2025 Volume 48 Issue 4 Pages 422-431
Mineralocorticoid receptor (MR) blockers reduce cardiovascular complications as MRs play a crucial role in cardiovascular regulation. Diabetic cardiovascular complications are caused by vascular endothelial dysfunction. This study used a type 2 diabetic mouse model (DM) to investigate whether esaxerenone (ESAX), an MR blocker, ameliorates vascular endothelial dysfunction. ESAX (3 mg/kg/d) was administered via diet to KK-Ay mice or C57BL/6J mice, a nondiabetic control (Control), for 8 weeks, and metabolic parameters and blood pressure were measured. Vascular responses of the aortic segments were analyzed with acetylcholine, sodium nitroprusside, UK14304, or phenylephrine (PE). The other aortas were used for Western blot analysis. DM mice exhibited higher plasma glucose, insulin, metabolic parameters, and blood pressure levels than those of the Control mice. Parameters that did not include blood pressure were unaltered by DM or ESAX-administered DM (DM + ESAX). However, DM impaired UK14304-induced endothelial-dependent relaxation and nitric oxide production and elevated PE-induced contraction. ESAX administration ameliorated endothelial dysfunction and improved the protein kinase B (Akt) phosphorylation under α2-agonist UK14304 stimulation in the aorta from DM mice compared with that of the Control mice. However, ESAX did not recover the increased G protein-coupled receptor kinase 2 (GRK2) expression and activity in the DM aorta. Furthermore, the DM-induced phosphorylation of serum and glucocorticoid-regulated kinase 1 (SGK1) was inhibited by ESAX. Overall, ESAX attenuates the development of DM-induced endothelial dysfunction by reducing SGK1 activity and enhancing Akt activity without affecting the GRK2 pathway. These results suggest that the vascular protective effects of ESAX could be employed for diabetic vascular complications.
The endothelium is a single layer of cells that forms a physical barrier between the vessel lumen and the vascular wall and plays a critical role in maintaining vascular homeostasis. This process is primarily regulated by the release of various mediators, such as nitric oxide (NO) from the endothelium, which controls blood coagulation and vascular tone.1–3) Endothelial dysfunction describes a state in which the endothelium loses its normal physiological functions, which leads to a predisposition toward vasoconstriction and proinflammatory conditions.2,3) This dysfunction is recognized as a precursor to atherosclerosis and is considered as a key pathophysiological feature associated with type 2 diabetes (DM).3,4)
Evidence has shown that endothelial dysfunction is frequently observed in patients with DM and, thus, serves as a predictor for the risk of developing DM.4–6) Moreover, endothelial dysfunction is understood to be a key initiating factor for the onset and progression of DM-related vascular complications.4,7) A characteristic of vascular endothelial dysfunction is the reduction in the bioavailability of NO, following the reduction of endothelial NO synthase (eNOS), which is involved in the regulation of vascular function.8) In the circulatory system, NO derived from eNOS is a critical signaling molecule and functions as a critical vasoactive factor related to endothelial-dependent relaxation.
Ca2+/calmodulin binding can activate eNOS. However, eNOS is also activated by phosphorylation at Ser1177 by protein kinase B (Akt), a serine/threonine kinase.9) Recent studies indicate that eNOS is highly regulated by posttranslational modifications, including Akt-induced phosphorylation and interaction with several regulatory proteins such as G protein-coupled receptor kinase-2 (GRK2).9–12) Furthermore, Akt activity is reduced in DM.13) We previously reported that an increase in GRK2 stimulates the GRK2-Akt complex and suppression of eNOS phosphorylation in DM.12,13) Therefore, in this study we surmised that there might be reduced Akt activity coupled to GRK2 in DM.
The mineralocorticoid receptor (MR) and its antagonists (MRAs) play a crucial role in drug therapies for cardiovascular conditions.14) Furthermore, recent preclinical and clinical research has demonstrated that DM impairs vascular endothelial-dependent relaxation, with MRs playing a key role in the onset of DM-associated vascular dysfunction.15) Moreover, MRAs increase Akt activation and reverse this vascular dysfunction.16) Esaxerenone (ESAX), a novel nonsteroidal MR blocker, ameliorates endothelial dysfunction induced by DM by enhancing eNOS phosphorylation.17,18) Multiple clinical studies have shown that ESAX provides substantial antihypertensive and cardiorenal protective benefits compared with other MRAs.19) However, there is limited research on the influence of ESAX on vascular endothelial-dependent function in DM.
