Sonic Hedgehog Signaling Mediates Epithelial Mesenchymal Communication and Promotes Renal Fibrosis

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1 Sonic Hedgehog Signaling Mediates Epithelial Mesenchymal Communication and Promotes Renal Fibrosis Hong Ding,* Dong Zhou,* Sha Hao,* Lili Zhou,* Weichun He,* Jing Nie, Fan Fan Hou, and Youhua Liu* *Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; Department of Medicine, First Affiliated Hospital, China Medical University, Shenyang, China; and Division of Nephrology, Nanfang Hospital, Southern Medical University, and Guangdong Provincial Institute of Nephrology, Guangzhou, China 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 Shhresponding cells. In cultured renal fibroblasts, recombinant Shh protein activated Gli1 and induced a-smoothmuscleactin(a-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, a-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 a-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 acriticalroleinpromotingfibroblast activation, production of extracellular matrix, and development of renal interstitial fibrosis. J Am Soc Nephrol 23: , doi: /ASN Hedgehog signaling is an evolutionarily conserved, developmental signal cascade that regulates a diverse array of biologic processes, such as embryonic development, tissue homeostasis, injury repair, and tumorigenesis. 1,2 Hedgehog transmits its signaling through binding to the plasma membrane receptor, Patched 1 (Ptch1). This leads to Ptch1 internalization and degradation, resulting in the de-repression of Smoothened (Smo). Activated Smo then moves from an intracellular vesicle to the cell membrane, where it activates the Gli family of transcription factors. Although the detailed molecular events that connect Smo to Gli activation remain poorly understood, activated Gli proteins are known to enter the nucleus and initiate the transcription of their target genes. 3 Direct targets of hedgehog signaling include several major components in its own pathway, such as Gli1, Ptch1, and hedgehog-interacting protein 1 (Hhip1), thereby providing both positive and negative feedback to ensure the delicate regulation of this system. Hedgehog signaling also controls, either directly or indirectly, many target genes involved in cell proliferation, cell-fate determination, and tissue homeostasis. 4 Received June 24, Accepted December 14, Published online ahead of print. Publication date available at. Correspondence: Dr. Youhua Liu, Department of Pathology, University of Pittsburgh School of Medicine, S-405 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA liuy@upmc.edu. Copyright 2012 by the American Society of Nephrology J Am Soc Nephrol 23: , 2012 ISSN : /

2 Among the three vertebrate hedgehog ligands, sonic hedgehog (Shh) is the most widely studied and best characterized. Synthesized as a precursor, Shh undergoes autocatalytic processing and post-translational modifications, which are important for its proper trafficking, secretion, and receptor interaction. Secreted Shh exercises decisive roles in development, acting as a morphogen involved in patterning many systems. 5 7 Shh is also an important regulator of adult stem cells from various tissues. 8 Not surprisingly, gene mutations and aberrant regulation of various components of Shh signaling lead to severe developmental abnormalities. 9,10 Likewise, dysregulated activation of hedgehog signaling has been implicated in the development of cancers in a variety of organs, including brain, lung, mammary gland, and skin. 11,12 Recent studies also implicate Shh signaling in regulating injury repair and wound healing after tissue damage. 13,14 Shh expression is upregulated in chronic liver and lung diseases, and it promotes epithelial to mesenchymal transition during biliary fibrosis. 13,15,16 However, whether hedgehog signaling plays a role in the pathogenesis of renal fibrosis, or kidney diseases in general, is completely unknown. Renal fibrosis, particularly tubulointerstitial fibrosis, is the common final outcome of a wide variety of progressive CKD, regardless of the initial causes Emerging evidence suggests that reactivation and dysregulation of key developmental signaling play a critical role in the pathogenesis of chronic tissue destruction and progressive loss of kidney function. 20,21 For instance, aberrant activation of Wnt/b-catenin signaling is implicated in the development and progression of renal fibrotic lesions in a variety of CKDs. 20,22 25 Similarly, Notch activation is also linked to renal fibrogenesis. 