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. 2012 May;23(5):801-13.
doi: 10.1681/ASN.2011060614. Epub 2012 Feb 2.

Sonic hedgehog signaling mediates epithelial-mesenchymal communication and promotes renal fibrosis

Hong Ding  1 , Dong Zhou, Sha Hao, Lili Zhou, Weichun He, Jing Nie, Fan Fan Hou, Youhua Liu
Affiliations

Sonic hedgehog signaling mediates epithelial-mesenchymal communication and promotes renal fibrosis

Hong Ding et al. J Am Soc Nephrol. 2012 May.

Abstract

Sonic hedgehog (Shh) signaling is a developmental signal cascade that plays an essential role in regulating embryogenesis and tissue homeostasis. Here, we investigated the potential role of Shh signaling in renal interstitial fibrogenesis. Ureteral obstruction induced Shh, predominantly in the renal tubular epithelium of the fibrotic kidneys. Using Gli1(lacZ) knock-in mice, we identified renal interstitial fibroblasts as Shh-responding cells. In cultured renal fibroblasts, recombinant Shh protein activated Gli1 and induced α-smooth muscle actin (α-SMA), desmin, fibronectin, and collagen I expression, suggesting that Shh signaling promotes myofibroblast activation and matrix production. Blockade of Shh signaling with cyclopamine abolished the Shh-mediated induction of Gli1, Snail1, α-SMA, fibronectin, and collagen I. In vivo, the kidneys of Gli1-deficient mice were protected against the development of interstitial fibrosis after obstructive injury. In wild-type mice, cyclopamine did not affect renal Shh expression but did inhibit induction of Gli1, Snail1, and α-SMA. In addition, cyclopamine reduced matrix expression and mitigated fibrotic lesions. These results suggest that tubule-derived Shh mediates epithelial-mesenchymal communication by targeting interstitial fibroblasts after kidney injury. We conclude that Shh/Gli1 signaling plays a critical role in promoting fibroblast activation, production of extracellular matrix, and development of renal interstitial fibrosis.

