Implication of dysregulation of the canonical wingless-type MMTV integration site (WNT) pathway in diabetic nephropathy
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The wingless-type MMTV integration site (WNT) pathway mediates multiple physiological and pathological processes, such as inflammation, angiogenesis and fibrosis. The aim of this study was to investigate whether canonical WNT signalling plays a role in the pathogenesis of diabetic nephropathy.
Expression of WNT ligands and frizzled receptors in the canonical WNT pathway in the kidney was compared at the mRNA level using real-time RT-PCR between Akita mice, streptozotocin-induced diabetic rats and db/db mice and their respective non-diabetic controls. Renal function was evaluated by measuring the urine albumin excretion. Human renal proximal tubular epithelial cells were treated with high-glucose medium and 4-hydroxynonenal (HNE). Levels of β-catenin, connective tissue growth factor and fibronectin were determined by western blot analysis.
Some of the WNT ligands and frizzled receptors showed increased mRNA levels in the kidneys of Akita mice, streptozotocin-induced diabetic rats and db/db mice compared with their non-diabetic controls. Renal levels of β-catenin and WNT proteins were upregulated in these diabetic models. Lowering the blood glucose levels by insulin attenuated the activation of WNT signalling in the kidneys of Akita mice. In cultured human renal proximal tubular epithelial cells, both high glucose and HNE activated WNT signalling. Inhibition of WNT signalling with a monoclonal antibody blocking LDL-receptor-related protein 6 ameliorated renal inflammation and fibrosis and reduced proteinuria in Akita mice.
The WNT pathway is activated in the kidneys of models of both type 1 and 2 diabetes. Dysregulation of the WNT pathway in diabetes represents a new pathogenic mechanism of diabetic nephropathy and renders a new therapeutic target.
KeywordsDiabetic nephropathy Fibrosis Insulin Kidney Oxidative stress WNT
Adenovirus expressing β-galactosidase
Adenovirus expressing a constitutively active mutant of β-catenin
Connective tissue growth factor
Human renal proximal tubular epithelial cells
Intercellular adhesion molecule 1
Plain L cell conditioned medium
Vascular endothelial growth factor
Wingless-type MMTV integration site
Diabetic nephropathy, one of the most common diabetic complications, is the leading cause of end-stage renal disease, and its prevalence is increasing worldwide . The renal lesions in type 1 and type 2 diabetes are similar , and involve multiple cell types, including glomerular podocytes, mesangial cells and tubular epithelial cells . Although the glomerulus has been the focus of intensive investigation, recently tubulo-interstitial injury has drawn more attention and interest in diabetic nephropathy research [4, 5].
Hyperglycaemia-induced AGE, cytokines, chemokines, growth factors and oxidative stress have been shown to mediate renal damage in human and experimental diabetes [1, 6]. Hyperglycaemia works through both metabolic and haemodynamic mediators to activate multiple signalling pathways and transcription factors, resulting in renal inflammation, angiogenesis, extracellular matrix accumulation/fibrosis, apoptosis/nephrin loss, proteinuria, hyperfiltration and eventual renal insufficiency .
The wingless-type MMTV integration site (WNT) signalling pathway is a multifunctional pathway that regulates cell proliferation and differentiation, stem cell maintenance, angiogenesis, inflammation, fibrosis and carcinogenesis . WNT ligands are secreted cysteine-rich glycosylated proteins that bind to a receptor complex comprised of frizzled (FZD) receptors and LDL-receptor-related protein (LRP) 5 or 6. Activated LRP5 or LRP6 transduces the signal from extracellular ligands to the intracellular cascade and leads to inactivation of the ‘destructive complex’, which is composed of glycogen synthase kinase-3β (GSK3β), axin and adenomatous polyposis coli. Inactivation of the ‘destructive complex’ prevents the proteasomal degradation of the transcription factor β-catenin and promotes its accumulation and nuclear translocation. Once β-catenin translocates into the nucleus, it dimerises with T cell factor (TCF) and regulates transcription of WNT target genes, including those encoding cyclin D1, vascular endothelial growth factor (VEGF), c-Myc and connective tissue growth factor (CTGF) [8, 9, 10].
Our previous studies have shown that the WNT pathway is overactivated in retinas from human patients with diabetic retinopathy and in those from animal models of diabetes . Furthermore, blockage of WNT signalling attenuates retinal inflammation and neovascularisation in diabetic retinopathy models . Documented studies suggest that the presence of either retinopathy or nephropathy may contribute to the development of the other, especially in type 1 diabetic patients . More importantly, WNT signalling has been shown to be a key regulator of kidney development  and has been implicated in certain kidney diseases [14, 15, 16]. It is reasonable to hypothesise that the WNT signalling pathway is implicated in the pathogenesis of diabetic nephropathy.