26 These findings suggest that the cellular and molecular responses in the injured kidneys largely recapitulate the evolutionarily conserved developmental programs in an attempt to repair and recover from initial damage. Because hedgehog signaling is an essential developmental pathway in regulating nephron formation and kidney development, 7,9 we hypothesized that aberrant activation of this signaling probably plays a role in renal fibrogenesis, a process often considered as a result of failed wound healing after injury. 17 In this study, we demonstrate that Shh is induced in renal tubular epithelium and specifically targets to interstitial fibroblasts in a mouse model of obstructive nephropathy. Genetic ablation or pharmacologic inhibition of this signaling attenuates matrix gene expression and mitigates renal fibrotic lesions. These results establish a critical role for Shh signaling in the pathogenesis of renal fibrosis. RESULTS Shh Is Induced Specifically in Renal Tubular Epithelium after Obstructive Injury To investigate the potential role of Shh signaling in regulating renal fibrosis, we first examined the expression of Shh in obstructive nephropathy, a classic model of renal interstitial fibrosis characterized by myofibroblast activation, tubular atrophy, and excessive matrix production. 27,28 As shown in Figure 1A, Shh mrna was markedly induced in the obstructed kidneys in a time-dependent manner. Quantitative determination revealed more than seven-fold induction of renal Shh mrna at 14 days after unilateral ureteral obstruction (UUO), compared with the sham controls (Figure 1C). Similarly, Shh protein was also induced in the obstructed kidneys at 7 days after UUO, as demonstrated by Western blot analyses of kidney lysates (Figure 1, C and D). We also examined the Shh protein expression in the fibrotic kidneys after obstructive injury by immunohistochemical staining. As shown in Figure 1E, little Shh protein was detected in sham-operated, control kidneys; however, a marked induction of Shh protein expression was observed in the fibrotic kidneys at 7 days after obstructive injury. Shh protein was predominantly localized in renal tubular epithelium (Figure 1E, arrows), whereas interstitial cells in the expanded interstitium were mostly negative for Shh staining. To further confirm the cell type specific expression pattern of Shh protein, we carried out double immunostaining for Shh and cell type specific markers. As shown in Figure 1F, Shh (red) was colocalized with aquaporin 1 (green), a marker for proximal tubular epithelium. In contrast, little co-staining for Shh (red) and a-smooth muscle actin (SMA) (green), a marker for activated interstitial fibroblasts, was observed (Figure 1G). These results indicate a specific induction of Shh protein in renal tubular epithelium in the pathogenesis of renal fibrosis. Shh Signaling Targets Renal Interstitial Fibroblasts in Injured Kidneys We next investigated the activation of Gli1, the principal downstream target and mediator of Shh signaling. As shown in Figure 2, A and B, the steady-state mrna level of Gli1 was dramatically induced in a time-dependent manner after obstructive injury. Similarly, Gli1 protein was also markedly induced in the obstructed kidneys at 7 days after UUO compared with findings in the controls (Figure 2, C and D). These results suggest that an increased Shh could be capable of triggering its signal transduction, leading to Gli1 activation in the fibrotic kidneys. To localize the Shh responding cells in vivo, weusedthe Gli1 lacz mutant mice, which harbor a b-galactosidase (b-gal) knock-in mutation that also abolishes endogenous Gli1 gene function (Figure 2E). Under the control of the native Gli1 upstream promoter/enhancer elements, lacz expression in these mice authentically recapitulates the expression pattern of the endogenous Gli1 mrna. 3 As shown in Figure 2, F and G, immunohistochemical staining using specific anti b-gal antibody revealed a marked increase in the Gli1 promoter driven b-gal expression after obstructive injury. Notably, the b-gal positive cells appeared in a spindle shape and were positioned in the interstitium, consistent with the characteristic features of renal interstitial fibroblasts (Figure 2G, arrows). Similarly, 802 Journal of the American Society of Nephrology J Am Soc Nephrol 23: , 2012

