Basic Research

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

Haiyan Fu, Yuan Tian, Lili Zhou, Dong Zhou, Roderick J. Tan, Donna B. Stolz and Youhua Liu
JASN March 2017, 28 (3) 785-801; DOI: https://doi.org/10.1681/ASN.2016020165

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.

Kidney fibrosis, characterized by the relentless deposition of extracellular matrix (ECM) leading to tissue scarring, represents the final common outcome of a wide variety of progressive CKD.1–3 Extensive studies demonstrate that renal fibrotic lesions are not homogeneous across the kidney parenchyma. Rather, they typically initiate at certain focal sites, in which interstitial fibroblasts become activated, proliferate, and produce a large amount of ECM components.4,5 Although it remains enigmatic why fibroblasts at certain sites respond differentially after injury, it has been long hypothesized that there is a specialized niche/microenvironment, which dictates the activation of fibroblasts in their discrete locations. The fibrogenic niche would be composed of a specialized ECM network consisting of many diverse constituents, as well as different cell types and unique soluble factors secreted into the extracellular space.6,7 Although the concept of the fibrogenic niche as a functional entity for supporting fibroblast activation and proliferation is intuitive, the molecular identities and composition of such a niche are currently poorly characterized.

Tenascin-C (TNC) is a large oligomeric ECM glycoprotein with cell signaling properties.8,9 Structurally, TNC contains an N-terminal oligomerization domain that leads to the formation of hexamers, a variable number of tandem EGF-like repeats, numerous fibronectin type 3 domains, and a fibrinogen homology domain at the C-terminus.10 TNC is mainly expressed during embryonic development with distinct spatial and temporal patterns. In adults, little or no TNC is detected in the kidney and other organs. However, prominent de novo expression of TNC is reported in the adult tissues in various pathophysiologic conditions, such as tissue injury/wound healing, inflammation, and tumorigenesis.11–13 As a so-called matricellular protein, TNC is not an obligatory structural element in the ECM, but it is able to bind to ECM structural proteins and cell surface receptors such as the EGF receptor (EGFR) and integrins.8,14,15 TNC binding to these receptors causes activation of their downstream pathways, thereby modulating cell adhesion, spreading, migration, and proliferation in a cell type– and context-dependent manner.9,16–18 Earlier studies demonstrate that TNC is present in several stem cell niches and plays a role in stem cell support, self-renewal, and maintenance.6,19–22 Whether TNC contributes to the functional organization of the fibrogenic niche in kidney fibrosis remains unknown.

In this study, we have investigated TNC expression, localization, and its role in fibroblast proliferation using in vivo, ex vivo, and in vitro strategies. We have also delineated its downstream signaling cascades. By using a decellularized matrix scaffold, we have illustrated a pivotal role for TNC in organizing the fibrogenic niche that favors fibroblast proliferation in kidney fibrosis.

Results

De Novo Upregulation of TNC in the Fibrotic Kidneys

To investigate TNC regulation after kidney injury in vivo, we examined its expression in two well characterized models of renal fibrosis induced by unilateral ureteral obstruction (UUO) and ischemia/reperfusion injury (IRI).23,24 As shown in Figure 1, A–E, TNC protein was virtually undetectable in normal control kidney by Western blot analyses and immunohistochemical staining, respectively. However, UUO caused a dramatic upregulation of TNC protein in the obstructed kidney at 7 days (Figure 1, A and B), suggesting de novo induction of its expression in response to injury in vivo. Similar results were obtained when renal TNC expression was analyzed at 10 days after IRI (Figure 1, C and D). We further examined the expression and localization of TNC in the fibrotic kidney by immunohistochemical staining. As shown in Figure 1E, marked upregulation of TNC protein was observed in the fibrotic kidneys after UUO, whereas it was undetectable in normal kidneys. Of interest, TNC protein was predominantly localized in renal interstitium with a characteristic focal distribution pattern (Figure 1E, circles with dashed lines). This suggests that TNC protein is enriched at the focal sites, creating a unique microenvironment, or niche, in the fibrotic kidney.

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.

We next investigated the dynamics of renal TNC induction after injury. To this end, the expression of TNC mRNA at different time points after UUO was assessed by quantitative, real-time RT-PCR. As shown in Figure 1F, TNC mRNA was significantly induced as early as 1 day after UUO. Immunohistochemical staining also revealed a focal staining of the TNC-positive cells in renal interstitium at 1 day after UUO (Figure 1G, arrows), suggesting that TNC induction is an early event that precedes renal fibrosis in this model. In a similar fashion, induction of TNC protein at the focal sites was also evident at 1 day after IRI (data not shown).

