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. 2017 Mar;28(3):785-801.
doi: 10.1681/ASN.2016020165. Epub 2016 Sep 9.

Tenascin-C Is a Major Component of the Fibrogenic Niche in Kidney Fibrosis

Haiyan Fu  1   2 , Yuan Tian  1 , Lili Zhou  1 , Dong Zhou  2 , Roderick J Tan  3 , Donna B Stolz  4 , Youhua Liu  5   2
Affiliations

Tenascin-C Is a Major Component of the Fibrogenic Niche in Kidney Fibrosis

Haiyan Fu et al. J Am Soc Nephrol. 2017 Mar.

Abstract

Kidney fibrosis initiates at certain focal sites in which the fibrogenic niche provides a specialized microenvironment that facilitates fibroblast activation and proliferation. However, the molecular identity of these fibrogenic niches is poorly characterized. Here, we determined whether tenascin-C (TNC), an extracellular matrix glycoprotein, is a component of the fibrogenic niche in kidney fibrosis. In vivo, TNC expression increased rapidly in kidneys subjected to unilateral ureteral obstruction or ischemia/reperfusion injury and predominantly localized at the foci rich in fibroblasts in renal interstitium. In vitro, TNC selectively promoted renal interstitial fibroblast proliferation, bromodeoxyuridine incorporation, and the expression of proliferation-related genes. The mitogenic activity of TNC required the integrin/focal adhesion kinase/mitogen-activated protein kinase signaling cascade. Using decellularized extracellular matrix scaffolds, we found that TNC-enriched scaffolds facilitated fibroblast proliferation, whereas TNC-deprived scaffolds inhibited proliferation. Matrix scaffold prepared from fibrotic kidney also promoted greater ex vivo fibroblast proliferation than did scaffolds prepared from healthy kidney. Conversely, small interfering RNA-mediated knockdown of TNC in vivo repressed injury-induced fibroblast expansion and renal fibrosis. These studies identify TNC as a major constituent of the fibrogenic niche that promotes fibroblast proliferation, and illustrate a pivotal role for the TNC-enriched microenvironment in kidney fibrogenesis.

Keywords: extracellular matrix; fibroblast; renal fibrosis.