Therefore, in this study, we investigated whether ESAX ameliorates diabetes-induced endothelial dysfunction in a DM mouse model. Specifically, we hypothesized that ESAX stimulates NO production via the Akt pathway.
Male mice (C57BL/6J normal mice and KK-Ay type 2 diabetes model mice) were obtained from CLEA Japan Inc. (Tokyo, Japan) at 4 weeks of age. Feed and water were provided ad libitum throughout the study. Animals at 8 weeks of age with no observable abnormalities in their general condition were selected for further experiments. This study was conducted in compliance with the principles and guidelines on animal care of Hoshi University Animal Care and Use Committee and reviewed by an ethics committee (Approval No. P24-108) (accredited by the Ministry of Education, Culture, Sports, Science and Technology of Japan).
An 8-week treatment with ESAX (Daiichi Sankyo Co., Ltd., Tokyo, Japan) at a dose of 3 mg/kg/d provides renoprotective benefits to DM mice.17) Therefore, the treatment dose of ESAX was determined based on this report. ESAX was mixed in a pelleted diet to obtain a concentration of 3 mg/kg from 28 to 30 weeks of age for 8 weeks. Normal C57BL/6J mice, a parent strain of KK-Ay mice, were used as the nondiabetic Control group, which were fed regular chow (n = 15) or regular chow containing ESAX (n = 15). The KK-Ay mice were also fed regular chow (n = 15) or ESAX-contained regular chow (n = 15).
Systolic blood pressure (SBP) was measured at 7 weeks after the initiation of ESAX administration. Tail-cuff blood pressure was measured using a BP-98A blood pressure system (Softron, Tokyo, Japan), and at 8 weeks after the initiation of ESAX administration, body weight was measured.
The mice were then anesthetized, and samples, including blood and thoracic aortas, were collected and used for further analyses. Each sample was subjected to centrifugation at 4°C at 3500 rpm for 10 min, followed by plasma collection. Plasma glucose (Cat. No. 439-90901), insulin (Cat. No. AKRIN-011T), total cholesterol (TC; Cat. No. LABCHO-M1), high-density lipoprotein (HDL; Cat. No. 431-52501), and triglyceride (TG; Cat. No. LABTRIG-M1) were measured using commercial kits according to the manufacturers’ instructions (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Low-density lipoprotein (LDL) levels were calculated using the Friedewald equation (LDL = TC – HDL – TG/5) for cases in which TG levels were below 400 mg/dL.
Vasoactivity AnalysisExperiments were performed as previously described.11,12,20–22) The thoracic aorta was isolated and placed in cold (4°C) modified Krebs–Henseleit solution (KHS) (mM: NaCl 118; KCl 4.7; NaHCO3 25; CaCl2 1.8; NaH2PO4 1.2; MgSO4 1.2; glucose 11). Aortic rings (2 mm long) were dissected, and care was taken not to damage the endothelium during preparation. The aortic rings were mounted under an optimal resting tension of 1.5 g in a 10-mL organ bath containing KHS aerated with 95% O2 and 5% CO2 and contracted with KCl (80 mM) to confirm that the ring generated a normal level of contraction and that it had fully recovered by washing with fresh KHS solution. Relaxation responses to acetylcholine (ACh, 1 × 10−9–1 × 10−5 M, Daiichi Sankyo Co., Ltd.), NO donor sodium nitroprusside (SNP, 1 × 10−10–1 × 10−5 M, Wako), and α2-agonist UK14304 (Tokyo Chemical Industry, Tokyo, Japan) were performed after the aortic rings were pre-contracted with prostaglandin F2α (PGF2α; 1 × 10−6–3 × 10−6 M) (Fuji pharma, Tokyo, Japan). This produced approximately 1 g force, and the results were expressed as a percentage of precontraction (ACh, SNP, and UK14304). The contractile response to the α1-agonist phenylephrine (PE, 1 × 10−10–1 × 10−5 M, Sigma-Aldrich, St. Louis, MO, U.S.A.) was assessed, and the results were expressed as a contraction percentage of the maximum response to KCl. To examine the effects of NO on UK14304-induced relaxation and PE-induced constriction, the NOS inhibitor NG-nitro-l-arginine (l-NNA; 1 × 10−4 M) (Sigma-Aldrich) was applied to the bath solution 30 min before the administration of PGF2α or PE. Furthermore, to examine the effects of Akt on UK14304-induced relaxation and PE-induced constriction, an Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2 ([R]-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate) (Akt inh; 1 × 10−6 M; Calbiochem, San Diego, CA, U.S.A.) was applied to the bath solution 30 min before the administration of PGF2α or PE.
