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Proteinuric chronic kidney disease (CKD) remains a major health problem worldwide. While it is well established that the progression of primary glomerular disease induces tubulointerstitial lesions, how tubular injury triggers glomerular damage is poorly understood. We hypothesized that injured tubules secrete mediators that adversely affect glomerular health. To test this, we used conditional knockout mice with tubule-specific ablation of β-catenin (Ksp-β-cat-/-) and subjected them to chronic angiotensin II (Ang II) infusion or Adriamycin. Compared with control mice, Ksp-β-cat-/- mice were dramatically protected from proteinuria and glomerular damage. MMP-7, a downstream target of β-catenin, was upregulated in treated control mice, but this induction was blunted in the Ksp-β-cat-/- littermates. Incubation of isolated glomeruli with MMP-7 ex vivo led to nephrin depletion and impaired glomerular permeability. Furthermore, MMP-7 specifically and directly degraded nephrin in cultured glomeruli or cell-free systems, and this effect was dependent on its proteolytic activity. In vivo, expression or infusion of exogenous MMP-7 caused proteinuria, and genetic ablation of MMP-7 protected mice from Ang II-induced proteinuria and glomerular injury. Collectively, these results demonstrate that β-catenin-driven MMP-7 release from renal tubules promotes glomerular injury via direct degradation of the key slit diaphragm protein nephrin.

Activation of β-catenin, the intracellular mediator of canonical Wnt signaling, has a critical role in mediating podocyte injury and proteinuria. However, the underlying mechanisms remain poorly understood. Here, we show that β-catenin triggers ubiquitin-mediated protein degradation of Wilms' tumor 1 (WT1) and functionally antagonizes its action. In mice injected with adriamycin, WT1 protein was progressively lost in glomerular podocytes at 1, 3, and 5 weeks after injection. Notably, loss of WT1 apparently did not result from podocyte depletion but was closely associated with upregulation of β-catenin. This change in WT1/β-catenin ratio was accompanied by loss of podocyte-specific nephrin, podocalyxin, and synaptopodin and acquisition of mesenchymal markers Snail1, α-smooth muscle actin, and fibroblast-specific protein 1. In vitro, overexpression of β-catenin induced WT1 protein degradation through the ubiquitin proteasomal pathway, which was blocked by MG-132. WT1 and β-catenin also competed for binding to common transcriptional coactivator CREB-binding protein and mutually repressed the expression of their respective target genes. In glomerular miniorgan culture, activation of β-catenin by Wnt3a repressed WT1 and its target gene expression. In vivo, blockade of Wnt/β-catenin signaling by endogenous antagonist Klotho induced WT1 and restored podocyte integrity in adriamycin nephropathy. These results show that β-catenin specifically targets WT1 for ubiquitin-mediated degradation, leading to podocyte dedifferentiation and mesenchymal transition. Our data also suggest that WT1 and β-catenin have opposing roles in podocyte biology, and that the ratio of their expression levels dictates the state of podocyte health and disease in vivo., (Copyright © 2015 by the American Society of Nephrology.)

Activation of the renin-angiotensin system (RAS) plays an essential role in the pathogenesis of CKD and cardiovascular disease. However, current anti-RAS therapy only has limited efficacy, partly because of compensatory upregulation of renin expression. Therefore, a treatment strategy to simultaneously target multiple RAS genes is necessary to achieve greater efficacy. By bioinformatics analyses, we discovered that the promoter regions of all RAS genes contained putative T-cell factor (TCF)/lymphoid enhancer factor (LEF)-binding sites, and β-catenin induced the binding of LEF-1 to these sites in kidney tubular cells. Overexpression of either β-catenin or different Wnt ligands induced the expression of all RAS genes. Conversely, a small-molecule β-catenin inhibitor ICG-001 abolished RAS induction. In a mouse model of nephropathy induced by adriamycin, either transient therapy or late administration of ICG-001 abolished established proteinuria and kidney lesions. ICG-001 inhibited renal expression of multiple RAS genes in vivo and abolished the expression of other Wnt/β-catenin target genes. Moreover, ICG-001 therapy restored expression of nephrin, podocin, and Wilms' tumor 1, attenuated interstitial myofibroblast activation, repressed matrix expression, and inhibited renal inflammation and fibrosis. Collectively, these studies identify all RAS genes as novel downstream targets of Wnt/β-catenin. Our results indicate that blockade of Wnt/β-catenin signaling can simultaneously repress multiple RAS genes, thereby leading to the reversal of established proteinuria and kidney injury., (Copyright © 2015 by the American Society of Nephrology.)

