Paracrine Wnt1 Drives Interstitial Fibrosis without Inflammation by Tubulointerstitial Cross-Talk.
ABSTRACT: AKI with incomplete epithelial repair is a major contributor to CKD characterized by tubulointerstitial fibrosis. Injury-induced epithelial secretion of profibrotic factors is hypothesized to underlie this link, but the identity of these factors and whether epithelial injury is required remain undefined. We previously showed that activation of the canonical Wnt signaling pathway in interstitial pericytes cell autonomously drives myofibroblast activation in vivo. Here, we show that inhibition of canonical Wnt signaling also substantially prevented TGF?-dependent myofibroblast activation in vitro. To investigate whether Wnt ligand derived from proximal tubule is sufficient for renal fibrogenesis, we generated a novel mouse strain with inducible proximal tubule Wnt1 secretion. Adult mice were treated with vehicle or tamoxifen and euthanized at 12 or 24 weeks postinjection. Compared with vehicle-treated controls, kidneys with tamoxifen-induced Wnt1 expression from proximal tubules displayed interstitial myofibroblast activation and proliferation and increased matrix protein production. PDGF receptor ?-positive myofibroblasts isolated from these kidneys exhibited increased canonical Wnt target gene expression compared with controls. Notably, fibrotic kidneys had no evidence of inflammatory cytokine expression, leukocyte infiltration, or epithelial injury, despite the close histologic correlation of each with CKD. These results provide the first example of noninflammatory renal fibrosis. The fact that epithelial-derived Wnt ligand is sufficient to drive interstitial fibrosis provides strong support for the maladaptive repair hypothesis in the AKI to CKD transition.
Project description:Acute kidney injury (AKI) is increasingly recognized as a cumulative risk factor for chronic kidney disease (CKD) progression. However, the underlying mechanisms remain unclear. Using an aristolochic acid (AA)-induced mouse model of AKI-to-CKD transition, we found that the development of tubulointerstitial fibrosis following AKI was accompanied with a strong activation of miR-21 and canonical Wnt signaling, whereas inhibition of miR-21 or selective silencing of Wnt ligands partially attenuated AKI-to-CKD transition. To explore the interaction between miR-21 and Wnt/β-catenin signaling, we examined the effects of genetic absence or pharmacologic inhibition of miR-21 on Wnt/β-catenin pathway expression. In miR-21<sup>-/-</sup> mice and in wild-type mice treated with anti-miR21 oligos, Wnt1 and Wnt4 canonical signaling in the renal tissue was significantly reduced, with partial reversal of renal interstitial fibrosis. Although the renal abundance of miR-21 remained unchanged after inhibition or activation of Wnt/β-catenin signaling, early intervention with ICG-001, a β-catenin inhibitor, significantly attenuated renal interstitial fibrosis. Moreover, early (within 24 h), but not late β-catenin inhibition after AA administration attenuated AA-induced apoptosis and inflammation. In conclusion, inhibition of miR-21 or β-catenin signaling may be an effective approach to prevent AKI-to-CKD progression.
Project description:BACKGROUND:Notch1 signalling is a stem-cell-related pathway that is essential for embryonic development, tissue regeneration and organogenesis. However, the role of Notch1 in the formation of myofibroblasts and fibrosis in kidneys following injury remains unknown. METHODS:The activity of Notch1 signalling was evaluated in fibrotic kidneys in CKD patients and in ureteral obstructive models in vivo and in cultured fibroblasts and TECs in vitro. In addition, the crosstalk of Notch1 with TGF-?1/Smad2/3 signalling was also investigated. RESULTS:Notch1 activity was elevated in fibrotic kidneys of rat models and patients with chronic kidney disease (CKD). Further study revealed that epithelial and interstitial Notch1 activity correlated with an ?-SMA-positive myofibroblastic phenotype. In vitro, injury stimulated epithelial Notch1 activation and epithelial-mesenchymal transition (EMT), resulting in matrix deposition in tubular epithelial cells (TECs). Additionally, interstitial Notch1 activation in association with fibroblast-myofibroblast differentiation (FMD) in fibroblasts mediated a myofibroblastic phenotype. These TGF-?1/Smad2/3-dependent phenotypic transitions were abolished by Notch1 knockdown or a specific antagonist, DAPT, and were exacerbated by Notch1 overexpression or an activator Jagged-1-Fc chimaera protein. Interestingly, as a major driving force behind the EMT and FMD, TGF-?1, also induced epithelial and interstitial Notch1 activity, indicating that TGF-?1 may engage in crosstalk with Notch1 signalling to trigger fibrogenesis. CONCLUSION:These findings suggest that epithelial and interstitial Notch1 activation in kidneys following injury contributes to the myofibroblastic phenotype and fibrosis through the EMT in TECs and to the FMD in fibroblasts by targeting downstream TGF-?1/Smad2/3 signalling.
