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. 2019 Jan;95(1):62-74.
doi: 10.1016/j.kint.2018.08.029. Epub 2018 Nov 6.

Tenascin-C protects against acute kidney injury by recruiting Wnt ligands

Shuangqin Chen  1 , Haiyan Fu  1 , Songzhao Wu  1 , Wenjuan Zhu  1 , Jinlin Liao  1 , Xue Hong  1 , Jinhua Miao  1 , Congwei Luo  1 , Yongping Wang  1 , Fan Fan Hou  1 , Lili Zhou  2 , Youhua Liu  3
Affiliations

Tenascin-C protects against acute kidney injury by recruiting Wnt ligands

Shuangqin Chen et al. Kidney Int. 2019 Jan.

Abstract

The development of acute kidney injury (AKI) is a complex process involving tubular, inflammatory, and vascular components, but less is known about the role of the interstitial microenvironment. We have previously shown that the extracellular matrix glycoprotein tenascin-C (TNC) is induced in fibrotic kidneys. In mouse models of AKI induced by ischemia-reperfusion injury (IRI) or cisplatin, TNC was induced de novo in the injured sites and localized to the renal interstitium. The circulating level of TNC protein was also elevated in AKI patients after cardiac surgery. Knockdown of TNC by shRNA in vivo aggravated AKI after ischemic or toxic injury. This effect was associated with reduced renal β-catenin expression, suggesting an impact on Wnt signaling. In vitro, TNC protected tubular epithelial cells against apoptosis and augmented Wnt1-mediated β-catenin activation. Co-immunoprecipitation revealed that TNC physically interacts with Wnt ligands. Furthermore, a TNC-enriched kidney tissue scaffold prepared from IRI mice was able to recruit and concentrate Wnt ligands from the surrounding milieu ex vivo. The ability to recruit Wnt ligands in this ex vivo model diminished after TNC depletion. These studies indicate that TNC is specifically induced at sites of injury and recruits Wnt ligands, thereby creating a favorable microenvironment for tubular repair and regeneration after AKI.

Keywords: Wnt/β-catenin; acute kidney injury; apoptosis; injury repair; tenascin-C; tissue scaffold.

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Conflict of interest statement

DISCLOSURES

All the authors declared no competing interests.