The present study investigates the pathogenic role of the canonical WNT pathway in diabetic nephropathy and identifies the cause for the activation of the WNT pathway in the kidneys of experimental diabetic models.
Materials and antibodies
l-glucose and d-glucose were purchased from Sigma (St. Louis, MO, USA), and 4-hydroxynonenal (HNE) was purchased from Calbiochem (Madison, WI, USA). Antibodies against β-catenin, cyclin D1, fibronectin and CTGF were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies against WNT3A and c-Myc were from Cell Signaling (Danvers, MA, USA). Antibodies against β-actin and horseradish peroxidase-conjugated secondary antibodies were obtained from Abcam (Cambridge, MA, USA). Conditioned media containing WNT3A were prepared from mouse L cells stably producing WNT3A (ATCC, Manassas, VA, USA). Control conditioned media were obtained from parental L cells (ATCC).
Animal models of diabetes
Male Akita mice and db/db mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and female Brown Norway rats were purchased from Harlan (Indianapolis, IN, USA). Care, use and treatment of all animals in this study were in strict agreement with the Guidelines for the Care and Use of Laboratory Animals set forth by the University of Oklahoma. Experimental diabetes was induced in the rats as described by Chen et al. .
Akita mice (blood glucose ≥19.5 mmol/l) were implanted with subcutaneous insulin pellets (LinShin Canada, Toronto, ON, Canada), according to the manufacturer’s protocol, to lower blood glucose levels for 8 weeks. Blood glucose levels were monitored weekly. Animals with poor glycaemic control received additional insulin pellets.
Monoclonal antibody 2F1 was raised using a recombinant peptide comprising the E1E2 domain of LRP6. The antibody was purified from a hybridoma cell line by affinity chromatography using a protein G column (Thermo Scientific, Waltham, MA, USA) and from the ascites fluid of BALB/c mice injected with the 2F1 hybridoma cells. The purified antibody was dialysed against PBS, sterile filtered and administrated intraperitoneally. Non-specific mouse IgG purchased from Vector Laboratories (Burlingame, CA, USA) was subjected to the same procedure.
Total RNA was isolated from kidneys using TRIzol according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). The cDNA was reverse transcribed using a reverse transcription reagent kit (Applied Biosystems, Foster City, CA, USA) and quantitative RT-PCR was conducted as described by Wang et al. .
Primary human renal proximal tubular epithelial cells (HRPTC) were purchased from ATCC and cultured according to the protocol recommended by ATCC.
Kidney tissue and cells were fractionated using FractPrep (BioVision, Mountain View, CA, USA) following the manufacturer’s protocol.
Western blot analysis
Western blot analysis was performed as described by Zhou et al. .
Immunocytochemistry was performed as described by Zhou et al. . Immunohistochemistry was performed as described by Chen et al. . Briefly, paraffin-embedded kidney sections were stained with the antibody for β-catenin at a dilution of 1:250 for 1 h. After thorough washes with PBS, immunosignals were developed using an ABC kit (Vectastain ABC; Vector Laboratories) according to the manufacturer’s protocol.
Measurement of microalbuminuria and creatinine
Mice were kept in individual metabolism cages. A 24 h urine sample was collected from each mouse. Urine albumin was measured by ELISA according to the manufacturer’s protocol (Exocell, Philadelphia, PA, USA). The total amount of albumin in the 24 h urine was calculated accordingly. Urine creatinine was measured by HPLC according to the protocol from the Animal Models of Diabetic Complications Consortium .
ELISA for fibronectin
The kidneys were dissected, homogenised and centrifuged at 800 g for 5 min. The total protein concentration in the supernatant fraction was measured using the bicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL, USA). A commercial mouse fibronectin ELISA kit (Assaypro, St Charles, MO, USA) was used to measure fibronectin levels according to the manufacturer’s instructions and normalised by total protein concentrations in the kidney.
Data are presented as mean±SD. Comparisons were performed by two-tailed Student’s t test. A difference of p < 0.05 was considered statistically significant.
Activation of the canonical WNT pathway in the kidneys of Akita mice
To confirm the upregulation of WNT ligands in the kidney, we have compared the protein levels of WNT3A, a commonly studied ligand in the canonical WNT pathway. As shown by western blot analysis, levels of WNT3A protein were also increased in the kidneys of Akita mice compared with those in the non-diabetic control mice (Fig. 1c, d). Further, to determine if the downstream WNT pathway is indeed activated, we examined the accumulation and nuclear translocation of β-catenin, a key effector of WNT signalling. As shown in Fig. 1e, f, both the cytosolic and nuclear β-catenin levels were increased significantly in the Akita kidneys, indicating the activation of β-catenin in the diabetic kidney.
