Sonic hedgehog is a novel tubule-derived growth factor for interstitial fibroblasts after kidney injury - PubMed (original) (raw)

Sonic hedgehog is a novel tubule-derived growth factor for interstitial fibroblasts after kidney injury

Dong Zhou et al. J Am Soc Nephrol. 2014 Oct.

Abstract

Tubular epithelium constitutes the majority of the renal parenchyma and is the primary target of various kidney injuries. However, how the injured tubules drive interstitial fibroblast activation and proliferation remains poorly understood. Here, we investigated the role of sonic hedgehog (Shh), a secreted extracellular signaling protein, in fibroblast proliferation. Shh was induced in renal tubular epithelia in animal models of CKD induced by ischemia/reperfusion injury (IRI), adriamycin, or renal mass ablation, and in renal tubules of kidney biopsy specimens from CKD patients with different etiologies. Using Gli1-CreER(T2) reporter mice, we identified interstitial fibroblasts as the principal targets of renal Shh signaling in vivo. In vitro, incubation with Shh promoted normal rat kidney fibroblast proliferation, which was assessed by cell counting, MTT assay, and BrdU incorporation assay, and stimulated the induction of numerous proliferation-related genes. However, Shh had no effect on the proliferation of renal tubular epithelial cells. In vivo, overexpression of Shh promoted fibroblast expansion and aggravated kidney fibrotic lesions after IRI. Correspondingly, blockade of Shh signaling by cyclopamine, a small molecule inhibitor of Smoothened, inhibited fibroblast proliferation, reduced myofibroblast accumulation, and attenuated renal fibrosis. These studies identify Shh as a novel, specific, and potent tubule-derived growth factor that promotes interstitial fibroblast proliferation and activation. Our data also suggest that blockade of Shh signaling is a plausible strategy for therapeutic intervention of renal fibrosis.

Keywords: cell proliferation; chronic renal disease; fibroblast; renal fibrosis; sonic hedgehog.

Copyright © 2014 by the American Society of Nephrology.

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Figures

Figure 1.

Figure 1.

Induction of Shh is a common finding in animal models of CKD. (A) qRT-PCR analyses show a significant induction of Shh mRNA expression in the fibrotic kidneys after renal IRI for 10 days. *P<0.05 versus sham controls (_n_=4–5). (B and C) Western blot analyses of renal expression of Shh protein in sham and injured kidneys at 10 days after IRI. Representative (B) Western blot and (C) quantitative data are presented. Numbers (1–3) indicate each individual animal in a given group. *P<0.05 versus sham controls (_n_=4–5). (D) Representative immunohistochemical staining shows an increased expression of Shh in mouse models of IRI (10 days) and ADR nephropathy (5 weeks). Boxed areas are enlarged in the lower panel. Arrows indicate Shh-positive tubules. (E) Renal Shh mRNA expression in ADR nephropathy. Shh mRNA expression was assessed in the kidneys at 5 weeks after ADR injection. *P<0.05 versus controls (_n_=5). (F and G) Western blot analyses of renal Shh protein at 5 weeks after ADR injection. Representative (F) Western blot and (G) quantitative data are presented. Numbers (1–5) indicate each individual animal in a given group. *P<0.05 versus controls (_n_=5). (H and I) Representative immunohistochemical staining showed induction of Shh protein in rat remnant kidney model after 5/6 nephrectomy at 12 weeks. Scale bar, 50 _µ_m. (J) Identification of the interstitial fibroblasts as hedgehog-responding cells in fibrotic kidneys. Transgenic Gli1-CreERT2 mice were subjected to IRI for 10 days, and kidneys were subjected to double immunostaining for Cre recombinase (red) and various cell type-specific markers (green). Arrows, CD45- or CD31-positive cells; arrowheads, Cre-positive cells; wide arrows, cells with positive staining for both vementin and Cre. Scale bar, 20 _µ_m. (K) Diagram shows that tubule-derived Shh specifically targets interstitial fibroblasts after kidney injury. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 2.

Figure 2.

