Vimentin expression is required for the development of EMT-related renal fibrosis following unilateral ureteral obstruction in mice

Abstract

Most renal transplants ultimately fail secondary to chronic allograft nephropathy (CAN). Vimentin (vim) is a member of the intermediate filament family of proteins and has been shown to be important in the development of CAN. One of the pathways leading to chronic renal fibrosis after transplant is thought to be epithelial to mesenchymal transition (EMT). Even though vim expression is one of the main steps of EMT, it is unknown whether vim expression is required for EMT leading to renal fibrosis and allograft loss. To this end, the role of vim in renal fibrosis was determined via unilateral ureteral obstruction (UUO) in vim knockout mice (129 svs6 vim -/-). Following UUO, kidneys were recovered and analyzed via Western blotting, immunofluorescence, and transcriptomics. Cultured human proximal renal tubular (HK-2) cells were subjected to lentiviral-driven inhibition of vim expression and then treated with transforming growth factor (TGF)-β to undergo EMT. Immunoblotting as well as wound healing assays were used to determine development of EMT. Western blotting analyses of mice undergoing UUO reveal increased levels of vim soon after UUO. As expected, interstitial collagen deposition increased in control mice following UUO but decreased in vim -/- kidneys. Immunofluorescence analyses also revealed altered localization of β-catenin in vim -/- mice undergoing UUO without significant changes in mRNA levels. However, RNA sequencing revealed a decrease in β-catenin-dependent genes in vim -/- kidneys. Finally, vim-silenced HK-2 cell lines undergoing EMT were shown to have decreased cellular migration during wound healing. We conclude that vim inhibition decreases fibrosis following UUO by possibly altering β-catenin localization and downstream signaling.

Keywords: CAN; EMT; fibrosis; renal transplant; vimentin; β-catenin.

Fig. 1.

Vimentin (vim) expression is important for the development murine renal fibrosis following unilateral ureteral obstruction (UUO). Following UUO, mice kidneys were harvested and underwent immunohistochemistry (IHC) with anticollagen 11α antibody (A–F) or Masson’s trichrome staining (H–L). Four weeks post-UUO, there was a significant decrease in the intensity of anticollagen signal comparing control mice (E) and vim −/− mice (F). Unligated control kidneys show very minimal staining (A), whereas 2 wk post-UUO, there is little difference in collagen staining comparing control vs. vim −/− mice (C vs. D). Masson’s trichrome staining also showed a decrease in collagen deposition (blue staining) comparing control (K) and vim −/− mice (L) 2 wk following UUO. Unligated control kidneys display no significant evidence of collagen deposition in the renal cortex. Size bars = 50 μm. Quantification of collagen deposition following UUO was carried out utilizing the ImageJ software as described previously. Signal intensity was calculated for both IHC and trichrome staining. Samples were then normalized to unligated control kidneys. Data represent three separate experiments, looking at five sections. Collagen deposition by IHC showed a >50% decrease in collagen staining at 4 wk (G). There was a smaller but significant decrease in intensity of trichrome staining at 2 wk (M). n = 3. Error bars = standard deviations, *P < 0.05.

Fig. 2.

Vimentin (vim) knockout mice undergoing unilateral ureteral obstruction (UUO) demonstrate altered β-catenin, E-cadherin, and keratin staining on Western blotting of whole kidneys 1, 2, and 4 wk following UUO. Whole kidneys from wild-type (WT) and vim −/− were recovered and homogenized in 10 mM Tris (pH 7.4) buffer without SDS. This SDS-deficient lysis buffer was used to minimize solubilization of membrane-associated proteins and to detect total cytosolic β-catenin levels (48). A: immunoblotting with anti-E-cadherin, -vim, -β-catenin, and -pan-keratin antibodies was performed. Vim was detected 1 wk following UUO in control mice. Nonmembrane-bound β-catenin was detected 2 wk following UUO in control mice but 1 wk after UUO in vim −/− mice. E-cadherin signaling was diminished when comparing vim −/− mice vs. control mice, whereas keratin staining increased from 1 to 2 wk in vim −/− mice and decreased in control mice. Actin staining was used as loading controls. Molecular weight is in kDa. B: Western blot staining was normalized against unligated control kidneys. Vim staining increased ~fourfold over control kidneys and peaks at 2 wk (~eightfold increase; P > 0.05). Nonmembrane-bound β-catenin increases fourfold over control kidneys 1 wk following UUO and peaks at 4 wk in vim −/− mice although β-catenin levels also increase in control kidneys over time following UUO. E-cadherin levels decrease in both WT and vim −/− kidneys 1 and 2 wk following UUO. Keratin staining increases in both vim −/− and WT mice, but keratin levels are higher in vim −/− mice. n = 4. Error bars = standard error, *P < 0.05.

Fig. 3.