To more precisely localize the TNC protein, we employed double immunofluorescence staining for TNC and various cell-specific markers. As illustrated in Figure 1, H and I, TNC protein (red) colocalized with vimentin (green), the intermediate filament protein specifically expressed in activated fibroblasts, and fibroblast-specific protein 1 (Fsp1) (green) in renal interstitium after either UUO or IRI. In contrast, little colocalization occurred between TNC (red) and aquaporin 1 (green), a specific marker for renal proximal tubular epithelial cells, after UUO or IRI (Figure 1, H and I). Of note, tubular cells occasionally stained positively for TNC as well in the advanced stage of renal fibrosis (data not shown). These results suggest that TNC is primarily upregulated in renal interstitial fibroblasts in mouse models of CKD in vivo.

Sonic Hedgehog Induces TNC Expression in Renal Fibroblasts In Vitro

We next sought to explore the possible upstream regulator of TNC expression in the kidney after injury in vivo. As shown in Figure 2A, sonic hedgehog (Shh), a protein that targets interstitial fibroblasts but originates in injured renal tubules,25 rapidly induced TNC mRNA expression in cultured normal rat kidney interstitial fibroblast (NRK-49F) cells. Similarly, Shh induced TNC protein expression in NRK-49F cells in a time-dependent manner, as demonstrated by Western blot analyses (Figure 2B). Immunofluorescence staining also revealed that Shh induced de novo TNC expression in renal fibroblasts (Figure 2C). These data suggest that, after kidney injury, tubule-derived Shh is able to act as an upstream regulator and promotes TNC expression in renal fibroblast cells.

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.

TNC Selectively Promotes Fibroblast Proliferation In Vitro

To investigate the potential roles of TNC in fibroblast biology, we next examined its effect on the proliferation and expansion of renal fibroblasts, a central event in tissue fibrogenesis.2,26 Hence, we incubated NRK-49F cells with different concentrations of recombinant human TNC protein for various periods of time as indicated. As shown in Figure 3A, TNC substantially increased the number of NRK-49F cells in a dose- and time-dependent manner, indicating that TNC is able to promote renal fibroblast proliferation. Similar results were obtained by using a quantitative colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay (Figure 3B). We further assessed the ability of TNC to promote cell cycle entry and subsequent DNA synthesis, which was reflected by bromodeoxyuridine (BrdU) incorporation. As shown in Figure 3, C and D, increased BrdU incorporation was observed in NRK-49F cells after incubation with TNC for 2 days, compared with the controls. Consistently, numerous proliferation-related proteins such as c-Myc, c-fos, and proliferating cell nuclear antigen (PCNA) were induced by TNC in NRK-49F cells (Figure 3, E–H). Taken together, these results suggest that TNC can activate multiple proliferation-related genes in renal interstitial fibroblasts, leading to augmented cell proliferation.

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.

We found that TNC is selective and specific in promoting the proliferation of renal fibroblasts, but not tubular epithelial cells. As shown in Figure 3, I–L, under the same condition as in NRK-49F cells, TNC failed to affect the replication of human kidney proximal tubular epithelial (HKC-8) cells, as assessed by cell counting, MTT assay, and BrdU incorporation assay, respectively. Similarly, TNC also did not induce the expression of several proliferation-related proteins including c-Myc and PCNA in HKC-8 cells (Figure 3L). These results clearly demonstrate that TNC selectively promotes the proliferation of renal fibroblasts, but not tubular epithelial cells.

Given that TNC promotes fibroblast proliferation in vitro, we further examined any correlation between local TNC abundance and interstitial cell proliferation in vivo. Kidney sections from UUO and IRI mice were double-immunostained for TNC and Ki67. As shown in Figure 3, M and N, there was a close association of local TNC abundance with the numbers of Ki67-positive interstitial cells in different regions of the kidney, suggesting that a TNC-enriched microenvironment is associated with an enhanced fibroblast proliferation in vivo as well.

TNC Activates Integrin/Focal Adhesion Kinase/MAP Kinase Signaling

We further explored the downstream signal pathway responsible for TNC promotion of fibroblast proliferation in vitro. TNC is known to bind to cell surface receptor integrins,15,17 and it also possesses EGF-like repeats which can directly trigger EGFR signaling by acting as a low affinity ligand.14 To delineate the mechanism underlying TNC-mediated fibroblast proliferation, we incubated NRK-49F cells with TNC for various periods of time. As shown in Figure 4, A and B, TNC rapidly induced focal adhesion kinase (FAK) phosphorylation at tyrosine-925 in NRK-49F cells, suggesting activation of integrin signaling. After FAK activation at 5–10 minutes after TNC treatment, extracellular signal-regulated kinase-1 and -2 (ERK1/2) were transiently phosphorylated at 30 minutes, and then their activities quickly returned to baseline at 60 minutes (Figure 4, A and C). TNC-triggered ERK1/2 activation was clearly mediated by FAK, because PF573228, a specific inhibitor of FAK, was able to fully repress their activation in NRK-49F cells (Figure 4D). However, we did not detect any EGFR tyrosine phosphorylation after TNC incubation (Figure 4E), whereas EGF itself dramatically stimulated EGFR activation. These results suggest that TNC may elicit its biologic activity primarily by activating integrin/FAK/ERK1/2 signaling in NRK-49F cells.

