AT1R-AT2R-RXFP1 Functional Crosstalk in Myofibroblasts: Impact on the Therapeutic Targeting of Renal and Cardiac Fibrosis

Abstract

Background Recombinant human H2 relaxin (serelaxin) has emerged as a potential agent to treat fibrosis, the pathological hallmark of chronic disease. As we now know that serelaxin requires the angiotensin II (Ang II) type 2 receptor (AT2R) to ameliorate renal fibrogenesis in vitro and in vivo, we sought to determine if its anti-fibrotic actions were affected by Ang II type 1 receptor (AT1R) modulation. Methods We examined the signal transduction mechanisms of serelaxin when applied to primary rat renal and human cardiac myofibroblasts in vitro, and in three models of renal-or cardiomyopathy-induced fibrosis in vivo. Results The anti-fibrotic signal transduction of serelaxin via its cognate receptor, relaxin family peptide receptor 1 (RXFP1), was abrogated by the AT1R blockers, irbesartan or candesartan in vitro and in vivo. Candesartan also ameliorated serelaxin's anti-fibrotic actions in the left ventricle of mice with cardiomyopathy, indicating that the inhibitory effects of candesartan were not confined to the kidney. In a transfected cell system, we demonstrated that serelaxin did not directly bind to AT1Rs but that constitutive AT1R-RXFP1 interactions could form. To potentially explain these findings, we also demonstrated that all three receptors were expressed by renal and cardiac (myo)fibroblasts and that antagonists acting at each receptor directly/allosterically blocked the anti-fibrotic effects of either serelaxin or the AT2R agonist, Compound 21. Conclusions These findings have significant implications for the concomitant use of RXFP1 or AT2R agonists with AT1R blockers and suggest that functional AT1R-AT2R-RXFP1 interactions on myofibroblasts may represent new targets for controlling fibrosis progression.

The anti‐fibrotic actions of relaxin are mediated through AT2R‐associated protein phosphatases via RXFP1‐AT2R functional crosstalk in human cardiac myofibroblasts

“…Primary human cardiac fibroblasts (HCFs; #6300; ScienCell Research Laboratories, Carlsbad, CA, USA) were obtained from fetusus of pregnant women (since AT 2 R expression is optimally expressed in embryonic and fetal tissues). Separate studies had shown that these cells expressed RXFP1 29,30 and AT 2 receptors 30 . These cells were maintained/expanded in filtered flasks with media containing Dulbecco's Modified Eagles Medium (DMEM; GIBCO‐Life Technologies, Grand Island, NY, USA), 10% (v/v) of fetal bovine serum (FBS; GIBCO‐Life Technologies), 1% (v/v) of fibroblast growth supplement‐2 (FGS; ScienCell Research Laboratories), penicillin (100 units/mL), and streptomycin (100 mg/mL) (GIBCO‐Life Technologies), in a 37ºC incubator with 95% of O 2 /5% of CO 2 .…”

Assessment of renal fibrosis and anti‐fibrotic agents using a novel diagnostic and stain‐free second‐harmonic generation platform

“…All samples were also exposed to the house‐keeping protein, α‐tubulin (ab52866; 1:2500 dilution; Abcam) to ensure equal loading. Densitometry of the relevant bands was performed using a Bio‐Rad GS710 Calibrated Imaging Densitomer and Quantity‐One software and expressed relative to values from the uninjured/untreated control group, which was expressed as 1 30,33 …”

“…Seven days post-UUO, all mice were weighed before being killed by anesthetic overdose, and their obstructed kidneys were isolated and fixed in 10% neutral-buffered formalin for further analysis. Serial sections from the mid-portion of paraffin-embedded tissues were subjected to either Masson's trichrome (MT) staining (performed by Monash Histological Services, Monash University, Clayton, Victoria, Australia); immunohistochemical (IHC) staining of collagen I, 24,33 using a polyclonal primary antibody (ab34710; 1:1000 dilution; Abcam, Cambridge, MA) and anti-rabbit secondary antibody via the use of a Dako anti-rabbit kit (Dako Corporation, Carpinteria, CA); or HistoIndex analysis.…”

The (apparent) sacubitril/valsartan sex interaction in heart failure with preserved ejection fraction: not the result of relaxin effects but of BNP action?!

“…At first sight, there could be the following link: angiotensin type-1 receptor (AT1R) blockers (ARBs) have recently been shown to completely block the anti-fibrotic effects of therapeutic relaxin, both in rodent models of renal and cardiac fibrosis and in human cardiac fibroblasts. 5,6 This is attributable to a direct interaction at receptor level-between RXFP1, the cognate receptor for relaxin-2, the AT1R, and the AT2R-and it enables antagonists to each receptor blocking agonist effects at the other receptors. 5 In other words, ARBs have been shown in these models to completely nullify relaxin's anti-fibrotic signalling.…”