NO ReleaseExperiments were performed as previously described.11,12,20–22) After an equilibration period of 30 min in KHS at 37°C, the aortic rings (4 mm long) were incubated with ACh (1 × 10−6 M) or UK14304 (1 × 10−6 M) for 20 min. The KHS was collected to measure the nonstimulated (basal) and the ACh or UK14304-induced NO release. Furthermore, other aortic rings were added to tubes containing KHS with an Akt inhibitor (1 × 10−6 M) and incubated for 30 min, then UK14034 was added to the tube for a further 20 min. Finally, the semidried aorta was weighed and frozen in liquid nitrogen. The frozen aortas were used for Western blot (WB) analysis. The KHSs were stored at −20°C until further analysis. The analysis was performed using an ENO20 NOx Analyzer (Eicom, Kyoto, Japan), based on the LC method with post-column derivatization and Griess reagent. The amount of NO released (NOx; nitrate + nitrite level) was expressed as an arbitrary value in mol/g (weight of the aorta)/min.
WB AnalysisThe protein expressions in the aortas were determined using WB analysis, as previously described.11,12,20–22) To ensure the repeatability of WB experiments in vivo, the aorta samples from six individual mice were obtained from the Control, Control + ESAX, DM, and DM + ESAX groups, respectively. Total proteins were extracted from mouse aortic tissues and subjected to WB analyses. The primary antibody dilutions used for WB analyses were as follows: Akt (Cat. No. 9272; Cell Signaling Technology, Danvers, MA, U.S.A.; 1 : 1000), p-Akt-Ser473 (Cat. No. 9271; Cell Signaling Technology; 1 : 1000), GRK2 (Cat. No. sc-13143; Santa Cruz Biotechnology, Dallas, TX, U.S.A.; 1 : 1000), p-GRK2-Ser670 (Cat. No. GTX24473; GeneTex, Inc., CA, U.S.A.; 1 : 1000), serum and glucocorticoid regulated kinase 1 (SGK1; Cat. No. GTX107750; GeneTex, Inc., CA, U.S.A.; 1 : 1000), p-SGK1-Ser78 (Cat. No. BS-3395R; Bioss Antibodies Inc., MA, U.S.A.; 1 : 1000), and β-actin (Cat. No. A5316; Sigma Chemical Co., St. Louis, MO, U.S.A.; 1 : 10000). Similar to previous reports, GRK2 activity was determined as the inverse of phosphorylated GRK2 expression.11,12)
Statistical AnalysisAll results are represented as mean ± standard error (S.E.); n values indicate the number of experiments. Significant intergroup differences were determined using the one-way or two-way repeated ANOVA. Tukey’s test was used for comparison of multiple means. Statistical significance was determined as a p-values < 0.05.
During feeding with the regular chow or ESAX-containing regular chow, DM mice exhibited a higher body weight and higher levels of plasma glucose, plasma insulin, TC, LDL-C, and TG compared with the Control mice (Table 1). ESAX administration only reduced HDL-C, which may occur through its effects on lipid metabolism.17) ESAX-induced MR blockade may alter lipid metabolism, leading to decreased HDL-C levels. In addition, ESAX may influence hepatic lipoprotein metabolism.
