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. 2014 Feb 12;9(2):e89233.
doi: 10.1371/journal.pone.0089233. eCollection 2014.

High glucose induces podocyte injury via enhanced (pro)renin receptor-Wnt-β-catenin-snail signaling pathway

Caixia Li  1 , Helmy M Siragy  1
Affiliations

High glucose induces podocyte injury via enhanced (pro)renin receptor-Wnt-β-catenin-snail signaling pathway

Caixia Li et al. PLoS One. .

Abstract

(Pro)renin receptor (PRR) expression is upregulated in diabetes. We hypothesized that PRR contributes to podocyte injury via activation of Wnt-β-catenin-snail signaling pathway. Mouse podocytes were cultured in normal (5 mM) or high (25 mM) D-glucose for 3 days. Compared to normal glucose, high glucose significantly decreased mRNA and protein expressions of podocin and nephrin, and increased mRNA and protein expressions of PRR, Wnt3a, β-catenin, and snail, respectively. Confocal microscopy studies showed significant reduction in expression and reorganization of podocyte cytoskeleton protein, F-actin, in response to high glucose. Transwell functional permeability studies demonstrated significant increase in albumin flux through podocytes monolayer with high glucose. Cells treated with high glucose and PRR siRNA demonstrated significantly attenuated mRNA and protein expressions of PRR, Wnt3a, β-catenin, and snail; enhanced expressions of podocin mRNA and protein, improved expression and reorganization of F-actin, and reduced transwell albumin flux. We conclude that high glucose induces podocyte injury via PRR-Wnt-β-catenin-snail signaling pathway.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effect of high glucose on PRR expression in podocytes.
A. Real time PCR analysis of PRR mRNA expression in response to high glucose for 72(n = 6); B. Western blot analysis of PRR protein expression in response to high glucose for 72 hours (n = 6); C, D and E. Immunohistochemistry staining of PRR shown in brown (n = 3); F and G. Immunofluorescence staining of PRR shown in red, DAPI shown in blue (n = 5).PRR, (Pro)renin receptor; normal glucose, 5 mM D-glucose (NG); high glucose, 25 mM D-glucose (HG). Data presented as mean ± SEM, *p<0.05 vs NG
Figure 2
Figure 2. Effect of high glucose on nephrin and podocin expression in podocytes.
A. Real time PCR analysis of nephrin mRNA expression (n = 5); B. Western blot analysis of nephrin protein expression (n = 6); C. Real time PCR analysis of Podocin mRNA expression (n = 5); D. Western blot analysis of Podocin protein expression (n = 4). Normal glucose, 5 mM D-glucose (NG); high glucose, 25 mM D-glucose (HG). Data presented as mean ± SEM, *p<0.05 vs NG
Figure 3
Figure 3. Effect of high glucose on structure and function injury in podocytes.
A and B Immunofluorescence staining of F-actin showed in red, DAPI showed in blue (n = 5); C. Analysis of the albumin flux across podocytes monolayer (n = 5). Normal glucose, 5 mM D-glucose (NG); high glucose, 25 mM D-glucose (HG). Data presented as mean ± SEM, *p<0.05 vs NG
Figure 4
Figure 4. Effect of high glucose on Wnt3a-β-catenin-snail signal pathway in podocytes.
A. Real time PCR analysis of Wnt3a mRNA expression (n = 5); B. Western blot analysis of Wnt3a protein expression (n = 4); C. Real time PCR analysis of β-catenin mRNA expression (n = 6); D. Western blot analysis of β-catenin mRNA expression (n = 5); E. Real time PCR analysis of snail mRNA expression (n = 5); F. Western blot analysis of snail protein expression (n = 4); Normal glucose, 5 mM D-glucose (NG); high glucose, 25 mM D-glucose (HG). Data presented as mean ± SEM, *p<0.05 vs NG
Figure 5
Figure 5. Effect of PRR siRNA on PRR expression in podocyte upon high glucose.
A. Real time PCR analysis of PRR mRNA expression (n = 6); B. Western blot analysis of PRR protein expression (n = 5–6); PRR, (Pro)renin receptor; Normal glucose, 5 mM D-glucose (NG); high glucose, 25 mM D-glucose (HG). Black bar, Scrambled siRNA; Grey bar, PRR siRNA. Data presented as mean ± SEM, *p<0.05 vs NG+ Scrambled siRNA; #p<0.05 vs HG+ Scrambled siRNA.
Figure 6
Figure 6. Effect of PRR siRNA on Wnt3a-β-catenin-snail signal pathway in podocytes in response to high glucose.
A. Real time PCR analysis of Wnt3a mRNA expression (n = 5); B. Western blot analysis of Wnt3a protein expression (n = 6); C. Real time PCR analysis of β-catenin mRNA expression (n = 6); D. Western blot analysis of β-catenin mRNA expression (n = 6); E. Real time PCR analysis of snail mRNA expression (n = 5); F. Western blot analysis of snail protein expression (n = 4); PRR, (Pro)renin receptor; Normal glucose, 5 mM D-glucose (NG); high glucose, 25 mM D-glucose (HG). Black bar, Scrambled siRNA; Grey bar, PRR siRNA. Data presented as mean ± SEM, *p<0.05 vs NG+ Scrambled siRNA; #p<0.05 vs HG+ Scrambled siRNA.
Figure 7
Figure 7. Effect of PRR siRNA on high glucose induced podocyte structure and function.
A. Real time PCR analysis of podocin mRNA expression (n = 5); B. Western blot analysis of podocin protein expression (n = 5); C. Analysis of the albumin flux across podocytes monolayer at 2 hours (n = 4); D, E, F and G. Immunofluorescence staining of F-actin shown in red, DAPI shown in blue (n = 5). PRR, (Pro)renin receptor; Normal glucose, 5 mM D-glucose (NG); high glucose, 25 mM D-glucose (HG). Black bar, Scrambled siRNA; Grey bar, PRR siRNA. Data presented as mean ± SEM, *p<0.05 vs NG+ Scrambled siRNA; #p<0.05 vs HG+ Scrambled siRNA.
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