Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Apr;29(4):1257-1271.
doi: 10.1681/ASN.2017080903. Epub 2018 Jan 17.

Fibroblast-Specific β-Catenin Signaling Dictates the Outcome of AKI

Dong Zhou  1 , Haiyan Fu  2 , Liangxiang Xiao  2 , Hongyan Mo  3 , Hui Zhuo  3 , Xiaojun Tian  4 , Lin Lin  3 , Jianhua Xing  4 , Youhua Liu  1   2
Affiliations

Fibroblast-Specific β-Catenin Signaling Dictates the Outcome of AKI

Dong Zhou et al. J Am Soc Nephrol. 2018 Apr.

Abstract

AKI is a devastating condition with high morbidity and mortality. The pathologic features of AKI are characterized by tubular injury, inflammation, and vascular impairment. Whether fibroblasts in the renal interstitium have a role in the pathogenesis of AKI is unknown. In this study, we investigated the role of fibroblast-specific β-catenin signaling in dictating the outcome of AKI, using conditional knockout mice in which β-catenin was specifically ablated in fibroblasts (Gli1-β-cat-/-). After ischemia-reperfusion injury (IRI), Gli1-β-cat-/- mice had lower serum creatinine levels and less morphologic injury than Gli1-β-cat+/+ littermate controls. Moreover, we detected fewer apoptotic cells, as well as decreased cytochrome C release; reduced expression of Bax, FasL, and p53; and increased phosphorylation of Akt, in the Gli1-β-cat-/- kidneys. Gli1-β-cat-/- kidneys also exhibited upregulated expression of proliferating cell nuclear antigen and Ki-67, which are markers of cell proliferation. Furthermore, Gli1-β-cat-/- kidneys displayed suppressed NF-κB signaling and cytokine expression and reduced infiltration of inflammatory cells. Notably, loss of β-catenin in fibroblasts induced renal expression of hepatocyte growth factor (HGF) and augmented the tyrosine phosphorylation of c-met receptor after IRI. In vitro, treatment with Wnt ligands or ectopic expression of active β-catenin inhibited HGF mRNA and protein expression and repressed HGF promoter activity. Collectively, these results suggest that fibroblast-specific β-catenin signaling can control tubular injury and repair in AKI by modulating HGF expression. Our studies uncover a previously unrecognized role for interstitial fibroblasts in the pathogenesis of AKI.

