Search

Extracellular superoxide dismutase (EC-SOD), also known as SOD3, is an antioxidant expressed at high levels in normal adult kidneys. Because oxidative stress contributes to a variety of kidney injuries, we hypothesized that EC-SOD may be protective in CKD progression. To study this hypothesis, we used a murine model of ADR nephropathy characterized by albuminuria and renal dysfunction. We found that levels of EC-SOD diminished throughout the course of disease progression and were associated with increased levels of NADPH oxidase and oxidative stress markers. EC-SOD null mice were sensitized to ADR injury, as evidenced by increases in albuminuria, serum creatinine, histologic damage, and oxidative stress. The absence of EC-SOD led to increased levels of NADPH oxidase and an increase in β-catenin signaling, which has been shown to be pathologic in a variety of kidney injuries. Exposure of EC-SOD null mice to either chronic angiotensin II infusion or to daily albumin injections also caused increased proteinuria. In contrast, EC-SOD null mice subjected to nonproteinuric CKD induced by unilateral ureteral obstruction exhibited no differences compared with wild-type mice. Finally, we also found a decrease in EC-SOD in human CKD biopsy samples, similar to our findings in mice. Therefore, we conclude that EC-SOD is protective in CKDs characterized by proteinuria., (Copyright © 2015 by the American Society of Nephrology.)

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.)

Tubular epithelium constitutes the majority of the renal parenchyma and is the primary target of various kidney injuries. However, how the injured tubules drive interstitial fibroblast activation and proliferation remains poorly understood. Here, we investigated the role of sonic hedgehog (Shh), a secreted extracellular signaling protein, in fibroblast proliferation. Shh was induced in renal tubular epithelia in animal models of CKD induced by ischemia/reperfusion injury (IRI), adriamycin, or renal mass ablation, and in renal tubules of kidney biopsy specimens from CKD patients with different etiologies. Using Gli1-CreER(T2) reporter mice, we identified interstitial fibroblasts as the principal targets of renal Shh signaling in vivo. In vitro, incubation with Shh promoted normal rat kidney fibroblast proliferation, which was assessed by cell counting, MTT assay, and BrdU incorporation assay, and stimulated the induction of numerous proliferation-related genes. However, Shh had no effect on the proliferation of renal tubular epithelial cells. In vivo, overexpression of Shh promoted fibroblast expansion and aggravated kidney fibrotic lesions after IRI. Correspondingly, blockade of Shh signaling by cyclopamine, a small molecule inhibitor of Smoothened, inhibited fibroblast proliferation, reduced myofibroblast accumulation, and attenuated renal fibrosis. These studies identify Shh as a novel, specific, and potent tubule-derived growth factor that promotes interstitial fibroblast proliferation and activation. Our data also suggest that blockade of Shh signaling is a plausible strategy for therapeutic intervention of renal fibrosis., (Copyright © 2014 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.

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.

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.

Integrin-linked kinase (ILK) is an intracellular serine/threonine protein kinase that regulates cell adhesion, survival, and epithelial-to-mesenchymal transition (EMT). In this study, we investigated the kinase activity of ILK during tubular EMT induced by TGF-beta1 and examined the therapeutic potential of an ILK inhibitor in obstructive nephropathy. TGF-beta1 induced a biphasic activation of ILK in renal tubular epithelial cells, with rapid activation starting at 5 min and the second wave of activation peaking at 24 h; the latter paralleled the induction of ILK protein expression. Pharmacologic inhibition of ILK with small-molecule inhibitor QLT-0267 abolished TGF-beta1-induced phosphorylation of Akt and glycogen synthase kinase-3beta, suppressed cyclin D1 expression, and largely restored the expression of E-cadherin and zonula occludens 1. Inhibition of ILK also blocked TGF-beta1-mediated induction of fibronectin, Snail1, plasminogen activator inhibitor 1, and matrix metalloproteinase 2. In a mouse model of obstructive nephropathy, administration of QLT-0267 inhibited beta-catenin accumulation; suppressed Snail1, alpha-smooth muscle actin, fibronectin, vimentin, and type I and type III collagen expression; and reduced total tissue collagen content. Inhibition of ILK did not affect kidney structure or function in normal mice. These findings suggest that increased ILK activity mediates EMT and the progression of renal fibrosis. Pharmacologic inhibition of ILK signaling may hold therapeutic potential for fibrotic kidney diseases.