Project description:Cell-cell communication <i>via</i> Wnt ligands is necessary in regulating embryonic development and has been implicated in CKD. Because Wnt ligands are ubiquitously expressed, the exact cellular source of the Wnts involved in CKD remains undefined. To address this issue, we generated two conditional knockout mouse lines in which Wntless (Wls), a dedicated cargo receptor that is obligatory for Wnt secretion, was selectively ablated in tubular epithelial cells or interstitial fibroblasts. Blockade of Wnt secretion by genetic deletion of Wls in renal tubules markedly inhibited myofibroblast activation and reduced renal fibrosis after unilateral ureteral obstruction. This effect associated with decreased activation of <i>?</i>-catenin and downstream gene expression and preserved tubular epithelial integrity. In contrast, fibroblast-specific deletion of Wls exhibited little effect on the severity of renal fibrosis after obstructive or ischemia-reperfusion injury. <i>In vitro</i>, incubation of normal rat kidney fibroblasts with tubule-derived Wnts promoted fibroblast proliferation and activation. Furthermore, compared with kidney specimens from patients without CKD, biopsy specimens from patients with CKD also displayed increased expression of multiple Wnt proteins, predominantly in renal tubular epithelium. These results illustrate that tubule-derived Wnts have an essential role in promoting fibroblast activation and kidney fibrosis <i>via</i> epithelial-mesenchymal communication.
Project description:Large epidemiological studies clearly demonstrate that multiple episodes of acute kidney injury contribute to the development and progression of kidney fibrosis. Although our understanding of kidney fibrosis has improved in the past two decades, we have limited therapeutic strategies to halt its progression. Myofibroblast differentiation and proliferation remain critical to the progression of kidney fibrosis. Although canonical Wnt signaling can trigger the activation of myofibroblasts in the kidney, mediators of Wnt inhibition in the resident progenitor cells are unclear. Recent studies demonstrate that the loss of a Krüppel-like factor 15 (KLF15), a kidney-enriched zinc-finger transcription factor, exacerbates kidney fibrosis in murine models. Here, we tested whether Klf15 mRNA and protein expression are reduced in late stages of fibrosis in mice that underwent unilateral ureteric obstruction, a model of progressive renal fibrosis. Knockdown of Klf15 in Foxd1-expressing cells (Foxd1-Cre Klf15fl/fl) increased extracellular matrix deposition and myofibroblast proliferation as compared to wildtype (Foxd1-Cre Klf15+/+) mice after three and seven days of ureteral obstruction. This was validated in mice receiving angiotensin II treatment for six weeks. In both these murine models, the increase in renal fibrosis was found in Foxd1-Cre Klf15fl/fl mice and accompanied by the activation of Wnt/?-catenin signaling. Furthermore, knockdown of Klf15 in cultured mouse embryonic fibroblasts activated canonical Wnt/?-catenin signaling, increased profibrotic transcripts, and increased proliferation after treatment with a Wnt1 ligand. Conversely, the overexpression of KLF15 inhibited phospho-?-catenin (Ser552) expression in Wnt1-treated cells. Thus, KLF15 has a critical role in attenuating kidney fibrosis by inhibiting the canonical Wnt/?-catenin pathway.