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).

We found that ERK1/2 activation was required for initiating the proliferative response of fibroblasts to TNC. Blockade of ERK1/2 activation by PD98059, a specific inhibitor of Mek-1, the kinase that lies upstream of ERK1/2, abolished TNC-induced c-Myc, c-fos, and PCNA expression in NRK-49F cells (Figure 4, F–J). Inhibition of ERK1/2 activation also abolished the TNC-induced NRK-49F cell proliferation, as demonstrated by cell counting and MTT assay (Figure 4, K and L). Conversely, ectopic expression of constitutively activated Mek-1 via adenoviral vector was able to activate ERK1/2 (Figure 4, M and N), and induced expression of several proliferation-related genes such as c-Myc, c-fos, and PCNA (Figure 4, O–R). Collectively, these observations suggest a pivotal role for the integrin/FAK/MAPK cascade in mediating the TNC-triggered fibroblast proliferation.

TNC-Enriched ECM Scaffold Acts As a Fibrogenic Niche In Vitro

TNC is a secreted protein that is integrated to ECM, being an integral element of the extracellular microenvironment. To test whether TNC is a constituent of the fibrogenic niche, we designed an in vitro strategy to validate the effect of a TNC-enriched extracellular microenvironment on fibroblast proliferation. As depicted in Figure 5A, NRK-49F cells were treated with Shh for 3 days to induce TNC production, and then subjected to decellularization protocol by EGTA. In this way, TNC-enriched ECM scaffold was prepared and tested for its ability to promote fibroblast proliferation. As shown in Figure 5B, the ECM scaffold prepared from Shh-treated NRK-49F cells did contain increased levels of TNC protein, compared with controls. Interestingly, when new NRK-49F cells were seeded on the TNC-enriched ECM scaffold and cultured for different periods of time, numbers of cells were increased in a time-dependent manner, compared with the controls (Figure 5C). Similar results were obtained when MTT assay and BrdU incorporation assessment were used (Figure 5, D–F). Not surprisingly, the TNC-enriched ECM scaffold also promoted the expression of proliferation-related proteins, including c-Myc and PCNA (Figure 5G).

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.

To further confirm the role of TNC in the ECM scaffold in facilitating fibroblast proliferation, we knocked down the expression of TNC by using an small interfering RNA (siRNA) strategy. As illustrated in Figure 6A, NRK-49F cells were transfected with control or TNC-specific siRNA in the presence of Shh. Efficient knockdown of TNC expression by siRNA was confirmed in whole-cell lysates (Figure 6B) or in the decellularized ECM scaffold (Figure 6C), respectively. Specific knockdown of TNC by siRNA did not affect other ECM components such as fibronectin expression (Figure 6C). As shown in Figure 6, D and E, deprivation of TNC in the ECM scaffold significantly inhibited fibroblast proliferation, compared with the controls. Similarly, TNC-deprived ECM scaffold also reduced BrdU incorporation (Figure 6, F and G) and repressed c-Myc, c-fos, and PCNA expression in NRK-49F cells (Figure 6H). Therefore, TNC is a principal component in the ECM scaffold that is responsible for promoting fibroblast proliferation.

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.

To provide more direct evidence that a TNC-enriched ECM niche promotes fibroblast proliferation, we transiently transfected NRK-49F cells with TNC expression vector (pCMV-TNC). Overexpression of TNC was confirmed by Western blot analyses of whole-cell lysates (Figure 6I) and the ECM scaffold (Figure 6J), respectively. As shown in Figure 6, K–M, increased TNC in the ECM scaffold alone was sufficient to induce fibroblast expansion and the expression of the proliferation-related genes.

Matrix Scaffold from Fibrotic Kidney Facilitates Fibroblast Proliferation Ex Vivo

Because de novo induction of TNC was observed after kidney injury (Figure 1), we reasoned that the fibrotic kidney could harbor a TNC-enriched environment that favors fibroblast proliferation. To test this hypothesis, we sought to prepare the matrix scaffold from normal and diseased kidneys. Fresh kidneys were harvested from mice (Figure 7A) and subjected to decellularization protocols.27,28 As shown in Figure 7B, decellularized kidney was nearly transparent and retained the weblike appearance of the basement membrane architecture. Scanning electron microscopy revealed that the ECM scaffold from normal kidney exhibited a continuous three-dimensional architecture with smooth surfaces (Figure 7C, left panel, arrow), whereas the scaffold from UUO kidneys displayed a disorganized coarse surface filled with holes (Figure 7C, right panel, arrow). The matrix scaffold from both normal and fibrotic kidneys displayed no nuclear staining by DAPI, confirming that all resident kidney cells were successfully removed (Figure 7D). Not surprisingly, immunofluorescence staining showed the presence of TNC protein in the matrix scaffold prepared from UUO kidney, but not from normal control kidney (Figure 7D).