Figures

Figure 1.
Figure 1.. Expression of TNC is induced in animal models of AKI and in humans.
(a, b) Representative micrographs show TNC protein expression in sham and ischemic kidney at 30 hours after IRI. Kidney sections were stained immunohistochemically with specific antibody against TNC. Boxed area was enlarged. Arrow indicates positive staining. Scale bar, 50 μm. (c, d) RT-PCR results show the relative mRNA abundances of TNC at different time points after IRI. Representative RT-PCR results (c) and quantitative data (fold induction over the controls) (d) are presented. *P < 0.05 versus controls (n = 5 to 6). (e, f) RTPCR results show the relative mRNA abundances of TNC at different time points after cisplatin. Representative RT-PCR results (e) and quantitative data (fold induction over the controls) (f) are presented. *P < 0.05 versus controls (n = 5 to 6). (g) The circulating levels of TNC are elevated in human AKI. TNC protein was detected by a specific ELISA in the plasma of normal healthy subjects and patients with mildand severe AKI after cardiac surgery, respectively. **P< 0.01 versus healthy subjects; ††P< 0.01 versusmild AKI. (h) Representative micrographs show the abundance and localization of TNC proteins in healthy control and AKI patients as indicated. Scale bar, 50 μm.
Figure 2.
Figure 2.. Knockdown of TNC deteriorates kidney injury after IRI.
(a) Experimental design. Green arrows indicate the injection of pLVX-shTNC plasmid. Red arrows indicate the timing of IRI surgery. (b) Representative micrographs show renal TNC expression in different groups as indicated. Arrow indicates positive TNC expression. Scale bar, 40 μm. (c) Representative Western blot shows renal TNC protein expression in different groups as indicated. (d) Graphic presentation shows the relative TNC protein levels (fold induction over the controls) in different groups. *P < 0.05 versus controls; †P< 0.05 versus IRI injected with Ctrl-shRNA (n = 5 to 6). (e) Graphic presentation shows serum creatinine levels in different groups as indicated. *P < 0.05 versus controls; †P< 0.05 versus IRI injected with Ctrl-shRNA (n = 5 to 6). (f) Graphic presentation shows blood urea nitrogen (BUN) levels in different groups as indicated. *P < 0.05 versus controls; †P< 0.05 versus IRI injected with Ctrl-shRNA (n = 5 to 6). (g) Representative micrographs show kidney injury in different groups as indicated. Images of PAS staining were shown. Scale bar, 50 μm. (h) Graphic presentation shows the injured tubules per high power field (HPF) in three groups as indicated. *P< 0.05 versus controls; †P< 0.05 versus IRI injected with Ctrl-shRNA (n = 5 to 6).
Figure 3.
Figure 3.. Knockdown of TNC aggravates tubular cell apoptosis and blocks β-catenin activation after IRI.
(a) Representative micrographs show TUNEL-positive cells and cleaved caspase-3 expression in different groups as indicated. Arrows indicate positive staining. Scale bar, 50 μm. (b) Graphic presentation shows the TUNEL-positive cells per high power field (HPF) in different groups as indicated. *P< 0.05 versus controls; †P< 0.05 versus IRI injected with Ctrl-shRNA (n = 5 to 6). (c) Representative Western blot analyses show renal expression of the cleaved caspase-3, Bax, Kim-1 and β-catenin proteins in different groups as indicated. Numbers (1, 2 and 3) indicate each individual animal in a given group. (d-g) Graphic presentations show the relative abundances of renal cleaved caspase-3 (d), Bax (e), Kim-1 (f) and β-catenin (g) proteins in different groups as indicated. *P < 0.05 versus controls; †P< 0.05 versus IRI injected with Ctrl-shRNA (n = 5 to 6). (h) Representative micrographs show β-catenin protein expression in different groups as indicated. Arrow indicates positive staining. Scale bar, 50 μm.
Figure 4.
Figure 4.. Knockdown of TNC aggravates kidney injury in cisplatin-induced AKI.
(a) Knockdown of TNC increases animal mortality rate after cisplatin. *P< 0.05 (n= 25). (b, c) Graphic presentations show serum creatinine (b) and blood urea nitrogen (BUN) (c) levels in different groups as indicated at 36 hours after cisplatin injection. *P < 0.05 versus controls; †P< 0.05 versus Ctrl-shRNA (n = 5 to 6). (d) Representative micrographs show kidney injury in different groups at 36 hours after cisplatin injection. Images of PAS staining were shown. Arrow indicates the injured tubule. Scale bar, 50 μm. (e) Immunohistochemical staining shows cleaved caspase-3 protein expression in different groups as indicated. Arrow indicates positive staining. Scale bar, 50 μm. (f) Graphic presentations show TUNEL-positive cells in different groups as indicated. *P < 0.05 versus controls; †P< 0.05 versus cisplatin injected with CtrlshRNA (n = 5 to 6). (g) Representative Western blot analyses show renal expression of TNC, Kim-1, cleaved caspase-3, Bax and β-catenin expression in different groups as indicated. (h-l) Graphic presentations show the relative levels of TNC (h), Kim-1 (i), cleaved caspase-3 (j), Bax (k) and β-catenin (l) protein expressions in different groups as indicated. *P < 0.05 versus controls; †P< 0.05 versus cisplatin injected with Ctrl-shRNA (n = 5 to 6).
Figure 5.
Figure 5.. TNC protects kidney tubular cells against apoptosis induced by hypoxia/reoxygenation injury in vitro.
