Relaxin Attenuates Organ Fibrosis via an Angiotensin Type 2 Receptor Mechanism in Aged Hypertensive Female Rats

Abstract

Background: The anti-fibrotic effects of recombinant human relaxin (RLX) in the kidney are dependent on an interaction between its cognate receptor (RXFP1) and the angiotensin type 2 receptor (AT2R) in male models of disease. Whether RLX has therapeutic effects, which are also mediated via the AT2R, in hypertensive adult and aged/reproductively senescent females is unknown. Thus, we determined whether treatment with RLX provides cardiorenal protection, via an AT2R-dependent mechanism, in adult and aged female stroke prone-spontaneously hypertensive rats (SHRSP). Methods: In 6- (6MO) and 15-month-old (15MO; reproductively senescent) female SHRSP, systolic blood pressure (SBP), glomerular filtration rate (GFR) and proteinuria were measured before and after 4 weeks treatment with vehicle (Veh), RLX (0.5 mg/kg/day s.c.) or RLX+PD123319 (AT2R antagonist; 3 mg/kg/day s.c.). Aortic endothelium-dependent relaxation and fibrosis of the kidney, heart and aorta were assessed. Results: In 6MO SHRSP, RLX significantly enhanced GFR by ~25% (P=0.001) and reduced cardiac fibrosis (P=0.01) as compared to vehicle-treated counterparts. These effects were abolished or blunted by PD123319 co-administration. In 15MO females, RLX reduced interstitial renal (P=0.02) and aortic (P=0.003) fibrosis, and lowered SBP (13±3 mmHg; P=0.04) relative to controls. These effects were also blocked by PD123319 co-treatment (all p<0.05 versus RLX treatment alone). RLX also markedly improved vascular function by ~40% (P<0.0001) in 15MO SHRSP, but this was not modulated by PD123319 co-treatment. Conclusion: The anti-fibrotic and organ-protective effects of RLX, when administered to a severe model of hypertension, conferred cardiorenal protection in adult and reproductively senescent female rats, to a great extent via an AT2R-mediated mechanism.

Relaxin Inhibits the Cardiac Myofibroblast NLRP3 Inflammasome as Part of Its Anti-Fibrotic Actions via the Angiotensin Type 2 and ATP (P2X7) Receptors

“…Whilst it was previously only demonstrated that the HCMFs used in this study expressed RXFP1 [34], the current study further validated that RXFP1 blockade (with the B-R13/17K H2 compound) was able to annul the myofibroblast NLRP3 inflammasomeinhibitory effects of RLX (from the level of TLR-4 to pro-IL-1β and pro-IL-18). Secondly, the results obtained align with growing evidence which has pointed out that RLX-RXFP1 signalling is able to interact (or potentially undergo crosstalk) with other receptors to mediate the therapeutic effects of RLX, including the AT 2 R [27,28,33], Notch-1 [32], glucocorticoid receptor [43,44] and PPAR Υ [45,46]. Furthermore, the results obtained from the in vitro studies conducted confirmed that RXFP1-AT 2 R crosstalk played a central role in facilitating the therapeutic effects of RLX [27,28,33], which now appear to include its ability to inhibit myofibroblast NLRP3 inflammasome activity (Figure 7).…”

“…Secondly, the results obtained align with growing evidence which has pointed out that RLX-RXFP1 signalling is able to interact (or potentially undergo crosstalk) with other receptors to mediate the therapeutic effects of RLX, including the AT 2 R [27,28,33], Notch-1 [32], glucocorticoid receptor [43,44] and PPAR Υ [45,46]. Furthermore, the results obtained from the in vitro studies conducted confirmed that RXFP1-AT 2 R crosstalk played a central role in facilitating the therapeutic effects of RLX [27,28,33], which now appear to include its ability to inhibit myofibroblast NLRP3 inflammasome activity (Figure 7).…”

“…Additionally, it was found that the anti-inflammatory (involving macrophage infiltration and MCP-1 expression levels), anti-hypertrophic (involving LV cardiomyocyte size) and anti-fibrotic (involving α-SMA-associated interstitial myofibroblast differentiation and interstitial LV collagen deposition) effects of RLX appeared to involve the P2X7R, since these therapeutic effects of RLX were blunted or significantly ameliorated by P2X7R antagonism. These findings warrant further investigation of these recently unravelled interactions between RLX-RXFP1 and the P2X7R, to determine: (i) the signal transduction mechanisms by which RXFP1 undergoes crosstalk with the P2X7; (ii) whether this receptor crosstalk only occurs in myofibroblasts or other NLRP3 inflammasome-expressing cells as well; and (iii) whether the crosstalk between RXFP1 and the P2X7R also requires the presence of the AT 2 R (which has been shown to be required for RLX to induce its antifibrotic effects [27,28,33], or can occur independently of the AT 2 R. Furthermore, the extent to which the effects of RLX on cardiac function are mediated via the P2X7R needs to be determined. Addressing these questions in additional studies will lead to a better understanding of the receptor crosstalk that is utilized by RLX to induce its pleiotropic effects as an effective anti-fibrotic therapy.…”

“…Recombinant human relaxin (RLX, also known as serelaxin) is the drug-based version of the naturally-occurring hormone, human gene-2 (H2) relaxin, and is well-known for its antifibrotic actions via its ability to inhibit TGF-β1 signal transduction and pro-fibrotic activity, via the relaxin family peptide receptor 1 (RXFP1) [24][25][26]. Furthermore, the anti-fibrotic actions of RLX appear to require the angiotensin II type 2 receptor (AT 2 R) [27,28], and can be mediated via the direct activation of cyclic guanosine monophosphate (cGMP) [29] or via the phosphorylation of extracellular signal-regulated kinase (pERK1/2) and a neuronal nitric oxide (NO) synthase (nNOS)-NO-soluble guanylate cyclase (sGC)-cGMP-dependent pathway to inhibit TGF-β1 signal transduction at the level of Smad2 [27,[29][30][31] and Smad3 [29,32] phosphorylation. This consistently results in the RLX-induced suppression of TGF-β1-induced myofibroblast differentiation and myofibroblast-mediated ECM/collagen deposition (which forms the basis of fibrosis), and also allows RLX to indirectly signal through AT 2 R-associated protein phosphatases [33].…”

Sex- and age-related differences in renal and cardiac injury and senescence in stroke-prone spontaneously hypertensive rats

“…SHRSP are salt sensitive and if fed a high salt diet, > 95% of SHRSPs die of stroke between 6 and 12 Mo [ 16 , 22 ]. However, previous reports indicate SHRSPs, in the absence of salt loading, survive past 9Mo of age for males [ 25 , 26 ], and past 15Mo of age for females [ 27 ]. In the present study, none of the male or female SHRSPs (of which all were fed a normal sodium diet) showed signs of stroke or died spontaneously by the final timepoint of 8.5Mo of age.…”