Aging is an independent risk factor for CKD, but the molecular mechanisms that link aging and CKD are not well understood. The antiaging protein Klotho may be an endogenous antagonist of Wnt/β-catenin signaling, which promotes fibrogenesis, suggesting that loss of Klotho may contribute to CKD through increased Wnt/β-catenin activity. Here, normal adult kidneys highly expressed Klotho in the tubular epithelium, but various models of nephropathy exhibited markedly less expression of Klotho. Loss of Klotho was closely associated with increased β-catenin in the diseased kidneys, suggesting an inverse correlation between Klotho and canonical Wnt signaling. In vitro, both full-length and secreted Klotho bound to multiple Wnts, including Wnt1, Wnt4, and Wnt7a. Klotho repressed gene transcription induced by Wnt but not by active β-catenin. Furthermore, Klotho blocked Wnt-triggered activation and nuclear translocation of β-catenin, as well as the expression of its target genes in tubular epithelial cells. Investigating potential mediators of Klotho loss in CKD, we found that TGF-β1 suppressed Klotho expression and concomitantly activated β-catenin; conversely, overexpression of Klotho abolished fibrogenic effects of TGF-β1. In two mouse models of CKD induced by unilateral ureteral obstruction or adriamycin, in vivo expression of secreted Klotho inhibited the activation of renal β-catenin and expression of its target genes. Secreted Klotho also suppressed myofibroblast activation, reduced matrix expression, and ameliorated renal fibrosis. Taken together, these results suggest that Klotho is an antagonist of endogenous Wnt/β-catenin activity; therefore, loss of Klotho may contribute to kidney injury by releasing the repression of pathogenic Wnt/β-catenin signaling.

Activation of β-catenin, the principal mediator of canonical Wnt signaling, is a common pathologic finding in a wide variety of chronic kidney diseases (CKD). While β-catenin is induced predominantly in renal tubular epithelium in CKD, surprisingly, depletion of tubular β-catenin had little effect on the severity of renal fibrosis. Interestingly, less apoptosis was detected in interstitial fibroblasts in knockout mice, which was accompanied by a decreased expression of Bax and Fas ligand (FasL). Tubule-specific knockout of β-catenin diminished renal induction of matrix metalloproteinase (MMP-7), which induced FasL expression in interstitial fibroblasts and potentiated fibroblast apoptosis in vitro. These results demonstrate that loss of tubular β-catenin resulted in enhanced interstitial fibroblast survival due to decreased MMP-7 expression. Our studies uncover a novel role of the tubular β-catenin/MMP-7 axis in controlling the fate of interstitial fibroblasts via epithelial-mesenchymal communication.

β-Catenin is a unique intracellular protein functioning as an integral component of the cell-cell adherens complex and a principal signaling protein mediating canonical Wnt signaling. Little is known about its function in adult kidneys in the normal physiologic state or after acute kidney injury (AKI). To study this, we generated conditional knockout mice in which the β-catenin gene was specifically disrupted in renal tubules (Ksp-β-cat-/-). These mice were phenotypically normal with no appreciable defects in kidney morphology and function. In the absence of β-catenin, γ-catenin functionally substituted for it in E-cadherin binding, thereby sustaining the integrity of epithelial adherens junctions in the kidneys. In AKI induced by ischemia reperfusion or folic acid, the loss of tubular β-catenin substantially aggravated renal lesions. Compared with controls, Ksp-β-cat-/- mice displayed higher mortality, elevated serum creatinine, and more severe morphologic injury. Consistently, apoptosis was more prevalent in kidneys of the knockout mice, which was accompanied by increased expression of p53 and Bax, and decreased phosphorylated Akt and survivin. In vitro activation of β-catenin by Wnt1 or stabilization of β-catenin protected tubular epithelial cells from apoptosis, activated Akt, induced survivin, and repressed p53 and Bax expression. Hence, endogenous β-catenin is pivotal for renal tubular protection after AKI by promoting cell survival through multiple mechanisms.