3 BASIC RESEARCH 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 mm. (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 a-sma (green). Arrows indicate Shh-positive tubules. histochemical staining for b-gal activity also demonstrated an increased Gli1/b-Gal positive cells in renal interstitium after obstructive injury (Figure 2, H and I). Figure 2J shows quantitative determination of the b-gal positive cells in the lacz knock-in mice at 7 days after obstructive injury. Although few b-gal positive cells were found in the interstitium of the sham control kidneys (Figure 2H, arrow), obstructive injury clearly caused an extensive increase in b-gal positive cells (Figure 2I, arrows), indicating an apparent activation of Shh/Gli1 signaling in this setting. To further characterize the identity of the Shh responding cells in vivo, we used double immunostaining for b-gal and a-sma, a marker of activated fibroblasts, in the fibrotic kidneys. As shown in Figure 2K, b-gal positive cells were costained with a-sma. Therefore, it seems that tubule-derived Shh protein targets interstitial fibroblasts in a paracrine fashion in the fibrotic kidneys. Shh Signaling Promotes Fibroblast Activation and Matrix Production We further investigated the potential effects of Shh signaling on renal interstitial fibroblasts using an in vitro model system. To that end, normal rat kidney interstitial fibroblasts (NRK-49F) were incubated with recombinant Shh protein. As shown in Figure 3, A and B, incubation of NRK-49F cells with Shh induced Gli1 mrna expression in a time- and dose-dependent manner, indicating that these cells are capable of responding to Shh stimulation. Interestingly, Shh was able to induce a-sma, J Am Soc Nephrol 23: , 2012 Shh Promotes Renal Fibrosis 803

4 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 b-galactosidase shows a marked induction of the Gli1 promoter/enhancer-driven b-gal expression in the obstructed kidney at 7 days after UUO (G) compared with the controls (F). Boxed areas were enlarged, in which b-gal positive cells were indicated by arrows. (H and I) Histochemical staining for b-galactosidase activity demonstrates a marked induction of the Gli1 promoter/enhancer-driven b-gal expression in the lacz knock-in mice after obstructive injury. Open arrowhead in panel (H, enlarged box) indicates nonspecific b-gal activity staining in renal tubules. (J) Quantitative determination of the Gli1-driven b-gal positive cells in the lacz knock-in mice after obstructive injury. The b-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 b-gal (red) and a-sma (green), respectively. Arrows indicate Gli1 promoter/enhancer-driven b-gal expression in a-sma positive myofibroblasts. 804 Journal of the American Society of Nephrology J Am Soc Nephrol 23: , 2012

5 BASIC RESEARCH 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, a-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 a-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 a-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. fibronectin, type I collagen, and desmin expression (Figure 3, A and B). Shh also induced Snail1 expression in renal interstitial fibroblasts in a time- and dose-dependent fashion (Figure 3, A and B). Expression of Gli1 protein, as demonstrated by Western blot analyses, was also induced by Shh in NRK-49F cells (Figure 3C). Similarly, Shh induced the protein expression of a-sma and desmin, as well as interstitial matrix proteins fibronectin and collagen I, in a dose-dependent fashion (Figure 3, C E). The induction of these matrix proteins by Shh in NRK-49F cells was time-dependent as well (data not shown). Immunofluorescence staining also illustrated that exogenous Shh induced the expression of a-sma, J Am Soc Nephrol 23: , 2012 Shh Promotes Renal Fibrosis 805

