Abstract
Introduction: The anti-fibrotic hormone, relaxin, has been inferred to disrupt transforming growth factor (TGF)-β1/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 μM) or the combined effects of RLX (16.8 nM) and DEA/NO (5 μM) over 72 h. The effects of RLX (16.8 nM) and DEA/NO (5 μM) were also evaluated in the presence of the NO scavenger, hydroxocobalamin (HXC; 100 μM) or sGC inhibitor, ODQ (5 μM) over 72 h. Furthermore, the effects of RLX (30 nM), DEA/NO (5 μM) and RLX (30 nM) + DEA/NO (5 μM) on cGMP levels were directly measured, in the presence or absence of ODQ (5 μM). Changes in matrix metalloproteinase (MMP)-2, MMP-9 (cell media), pSmad2 and α-smooth muscle actin (α-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 α-SMA expression by up to 50% (all p < 0.05 vs. untreated and vehicle-treated cells). However, 5μM 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-β1-inhibitory and gelatinase-promoting effects via a NO-sGC-cGMP-dependent pathway, which was additively augmented by co-administration of DEA/NO.
Highlights
The anti-fibrotic hormone, relaxin, has been inferred to disrupt transforming growth factor (TGF)-β1/Smad2 phosphorylation signal transduction and promote collagen-degrading gelatinase activity via a nitric oxide (NO)dependent pathway
We investigated the extent to which NO, soluble guanylate cyclase (sGC) and cyclic guanosine monophosphate (cGMP) were directly involved in the anti-fibrotic actions of relaxin, by evaluating the effects of a selective NO scavenger and sGC inhibitor (ODQ) on four well-known end-points associated with the anti-fibrotic effects of relaxin: pSmad2, α-smooth muscle actin (α-SMA), matrix metalloproteinase (MMP)-2 and MMP-9
The effects of RLX (16.8 nM) on renal pSmad2, α-SMA expression, MMP-2 and MMP-9 levels were firstly compared to increasing concentrations of DEA/NO (0.1–5 μM) (Figure 1); and combined with the highest concentration of DEA/NO evaluated (5 μM) (Figure 2)
Summary
The anti-fibrotic hormone, relaxin, has been inferred to disrupt transforming growth factor (TGF)-β1/Smad phosphorylation (pSmad2) signal transduction and promote collagen-degrading gelatinase activity via a nitric oxide (NO)dependent pathway. The rate of ECM synthesis which contributes to fibrosis is dependent on the activity of various pro-fibrotic cytokines and growth factors, primarily transforming growth factor (TGF)-β1, and on myofibroblast burden (Eddy, 2000; Hewitson, 2009; Wynn and Ramalingam, 2012). Recognized from their de novo expression of α-smooth muscle actin (α-SMA), myofibroblasts are mainly derived from the proliferation and differentiation of local fibroblasts following injury, and synthesize excessive amounts of collagen and other ECM proteins that form the basis of renal fibrosis. The effect of TGF-β1 on gelatinase activity has been demonstrated to be inhibitory (Howard et al, 2012), but can vary in a time- (Ogawa et al, 2011) and species/model (Saed et al, 2000; Ogawa et al, 2011; Ye et al, 2011; Bates et al, 2015)-dependent manner
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