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).

We next investigated the proliferation of renal fibroblasts inoculated in the matrix scaffold prepared from either normal or fibrotic kidneys. To visualize fibroblasts inoculated and grown on kidney matrix scaffold, we tracked the NRK-49F cells by green fluorescent probe. As shown in Figure 7E, fluorescence microscopy showed that fibroblasts could survive and grow on the kidney matrix scaffolds for an extended period of time. After 6 days of incubation, significant increase in the vimentin-positive cells was found in the TNC-enriched matrix scaffold prepared from UUO kidney by immunofluorescence staining, compared with that from normal kidney (Figure 7F, arrowhead). Quantification of the cells on kidney matrix scaffolds by MTT assay confirmed this finding (Figure 7G).

We also assessed cell proliferation by examining the levels of α-tubulin, the house-keeping protein that is often used for normalization in Western blotting, as a surrogate marker for cell mass. As shown in Figure 7, H and I, although a similar level of α-tubulin was detected when NRK-49F cells were seeded, marked induction of α-tubulin protein was evident in the cell population cultivated for 3 days in the UUO scaffold, compared with sham controls. In addition, the cells cultivated in the UUO scaffold expressed increased levels of c-fos and PCNA, even after normalizing with α-tubulin (Figure 7, J–L). These ex vivo data unambiguously indicate that the microenvironment endowed in the matrix scaffold from fibrotic kidney, in which TNC is enriched, provides a fibrogenic niche that favors fibroblast proliferation and expansion.

Knockdown of TNC Represses Fibroblast Proliferation and Renal Fibrosis In Vivo

To further investigate the relevance of the TNC-enriched niche to kidney fibrosis, we carried out in vivo studies by knocking down renal TNC expression using siRNA. As illustrated in Figure 8, A and B, renal expression of TNC was almost completely abolished in UUO mice by intravenous injection of TNC siRNA. We found that knockdown of TNC expression markedly repressed renal expression of the proliferation-related genes, such as PCNA and c-Myc (Figure 8, C–E). Such downregulation of TNC also reduced the PCNA-positive cells in both the interstitial and tubular compartment after UUO (Figure 8, F and G), suggesting that TNC promotes cell proliferation in vivo as well.

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.

We next examined the severity of renal fibrosis in UUO mice after injection of TNC-specific siRNA. We found that knockdown of TNC significantly reduced renal expression of fibronectin mRNA, as shown by RT-PCR analyses (data not shown). Similarly, fibronectin protein and α-smooth muscle actin were also dramatically repressed (Figure 9, A–C). Immunofluorescence staining also revealed a reduction of fibronectin protein in the interstitium of the obstructed kidney after UUO. Masson trichrome staining demonstrated a reduced collagen deposition and fibrotic lesions in the UUO kidney receiving TNC siRNA, compared with the controls (Figure 9, D–F).

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).

We finally examined whether depletion of TNC in the kidney affects fibroblast proliferation and expansion by using an ex vivo approach. As shown in Figure 9G, MTT assay demonstrated that deficiency of TNC in the matrix scaffold inhibited fibroblast expansion, compared with the controls. Similarly, less α-tubulin protein was detected in the cell population cultivated in the TNC-deficient matrix scaffold (Figure 9, H and I), indicating a decreased cell mass. Fibroblast cells cultivated in the TNC-deficient scaffold also exhibited a decreased expression of c-fos and PCNA (Figure 9, J–L). Taken together, these results indicate that depletion of TNC, a major component of the fibrogenic niche, is able to inhibit fibroblast proliferation and ameliorate kidney fibrosis in vivo.

Discussion

It is widely recognized that tissue fibrosis after injury is not homogeneous across affected organs.4 The factors that determine the focal allocation of fibrotic lesions within the kidney remain poorly characterized. However, it has long been thought that a specialized local environment, or niche, at the particular sites might play an instrumental role in the formation of fibrotic foci. In this study, we demonstrate that TNC, an evolutionarily conserved, multimodular glycoprotein of the ECM, is a key constituent of the fibrogenic niche, which provides the specialized microenvironment for fibroblast proliferation and expansion. This conclusion is supported by several lines of evidence, which include (1) de novo upregulation of TNC occurs very early after injury and is at the focal sites rich in fibroblasts in kidney parenchyma, (2) TNC promotes fibroblast proliferation in vitro, (3) TNC-enriched ECM scaffold acts as a fibrogenic niche that favors fibroblast expansion, and (4) depletion of TNC attenuates fibroblast expansion ex vivo and reduces kidney fibrosis in vivo. These studies establish that TNC is a major player in organizing the fibrogenic niche that provides a favorable environment for fibroblast activation and proliferation in kidney fibrogenesis.

