased renal HIF-1α expression is associated with tubulointerstitial injury in patients with chronic kidney disease. Thus, we provide clinical and genetic evidence that activation of HIF-1 signaling in renal epithelial cells is associated with the development of chronic renal disease and may promote fibrogenesis by increasing expression of extracellular matrix–modifying factors and lysyl oxidase genes and by facilitating EMT.

Authors

Debra F. Higgins, Kuniko Kimura, Wanja M. Bernhardt, Nikita Shrimanker, Yasuhiro Akai, Bernd Hohenstein, Yoshihiko Saito, Randall S. Johnson, Matthias Kretzler, Clemens D. Cohen, Kai-Uwe Eckardt, Masayuki Iwano, Volker H. Haase

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Figure 2

Hif-1 promotes EMT in PTECs.

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Hif-1 promotes EMT in PTECs. (A) Upper-left panel: Schematic illustratin...
(A) Upper-left panel: Schematic illustrating the genetic make-up of mice used for the in vitro EMT studies. Mice expressed the ROSA26RLacZ reporter (top) in conjunction with the PEPCK-cre transgene, in the presence (+) or absence (–) of the floxed Hif1a conditional allele (bottom). Triangles indicate the presence of loxP sites. Hif1a+/+ or Hif1a–/– PTECs were cultured under normoxia or hypoxia for 0–5 days. Cells were stained for β-gal (red) and the mesenchymal marker FSP-1 (green; original magnification, ×400). Epithelial cells undergoing EMT stained both red and green (arrows). (B) Percent FSP-1–positive epithelial cells in Hif1a+/+ or Hif1a–/– cultures exposed to normoxia (N) or hypoxia (H) for 1–5 days. Scale bars represent mean values ± SEM. *P < 0.01 (C) Western blot analysis for ZO-1, α-SMA, and CTGF in Hif1a+/+ or Hif1a–/– PTECs exposed to normoxia or hypoxia for 3 or 6 days. PTECs were stimulated with 3 ng/ml TGF-β1 for 3 days as a positive control for α-SMA and CTGF induction. β-Actin is included as loading control. (D) Neutralizing Ab against TGF-β (α-TGFβ Ab) (1 μg/ml) inhibits TGF-β1 induction of CTGF in PTECs. (E) Western blot analysis for α-SMA and CTGF in Hif1a+/+ or Hif1a–/– PTECs cultured for 6 days under either normoxia or hypoxia in the absence or presence (+) of neutralizing Ab against TGF-β.