Esaxerenone inhibits the macrophage-to-myofibroblast transition through mineralocorticoid receptor/TGF-β1 pathway in mice induced with aldosterone

Abstract

Renal fibrosis is the inevitable pathway of the progression of chronic kidney disease to end-stage renal disease, which manifests as progressive glomerulosclerosis and renal interstitial fibrosis. In a previous study, we observed severe interstitial fibrosis in the contralateral kidneys of 6-month unilateral ureteral obstruction (UUO) rats, which was accompanied by increased macrophage infiltration and phenotypic transformation; after eplerenone administration, these effects were reduced. Therefore, we hypothesized that this effect was closely related to mineralocorticoid receptor (MR) activation induced by the increased aldosterone (ALD) level. In this study, we used uninephrectomy plus continuous aldosterone infusion in mice to observe whether aldosterone induced macrophage-to-myofibroblast transition (MMT) and renal fibrosis and investigated the signaling pathways. Notably, aldosterone induced predominantly M1 macrophage-to-myofibroblast transition by activating MR and upregulating TGF-β1 expression, which promoted renal fibrosis. These effects were antagonized by the MR blocker esaxerenone. These findings suggest that targeting the MR/TGF-β1 pathway may be an effective therapeutic strategy for renal fibrosis.

Eplerenone reduces lymphangiogenesis in the contralateral kidneys of UUO rats

“…This finding was also validated in RAW264.7 cells cultured in vitro and suggested that UUO-induced macrophage infiltration generated a favorable microenvironment for lymphangiogenesis and was involved in lymphangiogenesis through the secretion of VEGFC. These macrophages, which may originate from the bone marrow or from in situ proliferation of kidney macrophages, may be activated by MR, the inflammatory cytokines IL-1β and TNF-α or hypoxia-inducible factor 21 , 23 – 26 . In addition, renal tubular epithelial cells can also be sources of VEGFC in response to renal injury 24 , 27 , which remains to be confirmed in future studies.…”

Esaxerenone Inhibits Renal Angiogenesis and Endothelial-Mesenchymal Transition via the VEGFA and TGF-β1 Pathways in Aldosterone-Infused Mice

“…In previous studies, we observed that macrophages in the kidney or heart were involved in fibrosis via macrophage-to-myofibroblast transition (MMT) in UUO rats and aldosterone-infused mice, which was associated with inflammatory injury induced by aldosterone-stimulated MR activation [45][46][47]. MR activation leads to macrophage infiltration and MMT, which participates in fibrosis by secreting the extracellular matrix.…”

“…Thirty mice were randomly assigned to the CON group, ALD group (aldosterone group), and ESA group (aldosterone + esaxerenone group) (n = 10 each). Aldosterone is infused with a mini-osmotic pump (0.75 mg/h, ALZET model 2006, DURECT Corporation, Cupertino, CA, USA) as described in a previous paper [47]. Esaxerenone (kindly provided by Daiichi Sankyo Co., Ltd., Tokyo, Japan) was administered to the ESA group via diet at a dose of 1 mg/kg diet for 12 weeks, and the other groups were fed regular chow.…”

“…The gene expression levels of VEGFA, IL-1β, TNF-α, and GAPDH were measured using real-time PCR in the RAW264.7 cells and BMDMs. Total RNA was extracted as described in a previous paper [47]. Primer sequences were as follows: forward 5 -GTAACGATGAAGCCCTGGAGTG-3 , reverse 5 -TCACAGTGAACGCTCCAGGAT-3 ; IL-1β: forward 5 -CTACAGGCTCCGAGATGAACAA-3 , reverse 5 -TTCTTCTTTGGGTATTG CTTGG-3 ; TNF-α: forward 5 -CTCTTCTGTCTACTGAACTTCGGG-3 , reverse 5 -GGTGG TTTGTGAGTGTGAGGGT-3 ; and GAPDH: forward 5 -CCTCGTCCCGTAGACAAAATG-3 , reverse 5 -TGAGGTCAATGAAGGGGTCGT-3 .…”

“…The source and culture of RAW264.7 cells and fresh bone marrow cells (BMDMs) were the same as described in a previous paper [47]. HUVECs (Sciencell, Carlsbad, CA, USA) were cultured in endothelial cell medium (ECM, Sciencell, Carlsbad, CA, USA) containing 10% FBS, 1% endothelial cell growth supplement (ECGS), 1% penicillin and streptomycin, and maintained in a humidified atmosphere with 5% CO 2 at 37 • C. After the density of the HUVECs reached 80%, the cells were incubated in serum-free medium for 24 h prior to the cell experiments.…”