Experimental inhibition of porcupine-mediated Wnt O-acylation attenuates kidney fibrosis
2016; Elsevier BV; Volume: 89; Issue: 5 Linguagem: Inglês
10.1016/j.kint.2016.01.017
ISSN1523-1755
AutoresBabita Madan, Mehul Patel, Jiandong Zhang, Ralph M. Bunte, Nathan P. Rudemiller, Robert Griffiths, David M. Virshup, Steven D. Crowley,
Tópico(s)Connective Tissue Growth Factor Research
ResumoActivated Wnt signaling is critical in the pathogenesis of renal fibrosis, a final common pathway for most forms of chronic kidney disease. Therapeutic intervention by inhibition of individual Wnts or downstream Wnt/β-catenin signaling has been proposed, but these approaches do not interrupt the functions of all Wnts nor block non-canonical Wnt signaling pathways. Alternatively, an orally bioavailable small molecule, Wnt-C59, blocks the catalytic activity of the Wnt-acyl transferase porcupine, and thereby prevents secretion of all Wnt isoforms. We found that inhibiting porcupine dramatically attenuates kidney fibrosis in the murine unilateral ureteral obstruction model. Wnt-C59 treatment similarly blunts collagen mRNA expression in the obstructed kidney. Consistent with its actions to broadly arrest Wnt signaling, porcupine inhibition reduces expression of Wnt target genes and bolsters nuclear exclusion of β-catenin in the kidney following ureteral obstruction. Importantly, prevention of Wnt secretion by Wnt-C59 blunts expression of inflammatory cytokines in the obstructed kidney that otherwise provoke a positive feedback loop of Wnt expression in collagen-producing fibroblasts and epithelial cells. Thus, therapeutic targeting of porcupine abrogates kidney fibrosis not only by overcoming the redundancy of individual Wnt isoforms but also by preventing upstream cytokine-induced Wnt generation. These findings reveal a novel therapeutic maneuver to protect the kidney from fibrosis by interrupting a pathogenic crosstalk loop between locally generated inflammatory cytokines and the Wnt/β-catenin signaling pathway. Activated Wnt signaling is critical in the pathogenesis of renal fibrosis, a final common pathway for most forms of chronic kidney disease. Therapeutic intervention by inhibition of individual Wnts or downstream Wnt/β-catenin signaling has been proposed, but these approaches do not interrupt the functions of all Wnts nor block non-canonical Wnt signaling pathways. Alternatively, an orally bioavailable small molecule, Wnt-C59, blocks the catalytic activity of the Wnt-acyl transferase porcupine, and thereby prevents secretion of all Wnt isoforms. We found that inhibiting porcupine dramatically attenuates kidney fibrosis in the murine unilateral ureteral obstruction model. Wnt-C59 treatment similarly blunts collagen mRNA expression in the obstructed kidney. Consistent with its actions to broadly arrest Wnt signaling, porcupine inhibition reduces expression of Wnt target genes and bolsters nuclear exclusion of β-catenin in the kidney following ureteral obstruction. Importantly, prevention of Wnt secretion by Wnt-C59 blunts expression of inflammatory cytokines in the obstructed kidney that otherwise provoke a positive feedback loop of Wnt expression in collagen-producing fibroblasts and epithelial cells. Thus, therapeutic targeting of porcupine abrogates kidney fibrosis not only by overcoming the redundancy of individual Wnt isoforms but also by preventing upstream cytokine-induced Wnt generation. These findings reveal a novel therapeutic maneuver to protect the kidney from fibrosis by interrupting a pathogenic crosstalk loop between locally generated inflammatory cytokines and the Wnt/β-catenin signaling pathway. Chronic injury to the kidney results in progressive scarring known as renal fibrosis. Renal fibrosis is a final common pathway for both immune-mediated glomerulonephritides and kidney diseases initiated by hemodynamic insults such as hypertension. The extent of renal