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Involvement of Cyclic Guanosine Monophosphate-Dependent Protein Kinase I in Renal Antifibrotic Effects of Serelaxin.

Wetzl V, Schinner E, Kees F, Hofmann F, Faerber L, Schlossmann J - Front Pharmacol (2016)

Bottom Line: The profibrotic connective tissue growth factor as well as myofibroblast differentiation were reduced and matrix metalloproteinases-2 and -9 were positively modulated after treatment.Moreover, Smad2 as well as extracellular signal-regulated kinase 1 (ERK1) phosphorylation were decreased, whereas phosphodiesterase (PDE) 5a phosphorylation was increased.However, these effects were not observed in cGKI-KO.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of RegensburgRegensburg, Germany; Novartis Pharma GmbHNuremberg, Germany.

ABSTRACT

Introduction: Kidney fibrosis has shown to be ameliorated through the involvement of cyclic guanosine monophosphate (cGMP) and its dependent protein kinase I (cGKI). Serelaxin, the recombinant form of human relaxin-II, increases cGMP levels and has shown beneficial effects on kidney function in acute heart failure patients. Antifibrotic properties of serelaxin are supposed to be mediated via relaxin family peptide receptor 1 and subsequently enhanced nitric oxide/cGMP to inhibit transforming growth factor-β (TGF-β) signaling. This study examines the involvement of cGKI in the antifibrotic signaling of serelaxin.

Methods and results: Kidney fibrosis was induced by unilateral ureteral obstruction in wildtype (WT) and cGKI knock-out (KO) mice. After 7 days, renal antifibrotic effects of serelaxin were assessed. Serelaxin treatment for 7 days significantly increased cGMP in the kidney of WT and cGKI-KO. In WT, renal fibrosis was reduced through decreased accumulation of collagen1A1, total collagen, and fibronectin. The profibrotic connective tissue growth factor as well as myofibroblast differentiation were reduced and matrix metalloproteinases-2 and -9 were positively modulated after treatment. Moreover, Smad2 as well as extracellular signal-regulated kinase 1 (ERK1) phosphorylation were decreased, whereas phosphodiesterase (PDE) 5a phosphorylation was increased. However, these effects were not observed in cGKI-KO.

Conclusion: Antifibrotic renal effects of serelaxin are mediated via cGMP/cGKI to inhibit Smad2- and ERK1-dependent TGF-β signaling and increased PDE5a phosphorylation.

No MeSH data available.


Related in: MedlinePlus

Effect of serelaxin on (C), (E) mRNA and (A), (B), (D), (F) protein expression of MMP-2, MMP-9 determined by zymography (each 70 μg protein) in kidneys from WT and cGKI-KO mice. (A) Representative blot from untreated WT, which illustrates active and latent MMP expression in healthy and fibrotic renal tissue; (B) representative blot from fibrotic WT kidneys untreated or treated with serelaxin; mRNA was determined with RT-qPCR and 18s rRNA as housekeeping gene. Results are shown as fold change of mRNA expression (2ΔΔct) in the fibrotic kidney relative to contralateral kidney, which was set as 1 [(C) MMP2; (E) MMP9]; latent and active MMP expression determined by zymography (70 μg protein) and is shown only in fibrotic tissue. Each value of WT and cGKI-KO is related to untreated fibrotic WT, which was set as 1 [(D) MMP2; (F) MMP9]; cGKI, cGMP-dependent protein kinase I; KO, knock out; MMP, matrix metalloproteinase; RLX, serelaxin; UUO, unilateral ureteral obstruction gibt es nicht; WT, wildtype, ∗p < 0.05, ∗∗∗p < 0.001.
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Figure 5: Effect of serelaxin on (C), (E) mRNA and (A), (B), (D), (F) protein expression of MMP-2, MMP-9 determined by zymography (each 70 μg protein) in kidneys from WT and cGKI-KO mice. (A) Representative blot from untreated WT, which illustrates active and latent MMP expression in healthy and fibrotic renal tissue; (B) representative blot from fibrotic WT kidneys untreated or treated with serelaxin; mRNA was determined with RT-qPCR and 18s rRNA as housekeeping gene. Results are shown as fold change of mRNA expression (2ΔΔct) in the fibrotic kidney relative to contralateral kidney, which was set as 1 [(C) MMP2; (E) MMP9]; latent and active MMP expression determined by zymography (70 μg protein) and is shown only in fibrotic tissue. Each value of WT and cGKI-KO is related to untreated fibrotic WT, which was set as 1 [(D) MMP2; (F) MMP9]; cGKI, cGMP-dependent protein kinase I; KO, knock out; MMP, matrix metalloproteinase; RLX, serelaxin; UUO, unilateral ureteral obstruction gibt es nicht; WT, wildtype, ∗p < 0.05, ∗∗∗p < 0.001.

Mentions: MMPs are relevant for the degradation of ECM. mRNA of MMP2 was 6.8-fold (± 0.58) increased in fibrotic tissue, whereas MMP9 was not increased in that pathological condition (0.88-fold ± 0.53). Through RLX treatment, only the elevated levels of MMP2 mRNA were significantly decreased (4.3-fold ± 0.61), no changes were found for MMP9 mRNA after treatment (0.76-fold ± 0.067; Figures 5C,E). Zymography distinguishes between latent and active MMP proteins (Figures 5A,B). In WT, latent and active forms of MMP2 were elevated in fibrosis (5.9-fold ± 1.3; 4.2-fold ± 0.84), protein expression of latent MMP9 was significantly enhanced in fibrotic tissue (3.0-fold ± 0.42), active form significantly decreased (0.69-fold ± 0.098) compared to contralateral kidneys (n = 12, respectively).


