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-1alpha 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-1alpha enhanced epithelial-to-mesenchymal transition (EMT) in vitro and induced epithelial cell migration through upregulation of lysyl oxidase genes. Genetic ablation of epithelial Hif-1alpha 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-1alpha 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.

Figure 1. Hypoxia induces morphological and phenotypic changes in PTECs consistent with an EMT phenotype.

PTECs cultured under hypoxic (1% O₂; right panels) compared with normoxic (21% O₂; left panels) conditions displayed altered cell shape (phase contrast, original magnification, ×100), reduced staining for the epithelial junction protein ZO-1 (green; original magnification, ×400), and increased staining for α-SMA (red; original magnification, ×200), nuclei were stained with DAPI (blue).

Figure 2. Hif-1 promotes EMT in PTECs.

(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.

Figure 3. Hif-1 enhances epithelial cell migration through induction of lysyl oxidases.

(A) Hif1a^+/+ or Hif1a^–/– PTECs were cultured under normoxia or hypoxia for 6 days. Migration was analyzed by introducing a scratch into the epithelial layer and measuring scratch width, i.e., migration distance, at 0, 4, and 24 hours. Hypoxic Hif1a^+/+ cells (top panel) displayed complete scratch closure after 24 hours, whereas the scratch was still apparent in hypoxic Hif1a^–/– cells (lower panel); compare images labeled with #. Original magnification, ×100. Graph shows fold increase of hypoxic (Hx) Hif1a^+/+ (black bars) and Hif1a^–/– (gray bars) PTECs compared with their respective normoxic (Nx) migration. *P < 0.05. (B) Quantitative real-time PCR analysis of Pgk (4.2-fold induction at 12 hours), Vegf (13.8-fold increase at 12 hours), Mdr-1 (4.6-fold increase), Pai-1 (3.2-fold induction at 12 hours), Lox and LoxL2 (2.4- and 2.5-fold induction at 12 and 6 hours, respectively) mRNA in Hif1a^+/+ (+/+) or Hif1a^–/– (–/–) PTECs exposed to hypoxia for 0, 6, and 12 hours. Gene expression was normalized to 18S mRNA. (C) Hif1a^+/+ PTECs were cultured under hypoxia for 6 days; 2 hours prior to wounding, lysyl oxidase inhibitors BAPN or BCS were added and cell migration monitored over 30 hours. Hx, untreated control cells (hypoxia without inhibitor). Original magnification, ×100.

Figure 4. UUO kidneys are hypoxic prior to development of tubulointerstitial fibrosis.

(A) Hypoxyprobe (Chemicon) was used to detect hypoxic regions in obstructed kidneys (UUO) 1 day and 8 days after ligation of the ureter (upper 4 panels). In contrast, hypoxyprobe staining was not detected in contralateral kidneys (CTL) 1 day after ligation but was apparent at low levels by day 8; original magnification ×200. Lower panels show cortical immunostaining for Hif-1α and Hif-2α in 8-day UUO and CTL kidneys; original magnification, ×400; arrows indicate cells with positive nuclear staining. (B) Quantitative real-time PCR analysis for Mdr-1, Pai-1, Lox, LoxL2, Collagen 1α1, and Collagen 18α1 in cortex of Hif1a^+/+ CTL and UUO kidneys 8 days after ligation. Shown are relative expression values normalized to 18S rRNA. Data points represent individual kidneys; red bars represent mean values; n = 7; *P < 0.05, **P < 0.01, ^#P < 0.001.

Figure 5. HIF-1α in renal biopsies from patients with CKD.

(A) HIF-1α immunostaining in formalin-fixed, paraffin-embedded renal biopsy tissues from patients with DN analyzed by differential interference contrast microscopy. Top row: Tissue from a normal control kidney (nl.) and from a DN kidney, both with 1+ staining (≤25% cells positive per visual field). Bottom row: Representative photographs from DN kidneys with 3+ staining (>50% cells stained positive). A glomerulus (gl.) with HIF-1α–positive cells is shown on the left; the tubulointerstitial compartment from a different DN kidney is shown on the right. Arrows highlight cells with nuclear HIF-1α staining. The asterisk indicates area with nodular sclerosis. (B) Summary of HIF-1α expression analysis in DN. DN cases are grouped according to tubulointerstitial injury score as described by Hohenstein et al. (41). The number of biopsies with glomerular or tubular staining (t) is shown in parentheses. –, absence of staining; +, 1%–25% of cells per visual field with positive staining; ++, >25%–50%; +++, >50% of cells with positive staining. (C) Expression analysis of LOXL2 in microdissected tubulointerstitium from patients with DN, IgA nephropathy (IgAN), and hypertensive nephrosclerosis (NS) by real-time PCR. Shown are relative expression values normalized to 18S. Pretransplant biopsies from living donor kidneys (LD) were used as control. **P < 0.01 by Mann-Whitney U test.

Figure 6. Deletion of Hif1a in PTECs attenuates renal fibrogenesis.

Hif1a^+/+ and Hif1a^–/– kidneys were stained for collagen content (sirius red staining of collagen fibers shown in red; original magnification, ×200; n = 8 for mutant and n = 7 for control), macrophage marker F4/80 (original magnification, ×400; n = 3 in each group), and EMT marker FSP-1 (original magnification, ×400; n = 9 for mutant and n = 5 for control). For statistical analysis, sirius red–positive areas from 10 individual measurements per mouse were averaged across control and mutant cohorts. Morphometric analysis showed a reduction of all 3 stains in Hif1a mutant tissues. Scale bars represent mean values ± SEM; *P < 0.05, **P < 0.01.

Figure 7. Inhibition of lysyl oxidases reduces fibrosis in UUO kidneys.

Shown are the number of FSP-1–positive cells (top panel) and the area stained for collagen by sirius red (bottom panel) in vehicle-treated (VEH-UUO) and BAPN-treated UUO (BAPN-UUO) kidneys; original magnification, ×100 (top panel) and ×400 (bottom panel). Scale bars represent mean values ± SEM; *P < 0.05, **P < 0.001; n = 6.

Figure 8. Model proposing a role for HIF-1 in the progression of CKD.

Tubulointerstitial hypoxia is due to glomerulosclerosis and capillary rarefaction, which is commonly found in kidneys from patients with CKD. As a consequence of hypoxia in renal epithelial cells HIF-1α is stabilized, resulting in increased expression of lysyl oxidase genes and other profibrogenic factors, thus promoting EMT and the accumulation of ECM.