A variety of chronic kidney diseases exhibit reactivation of Wnt/β-catenin signaling. In some tissues, β-catenin transcriptionally regulates matrix metalloproteinase-7 (MMP-7), but the association between MMP-7 and Wnt/β-catenin signaling in chronic kidney disease is unknown. Here, in mouse models of both obstructive nephropathy and focal segmental glomerulosclerosis (adriamycin nephropathy), we observed upregulation of MMP-7 mRNA and protein in a time-dependent manner. The pattern and extent of MMP-7 induction were positively associated with Wnt/β-catenin signaling in these models. Activation of β-catenin through ectopic expression of Wnt1 promoted MMP-7 expression in vivo, whereas delivery of the gene encoding the endogenous Wnt antagonist Dickkopf-1 abolished its induction. Levels of MMP-7 protein detected in the urine correlated with renal Wnt/β-catenin activity. Pharmacologic blockade of Wnt/β-catenin signaling by paricalcitol inhibited MMP-7 expression in diseased kidneys and reduced the levels detected in the urine. In vitro, β-catenin activation induced the expression and secretion of MMP-7 and promoted the binding of T cell factor to the MMP-7 promoter in kidney epithelial cells. We also observed higher levels of MMP-7 expression, which correlated with β-catenin, in kidney tissue from patients with various nephropathies. In summary, levels of renal MMP-7 correlate with Wnt/β-catenin activity, and urinary MMP-7 may be a noninvasive biomarker of this profibrotic signaling in the kidney.

Transforming growth factor-β1 (TGF-β1) upregulation occurs in virtually all chronic kidney diseases and is associated with podocyte injury and proteinuria; however, the mechanisms contributing to this in vivo are ambiguous. In vitro, incubation of podocytes with TGF-β1 induced Wnt1 expression, β-catenin activation, and stimulated the expression of Wnt/β-catenin downstream target genes. Ectopic expression of Wnt1 or β-catenin mimicked TGF-β1, induced Snail1, and suppressed nephrin expression. The Wnt antagonist, Dickkopf-1, blocked TGF-β1-induced β-catenin activation, Snail1 induction, and nephrin suppression. In vivo, ectopic expression of TGF-β1 induced Wnt1 expression, activated β-catenin, and upregulated Wnt target genes such as Snail1, MMP-7, MMP-9, desmin, Fsp1, and PAI-1 in mouse glomeruli, leading to podocyte injury and albuminuria. Consistently, concomitant expression of Dickkopf-1 gene abolished β-catenin activation, inhibited TGF-β1-triggered Wnt target gene expression, and mitigated albuminuria. Thus, canonical Wnt/β-catenin signaling mediates TGF-β1-driven podocyte injury and proteinuria. These studies suggest that Wnt/β-catenin signaling may be exploited as a therapeutic target for the treatment of proteinuric kidney diseases.

Because fibrotic kidneys exhibit aberrant activation of β-catenin signaling, this pathway may be a potential target for antifibrotic therapy. In this study, we examined the effects of β-catenin activation on tubular epithelial-mesenchymal transition (EMT) in vitro and evaluated the therapeutic efficacy of the peptidomimetic small molecule ICG-001, which specifically disrupts β-catenin-mediated gene transcription, in obstructive nephropathy. In vitro, ectopic expression of stabilized β-catenin in tubular epithelial (HKC-8) cells suppressed E-cadherin and induced Snail1, fibronectin, and plasminogen activator inhibitor-1 (PAI-1) expression. ICG-001 suppressed β-catenin-driven gene transcription in a dose-dependent manner and abolished TGF-β1-induced expression of Snail1, PAI-1, collagen I, fibronectin, and α-smooth muscle actin (α-SMA). This antifibrotic effect of ICG-001 did not involve disruption of Smad signaling. In the unilateral ureteral obstruction model, ICG-001 ameliorated renal interstitial fibrosis and suppressed renal expression of fibronectin, collagen I, collagen III, α-SMA, PAI-1, fibroblast-specific protein-1, Snail1, and Snail2. Late administration of ICG-001 also effectively attenuated fibrotic lesions in obstructive nephropathy. In conclusion, inhibiting β-catenin signaling may be an effective approach to the treatment of fibrotic kidney diseases.

