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Uremic toxins induce kidney fibrosis by activating intrarenal renin-angiotensin-aldosterone system associated epithelial-to-mesenchymal transition.

Sun CY, Chang SC, Wu MS - PLoS ONE (2012)

Bottom Line: IS and PCS significantly increased transforming growth factor-β1 (TGF-β1) expression and activated the TGF-β pathway by increasing Smad2/Smad2-P, Smad3/Smad3-P, and Smad4 expression.The expression of the EMT-associated transcription factor Snail was increased by IS and PCS treatment.IS and PCS may increase Snail expression and induce EMT-like transition.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Chang Gung University, Taoyuan, Taiwan.

ABSTRACT

Background: Uremic toxins are considered to have a determinant pathological role in the progression of chronic kidney disease. The aim of this study was to define the putative pathological roles of the renal renin-angiotensin-aldosterone system (RAAS) and renal tubular epithelial-to-mesenchymal transition (EMT) in kidney fibrosis induced by (indoxyl sulfate) IS and (p-cresol sulfate) PCS.

Methods: Mouse proximal renal tubular cells (PKSV-PRs) treated with IS or PCS were used. Half-nephrectomized B-6 mice were treated with IS or PCS for 4 weeks. In the losartan treatment study, the study animal was administrated with IS+losartan or PCS+losartan for 4 weeks.

Results: IS and PCS significantly activated the intrarenal RAAS by increasing renin, angiotensinogen, and angiotensin 1 (AT1) receptor expression, and decreasing AT2 receptor expression in vitro and in vivo. IS and PCS significantly increased transforming growth factor-β1 (TGF-β1) expression and activated the TGF-β pathway by increasing Smad2/Smad2-P, Smad3/Smad3-P, and Smad4 expression. The expression of the EMT-associated transcription factor Snail was increased by IS and PCS treatment. IS and PCS induced the phenotype of EMT-like transition in renal tubules by increasing the expression of fibronectin and α-smooth muscle actin and decreasing the expression of E-cadherin. Losartan significantly attenuated the expression of TGF-β1 and Snail, and decreased kidney fibrosis induced by IS and PCS in vivo.

Conclusion: Activating the renal RAAS/TGF-β pathway has an important pathological role in chronic kidney injury caused by IS and PCS. IS and PCS may increase Snail expression and induce EMT-like transition.

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Indoxyl sulfate and p-cresol sulfate activated transforming growth factor-β pathway and increased Snail expression in vitro.A: Results of western blotting show that mouse proximal renal tubular cells (PKSV) cells had significantly increased protein expression of Sma- and Mad-related protein (Smad)2/Smad2-P, Smad3/Smad3-P, and Smad4, when treated with indoxyl sulfate (IS) and p-cresol sulfate (PCS). B: Western blotting and immunostaining for Snail show that PKSV cells had significantly increased nuclolear expression of Snail, when treated with IS and PCS. In the immunostaining study of Snail, PKSV cells were treated with 5 mg/dL IS or PCS for 3 days. C: The transforming growth factor-β1 (TGF-β1) concentrations in the culture medium were measured by ELISA method. PKSV cells treated with IS (5 and 50 mg/L) and PCS (1, 5 and 50 mg/L) had significantly higher TGF-β1 concentration in culture medium than control cells. (600×) (*: P<0.05; **: P<0.01; ***: P<0.001, vs. control).
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pone-0034026-g003: Indoxyl sulfate and p-cresol sulfate activated transforming growth factor-β pathway and increased Snail expression in vitro.A: Results of western blotting show that mouse proximal renal tubular cells (PKSV) cells had significantly increased protein expression of Sma- and Mad-related protein (Smad)2/Smad2-P, Smad3/Smad3-P, and Smad4, when treated with indoxyl sulfate (IS) and p-cresol sulfate (PCS). B: Western blotting and immunostaining for Snail show that PKSV cells had significantly increased nuclolear expression of Snail, when treated with IS and PCS. In the immunostaining study of Snail, PKSV cells were treated with 5 mg/dL IS or PCS for 3 days. C: The transforming growth factor-β1 (TGF-β1) concentrations in the culture medium were measured by ELISA method. PKSV cells treated with IS (5 and 50 mg/L) and PCS (1, 5 and 50 mg/L) had significantly higher TGF-β1 concentration in culture medium than control cells. (600×) (*: P<0.05; **: P<0.01; ***: P<0.001, vs. control).

Mentions: Western blotting analysis of cultured PKSV cells showed that IS and PCS significantly increased TGF-β1 expression. The expression of downstream signals of the TGF-β pathway, such as Sma- and Mad-related protein (Smad)2/Smad2-P, Smad3/Smad3-P, and Smad4, were also increased significantly in vitro (Figure 3A). In addition, the expression of the EMT-associated transcription factor Snail was increased by IS and PCS treatment. The results of western blotting and immunostaining for Snail are shown in Figure 3B. The TGF-β1 concentrations in the culture medium were shown in the Figure 3C. The animal study revealed that chronic IS and PCS injection could significantly increase renal TGF-β1 and Snail expression in vivo (Figure 4).


