Spironolactone ameliorates endothelial dysfunction through inhibition of the AGE/RAGE axis in a chronic renal failure rat model
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Spironolactone can improve endothelial dysfunction in the setting of heart failure and diabetes models. However, its beneficial effect in the cardiovascular system is not clear in the setting of non-diabetic renal failure. We conducted this study to investigate whether spironolactone can ameliorate endothelial dysfunction in a 5/6 nephrectomy model, and to determine the underlying mechanism.
Twenty-four Sprague-Dawley rats were divided into four groups. A renal failure model was created using the 5/6 nephrectomy method. The four groups included: Sham-operation group (Group1), chronic kidney disease (CKD; Group2), CKD + ALT-711 (advanced glycation end products [AGEs] breaker; Group 3), and CKD + spironolactone group (Group4). Acetylcholine (Ach)-mediated vasodilatation responses were compared between the four groups. To investigate the underlying mechanism, we cultured human aortic endothelial cells (HAECs) for in-vitro assays.
Differences between two groups were determined with the paired student’s t test. Differences between three or more groups were determined through one-way analysis of variance (ANOVA) with post-hoc analysis with LSD method.
Compared with Group 1, Group 2 has a significantly impaired Ach-mediated vasodilatation response. Group 3 and 4 exhibited improved vasoreactivity responses. To determine the underlying mechanism, we performed an in-vitro study using cultured HAECs. We noted significant sirtuin-3 (SIRT3) protein downregulation, reduced phosphorylation of endothelial nitric oxide synthase at serine 1177 (p-eNOS), and increased intracellular oxidative stress in cultured HAECs treated with AGEs (200 μg/mL). These effects were counter-regulated when cultured HAECs were pretreated with spironolactone (10 μM). Furthermore, the increased p-eNOS production by spironolactone was abrogated when the HAECs were pretreated with tenolvin (1 μM), a SIRT3 inhibitor.
Spironolactone could ameliorate endothelial dysfunction in a 5/6 nephrectomy renal failure model through AGEs/Receptor for AGEs (RAGEs) axis inhibition, SIRT3 upregulation, and nicotinamide adenine dinucleotide phosphate oxidase-2 (NOX-2) and its associated intracellular oxidative stress attenuation.
KeywordsAdvanced glycation end products Spironolactone Endothelial dysfunction
Advanced glycation end products
Analysis of variance
Blood urea nitrogen
Chronic kidney disease
enhanced chemiluminescence detection
endothelial nitric oxide synthase
Human aortic endothelial cells
nicotinamide adenine dinucleotide phosphate oxidase
Receptor for advanced glycation end products
ROS-induced ROS release
reactive oxygen species
sodium dodecyl sulphate-polyacrylamide gel electrophoresis
Chronic kidney disease (CKD) is a well-known risk factor for cardiovascular disease. Approximately half of the uremia patients die of cardiovascular disease.  The association between CKD and cardiovascular disease cannot be explained by conventional cardiovascular disease risk factors. Accelerated atherosclerosis has been observed in the setting of CKD and has been proposed as a reason for higher-than expected death rate in patients with CKD.  The explanation for the association between CKD and cardiovascular disease may be attributed to increased oxidative stress. [3, 4] Advanced glycation end products (AGEs) are formed by a non-enzymatic reaction between amino acids and sugar called the Maillard reaction.  The formation of AGEs requires the presence of increased oxidative stress.  Clinically, increased AGEs can be observed in the setting of aging, smoking, diabetes, and CKD. Studies have implied that tissue accumulation of AGEs could result in arterial stiffness, endothelial dysfunction and atherosclerosis. [7, 8, 9, 10] Hence, AGEs may play a major role in CKD-related cardiovascular disease.
Spironolactone is a mineralocorticoid receptor antagonist. Previous study proposed that spironolactone could ameliorate endothelial dysfunction in a heart failure rat model.  However, the question whether spironolactone can have overall long-term beneficial outcomes in patients with CKD remains controversial. The potential beneficial cardiovascular effect should weigh against the risk of hyperkalemia in the setting of renal failure.  Recently, a clinical trial indicated that low-dose spironolactone (25 mg daily) could provide better cardiovascular outcomes in uremia patients and the plasma potassium levels were not significantly increased.  However, the potential mechanism through which spironolactone engenders cardiovascular benefits in patients with CKD remained elusive. To investigate this issue, we conducted an animal study and used the Sprague-Dawley rat, a model which has been applied in all aspects of biomedical research. We used 5/6 radical nephrectomy as an experimental renal failure model.
We hypothesized that treatment with spironolactone in a 5/6 nephrectomy renal failure rat model could ameliorate endothelial dysfunction, and that the beneficial effect of spironolactone may involving inhibition of the AGEs/Receptor for AGEs (RAGE) axis and attenuation of cellular oxidative stress.
Animals and experimental design
Twenty-four male Sprague-Dawley rats, weighing 180–200 g, from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan) were housed at the animal facility of China Medical University under a 12-h light-dark cycle and had free access to a standard rat chow diet and tap water. The food and water intake, their activity, and urine output were observed daily throughout the experiment. After an acclimization period of 2 weeks, the animals were randomly divided into four groups.
Group 1 (Control group, n = 6) comprised rats that received a sham-operation.