fibrosis predicts progression to end-stage kidney disease in most forms of chronic kidney disease.1Wynn T.A. Ramalingam T.R. Mechanisms of fibrosis: therapeutic translation for fibrotic disease.Nat Med. 2012; 18: 1028-1040Crossref PubMed Scopus (2087) Google Scholar Despite an intense focus of research in this area, no specific therapies are currently available to abrogate or reverse fibrosis in human chronic kidney disease. In recent years, the Wingless-related integration site (Wnt) family of signaling proteins have emerged as potential targets of inhibition to control fibrosis in several target organs.1Wynn T.A. Ramalingam T.R. Mechanisms of fibrosis: therapeutic translation for fibrotic disease.Nat Med. 2012; 18: 1028-1040Crossref PubMed Scopus (2087) Google Scholar, 2Hao S. He W. Li Y. et al.Targeted inhibition of β-catenin/CBP signaling ameliorates renal interstitial fibrosis.J Am Soc Nephrol. 2011; 22: 1642-1653Crossref PubMed Scopus (192) Google Scholar, 3He W. Dai C. Li Y. et al.Wnt/β-catenin signaling promotes renal interstitial fibrosis.J Am Soc Nephrol. 2009; 20: 765-776Crossref PubMed Scopus (452) Google Scholar, 4Akhmetshina A. Palumbo K. Dees C. et al.Activation of canonical Wnt signalling is required for TGF-β-mediated fibrosis.Nat Commun. 2012; 3: 735Crossref PubMed Scopus (568) Google Scholar, 5Satoh M. Nagasu H. Morita Y. et al.Klotho protects against mouse renal fibrosis by inhibiting Wnt signaling.Am J Physiol Renal Physiol. 2012; 303: F1641-F1651Crossref PubMed Scopus (115) Google Scholar, 6Henderson W.R. Chi E.Y. Ye X. et al.Inhibition of Wnt/β-catenin/CREB binding protein (CBP) signaling reverses pulmonary fibrosis.Proc Natl Acad Sci U S A. 2010; 107: 14309-14314Crossref PubMed Scopus (357) Google Scholar In the so-called "canonical" pathway, Wnt triggers activation of β-catenin signaling that drives progressive fibrosis in various models of kidney disease.1Wynn T.A. Ramalingam T.R. Mechanisms of fibrosis: therapeutic translation for fibrotic disease.Nat Med. 2012; 18: 1028-1040Crossref PubMed Scopus (2087) Google Scholar, 3He W. Dai C. Li Y. et al.Wnt/β-catenin signaling promotes renal interstitial fibrosis.J Am Soc Nephrol. 2009; 20: 765-776Crossref PubMed Scopus (452) Google Scholar As expected, β-catenin inhibitor molecules have shown some efficacy in slowing the progression of renal fibrosis.2Hao S. He W. Li Y. et al.Targeted inhibition of β-catenin/CBP signaling ameliorates renal interstitial fibrosis.J Am Soc Nephrol. 2011; 22: 1642-1653Crossref PubMed Scopus (192) Google Scholar, 5Satoh M. Nagasu H. Morita Y. et al.Klotho protects against mouse renal fibrosis by inhibiting Wnt signaling.Am J Physiol Renal Physiol. 2012; 303: F1641-F1651Crossref PubMed Scopus (115) Google Scholar However, as there are 19 different Wnt ligands that may signal via the canonical pathways, the alternative "non-canonical" pathways, or both, the efficacy of these direct β-catenin inhibitors may be limited.7Emami K.H. Nguyen C. Ma H. et al.A small molecule inhibitor of β-catenin/cyclic AMP response element-binding protein transcription.Proc Natl Acad Sci U S A. 2004; 101: 12682-12687Crossref PubMed Scopus (706) Google Scholar To overcome the limitations of the available Wnt pathway inhibitors and to block all Wnt-dependent pathways concomitantly by preventing the secretion of all Wnts, we have employed a small-molecule inhibitor of Porcupine named Wnt-C59 (C59).8Proffitt K.D. Madan B. Ke Z. et al.Pharmacological inhibition of the Wnt acyltransferase PORCN prevents growth of WNT-driven mammary cancer.Cancer Res. 2013; 73: 502-507Crossref PubMed Scopus (261) Google Scholar Porcupine (PORCN) is a membrane-bound O-acyl transferase that resides in the endoplasmic reticulum and catalyzes the acylation of all mammalian Wnts at a conserved serine residue. This acylation is essential for the secretion and binding of all Wnts to their receptors.9Coombs G.S. Yu J. Canning C.A. et al.WLS-dependent secretion of WNT3A requires Ser209 acylation and vacuolar acidification.J Cell Sci. 2010; 123: 3357-3367Crossref PubMed Scopus (145) Google Scholar, 10Janda C.Y. Waghray D. Levin A.M. et al.Structural basis of Wnt recognition by Frizzled.Science. 