| Control (n = 15) |
Control + ESAX (n = 15) |
DM (n = 15) |
DM + ESAX (n = 15) |
|
|---|---|---|---|---|
| Body weight (g) | 31.5 ± 0.6 | 33.5 ± 0.5 | 49.8 ± 1.3*** | 50.1 ± 0.6*** |
| Plasma glucose (mg/dL) | 254.7 ± 15.8 | 243.4 ± 24.7 | 554.4 ± 43.9*** | 630.4 ± 46.3*** |
| Plasma insulin (ng/mL) | 1.0 ± 0.1 | 0.8 ± 0.1 | 50.9 ± 7.7*** | 54.7 ± 10.2*** |
| TC (mg/dL) | 66.8 ± 3.9 | 91.2 ± 14.8 | 132.5 ± 9.5** | 173.8 ± 17.9*** |
| HDL-C (mg/dL) | 24.3 ± 0.7 | 21.4 ± 1.0 | 20.5 ± 0.9 | 15.9 ± 1.5***, # |
| LDL-C (mg/dL) | 38.9 ± 4.6 | 44.7 ± 7.2 | 73.4 ± 9.9* | 102.3 ± 8.5*** |
| TG (mg/dL) | 34.9 ± 2.8 | 36.7 ± 5.0 | 238.4 ± 38.2*** | 220.3 ± 42.5*** |
| SBP (mmHg) | 104 ± 1 | 106 ± 1 | 139 ± 2*** | 113 ± 1***, ### |
Data are expressed as mean ± standard error (S.E.). (n = 15, *p < 0.05, **p < 0.01, ***p < 0.001 Control vs. DM, #p < 0.05, ###p < 0.001 DM vs. DM + ESAX, Tukey’s post hoc test.) ESAX, esaxerenone; TC, total cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein; LDL-C, low-density lipoprotein; SBP, systolic blood pressure.
DM mice also showed elevated SBP compared with age-matched Control mice; however, feed containing ESAX decreased the SBP in DM mice (Table 1).
Effect of ESAX on the Vascular ResponsesFigure 1 and Table 2 illustrate the effect of ESAX on the ACh-(Fig. 1A), SNP-(Fig. 1B), and UK14304-induced (Fig. 1C) relaxation and PE-induced contraction (Fig. 1D) in the aortic rings from the Control, Control + ESAX, DM, and DM + ESAX mice. The ACh- and SNP-induced relaxations were not different between the aortic rings from the Control and DM mice, whether the mice were fed with or without ESAX (Fig. 1A, Table 2). The UK14304-induced relaxation was very weak in the aortic rings of the DM mice compared with the Control mice (Fig. 1C, Table 2). However, in the DM mice, the UK14304-induced relaxation response was significantly improved after ESAX feeding. The PE-induced contraction was enhanced in the DM mice compared with the Control mice (Fig. 1D, Table 2). However, in DM mice, PE-induced contraction response was significantly improved after ESAX feeding.
The values are shown as mean ± standard error (S.E.) (n = 6). ***p < 0.001, compared with the Control. ##p < 0.01, ###p < 0.001 compared with DM. ESAX, esaxerenone; DM, type 2 diabetes.
| Agonist | Control (n = 6) |
Control + ESAX (n = 6) |
DM (n = 6) | DM + ESAX (n = 6) |
|---|---|---|---|---|
| ACh | 92.5 ± 0.7 | 99.0 ± 0.6 | 91.5 ± 2.6 | 89.6 ± 0.6 |
| SNP | 99.1 ± 0.4 | 100.0 ± 0.0 | 100.0 ± 0.0 | 100.0 ± 0.0 |
| UK | 80.3 ± 3.8 | 80.2 ± 4.2 | 10.1 ± 2.7 *** | 31.7 ± 2.1***, ### |
| PE | 10.8 ± 2.6 | 6.4 ± 0.8 | 79.5 ± 4.4 *** | 44.0 ± 1.6***, ### |
Data are expressed as mean ± S.E. (n = 6). Emax, maximal agonist-induced response expressed as a percentage of precontraction (ACh and SNP, and UK) or 80 mM high K (PE-induced). ***p < 0.001 vs. the Control (Tukey’s post-hoc test). ###p < 0.001 vs. DM (Tukey’s post-hoc test). ACh, acetylcholine; SNP, sodium nitroprusside; UK, UK14304; PE, phenylephrine; DM, DM type 2 diabetes.
