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Angiotensin-(1-7) activates a tyrosine phosphatase and inhibits glucose-induced signalling in proximal tubular cells.

Gava E, Samad-Zadeh A, Zimpelmann J, Bahramifarid N, Kitten GT, Santos RA, Touyz RM, Burns KD - Nephrol. Dial. Transplant. (2009)

Bottom Line: Ang-(1-7) inhibited high glucose-stimulated protein synthesis, and blocked the stimulatory effect of glucose on TGF-beta1.Conversely, Ang-(1-7) had no effect on glucose-stimulated synthesis of fibronectin or collagen IV.In diabetic nephropathy, Ang-(1-7) may partly counteract the profibrotic effects of high glucose.

View Article: PubMed Central - PubMed

Affiliation: Division of Nephrology, Department of Medicine, Kidney Research Centre, Ottawa Health Research Institute, University of Ottawa, Ottawa, Ontario, Canada.

ABSTRACT

Background: In the diabetic kidney, stimulation of mitogen-activated protein kinases (MAPKs) leads to extracellular matrix protein synthesis. In the proximal tubule, angiotensin-(1-7) [Ang-(1-7)] blocks activation of MAPKs by angiotensin II. We studied the effect of Ang-(1-7) on signalling responses in LLC-PK(1) cells in normal (5 mM) or high (25 mM) glucose.

Methods: The p38 MAPK was assayed by immunoblot, Src homology 2-containing protein-tyrosine phosphatase-1 (SHP-1) activity was measured after immunoprecipitation, cell protein synthesis was determined by [(3)H]-leucine incorporation and transforming growth factor-beta1 (TGF-beta1), fibronectin and collagen IV were assayed by immunoblots and/or ELISA.

Results: High glucose stimulated p38 MAPK. This response was inhibited by Ang-(1-7) in a concentration-dependent fashion, an effect reversed by the receptor Mas antagonist A-779. Ang-(1-7) increased SHP-1 activity, via the receptor Mas. An inhibitor of tyrosine phosphatase, phenylarsine oxide, reversed the inhibitory effect of Ang-(1-7) on high glucose-stimulated p38 MAPK. Ang-(1-7) inhibited high glucose-stimulated protein synthesis, and blocked the stimulatory effect of glucose on TGF-beta1. Conversely, Ang-(1-7) had no effect on glucose-stimulated synthesis of fibronectin or collagen IV.

Conclusions: These data indicate that in proximal tubular cells, binding of Ang-(1-7) to the receptor Mas stimulates SHP-1, associated with the inhibition of glucose-stimulated p38 MAPK. Ang-(1-7) selectively inhibits glucose-stimulated protein synthesis and TGF-beta1. In diabetic nephropathy, Ang-(1-7) may partly counteract the profibrotic effects of high glucose.

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Effect of Ang-(1–7) on p38 MAPK phosphorylation in normal glucose. (A) Graph depicts the concentration-dependent stimulatory effect of incubation of Ang-(1–7) (10−11–10−7 M) on phosphorylation of p38 MAPK in normal glucose (NG, 5 mM). By ANOVA, the effect of Ang-(1–7) (10−7 M) was not significant compared to NG alone in this series of experiments; n = 7–8. Results are presented as the ratio of phosphorylated p38/total p38, in arbitrary units. Representative blot is depicted above the graph, showing phosphorylated p38 (pp38) and total p38 (p38). (B) Graph depicts the effect of Ang-(1–7) (10−7 M) in normal glucose (NG, 5 mM) with or without A-779 (10−5 M), Losartan (Los, 10−6 M) or PD123319 (PD, 10−6 M). *P < 0.05 versus control; n = 5–6. To control for the possible effects of osmolality in these experiments, incubations in NG were supplemented with l-glucose (20 mM). Representative blot is depicted above the graph, showing phosphorylated p38 (pp38) and total p38 (p38). (C) Graph depicts the effect of Ang-(1–7) (10−7 M) and bradykinin (BK) (10−7 M) in normal glucose (NG, 5 mM) with or without the B2 receptor antagonist HOE-140 (HOE, 10−6 M). *P < 0.05 versus control, **P < 0.001 versus control ***P < 0.01 versus BK; n = 7–16. Representative blot is depicted above the graph, showing phosphorylated p38 (pp38) and total p38 (p38).
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Figure 2: Effect of Ang-(1–7) on p38 MAPK phosphorylation in normal glucose. (A) Graph depicts the concentration-dependent stimulatory effect of incubation of Ang-(1–7) (10−11–10−7 M) on phosphorylation of p38 MAPK in normal glucose (NG, 5 mM). By ANOVA, the effect of Ang-(1–7) (10−7 M) was not significant compared to NG alone in this series of experiments; n = 7–8. Results are presented as the ratio of phosphorylated p38/total p38, in arbitrary units. Representative blot is depicted above the graph, showing phosphorylated p38 (pp38) and total p38 (p38). (B) Graph depicts the effect of Ang-(1–7) (10−7 M) in normal glucose (NG, 5 mM) with or without A-779 (10−5 M), Losartan (Los, 10−6 M) or PD123319 (PD, 10−6 M). *P < 0.05 versus control; n = 5–6. To control for the possible effects of osmolality in these experiments, incubations in NG were supplemented with l-glucose (20 mM). Representative blot is depicted above the graph, showing phosphorylated p38 (pp38) and total p38 (p38). (C) Graph depicts the effect of Ang-(1–7) (10−7 M) and bradykinin (BK) (10−7 M) in normal glucose (NG, 5 mM) with or without the B2 receptor antagonist HOE-140 (HOE, 10−6 M). *P < 0.05 versus control, **P < 0.001 versus control ***P < 0.01 versus BK; n = 7–16. Representative blot is depicted above the graph, showing phosphorylated p38 (pp38) and total p38 (p38).