2012; 337: 59-64Crossref PubMed Scopus (568) Google Scholar C59 is orally bioavailable and by inhibiting Wnt acylation blocks the secretion and activity of all Wnts.8Proffitt K.D. Madan B. Ke Z. et al.Pharmacological inhibition of the Wnt acyltransferase PORCN prevents growth of WNT-driven mammary cancer.Cancer Res. 2013; 73: 502-507Crossref PubMed Scopus (261) Google Scholar Here we report that preventing Wnt secretion with C59 therapy downregulates Wnt signaling and as a consequence dramatically reduces kidney fibrosis in the mouse unilateral ureteral obstruction (UUO) model. Preventing the secretion of Wnts using the PORCN inhibitor C59 blunts the proliferation of fibroblasts and the expression of key proinflammatory cytokines that are in turn capable of eliciting Wnt generation. Our results provide a compelling rationale for the clinical evaluation of PORCN inhibitors for the treatment of kidney fibrosis and other progressive scarring disorders. Wnt signaling is known to potentiate kidney fibrosis.3He W. Dai C. Li Y. et al.Wnt/β-catenin signaling promotes renal interstitial fibrosis.J Am Soc Nephrol. 2009; 20: 765-776Crossref PubMed Scopus (452) Google Scholar We therefore tested whether inhibition of Wnt signaling with a PORCN inhibitor could limit the extent of renal fibrosis following injury in mice. We evaluated the severity of fibrosis in kidneys from mice treated with C59 or vehicle for 7 days following UUO. As illustrated by polarized images of the kidney sections stained with picrosirius red (Figure 1a), the obstructed kidneys from the vehicle group had markedly increased fibrosis compared to the contralateral kidneys. However, treatment with C59 significantly attenuated the level of fibrosis in the obstructed kidneys. Blinded morphometric analysis of the kidney sections (Figure 1b) confirmed that C59 therapy reduced the deposition of collagen fibrils in the obstructed kidney by half compared to vehicle-treated controls. Thus, treatment with C59 substantially mitigates renal fibrosis in the mouse UUO model. Wnts act via the canonical signaling pathway to induce nuclear translocation of β-catenin. To determine if the beneficial effects of PORCN inhibition to prevent tissue fibrosis were mediated by downregulation of Wnt signaling pathways, we analyzed the cellular localization of β-catenin. In the vehicle-treated group, ureteral obstruction induced robust nuclear localization of β-catenin within the interstitial and epithelial cells of the kidney (Figure 2a). By contrast, treatment with C59 nearly eliminated Wnt-driven nuclear localization of β-catenin in cells of the obstructed kidney. Upon translocation to the nucleus, β-catenin forms a complex with transcription factors T cell–specific transcription factor (TCF)/lymphoid enhancer binding factor (LEF) to drive transcription of key Wnt target genes including Axin2 and Nkd1. Accordingly, obstructed kidneys from the vehicle group had markedly increased expression of Axin2 and Nkd1, whereas this was significantly attenuated in the obstructed kidneys from C59-treated mice (Figure 2b and c). Axin2-LacZ reporter mice have an integrated genetic reporter so that cells and tissues with Axin2 induction express β-galactosidase, with activity that is readily visible with a specific histochemical stain. Following UUO, Axin2-LacZ mice showed marked reporter activity in the obstructed kidney (Figure 2d). However, treatment with C59 nearly abrogated the UUO-mediated induction of Axin2 as detected in the reporter model (Figure 2d). Thus, Wnt signaling is critical to the pathogenesis of kidney fibrosis, and comprehensive inhibition