The niche is usually composed of a specialized ECM network, a unique set of secreted factors and particular cell types involved.20,29 As depicted in Figure 10, we propose that TNC is a major element in the formation of the fibrogenic niche in kidney fibrosis. As previously reported,30,31 kidney injury often causes tubular epithelial cell damage or stress (Figure 10), which triggers the expression and secretion of various fibrogenic cues such as Shh, Wnt ligands, and TGF-β1, which can target interstitial fibroblasts in a paracrine manner. Upon stimulation by these extracellular cues, fibroblasts produce and secrete TNC (Figure 5), as well as other ECM components such as collagen and fibronectin, which are assembled into a TNC-enriched ECM network, thereby forming the fibrogenic niche (Figure 10). Such a fibrogenic niche provides a favorable environment for fibroblast proliferation and expansion, and eventually leads to the formation of fibrotic foci (Figure 10). According to this model, it becomes apparent that TNC, a unique ECM component whose expression is induced de novo when kidney homeostasis is challenged by injury (Figure 1), markedly contributes to the framework of the fibrogenic niche.

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.

TNC belongs to a family of matricellular proteins that primarily regulate the interaction of cells with other ECM components, growth factors, and plasma membrane receptors, thereby modulating multiple cellular functions including cell adhesion, proliferation, survival, migration, and differentiation.13,32,33 Unlike other ECM proteins such as collagen, fibronectin, and laminin, TNC is not an obligatory structural element of the ECM, and its expression is uniquely tuned temporally and spatially within local areas during particular moments of development, wound healing, remodeling, or tumorigenesis.8,34–36 Consistent with earlier reports,37 it is apparent that TNC is mainly produced by interstitial fibroblasts, especially in the earlier stage after kidney injury (Figure 1G). Notably, the colocalization between TNC and fibroblast markers including vimentin and Fsp1 appears not impressive, partially because much of the TNC being stained is already integrated into the ECM. Besides fibroblasts, other cells in renal interstitium such as macrophages or even tubular epithelial cells could also express TNC, especially in the advanced stage of CKD. Along this line, TNC is reported to be expressed in kidney tubular epithelial cells under profibrotic conditions in vitro,38 and is occasionally found in renal tubules at 7 days after UUO in vivo.

Using both in vitro and ex vivo decellularized matrix scaffolds, we have unambiguously demonstrated that TNC in the extracellular microenvironment is both sufficient and necessary for fibroblast proliferation (Figures 5–7). In this regard, TNC-enriched matrix scaffolds provide a unique fibrogenic microenvironment in which fibroblast proliferation is autonomous and self-sufficient, independent of stimulation by other mitogens. Consistent with this speculation, TNC expression is induced as early as 1 day after UUO, a time point that significantly precedes robust fibroblast proliferation in this model, and it is predominantly localized at the focal sites of the injured kidney where fibroblast accumulation is evident (Figure 1), and depletion of TNC inhibits fibroblast proliferation and reduces renal fibrosis ex vivo and in vivo (Figures 6, 8, and 9). It should be pointed out that depletion of TNC also reduced tubular cell PCNA expression in the obstructed kidney. This probably is a consequence of the improved histologic lesion after TNC inhibition, because TNC per se did not affect tubular cell proliferation in vitro (Figure 3). However, the possibility also exists that inhibition of TNC may also have some direct effects on tubular epithelial cells, especially in light of that in the advanced stage of CKD tubular cells occasionally express TNC as well.

The molecular mechanism by which TNC promotes fibroblast proliferation appears to be associated with its activation of the integrin/FAK/MAPK cascade (Figure 4). Because FAK is the key molecular mediator in the integrin signaling,39,40 TNC-triggered FAK phosphorylation illustrates that the integrin signaling in renal fibroblasts within the TNC-enriched microenvironment is activated. FAK activation leads to ERK1/2 activation,41 which in turn promotes multiple proliferation-related genes’ expression and fibroblast proliferation (Figure 4). Of note, the involvement of integrin signaling in mediating TNC action is not without precedent, as a previous report shows that TNC induces epithelial-mesenchymal transition in breast cancer cells by binding to αvβ1 and αvβ6 integrins.10 Although TNC has been shown to elicit its cellular activities by binding to and activating EGFR,42 we did not observe any stimulation of EGFR phosphorylation and activation by TNC in renal fibroblasts (Figure 4), suggesting a predominant role of the integrin/FAK/MAPK cascade in transducing TNC signaling. It should be pointed out that, although not tested, other signal pathways may also be involved in mediating TNC-induced fibroblast proliferation. In that regard, previous studies have shown that deficiency of TNC protects lung and liver against tissue fibrosis after injury by disrupting TGF-β/Smad signaling.43,44

In summary, we show here that TNC, a matricellular protein that binds cell surface receptors and triggers intracellular signaling, is primarily induced in focal areas of the injured kidney, thereby instigating the formation of a dominant fibrogenic microenvironment. Fibroblasts in this TNC-enriched niche are prompted to proliferate in an autonomous and self-sufficient manner. These studies represent the first attempt to molecularly characterize the components and functional organization of the fibrogenic niche in renal fibrogenesis, and should have wide implications in designing future remedies for the treatment of fibrotic kidney diseases.