Mentions: In normal glucose (5 mM), incubation of cells with Ang-(1–7) caused small concentration-dependent activation of p38 MAPK, with a peak effect at 10−7 M (Figure 2A). Pretreatment with A-779 (10−5 M), the AT1 receptor antagonist losartan (10−6 M), or the AT2 receptor antagonist PD123319 (10−6 M) did not reverse this activation (Figure 2B). In contrast, the bradykinin B2 receptor antagonist HOE-140 (10−6 M) partly inhibited the Ang-(1–7)-stimulated p38 MAPK phosphorylation in normal glucose. Bradykinin also stimulated p38 MAPK phosphorylation in normal glucose, and this was reversed by HOE-140 (Figure 2C).


Angiotensin-(1-7) activates a tyrosine phosphatase and inhibits glucose-induced signalling in proximal tubular cells.

Gava E, Samad-Zadeh A, Zimpelmann J, Bahramifarid N, Kitten GT, Santos RA, Touyz RM, Burns KD - Nephrol. Dial. Transplant. (2009)

Effect of Ang-(1–7) on p38 MAPK phosphorylation in normal glucose. (A) Graph depicts the concentration-dependent stimulatory effect of incubation of Ang-(1–7) (10−11–10−7 M) on phosphorylation of p38 MAPK in normal glucose (NG, 5 mM). By ANOVA, the effect of Ang-(1–7) (10−7 M) was not significant compared to NG alone in this series of experiments; n = 7–8. Results are presented as the ratio of phosphorylated p38/total p38, in arbitrary units. Representative blot is depicted above the graph, showing phosphorylated p38 (pp38) and total p38 (p38). (B) Graph depicts the effect of Ang-(1–7) (10−7 M) in normal glucose (NG, 5 mM) with or without A-779 (10−5 M), Losartan (Los, 10−6 M) or PD123319 (PD, 10−6 M). *P < 0.05 versus control; n = 5–6. To control for the possible effects of osmolality in these experiments, incubations in NG were supplemented with l-glucose (20 mM). Representative blot is depicted above the graph, showing phosphorylated p38 (pp38) and total p38 (p38). (C) Graph depicts the effect of Ang-(1–7) (10−7 M) and bradykinin (BK) (10−7 M) in normal glucose (NG, 5 mM) with or without the B2 receptor antagonist HOE-140 (HOE, 10−6 M). *P < 0.05 versus control, **P < 0.001 versus control ***P < 0.01 versus BK; n = 7–16. Representative blot is depicted above the graph, showing phosphorylated p38 (pp38) and total p38 (p38).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Effect of Ang-(1–7) on p38 MAPK phosphorylation in normal glucose. (A) Graph depicts the concentration-dependent stimulatory effect of incubation of Ang-(1–7) (10−11–10−7 M) on phosphorylation of p38 MAPK in normal glucose (NG, 5 mM). By ANOVA, the effect of Ang-(1–7) (10−7 M) was not significant compared to NG alone in this series of experiments; n = 7–8. Results are presented as the ratio of phosphorylated p38/total p38, in arbitrary units. Representative blot is depicted above the graph, showing phosphorylated p38 (pp38) and total p38 (p38). (B) Graph depicts the effect of Ang-(1–7) (10−7 M) in normal glucose (NG, 5 mM) with or without A-779 (10−5 M), Losartan (Los, 10−6 M) or PD123319 (PD, 10−6 M). *P < 0.05 versus control; n = 5–6. To control for the possible effects of osmolality in these experiments, incubations in NG were supplemented with l-glucose (20 mM). Representative blot is depicted above the graph, showing phosphorylated p38 (pp38) and total p38 (p38). (C) Graph depicts the effect of Ang-(1–7) (10−7 M) and bradykinin (BK) (10−7 M) in normal glucose (NG, 5 mM) with or without the B2 receptor antagonist HOE-140 (HOE, 10−6 M). *P < 0.05 versus control, **P < 0.001 versus control ***P < 0.01 versus BK; n = 7–16. Representative blot is depicted above the graph, showing phosphorylated p38 (pp38) and total p38 (p38).
