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The Anti-fibrotic Actions of Relaxin Are Mediated Through a NO-sGC-cGMP-Dependent
Pathway in Renal Myofibroblasts In Vitro and Enhanced by the NO Donor,
Diethylamine NONOate
#MMPMID27065874
Wang C
; Kemp-Harper BK
; Kocan M
; Ang SY
; Hewitson TD
; Samuel CS
Front Pharmacol
2016[]; 7
(?): 91
PMID27065874
show ga
INTRODUCTION: The anti-fibrotic hormone, relaxin, has been inferred to disrupt
transforming growth factor (TGF)-?1/Smad2 phosphorylation (pSmad2) signal
transduction and promote collagen-degrading gelatinase activity via a nitric
oxide (NO)-dependent pathway. Here, we determined the extent to which NO, soluble
guanylate cyclase (sGC) and cyclic guanosine monophosphate (cGMP) were directly
involved in the anti-fibrotic actions of relaxin using a selective NO scavenger
and sGC inhibitor, and comparing and combining relaxin's effects with that of an
NO donor. METHODS AND RESULTS: Primary renal cortical myofibroblasts isolated
from injured rat kidneys were treated with human recombinant relaxin (RLX; 16.8
nM), the NO donor, diethylamine NONOate (DEA/NO; 0.5-5 ?M) or the combined
effects of RLX (16.8 nM) and DEA/NO (5 ?M) over 72 h. The effects of RLX (16.8
nM) and DEA/NO (5 ?M) were also evaluated in the presence of the NO scavenger,
hydroxocobalamin (HXC; 100 ?M) or sGC inhibitor, ODQ (5 ?M) over 72 h.
Furthermore, the effects of RLX (30 nM), DEA/NO (5 ?M) and RLX (30 nM) + DEA/NO
(5 ?M) on cGMP levels were directly measured, in the presence or absence of ODQ
(5 ?M). Changes in matrix metalloproteinase (MMP)-2, MMP-9 (cell media), pSmad2
and ?-smooth muscle actin (?-SMA; a measure myofibroblast differentiation) (cell
layer) were assessed by gelatin zymography and Western blotting, respectively. At
the highest concentration tested, both RLX and DEA/NO promoted MMP-2 and MMP-9
levels by 25-33%, while inhibiting pSmad2 and ?-SMA expression by up to 50% (all
p < 0.05 vs. untreated and vehicle-treated cells). However, 5?M of DEA/NO was
required to produce the effects seen with 16.8 nM of RLX over 72 h. The
anti-fibrotic effects of RLX or DEA/NO alone were completely abrogated by HXC and
ODQ (both p < 0.01 vs. RLX alone or DEA/NO alone), but were significantly
enhanced when added in combination (all p < 0.05 vs. RLX alone). Additionally,
the direct cGMP-promoting effects of RLX, DEA/NO and RLX+DEA/NO (which all
increased cGMP levels by 12-16-fold over basal levels; all p < 0.01 vs.
vehicle-treated cells) were significantly inhibited by pre-treatment of ODQ (all
p < 0.05 vs. the respective treatments alone). CONCLUSION: These findings
confirmed that RLX mediates its TGF-?1-inhibitory and gelatinase-promoting
effects via a NO-sGC-cGMP-dependent pathway, which was additively augmented by
co-administration of DEA/NO.
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