Concise Methods

Animal Models

Male C57/BL6 mice weighing about 21–24 g were obtained from Harlan Industries (Indianapolis, IN). UUO was performed as described previously.24 At 7 days after surgery, groups of mice were euthanized, and kidney tissues were collected for subsequent analyses. Renal IRI was performed by using an established protocol as described elsewhere.30 Briefly, unilateral renal pedicles were clamped for 35 minutes using microaneurysm clamps. During the ischemic period, body temperature was maintained between 37°C and 38°C by using a temperature-controlled heating system. At day 9, the contralateral, intact kidney was removed. Mice were euthanized at 10 days after IRI, and kidney tissues collected for various analyses. All animal experiments were performed by procedures approved by the Ethics Committee for Animal Research at the Nanfang Hospital, Southern Medical University, and the Institutional Animal Care and Use Committee at the University of Pittsburgh.

Knockdown of TNC In Vivo by siRNA Strategy

Male C57/BL6 mice weighing 21–24 g were divided into three groups (n=6 in each group): (1) sham-operated mice, (2) UUO mice receiving control siRNA, and (3) UUO mice receiving TNC-specific siRNA. UUO was performed using an established procedure as described.24 Administration of TNC siRNA or control siRNA into mice was performed as described previously.45 Briefly, synthetic TNC or control siRNA (50 μg dissolved in 1 ml of PBS) was rapidly administered (within 10 seconds) into mice via tail-vein injection once a day for 3 consecutive days before UUO surgery and every other day after surgery. Mice were euthanized at 7 days after surgery. Phosphoramidite modified siRNA against TNC and the negative control siRNA was synthesized by Genepharm, Inc. (Sunnyvale, CA). The target sequences used for knockdown of TNC in this study were as follows – sense: 5′- CGC ACA GUG UCU GGA AAU ATT-3′, and antisense: 5′-UAU UUC CAG ACA CUG UGC GTT-3′.

TNC-Coating Procedure

Cell culture plates were incubated with purified recombinant human TNC protein (CC065; Millipore, Billerica, MA) at a concentration of 0.5, 1.0, or 2.0 µg/cm2 for 3 hours at 37°C. They were then washed with PBS, blocked for 1 hour with a solution of heat-denatured BSA (1 mg/ml) and washed again.18 Uncoated wells were used as controls.

Cell Culture and Treatment

NRK-49F cells were obtained from the American Type Culture Collection (Manassas, VA). HKC-8 cells were provided by L. Racusen (Johns Hopkins University, Baltimore, MD). Cells were maintained as described previously.31 Serum-starved NRK-49F cells were treated with various recombinant human proteins such as Shh (SHH-025; StemRD, Inc., Burlingame, CA) and TNC (CC065; Millipore) at different concentrations in the serum-free medium for various periods of time as indicated. In some experiments, cells were pretreated for 30 minutes with various chemical inhibitors at the specified concentrations, followed by incubation with vehicle or TNC for an additional 30 minutes and 3 days, respectively. Control cells were treated with an equal amount of vehicle. Cells were then collected and subjected to various analyses. The chemical inhibitors used in the experiments were as follows: FAK inhibitor PF573228 (3239; Tocris Bioscience, Bristol, UK), Mek1 inhibitor PD98059 (9900L; Cell Signaling Technology, Danvers, MA).

Adenovirus Infection, Plasmid Transfection, and siRNA Inhibition

Infection of renal fibroblasts with recombinant adenovirus harboring constitutively active MEK1 (Ad.Mek1-CA) was performed as described previously.46 NRK-49F cells were infected by 2×107 particles/ml with either control adenovirus (Ad.LacZ) or Ad.Mek1-CA overnight, and then incubated for various periods of time as indicated. The expression of the related genes was assessed by Western blot analysis. In some experiments, NRK-49F cells were transfected with pCMV-TNC expression vector or pcDNA3 empty vector, and then subjected to decellularization protocol for preparing the ECM scaffold. These scaffolds were then used in the subsequent various cell proliferation assays. For knockdown of endogenous TNC expression, NRK-49F cells were transfected with either control siRNA or TNC-specific siRNA (Invitrogen, Carlsbad, CA), as previously reported.47 At 6 hours after transfection, cells were treated with or without Shh (50 ng/ml) for another 48 hours. Whole-cell lysate or the ECM was prepared for Western blot analysis or further experiments.