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Figures

Figure 1.
Figure 1.
TNC is induced and localized focally in the fibrotic kidneys. (A and B) Western blot analyses of renal expression of TNC protein in sham and obstructed kidneys at 7 days after UUO. Representative Western blot (A) and quantitative data (B) are presented. Relative TNC protein levels (fold induction over the controls) after normalization with actin are reported. Numbers (1–4) indicate each individual animal in a given group. **P<0.01 versus sham controls. (C and D) Western blot analyses of renal TNC protein at 10 days after IRI. Representative (C) Western blot and (D) quantitative data are presented. Numbers (1–3) indicate each individual animal in a given group. **P<0.01 versus sham controls. (E) Representative micrographs showing the expression and localization of TNC in mouse models of UUO (7 days). Kidney sections were immunohistochemically stained with specific antibody against TNC. Circles with dashed lines indicate the predominantly focal distribution of TNC protein. Arrow in the enlarged box indicates the TNC-enriched foci. Scale bar, 50 µm. (F) Quantitative, real-time RT-PCR demonstrated an early induction of TNC mRNA in the obstructed kidneys after UUO. TNC mRNA levels were assessed at 1, 3, and 7 days after UUO. *P<0.05, **P<0.01 versus sham controls (n=4). (G) TNC protein was induced in the obstructed kidneys as early as 1 day after UUO. Kidney sections obtained at 1 day after UUO was subjected to immunohistochemical staining for TNC. Arrows indicate positive staining. Scale bar, 50 µm. (H and I) Renal interstitial fibroblasts are the major TNC-producing cells in vivo. Kidney sections after UUO (H) and IRI (I) were subjected to double immunofluorescence staining for TNC (red) and vimentin (Vim), fibroblast-specific protein 1 (Fsp1), or aquaporin 1 (AQP1) (green), respectively. Costaining of TNC and fibroblast markers (vimentin and Fsp1) but not tubular cell marker (AQP1) was evident in diseased kidneys after UUO (7 days) (H) and IRI (10 days) (I). Arrows indicate TNC-positive cells. Scale bar, 50 µm.
Figure 2.
Figure 2.
Shh induces TNC expression in renal interstitial fibroblasts. (A) Quantitative, real-time RT-PCR analyses show that Shh (50 ng/ml) induced TNC mRNA expression in NRK-49F cells. **P<0.01, *P<0.05 versus control. (B) Western blot analyses demonstrate that Shh induced TNC protein expression in NRK-49F cells in a time-dependent manner as indicated. (C) Immunofluorescence staining for TNC expression in NRK-49F cells. Cells were stained for TNC expression at 2 days after incubation with Shh.
Figure 3.
Figure 3.
TNC selectively promotes fibroblast proliferation. (A) TNC promotes fibroblast proliferation in a dose- and time-dependent manner. NRK-49F cells were incubated with different concentrations of TNC, and then cell numbers were counted for various periods of time as indicated. *P<0.05, TNC (both 1 µg/cm2 and 2 µg/cm2) versus controls (n=3). (B) Graphic presentation shows that TNC promoted NRK-49F cell proliferation as assessed by a colorimetric MTT assay. **P<0.01 versus controls (n=3). (C) Representative micrographs show that TNC promoted fibroblast DNA synthesis as shown by BrdU incorporation. NRK-49F cells were incubated with 1 µg/cm2 TNC for 2 days. Cells were immunostained with mouse anti-BrdU antibody (red). SYTOX-Green (green) was used to visualize the nuclei. Arrows indicate BrdU-positive cells. Scale bar, 20 µm. (D) Quantitative determination of the percentage of BrdU-positive cells after TNC treatment. **P<0.01 versus controls (n=3). (E) Western blots show that TNC promotes the expression of numerous proliferation-related genes in fibroblasts. NRK-49F cells were incubated with TNC (1 µg/cm2) for various periods of time as indicated. Cell lysates were subjected to Western blot analyses for c-Myc, c-fos, PCNA, and α-tubulin. (F–H) Graphic presentation of the quantitative data demonstrated TNC induction of c-Myc (F), c-fos (G), and PCNA (H) in NRK-49F cells. Relative protein levels (fold induction over the controls) after normalization with α-tubulin are reported. *P<0.05, **P<0.01 versus controls (n=3). (I–L) TNC shows no effect on HKC-8 cell proliferation. HKC-8 cells were incubated with TNC (1 µg/cm2) for variable durations, then cell number count (I), MTT assay (J), BrdU incorporation (K), and proliferation-related gene expression (L) were assessed. (M and N) Renal interstitial cell proliferation was correlated with TNC abundance in vivo. Kidney sections from UUO (M) or IRI (N) were double stained with antibodies against Ki67 and TNC, respectively. Images from areas with different levels of TNC expression were taken and Ki67-positive interstitial cells were counted and plotted. TNC abundance was assessed and expressed as positive cells per high power field.
