The Wnt/β-catenin pathway in podocytes integrates cell adhesion, differentiation and survival

Diabetic kidney disease (DKD) is the single most common cause of albuminuria and end-stage kidney disease in the United States. We found increased expression of Wnt/β-catenin (Ctnnb1) pathway transcripts and proteins in glomeruli and podocytes of patients and mouse models of DKD. Mice with podocyte-specific expression of stabilized Ctnnb1 exhibited basement membrane abnormalities, albuminuria, and increased susceptibility to glomerular injury. Mice with podocyte-specific deletion of Ctnnb1 or podocyte-specific expression of the canonical Wnt inhibitor Dickkopf-related protein 1 (Dkk1) also showed increased susceptibility to DKD. Podocytes with stabilized Ctnnb1 were less motile and less adhesive to different matrices. Deletion of Ctnnb1 in cultured podocytes increased the expression of podocyte differentiation markers and enhanced cell motility; however, these cells were more susceptible to apoptosis. These results indicate that Wnt/Ctnnb1 signaling in podocytes plays a critical role in integrating cell adhesion, motility, cell death, and differentiation. Balanced Ctnnb1 expression is critical for glomerular filtration barrier maintenance.

The glomerular filtration barrier consists of three layers: glomerular endothelial cells, the glomerular basement membrane (GBM), and the glomerular epithelial cell (or podocyte) layer (2,3,4). Decreased glomerular podocyte density is shown to be the strongest predictor for ESRD development in patients with diabetes (5).
Hyperglycemia via the generation of reactive oxygen species induces podocyte apoptosis and loss, which has been well documented in many different mouse DKD models (6,7). As podocytes are terminally differentiated cells, they are unable to proliferate; therefore, apoptosis or detachment can lead to podocyte deficiency, which in turn will lead to glomerulosclerosis development (8,9). Early administration of drugs that prevent podocyte apoptosis has been shown to ameliorate DKD in rodent models, however, this may not be a clinically translatable strategy (6,10). Another early lesion in diabetes is the thickening of the basement membrane. The role and mechanism of GBM thickening are not fully understood. It is speculated that GBM thickening could cause alterations in integrin expression, which could interfere with podocyte adhesiveness. Genetic deletion of podocyte-specific integrins, Itgb1 and Itga3, cause albuminuria and glomerulosclerosis, however, the contribution of podocyte adhesion to DKD development has not been demonstrated (11,12). adriamycin-induced proteinuria model (18).  (19). They also proposed that sustaining Wnt/Ctnnb1 signaling is beneficial for promoting survival of high glucose stressed cells and protects mice from DKD (20). These contradictory results highlight the importance of the use of in vivo cell type-specific transgenic animals to define the role of Wnt/Ctnnb1 pathway in the glomerulus.
Here we analyzed the role of the Wnt/Ctnnb1 pathway in podocytes at baseline and in DKD. In vitro and in vivo studies indicated that Wnt/Ctnnb1 pathway plays a key role in determining podocyte differentiation, motility, cell-matrix adhesion and cell death.

EXPERIMENTAL PROCEDURES
Human kidney samples were collected from kidney biopsies and nephrectomies. The study was approved by the Institutional Board Review.
The biopsy tissue was manually microdissected at 4°C in RNALater as described previously (21).
Quantitative real-time PCR was performed as described earlier (10). Supplemental Table 4 contains primer sequences used in the study.

Statistical Analysis
Results are presented as mean standard deviation. Student t-test was used to analyze the difference between two groups.
Values were regarded significant at P <0.05.   2D). GBM abnormalities were evident as early as 4 weeks of age and became progressively severe by 20 weeks (Fig. 2D).