Next, we determined whether improvement in the inhibition of UK14304-induced endothelial-dependent relaxation and enhancement of PE-induced contraction by ESAX resulted from increased NO signaling. As α2-agonist-induced vasorelaxation largely depends on endothelial-derived NO in mice aorta,11) this relaxation was inhibited by pretreatment with l-NNA (Fig. 2A). Furthermore, l-NNA-treated Control + ESAX and DM + ESAX aortic rings inhibited UK14304-induced vasorelaxation to the same extent as that observed in the l-NNA-treated Control aortic rings (Fig. 2A). It is well known that the impedance of endothelial-derived NO induces the hypercontractility of blood vessels.21) The contractile force of the DM aortas did not increase further in these NO-blocked experiments, and the other groups exhibited significant contractility, showing a similar level of contractile force across all groups (Fig. 2B).
(C) Effects of 20 min of incubation with ACh (10−6 M) and UK14304 (10−6 M) on the NO production in aortic rings from the Control, Control + ESAX, DM, DM + ESAX mice. UK14304-induced NO production levels were significantly reduced in the DM group. The values are shown as mean ± S.E. (n = 6). **p < 0.01, compared with the UK-stimulated Control. ESAX, esaxerenone; DM, type 2 diabetes.
ACh and UK14304 induce vascular smooth muscle relaxation by triggering the production of NO.20,22) The changes in NO production in the aortas from the Control, Control + ESAX, DM, and DM + ESAX mice are shown in Fig. 2C. NO production in response to ACh was similar across all groups, whereas NO production in response to UK14304 in aortas from the DM group was significantly lower than that from the Control group, which is consistent with the results of ACh- and UK14304-induced endothelial-dependent relaxation. However, in the DM group, UK14304-induced NO production tended to increase after ESAX feeding. These results suggest that the negative endothelial modulation of some NO production was reduced by ESAX treatment.
ESAX Increased the UK14304-Induced Akt/NO Production Pathwayα2-Agonists induce vasorelaxation via the Akt/eNOS pathway.20) To clarify the mechanism by which ESAX increased UK14304-induced NO production in the DM mice, we tested whether improvement in the inhibition of UK14304-induced relaxation and enhancement of PE-induced contraction by ESAX resulted from increased Akt activation. UK14304-induced relaxation was inhibited by pretreatment with an Akt inhibitor (Fig. 3A). Akt inhibitor-treated Control + ESAX and DM ESAX aortic rings inhibited UK14304-induced vasorelaxation to the same extent as that observed in the Akt inhibitor-treated Control mice aorta rings (Fig. 3A). Furthermore, the contractile force in the Control and Control + ESAX aortas did not change with this Akt inhibitor pretreatment (Fig. 3B). However, the PE-induced contraction decreased in the DM + ESAX group compared with the DM group (Fig. 3C). Pretreatment with the Akt inhibitor significantly increased PE-induced contraction in the DM + ESAX group (Fig. 3C), and the contractile force in the aortas of the DM group was not further increased by Akt inhibition (Fig. 3C).
(B, C) Cumulative concentration–response curves for the phenylephrine (PE)-induced contraction of aortic rings from Control and Control + ESAX (B), DM, and DM + ESAX mice before and after 30 min of incubation with 10−6 M Akt inhibitor. The values are shown as mean ± S.E. (n = 6). (D) NO production under nonstimulated conditions in aortic rings from the Control, Control + ESAX, DM, and DM + ESAX mice. The aortic rings were incubated for 50 min. (E) Effects of 20 min of incubation with UK14304 (10−6 M) in the presence of Akt inhibitor on NO production in aortic rings from the Control, Control + ESAX, DM, and DM + ESAX mice. The aortic rings were treated with vehicle or Akt inhibitor 10−6 M for 30 min and then treated with UK14304 10−6 M for 20 min. The values are shown as mean ± S.E. (n = 6). ***p < 0.001 compared with the Control. ##p < 0.01 compared with DM. !!p < 0.01, !!!p < 0.001 compared with the Control + ESAX. $p < 0.05, $$$p < 0.001 compared with DM + ESAX. ESAX, esaxerenone; DM, type 2 diabetes.