6 fibronectin, and type I collagen proteins in NRK-49F cells (Figure 3F). Together, these results indicate that Shh signaling directly promotes renal interstitial fibroblast activation and matrix production. Genetic Ablation of Gli1 Gene Attenuates Renal Fibrosis after Obstructive Injury Given the critical role for Shh signaling in promoting renal fibroblast activation (Figure 3), we next investigated whether genetic ablation of Gli1 affects renal interstitial fibrosis in vivo. To this end, we again used the Gli1 lacz mutant mice because exons 2 7of the Gli1 gene encoding the entire N-terminal and zinc-finger domains in these mice were replaced by LacZ, leading to functional ablation of endogenous gene. 3 The Gli1 null mice (knockout) and their wild-type (WT) control littermates underwent UUO for 7 days, and renal mrna levels of various fibrosis-related genes, such as type I collagen, fibronectin, a-sma, and connective tissue growth factor, were determined by quantitative, real time reverse transcriptase (RT) PCR analyses. As shown in Figure 4, A D, genetic ablation of Gli1 significantly suppressed the expression of these fibrosisrelated genes in the obstructed kidneys compared with the WT controls. Immunofluorescence staining also illustrated an attenuated protein expression of type I collagen, fibronectin, and a-sma in the obstructed kidneys of the Gli1 null mice at 7 days after UUO compared with WT controls (Figure 4, E 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 (Gli12/2), compared with WT controls (Gli1+/+). Both Gli12/2 and Gli1+/+ were on 129S1/SvImJ background. Renal mrna levels of type I collagen (A), fibronectin (B), a-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 b-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 a-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 a-sma, respectively. Scale bar, 50 mm. **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 mm. 806 Journal of the American Society of Nephrology J Am Soc Nephrol 23: , 2012

7 BASIC RESEARCH and F). Masson trichrome staining (MTS) revealed that disruption of Gli1 gene significantly reduced collagen deposition and fibrotic lesion in the obstructed kidneys (Figure 4, G and H). These results obtained from the Gli1 null mice clearly suggest a role of Shh/Gli1 signaling in mediating fibroblast activation and renal interstitial fibrosis in vivo. Inhibition of Shh Signaling Prevents Fibroblast Activation and Matrix Production In Vitro We next sought to explore whether pharmacologic inhibition of Shh signaling modulates fibroblast activation and matrix production. To this end, NRK-49F cells were treated with cyclopamine (CPN), a small molecule inhibitor of the hedgehog signaling pathway, by inhibiting Smo protein. As shown in Figure 5, A and B, incubation of NRK-49F cells with CPN at 5 mm completely abolished Gli1 mrna expression induced by Shh. Because Gli1 transcription is a reliable readout of the hedgehog signaling, these results indicate that CPN is effective in blocking Shh signal transduction. Intriguingly, CPN also effectively blocked Shh-induced Snail1 (Figure 5C), type I collagen (Figure 5D), desmin (Figure 5E), fibronectin (Figure 5F), and a-sma (Figure 5G) expression in NRK-49F cells, as demonstrated by quantitative, real-time RT-PCR analyses. Notably, CPN at 10 mm exhibited the tendency to reduce the mrna levels of Snail1, collagen I, fibronectin, and a-sma even below the baseline in the controls, suggesting a potential importance of endogenous Shh signaling in controlling the basal expression of these genes. Similarly, immunofluorescence Figure 5. Inhibition of Shh/Gli1 signaling prevents fibroblast activation and matrix production in vitro. (A and B)CPN inhibits Shhinduced 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 a-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 a-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. J Am Soc Nephrol 23: , 2012 Shh Promotes Renal Fibrosis 807

8 staining revealed a dose-dependent inhibition of the Shh-induced type I collagen and a-sma protein expression by CPN in NRK-49F cells (Figure 5H). Blockade of Shh Signaling Suppresses Gli1 and Snail1 Expression We next explored the possibility of pharmacologic inhibition of Shh signaling in vivo. Therefore, mice were administered CPN at 5 mg/kg body weight via daily intraperitoneal injection. As shown in Figure 6, A D, CPN did not affect the Shh expression in the obstructed kidneys, but it significantly inhibited Gli1 mrna expression after obstructive injury. These results are not unexpected because CPN directly targets Smo, an intermediate receptor that is positioned at the downstream of Shh and upstream of Gli1. CPN not only inhibited Gli1 but also repressed the expression of Snail1 (Figure 6E), a target of Shh/Gli1 signaling. Hence, administration of CPN can effectively block Shh/Gli1 signaling in the obstructed kidneys in vivo. Blockade of Shh Signaling Represses Matrix Gene Expression and Reduces Renal Interstitial Fibrosis We further analyzed the effect of Shh signaling