Involvement of Cyclic Guanosine Monophosphate-Dependent Protein Kinase I in Renal Antifibrotic Effects of Serelaxin.

Wetzl V, Schinner E, Kees F, Hofmann F, Faerber L, Schlossmann J - Front Pharmacol (2016)

Effect of serelaxin on (C), (E) mRNA and (A), (B), (D), (F) protein expression of MMP-2, MMP-9 determined by zymography (each 70 μg protein) in kidneys from WT and cGKI-KO mice. (A) Representative blot from untreated WT, which illustrates active and latent MMP expression in healthy and fibrotic renal tissue; (B) representative blot from fibrotic WT kidneys untreated or treated with serelaxin; mRNA was determined with RT-qPCR and 18s rRNA as housekeeping gene. Results are shown as fold change of mRNA expression (2ΔΔct) in the fibrotic kidney relative to contralateral kidney, which was set as 1 [(C) MMP2; (E) MMP9]; latent and active MMP expression determined by zymography (70 μg protein) and is shown only in fibrotic tissue. Each value of WT and cGKI-KO is related to untreated fibrotic WT, which was set as 1 [(D) MMP2; (F) MMP9]; cGKI, cGMP-dependent protein kinase I; KO, knock out; MMP, matrix metalloproteinase; RLX, serelaxin; UUO, unilateral ureteral obstruction gibt es nicht; WT, wildtype, ∗p < 0.05, ∗∗∗p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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Figure 5: Effect of serelaxin on (C), (E) mRNA and (A), (B), (D), (F) protein expression of MMP-2, MMP-9 determined by zymography (each 70 μg protein) in kidneys from WT and cGKI-KO mice. (A) Representative blot from untreated WT, which illustrates active and latent MMP expression in healthy and fibrotic renal tissue; (B) representative blot from fibrotic WT kidneys untreated or treated with serelaxin; mRNA was determined with RT-qPCR and 18s rRNA as housekeeping gene. Results are shown as fold change of mRNA expression (2ΔΔct) in the fibrotic kidney relative to contralateral kidney, which was set as 1 [(C) MMP2; (E) MMP9]; latent and active MMP expression determined by zymography (70 μg protein) and is shown only in fibrotic tissue. Each value of WT and cGKI-KO is related to untreated fibrotic WT, which was set as 1 [(D) MMP2; (F) MMP9]; cGKI, cGMP-dependent protein kinase I; KO, knock out; MMP, matrix metalloproteinase; RLX, serelaxin; UUO, unilateral ureteral obstruction gibt es nicht; WT, wildtype, ∗p < 0.05, ∗∗∗p < 0.001.
Mentions: MMPs are relevant for the degradation of ECM. mRNA of MMP2 was 6.8-fold (± 0.58) increased in fibrotic tissue, whereas MMP9 was not increased in that pathological condition (0.88-fold ± 0.53). Through RLX treatment, only the elevated levels of MMP2 mRNA were significantly decreased (4.3-fold ± 0.61), no changes were found for MMP9 mRNA after treatment (0.76-fold ± 0.067; Figures 5C,E). Zymography distinguishes between latent and active MMP proteins (Figures 5A,B). In WT, latent and active forms of MMP2 were elevated in fibrosis (5.9-fold ± 1.3; 4.2-fold ± 0.84), protein expression of latent MMP9 was significantly enhanced in fibrotic tissue (3.0-fold ± 0.42), active form significantly decreased (0.69-fold ± 0.098) compared to contralateral kidneys (n = 12, respectively).

Bottom Line: The profibrotic connective tissue growth factor as well as myofibroblast differentiation were reduced and matrix metalloproteinases-2 and -9 were positively modulated after treatment.Moreover, Smad2 as well as extracellular signal-regulated kinase 1 (ERK1) phosphorylation were decreased, whereas phosphodiesterase (PDE) 5a phosphorylation was increased.However, these effects were not observed in cGKI-KO.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of RegensburgRegensburg, Germany; Novartis Pharma GmbHNuremberg, Germany.

ABSTRACT

Introduction: Kidney fibrosis has shown to be ameliorated through the involvement of cyclic guanosine monophosphate (cGMP) and its dependent protein kinase I (cGKI). Serelaxin, the recombinant form of human relaxin-II, increases cGMP levels and has shown beneficial effects on kidney function in acute heart failure patients. Antifibrotic properties of serelaxin are supposed to be mediated via relaxin family peptide receptor 1 and subsequently enhanced nitric oxide/cGMP to inhibit transforming growth factor-β (TGF-β) signaling. This study examines the involvement of cGKI in the antifibrotic signaling of serelaxin.

Methods and results: Kidney fibrosis was induced by unilateral ureteral obstruction in wildtype (WT) and cGKI knock-out (KO) mice. After 7 days, renal antifibrotic effects of serelaxin were assessed. Serelaxin treatment for 7 days significantly increased cGMP in the kidney of WT and cGKI-KO. In WT, renal fibrosis was reduced through decreased accumulation of collagen1A1, total collagen, and fibronectin. The profibrotic connective tissue growth factor as well as myofibroblast differentiation were reduced and matrix metalloproteinases-2 and -9 were positively modulated after treatment. Moreover, Smad2 as well as extracellular signal-regulated kinase 1 (ERK1) phosphorylation were decreased, whereas phosphodiesterase (PDE) 5a phosphorylation was increased. However, these effects were not observed in cGKI-KO.

Conclusion: Antifibrotic renal effects of serelaxin are mediated via cGMP/cGKI to inhibit Smad2- and ERK1-dependent TGF-β signaling and increased PDE5a phosphorylation.

No MeSH data available.


Related in: MedlinePlus