Cell Proliferation Assay

Cell proliferation was assessed by counting cells and the MTT assay.30 For counting cell numbers, at least five pictures from each well of a 12-well plate were taken and cell numbers in five wells were counted for a sample under the microscope. For the MTT assay, MTT was added at the final concentration of 0.5 mg/ml to individual cultures for 4 hours at the end of experiments. Tetrazolium was released by dimethyl sulfoxide, and the optical density was determined with an ELx800 Absorbance Reader (BioTek Instruments, Winooski, VT) at 570 nm.

BrdU Incorporation Assay

BrdU incorporation assay was performed according to our previous protocols.30 Briefly, cells were seeded onto 24-well plates and treated as indicated, and then they were pulsed with BrdU (10 mM) for 24 hours. At the end of incubation, cells were fixed after removing the labeling medium, and cellular DNA was then denatured. Nonspecific binding was blocked by incubating the cells with 10% donkey serum for 10 minutes at room temperature. Incorporated BrdU was detected with mouse monoclonal anti-BrdU antibody (B2531; Sigma-Aldrich, St. Louis, MO) followed by incubation with cyanine Cy3-conjugated, affinity-purified secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Stained cells were mounted with Vectashield antifade mounting media by using SYTOX-Green to visualize the nuclei. Stained samples were viewed under an Eclipse E600 epifluorescence microscope equipped with a digital camera (Nikon, Tokyo, Japan).

RT-PCR and Real-Time RT-PCR

Total RNA was extracted using the TRIzol RNA isolation system (Invitrogen). First-strand cDNA synthesis was carried out by using a reverse transcription system kit according to the instructions of the manufacturer (Promega, Madison, WI). PCR was carried out using a standard PCR kit and 1 μl aliquot of cDNA HotStar Taq polymerase (Qiagen, Germantown, MD) with specific primer pairs, and 25 cycles for amplification in the linear range were used, as described previously.31 Real-time, quantitative RT-PCR was run with a cycle of 40, performed on an ABI PRISM7000 sequence detection system (Applied Biosystems, Foster City, CA) as described previously.31 The sequences of primer pairs were as follows – TNC sense: 5′-TGAACCACAAGAAATAACCCTC-3′, antisense: 5′-GTTGCTATGGCACTGACTGG-3′; β-actin sense: 5′-CAGCTGAGAGGGAAATCGTG-3′, antisense: 5′- CGTTGCCAATAGTGATGACC-3′. The mRNA levels of various genes were calculated after normalizing with β-actin.

Western Blot Analysis

Protein expression was analyzed by Western blot analysis as described previously.30 The primary antibodies used were as follows: anti-TNC (ab108930; Abcam, Inc., Cambridge, MA), anti–p-MEK1/2 (Ser217/221) (#9121; BioLabs), anti–p-ERK1/2 (Thr202/Tyr204) (#4370S; Cell Signaling Technology), anti–p-EGFR (#2231S), anti-EGFR (#2646S), anti–p-FAK (Y925) (#3284S), anti-FKA (#3285), anti-PCNA (sc-56), anti–c-Myc (sc-764) and anti-actin (sc-1616) (Santa Cruz Biotechnology, Santa Cruz, CA), anti–total-ERK (#M5670), anti–α-tubulin (#T9026) (Sigma-Aldrich), anti–c-fos (#PC05) (Calbiochem, San Diego, CA), and anti-GAPDH (AM4300; Ambion, Austin, TX).

Histology and Immunohistochemical Staining

Paraffin-embedded mouse kidney sections (3 µm thickness) were prepared by a routine procedure. The sections were stained with Masson trichrome staining reagents by standard protocol. Immunohistochemical staining was performed according to the established protocol as described previously.30 The antibodies against TNC (ab6346; Abcam) and PCNA (sc-56; Santa Cruz Biotechnology) were used.

Double Immunofluorescence Staining and Confocal Microscopy

Kidney cryosections were fixed with 3.7% paraformaldehyde for 15 minutes at room temperature and immersed in 0.2% Triton X-100 for 10 minutes. After blocking with 10% donkey serum in PBS for 1 hour, slides were immunostained with the following antibodies: anti-TNC (ab6346; Abcam), anti-vimentin (#5741s; Cell Signaling Technology), anti-Fsp1(A5114; Dako), anti–aquaporin 1 (AB2219; Millipore), and anti-Ki-67 (ab66155; Abcam). To visualize the primary antibodies, slides were stained with cyanine Cy2- or Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). Stained slides were viewed under a Leica TCS-SL confocal microscope equipped with a digital camera. The TNC abundance was assessed in randomly selected fields by counting the positively stained cells. Three to four randomly selected high power field images per animal were used for determining the correlation of TNC abundance with Ki-67.

