Wnt signaling in kidney development and disease

Abstract

The Wnt gene family, which encodes secreted growth and differentiation factors, has been implicated in kidney organogenesis. The Wnts control both ureteric bud development and signalling, but they also serve as inductive factors to regulate nephrogenesis in the mesenchcymal cells. Several of the Wnt genes are expressed in the developing kidney, and gene knock-out studies have revealed specific developmental functions for these. Consistent with this, changes in Wnt ligands and pathway components are associated with many kidney diseases, including kidney cancers, renal fibrosis, cystic kidney diseases, acute renal failure, diabetic nephropathy and ischaemic injury. It is these associations of the Wnt signalling system with kidney development and kidney diseases that form to topic of this review.

Introduction

The permanent mammalian kidney, or metanephros, is a typical organ that develops on the basic of interactions between epithelial and mesenchymal tissues. Kidney development initiates when the epithelial Wolffian duct generates the ureteric bud at its extreme caudal end. The ureteric duct then grows into the predetermined kidney mesenchyme, starts to branch and induces nephrogenesis in a sequential manner in the metanephric blastema, leading via mesenchymal epithelial transition and simple morphogenesis steps to formation of the nephrons. Following these processes the endothelial cells are required for glomerulogenesis in order to form a functional nephron.1

Several studies have revealed a critical role for the Wnt family of secreted growth and differentiation factors in kidney development. Many of the Wnts are expressed in the specific tissue compartment during kidney ontogeny, and Wnt knock-outs performed on gene-targeted embryonic stem (ES) cell-derived embryos have revealed critical roles for the Wnts in certain developmental steps. Consequently, the Wnt pathway is expected to be involved in many kidney diseases.

The Wnt Signaling Pathway

Wnts are thought to signal via at least three distinct pathways. The canonical pathway involves the critical cytoplasmic protein β-catenin.2 In the absence of Frizzled (Fz) receptor binding of a Wnt, cytoplasmic protein β-catenin is phosphorylated by glycogen synthase kinase 3 (GSK3) and degraded by a destruction complex composed of the adenomatous polyposis coli (APC) protein and axin. The most typical Wnt signaling occurs when a Wnt binds to a Fz and a low density lipoprotein (LRP) co-receptor, leading to the phosphorylation of dishevelled (dvl) protein in the cytoplasm. The activated dvl binds to axin in order to antagonize the action of GSK3β, thus preventing phosphorylation and ubiquitination of β-catenin. This causes unphosphorylated β-catenin to accumulate in the cytoplasm and translocate to the nucleus along with the TCF/LEF family of HMG-domain transcription factors, thus regulating the Wnt target genes. The biochemistry of the non-canonical Wnt signaling pathways is less clear at present (see the Wnt web page www.stanford.edu/∼rnusse/wntwindow.html).2,3

Wnt Expression and Function During Kidney Organogenesis

A number of Wnt family members are expressed in the embryonic kidney (for a more detailed discussion of their expression and function, see a recent review by Carroll et al., 2007).4 Observations made so far suggest that Wnt-2b, -4, -5b, -6, -7b, -9b and -11 are expressed during kidney ontogeny.5–9 Wnt-6, -7b, -9b and 11 are all expressed in the branching ureteric bud during the early stages of organogenesis, while Wnt-2b and -4 are transcribed in the kidney mesenchymal cells.5,6 In vivo gene inactivation studies with ES cells have revealed the roles for Wnt-4, 9b and 11. The Wnts expressed in the ureteric bud may have some redundant roles, while Wnt-4 appears to be the only one of the currently characterized Wnts that is expressed in the nephron precursor cells, being important for their development to nephrons.6 Of the epithelial Wnts, Wnt-11 is involved in the control of ureteric bud branching,8 while Wnt-9b signaling is implicated as a ureteric bud-derived inducer of nephrogenesis.9

Role of Wnt Signaling in Kidney Cancers

Given the similarities between embryonic growth control, dysregulated cell proliferation in development of cancer and the findings that Wnt signaling regulates kidney organogenesis, the Wnts were candidates for involvement in the development of kidney cancers. The adult kidney cancers, referred to collectively as renal cell carcinoma (RCC), account for around 3% of the malignancies observed in humans (reviewed in ref. 10). Metastases are typically observed in 25–30% of the RCC patients, 75% of these being found in the lungs or lymph nodes (36%). The survival rate for patients with metastases is close to 48% one year after diagnosis but only 9% after five years (reviewed in ref. 10). Because of our rather poor understanding of the promoting molecular factors, surgery is still the most efficient treatment for RCC.