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Figures

Figure 1.
Figure 1.
Shh is specifically induced in renal tubular epithelium after obstructive injury. (A) Representative RT-PCR analyses show the expression of Shh mRNA in sham control and the obstructed kidneys after UUO. CD-1 mice were subjected to UUO, and kidney tissues were collected at different time points after surgery as indicated. Numbers (1, 2, and 3) indicate each individual animal in a given group. (B) mRNA levels of Shh in the obstructed kidneys at different time points after UUO. Relative Shh mRNA levels were expressed as fold induction over sham controls after normalization with glyceraldehyde 3-phosphate dehydrogenase, respectively. *P<0.05 versus sham controls (n=5). (C and D) Western blot analyses demonstrate Shh protein induction in obstructive nephropathy. Western blot of the kidney lysates at 7 days after UUO (C) and graphic presentation (D) are shown. **P<0.01 versus sham controls (n=4). (E) Immunohistochemical staining shows the induction and localization of Shh protein in obstructive nephropathy. Kidney sections from sham and UUO mice (at 7 days) were stained with specific anti-Shh antibody. Arrows indicate positive staining. Scale bar, 35 µm. (F) Double immunofluorescence staining for Shh and aquaporin 1 (AQP1) shows that Shh was induced predominantly in renal tubular epithelium. Kidney cryosections were stained for Shh (red) and AQP1 (green), a proximal tubular marker. Arrows indicate positive staining. (G) Little Shh staining was found in the activated interstitial fibroblasts in obstructive nephropathy. Cryosections from the obstructed kidneys at 7 days after UUO were stained for Shh (red) and α-SMA (green). Arrows indicate Shh-positive tubules.
Figure 2.
Figure 2.
Gli1 is specifically activated in renal interstitial fibroblasts after obstructive injury. (A and B) Representative RT-PCR analyses (A) and graphic presentation (B) show renal Gli1 mRNA induction at different time points after UUO. Numbers (1, 2, and 3) indicate each individual animal in a given group. Relative Gli1 mRNA levels were expressed as fold induction over sham controls after normalization with glyceraldehyde 3-phosphate dehydrogenase, respectively. **P<0.01 versus sham controls (n=5). (C and D) Western blot analyses (C) and graphic presentation (D) show the induction of renal Gli1 protein at 7 days after UUO. **P<0.01 versus sham controls (n=3). (E) Diagram shows the construction of Gli1-LacZ knock-in mice. (F and G) Immunohistochemical staining for β-galactosidase shows a marked induction of the Gli1 promoter/enhancer-driven β-Gal expression in the obstructed kidney at 7 days after UUO (G) compared with the controls (F). Boxed areas were enlarged, in which β-Gal–positive cells were indicated by arrows. (H and I) Histochemical staining for β-galactosidase activity demonstrates a marked induction of the Gli1 promoter/enhancer-driven β-Gal expression in the lacZ knock-in mice after obstructive injury. Open arrowhead in panel (H, enlarged box) indicates nonspecific β-Gal activity staining in renal tubules. (J) Quantitative determination of the Gli1-driven β-Gal–positive cells in the lacZ knock-in mice after obstructive injury. The β-Gal–positive cells per high-power field (HPF) were counted in the obstructed kidney at 7 days after UUO. **P<0.01 versus controls (n=3). (K) Double immunostaining demonstrates a selective activation of Gli1 signaling in the interstitial fibroblasts in obstructed kidneys after injury. Kidney sections were co-stained for β-Gal (red) and α-SMA (green), respectively. Arrows indicate Gli1 promoter/enhancer-driven β-Gal expression in α-SMA–positive myofibroblasts.
Figure 3.
Figure 3.
Shh signaling promotes fibroblast activation and matrix production. (A and B) RT-PCR demonstrates that recombinant Shh activated Gli1 and promoted matrix expression in NRK-49F cells in a time-dependent (A) and dose-dependent (B) manner. NRK-49F cells were incubated with Shh, 50 ng/ml, for various periods of time (A) or different concentrations of Shh for 48 hours (B) as indicated. (C) Shh activates Gli1 and induces matrix expression in renal interstitial fibroblasts in vitro. NRK-49F cells were incubated for 48 hours with different concentrations of Shh as indicated. Cell lysates were immunoblotted with specific antibodies against Gli1, α-SMA, fibronectin, collagen I, desmin, and actin, respectively. (D and E) Quantitative determination of protein expression levels in NRK-49F fibroblasts after Shh stimulation. Relative levels (fold induction over the controls) of α-SMA and desmin (D) and collagen I and fibronectin (E) are presented. FN, fibronectin. *P<0.05 versus controls (n=3). (F) Immunofluorescence staining shows that Shh induced α-SMA, fibronectin, and type I collagen protein expression in NRK-49F cells. Cells were incubated with different concentrations of Shh as indicated for 48 hours.