of Wnt secretion by the PORCN inhibitor prevents kidney scarring in a robust model of renal fibrogenesis. Fibroblasts are the key effector cells in collagen deposition, and they accumulate in areas of severe interstitial fibrosis.1Wynn T.A. Ramalingam T.R. Mechanisms of fibrosis: therapeutic translation for fibrotic disease.Nat Med. 2012; 18: 1028-1040Crossref PubMed Scopus (2087) Google Scholar, 11Nishitani Y. Iwano M. Yamaguchi Y. et al.Fibroblast-specific protein 1 is a specific prognostic marker for renal survival in patients with IgAN.Kidney Int. 2005; 68: 1078-1085Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 12Zhang J. Chen L. Xiao M. et al.FSP1+ fibroblasts promote skin carcinogenesis by maintaining MCP-1-mediated macrophage infiltration and chronic inflammation.Am J Pathol. 2011; 178: 382-390Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar β-catenin/TCF-dependent transcription promotes fibrogenesis by enhancing fibroblast proliferation and function.4Akhmetshina A. Palumbo K. Dees C. et al.Activation of canonical Wnt signalling is required for TGF-β-mediated fibrosis.Nat Commun. 2012; 3: 735Crossref PubMed Scopus (568) Google Scholar, 13DiRocco D.P. Kobayashi A. Taketo M.M. et al.Wnt4/β-catenin signaling in medullary kidney myofibroblasts.J Am Soc Nephrol. 2013; 24: 1399-1412Crossref PubMed Scopus (130) Google Scholar, 14Beyer C. Schramm A. Akhmetshina A. et al.β-catenin is a central mediator of pro-fibrotic Wnt signaling in systemic sclerosis.Ann Rheum Dis. 2012; 71: 761-767Crossref PubMed Scopus (160) Google Scholar As treatment with C59 prevents nuclear accumulation of β-catenin and fibrosis, we examined whether blocking Wnt secretion impacts the proliferation of collagen-producing fibroblasts in the obstructed kidneys. To identify the fibroblasts, kidney sections were stained with an antibody against fibroblast-specific protein (Fsp-1) that primarily stains fibroblasts.11Nishitani Y. Iwano M. Yamaguchi Y. et al.Fibroblast-specific protein 1 is a specific prognostic marker for renal survival in patients with IgAN.Kidney Int. 2005; 68: 1078-1085Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 15Strutz F. Identification and characterization of a fibroblast marker: FSP1.J Cell Biol. 2015; 130: 1-13Google Scholar In the vehicle-treated group, UUO strongly increased the level of Fsp-1 staining in the kidney (Figure 3a and b ). By contrast, C59 treatment markedly reduced Fsp-1 staining in the obstructed kidney, consistent with an attenuated number of fibroblasts. At the mRNA level, C59 similarly reduced Fsp1 expression in the kidney following UUO (Figure 3c). These data indicate that PORCN inhibition limits UUO-induced kidney fibrosis in part by blocking the local accumulation of fibroblasts. Fibrosis is characterized by excessive scarring due to production, deposition, and contraction of extracellular matrix by proliferating fibroblasts. To characterize how the inhibition of Wnt secretion provides benefit in the mouse model of kidney fibrosis, we analyzed global changes in gene expression programs within the obstructed kidneys from vehicle- and C59-treated animals at day 7 of UUO. The disruption in Wnt signaling by C59 therapy had a broad impact on the expression of genes linked to the pathogenesis of kidney fibrosis. Five hundred fifty-six genes were differentially expressed in the control versus C59-treated UUO kidneys using a cutoff of 1.5-fold and false discovery rate P value < 0.05 (Figure 4a). Ingenuity pathway analysis of the differentially expressed genes showed that UUO led to marked upregulation of genes associated with fibroblast-mediated deposition of extracellular matrix such as matrix metalloproteases and collagens (Figure 4b). C59 treatment also resulted in a reduction in the expression of fibroblast-associated genes including various collagens, matrix metalloprotease 2, fibronectin 1 (FN1), and intercellular