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.

PINCH-1 is an adaptor protein that binds to the integrin-linked kinase (ILK), an intracellular serine/threonine protein kinase that plays a critical role in mediating tubular epithelial-to-mesenchymal transition (EMT). To determine whether PINCH-1 is also involved in the EMT process, we investigated its regulation and function during TGF-beta1-stimulated EMT. TGF-beta1 induced PINCH-1 mRNA and protein expression in human proximal tubular epithelial cells in a time-dependent fashion, an effect that was largely dependent on intracellular Smad signaling. Overexpression of PINCH-1 suppressed epithelial markers E-cadherin and ZO-1 and increased fibronectin expression and extracellular assembly, whereas knockdown of PINCH-1 via small interfering RNA reduced TGF-beta1-mediated fibronectin expression and partially restored E-cadherin. PINCH-1 formed a ternary complex with ILK at the focal adhesion sites of tubular epithelial cells. Treatment with an ILK inhibitor or disruption of the ILK/PINCH-1 interaction by overexpressing a dominant-negative N-terminal ankyrin domain of ILK resulted in reduced fibronectin deposition, indicating that the ability of PINCH-1 to stimulate EMT is ILK-dependent. In a mouse model of obstructive nephropathy, PINCH-1 expression increased in a time-dependent manner, suggesting that it may play a role in EMT and renal fibrosis in vivo. We conclude that PINCH-1, through its interaction with ILK, plays an important role in regulating TGF-beta1-mediated EMT and could be a potential future therapeutic target to prevent progression of renal disease.

Hepatocyte growth factor (HGF) is a potent antifibrotic cytokine that antagonizes the TGF-beta1/Smad signaling in diverse types of kidney cells by different mechanisms. HGF is shown to induce Smad co-repressor Sloan-Kettering Institute proto-oncogene-related novel gene, non-Alu-containing (SnoN) expression in proximal tubular epithelial cells (HKC-8) but not in glomerular mesangial cells and interstitial fibroblasts. This study investigated the molecular mechanisms underlying the cell type-specific induction of SnoN by HGF. Treatment of HKC-8 cells with actinomycin D completely abolished HGF-mediated SnoN induction, suggesting its dependence on gene transcription. Although HGF activated several signal pathways in HKC-8 cells, blockade of extracellular signal-regulated kinase-1 and -2 (Erk-1/2) activation but not Akt and p38 mitogen-activated protein kinase abolished SnoN induction. HGF rapidly activated both upstream and downstream signaling of Erk-1/2, which led to the activation of the cAMP response element-binding protein (CREB). In the promoter region of human SnoN gene, two cAMP response elements were located in close proximity to Sp1 sites. Chromatin immunoprecipitation assay revealed that activated CREB and Sp1 bound to their cognate cis-acting elements in SnoN promoter in response to HGF stimulation. Ectopic expression of wild-type CREB promoted SnoN expression, whereas dominant negative mutant CREB abrogated SnoN induction by HGF. Likewise, chemical blockade of Sp1 binding abolished HGF-mediated SnoN induction. Furthermore, HGF selectively induced CREB phosphorylation in HKC-8 cells but not in mesangial cells and fibroblasts. In vivo, administration of HGF gene induced renal Erk-1/2 phosphorylation, CREB activation, and SnoN expression in obstructive nephropathy. Collectively, these results suggest that CREB activation, in concert with Sp1, constitutes a molecular switch that confers the cell type-specific induction of SnoN in response to HGF stimulation.