(a) Representative FACS analyses show that TNC reduced hypoxia/reoxygenation (H/R)-induced cell apoptosis. HKC-8 cells were pre-incubated with recombinant TNC (50 ng/ml) for 1 hour, followed by incubation in hypoxic condition for 24 hours and then reoxygenation for 2 hours. (b) Graphic presentation shows the percentage of apoptotic cells in different groups as indicated. HKC-8 cells were treated with H/R injury in the absence or presence of TNC. The PE-labeled Annexin V-positive cells were counted by flow cytometry. *P < 0.05 versus controls; †P< 0.05 versus H/R (n = 3). (c) Representative Western blot analyses show protein expression of P53, Bax, FasL, Kim-1 and Parp-1 after various treatments in HKC-8 cells. (d-h) Graphic presentations show the relative abundances of P53 (d), Bax (e), FasL (f), Kim-1 (g) and Parp-1 (h) proteins in different groups as indicated. *P < 0.05 versus controls; †P< 0.05 versus H/R (n = 3).
Figure 6.
Figure 6.. TNC protects kidney tubular cells against apoptosis induced by cisplatin in vitro.
(a) Representative micrographs show TUNEL-positive cells in different groups as indicated. Human kidney proximal tubular epithelial cells (HKC-8) were pre-incubated with recombinant TNC (50 ng/ml) for 1 hour, followed by incubating with cisplatin (25 μg/ml) for 36 hours. Arrows indicate TUNEL-positive apoptotic cells. (b) Graphic presentation shows the percentage of apoptotic cells in different groups as indicated. HKC-8 cells were treated with cisplatin in the absence or presence of TNC. The PE-labeled Annexin V-positive cells were counted by flow cytometry. *P < 0.05 versus controls; †P< 0.05 versus cisplatin alone (n = 3). (c) Representative Western blot analyses show protein expression of cleaved caspase-3, FasL, Bax, P53 and active β-catenin after various treatments in HKC-8 cells. (d-h) Graphic presentations show the relative abundances of cleaved caspase-3 (d), FasL (e), Bax (f), P53 (g) and active β-catenin (h) protein expressions in different groups as indicated. *P < 0.05 versus controls; †P< 0.05 versus cisplatin alone (n = 3). (i) Representative Western blot analyses show that TNC potentiated Wnt3a-mediated down-regulation of P53 and Bax expression induced by cisplatin. (j, k) Graphic presentations show the relative abundances of P53 (j) and Bax (k) protein expressions in different groups as indicated. *P < 0.05 versus controls; †P< 0.05 versus cisplatin alone; #P < 0.05 versus cisplatin plus Wnt3a; ǂ P< 0.05 versus cisplatin plus TNC (n = 3).
Figure 7.
Figure 7.. TNC potentiates Wnt/β-catenin signaling by physically interacting with Wnt ligands.
(a, b) Western blot analyses show that TNC augmented Wnt1-triggered β-catenin activation. HKC-8 cells were transfected with Wnt1 expression vector (pHA-Wnt1) or empty vector in the absence or presence of TNC as indicated. Whole cell lysates were analyzed by Western blotting with specific antibodies against dephosphorylated, active β-catenin, or Wnt1. Representative Western blot (a) and graphic presentation of active β-catenin abundance (b) were presented. *P < 0.05 versus empty vector controls (n = 3); †P< 0.05 versus pHA-Wnt1 alone (n = 3). (c, d) TNC augmented Wnt1-triggered β-catenin nuclear translocation. Nuclear (c) and cytoplasmic (d) proteins were prepared from HKC-8 cells after various treatments as indicated, and immunoblotted with antibodies against β-catenin. Nuclear β-catenin (n-β-catenin) and cytoplasmic β-catenin (c-β-catenin) were normalized with TATA-binding protein (TBP) and α-tubulin, respectively. (e) TNC promotes β-catenin-driven gene transcription. HKC-8 cells were transfected with TOP-Flash reporter plasmid and pHA-Wnt1 plasmid in the absence or presence of TNC. TOP-Flash reporter luciferase activities were assessed, and relative luciferase activity (fold induction) was reported. *P < 0.05 versus empty vector controls (n = 3); †P< 0.05 versus pHA-Wnt1 alone (n = 3). (f-h) Coimmunoprecipitation (Co-IP) demonstrates that TNC bound to Wnt1 and Wnt4 in tubular epithelial cells. HKC-8 cells were transfected with HA-tagged Wnt1 (pHA-Wnt1) or Wnt4 expression vectors (pHA-Wnt4), followed by incubation with TNC. Cell lysates were immunoprecipitated with anti-HA or anti-TNC, followed by immunoblotting with anti-TNC, anti-Wnt1 or anti-Wnt4, respectively.
Figure 8.
Figure 8.. TNC-enriched kidney tissue scaffold (KTS) recruits Wnt ligands.
(a) Decellularized KTS. (b) Micrograph shows TNC-positive region in the IRI-KTS. Arrow indicates TNC-positive region, whereas arrowhead denotes TNC-negative area. CMJ, corticomedullary junction. (c) Experimental design for testing TNC recruiting Wnt ligands. Sham-KTS or IRI-KTS were placed in medium containing Wnt3a or GFPWnt8a fusion protein, and incubated for various periods of time as indicated. (d) TNC-enriched IRI-KTS recruits Wnt3a from surrounding medium. Sham-KTS or IRI-KTS were incubated in the medium containing recombinant Wnt3a for different time periods as indicated, followed by Western blotting with anti-Wnt3a antibody. (e) TNC is required for IRI-KTS recruiting Wnt ligands. KTS was prepared from different groups as indicated, and incubated in the medium containing GFP-Wnt8a fusion protein, then followed by Western blotting. (f) Diagram shows that TNC, acting as a sponge, recruits and concentrates Wnt ligands from surrounding extracellular mellitus. This creates a unique tissue microenvironment in which Wnt ligands are enriched and presented to the responsive cells.
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