Shh is induced specifically in renal tubular epithelium in human CKD. Human kidney biopsies samples were immunohistochemically stained with specific antibody against Shh protein. Representative micrographs show the expression and localization of Shh protein in human CKD. Nontumor kidney tissue from the patients who had renal cell carcinoma and underwent nephrectomy was used as normal controls. Boxed areas are enlarged in the right panels. Arrows indicate Shh-positive tubules. Scale bar, 50 _μ_m.

Figure 3.

Figure 3.

Shh selectively promotes fibroblast cell proliferation in vitro. (A) Representative micrographs show the phase-contrast images of renal interstitial fibroblasts after incubation with recombinant Shh. NRK-49Fs were incubated for 3 days with different concentrations of Shh as indicated. (B and C) Shh promotes fibroblast proliferation in a dose- and time-dependent manner. NRK-49F cells were incubated with (B) different concentrations of Shh for 3 days or (C) a fixed dose of Shh (100 ng/ml) for various periods of time as indicated. Cell numbers (×1000 per well) were counted and presented. *P<0.05 versus controls (_n_=3). (D) Graphic presentation shows that Shh promotes NRK-49F cell proliferation assessed by a colorimetric MTT assay. *P<0.05 versus controls (_n_=3). (E) Representative micrographs show that Shh promotes fibroblast DNA synthesis as shown by BrdU incorporation. NRK-49F cells were incubated with 50 and 100 ng/ml Shh for 2 days. Cells were immunostained with mouse anti-BrdU antibody (red). SYTO-Green (green) was used to visualize the nuclei. Arrows indicate BrdU-positive cells. (F) Quantitative determination of the percentage of BrdU-positive cells after Shh treatment. *P<0.05 versus controls (_n_=3). (G) Ptch1 is expressed in renal fibroblasts and induced by Shh. NRK-49 F cells were treated with different concentrations of Shh for 24 hours or 50 ng/ml Shh for various periods of time as indicated. (H–J) Shh does not promote renal proximal tubular cell proliferation. HKC-8 cells were incubated with (H) 100 ng/ml Shh for various periods of time or (I and J) different concentrations of Shh for 3 days. Cell proliferation was assessed by (H and I) cell number counting or (J) a colorimetric MTT assay. (K) Western blots show that Shh promotes the expression of numerous proliferation-related genes in fibroblasts. NRK-49F cells were incubated with Shh (50 ng/ml) for various periods of time as indicated. Cell lysates were subjected to Western blot analyses for c-myc, c-fos, PCNA, and actin.

Figure 4.

Figure 4.

Shh is expressed in renal tubular epithelium in vivo after hydrodynamic-based gene transfer. (A) Experimental design. Red arrows indicate the time point of renal IRI. White and red arrowheads indicate the time points when pcDNA3 or pFlag-Shh was injected, respectively. (B) RT-PCR shows Shh and Gli1 mRNA expression in the kidneys at 7 days after single plasmid injection. (C and D) Western blot analyses of renal Shh protein expression at 7 days after plasmid injection. Kidney lysates were immunoblotted with antibodies against Shh, Flag, or _α_-tubulin. Representative (C) Western blot and (D) quantitative data are presented. *P<0.05 versus pcDNA3. Numbers (1–4) indicate each individual animal in a given group. (E) Representative micrographs show renal Shh protein expression and localization at 7 days after plasmid injection. Boxed areas are enlarged in the right panels. Scale bar, 50 _μ_m. (F) Representative Western blots show renal Gli1 protein expression at 7 days after plasmid injection.

Figure 5.

Figure 5.