Keywords: HGF; Wnt; acute renal failure; apoptosis; fibroblast; signaling.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Pharmacologic inhibition of β-catenin signaling has little effect on the severity of AKI. (A) Experimental design. Cartoon syringes indicate the administration of ICG-001. (B and C) ICG-001 blocks β-catenin activation in the kidney at 1 day after IRI. Kidney lysates were immunoblotted with antibodies against active (dephosphorylated) β-catenin and α-tubulin. (B) Representative western blots and (C) quantitative data are presented. Numbers (1–4) indicate each individual animal in a given group. **P<0.01, *P<0.05 (n=6). (D) qRT-PCR analyses reveal that ICG-001 suppressed PAI-1 mRNA expression at 1 day after IRI. *P<0.05 versus shams, †P<0.05 versus vehicles (n=4–6). (E) Serum creatinine level in the mice with or without ICG-001 at 1 day after IRI, compared with shams. *P<0.05 versus shams (n=4–6). (F) Representative micrographs show morphologic injury and apoptosis at 1 day after IRI. Boxed areas are enlarged and presented. Apoptotic cell death was assessed by TUNEL staining. Arrows indicate apoptotic cells. Scale bar, 100 µm. (G and H) Quantitative data on (G) histologic injury and (H) apoptosis are presented. Data are presented as (G) percentage of injured tubules or (H) numbers of apoptotic cells per high power field (HPF), respectively. *P<0.05 versus shams (n=3). (I–K) Western blot analyses of renal expression of Bax and PCNA proteins at 1 day after IRI. Kidney lysates were immunoblotted with specific antibody against Bax, PCNA, and α-tubulin, respectively. (I) Representative western blot and (J and K) quantitative data are presented. Numbers (1–4) indicate each individual animal in a given group (n=6). Sac, sacrifice.
Figure 2.
Figure 2.
Specific ablation of β-catenin in fibroblasts reduces kidney injury after AKI. (A–C) Using Gli1-LacZ reporter mice determinates the efficacy and specificity of Gli1-driven gene expression after IRI. X-Gal staining shows the Gli1-LacZ+ cell population and distribution at 1 day after IRI. (A) Boxed area was enlarged. Scale bar, 100 µm. (B) Intensive staining was found in the corticomedullary junction region of the kidney. Arrows indicate positive staining. (C) Pie chart shows the relative distribution of the Gli1-LacZ+ fibroblasts in renal interstitium. (D) Schematic diagram depicts the generation of conditional knockout mice with fibroblast-specific deletion of β-catenin by using Cre-LoxP system. The β-catenin–floxed mice (β-catfl/fl) were crossbred with tamoxifen-inducible Cre transgenic mice under the control of endogenous Gli1 promoter/enhancer elements. Black boxes indicate the exons of the β-catenin gene. Orange boxes denote LoxP site. (E) Experimental design. The mice were injected with tamoxifen at 30 mg/kg body wt for 5 consecutive days to activate Cre recombinase in vivo. After wash out for 2 weeks, the mice were subjected to IRI for 1 day. (F) Representative micrographs show kidney morphology in Gli-β-cat−/− and Gli-β-cat+/+ mice. Sections of PAS staining are shown. Scale bar, 50 µm. (G and H) Ablation of β-catenin does not affect fibroblast survival in the kidney in vivo. Immunostaining for Fsp1 shows similar fibroblast density in Gli-β-cat−/− and Gli-β-cat+/+ kidneys under basal conditions. (G) Representative micrographs and (H) quantitative data are presented. Arrows indicate positive Fsp1 staining. Scale bar, 50 µm. (I) Knockdown of β-catenin in vitro does not affect PCNA and Bax expression in renal fibroblasts. NRK-49F cells were transfected with control or β-catenin–specific siRNA for 3 days. Cell lysates were then immunoblotted with antibodies against β-catenin, PCNA, Bax, and α-tubulin, respectively. (J) Serum creatinine level in the Gli1-β-cat+/+ and Gli1-β-cat−/− mice at 1 day after IRI. *P<0.05 (n=9). (K and L) Representative micrographs show the kidneys at 1 day after IRI in the Gli1-β-cat+/+ and Gli1-β-cat−/− mice. Boxed areas were enlarged and presented. Black asterisks in the enlarged boxed areas indicate the injured tubules. Scale bar, 100 µm. (F) Quantitative assessment of injury is presented. *P<0.05 (n=4). (M) qRT-PCR analyses show an increased Klotho mRNA expression in the Gli1-β-cat−/− mice at 1 day after IRI, compared with the Gli1-β-cat+/+ controls. *P<0.05 (n=9). Ctrl, control; PAS, periodic acid–Schiff.
