Anti-apoptotic and Matrix Remodeling Actions of a Small Molecule Agonist of the Human Relaxin Receptor, ML290 in Mice With Unilateral Ureteral Obstruction

Diseases, such as diabetes and hypertension, often lead to chronic kidney failure. The peptide hormone relaxin has been shown to have therapeutic effects in various organs. In the present study, we tested the hypothesis that ML290, a small molecule agonist of the human relaxin receptor (RXFP1), is able to target the kidney to remodel the extracellular matrix and reduce apoptosis induced by unilateral ureteral obstruction (UUO). UUO was performed on the left kidney of humanized RXFP1 mice, where the right kidneys served as contralateral controls. Mice were randomly allocated to receive either vehicle or ML290 (30 mg/kg) via daily intraperitoneal injection, and kidneys were collected for apoptosis, RNA, and protein analyses. UUO significantly increased expression of pro-apoptotic markers in both vehicle- and ML290-treated mice when compared to their contralateral control kidneys. Specifically, Bax expression and Erk1/2 activity were upregulated, accompanied by an increase of TUNEL-positive cells in the UUO kidneys. Additionally, UUO induced marked increase in myofibroblast differentiation and aberrant remodeling on the extracellular matrix. ML290 suppressed these processes by promoting a reduction of pro-apoptotic, fibroblastic, and inflammatory markers in the UUO kidneys. Finally, the potent effects of ML290 to remodel the extracellular matrix were demonstrated by its ability to reduce collagen gene expression in the UUO kidneys. Our data indicate that daily administration of ML290 has renal protective effects in the UUO mouse model, specifically through its anti-apoptotic and extracellular matrix remodeling properties.

Abstract 17021: A Novel Human Relaxin Receptor Agonist, ML290, Attenuates Atherosclerosis- and Chronic Kidney Disease-Induced Vascular Calcification in Mice

Introduction: Vascular calcification is the most significant predictor of cardiovascular morbidity and mortality, but therapeutic options are unavailable. Relaxin has emerged as a vasoprotective molecule, but several drawbacks prevent therapeutic translation. Targeting the relaxin receptor, RXFP1, is safe and well-tolerated in animal models of vascular disease and humans. We identified a biased allosteric agonist of human RXFP1, ML290, and aimed to test the hypothesis that ML290 arrests the progression of vascular calcification in mouse models of atherosclerosis and chronic kidney disease (CKD). Methods and Results: Recurrent treatment with ML290 significantly prevented ( P = 0.0422, n = 8) and reversed ( P = 0.0489, n = 6) atherosclerotic calcification in humanized ( hRXFP1/hRXFP1 ) Apoe -/- mice fed an atherogenic diet. Longitudinal tracing of mineral formation in the aortic arch of these mice revealed the presence of mineral in vehicle- but not ML290-treated mice after 15 weeks of diet. Accelerated mineral growth was observed in vehicle-treated mice after 20 weeks of the diet, which was reduced by ML290 treatment. In humanized mice with CKD, ML290 significantly prevented ( P = 0.0344, n = 9) medial calcification. In vitro , ML290 reduced ( P = 0.0005, n = 3) superoxide production under osteogenic conditions in vascular smooth muscle cells (VSMCs). Osteogenic changes in VSMC phenotype associate with a release of alkaline phosphatase (ALP) in extracellular vesicles (EVs), which promote mineralization. ML290 treatment significantly ( P = 0.0001, n = 3) suppressed the formation of ALP-loaded EVs in vitro . Bone morphogenetic protein-4, an inducer of osteogenic transitions, and caveolin-1, a scaffolding protein required for calcifying EV formation, were significantly ( P = 0.0059, n = 4) down-regulated after 24 h treatment with ML290 compared to vehicle-treated VSMCs under osteogenic conditions. Conclusions: We demonstrate the therapeutic potential for ML290 to mitigate atherosclerosis and CKD-induced vascular calcification in vivo . The actions of ML290 to prevent medial calcification are in part attributed to its ability to limit the release of calcifying EVs as a result of osteogenic differentiation, and to reduce vascular superoxide production.