inhibition on renal fibrosis in obstructive nephropathy. As shown in Figure 7, A C, administration of CPN significantly inhibited the expression of type I collagen, fibronectin, and a-sma, as demonstrated by quantitative, real-time RT-PCR analyses. Immunofluorescence staining also revealed that CPN inhibited type I collagen and fibronectin expression and myofibroblast activation in the obstructed kidneys at 7 days after UUO (Figure 7, D and E). Collagen deposition and renal fibrotic lesion was significantly reduced after CPN administration (Figure 7, G and H). These results indicate that inhibition of Shh signaling by small molecule inhibitor represses matrix gene expression and reduces renal fibrosis after obstructive injury. DISCUSSION Activation of a-sma positive, matrix-producing myofibroblasts arguably is the central event in renal fibrogenesis; it results in excessive production and deposition of extracellular matrix, leading to scar formation. 18,29,30 The studies presented here demonstrate that developmental signal Shh/Gli1 plays an important role in regulating myofibroblast activation and matrix production in diseased kidneys. Shh is predominantly induced in renal tubular epithelium after obstructive injury, and it apparently targets renal interstitial fibroblasts and activates Gli1. Furthermore, Shh is able to activate Snail1 transcription factor and is sufficient to induce a-sma expression and matrix production in renal fibroblasts in vitro. Moreover, genetic ablation or pharmacologic inhibition of Gli1 ameliorates renal interstitial fibrosis in obstructive nephropathy. These results suggest that tubule-derived Shh mediates epithelial mesenchymal communication by virtue of its ability to target interstitial fibroblasts in a paracrine fashion. Our studies have also established a role for Shh/Gli1 signaling in the 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 b-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). 808 Journal of the American Society of Nephrology J Am Soc Nephrol 23: , 2012

9 BASIC RESEARCH 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 a-sma mrna expression in CD-1 mice. Renal mrna levels of type I collagen (A), fibronectin (B), and a-sma (C) were determined at 7 days after UUO. Relative mrna levels are reported after normalization with b-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, a-sma, and fibronectin in the obstructed kidneys at 7 days after UUO. Scale bar, 50 mm. **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). pathogenesis of fibroblast activation, matrix production, and renal fibrosis. Therefore, targeting hedgehog signaling could be a novel strategy for therapeutic intervention of fibrotic kidney disorders. Renal fibrosis is generally regarded as a failed woundhealing process provoked by chronic or repetitive injury. 30,31 As the major constituent of renal parenchyma, tubular epithelium is the primary target of a variety of metabolic, immunologic, ischemic, and toxic insults. How an injured tubular epithelium communicates with interstitial fibroblasts remains poorly understood. The present study illustrates a critical role for Shh/Gli1 signaling in mediating epithelial mesenchymal communication in renal fibrogenesis. Shh is induced predominantly in renal tubular epithelium, but not in the expanded interstitium, in the fibrotic kidneys after obstructive injury, as demonstrated by immunohistochemical staining, as well as double staining for Shh and tubular or fibroblast markers, respectively (Figure 1). This finding underscores that tubular epithelium responds to obstructive injury by recapitulating a key developmental program through induction of Shh. Despite the high level of tubular Shh expression, however, tubular epithelium seems not to be the primary target of Shh, as demonstrated by J Am Soc Nephrol 23: , 2012 Shh Promotes Renal Fibrosis 809