Figure 4.
Figure 4.
TNC promotes renal fibroblast proliferation through activating the FAK/MAPK signaling pathway. NRK-49F cells were treated with TNC (1 µg/cm2) for various periods of time as indicated. Cell lysates were immunoblotted with specific antibodies. (A–C) TNC induced a rapid FAK and ERK1/2 phosphorylation and activation. Representative Western blots (A) and quantitative data (B and C) are presented. **P<0.01 versus controls (n=3). (D) TNC-mediated ERK1/2 activation was dependent on FAK signaling. NRK-49F cells were pretreated with specific FAK inhibitor PF573228 (10 μM) for 30 minutes, followed by incubation with TNC. Representative Western blots and quantitative data are presented. **P<0.01 versus controls (n=3). (E) TNC did not induce EGFR activation in renal fibroblasts, whereas EGF itself markedly stimulated EGFR phosphorylation at tyrosine-845. (F) TNC-mediated ERK1/2 activation was dependent on Mek1 signaling. NRK-49F cells were pretreated with specific Mek1 inhibitor PD98059 (20 μM) for 30 minutes, followed by incubation with TNC. Representative Western blots and quantitative data are presented. **P<0.01 versus controls; †P<0.05 versus TNC alone (n=3). (G–J) Blockade of FAK/Mek1/ERK1/2 signaling abolished the induction of proliferation-related genes by TNC. NRK-49F cells were treated with specific Mek1 inhibitor PD98059 (20 μM) for 30 min, followed by incubation with TNC, and then cell lysates were subjected to Western blot analyses for c-Myc, c-fos, and α-tubulin, respectively. Representative Western blots (G) and quantitative data (H, I, and J) are presented. *P<0.05, **P<0.01 versus controls; †P<0.05 versus TNC alone (n=3). (K and L) Blockade of ERK1/2 signaling abolished the induction of fibroblast proliferation by TNC. Cell number counting (K) and MTT assay (L) were used to assess cell proliferation. *P<0.05 versus controls; †P<0.05 versus TNC alone (n=3). (M and N) Infection with constitutively active Mek1 adenovirus (Ad.Mek1-CA) was sufficient to induce ERK1/2 phosphorylation. Representative Western blots (M) and quantitative data (N) are presented. **P<0.01 versus controls (n=3). (O–R) Infection with constitutively active Mek1 adenovirus (Ad.Mek1-CA) was sufficient to induce the expression of proliferation-related genes in NRK-49F cells. Representative Western blots (O) and quantitative data (P, Q, and R) are presented. Relative protein levels (fold induction over the controls) after normalization with α-tubulin are reported. *P<0.05, **P<0.01 versus controls (n=3).
Figure 5.
Figure 5.
TNC-enriched ECM constitutes a fibrogenic niche promoting fibroblast proliferation. (A) Flow chart shows the experimental design and procedures. Normal NRK-49F cells were cultured in a 6 cm dish and incubated with or without Shh to induce TNC expression. Three days later, the cultures were decellularized by EGTA, and the ECM scaffold was prepared. New NRK-49F cells were replated to ECM scaffold. (B) Western blot analysis of TNC expression in the ECM scaffold of NRK-49F cells after incubation with or without Shh. (C–G) The TNC-enriched ECM scaffold promoted fibroblast proliferation. NRK-49F cells were inoculated on the ECM scaffold prepared after incubation with or without Shh, and cultured for various periods of time as indicated. (C) Cell numbers were counted and presented at different time points as indicated. *P<0.05 versus controls (n=3). (D) MTT and (E and F) BrdU incorporation assay were performed at 2 days after NRK-49F cells were inoculated on the ECM scaffold. *P<0.05 versus controls (n=3). (G) Western blots show that the TNC-enriched ECM scaffold promoted the expression of numerous proliferation-related genes in fibroblasts.
Figure 6.
Figure 6.