As shown in Fig. 3D, NO production was minimal in all four groups and no significant differences were observed among them under nonstimulating conditions. NO production in the DM group under UK14304 stimulation was significantly lower than that of the Control group (Fig. 3E). As expected, ESAX administration significantly increased NO production in the DM group under UK14304 stimulation. Compared with the UK14304 stimulation alone-treated group, the NO production in the Control, Control + ESAX, and DM + ESAX groups was inhibited due to the Akt inhibitor. Thus, it does not appear that the Akt inhibitor suppresses basal NO production. Our results indicated that Akt activation of ESAX could lead to positive effects on the impaired UK14304-induced Akt/NO production pathway.
To further elucidate the molecular mechanisms underlying the regulation of ESAX on NO production in the DM group under UK14304-stimulating or nonstimulating conditions, we tested the expression of Akt by WB. These results revealed that DM significantly decreased Akt phosphorylation without modifying Akt protein expression (Figs. 4A and 4B). Furthermore, ESAX significantly induced Akt phosphorylation in DM (Figs. 4A and 4B); however, ESAX did not alter Akt phosphorylation under nonstimulating conditions in DM (Figs. 4C and 4D).
The aortas of the Control, Control + ESAX, DM, and DM + ESAX mice were measured for the expression levels of the p-Akt, Akt, and β-actin by Western blots under UK14304 stimulation (10−6 M, 20 min) (A, B) or nonstimulation (C, D). (A, C) Representative Western blots. (B, D) The expression of p-Akt and Akt was measured. All data are shown as mean ± S.E. (n = 6). ***p < 0.001 compared with the Control. ##p < 0.01 compared with DM. ESAX, esaxerenone; DM, type 2 diabetes.
Previously, we reported that GRK2 is an important negative regulator of the Akt/eNOS/NO production signaling pathway in vascular endothelial cells.11,12) We demonstrated that the inhibition of GRK2 activity activates the Akt/eNOS pathway, thereby improving endothelial function.11,12) Therefore, to investigate whether ESAX improves endothelial function by suppressing the increased expression of GRK2 and GRK2 activity in DM, we examined GRK2 expression and GRK2 activity. The results indicate that the upregulation of GRK2 expression and GRK2 activity in the aortas of DM mice was not altered by ESAX treatment (Fig. 5).
The aortas of the Control, Control + ESAX, DM, and DM + ESAX mice were measured for the expression levels of GRK2 and β-actin by (A) Western blot analysis and (B) GRK2 activity under UK14304 stimulation (10−6 M, 20 min). All data are shown as mean ± S.E. (n = 6). **p < 0.01, ***p < 0.001 compared with the Control. ESAX, esaxerenone; DM, type 2 diabetes; GRK2, G protein-coupled receptor kinase 2.
SGK1 expression is upregulated in vascular cells following exposure to high glucose levels.23) Previous studies have demonstrated that SGK1, a classical target gene of MR, plays a crucial role in vascular remodeling and regulates inflammatory responses.24) Therefore, to elucidate the underlying mechanisms of the improved UK14304-induced Akt/NO production in the DM + ESAX group, the experiments that followed explored the effects on SGK1 activation and expression. ESAX completely and significantly abolished the upregulation of SGK1 phosphorylation under UK14304 stimulation in DM without modifying SGK1 protein expression (Figs. 6A and 6B). In addition, ESAX specifically reduced SGK1 phosphorylation under nonstimulating conditions in DM (Figs. 6C and 6D).
The aortas of the Control, Control + ESAX, DM, and DM + ESAX mice were measured for the expression levels of p-SGK1, SGK1, and β-actin by Western blot analysis under UK14304 stimulation (10–6 M, 20 min) (A, B) or nonstimulation (C, D). (A, C) Representative Western blots. (B, D) The expression of p-SGK1 and SGK1 was measured. All data are shown as mean ± S.E. (n = 6). *p < 0.05 compared with the Control. #p < 0.05, ###p < 0.001 compared with DM. ESAX, esaxerenone; DM, type 2 diabetes; SGK1, serum and glucocorticoid-regulated kinase 1.
These results are the first to demonstrate a role for ESAX in ameliorating endothelial function in DM. Furthermore, the mechanism by which ESAX improved endothelial dysfunction was elucidated, revealing that it inhibited SGK1 and increased Akt activity without involving the GRK2 pathway.