Figure 3.
Figure 3.
Loss of β-catenin in fibroblasts reduces tubular cell apoptosis after AKI. (A) Representative micrographs show apoptotic cells detected by TUNEL staining after IRI at 1 day after IRI. Scale bar, 50 µm. Boxed areas were enlarged. Arrows indicate apoptotic cells. (B) Representative micrographs show renal expression of cytochrome C in the Gli1-β-cat+/+ and Gli1-β-cat−/− mice at 1 day after IRI. Scale bar, 50 µm. The images in the blue channel were shown in the bottom panels. (C and D) Quantitative assessments of apoptotic cells are presented. Data are presented as (C) numbers of apoptotic cells per HPF or (D) area of positive staining of cytochrome C. *P<0.05 (n=4). (E–G) Loss of β-catenin in fibroblasts reduced renal Bax protein expression and activated Akt. (E) Representative western blots and (F and G) quantitative analyses are presented. Numbers (1–4) indicate each individual animal in a given group. **P<0.01, *P<0.05 (n=6). (H–J) Fibroblast-specific ablation of β-catenin suppressed FasL and p53 protein expression. (H) Representative western blots and (I and J) quantitative data are presented. Numbers (1–4) indicate each individual animal in a given group. **P<0.01, *P<0.05 (n=6). Ctrl, control; Cyto C, Cytochrome C
Figure 4.
Figure 4.
Loss of β-catenin in fibroblasts promotes tubular cell proliferation and tubular regeneration after AKI. (A–C) Western blot analyses show that fibroblast-specific ablation of β-catenin promoted renal PCNA and Na+/K+-ATPase expression at 1 day after IRI. (A and B) Representative western blots and (C) quantitative data are presented. Numbers (1–4) indicate each individual animal in a given group. **P<0.01, *P<0.05 (n=6). (D–F) Representative immunohistochemical micrographs in blue channel show (D) Ki-67 and Na+/K+-ATPase protein expression in Gli1-β-cat+/+ and Gli1-β-cat−/− mice at 1 day after IRI. Kidney sections were immunostained with specific antibodies against Ki-67 and Na+/K+-ATPase. Boxed areas are enlarged. Scale bar, 50 µm. Quantitative determination of (E) positive Ki-67 cells and (F) areas of positive Na+/K+-ATPase are presented. *P<0.05 (n=4). Ctrl, control.
Figure 5.
Figure 5.
Loss of β-catenin in fibroblasts attenuates renal inflammation after AKI. (A and B) qRT-PCR demonstrates a decreased mRNA expression of (A) proinflammatory cytokine TNF-α and (B) MCP-1 in Gli1-β-cat−/− kidneys, compared to Gli1-β-cat+/+ controls. *P<0.05 (n=9). (C and D) Western blot analyses show phosphorylated p65 (p-p65) and total p65 expression in the Gli1-β-cat+/+ and Gli1-β-cat−/− mice at 1 day after IRI. Graphic presentations show the p-p65 and p65 protein abundances in different groups. Relative protein levels over the Gli1-β-cat+/+ controls are reported. *P<0.05 (n=6). (E–G) Immunofluorescence staining revealed a decreased infiltration of CD3+ T cells and CD45+ cells in the kidneys at 1 day after IRI. Boxed areas are enlarged. Arrows indicate positive staining. Scale bar, 50 µm. Quantitative data are presented as the percentage of the areas of (F) CD3+ T cells and (G) CD45+ cells. *P<0.05 (n=4). Ctrl, control.
Figure 6.
Figure 6.
Fibroblast-specific ablation of β-catenin promotes HGF/c-met signaling after AKI in vivo. (A) qRT-PCR revealed an increased expression of HGF mRNA in Gli1-β-cat−/− kidneys at 1 day after IRI, compared with the Gli1-β-cat+/+ controls. *P<0.05 (n=6). (B and C) Western blot analyses show an increased HGF receptor, c-met, phosphorylation at Tyr1234/1235 in the Gli1-β-cat−/− kidneys at 1 day after IRI, compared with the Gli1-β-cat+/+ controls. (B) Representative western blot and (C) quantitative data are presented. Numbers (1–4) indicate each individual animal in a given group. *P<0.05 (n=6). (D and E) Representative micrographs show renal localization of phosphorylated c-met in the Gli1-β-cat+/+ and Gli1-β-cat−/− kidneys at 1 day after IRI. The images in the blue channel were shown in the bottom panels. The phosphorylated c-met protein was detected by immunohistochemical staining. Boxed areas are enlarged. Scale bar, 50 µm. (E) Quantitative data are presented as the percentage of the areas of phosphorylated c-met. *P<0.05 (n=4). (F and G) Western blots demonstrate ERK1/2 phosphorylation in the kidneys of the Gli1-β-cat+/+ and Gli1-β-cat−/− mice at 1 day after IRI. (F) Representative western blot and (G) quantitative data are presented. *P<0.05 (n=4). Ctrl, control.