adhesion molecule 1 (ICAM-1), consistent with its effects on fibroblast proliferation (Figure 4b). The changes in expression of Col1a1 and FN1 were validated using real-time reverse transcriptase quantitative polymerase chain reaction (qRT-PCR) (Figure 4c and d). PORCN inhibition also reduced protein levels of FN1 as examined by immunohistochemistry (Figure 4e). Taken together, these data confirm that inhibition of Wnt secretion comprehensively interrupts profibrotic signaling pathways in the obstructed kidney by blunting the proliferation of fibroblasts. Chronic kidney disease ultimately culminates in kidney fibrosis that in turn predicts end-stage kidney disease. To determine whether C59 therapy preserves the architecture of the kidney tubules, we evaluated the severity of pathologic changes in the renal tubules at 7 days following UUO. The tubules in the control kidneys from both the groups were normal with eosinophilic cytoplasm. In the obstructed kidneys from the vehicle-treated group, most of the tubules were atrophic, having smaller cells and nuclei with basophilic cytoplasm, or dilated and filled with proteinaceous casts (Figure 5a). The severity of nephropathy was scored on a scale from 0 to 5 based on the extent of tubular atrophy, dilation, necrosis, and cell loss, with 0 representing no damage, and 1 to 5 marking a spectrum from minimal up to severe tubular damage. Here again, treatment with C59 yielded a quantitative reduction in the level of tubular damage induced by UUO (Figure 5a and b). At the mRNA level, microarray analysis confirmed the preservation of tubular integrity by C59 following UUO (Figure 5c). Specifically, expression of solute carriers expressed in the proximal nephron (Slc5a10/Sglt5), the thick ascending limb (Slc12a1/Nkcc2), and the collecting tubule (Slc4a1/AE1) was downregulated in obstructed kidneys but was restored toward normal by C59 therapy. Thus blocking Wnt secretion with a PORCN inhibitor mitigates the renal tubular injury that triggers the progression of kidney fibrosis. Mitigation of fibrosis with PORCN inhibition was accompanied by differential expression not only of multiple collagens but also of immune-signaling genes (Figure 4b). As proinflammatory cytokines produced in the kidney by intrinsic and/or infiltrating immune cells are key instigators of fibrosis, we measured the expression of several profibrotic cytokines in the kidneys from both vehicle- and PORCN inhibitor–treated animals (Figure 6).4Akhmetshina A. Palumbo K. Dees C. et al.Activation of canonical Wnt signalling is required for TGF-β-mediated fibrosis.Nat Commun. 2012; 3: 735Crossref PubMed Scopus (568) Google Scholar, 16Wynn T.A. Fibrotic disease and the TH1/TH2 paradigm.Nat Rev Immunol. 2004; 4: 583-594Crossref PubMed Scopus (1233) Google Scholar, 17Nee L.E. McMorrow T. Campbell E. et al.TNF-alpha and IL-1beta-mediated regulation of MMP-9 and TIMP-1 in renal proximal tubular cells.Kidney Int. 2004; 66: 1376-1386Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar Indeed, there was a marked upregulation of Tnfα, Tgfβ, and IL1β in the obstructed kidneys, effects that were reduced by treatment with C59 (Figure 6a–c). Moreover, the expression of Tnfrsf1a, one of the major receptors for tumor necrosis factor-α (TNF-α) involved in activation of nuclear factor-κB signaling, was also significantly downregulated by C59 treatment (Figure 6d). These results indicate that preventing Wnt secretion reduces fibrosis by mitigating the expression of profibrotic cytokines and downstream signaling in the obstructed kidney. Circulating mononuclear cells, particularly macrophages that infiltrate the kidney, have been reported to be a key source of profibrotic cytokines.1Wynn T.A. Ramalingam T.R. Mechanisms of fibrosis: therapeutic translation for fibrotic disease.Nat Med. 2012; 18: 1028-1040Crossref PubMed Scopus (2087) Google Scholar, 18Lin S.L. Castano A.P. Nowlin B.T. et al.Bone marrow Ly6Chigh monocytes are selectively recruited to injured kidney and differentiate into functionally distinct populations.J Immunol. 