β-catenin mRNA production is not affected in vimentin (vim) −/− mice undergoing unilateral ureteral obstruction (UUO). qPCR primers for vim, β-catenin, and keratin 8 were designed and utilized to probe wild-type (WT) and vim −/− kidneys 1, 2, and 4 wk following UUO. qPCR readouts were normalized to ribosome 18 s (Rn18s) to account for variability in loading and then expressed as fold changes of the internal control, nonligated right kidney, for each individual mouse that was analyzed. Relative mRNA expression to Rn18s shows increase in vim expression at 1 and 2 wk following UUO in WT kidneys, whereas vim expression is detected relative to Rn18s 4 wk following UUO in the unligated WT control kidney (A). β-catenin RNA expression is not statistically significant among all kidneys 1, 2, and 4 wk following UUO (B). Keratin 8 mRNA levels are also increased 1 wk post-UUO in WT ligated kidneys. At 4 wk, there is an increase of keratin 8 when compared with the other samples (C). mRNA levels were then normalized against nonligated right kidney (taken as 1). Histogram shows the mRNA levels of ligated left kidney. Vim expression was detected 1 and 2 wk following UUO and decreased sharply 4 wk post-UUO (D). There is no statistical significance in the detection of β-catenin mRNA 1, 2, and 4 wk post-UUO between WT and vim −/− mice (E). Keratin 8 mRNA levels are slightly higher in vim −/− mice 2 and 4 wk post-UUO (F). Data are representative of n = 4 independent experiments. Error bars = Standard error; *P < 0.05.

Fig. 4.

Vimentin (vim) −/− mice undergoing unilateral ureteral obstruction (UUO) show altered cellular localization of β-catenin and E-cadherin in proximal renal tubules. OCT-embedded kidneys following UUO underwent immunofluorescence with antibodies directed against vim, β-catenin. DAPI was used for nuclear staining. All images were obtained on with a Leica DMI4000 B confocal microscope and analyzed using Leica Advanced Fluorescence Application Suite. Wild-type (WT) kidneys 1 wk post-UUO have an increased expression of vim in the cytoplasm of proximal renal tubular cells (C). However, β-catenin staining colocalizes with DNA in the nucleus (D, arrow). Vim −/− kidneys 1-wk post-UUO kidneys result in β-catenin signals more prominently in the cytoplasm (F, H; arrow), with no detectable vim staining (G). Mander’s colocalization coefficients calculation reveals a statically significant increase in colocalization between DAPI and β-catenin staining in WT vs. vim −/− mice (0.34 vs. 1.62). Size bars = 10 μm. *P < 0.05.

Fig. 5.

Vimentin (vim) −/− mice undergoing unilateral ureteral obstruction (UUO) display no difference in E-cadherin in proximal renal tubules following UUO. OCT-embedded kidneys following UUO underwent immunofluorescence with antibodies directed against E-cadherin and DAPI. All images were obtained on with a Leica DMI4000 B confocal microscope and analyzed using Leica advanced fluorescence application suite. E-cadherin localization 1 wk following UUO in vim −/− mice reveals a strong peripheral staining pattern along the cell membrane (D and F), whereas wild-type (WT) mice E-cadherin is seen both in at the cell membrane as well as the cytoplasm (A and C). Size bars = 10 μm.

Fig. 6.

Vimentin (vim) mediates transforming growth factor (TGF)-β-induced epithelial to mesenchymal transition (EMT) of human renal tubular epithelial cells (A). Sequence of shRNA to human vim. shRNA specially targeting (shVIM) downregulated vim expression efficiently in human proximal renal tubular (HK-2) cells (B). Two and 3 days following induction with TGF-β, control HK-2 cell lines express vim, whereas shVIM cell lines (VIM) do not express vim in appreciable amounts. All lanes were normalized to β-actin to ensure proper loading. Time lapse phase contrast microscopy was used to visualize wound healing in control cells (shCON) and shVIM cells following TGF-β induction (C). Quantification of cellular migration was carried out by measuring the changes in the wound area after 24 h (D). There was a ~30% reduction in migration following EMT induction in shVIM cell lines. Experiments were performed in triplicate and P values calculated. Immunoblotting using antibodies against vim, pan-keratin, and EMT markers, including zona occludens (ZO)-1, N-cadherin, E-cadherin, and β-catenin was carried out on shVIM HK-2 cells following TGF-β induction (E). Signals were normalized to GAPDH to account for variability in loading and then expressed as fold changes of the negative control, shCON. shVIM HK-2 cells demonstrate “sheet-like” motility following TGF-β stimulation. Vim-silenced HK-2 cells were treated with TGF-β and then underwent wound healing assay. Time lapse phase contrast microscopy for 24 h demonstrate sheet-like movement when compared with control cell lines. Time lapse assembled from 1-h images over 24-h period. Supplemental Material for this article is available online at the Journal website.