Several studies have raised the possibility that the Wnt signaling pathway may be implicated in RCC. One link may be provided by hypoxia-inducible protein-2 (HIG2), a recently identified marker of RCC detected early in its onset, especially in the case of clear cell RCC and papillary cell carcinomas.11 A gain of HIG2 function stimulates cell proliferation, which suggests an oncogenic potential for this molecule. HIG2 binds to the extracellular domain of the Wnt receptor Fzd10, leading to induction of certain genes typically associated with enhanced Wnt signaling.11 In addition to this, the β-catenin/Tcf4 complex binds to the promoter of the HIG2 gene, which suggests that the HIG2 itself may be a target of Wnt-mediated regulation. Given these associations, it has been speculated that a humanized anti-HIG2 monoclonal antibody may provide a potential therapy for RCC via the modulation of Wnt signaling.11

Apart from the indirect association of Wnt signaling with RCC, changes in the Wnt Frizzled receptors have also been implicated in this form of cancer, since Fzd5 and Fzd8 gene expression is increased, possibly promoting susceptibility of the RCC cells to the Wnt ligands expressed by them.12 Changes in Wnt signaling may also occur via lowered expression of the secreted antagonists of Wnt ligands, such as the frizzled-related proteins (FRPs). The levels of secretion of frizzled-related protein 1 (sFRP1) are indeed reduced in RCC.13

This reduced sFRP1 expression correlates especially well with the clear renal cell carcinoma (cRCC) type of RCC,14 and levels of sFRP1 mRNA have been found to be reduced in human cRCC samples taken at different stages of the disease. The reduction in sFRP1 is still more striking at the protein level, since sFRP1 protein was lost in more than 70% of the samples analyzed, this not being the case in normal samples.15 The reason for the reduction in sFRP1 expression may lie in epigenetic changes in the sFRP2 gene, since the promoter of sFRP1 was methylated in 8/10 of the patients' samples analyzed in the study, apparently inactivating the sFRP-1 gene.15 Consistent with the reduction in sFRP-1 expression, the typical Wnt target genes are in turn upregulated in these cRCCs, including cyclin D1, c-myc, VEGF and fibronectin, which were also reduced in the presence of excess sFRP1. The function of sFRP-1 may normally be to inhibit cell proliferation, but reduced sFRP-1 expression may result in enhanced cell proliferation.15 It is also striking that injected sFRP1 significantly reduces tumor size in the mouse model for RCC and can even abolish the tumors completely in some cases. These findings together suggest that changes in Wnt antagonists are critical for the development of RCC.

In addition to the factors that modulate Wnt signaling at the receptor level, the cytoplasmic Wnt pathway components may also be involved in the development of RCC. This suggestion is based on the observations that compound loss-of-function mutants of p53 and Apc lead to related phenotypes typical of RCC.16 The kidney cells of Ah Cre⁺ Apc^fl/flp53^-/- mice, for example, had more pronounced nuclear staining for β-catenin than controls, suggesting promoted canonical Wnt signaling. Hence loss of synergy between p53 and the Wnt signaling component Apc protein appears to be connected with the development of RCC in the rodent model.16

In addition to the role of the Wnts in the positive control of cell proliferation in RCC, Wnt signaling may contribute to the development of the disease by influencing apoptosis.17 These properties of the Wnt pathway may be mediated in part by the splicing isoforms of the TCF, since the lack of exon 15 in one human TCF is associated with the probability of survival in RCC patients. These splice forms may function by controlling the expression of proteins involved in apoptosis. The presence of the specific isoforms of TCF in RCC, for example, is associated with reduced levels of expression of the anti-apoptotic factors Bcl-2 and Bcl-x_L and the pro-apoptotic factor Bak.17 These findings should be modelled in mice in order to obtain a better view of the roles of specific isoforms of TCFs in the generation of RCC. Collectively, the findings of various studies have implicated that changes in the Wnt signaling pathway are associated with the development of RCCs at several levels.