Overexpression of exogenous Shh in vivo promotes interstitial cell proliferation and accelerates the progression of renal fibrosis after AKI. (A) Representative micrographs show immunohistochemical staining for Ki67 at 7 days after plasmid injection. Boxed areas are enlarged in the right panels. Yellow arrows indicate Ki67-positive cells. Scale bar, 50 _μ_m. (B) Quantitative determination of the Ki67-positive cells in renal tubular and interstitial compartments. *P<0.05 versus pcDNA3. HPF, high power field. (C and D) qRT-PCR analyses show that overexpression of Shh in vivo promoted renal expression of (C) collagen I and (D) collagen III after IRI. *P<0.05 versus pcDNA3 (_n_=4–6). (E) Western blot analyses of renal expression of various fibrosis-related genes. Kidney lysates were immunoblotted with specific antibodies against PCNA, PDGFR-β, desmin, fibronectin, _α_-SMA, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Numbers (1–4) indicate each individual animal in a given group. (F and G) Graphic presentation of renal protein levels of various fibrosis-related genes. Data are presented as fold induction over pcDNA3 controls. *P<0.05 versus pcDNA3; **P<0.01 versus pcDNA3. (H) Representative micrographs show collagen deposition in the kidneys at 7 days after plasmid injection. Paraffin sections were subjected to MTS. Boxed areas are enlarged in the right panels. Arrows indicate collagen-deposited area with blue staining. Scale bar, 150 _μ_m. (I) Graphical presentation shows kidney fibrotic lesions at 7 days after plasmid injection after quantitative determination. *P<0.05 versus pcDNA3.

Figure 6.

Figure 6.

Blockade of Shh signaling reduces renal fibrosis after IRI. (A) Experimental design. Green arrowheads indicate the injection of CPN, whereas white arrowheads indicate vehicle injection. (B) qRT-PCR analyses show that CPN inhibited renal Gli1 mRNA expression. *P<0.05 versus sham controls; †P<0.05 versus vehicles (_n_=3). (C) Representative Western blots show renal Gli1 protein expression at different groups as indicated. Kidney lysates were immunoblotted with antibodies against Gli1 and actin. Veh, vehicle. (D) Representative micrographs show that CPN ameliorated renal fibrotic lesions after IRI. Kidney sections were subjected to MTS. Boxed areas are enlarged and presented in lower panels. Arrows indicate fibrotic areas with blue staining. Scale bar, 50 μ_m. (E) Quantitative determination of renal fibrotic lesions in different groups. Renal fibrotic lesions (defined as percentage of MTS-positive fibrotic area) were quantified by computer-aided morphometric analyses. †_P<0.05 versus vehicles. (F–I) qRT-PCR analyses show that CPN inhibited renal expression of (F) collagen I, (G) collagen III, (H) fibronectin, and (I) α_-SMA after IRI. *P<0.05 versus sham controls; †_P<0.05 versus vehicles (_n_=3–5).

Figure 7.

Figure 7.

Blockade of Shh signaling selectively inhibits renal interstitial fibroblast proliferation. (A and B) Western blot analyses show that CPN inhibited renal PCNA expression at 10 days after IRI. Representative (A) Western blot and (B) quantitative data are presented. *P<0.05 versus vehicle. Numbers (1–3) indicate each individual animal in a given group. (C) Representative micrographs show that CPN inhibited renal interstitial cells proliferation after IRI. Double immunofluorescence staining for Ki67 (red) and laminin (green) shows that Ki67-positive cells were predominantly localized in the interstitial compartment. Boxed areas are enlarged and presented in right panels. Arrows indicate Ki67-positive cells in the interstitium, whereas arrowheads denote tubular cells with Ki67-positive staining. Scale bar, 50 _μ_m. (D and E) CPN selectively inhibits interstitial but not tubular cell proliferation in vivo after IRI. Quantitative data on Ki67-positive cells in the (D) interstitial and (E) tubular compartments are presented. *P<0.05 versus vehicle. (F and G) Double immunofluorescence staining shows the costaining of Ki67 (red) and different cell markers (green) in the kidneys at 10 days after IRI. Cell type-specific markers used are CD31 (endothelial cells), CD45 (leukocytes), and _α_-SMA (myofibroblasts). Quantitative data on costaining for Ki67 and different cell type-specific markers are presented in G. DAPI, 4′,6-diamidino-2-phenylindole. (H–J) Western blot analyses of renal PDGFR-β, desmin, fibronectin. and _α_-SMA expression at 10 days after IRI. Representative (H) Western blot and (I and J) quantitative data are presented. *P<0.05 versus vehicle. Numbers (1–3) indicate each individual animal in a given group. Fn, fibronectin. (K) Representative micrographs of immunohistochemical staining show that CPN inhibited renal PDGFR-β and desmin expression after IRI. Arrows indicate positive cells. Scale bar, 50 _μ_m. HPF, high power field.

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