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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

(A) Representative western blots (each 50 g protein) of signaling molecules in kidney tissue of WT mice with or without UUO untreated or treated with serelaxin; (B) effect of serelaxin on protein expression of signaling molecules (P-ERK2/ERK2, P-ERK1/ERK1, PDE5a, P-PDE5a, TGF-β, CTGF, P-SMAD2) in fibrotic kidney from WT and cGKI-KO mice; in Figure 5 (B) effects of serelaxin on protein expression of markers (normalized to GAPDH) was determined only in fibrotic tissue. Each value of WT and cGKI-KO is related to untreated fibrotic WT, which was set as 1; α-SMA, α-smooth muscle actin; cGKI, cGMP-dependent protein kinase I; cGMP, cyclic guanosine monophosphate; CTGF, connective tissue growth factor; ECM, extracellular matrix; eNOS, endothelial nitric oxide synthase; ERK, extracellular-signal regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GMP, guanosine monophosphate; KO, knock out; MMPs, matrix metalloproteinases; PDE5a, phosphodiesterase 5a; RLX, serelaxin; RXFP1, relaxin receptor; Smad, small mothers against decapentaplegic protein; TGF-β, transforming growth factor-β; UUO, unilateral ureteral obstruction; WT, wildtype, ∗p < 0.05, ∗∗p < 0.01.
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Figure 6: (A) Representative western blots (each 50 g protein) of signaling molecules in kidney tissue of WT mice with or without UUO untreated or treated with serelaxin; (B) effect of serelaxin on protein expression of signaling molecules (P-ERK2/ERK2, P-ERK1/ERK1, PDE5a, P-PDE5a, TGF-β, CTGF, P-SMAD2) in fibrotic kidney from WT and cGKI-KO mice; in Figure 5 (B) effects of serelaxin on protein expression of markers (normalized to GAPDH) was determined only in fibrotic tissue. Each value of WT and cGKI-KO is related to untreated fibrotic WT, which was set as 1; α-SMA, α-smooth muscle actin; cGKI, cGMP-dependent protein kinase I; cGMP, cyclic guanosine monophosphate; CTGF, connective tissue growth factor; ECM, extracellular matrix; eNOS, endothelial nitric oxide synthase; ERK, extracellular-signal regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GMP, guanosine monophosphate; KO, knock out; MMPs, matrix metalloproteinases; PDE5a, phosphodiesterase 5a; RLX, serelaxin; RXFP1, relaxin receptor; Smad, small mothers against decapentaplegic protein; TGF-β, transforming growth factor-β; UUO, unilateral ureteral obstruction; WT, wildtype, ∗p < 0.05, ∗∗p < 0.01.

Mentions: As mentioned above, cGMP levels were elevated through RLX and, so far, cGKI-KO mice showed no antifibrotic effects, which suggested the involvement of the NO/cGMP/cGKI pathway in the antifibrotic effect of RLX. Subsequently, several signaling molecules were analyzed by western blotting, which are involved in fibrosis. Representative western blots demonstrated modulation of the selected markers in fibrotic conditions compared to healthy (Figure 6A).


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)

(A) Representative western blots (each 50 g protein) of signaling molecules in kidney tissue of WT mice with or without UUO untreated or treated with serelaxin; (B) effect of serelaxin on protein expression of signaling molecules (P-ERK2/ERK2, P-ERK1/ERK1, PDE5a, P-PDE5a, TGF-β, CTGF, P-SMAD2) in fibrotic kidney from WT and cGKI-KO mice; in Figure 5 (B) effects of serelaxin on protein expression of markers (normalized to GAPDH) was determined only in fibrotic tissue. Each value of WT and cGKI-KO is related to untreated fibrotic WT, which was set as 1; α-SMA, α-smooth muscle actin; cGKI, cGMP-dependent protein kinase I; cGMP, cyclic guanosine monophosphate; CTGF, connective tissue growth factor; ECM, extracellular matrix; eNOS, endothelial nitric oxide synthase; ERK, extracellular-signal regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GMP, guanosine monophosphate; KO, knock out; MMPs, matrix metalloproteinases; PDE5a, phosphodiesterase 5a; RLX, serelaxin; RXFP1, relaxin receptor; Smad, small mothers against decapentaplegic protein; TGF-β, transforming growth factor-β; UUO, unilateral ureteral obstruction; WT, wildtype, ∗p < 0.05, ∗∗p < 0.01.
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Figure 6: (A) Representative western blots (each 50 g protein) of signaling molecules in kidney tissue of WT mice with or without UUO untreated or treated with serelaxin; (B) effect of serelaxin on protein expression of signaling molecules (P-ERK2/ERK2, P-ERK1/ERK1, PDE5a, P-PDE5a, TGF-β, CTGF, P-SMAD2) in fibrotic kidney from WT and cGKI-KO mice; in Figure 5 (B) effects of serelaxin on protein expression of markers (normalized to GAPDH) was determined only in fibrotic tissue. Each value of WT and cGKI-KO is related to untreated fibrotic WT, which was set as 1; α-SMA, α-smooth muscle actin; cGKI, cGMP-dependent protein kinase I; cGMP, cyclic guanosine monophosphate; CTGF, connective tissue growth factor; ECM, extracellular matrix; eNOS, endothelial nitric oxide synthase; ERK, extracellular-signal regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GMP, guanosine monophosphate; KO, knock out; MMPs, matrix metalloproteinases; PDE5a, phosphodiesterase 5a; RLX, serelaxin; RXFP1, relaxin receptor; Smad, small mothers against decapentaplegic protein; TGF-β, transforming growth factor-β; UUO, unilateral ureteral obstruction; WT, wildtype, ∗p < 0.05, ∗∗p < 0.01.
Mentions: As mentioned above, cGMP levels were elevated through RLX and, so far, cGKI-KO mice showed no antifibrotic effects, which suggested the involvement of the NO/cGMP/cGKI pathway in the antifibrotic effect of RLX. Subsequently, several signaling molecules were analyzed by western blotting, which are involved in fibrosis. Representative western blots demonstrated modulation of the selected markers in fibrotic conditions compared to healthy (Figure 6A).

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