Significance of the Wnt Signaling Pathway for the Development of Wilms' Tumor

There is conclusive evidence that Wnt signaling not only influences kidney cancer development in the adult but is also involved in the prognosis for the foetal kidney tumors, namely Wilms' tumor. Also known as nephroplastoma, this is a cancer that is typically observed in children, 80% of diagnosed patients being younger than five years (reviewed in ref. 18). Like RCCs, Wilms' tumor can generate metastases to the lungs (85%), lymph nodes and liver. The Wilms' tumor protein (WT) I mutations lying behind some Wilms' tumor cases can lead to other diseases such as the WAGR and Drash syndromes, while a mutation in another WT protein, WT2, lies behind the Beckwith-Wiedemann syndrome (reviewed in ref. 18). Even though the role of WT proteins in Wilms' tumor is well established, the mutations in these do not explain the whole array of observed kidney tumor cases, and additional mutations, e.g., in chromosomes 1 and 12, are likely to be involved (reviewed in ref. 18).

The association of the Wnt pathway with Wilms' tumors has become clear in recent years. Typically mutations that change the coding region for β-catenin have been detected,19 and a mutation in the β-catenin gene leading to stabilization of the protein is a typical finding in 75% of the Wilms' tumor cases analyzed involving a WT1 mutation.20 Associations with the human CTNNB1 gene mutations that also encode β-catenin and with WT1 mutations also lie behind rhabdomyogenesis.21 Since β-catenin mutations are typically associated with Wilms' tumors, screening for these mutations could lead to an earlier diagnosis in Wilms' tumor patients.22

In addition to mutations in the WT proteins, the “Wilms' tumor gene in the X chromosome” (WTX) undergoes mutation in approximately 30% of Wilms' tumors.23 Mutations in WTX, including ones in the WT-I and β-catenin genes, are typically detected in sporadic Wilms' tumors.23 The familiar Wilms' tumor mutation represents only 1% of cases.

The function of the WTX protein is also connected with the canonical Wnt β-catenin signaling.24 In the HEK293T cells, for example, WTX interacts both with the wild-type of β-catenin and with the stabilized form, and it can negatively regulate β-catenin, apparently by inducing its degradation.24

Wilms' tumorigenesis may also involve enhanced expression of the Wnt ligands, since the WT-1 protein acts as a transcription factor connected with the regulation of Wnt-4 gene expression.25 Out of the four splicing isoforms that are expressed during mouse kidney development,26 the most common mutations associated with the Denys-Drash syndrome occur in the zinc-finger region of the WT1-C isoform protein, affecting DNA binding activity. This mutant WT1-C interacts with the normal WT1 sites in a dominant-negative fashion27 and reduces the expression of the Wnt-4 gene in model cells.25 The mode of action of WT-I in controlling the Wnt-4 gene, given that both are critical regulators of nephrogenesis during embryogenesis,28 remains to be analyzed in detail.

Association of the Wnt Signaling Pathway with Invasive Behavior of Renal Cells

The association of Wnt signals with ureteric bud branching during kidney development represents a situation in which an epithelial tissue is invading the mesenchymal cells as they are transforming to epithelial cells.29 Given these associations, the Wnt signaling pathway may also play a role in invasion by renal cancer cells, which is critical for the development of their metastasis potential. Indeed, overexpression of dishevelled-2 (Dvl-2) and axin having only the GSK-3β binding domain increases transcription of the LEF/TCF reporter in kidney- associated transformed model cells, as expected. Interestingly, when these cells were manipulated with respect to Wnt signaling by means of GSK-3β inhibitors this promoted their invasiveness with respect to the type 1 collagen matrix in a dose-dependent manner. The ligand that may regulate cell invasiveness is Wnt-2. This suggestion is based on the findings that the HEK-293T cells that had been engineered to express the Wnt-2 protein invaded the type 1 collagen matrix more efficiently than did the controls. Cell invasiveness probably involves GSK function, since RNAi against GSK-3β almost completely prevented such Wnt-2-induced invasion.30 Taken together, these observations raise the possibility that the Wnt signaling pathway may be connected with the invasive behavior of transformed cells, but precisely defined in vivo kidney cancer disease models are required in order to address this possibility in more detail.