Figure 4.
Figure 4.
Knockout of Gli1 gene attenuates renal interstitial fibrosis in obstructive nephropathy. (A–D) Quantitative, real-time RT-PCR analyses show a significant suppression of various fibrosis-related genes in Gli1 null mice (Gli1−/−), compared with WT controls (Gli1+/+). Both Gli1−/− and Gli1+/+ were on 129S1/SvImJ background. Renal mRNA levels of type I collagen (A), fibronectin (B), α-SMA (C), and connective tissue growth factor (CTGF) (D) were determined and compared between WT and Gli1 knockout (KO) mice at 7 days after UUO. Relative mRNA levels are reported after normalization with β-actin. *P<0.05, **P<0.01 versus WT controls (n=6). (E and F) Representative micrographs (E) and graphic presentation (F) illustrate a reduced protein expression of type I collagen, fibronectin, and α-SMA in the obstructed kidneys of Gli1 null mice at 7 days after UUO. Kidney sections from WT and knockout mice were immunostained with specific antibodies against collagen I, fibronectin, and α-SMA, respectively. Scale bar, 50 μm. **P<0.01 versus WT controls. (G and H) Knockout of Gli1 gene reduces renal interstitial fibrosis after obstructive injury. MTS revealed a reduced collagen deposition in the obstructed kidney of Gli1 null mice, compared with WT controls. Representative micrographs of MTS (G) and quantitative determination of the relative fibrotic area in knockout and WT mice at 7 days after UUO (H) are presented. **P<0.01 versus WT controls (n=6). Scale bar, 50 μm.
Figure 5.
Figure 5.
Inhibition of Shh/Gli1 signaling prevents fibroblast activation and matrix production in vitro. (A and B) CPN inhibits Shh-induced Gli1 expression in renal interstitial fibroblasts. NRK-49F cells were treated with Shh (50 ng/ml) in the absence or presence of different concentrations of CPN for 24 hours as indicated. Representative RT-PCR (A) and quantitative determination (B) demonstrate that CPN blocked Shh-induced Gli1 expression in NRK-49F cells. **P<0.01 versus controls; ††P<0.01 versus Shh alone (n=3). (C–G) Quantitative, real-time RT-PCR shows the relative mRNA levels of Snail1 (C), type I collagen (D), desmin (E), fibronectin (F), and α-SMA (G) after various treatments as indicated. NRK-49F cells were treated with Shh (50 ng/ml) in the absence or presence of different concentrations of CPN for 24 hours as indicated. Relative mRNA levels (fold induction over the controls) are presented. **P<0.01, *P<0.05 versus controls; ††P<0.01, †P<0.05 versus Shh alone (n=3). (H) Immunofluorescence staining demonstrates that CPN suppressed α-SMA and type I collagen expression induced by Shh in NRK-49F cells. Cells were treated with Shh (50 ng/ml) for 48 hours in the absence or presence of different concentrations of CPN as indicated.
Figure 6.
Figure 6.
Inhibition of Shh signaling by CPN suppresses Gli1 and Snail1 expression in vivo. (A and B) Representative RT-PCR analyses show that CPN inhibited renal mRNA expression of Gli1 (B), but not Shh (A), in CD-1 mice at 7 days after UUO. Numbers (1, 2, and 3) indicate each individual animal in a given group. GADPH, glyceraldehyde 3-phosphate dehydrogenase. (C and D) Graphic presentation shows the mRNA levels of Shh (C) and Gli1 (D) in the obstructed kidneys in various groups as indicated. Relative Shh and Gli1 mRNA levels were expressed as fold induction over sham controls after normalization with β-actin, respectively. **P<0.01 versus sham controls; †P<0.05 versus vehicle controls (n=5). (E) Quantitative, real-time RT-PCR demonstrates that CPN suppressed renal Snail1 mRNA expression after obstructive injury. **P<0.01 versus sham controls; ††P<0.01 versus vehicle controls (n=5).
Figure 7.
Figure 7.
Inhibition of Shh/Gli1 signaling represses matrix gene expression and reduces renal interstitial fibrosis. (A–C) Quantitative, real-time RT-PCR analyses demonstrate that CPN suppressed renal type I collagen, fibronectin, and α-SMA mRNA expression in CD-1 mice. Renal mRNA levels of type I collagen (A), fibronectin (B), and α-SMA (C) were determined at 7 days after UUO. Relative mRNA levels are reported after normalization with β-actin. **P<0.01 versus sham controls; †P<0.05, ††P<0.01 versus vehicle controls (n=5). (D–F) Representative micrographs (D) and graphic presentation (E and F) illustrate reduced protein expression of type I collagen, α-SMA, and fibronectin in the obstructed kidneys at 7 days after UUO. Scale bar, 50 μm. **P<0.01 versus sham controls; †P<0.05 versus vehicle controls (n=5). Col I, collagen I; Fn, fibronectin. (G and H) inhibition of Shh/Gli1 signaling reduces renal interstitial fibrosis after obstructive injury. Representative micrographs of MTS (G) and quantitative determination of the relative fibrotic area in different groups (H) are presented. **P<0.01 versus sham controls; †P<0.05 versus vehicle controls (n=5).
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