Esaxerenone Attenuates Aldosterone-Induced Mitochondrial Damage-Mediated Pyroptosis in Mouse Aorta and Rat Vascular Smooth Muscle Cells.

BACKGROUND: Vascular smooth muscle cell (VSMC) injury caused by the inflammatory response plays a key role in cardiovascular disease (CVD), and the vasoprotective effects of mineralocorticoid receptor blockers (MRBs) support the role of mineralocorticoid receptor (MR) activation. METHODS: C57BL/6 mice and VSMCs isolated from rats were treated with aldosterone and esaxerenone. Caspase-1, GSDMD-N, IL-1ß, and NR3C2 expression and DNA damage in aortic VSMCs were detected using immunohistochemistry, Western blotting, and TUNEL staining. Mitochondrial changes were detected by transmission electron microscopy (TEM). Reactive oxygen species (ROS), MitoTracker, JC-I, mitochondrial respiratory chain complexes I-V, and NR3C2 were detected using immunofluorescence and flow cytometry. Pyroptosis was detected with scanning electron microscopy (SEM). RESULTS: After aldosterone treatment, the number of TUNEL-positive cells increased significantly, and the expression of caspase-1, GSDMD-N, and IL-1ß increased. TEM revealed mitochondrial damage, and SEM revealed specific pyroptotic changes, such as cell membrane pore changes and cytoplasmic extravasation. Increased ROS levels and nuclear translocation of NR3C2 were also observed. These pyroptosis-related changes were reversed by esaxerenone. CONCLUSIONS: Aldosterone activates the MR and mediates mitochondrial damage, thereby inducing pyroptosis in VSMCs via the NLRP3/caspase-1 pathway. Esaxerenone inhibits MR activation and reduces mitochondrial damage and oxidative stress, thereby inhibiting pyroptosis.

Eplerenone reduces lymphangiogenesis in the contralateral kidneys of UUO rats.

Inflammation and fibrosis often occur in the kidney after acute injury, resulting in chronic kidney disease and consequent renal failure. Recent studies have indicated that lymphangiogenesis can drive renal inflammation and fibrosis in injured kidneys. However, whether and how this pathogenesis affects the contralateral kidney remain largely unknown. In our study, we uncovered a mechanism by which the contralateral kidney responded to injury. We found that the activation of mineralocorticoid receptors and the increase in vascular endothelial growth factor C in the contralateral kidney after unilateral ureteral obstruction could promote lymphangiogenesis. Furthermore, mineralocorticoid receptor activation in lymphatic endothelial cells resulted in the secretion of myofibroblast markers, thereby contributing to renal fibrosis. We observed that this process could be attenuated by administering the mineralocorticoid receptor blocker eplerenone, which, prevented the development of fibrotic injury in the contralateral kidneys of rats with unilateral ureteral obstruction. These findings offer valuable insights into the intricate mechanisms underlying kidney injury and may have implications for the development of therapeutic strategies to mitigate renal fibrosis in the context of kidney disease.

Esaxerenone Inhibits Interferon-γ Induced Pyroptosis of Macrophages in the Lungs of Aldosterone-treated Mice.

Lung immune cells such as lymphocytes and macrophages can induce an inflammatory response due to the activation of mineralocorticoid receptor (MR), which is manifested by the infiltration of inflammatory cells and the secretion of inflammatory cytokines and subsequent apoptosis, pyroptosis and necrosis of intrinsic lung cells and immune cells. Macrophages are immune cells that are abundant in the lung and act as the first line of defense against pathogens but are also aggravating factors of infection. The activation of the renin-angiotensin-aldosterone system (RAAS), especially aldosterone-stimulated MR activation, can induce macrophage and CD8+ T cell aggregation and the secretion of cytokines such as tumor necrosis factor-α (TNF-α) and interferon-gamma (IFN-γ). Increased IFN-γ secretion can induce macrophage pyroptosis and the release of interleukin 1-ß (IL-1ß), aggravating lung injury. In this study, lung injury in C57BL/6 mice was induced by subcutaneous micro-osmotic pump infusion of aldosterone. After 12 weeks of administration, the kidney, heart, blood vessels and lungs all showed obvious inflammatory injury, which manifested as rapid accumulation of macrophages. The overexpression of IFN-γ in the lungs of aldosterone-treated mice and the stimulation of MH-S and RAW264.7 alveolar macrophages (AMs) with aldosterone in vitro showed that IFN-γ induced pyroptosis of macrophages via the activation of the inflammasome, and the MR blocker esaxerenone effectively inhibited this effect and alleviated lung injury. In addition, IFN-γ secreted by CD8+ T cells is associated with macrophage pyroptosis. In conclusion, the inhibition of macrophage pyroptosis can effectively alleviate lung injury.

Eplerenone inhibits the macrophage-to-myofibroblast transition in rats with UUO-induced type 4 cardiorenal syndrome through the MR/CTGF pathway.