Wnt Signaling in Kidney Diseases Involving Cystogenesis

There are indications that deregulated Wnt signaling is connected with the development of cystic kidney diseases. When a patient has a frame shift mutation in the gene for the Wnt pathway component TCF2 this will be associated with enlarged kidneys displaying cysts, reduced renal function and pancreatic hypoplasia that can already be diagnosed in utero.31 These phenotypes are connected with elevated β-catenin expression, suggesting that the TCF2 frame shift mutation leads to enhanced Wnt signaling that contributes to the disease phenotypes. The mechanism that may promote cystogenesis in the kidney at the cellular level may involve not only changes in the canonical Wnt signaling but also alterations in cell adhesion and planar cell polarity in the collecting duct, which can be regulated by Wnt signaling as well in some contexts.31

Some other cytoplasmic Wnt pathway components as well as TCF have been implicated in the generation of cysts in the kidney. Ksp-Cre; Apc^580S/580S mice, in which Apc function is conditionally inactivated in the collecting duct epithelium, frequently die, probably due to kidney dysfunction, while some surviving Apc knock-out mice have been reported to have cysts, nephroblastoma and renal adenoma.32 The cysts may originate from some portion of the nephron or collecting duct, but this remains to be defined in detail.

Cystogenesis has also been induced by the expression of dominant- positive β-catenin in the kidney,33 in which case it is connected with high levels of ΔN131-β-catenin expression, as lower levels do not induce cystogenesis in the same way.33 The renal cysts in this mouse model may originate from either the glomerulus, the tubules of the nephron or the collecting duct, but this remains to be analyzed by in vivo fate mapping approaches.

Changes in Wnt signaling may not necessarily be connected only with polycystic kidney disease, as studies with mouse models are also starting to reveal that this pathway may contribute to renal medullary cystic dysplasias as well. This idea is based on the finding that the level of β-catenin expression is increased in a related mouse model. It has been speculated that such changes in the Wnt signaling pathway may be connected with changes in bone morphogenetic protein (BMP) signaling, since β-catenin can form a complex with the BMP pathway component SMAD1, so that these may perhaps be jointly involved.34 Taken together, the mouse disease models of renal cystogenesis that have been generated so far are expected to serve as useful tools for addressing the detailed mechanisms by which changes in Wnt signaling contribute to the generation of cystic diseases of the kidney, perhaps providing new avenues for developing novel therapies.

Connection of Wnt Signaling with Renal Fibrosis

The formation of fibrotic lesions in the kidney involves distinct transition points that are well characterized at the cellular level, including the epithelial-to-mesenchymal transition (EMT) (reviewed in ref. 35), which is likely to bear a close similarity to the process of mesenchymal-to-epithelial transition (MET) that takes place during normal kidney development and is regulated by Wnt signaling, namely Wnt-4.28,36 There is some circumstantial evidence that the Wnt pathway is indeed connected with renal fibrosis, based on the finding of nuclear accumulation of β-catenin during induction of the EMT in association with induced fibrosis response.37

Additional observations suggest roles for Wnt signaling in renal fibrosis. Besides β-catenin, expression of the Wnt inhibitors Dkk1 and sFRP is increased in an animal model of renal fibrosis in which the kidney has a unilateral obstruction.38 More specifically, sFRP4 accumulates in the perivasculature of the kidney but in spite of this the total amount of protein in the affected kidney decreases. This may be due to the reduced potential of the damaged kidney cells for utilizing the circulating sFRP4.38 Interestingly, when recombinant sFRP4 was injected into the kidney it transiently downregulated the β-catenin and fibronectin levels and also reduced the number of myofibroblasts associated with the development of renal fibrosis, the latter suggesting that secreted Wnt inhibitors may suppress the development of an induced fibrosis response. Other studies involving chemically induced renal fibrosis have suggested further roles for Wnt-4 in the fibrotic disease process.39–41 The potential beneficial roles of Wnt-4 in experimental fibrosis may be connected with the functions of matrilysin.41 Collectively, inhibition of the action of the Wnt pathway may contribute to renal fibrosis, perhaps by controlling the EMT.38 Whether these associations are significant would need to be verified in studies with conditional knock-out mouse models.

Renal Wnt Signaling in Acute Renal Failure and Diabetes

Ischaemia can lead to severe acute renal failure, and it is important to develop new ways to manage this condition, including exploitation of the induced cell proliferation connected with the regenerative response to ischaemia. Wnt-4 expression has been shown in a rat model to be increased after ischaemia, to co-localize with aquaporin-1 expression in the proximal tubules and to be downregulated after perfusion. The action of Wnt-4 may be to promote cell proliferation, perhaps via the control of cyclin D1 in the tubular cells, in order to promote recovery from ischaemia.42