Vascular endothelial dysfunction is a key factor that leads to increased mortality and morbidity from cardiovascular disease in individuals with DM.25) Consistent with our findings, both preclinical and clinical studies have shown that MR activity contributes to an increased risk of developing diabetes.15,26) In addition, MRAs improved vascular endothelial dysfunction in DM models.26,27) This suggests that MR antagonism using ESAX may serve as an effective vasoprotective treatment for DM.
In this study, we used ESAX, which possesses greater specificity for MR binding and has a nonsteroidal structure.18) Previous preclinical and clinical studies have shown that ESAX is more potent than spironolactone or eplerenone in treating hypertension.18,28) The nonrenal effects of MRAs have been garnering increasing attention. Notably, multiple studies have demonstrated the anti-atherosclerotic effects of MRAs that target vascular cells.29) Endothelial dysfunction is a key underlying factor in the development of vascular complications in patients with DM.30) Therefore, preventing the development of endothelial dysfunction is essential to avoid vascular complications. Accordingly, we concentrated on examining the effects of ESAX on vascular function in a DM mouse model. The KK-Ay mouse is a genetically modified rodent model used to study diabetic vascular complications as it spontaneously develops obesity, insulin resistance, and severe DM. Therefore, we selected 28-week-old KK-Ay mice, in which only the UK14304-induced vascular relaxation response, but not ACh-induced vascular relaxation, was attenuated.
Various stimuli, such as α2-agonists modulate NO production by promoting Akt/eNOS.11,12,20,22,31) ACh triggers the release of Ca2+ from intracellular stores and facilitates the influx of extracellular Ca2+ into endothelial cells, resulting in the activation of eNOS through calcium/calmodulin-dependent mechanisms.10,20) As we have previously reported, the α2-agonist-induced endothelial-dependent relaxation (the endothelial-dependent relaxation response mediated via the Akt/eNOS pathway) becomes chronically insufficiently relaxed without a decrease in the ACh-induced endothelial-dependent relaxation response (the endothelial-dependent relaxation response via the classical pathway) under DM conditions,11,20,22) so we measured the endothelial-dependent vasorelaxation. To measure endothelial-dependent vasorelaxation, we used ACh and UK14304, which causes NO-mediated vasodilation in the artery. Moreover, we previously reported that only clonidine-induced relaxation responses are impaired under DM conditions,11,12,20,22) and UK14304 is an α2-adrenergic receptor agonist, similar to clonidine. In this study, our results revealed that only the UK14304-induced endothelial-dependent vasorelaxation was attenuated in the aorta from DM mice (Fig. 1). There was no change in vasorelaxation in response to ACh- and the NO donor SNP-stimulation among the Control, Control + ESAX, and DM + ESAX group mice, indicating that endothelial dysfunction, especially the downregulation of the Akt/eNOS pathway in the endothelium, only occurred in DM mice. When an Akt inhibitor was administered to mouse aortas in the previous study, there was no significant effect on ACh-induced relaxation or ACh-stimulated NO production in any group, whereas the α2-agonist-induced responses in control aortas were reduced by the Akt inhibitor.11,12,20,22) This suggests that UK14304-induced vascular relaxation is regulated by the Akt/eNOS pathway and that UK14304-induced vascular endothelial relaxation responses are regulated by the Akt/eNOS pathway. Moreover, ESAX only prevents impaired UK-induced relaxation, but not ACh-induced relaxation. However, ESAX-induced changes were observed in the aortic contractile response (Fig. 1D). The PE-induced contractile response in the aorta from DM mice was enhanced compared with that of the Control mice. In DM, it is believed that more intense vasoconstriction occurs because of an increase in the ability of blood vessels to contract, along with a reduced capacity to relax. α1-receptor expression and sensitivity are upregulated in DM, suggesting that increased α1-receptor sensitivity is the primary cause of the enhanced contractile response in the DM group. A decrease in aortic contraction was observed in DM after ESAX administration. In addition, enhanced vasoconstriction in the aorta of the Control, Control + ESAX, DM, and DM + ESAX groups resulted in a further increase under the NOS inhibitor l-NNA (Fig. 2). Therefore, the increase in NO production by ESAX may be influenced by PE-induced contraction. Furthermore, we found that an Akt inhibitor enhanced the PE-induced aortic contraction in the Control, Control + ESAX, and DM + ESAX mice (Fig. 3). These results suggest that ESAX may have improved the vasorelaxation and contraction responses by increasing NO production via the Akt/eNOS pathway. Moreover, we observed the direct effects of ESAX on the UK14304-induced NO production. Data obtained in the present study revealed that although ACh-stimulated NO production did not change in all groups, UK14304-induced NO production resulted in a clear decrease in the DM group, whereas ESAX administration markedly increased the UK14304-induced NO production in the DM group. Furthermore, UK14304-induced NO production was inhibited by an Akt inhibitor in the Control, Control + ESAX, and DM + ESAX groups. Furthermore, our data also indicate that the UK14304-induced NO production occurred via the Akt/eNOS pathway, and ESAX administration in the DM group activated this pathway.