Figure 7.
Figure 7.
Pharmacologic inhibition of β-catenin signaling in Ksp-β-cat mice attenuates kidney injury after AKI. (A) Experimental design. ICG-001 was administrated in conditional knockout mice with tubule-specific ablation of β-catenin 2 days before IRI, and the mice were euthanized at 1 day after IRI. (B) Serum creatinine level in Ksp-β-cat+/+ and Ksp-β-cat−/− mice in the absence or presence of ICG-001 at 1 day after IRI. *P<0.05 (n=8–9). (C) Quantitative assessment of renal injury in Ksp-β-cat+/+ and Ksp-β-cat−/− mice after ICG-001 treatments. Injury score (% of injured tubules) is presented (n=4). (D and E) Representative micrographs showed immunohistochemical staining (blue channel) for Bax in the kidneys at 1 day after IRI. Red color indicates Bax-positive tubules. Scale bar, 50 µm. (E) Quantitative data is presented as the percentage of the areas of Bax staining (n=4). (F and G) Western blot analysis showed little difference in Bax expression between Ksp-β-cat+/+ mice and Ksp-β-cat−/− mice administrated with ICG-001. (F) Representative western blot and (G) quantitative data are presented (n=6). (H and I) Western blot analysis demonstrated Cyclin D1 expression also has no changes between Ksp-β-cat+/+ and Ksp-β-cat−/− mice with ICG-001. (H) Representative western blot and (I) quantitative data are presented (n=6). (J and K) Representative micrographs show renal expression of (J) Cyclin D1 in Ksp-β-cat+/+ and Ksp-β-cat−/− mice in the absence or presence of ICG-001. Scale bar, 50 µm. (K) Quantitative data is presented as positive cell number of Cyclin D1 per HPF. *P<0.05; †P<0.05 (n=4). Sac, Sacrifice.
Figure 8.
Figure 8.
Wnt/β-catenin suppresses HGF expression in vitro. (A) RT-PCR analyses show that Wnt-enriched conditioned medium suppressed HGF gene expression in cultured NRK-49F fibroblasts, whereas blockade of Wnt/β-catenin signaling by ICG-001 restored, at least partially, HGF expression in vitro. (B) Western blot analyses reveal that Wnt-enriched conditioned medium inhibited HGF protein expression in NRK-49F cells in vitro. (C) RT-PCR analyses show that recombinant Wnt3a and Wnt5a repressed HGF gene expression in cultured NRK-49F fibroblasts in vitro. (D) Western blot analyses demonstrate that constitutive activation of β-catenin also inhibited HGF expression in human cervical cancer cells (C-33A). C-33A cells were transfected with N-terminally truncated, stabilized β-catenin (pDel-β-cat) or empty vector (pcDNA3), respectively. (E) Expression of stabilized β-catenin suppressed HGF gene transcription in luciferase assay. NRK-49F cells were cotransfected with the pGL3-HGF promoter reporter plasmid (HGF promoter region, −1037 to +56) or pGL3-Basic vector, and N-terminally truncated, stabilized β-catenin expression vector (pDel-β-cat). Luciferase activity was assessed and reported as fold induction over the controls. **P<0.01 versus pcDNA3 control (n=3). (F) Schematic diagram depicts the potential mechanisms accounting for fibroblast-specific β-catenin ablation protecting against tubular injury after AKI by promoting HGF signaling. Ctrl, control.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Vanmassenhove J, Kielstein J, Jörres A, Biesen WV: Management of patients at risk of acute kidney injury. Lancet 389: 2139–2151, 2017 - PubMed
    1. Odutayo A, Wong CX, Farkouh M, Altman DG, Hopewell S, Emdin CA, et al. .: AKI and long-term risk for cardiovascular events and mortality. J Am Soc Nephrol 28: 377–387, 2017 - PMC - PubMed
    1. Xu X, Nie S, Liu Z, Chen C, Xu G, Zha Y, et al. .: Epidemiology and clinical correlates of AKI in Chinese hospitalized adults. Clin J Am Soc Nephrol 10: 1510–1518, 2015 - PMC - PubMed
    1. Bonventre JV, Yang L: Cellular pathophysiology of ischemic acute kidney injury. J Clin Invest 121: 4210–4221, 2011 - PMC - PubMed
    1. Yang L, Humphreys BD, Bonventre JV: Pathophysiology of acute kidney injury to chronic kidney disease: Maladaptive repair. Contrib Nephrol 174: 149–155, 2011 - PubMed

Publication types

MeSH terms

LinkOut - more resources