Project description:AKI is increasingly recognized as a major risk factor for progression to CKD. However, the factors governing AKI to CKD progression are poorly understood. In this study, we investigated this issue using moderate (20 minutes) and severe (30 minutes) ischemia/reperfusion injury (IRI) in mice. Moderate IRI led to acute kidney failure and transient Wnt/?-catenin activation, which was followed by the restoration of kidney morphology and function. However, severe IRI resulted in sustained and exaggerated Wnt/?-catenin activation, which was accompanied by development of renal fibrotic lesions characterized by interstitial myofibroblast activation and excessive extracellular matrix deposition. To assess the role of sustained Wnt/?-catenin signaling in mediating AKI to CKD progression, we manipulated this signaling by overexpression of Wnt ligand or pharmacologic inhibition of ?-catenin. In vivo, overexpression of Wnt1 at 5 days after IRI induced ?-catenin activation and accelerated AKI to CKD progression. Conversely, blockade of Wnt/?-catenin by small molecule inhibitor ICG-001 at this point hindered AKI to CKD progression. In vitro, Wnt ligands induced renal interstitial fibroblast activation and promoted fibronectin expression. However, activated fibroblasts readily reverted to a quiescent phenotype after Wnt ligands were removed, suggesting that fibroblast activation requires persistent Wnt signaling. These results indicate that sustained, but not transient, activation of Wnt/?-catenin signaling has a decisive role in driving AKI to CKD progression.
Project description:The activation of Wnt/?-catenin pathway plays a pivotal role in promoting renal fibrosis. The activation of Wnt/?-catenin pathway relies on the binding of Wnts to Frizzled receptors on cell membrane. However, the factor regulating Wnts production remains unclear. Here, we demonstrated that transcriptional factor FoxM1 was significantly increased in obstructed kidneys and patients' kidneys with fibrosis. The up-regulation of FoxM1 mainly distributed in tubular epithelial cells. Pharmacological inhibition of FoxM1 down-regulated multi-Wnts elevation in UUO mice and attenuated renal fibrosis. In cultured renal tubular epithelial cells, overexpression of FoxM1 promoted 8 Wnts expression, while knock-down on FoxM1-suppressed multi-Wnts including Wnt1, Wnt2b and Wnt3 expression induced by Ang II. Chromatin immunoprecipitation PCR confirmed that FoxM1 bound to Wnt1, Wnt2b, Wnt3 promoters and luciferase assay further identified that the transcriptions of Wnt1, Wnt2b and Wnt3 were regulated by FoxM1. Thus, our findings show that multi-Wnt family members were regulated by transcriptional factor FoxM1. FoxM1 might be a key switch for activating ?-catenin pathway and renal fibrosis. Therefore, FoxM1 might be a potential therapeutic target in manipulating renal fibrosis.
Project description:Chronic tubulointerstitial damage with tubular epithelial atrophy and interstitial fibrosis is the hallmark of chronic kidney disease (CKD) and a predictor for progression of CKD.Several experiments have now provided evidence that the Wnt signaling pathways are significantly contributing to atrophy and fibrosis; in contrast, it also has been shown that the Wnt system fosters regenerative processes in acute tubular injury.We now have demonstrated that Dickkopf 3 (DKK3) is an agonist for canonical Wnt signaling in CKD and fosters chronic fibrosing inflammation of the tubulointerstitial compartment. Genetic- and antibody-mediated inhibition of DKK3 leads to a pronounced improvement of tubular differentiation and a reduction in fibrosis.In addition, the secreted glycoprotein DKK3 can be used as a non-invasive urinary marker for the extent of CKD in man.