The current data revealed that ESAX augmented the DM-suppressed Akt phosphorylation under UK14304 stimulation. Akt plays a vital role in regulating endothelial cell functions.32) In addition to controlling endothelial cell migration, survival, and tube formation, Akt sustains NO production, thereby supporting endothelial cell homeostasis via the direct phosphorylation of eNOS.31) However, in DM, both Akt phosphorylation and its ability to effectively phosphorylate eNOS are reduced.11,12,20,22,33) The disruption of the Akt/eNOS pathway contributes to endothelial dysfunction in DM.11,12,20,22) Conversely, treatments that enhance Akt activation are considered beneficial for vascular health by stimulating eNOS activity.34) The present data indicate that the effect of ESAX on endothelial cells is mediated via Akt signaling.
GRK2 is a serine-threonine kinase involved in controlling the intracellular signaling pathways of multiple G protein-coupled receptors.35) GRK2 can interact with various proteins that have roles in signaling and receptor trafficking, including Akt.35,36) Furthermore, studies have shown that the interaction between GRK2 and Akt suppresses the kinase activity of Akt.36) Recently, we demonstrated that GRK2-Akt interactions reduced the α2-agonist-induced relaxation response in DM aortas.11,12) In this study, we investigated whether ESAX functions as a checkpoint to limit the GRK2-mediated inhibition of the Akt/eNOS/NO production signal pathway. However, we found that both the expression and activity of GRK2 in the aorta increased under DM conditions and remained unchanged following ESAX administration. Given these findings, ESAX activated the Akt/eNOS signaling pathway independently of the GRK2 pathway.
The inflammatory signaling of mineralocorticoids involves the activation of SGK1, and mineralocorticoids strongly enhance the expression of SGK1 and activate the Akt pathway, which subsequently leads to SGK1 activation.37) Akt and SGK1 share a large number of target proteins,38) and both Akt and SGK1 are involved in promoting cell proliferation and survival responses.39) Furthermore, previous studies have shown that SGK1 expression is increased in patients with obesity and DM, as well as in animal models thereof.40) In our study, ESAX successfully reversed the UK14304-stimulated Akt phosphorylation and SGK1 phosphorylation in DM. However, ESAX did not alter Akt phosphorylation under nonstimulated conditions in DM, whereas it specifically reduced SGK1 phosphorylation in DM. This suggests that MRs might function upstream of SGK1 and that the increased expression of SGK1 might suppress the Akt/eNOS pathway. Aldosterone activates MRs to induce inflammatory changes in the vasculature. However, we did not directly examine the impact of aldosterone and MRs on vascular endothelial cells in this study. Thus, further studies are required to verify this hypothesis and elucidate the mechanism of MRs in vascular endothelial cells. However, from this perspective, ESAX may improve vascular endothelial function by reducing SGK1 activity, which results in vascular relaxation and subsequent blood pressure reduction.
In conclusion, our findings indicate that ESAX administration improves DM-induced endothelial dysfunction by enhancing Akt activity in DM mice. Moreover, the DM-induced Akt inactivation was ameliorated by ESAX administration by reducing SGK1 activity without affecting GRK2 activity in mice aortas. Our findings support the concept that MR significantly contributes to vascular endothelial dysfunction, and the blocking of MR with ESAX exhibits potential as an effective treatment for vascular issues in DM.
Esaxerenone was provided by Daiichi Sankyo Co., Ltd.
This study was a collaborative study supported by Daiichi Sankyo Co., Ltd. The funder had no role in finalizing the study design, execution of experiments, decision to publish, or preparation of the manuscript.