Uremic toxins induce kidney fibrosis by activating intrarenal renin-angiotensin-aldosterone system associated epithelial-to-mesenchymal transition.

Sun CY, Chang SC, Wu MS - PLoS ONE (2012)

Indoxyl sulfate and p-cresol sulfate activated transforming growth factor-β pathway and increased Snail expression in vitro.A: Results of western blotting show that mouse proximal renal tubular cells (PKSV) cells had significantly increased protein expression of Sma- and Mad-related protein (Smad)2/Smad2-P, Smad3/Smad3-P, and Smad4, when treated with indoxyl sulfate (IS) and p-cresol sulfate (PCS). B: Western blotting and immunostaining for Snail show that PKSV cells had significantly increased nuclolear expression of Snail, when treated with IS and PCS. In the immunostaining study of Snail, PKSV cells were treated with 5 mg/dL IS or PCS for 3 days. C: The transforming growth factor-β1 (TGF-β1) concentrations in the culture medium were measured by ELISA method. PKSV cells treated with IS (5 and 50 mg/L) and PCS (1, 5 and 50 mg/L) had significantly higher TGF-β1 concentration in culture medium than control cells. (600×) (*: P<0.05; **: P<0.01; ***: P<0.001, vs. control).
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pone-0034026-g003: Indoxyl sulfate and p-cresol sulfate activated transforming growth factor-β pathway and increased Snail expression in vitro.A: Results of western blotting show that mouse proximal renal tubular cells (PKSV) cells had significantly increased protein expression of Sma- and Mad-related protein (Smad)2/Smad2-P, Smad3/Smad3-P, and Smad4, when treated with indoxyl sulfate (IS) and p-cresol sulfate (PCS). B: Western blotting and immunostaining for Snail show that PKSV cells had significantly increased nuclolear expression of Snail, when treated with IS and PCS. In the immunostaining study of Snail, PKSV cells were treated with 5 mg/dL IS or PCS for 3 days. C: The transforming growth factor-β1 (TGF-β1) concentrations in the culture medium were measured by ELISA method. PKSV cells treated with IS (5 and 50 mg/L) and PCS (1, 5 and 50 mg/L) had significantly higher TGF-β1 concentration in culture medium than control cells. (600×) (*: P<0.05; **: P<0.01; ***: P<0.001, vs. control).
Mentions: Western blotting analysis of cultured PKSV cells showed that IS and PCS significantly increased TGF-β1 expression. The expression of downstream signals of the TGF-β pathway, such as Sma- and Mad-related protein (Smad)2/Smad2-P, Smad3/Smad3-P, and Smad4, were also increased significantly in vitro (Figure 3A). In addition, the expression of the EMT-associated transcription factor Snail was increased by IS and PCS treatment. The results of western blotting and immunostaining for Snail are shown in Figure 3B. The TGF-β1 concentrations in the culture medium were shown in the Figure 3C. The animal study revealed that chronic IS and PCS injection could significantly increase renal TGF-β1 and Snail expression in vivo (Figure 4).

Bottom Line: IS and PCS significantly increased transforming growth factor-β1 (TGF-β1) expression and activated the TGF-β pathway by increasing Smad2/Smad2-P, Smad3/Smad3-P, and Smad4 expression.The expression of the EMT-associated transcription factor Snail was increased by IS and PCS treatment.IS and PCS may increase Snail expression and induce EMT-like transition.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Chang Gung University, Taoyuan, Taiwan.

ABSTRACT

Background: Uremic toxins are considered to have a determinant pathological role in the progression of chronic kidney disease. The aim of this study was to define the putative pathological roles of the renal renin-angiotensin-aldosterone system (RAAS) and renal tubular epithelial-to-mesenchymal transition (EMT) in kidney fibrosis induced by (indoxyl sulfate) IS and (p-cresol sulfate) PCS.

Methods: Mouse proximal renal tubular cells (PKSV-PRs) treated with IS or PCS were used. Half-nephrectomized B-6 mice were treated with IS or PCS for 4 weeks. In the losartan treatment study, the study animal was administrated with IS+losartan or PCS+losartan for 4 weeks.

Results: IS and PCS significantly activated the intrarenal RAAS by increasing renin, angiotensinogen, and angiotensin 1 (AT1) receptor expression, and decreasing AT2 receptor expression in vitro and in vivo. IS and PCS significantly increased transforming growth factor-β1 (TGF-β1) expression and activated the TGF-β pathway by increasing Smad2/Smad2-P, Smad3/Smad3-P, and Smad4 expression. The expression of the EMT-associated transcription factor Snail was increased by IS and PCS treatment. IS and PCS induced the phenotype of EMT-like transition in renal tubules by increasing the expression of fibronectin and α-smooth muscle actin and decreasing the expression of E-cadherin. Losartan significantly attenuated the expression of TGF-β1 and Snail, and decreased kidney fibrosis induced by IS and PCS in vivo.

Conclusion: Activating the renal RAAS/TGF-β pathway has an important pathological role in chronic kidney injury caused by IS and PCS. IS and PCS may increase Snail expression and induce EMT-like transition.

Show MeSH
Related in: MedlinePlus