10 lacking of b-gal expression in tubular epithelium after obstructive injury in the Gli1 lacz knock-in mice (Figure 2). These findings are in line with the mode of action of Shh signaling in embryonic development, in which Shh, as a morphogen, regulates tissue patterning in a paracrine- and concentration-dependent fashion. 1,6 Using Gli1 lacz knock-in mice, we found that the Shhresponding cells are located at the peritubular interstitium, with spindle shape and co-localization with a-sma (Figure 2). These characteristic features define fibroblasts as the principal target cells of Shh signaling in the diseased kidneys. Consistent with this notion, recombinant Shh per se is sufficient to induce fibroblast activation and matrix production in vitro (Figure 3). Furthermore, genetic Gli1 ablation or its inhibition by CPN is able to ameliorate fibroblast activation, matrix production, and renal fibrosis (Figures 4 and 7), illustrating a crucial role for Shh/ Gli1 signaling in controlling fibroblast activation in vivo. Although activated fibroblasts in the injured kidneys can derive from different sources, 19,31 33 the majority of them probably come from phenotypic activation of resident, quiescent fibroblasts in the renal interstitium. 30 Because fibroblast activation is closely correlated with the severity of renal fibrosis and often predicts the decline of kidney functions, tubule-derived Shh could play a pivotal role in renal fibrogenesis by promoting fibroblast activation and matrix production. Shh signaling exerts its action by regulating the transcription of its target genes. 4 The best-characterized direct target and downstream mediator of Shh signaling is Gli1, which is induced after incubation of renal fibroblasts with exogenous Shh (Figure 3). Another well-known target of Shh signaling is Snail1, a key transcription factor for mediating epithelial to mesenchymal transition and fibroblast migration. 37,38 Interestingly, Shh not only stimulates the expression of these knowntargetgenesbutalsoinducestheexpressionof a-sma, fibronectin, collagen I, and desmin, genes that are directly implicated in fibroblast activation and interstitial matrix production. Judged from the kinetics of mrna expression(figure3a),itappearsthattheupregulationofthese fibrosis-related genes may not be a result of Gli1 induction. These data suggest that Shh signaling might directly control the expression of a-sma, fibronectin,collagen I,anddesmin in renal interstitial fibroblasts. Therefore, it is plausible that Shh signaling promotes myofibroblast activation and matrix production by directly controlling the expression of a battery of fibrogenic genes, leading to excessive matrix deposition and scar formation. Evidence is emerging that key developmental signals, such as Wnt/b-catenin and Notch pathways, play a critical role in the onset and progression of renal fibrosis in a variety of CKDs. 20,26 However, Shh signaling is unique in that it specifically targets on the activation of renal fibroblast, a central event in renal fibrogenesis. In contrast, both Wnt/b-catenin and Notch signaling predominantly target on tubular epithelial cells in the diseased kidneys. 23,26 Although tubular activation of these developmental signals could contribute to renal fibrosis by inducing epithelial to mesenchymal transition or mediating the expression of fibrogenic factors, fibroblast activation induced by Shh signaling would have an immediate, direct, and profound effect on renal fibrogenesis. It should be noted that these developmental signals can cross-talk, thereby working in concert to elicit biologic actions in a coordinated fashion. For instance, hedgehog signals are known to upregulate Wnt2b and Wnt5a, as well as Notch ligand Jaggad2, 4,39 and both Wnt/b-catenin and Shh signaling can induce Snail1 expression. 24 It is conceivable that Shh signaling may promote renal fibrosis by regulating other fibrogenic signaling as well. The importance of Shh signaling in renal fibrogenesis is also illustrated by the therapeutic efficacy of its inhibitor. Targeting Smo by the small molecule inhibitor CPN can inhibit fibroblast activation and matrix production in vitro and attenuate renal fibrosis in vivo in a mouse model of obstructive nephropathy. Intriguingly, Shh expression is not affected, but its downstream Gli1 and Snail1 induction is largely blocked by CPN (Figure 6), findings consistent with its ability to inhibit Smo activity. Notably, the ameliorative effect of CPN appears greater than that of Gli1 deficiency (Figures 4 and 7), suggesting that Gli1 may not be the only mediator downstream of hedgehog signaling that is implicated in renal fibrotic response after obstructive injury. However, these results should be interpreted with caution because these two sets of experiments were carried out in mice with different genetic backgrounds. In addition, we cannot completely exclude the possibility that CPN may have some offtarget effects in the dosage used in vivo.clearly,morestudiesare