Activation of the canonical WNT pathway in the kidney of streptozotocin-induced diabetic rats
Activation of the canonical WNT pathway in the kidney of db/db mice
Next, we examined β-catenin activation in the db/db kidney. Western blot analysis showed that the cytosolic and nuclear levels of β-catenin were both increased in the db/db mouse kidney compared with the age-matched non-diabetic controls (Fig. 4e, f), demonstrating the accumulation and nuclear translocation of β-catenin. Furthermore, renal levels of WNT target genes for cyclin D1, c-Myc and CTGF were increased in db/db mice compared with controls (Fig. 4g, h), reflecting activation of the WNT signalling in the kidneys of db/db mice.
Insulin treatment attenuated the activation of the canonical WNT pathway in the Akita mouse kidney
Activation of the WNT pathway by high glucose and HNE in HRPTC
Activation of WNT signalling alone induced fibrogenic factors in HRPTC
Antagonists of the WNT signalling pathway ameliorate diabetic nephropathy in Akita mice
The present study reports a novel systematic evaluation of the expression of genes encoding components of the WNT signalling pathway in the kidneys of animal models of both type 1 and type 2 diabetes. The results show that WNT signalling is overactivated in the kidneys of models of both type 1 and type 2 diabetes. In addition, hyperglycaemia and oxidative stress were found to play causative roles in the WNT pathway activation in the kidney of diabetic animals. Moreover, blockage of WNT signalling by a monoclonal antibody to LRP6 ameliorated diabetic nephropathy. These results suggest that dysregulation of the WNT pathway in the diabetic kidney plays a pathogenic role in diabetic nephropathy.
We have determined the expression levels of all known WNT genes encoding WNT ligands involved in the canonical WNT pathway. One interesting observation is that there is concurrent upregulation of multiple WNT ligands. Although different diabetic animal models show different profiles of WNT ligand upregulation, none of the canonical WNT ligands analysed was found to be downregulated in the kidney of any of the diabetic models. This suggests that most WNT ligands are positively regulated in the kidney by diabetes. Of note, the mRNA expression of Wnt3a and Wnt10b in the kidney was upregulated in both the type 1 (Akita mice and STZ-induced diabetic rats) and type 2 (db/db mice) diabetic models, indicating that induction of Wnt3a and Wnt10b expression is common in diabetic kidneys. Compared with the STZ-induced diabetic rats, Akita mice showed upregulation of more WNT ligands. In parallel, it has been reported that Akita mice develop more severe diabetic nephropathy compared with STZ-treated animals . However, it has been reported that overexpression of Wnt10b was accompanied by a reduction of plasma levels of triacylglycerol as well as improved glucose homeostasis . This disparity suggests that local Wnt10b regulation in the kidney is different from its systemic regulation.
Moreover, there are apparent differences in WNT ligand and receptor gene expression between the three rodent models. We speculated that the differences may be due to differences in species, genetic backgrounds and ages of the animal models. Different severities and durations of diabetes may also contribute to the differences in WNT ligand upregulation. In addition to diabetic nephropathy, obstructive kidney injury and ischaemia–reperfusion injury have also been shown to induce overexpression of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt8a, Wnt16, Fzd3, Fzd4, Fzd9 and Fzd10 gene expression in kidney [14, 25], suggesting that the activation of the WNT signalling pathway may be a common pathogenic mechanism for some kidney diseases.
It has been well documented that good glycaemic control reduces the risk or arrests the progression of diabetic complications [32, 33]. The present study showed that levels of blood glucose negatively correlated with renal WNT signalling activities in Akita mice treated with insulin. This result suggests a causative role of hyperglycaemia in WNT pathway activation in diabetic nephropathy. This notion is supported by the observation that high-glucose medium induces WNT signalling in cultured renal tubular epithelial cells. Reactive oxygen species are considered the common denominator and amplifier of the cellular pathway activated by hyperglycaemia [34, 35, 36]. Our previous study also showed that oxidative stress is responsible for WNT pathway activation in cultured retinal cells and in the retina of diabetic rats . Consistent with this, the present study showed that HNE activated the WNT pathway in renal tubular epithelial cells, suggesting that oxidative stress in diabetes also contributes to WNT pathway activation in the diabetic kidney.