2009; 183: 6733-6743Crossref PubMed Scopus (268) Google Scholar, 19Lee S.B. Kalluri R. Mechanistic connection between inflammation and fibrosis.Kidney Int. 2010; 78: S22-S26Abstract Full Text Full Text PDF Scopus (221) Google Scholar, 20Vielhauer V. Anders H.J. Mack M. et al.Obstructive nephropathy in the mouse: progressive fibrosis correlates with tubulointerstitial chemokine expression and accumulation of CC chemokine receptor 2- and 5-positive leukocytes.J Am Soc Nephrol. 2001; 12: 1173-1187PubMed Google Scholar, 21Kitagawa K. Wada T. Furuichi K. et al.Blockade of CCR2 ameliorates progressive fibrosis in kidney.Am J Pathol. 2004; 165: 237-246Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar However, we found similar levels of F4/80+ macrophage infiltration in the UUO kidneys from our vehicle-treated (4.6% ± 0.6%) and C59-treated groups (4.7% ± 0.5%). Importantly, in situ hybridization showed that transforming growth factor-β (TGF-β) was predominantly expressed in the interstitial cells and that C59 suppressed the expression of TGF-β (Figure 6e and f), suggesting that Wnts may promote renal fibrosis through the induction of profibrotic cytokines directly in renal stromal cells. Upregulated expression of plasminogen activator inhibitor-1 (PAI-1) is a convergent pathway that mediates the fibrogenic actions of the Wnt/β-catenin and TGF-β pathways.22He W. Tan R. Dai C. et al.Plasminogen activator inhibitor-1 is a transcriptional target of the canonical pathway of Wnt/β-catenin signaling.J Biol Chem. 2010; 285: 24665-24675Crossref PubMed Scopus (89) Google Scholar PAI-1 promotes extracellular matrix deposition, and mice lacking PAI-1 are protected against fibrosis induced by various pathogenic insults.23Huang Y. Noble N. An unexpected role of plasminogen activator inhibitor-type 1 (PAI-1) in renal fibrosis.Kidney Int. 2005; 67: 1-2Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 24Oda T. Jung Y.O. Kim H.S. et al.PAI-1 deficiency attenuates the fibrogenic response to ureteral obstruction.Kidney Int. 2001; 60: 1-10Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar As inhibition of Wnt secretion using C59 prevents the activation of Wnt/β-catenin target genes by preventing nuclear accumulation of β-catenin and decreases the expression of TGF-β, we measured renal expression of PAI-1 in the experimental groups. PAI-1 expression was high in the obstructed kidneys and was significantly decreased after treatment with C59 (Figure 6g). Thus inhibition of Wnt signaling by C59 interrupts PAI-1-mediated renal fibrogenesis. Given the magnitude of protection from renal fibrosis afforded by PORCN inhibition, we posited that profibrotic cytokines could conversely stimulate Wnt/β-catenin activity. In that case, comprehensive Wnt inhibition by C59 would interrupt a positive feedback loop in which Wnts trigger the expression of cytokines that in turn provoke generation of Wnts. We therefore assessed the capacity of exogenous TGF-β and TNF-α to regulate the expression of all 19 Wnt isoforms in primary kidney fibroblasts and a kidney epithelial cell line in vitro. Treatment with TNF-α upregulated Wnt1 and Wnt10a in the kidney epithelial cells but had no measurable effect on the expression of Wnt isoforms in kidney fibroblasts (Figure 7a and b ). By contrast, TGF-β increased the expression of Wnt5a and Wnt5b in the kidney epithelial cells and led to an upregulation of Wnt1 and Wnt10b in the kidney fibroblasts. An increase in expression of Wnt5a, Wnt4, and Wnt7a in the epithelial stromal cells of the ligated kidneys was also observed in vivo, using in situ hybridization assay (Figure 7c). In acute kidney injury models, macrophages infiltrating the kidney constitute another source of Wnt ligands.25Lin S.L. Li B. Rao