Limits...
Wnt/ β -Catenin Signaling Mediated-UCH-L1 Expression in Podocytes of Diabetic Nephropathy

View Article: PubMed Central - PubMed

ABSTRACT

Increasing studies identified podocyte injury as a key early risk factor resulting in diabetic nephropathy (DN). The ubiquitin carboxy-terminal hydrolase 1 (UCH-L1) participates in podocyte differentiation and injury, which is elevated in the podocytes of a variety of nephritis. Whether UCH-L1 expression is positively related to podocyte injury of DN remains unclear. In this study, elevated expression of UCH-L1 and its intrinsic mechanism in high glucose (HG)-stimulated murine podocytes were investigated using western blot and real-time quantitative PCR. Kidney biopsies of DN patients and health individuals were stained by immunofluorescence (IF) method. The morphological and functional changes of podocytes were tested by F-actin staining and cell migration assay. Results demonstrated that HG induced upregulation of UCH-L1 and activation of the Wnt/β-catenin signaling pathway in podocytes. However, blocking of the Wnt pathway by dickkopf related protein 1 (DKK1) eliminated the above changes. Furthermore, IF staining confirmed that, compared with healthy individuals, the expression of UCH-L1 and β-catenin were obviously increased in kidney biopsy of DN patients. Overexpression of UCH-L1 remodeled its actin cytoskeleton, increased its cell migration and impacted its important proteins. All the findings manifested that Wnt/β-catenin/UCH-L1 may be a new potential therapy method in the treatment of DN in future.

No MeSH data available.


Inhibition of the Wnt/β-catenin pathway downregulated HG-induced UCH-L1 expression. Podocytes were stimulated with HG or HG + DKK1 (200 ng/mL) for 48 h. (A) Western blot assay of UCH-L1 protein level; (B) corresponding statistic histogram of UCH-L1 protein expression; (C) western blot assay of Wnt5a and β-catenin protein level; (D) corresponding statistic histogram of Wnt5a and β-catenin expression. Data representative of three independent experiments. * p < 0.05, ** p < 0.01 compared to control group. #p < 0.05 compared to HG + DKK1 group.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5037684&req=5

ijms-17-01404-f003: Inhibition of the Wnt/β-catenin pathway downregulated HG-induced UCH-L1 expression. Podocytes were stimulated with HG or HG + DKK1 (200 ng/mL) for 48 h. (A) Western blot assay of UCH-L1 protein level; (B) corresponding statistic histogram of UCH-L1 protein expression; (C) western blot assay of Wnt5a and β-catenin protein level; (D) corresponding statistic histogram of Wnt5a and β-catenin expression. Data representative of three independent experiments. * p < 0.05, ** p < 0.01 compared to control group. #p < 0.05 compared to HG + DKK1 group.

Mentions: In the transformed cells, β-catenin can upregulate the expression of endogenous UCH-L1 mRNA and protein [18]. However, it is unknown whether the canonical Wnt/β-catenin signaling mediates high glucose induced upregulation of UCH-L1 in podocytes. To provide the intrinsic mechanism of UCH-L1 upregulation in HG-stimulated podocytes, podocyte cells were treated with HG for 24 or 48 h, after preincubated with or without recombinant Dickkopf-1 (DKK1) protein, a secreted Wnt antagonist that can specially block the canonical Wnt signaling [19]. As shown in Figure 3A,B, preincubation with DKK1 obviously reduced UCH-L1 expression in HG-stimulated podocytes at the protein levels by 48 h. Meanwhile, western blot results demonstrated that the expression of Wnt5a and β-catenin were obviously elevated in HG-stimulated podocytes. However, preincubation with DKK1 significantly lowed the expression of the aforementioned two proteins (Figure 3C,D), which confirmed that the canonical Wnt/β-catenin pathway was really blocked and such blockage reduced UCH-L1. All the above results suggested an important upstream role for Wnt/β-catenin in mediating the upregulation of UCH-L1 in HG environment.