needed in this area. In summary, the studies presented here demonstrate that Shh is induced predominantly in renal tubular epithelium after obstructive injury, but it targets interstitial fibroblasts. This epithelial mesenchymal communication, mediated by Shh, probably plays a critical role in the pathogenesis of fibroblast activation, matrix production, and renal fibrosis. These studies also provide a proof-of-principle that targeting Shh/Gli1 signaling could be a novel strategy for the therapeutic intervention of a variety of fibrotic kidney disorders. CONCISE METHODS Animal Model Male CD-1 mice weighing approximately g were obtained from Harlan Sprague-Dawley (Indianapolis, IN). For time-course studies of Shh and Gli1 expression after obstructive injury, four groups of animals (n=5 per group) were used: (1)shamcontrol,(2)UUOfor3 days, (3) UUOfor7days,and(4) UUO for 14 days. UUO was performed using an established protocol, as described previously. 40 Mice were killed at different time points after UUO as indicated, and kidney tissues were removed for various analyses. For evaluating the efficacy of Shh/Gli1 signaling inhibition on renal fibrosis, three groups of animals (n=5 per group) were used: (1)shamcontrol,(2)UUOmice injected with vehicle, and (3) UUO mice injected with CPN (Sigma, St. Louis, MO). CPN was complexed with 2-hydroxypropyl-b-cyclodextrin 810 Journal of the American Society of Nephrology J Am Soc Nephrol 23: , 2012

11 BASIC RESEARCH (HBC; Sigma). A CPN/HBC stock solution was produced by suspending 1 mg CPN in 1 ml 45% HBC in sterile PBS and stirring at 65 C for 60 minutes. CPN was administered by daily intraperitoneal injection at a dose of 5 mg/kg body weight beginning from 1 day before UUO. Mice were killed at 7 days after UUO, and the kidney tissues were removed for various analyses. The Gli1 lacz mutant mice in 129S1/ SvImJ background, which harbor a b-galactosidase knock-in mutation that also abolishes endogenous Gli1 gene function, 3 was obtained from the Jackson Laboratory (Bar Harbor, ME). Heterozygous mice were mated, and the offspring were genotyped by PCR according to the protocol specified by the Jackson Laboratory. Mutant mice (Gli12/2) and their WT (Gli1+/+) littermates from the colony at the age of 5 weeks underwent UUO. Kidney tissues were collected for various analyses at 7 days after UUO. All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh. Cell Culture and Treatment Normal rat kidney interstitial fibroblast cell lines (NRK-49F) were purchased from the American Type Culture Collection (Manassas, VA) and maintained as described previously. 36 Cells were seeded onto six-well plates and maintained in complete medium overnight. After serum starvation for 16 hours, human Shh protein (StemRD Inc., Burlingame, CA) was added to the serum-free medium at different concentrations for various periods of time as indicated. In some experiments, cells were pretreated with CPN (5 or 10 mm) for 30 minutes before incubation with Shh. At 24 or 48 hours after treatment with Shh, cells were subjected to various analyses. RT-PCR and Real-Time RT-PCR Total RNA was extracted using TRIzol RNA isolation system (Invitrogen, Carlsbad, CA). The first strand of cdna was synthesized using 2 mg of RNA in 20 ml of reaction buffer by reverse transcription using AMV-RT (Promega, Madison, WI) and random primers at 42 C for 30 minutes. PCR was carried out using a standard PCR kit and 1-mL aliquot of cdna, HotStar Taq polymerase (Qiagen, Valencia, CA) with specific primer pairs. The sequences of the primer pairs are given in Supplemental Table 1. For quantitative determination of mrna levels, real-time RT-PCR was performed on an ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA), as described previously. 28 The PCR reaction mixture in a 25-mL volume contained 12.5 ml 23 SYBR Green PCR Master Mix (Applied Biosystems), 5 ml diluted RT product (1:10), and 0.5 mm sense and antisense primer sets (Supplemental Table 1). PCR reaction was run by using standard conditions. After sequential incubations at 50 C for 2 minutes and 95 C for 10 minutes, respectively, the amplification protocol consisted of 50 cycles of denaturing at 95 C for 15 seconds and annealing and extension at 60 C for 60 seconds. The mrna levels of various genes were calculated after normalizing with b-actin. Western Blot Analysis Western blot analysis for specific protein expression was performed essentially according to an established procedure. 