Project description:<h4>Background</h4>Fibrosis is a major cause of loss of renal function in autosomal dominant polycystic kidney disease (ADPKD). In this study, we examined whether vasopressin type-2 receptor (V2R) activity in cystic epithelial cells can stimulate interstitial myofibroblasts and fibrosis in ADPKD kidneys.<h4>Methods</h4>We treated <i>Pkd1</i> gene knockout (<i>Pkd1</i>KO) mice with dDAVP, a V2R agonist, for 3 days and evaluated the effect on myofibroblast deposition of extracellular matrix (ECM). We also analyzed the effects of conditioned media from primary cultures of human ADPKD cystic epithelial cells on myofibroblast activation. Because secretion of the profibrotic connective tissue growth factor (CCN2) increased significantly in dDAVP-treated <i>Pkd1</i>KO mouse kidneys, we examined its role in V2R-dependent fibrosis in ADPKD as well as that of yes-associated protein (YAP).<h4>Results</h4>V2R stimulation using dDAVP increased the renal interstitial myofibroblast population and ECM deposition. Similarly, conditioned media from human ADPKD cystic epithelial cells increased myofibroblast activation <i>in vitro</i>, suggesting a paracrine mechanism. Renal collecting duct-specific gene deletion of <i>CCN2</i> significantly reduced cyst growth and myofibroblasts in <i>Pkd1</i>KO mouse kidneys. We found that YAP regulates <i>CCN2</i>, and YAP inhibition or gene deletion reduces renal fibrosis in <i>Pkd1</i>KO mouse kidneys. Importantly, YAP inactivation blocks the dDAVP-induced increase in myofibroblasts in <i>Pkd1</i>KO kidneys. Further <i>in vitro</i> studies showed that V2R regulates YAP by an ERK1/2-dependent mechanism in human ADPKD cystic epithelial cells.<h4>Conclusions</h4>Our results demonstrate a novel mechanism by which cystic epithelial cells stimulate myofibroblasts in the pericystic microenvironment, leading to fibrosis in ADPKD. The V2R-YAP-CCN2 cell signaling pathway may present a potential therapeutic target for fibrosis in ADPKD.
Project description:Renal tubular atrophy and interstitial fibrosis are common hallmarks of etiologically different progressive chronic kidney diseases (CKD) that eventually result in organ failure. Even though these pathological manifestations constitute a major public health problem, diagnostic tests, as well as therapeutic options, are currently limited. Members of the dickkopf (DKK) family, DKK1 and -2, have been associated with inhibition of Wnt signaling and organ fibrosis. Here, we identify DKK3 as a stress-induced, tubular epithelia-derived, secreted glycoprotein that mediates kidney fibrosis. Genetic as well as antibody-mediated abrogation of DKK3 led to reduced tubular atrophy and decreased interstitial matrix accumulation in two mouse models of renal fibrosis. This was facilitated by an amplified, antifibrogenic, inflammatory T cell response and diminished canonical Wnt/?-catenin signaling in stressed tubular epithelial cells. Moreover, in humans, urinary DKK3 levels specifically correlated with the extent of tubular atrophy and interstitial fibrosis in different glomerular and tubulointerstitial diseases. In summary, our data suggest that DKK3 constitutes an immunosuppressive and a profibrotic epithelial protein that might serve as a potential therapeutic target and diagnostic marker in renal fibrosis.
Project description:Interstitial fibrosis is an inevitable outcome of all kinds of progressive chronic kidney disease (CKD). Emerging data indicate that rapamycin can ameliorate kidney fibrosis by reducing the interstitial infiltrates and accumulation of extra cellular matrix (ECM). However, the cellular mechanism that regulates those changes has not been well understood yet. In this study, we revealed the persistent activation of mammalian target of rapamycin (mTOR) signaling in the interstitial macrophages and myofibroblasts, but rarely in injured proximal epithelial cells, CD4+ T cells, neutrophils, or endothelial cells, during the development of kidney fibrosis. Administration of rapamycin to unilateral ureteral obstruction (UUO) mice significantly suppressed the immunoreactivity of mTOR signaling, which decreased the inflammatory responses and ECM accumulation in the obstructed kidneys. Isolated macrophages from rapamycin-treated obstructed kidneys presented less inflammatory activity than vehicle groups. In vitro study confirmed that rapamycin significantly inhibited the fibrogenic activation of cultured fibroblasts (NIH3T3 cells), which was induced by the stimulation of TGF-?(1). Further experiment revealed that rapamycin did not directly inhibit the fibrogenesis of HK2 cells with aristolochic acid treatment. Our findings clarified that rapamycin can ameliorate kidney fibrosis by blocking the mTOR signaling in interstitial macrophages and myofibroblasts.