Plasminogen activator inhibitor-1 (PAI-1) is a multifunctional glycoprotein that plays a critical role in the pathogenesis of chronic kidney and cardiovascular diseases. Although transforming growth factor (TGF)-beta1 is a known inducer of PAI-1, how it controls PAI-1 expression remains enigmatic. Here we investigated the mechanism underlying TGF-beta1 regulation of PAI-1 in kidney tubular epithelial cells (HKC-8). Surprisingly, overexpression of Smad2 or Smad3 in HKC-8 cells blocked PAI-1 induction by TGF-beta1, whereas knockdown of them sensitized the cells to TGF-beta1 stimulation, suggesting that Smad signaling is not responsible for PAI-1 induction. Blockade of several TGF-beta1 downstream pathways such as p38 MAPK or JNK, but not phosphatidylinositol 3-kinase/Akt and ERK1/2, only partially inhibited PAI-1 expression. TGF-beta1 stimulated beta-catenin activation in tubular epithelial cells, and ectopic expression of beta-catenin induced PAI-1 expression, whereas inhibition of beta-catenin abolished its induction. A functional T cell factor/lymphoid enhancer-binding factor-binding site was identified in the promoter region of the PAI-1 gene, which interacted with T cell factor upon beta-catenin activation. Deletion or site-directed mutation of this site abolished PAI-1 response to beta-catenin or TGF-beta1 stimulation. Similarly, ectopic expression of Wnt1 also activated PAI-1 expression and promoter activity. In vivo, PAI-1 was induced in kidney tubular epithelia in obstructive nephropathy. Delivery of Wnt1 gene activated beta-catenin and promoted PAI-1 expression after obstructive injury, whereas blockade of Wnt/beta-catenin signaling by Dickkopf-1 gene inhibited PAI-1 induction. Collectively, these studies identify PAI-1 as a direct downstream target of Wnt/beta-catenin signaling and demonstrate that PAI-1 induction could play a role in mediating the fibrogenic action of this signaling.

Wnts compose a family of signaling proteins that play an essential role in kidney development, but their expression in adult kidney is thought to be silenced. Here, we analyzed the expression and regulation of Wnts and their receptors and antagonists in normal and fibrotic kidneys after obstructive injury. In the normal mouse kidney, the vast majority of 19 different Wnts and 10 frizzled receptor genes was expressed at various levels. After unilateral ureteral obstruction, all members of the Wnt family except Wnt5b, Wnt8b, and Wnt9b were upregulated in the fibrotic kidney with distinct dynamics. In addition, the expression of most Fzd receptors and Wnt antagonists was also induced. Obstructive injury led to a dramatic accumulation of beta-catenin in the cytoplasm and nuclei of renal tubular epithelial cells, indicating activation of the canonical pathway of Wnt signaling. Numerous Wnt/beta-catenin target genes (c-Myc, Twist, lymphoid enhancer-binding factor 1, and fibronectin) were induced, and their expression was closely correlated with renal beta-catenin abundance. Delivery of the Wnt antagonist Dickkopf-1 gene significantly reduced renal beta-catenin accumulation and inhibited the expression of Wnt/beta-catenin target genes. Furthermore, gene therapy with Dickkopf-1 inhibited myofibroblast activation; suppressed expression of fibroblast-specific protein 1, type I collagen, and fibronectin; and reduced total collagen content in the model of obstructive nephropathy. In summary, these results establish a role for Wnt/beta-catenin signaling in the pathogenesis of renal fibrosis and identify this pathway as a potential therapeutic target.