Preparation of the Fibroblast-Derived ECM Scaffold

Serum-starved NRK-49F cells were treated without or with human recombinant Shh protein at 50 ng/ml for 3 days. Cells were then treated with EGTA (#E3889; Sigma-Aldrich) (5×10−4 M; pH 7.4) in calcium-free PBS, followed by shaking at 4°C for 1 hour. The treatment was repeated 3–4 times until all of the cells were lifted from their underlying matrix. The fibroblast-derived ECM scaffold was washed with PBS and then stored in the refrigerator until the next experiment. Some ECM scaffold was collected by scraping with a rubber policeman in PBS for subsequent Western blot analysis.

Preparation of Kidney Tissue Matrix Scaffold

Kidney tissue matrix scaffold was prepared according to an established protocol with modification.27,28 Briefly, at 7 days after UUO, groups of mice were euthanized, and kidney was arterially perfused in situ using a saline solution to remove the blood. The organ was observed to confirm uniform blanching indicative of even distribution of perfusion and then harvested. Each kidney was cut into 3–4 slices of uniform thickness along the sagittal plane. The kidney slices were then immersed in 100 ml of ultrapure water, followed by shaking for 30 minutes. After changing with fresh ultrapure water, this process was repeated two times. The kidney slices were then placed on the screen of a sieve, and gently massaged using a rubber rolling pin to hasten the lysis of cells. The slices were then incubated with 100 ml of 0.02% trypsin/0.05% EDTA solution in the water bath for 1 hour at 37°C. After straining off the solution using a sieve, the slices were rinsed momentarily under a stream of water, and transferred to a flask with 100 ml of 3% Triton X-100 solution, followed by shaking for 12 hours. After brief washes, the kidney slices were incubated with 100 ml of 4% deoxycholic acid solution on a shaker for 5 hours. The kidney scaffolds were thoroughly rinsed with distilled water for 3–5 minutes to remove as much residual surfactant as possible, and stored in ultrapure water in the refrigerator until further usage.

Cell Culture on Kidney Matrix Scaffolds

Kidney scaffolds were washed three times with sterile PBS, and then transferred into 24-well culture plates. NRK-49F cells were labeled with or without Cell Tracker Green CMFDA (C7025; Invitrogen) for 4 hours and then digested with 0.25% trypsin, and cell suspension containing 5×104 cells in 100 μl was seeded into each kidney scaffold slice. After 3 hours, another 900 μl culture medium was added to each well, and incubated at 37°C in 5% CO2 under 95% humidity. Six days after initial seeding, the scaffolds inoculated with Cell Tracker Green-labeled NRK-49F cells were viewed under an Eclipse E600 epifluorescence microscope equipped with a digital camera (Nikon). Cell proliferation in the scaffolds was determined by MTT assay as described earlier. Cryosections of the kidney scaffolds were made after embedding with Optimal Cutting Temperature (O.C.T. #4583; Sakura Finetek, Inc., Torrance, CA) for further immunofluorescence staining. In a separate experiment, cell mass in the kidney scaffolds was determined by MTT assay and Western blot analyses of housekeeping protein α-tubulin as described earlier.

Scanning Electron Microscopy

Decellularized kidney scaffold samples were freeze-dried, cut along the transverse plane by use of a sharp blade, then loaded onto aluminum studs, coated with gold, and examined under a field emission scanning electron microscope (1530VP; LEO, Germany). Morphologic changes were compared among different groups.

Statistical Analyses

All data were expressed as mean±SEM. Statistical analyses of the data were performed using SigmaStat software (Jandel Scientific Software, San Rafael, CA). Comparisons between groups were made by t test, or using one-way ANOVA followed by the Newman–Keuls test. P<0.05 was considered significant.

Disclosures

None.

Acknowledgments

This work was supported by the National Science Foundation of China grants 81130011 and 81521003, the National Institutes of Health grants DK064005 and DK106049, Guangdong Science Foundation Innovative Group grant 2014A030312014, and Guangzhou Research Fund 15020025. H.F. was supported by the National Science Foundation of China Grant 31371394. R.J.T. was supported by the American Heart Association Fellow-to-Faculty Transition grant 13FTF16990086.

Footnotes

  • H.F. and Y.T. contributed equally to this work.

  • Published online ahead of print. Publication date available at www.jasn.org.

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Tenascin-C Is a Major Component of the Fibrogenic Niche in Kidney Fibrosis
Haiyan Fu, Yuan Tian, Lili Zhou, Dong Zhou, Roderick J. Tan, Donna B. Stolz, Youhua Liu
JASN Mar 2017, 28 (3) 785-801; DOI: 10.1681/ASN.2016020165
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