Wnt/ β -Catenin Signaling Mediated-UCH-L1 Expression in Podocytes of Diabetic Nephropathy
Inhibition of the Wnt/β-catenin pathway downregulated HG-induced UCH-L1 expression. Podocytes were stimulated with HG or HG + DKK1 (200 ng/mL) for 48 h. (A) Western blot assay of UCH-L1 protein level; (B) corresponding statistic histogram of UCH-L1 protein expression; (C) western blot assay of Wnt5a and β-catenin protein level; (D) corresponding statistic histogram of Wnt5a and β-catenin expression. Data representative of three independent experiments. * p < 0.05, ** p < 0.01 compared to control group. #p < 0.05 compared to HG + DKK1 group.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037684&req=5

ijms-17-01404-f003: Inhibition of the Wnt/β-catenin pathway downregulated HG-induced UCH-L1 expression. Podocytes were stimulated with HG or HG + DKK1 (200 ng/mL) for 48 h. (A) Western blot assay of UCH-L1 protein level; (B) corresponding statistic histogram of UCH-L1 protein expression; (C) western blot assay of Wnt5a and β-catenin protein level; (D) corresponding statistic histogram of Wnt5a and β-catenin expression. Data representative of three independent experiments. * p < 0.05, ** p < 0.01 compared to control group. #p < 0.05 compared to HG + DKK1 group.
Mentions: In the transformed cells, β-catenin can upregulate the expression of endogenous UCH-L1 mRNA and protein [18]. However, it is unknown whether the canonical Wnt/β-catenin signaling mediates high glucose induced upregulation of UCH-L1 in podocytes. To provide the intrinsic mechanism of UCH-L1 upregulation in HG-stimulated podocytes, podocyte cells were treated with HG for 24 or 48 h, after preincubated with or without recombinant Dickkopf-1 (DKK1) protein, a secreted Wnt antagonist that can specially block the canonical Wnt signaling [19]. As shown in Figure 3A,B, preincubation with DKK1 obviously reduced UCH-L1 expression in HG-stimulated podocytes at the protein levels by 48 h. Meanwhile, western blot results demonstrated that the expression of Wnt5a and β-catenin were obviously elevated in HG-stimulated podocytes. However, preincubation with DKK1 significantly lowed the expression of the aforementioned two proteins (Figure 3C,D), which confirmed that the canonical Wnt/β-catenin pathway was really blocked and such blockage reduced UCH-L1. All the above results suggested an important upstream role for Wnt/β-catenin in mediating the upregulation of UCH-L1 in HG environment.

View Article: PubMed Central - PubMed

ABSTRACT

Increasing studies identified podocyte injury as a key early risk factor resulting in diabetic nephropathy (DN). The ubiquitin carboxy-terminal hydrolase 1 (UCH-L1) participates in podocyte differentiation and injury, which is elevated in the podocytes of a variety of nephritis. Whether UCH-L1 expression is positively related to podocyte injury of DN remains unclear. In this study, elevated expression of UCH-L1 and its intrinsic mechanism in high glucose (HG)-stimulated murine podocytes were investigated using western blot and real-time quantitative PCR. Kidney biopsies of DN patients and health individuals were stained by immunofluorescence (IF) method. The morphological and functional changes of podocytes were tested by F-actin staining and cell migration assay. Results demonstrated that HG induced upregulation of UCH-L1 and activation of the Wnt/&beta;-catenin signaling pathway in podocytes. However, blocking of the Wnt pathway by dickkopf related protein 1 (DKK1) eliminated the above changes. Furthermore, IF staining confirmed that, compared with healthy individuals, the expression of UCH-L1 and &beta;-catenin were obviously increased in kidney biopsy of DN patients. Overexpression of UCH-L1 remodeled its actin cytoskeleton, increased its cell migration and impacted its important proteins. All the findings manifested that Wnt/&beta;-catenin/UCH-L1 may be a new potential therapy method in the treatment of DN in future.

No MeSH data available.