Activation of the canonical
Podocyte foot processes appeared normal with occasional effacement, and that was prominent   Immunofluorescence studies confirmed the decreased expression of Nphs1 in mice with podocyte-specific Ctnnb1 expression (Fig. 3L). (podocyte-specific collagen), Mmp16 and Mmp23 (Fig. 4H, I). In addition, we observed a significant decrease in Itgb1 expression following Cre recombination (Fig. 4F). Total Ilk or Fak levels did not change significantly (Fig. 4F), while phospho-Fak Tyr397 was lower following Cre recombination. These results suggest that active Ctnnb1 expression was associated with decreased adhesiveness in podocytes, which could be due to the decreased in Itgb1 expression and FAK phosphorylation (38).
We also examined whether canonical wnt ligand treatment leads to a similar phenotype. Recombinant Wnt3a treatment increased active Ctnnb1 levels, without significantly affecting total Ctnnb1 levels ( Fig.   5A). Immunofluorescence studies showed nuclear accumulation of Ctnnb1 following Wnt3a treatment (Fig. 5B) 5C). Treatment with GSK3β inhibitors also significantly lowered cell motility compared to control PBS (Fig. 5D, E). These results suggest that Wnt/Ctnnb1 activation in podocytes is associated with decreased adhesiveness and migration.  (Fig. 6A).
Kidney histology of the NPHS2 Cre /Ctnnb1 KO/KO mice appeared normal (Fig. 6B). and mice did not show signs of albuminuria even at 20 weeks of age (Fig. 6D). Electron microscopic analysis indicated minor GBM alterations, evident mainly as occasional GBM splitting (Fig. 5D).
Quantitative analysis of GBM width, however, did not reveal statistically significant differences in overall GBM thickness (Fig. 6E).
As we observed increased activation of the  (Fig. 6F), when they were sacrificed.
Control diabetic mice had only a minimal increase in albuminuria compared to nondiabetic mice (Fig. 6D) (Fig. 7B, D).
Transmission EM analysis showed occasional GBM splitting, similar to that observed in NPHS2 Cre /Ctnnb1 KO/KO mice (Fig. 7C).
Next, we studied the effect of diabetes in uninephrectomized male mice following lowdose STZ injection. At 20 weeks of age, diabetic NPHS2 rtTA / TRE -Dkk1 mice had significantly increased albuminuria and glomerulosclerosis compared to control diabetic mice (Fig. 7D, E).
These changes were similar to those observed in NPHS2 Cre /Ctnnb1 KO/KO mice. These results suggest that inhibiting podocyte Wnt signaling in the context of diabetes is deleterious.

Ctnnb1 deletion from podocytes promotes cell differentiation and adhesion, but increases apoptosis susceptibility
We generated multiple podocyte clones and Nphs2 after Ctnnb1 silencing (Fig. 8F).

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The role of wnt/β-catenin in podocytes

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Consistent with these findings, mRNA levels of podocyte-specific genes, including Wt1, Nphs1, Plce1, Pdxl and Synpo, were slightly but significantly increased in podocyte-specific Ctnnb1 knockout animals as compared to control mice (Supplemental Table 2).
Ctnnb1 KO/KO podocytes showed increased adhesiveness to type IV collagen (Fig.   8G). In addition there was a minor, but consistent increase in phospho-Fak levels following Cre infection, while total Ilk and total Fak levels were unchanged (Fig. 8H).

Further analysis indicated that cells with
Ctnnb1 deletion had increased mRNA levels of Cdkn1a, Tp53 and Apaf1 (Fig. 8I). Protein expression of Cdkn1a and phosphorylated p53 were significantly increased following Ctnnb1 deletion (Fig. 8H). Moreover, the increase in cleaved PARP levels following Ctnnb1 deletion confirmed the increased apoptotic rate (Fig. 8H).
In summary, podocyte-specific Ctnnb1 deletion in vitro was associated with increased expression of differentiation markers of podocyte (Wt1, Nphs2) and increased adhesiveness, however, these cells appeared to be more susceptible to apoptosis.          Our results indicate that the Wnt/Ctnnb1 pathway plays a key role in podocyte adhesion, motility, differentiation and survival. Increased activation of the pathway in DKD might occur to promote podocyte survival, but it also leads to cell detachment and podocyte loss. Downregulation of the