S. et al.Macrophage Wnt7b is critical for kidney repair and regeneration.Proc Natl Acad Sci U S A. 2010; 107: 4194-4199Crossref PubMed Scopus (330) Google Scholar We therefore profiled the expression of Wnt ligands in the CD11b+ macrophages infiltrating the kidney in our UUO model and observed an upregulation of Wnt1, Wnt7a, and Wnt10a in these cells compared to macrophages in the contralateral kidney (Figure 7d). These data suggest that both intrinsic kidney cells and infiltrating mononuclear cells generate Wnts in the obstructed kidney. We posited that by disrupting a positive feedback cycle in which Wnt signaling promotes the expression of profibrotic cytokines that in turn induce additional Wnt expression, PORCN inhibition with C59 yields profound protection from kidney fibrosis. Consistent with that hypothesis, expression of multiple Wnts, including Wnt1, 2, 4, 7a, and 10a, that is increased in the obstructed kidneys was significantly reduced with C59 treatment (Figure 8a). By contrast, while Wnt5a levels were increased in the obstructed kidneys, treatment with C59 did not alter its expression. We also tested whether C59 treatment influences the surface abundance of Wnt receptors by immunohistochemistry, but the expression of Frizzleds was not altered with C59 treatment (data not shown). Finally, to examine if cytokine-mediated Wnt induction in intrinsic kidney cells could drive expression of Wnt target genes in those cells, we quantitated Axin2 mRNA levels in primary kidney fibroblasts or HK-2 epithelial cells exposed to TGF-β in culture. As shown in Figure 8b, TGF-β induced Axin2 in the kidney fibroblasts. Moreover, Axin2 induction was abrogated by treatment with C59 (Figure 8b). By contrast, we did not observe a significant change in Axin2 expression in epithelial cells following TGF-β or C59 treatment. Taken together, these data indicate that PORCN inhibition with C59 treatment attenuates the induction of profibrotic cytokines, reducing Wnt expression and disrupting a pathogenic positive feedback cycle to provide protection from kidney fibrosis. Wnt/β-catenin signaling plays a fundamental role in the developing organism through the regulation of diverse cell growth and proliferation pathways.26Grouls S. Iglesias D.M. 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Dai C. et al.Blockade of Wnt/β-catenin signaling by paricalcitol ameliorates proteinuria and kidney injury.J Am Soc Nephrol. 2011; 22: 90-103Crossref PubMed Scopus (231) Google Scholar Nevertheless, these antagonists are not capable of concomitantly interrupting the actions of all 19 Wnt isoforms signaling via canonical and non-canonical pathways. Consistent with an important role for canonical Wnt signaling in kidney fibrosis, our study shows that blocking all Wnt secretion with PORCN inhibition profoundly inhibits the progression of kidney fibrosis in the UUO model. The striking efficacy of PORCN inhibition is likely mediated by its effect on a diverse cascade of profibrotic signaling pathways in multiple cell lineages. Inhibition of Wnt secretion minimized the susceptibility to scarring in the obstructed kidney both by limiting the renal accumulation of fibroblasts and by restoration of gene expression of solute transporters expressed throughout the nephron, thus preserving normal tubular structure and function. One of the most profound effects of inhibiting the Wnt secretion was the subduing of the proinflammatory milieu composed of key profibrotic cytokines such as TGF-β, interleukin-1β, and TNF-α, all of which have been implicated in the pathogenesis of kidney scarring.32Meldrum K.K. Misseri R. Metcalfe P. et al.TNF-α neutralization ameliorates obstruction-induced renal fibrosis and dysfunction.Am J Physiol Regul Integr Comp Physiol. 2006; 292: R1456-R1464Crossref PubMed Scopus (75) Google Scholar, 33Border W.A. Noble N.A. 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