Cardiovascular complications are the leading causes of death in patients with chronic kidney disease (CKD), accounting for approximately 50% of deaths. Despite significant advances in the understanding of cardiac disease due to CKD, the underlying mechanisms involved in many pathological changes have not been fully elucidated. In our previous study, we observed severe fibrosis in the contralateral kidney of a 6-month-old rat UUO model. In the present experiment, we also observed severe fibrosis in the hearts of rats subjected to UUO and the macrophage-to-myofibroblast transition (MMT). These effects were inhibited by the mineralocorticoid receptor (MR) blocker eplerenone. Notably, in vitro, aldosterone-activated MR induced the MMT and subsequently promoted the secretion of CTGF, the target of MR, from macrophages; these changes were inhibited by eplerenone. The CTGF also induced the MMT and both the aldosterone and CTGF-induced MMT could be alleviated by the CTGF blocker. In conclusion, our results suggest that targeting the MR/CTGF pathway to inhibit the MMT may be an effective therapeutic strategy for the treatment of cardiac fibrosis.

Eplerenone Prevents Cardiac Fibrosis by Inhibiting Angiogenesis in Unilateral Urinary Obstruction Rats.

Introduction: Cardiovascular disease constitutes the leading cause of mortality in patients with chronic kidney disease (CKD), which is termed cardiorenal syndrome type 4 (CRS-4). Here, we report the development of pathological cardiac remodeling and fibrosis in unilateral urinary obstruction (UUO) rats. Methods: Hematoxylin and eosin (H&E) staining was performed to observe the pathology of myocardial tissue. The degree of myocardial tissue fibrosis was observed by Masson and Sirius red staining. Immunohistochemical staining was applied to detect the expression of CD34 and CD105 in myocardial tissue, and immunofluorescent staining was performed to examine the expression of CD34, collagen I/collagen III, and alpha smooth muscle actin (α-SMA). The expression of the signal pathway-related proteins vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptor 2 (VEGFR2), nuclear factor κB (NF-κB), and interleukin (IL)-1ß was tested by western blotting. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA levels of serum and glucocorticoid-inducible kinase (SGK)-1, NF-κB, and interleukin-1ß (IL-1ß). Results: The results showed the development of pathological cardiac remodeling and cardiac dysfunction in UUO rats. Moreover, there was more angiogenesis and endothelial-mesenchymal transition (End-MT) in the UUO group, and these effects were inhibited by eplerenone. Conclusions: The results indicated that this cardiac fibrosis was associated with angiogenesis and that End-MT was related to aldosterone and mineralocorticoid receptor (MR) activation. Moreover, in association with the MR/IL-1ß/VEGFA signaling pathway, early treatment with the MR antagonist eplerenone in rats with UUO-induced CKD may significantly attenuate MR activation and cardiac fibrosis.

Esaxerenone inhibits the macrophage-to-myofibroblast transition through mineralocorticoid receptor/TGF-ß1 pathway in mice induced with aldosterone.

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-ß1 expression, which promoted renal fibrosis. These effects were antagonized by the MR blocker esaxerenone. These findings suggest that targeting the MR/TGF-ß1 pathway may be an effective therapeutic strategy for renal fibrosis.

Eplerenone inhibits UUO-induced lymphangiogenesis and cardiac fibrosis by attenuating inflammatory injury.

Cardiorenal syndrome (CRS) is the leading cause of death associated with chronic kidney disease (CKD) and end-stage renal disease (ESRD). However, the underlying mechanisms of CRS are still poorly understood. Here, we studied a CKD model of unilateral ureteral obstruction (UUO) and observed pathological cardiac fibrosis and lymphangiogenesis in 180-day old UUO rats, in which inflammatory injury plays a major role. In addition, treatment of UUO rats with eplerenone, a mineralocorticoid receptor blocker (MRB), significantly reduced cardiac lymphangiogenesis and fibrosis. In conclusion, our experimental results showed that cardiac lymphangiogenesis in long-term UUO rats may be involved in the formation of cardiac fibrosis and that eplerenone can alleviate lymphangiogenesis and cardiac fibrosis by inhibiting inflammation.

Huoxue Jiedu Huayu Formula Alleviates Cell Pyroptosis in Contralateral Kidneys of 6-Month-Old UUO Rats through the NLRP3/Caspase-1/IL-1ß Pathway.

OBJECTIVES: To study the protective effects and mechanisms of Huoxue Jiedu Huayu formula on cell pyroptosis through the NLRP3/caspase-1/IL-1ß pathway in contralateral kidneys in 6-month-old unilateral ureteral obstruction (UUO) rats. METHODS: Wistar rats were randomly assigned to 5 groups: a Sham group, a unilateral nephrectomy group (UNX group), a UUO group, a UUO treated with spironolactone group (Spi group), and a UUO treated with Huoxue Jiedu Huayu formula group (HJHF group). After 6 months of oral drug intervention, blood and contralateral kidneys were collected for research. RESULTS: The morphology and function of the contralateral kidneys were essentially normal after unilateral nephrectomy. HJHF obviously decreased serum creatinine, urea, and inflammatory lesions and depressed cell pyroptosis based on the NLRP3/caspase-1/IL-1ß pathway. Moreover, spironolactone, a mineralocorticoid receptor (MR) blocker, suppressed cell pyroptosis through SGK-1 and NF-кB. CONCLUSION: HJHF and spirolactone inhibited excessive activation of MR and then reduced cell pyroptosis, which was dependent on the NLRP3/caspase-1/IL-1ß pathway, to protect the contralateral kidneys of 6-month-old UUO rats.