In the fibrotic kidney, tubular cells undergo epithelial-to-mesenchymal transition (EMT), a phenotypic conversion that is characterized by sequential loss of epithelial markers and gain of mesenchymal features. For understanding of the molecular mechanism that governs this process, a high-throughput gene expression microarray analysis was used to identify the critical genes in the initial phase of the TGF-beta1-mediated EMT. Inhibitor of differentiation-1 (Id1), a dominant negative antagonist of the basic helix-loop-helix transcription factors, was found to be induced rapidly in human proximal tubular epithelial cells after TGF-beta1 treatment. This induction of Id1 depended on intracellular Smad signaling. Ectopic expression of Id1 suppressed epithelial E-cadherin and zonula occludens-1 expression. Id1 physically formed complex with basic helix-loop-helix transcription factor HEB (Hela E-box binding factor), sequestered its ability to bind to E-box, and repressed the trans-activation of E-cadherin promoter. However, overexpression of Id1 failed to induce alpha-smooth muscle actin, matrix metalloproteinase-2, fibronectin, and integrin-linked kinase (ILK), indicating its inability to confer a complete EMT. Overexpression of ILK or inhibition of ILK activity had no effect on Id1 induction by TGF-beta1, suggesting that Id1 and ILK have independent roles in epithelial dedifferentiation and EMT. In vivo, Id1 was induced exclusively in the degenerated, dilated renal tubular epithelium after unilateral ureteral obstruction. These studies identify Id1 transcriptional inhibitor as a crucial player in mediating cell dedifferentiation of renal tubular epithelium and suggest that EMT is a multistep process in which loss of epithelial adhesion does not necessarily lead to an autonomous mesenchymal transition.

Deficiency in vitamin D and its active metabolites is a pathologic feature of chronic kidney diseases. Despite that tubular epithelial cells are the major sites of active vitamin D synthesis, little is known about the role of vitamin D in maintaining the structural and functional integrity of tubular epithelium. This study investigated the effects of paricalcitol (19-nor-1,25-hydroxy-vitamin D(2)), a synthetic vitamin D analogue, on obstructive nephropathy, a model that is characterized by predominant tubulointerstitial lesions. Compared with vehicle controls, paricalcitol significantly attenuated renal interstitial fibrosis in mouse kidney after ureteral obstruction, as demonstrated by a reduced interstitial volume, decreased collagen deposition, and repressed mRNA expression of fibronectin and type I and type III collagens. Paricalcitol largely preserved E-cadherin and reduced alpha-smooth muscle actin expression in vivo. In addition, paricalcitol suppressed renal TGF-beta1 and its type I receptor expression, restored vitamin D receptor abundance, and inhibited cell proliferation and apoptosis after obstructive injury. In vitro, paricalcitol abolished TGF-beta1-mediated E-cadherin suppression and alpha-smooth muscle actin and fibronectin induction in tubular epithelial cells, underscoring its ability to block directly the epithelial to mesenchymal transition (EMT). It is interesting that paricalcitol almost completely suppressed renal induction of Snail, a critical transcription factor that is implicated in EMT programming. Furthermore, paricalcitol inhibited the TGF-beta1-mediated Snail induction in vitro, and ectopic expression of Snail repressed E-cadherin promoter activity and downregulated E-cadherin expression in tubular epithelial cells. These studies suggest that paricalcitol is able to ameliorate renal interstitial fibrosis in obstructive nephropathy, possibly by preserving tubular epithelial integrity through suppression of EMT.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a ligand-dependent transcription factor that plays an important role in the regulation of insulin sensitivity and lipid metabolism. Evidence shows that PPAR-gamma agonists also ameliorate renal fibrotic lesions in both diabetic nephropathy and nondiabetic chronic kidney disease. However, little is known about the mechanism underlying their antifibrotic action. This study demonstrated that PPAR-gamma agonists could exert their actions by inducing antifibrotic hepatocyte growth factor (HGF) expression. Incubation of mesangial cells with natural or synthetic PPAR-gamma agonists 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) or troglitazone and ciglitazone suppressed TGF-beta1-mediated alpha-smooth muscle actin, fibronectin, and plasminogen activator inhibitor-1 expression. PPAR-gamma agonists also induced HGF mRNA expression and protein secretion. Transfection studies revealed that 15d-PGJ2 stimulated HGF gene promoter activity, which was dependent on the presence of a novel peroxisome proliferator response element. Treatment of mesangial cells with 15d-PGJ2 induced the binding of PPAR-gamma to the peroxisome proliferator response element in the HGF promoter region. PPAR-gamma agonists also activated c-met receptor tyrosine phosphorylation, induced Smad transcriptional co-repressor TG-interacting factor expression, and blocked TGF-beta/Smad-mediated gene transcription in mesangial cells. Furthermore, ablation of c-met receptor through the LoxP-Cre system in mesangial cells abolished the antifibrotic effect of 15d-PGJ2. PPAR-gamma activation also induced HGF expression in renal interstitial fibroblasts and repressed TGF-beta1-mediated myofibroblast activation. Both HGF and 15d-PGJ2 attenuated Smad nuclear translocation in response to TGF-beta1 stimulation in renal fibroblasts. Together, these findings suggest that HGF may act as a downstream effector that mediates the antifibrotic action of PPAR-gamma agonists.