In addition to the roles of the Wnt-4 signal, β-catenin and TCF/LEF-1 seem to be associated with recovery from sub-lethal renal ischaemic injury, since both β-catenin and TCF proteins transiently accumulate in the nucleus in response to ischaemia and the action of catenins (α and β) and E-cadherin may concurrently be translocated from the basolateral cellular location to be connected more closely with the cytoskeleton and may involve Wnt signaling.43

A few studies have started to raise the possibility that changes in Wnt signaling may also be connected with diabetic nephropathy, a condition that severely compromises kidney function. Cultured mesangial cells exposed to high glucose become apoptotic, thus proliferating less in association with lower Wnt-1, -3a, -4 and -5a expression. This glucose-induced apoptosis can be reduced by overexpression of Wnt-4 or -5a in association with increased β-catenin stability.44 Collectively, the available evidence raises the possibility that Wnt signaling may be associated with both acute experimental ischaemia and diabetic nephropathy, but more precisely defined functional studies with conditional knock-out or gain-of-function mouse models will certainly be needed in order to reveal these possibilities in more detail.

The research carried out so far is starting to suggest that changes in Wnt signaling are connected with the development of several severe kidney diseases. Following the discovery that Wnt signals play a critical role in controlling the tubular ureteric bud and nephron differentiation processes, the pathway has been a candidate for association with the prognosis for kidney diseases. Our current sophisticated functional genomic mouse technologies mean that these associations can be now studied in detail to reveal the causal mechanisms behind the disease process. The discovery that the Wnt signaling pathway is connected with the development of kidney diseases should provide ways of generating new therapies for these kidney diseases in the future.

Abbreviations

ADPKD	=	autosomal dominant polycystic kidney disease
ARPKD	=	autosomal recessive polycystic kidney disease
ALK3	=	activin-like kinase 3
Apc	=	adenomatous polyposis coli
Bmp	=	bone morphogenic protein
CTNNB1	=	gene encoding β-catenin
DDS	=	denysdrash syndrome
Dkk	=	dickkopf
FZD	=	frizzled
GAD1	=	glutamic acid decarboxylase
GSK-3β	=	glycogen synthase kinase
HEK	=	human embryonic kidney cells
HGKD	=	hypoplastic glomerulocystic kidney disease
HIG-2	=	hypoxia-inducible protein-2
HNF-1β	=	hepatocyte nuclear factor
jck	=	juvenile cystic kidneys
MADH	=	methylamine dehydrogenase
MDCK	=	madin-darby canine kidney epithelial cells
MMP	=	matrilysin, matrix metalloproteinase
sFRP	=	secreted frizzled-related protein
TCF	=	T-cell factor
UUO	=	unilateral ureteric obstruction
WAGR syndrome	=	wilms' tumor, aniridia, genitourinary abnormalities, mental retardation
WTX	=	wilms' tumor gene in the X chromosome

Figures and Tables

Figure 1 Schematic representation of the expression pattern of the Wnt family members in the embryonic kidney. The Wnt-6, -7b, -9b and -11 genes are expressed in the epithelial ureteric bud that will form the future collecting duct of the kidney. Of these, Wnt-9b serves as a signal mediating reciprocal inductive signalling from the ureteric bud to the kidney mesenchyme to trigger nephrogenesis, while Wnt-11 controls ureteric bud branching morphogenesis to create the collecting duct tree. Wnt-4 is the only Wnt family member that has so far been found to be expressed in the pretubular mesenchymal cells. Wnt-4 is essential for controlling nephrogenesis. Wnt-2b gene is transiently expressed in the perinephric mesenchyme and may control ureteric branching from the mesenchymal periphery.5

Table 1 Association of the Wnt pathway components with kidney diseases

RCC	sFRP1,13,45 Fzd5 and Fzd8,12 HIG2 and Fzd10,11 β-catenin and HIG2 promoter,11 p53 and Apc,16 TCF17
Wilms' tumor	β-catenin,19,20,22 WTX,23,24 Wnt-4 and WT-125–28
Cell invasiveness	Wnt-2 and/or GSK-3β30
Disease cysts	TCF2,31 Apc,32 dominant positive β-catenin,33 Wnt and BMP pathways34
Renal fibrosis	β-catenin,37 Wnt inhibitors,38 Wnt-4,39,40 Wnt-4 and matrilysin41
Recovery from ischaemia	Wnt-4,42 β-catenin and TCF43
Survival of high glucose-stressed mesangial cells	GSK-3β, Wnt4 and Wnt5a44