TNC-depleted ECM scaffold inhibits fibroblast proliferation. (A) Flow chart shows the experimental design and procedures. NRK-49F cells were transfected with either control siRNA or TNC siRNA to knockdown TNC expression and secretion. Three days later, the cells were decellularized by EGTA, and the ECM scaffold was prepared. New NRK-49F cells were replated as indicated. (B) Knockdown of TNC expression in NRK-49F cells by siRNA. NRK-49F cells were transfected with either control siRNA or TNC-specific siRNA, followed by treatment with Shh for 3 days as indicated. Cell lysates were immunoblotted with antibodies against TNC and α-tubulin. (C) TNC depletion in the ECM scaffold. Western blot analysis showed a decreased TNC protein in the ECM scaffold prepared from NRK-49F cells that were transfected with either control siRNA or TNC-specific siRNA for 3 days. The ECM scaffold proteins were immunoblotted with specific antibodies against TNC or fibronectin, respectively. (D–H) Depletion of TNC in the ECM scaffold repressed fibroblast proliferation. NRK-49F cells were cultured on the ECM scaffold prepared from the Shh-treated cells transfected with either control siRNA or TNC-specific siRNA for various periods of time as indicated. (D) Cell numbers were counted and presented at different time points as indicated. *P<0.05 versus controls (n=3). (E) MTT and (F and G) BrdU incorporation assay was performed at 2 days after culture. *P<0.05 versus controls (n=3). (H) Western blots show that depletion of TNC in the ECM scaffold suppressed the expression of numerous proliferation-related genes in fibroblasts. (I–M) Increase of TNC expression in the ECM scaffold promoted fibroblast proliferation. NRK-49F cells were transfected with TNC expression vector (pCMV-TNC). (I) Whole-cell lysates were immunoblotted with antibodies against TNC and α-tubulin. (J) The ECM scaffolds were prepared from NRK-49F cells transfected with pCMV-TNC, and immunoblotted with antibodies against TNC and fibronectin. NRK-49F cells were cultured on the ECM scaffold prepared from NRK-49F cells transfected with pcDNA3 or pCMV-TNC. (K) Cell numbers were counted and presented at different time points as indicated. *P<0.05 versus pcDNA3 (n=3). (L) MTT assay was performed at 2 days after culture. *P<0.05 versus pcDNA3 (n=3). (M) Western blots show that an increase of TNC in the ECM scaffold promoted the expression of numerous proliferation-related genes in fibroblasts.
Figure 7.
Figure 7.
ECM scaffold prepared from the fibrotic kidney promotes fibroblast proliferation ex vivo. (A) Photograph of mouse freshly isolated kidney and (B) translucent ECM scaffold prepared from mouse kidney by decellularization protocol. (C) Scanning electron microscopic images of the ECM scaffold prepared from normal and fibrotic kidney at 7 days after UUO. Boxed areas are enlarged and presented in the lower panel. (D) ECM scaffold from the fibrotic kidney was rich in TNC. DAPI staining for nuclei confirmed no residual cells left in the ECM scaffold prepared from sham and UUO kidneys. TNC abundance in the ECM scaffold was assessed by immunostaining. Arrow indicates the TNC staining. Scale bar, 50 µm. (E) Fluorescence micrograph of fibroblasts grown in the ECM scaffold for 6 days ex vivo. NRK-49F cells were labeled with cell tracker (green CMFDA) probes and then plated in the ECM scaffold and cultured for 6 days. Boxed area is enlarged and presented in the right panel. Arrow indicates the labeled NRK-49F cell. Scale bar, 50 µm. (F) ECM scaffold from the fibrotic kidney facilitates fibroblast growth ex vivo. Cryosections of the ECM scaffold were immunostained for vimentin (red, active fibroblast marker) and subjected to DAPI staining for the nucleus (blue). Boxed areas are enlarged and presented in the right panel. Arrows indicate cells with positive staining for vimentin and DAPI. Scale bar, 20 µm. (G) Quantitative determination of fibroblast cells grown in the ECM scaffold prepared from sham and UUO kidney. Cell growth was assessed by a colorimetric MTT assay. *P<0.05 versus controls (n=3). (H and I) Assessment of the housekeeping gene α-tubulin demonstrated an increased fibroblast proliferation in the ECM scaffold prepared from UUO kidney. The abundances of α-tubulin in cell populations were assessed when fibroblasts were seeded (Input, 0 day) or after 3 days of cultivation in the ECM scaffold prepared from sham and UUO kidney. Representative Western blot (H) and quantitative data (I) are presented. *P<0.05 versus sham (n=3). (J–L) Western blotting showed the induction of c-fos and PCNA expression in the fibroblasts cultivated in the UUO scaffold, comparing with sham controls. Representative Western blot (J) and quantitative data on c-fos (K) and PCNA (L) are presented. Relative protein levels (fold induction over the controls) after normalization with α-tubulin are reported. *P<0.05 versus sham (n=3).