Mentions: In normal glucose (5 mM), incubation of cells with Ang-(1–7) caused small concentration-dependent activation of p38 MAPK, with a peak effect at 10−7 M (Figure 2A). Pretreatment with A-779 (10−5 M), the AT1 receptor antagonist losartan (10−6 M), or the AT2 receptor antagonist PD123319 (10−6 M) did not reverse this activation (Figure 2B). In contrast, the bradykinin B2 receptor antagonist HOE-140 (10−6 M) partly inhibited the Ang-(1–7)-stimulated p38 MAPK phosphorylation in normal glucose. Bradykinin also stimulated p38 MAPK phosphorylation in normal glucose, and this was reversed by HOE-140 (Figure 2C).

Bottom Line: Ang-(1-7) inhibited high glucose-stimulated protein synthesis, and blocked the stimulatory effect of glucose on TGF-beta1.Conversely, Ang-(1-7) had no effect on glucose-stimulated synthesis of fibronectin or collagen IV.In diabetic nephropathy, Ang-(1-7) may partly counteract the profibrotic effects of high glucose.

View Article: PubMed Central - PubMed

Affiliation: Division of Nephrology, Department of Medicine, Kidney Research Centre, Ottawa Health Research Institute, University of Ottawa, Ottawa, Ontario, Canada.

ABSTRACT

Background: In the diabetic kidney, stimulation of mitogen-activated protein kinases (MAPKs) leads to extracellular matrix protein synthesis. In the proximal tubule, angiotensin-(1-7) [Ang-(1-7)] blocks activation of MAPKs by angiotensin II. We studied the effect of Ang-(1-7) on signalling responses in LLC-PK(1) cells in normal (5 mM) or high (25 mM) glucose.

Methods: The p38 MAPK was assayed by immunoblot, Src homology 2-containing protein-tyrosine phosphatase-1 (SHP-1) activity was measured after immunoprecipitation, cell protein synthesis was determined by [(3)H]-leucine incorporation and transforming growth factor-beta1 (TGF-beta1), fibronectin and collagen IV were assayed by immunoblots and/or ELISA.

Results: High glucose stimulated p38 MAPK. This response was inhibited by Ang-(1-7) in a concentration-dependent fashion, an effect reversed by the receptor Mas antagonist A-779. Ang-(1-7) increased SHP-1 activity, via the receptor Mas. An inhibitor of tyrosine phosphatase, phenylarsine oxide, reversed the inhibitory effect of Ang-(1-7) on high glucose-stimulated p38 MAPK. Ang-(1-7) inhibited high glucose-stimulated protein synthesis, and blocked the stimulatory effect of glucose on TGF-beta1. Conversely, Ang-(1-7) had no effect on glucose-stimulated synthesis of fibronectin or collagen IV.

Conclusions: These data indicate that in proximal tubular cells, binding of Ang-(1-7) to the receptor Mas stimulates SHP-1, associated with the inhibition of glucose-stimulated p38 MAPK. Ang-(1-7) selectively inhibits glucose-stimulated protein synthesis and TGF-beta1. In diabetic nephropathy, Ang-(1-7) may partly counteract the profibrotic effects of high glucose.

Show MeSH
Related in: MedlinePlus