Diabetic nephropathy is characterized by progressive loss of renal function, persistent proteinuria, and relentless accumulation of extracellular matrix leading to glomerulosclerosis and interstitial fibrosis. This study investigated the potential effects of long-term expression of exogenous hepatocyte growth factor (HGF) on normal and diabetic kidneys. Intravenous injection of human HGF gene via naked plasmid vector resulted in abundant HGF protein specifically localized in renal glomeruli, despite an extremely low level of transgene mRNA in the kidney. In uninephrectomized mice made diabetic with streptozotocin, delivery of exogenous HGF gene ameliorated the progression of diabetic nephropathy. HGF attenuated urine albumin and total protein excretion in diabetic mice. Exogenous HGF also mitigated glomerular mesangial expansion, reduced fibronectin and type I collagen deposition, and prevented interstitial myofibroblast activation. In addition, HGF prevented kidney cells from apoptotic death in the glomeruli and tubulointerstitium. Moreover, expression of HGF inhibited renal expression of TGF-beta1 and reduced urine level of TGF-beta1 protein. Therefore, despite the effects of HGF on diabetic nephropathy being controversial, these observations suggest that supplementation of HGF is beneficial in ameliorating diabetic renal insufficiency in mice.

TGF-beta1 is a profibrotic cytokine that plays a central role in the onset and progression of chronic renal diseases. The activity of TGF-beta1 is tightly controlled by multiple mechanisms, in which antagonizing Smad-mediated gene transcription by co-repressors is an important regulatory component. This study examined the expression of Smad transcriptional co-repressors in the fibrotic kidney and investigated their potential functions in controlling TGF-beta1 response. Western blot analysis demonstrated that the protein levels of Smad transcriptional co-repressors SnoN and Ski were progressively reduced in a time-dependent manner in the fibrotic kidney induced by unilateral ureteral obstruction in mice, whereas renal Smad abundance was relatively unaltered. Consistently, SnoN and Ski staining was diminished in the nuclei of renal tubular epithelium and interstitium after obstructive injury. In vitro, knockdown of SnoN expression by RNA interference in tubular epithelial cells dramatically sensitized their responsiveness to TGF-beta1 stimulation. Conversely, ectopic expression of exogenous SnoN or Ski after transfection conferred tubular epithelial cell resistance to TGF-beta1-induced epithelial to myofibroblast transition. Both SnoN and Ski could block Smad-mediated activation of TGF-beta1-responsive promoter and exhibited additive effect in abrogating the profibrotic actions of TGF-beta1. These results indicate that as a result of loss of Smad transcriptional co-repressors, the profibrotic TGF-beta1 signaling in diseased kidney is markedly amplified in a magnitude much greater than previously thought. Therefore, new strategy aimed to increase Smad transcriptional co-repressors expression may be effective in antagonizing TGF-beta1 signaling and thereby blocking the progression of chronic renal fibrosis.