Figure 8.
Figure 8.
Knockdown of TNC inhibits interstitial cell proliferation in vivo. (A) Western blot analysis of renal TNC expression in the obstructed kidneys at 7 days after UUO. Mice were injected with either control siRNA or TNC siRNA. (B) Representative micrographs showed an effective knockdown of TNC protein in the obstructed kidneys by TNC siRNA, as shown by immunohistochemical staining. (C) Western blot analyses of the cell proliferation-related proteins at 7 days after UUO. Kidney homogenates from various groups as indicated were immunoblotted for PCNA, c-Myc, and GAPDH, respectively. (D and E) Quantitative data are presented as fold induction versus controls. Numbers (1–3) indicate each individual animal in a given group. Relative protein levels (fold induction over sham controls) after normalization with GAPDH are reported. *P<0.05 versus sham; †P<0.05 versus control siRNA. (F) Representative micrographs show immunohistochemical staining for PCNA in the obstructed kidney at 7 days after UUO. Arrowheads indicate PCNA-positive cells in the interstitium whereas arrows show PCNA-positive cells in renal tubules. Scale bar, 50 µm. (G) Quantitative determination of the PCNA-positive cells in renal tubular and interstitial compartments. *P<0.05 versus sham; †P<0.05 versus control siRNA; siR, siRNA.
Figure 9.
Figure 9.
Knockdown of TNC ameliorates renal fibrosis in vivo and reduces fibroblast proliferation ex vivo. (A–C) Western blot analyses of renal fibronectin and α-smooth muscle actin expression in mice injected with either control siRNA or TNC siRNA. Representative Western blot (A) and quantitative data (B and C) are shown. Numbers (1–3) indicate each individual animal in a given group. Relative protein levels (fold induction over sham controls) after normalization with GAPDH are reported. *P<0.05 versus sham; †P<0.05 versus control siRNA (n=4). (D) Representative micrographs show renal fibronectin expression and collagen deposition at 7 days after UUO in various groups as indicated. Kidney sections were subjected to immunofluorescence staining for fibronectin and Masson Trichrome staining for collagen deposition, respectively. Arrows indicate positive staining. Scale bar, 50 µm. (E and F) Graphical presentation shows kidney fibrotic lesions at 7 days after UUO in various groups. *P<0.05 versus sham; †P<0.05 versus control siRNA (n=4); siR, siRNA. (G) Knockdown of TNC in vivo reduced fibroblast proliferation ex vivo. Kidney ECM scaffolds were prepared from different groups as indicated. NRK-49F cells were seeded and cultivated for 3 days. Cell numbers were estimated by MTT assay. *P<0.05 versus sham; †P<0.05 versus control siRNA (n=4–5). (H and I) Depletion of TNC in the kidney ECM scaffolds reduced cell proliferation ex vivo. The abundances of α-tubulin in cell populations were assessed when fibroblasts were seeded (Input, 0 day) or after 3 days of cultivation in the ECM scaffold prepared from different groups, as indicated. Representative Western blot (H) and quantitative data (I) are presented. *P<0.05 versus sham; †P<0.05 versus control siRNA (n=4–5). (J–L) Levels of c-fos and PCNA expression in the fibroblasts cultivated in the ECM scaffolds prepared from different groups, as indicated. Representative Western blot (J) and quantitative data on c-fos (K) and PCNA (L) are presented. Relative protein levels (fold induction over sham controls) after normalization with α-tubulin are reported. *P<0.05 versus sham; †P<0.05 versus control siRNA (n=4–5).
Figure 10.
Figure 10.
Schematic diagram shows that TNC is a major component of the fibrogenic niche promoting fibroblast proliferation and renal fibrosis. In response to kidney injury, tubular cells produce and secrete various fibrogenic factors such as Shh, Wnt ligands, and TGF-β1. These fibrogenic cues (yellow dots) will then stimulate the induction of TNC (orange dots) in fibroblasts, which is secreted into the extracellular space and forms a fibrogenic niche. Such a microenvironment will further promote fibroblast proliferation and facilitate matrix protein production and deposition, leading to formation of fibrotic foci.
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