41 The primary antibodies used were as follows: anti-shh (sc-9024; Santa Cruz Biotechnology, Santa Cruz, CA), anti-gli1 (ab92611; Abcam, Cambridge, MA), anti a-sma (A2547; Sigma, St. Louis, MO), anti-fibronectin (F3648; Sigma), anti-collagen I (#234168; Millipore, Danvers, MA), anti-desmin (D1033; Sigma), and anti-actin (MAB1501; Millipore). Quantification was performed by measurement of the intensity of the signals with the use of National Institutes of Health Image analysis software. b-galactosidase Staining Histochemical staining for b-galactosidase was carried out using b-gal Staining Kit (Mirus, Madison, WI), according to the procedures specified by the manufacturer. Briefly, kidney cryosections were prepared at 16-mm thickness and thoroughly dried before use. Sections were then incubated with fixative Working Solution for 5 minutes at room temperature, followed by washing with PBS at least three times. Sections were completely covered with Cell Staining Working Solution in a humidified chamber at 37 C for 12 hours in the dark. The specimens were washed three times with PBS. Tissue sections were viewed using light microscopy. Histology and Immunohistochemical Staining Paraffin-embedded mouse kidney sections (4-mm thickness) were prepared by a routine procedure. Sections were stained with MTS using a standard protocol. Tissue fibrotic lesions were semi-quantified by a computer-aided, point-counting morphometric analysis (MetaMorph, Universal Imaging Co., Downingtown, PA). Briefly, a grid containing 117 (1339) sampling points was superimposed on MTS images of cortical high-power field (4003). The number of grid points overlying MTS-positive area (except tubular lumen and glomeruli) was counted and expressed as a percentage of all sampling points (percentage of fibrotic area), as described elsewhere. 42 For immunohistochemical staining, paraffin-embedded sections were stained with rabbit polyclonal b-galactosidase antibody (ab616; Abcam Inc., Cambridge, MA) and anti-shh antibody (sc-9024, Santa Cruz Biotechnology) using the methods as described previously. 42 Immunofluorescence Staining Indirect immunofluorescence staining was performed using an established procedure. 41 Briefly, cells cultured on coverslips were fixed with cold methanol:acetone (1:1) for 10 minutes and blocked with 10% donkey serum in PBS for 30 minutes. Kidney cryosections were prepared at 5-mm thickness, then fixed and blocked in the same fashion. Sections were then incubated with anti a-sma (clone 1A4; Sigma), anti-fibronectin (catalog no , BD Transduction Laboratories, San Jose, CA), anti-collagen 1 (catalog no ; Southern Biotech, Birmingham, AL). For some samples, sections were double stained with primary antibodies against b-gal (ab616, Abcam) and a-sma (A2549, Sigma), Shh (sc-9024, Santa Cruz) and aquaporin 1 (sc-9878, Santa Cruz), and Shh and a-sma, respectively. To visualize the primary antibodies, sections were stained with cyanine Cy2- or Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). Cells were also stained with DAPI (49, 6-diamidino-2-phenylindole, HCl) to visualize the nuclei. Stained cells and cryosections were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA) and viewed with a Nikon Eclipse E600 microscope equipped with a digital camera (Melville, NY). Staining was semi-quantified by a computer-aided, point-counting J Am Soc Nephrol 23: , 2012 Shh Promotes Renal Fibrosis 811

12 morphometric analysis (MetaMorph), as described elsewhere. 42 The percentage of the positive area (except tubular lumen and glomeruli) was expressed, as described elsewhere. 42 Statistical Analyses All data examined are expressed as mean 6 SEM. Statistical analyses of the data were performed using SigmaStat software (Jandel Scientific Software, San Rafael, CA). Comparisons between groups were made using one-way ANOVA, followed by Student-Newman-Keuls test. P,0.05 was considered to represent a statistically significant difference. ACKNOWLEDGMENTS This work was supported by National Institutes of Health Grant DK064005, 973 Program Grant 2012CB517700, and National Natural Science Foundation of China Grant DISCLOSURES None. REFERENCES 1. Briscoe J: Making a grade: Sonic Hedgehog signalling and the control of neural cell fate. EMBO J 28: , Jenkins D: Hedgehog signalling: Emerging evidence for non-canonical pathways. 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