The canonical WNT signalling pathway is known to mediate inflammation, angiogenesis and fibrosis through upregulation of intercellular adhesion molecule 1 (ICAM-1), TNF-α, VEGF and CTGF, which are all products of WNT target genes [10, 18]. It has been reported that WNT1 protein levels are increased in the podocytes of human kidney biopsies from patients with diabetic nephropathy . In addition, expression of MMP7, a WNT target gene, was strongly increased in the tubular epithelial cells in human kidneys with diabetic nephropathy . Expression of osteopontin, another WNT target gene, was induced in proximal tubular cells of diabetic rats . Compared with other cell types in the kidneys, proximal tubular cells are exposed to conditions of elevated glucose concentrations both apically, as a result of glycosuria, and basally, as a result of elevated interstitial tissue concentrations of glucose . In addition, they express most of the FZD receptors  and are the major WNT target cells . Tubular epithelial cells are the predominant cell type in the normal renal interstitium. They not only produce various pro-fibrogenic factors in response to high glucose and increased reactive oxygen species, but also serve as a target for cytokines and relay fibrogenic signals to cortical fibroblasts in the diseased kidney .
It is postulated that activated proximal tubular cells indirectly contribute to interstitial fibrosis by activation of interstitial fibroblasts. In turn, these stimulated fibroblasts amplify this response by their reciprocal action on the proximal tubular epithelial cells . In diabetic nephropathy, the expression of mesenchymal markers in tubular epithelial cells is well correlated with declining renal function . It is evident that macrophages could be a source of the WNT ligands to which epithelial cells respond in ischaemia–reperfusion injury . It was recently reported that WNT/β-catenin signalling promotes renal interstitial fibrosis after obstructive injury . However, the implication of WNT signalling in interstitial fibrosis in diabetic nephropathy is largely unknown. Here, we first reported that activation of WNT signalling alone without high glucose can induce the production of CTGF and fibronectin, which are key players in fibrosis in diabetic nephropathy [1, 6, 41]. These results render new evidence to emphasise the importance of WNT signalling and tubular cells in the fibrosis associated with diabetic nephropathy.
WNT signalling initiates when a WNT ligand binds to FZD and LRP5/6 co-receptors . As there are multiple WNT ligands and FZD receptors, but only two co-receptors—LRP5 and LRP6—we used an antibody specific for the ligand-binding domain of LRP6 to inhibit WNT signalling . Injection of this antibody into Akita mice inhibited the activation of the WNT pathway and ameliorated diabetic nephropathy as shown by fibrogenic factor production, extracellular matrix accumulation and proteinuria. Meanwhile, delivery of serine (or cysteine) peptidase inhibitor, clade A, member 3K (SERPINA3K), an endogenous antagonist of LRP6 , also reduced albuminuria in db/db mice (Electronic supplementary material [ESM] Fig. 1). These observations indicate a pathogenic role for WNT pathway activation in diabetic nephropathy.
Moreover, our study also suggests that targeting WNT signalling is a promising strategy to hinder the progression of diabetic nephropathy. It is emerging that anti-TGF-β antibody and a CTGF antisense oligonucleotide ameliorate renal fibrosis [43, 44]. Our results suggest that an antagonist of WNT signalling may have advantages over those treatments targeting individual growth factors, because WNT signalling regulates the production of a spectrum of pathogenic factors of diabetic retinopathy, including VEGF, CTGF, ICAM-1, TNF-α, Twist homologue 1 (TWIST) and matrix metalloproteinase 7 [10, 14, 37, 45]. This notion is substantiated by previous reports demonstrating that dickkopf homologue 1 (DKK1) and secreted frizzled-related protein 4 (SFRP4) were able to ameliorate renal fibrosis [14, 46].
In conclusion, our study demonstrates dysregulation of the WNT signalling pathway in diabetic nephropathy and establishes oxidative stress and hyperglycaemia as activators of the canonical WNT pathway in the kidneys of diabetic rodent models. Furthermore, blocking WNT signalling ameliorated the renal dysfunction. Therefore, our observation underscores the pivotal role of the canonical WNT pathway in the pathogenesis of diabetic nephropathy.
This study was supported by: National Institutes of Health Grants EY018659, EY012231, EY019309 and P20RR024215; and a research award from the American Diabetes Association.
All authors had substantial contribution to: conception and design, or analysis and interpretation of data; drafting the article or revising it critically for important intellectual content; and final approval of the version to be published.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
- 43.Ziyadeh FN, Hoffman BB, Han DC et al (2000) Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor-beta antibody in db/db diabetic mice. Proc Natl Acad Sci USA 97:8015–8020PubMedCrossRefGoogle Scholar
- 45.Elmarakby AA, Sullivan JC (2011) Relationship between oxidative stress and inflammatory cytokines in diabetic nephropathy. Cardiovasc Ther. doi: 10.1111/j.1755-5922.2010.00218.x