Group 2 (CKD group, n = 6) contained rats that were anesthetized with pentobarbital sodium (35 mg/kg, administered intraperitoneally) and received a two-stage 5/6 nephrectomy operation. (The upper and the lower poles of the left kidney, as well as the whole right kidney were removed) [14, 15] The interval between the two index operation was 2 weeks. After the procedure, the rats were given a standard rat chow diet and tap water ad libitum for 2 months. After the two-month post-operation period, blood samples were collected to measure plasma blood urea nitrogen (BUN), creatinine, and AGEs levels. The rats were then treated with same volume of vehicle by oral gavage for the subsequent one month.
Group 3 (CKD + AGEs breaker [ALT-711], n = 6) comprised rats that received the same two-stage 5/6 nephrectomy operation. The rats were given a standard rat chow diet and tap water ad libitum for 2 months post-operation. Blood samples were collected. After the 2-month post-operation period, the rats were treated with ALT-711 (3 mg/kg/day) by oral gavage for 1 month.
At the end of the experiment, body weights were measured in all animals, and blood pressure levels were measured in all conscious animals by using the tail-cuff method. The rats were euthanized with CO2 inhalation. The remnant kidneys and hearts were removed and weighed. The thoracic aorta was immediately removed for vascular reactivity study, and a portion of the thoracic aorta was fixed in 10% neutral buffered formalin for immunohistochemical analysis.
All animal studies adhere to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, Eighth Edition. All animal study protocols were approved by the Institutional Animal Use and Care Committee of China Medical University (protocol number: 104–272).
Vascular reactivity study
The thoracic aorta was rapidly removed and excised into 3 mm segments. A sample was transferred onto an arteriograph system (Radnoti M1000; Covina, California, USA) for measuring vascular tension force. To mount the vessel segments, two individual parallel wires were passed through the vessel lumen, and attached to the support brackets. One bracket was connected to an isometric force transducer. The vessel ring was bathed in a 5-mL chamber containing Kreb’s ringer solution at 37 °C. Determining arterial reactivity.
To evaluate endothelium-dependent arterial relaxation, aortic rings were precontracted with phenylephrine at a concentration of 10− 6 mol/L. As the arterial contraction reached a plateau and stabilized, acetylcholine (Ach) was added cumulatively at concentrations in the range of 10− 8 - 10− 5 mol/L. To evaluate endothelium-independent arterial relaxation, the endothelial layer of the aortic ring was denuded, and precontracted with phenylephrine at a concentration of 10− 6 mol/L. Sodium nitroprusside (SNP) was then added cumulatively at concentrations in the range of 10− 9 - 10− 6 mol/L.
Sections of the thoracic aorta were fixed in 10% neutral buffered formalin for 24 h. After fixation, a tissue block was embedded in paraffin and sectioned with a microtome. Subsequently, the tissue sections were mounted on glass slides and dried to remove any water. Before being stained with primary and secondary antibodies, the tissue sections on the glass slides were deparaffinized with xylene, ethanol before rehydration. To remove the formation of methylene bridges during fixation, heat- induced epitope retrieval was applied. The tissue sections were stained with a primary antibody such as mouse anti-endothelial nitric oxide synthase (eNOS) antibody (1:50; Abcam, Cambridge, MA, USA) or mouse anti–phospho-eNOS antibody (p-eNOS) (1:50; Abcam, Cambridge, MA, USA), or mouse anti-RAGEs antibody (1:50; Abcam, Cambridge, MA, USA) at 4 °C overnight. Subsequently, the stained sections were washed with phosphate-buffered saline (PBS) twice and incubated with a horseradish peroxidase (HRP)-conjugated mouse IgG secondary antibody (1500; GeneTex, California, USA) at room temperature for 1 h. Images were acquired using a Nikon E600 light microscope (Nikon, Tokyo, Japan; magnification, 200X).
Human aortic endothelial cells (HAECs) were purchased from PromoCell (Heidelberg, Germany). The HAECs were cultured in the endothelial cell growth medium MV2 (PromoCell GmbH, Heidelberg, Germany) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin(Thermo Fisher Scientific, MA, USA) at 37 °C in a 5% CO2 atmosphere. The medium was replaced every 2 days. Passage 5–6 was used for this study.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
The HAECs were plated on a 96-well plate at a density of 5000–10,000/well. The cells were treated with the vehicle, AGEs (Sigma Aldrich, Merck KGaA, Darmstadt, Germany) or bovine-serum albumin (BSA) (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) at concentrations of 100, 200 and 500 μg/ml and were incubated for 24 h. After the incubation period, the HAECs were treated with 20 μL of 5 mg/mL MTT reagent and incubated for 4–6 h at 37 °C. The media were carefully removed subsequently. Dimethyl sulfoxide (DMSO)(Sigma Aldrich, Merck KGaA, Darmstadt, Germany) 100 μL was added to each well and followed by thorough mixing to dissolve formazan crystals. Subsequently, the cells were agitated on a plate shaker for 5 min. Absorbance was measured at 570 nm using a TECAN Infinite M1000 plate reader (Thermo Fisher Scientific, MA, USA). Results were compared with the control group.
Measurements of cytosolic and mitochondrial oxidative stress
The HAECs were seeded in a 96-well plate at a density of 1.4 × 104 cells per well. The cells were pretreated with AGEs neutralizing antibody 50 μg/mL, N-acetylcysteine 20 mM, and spironolactone (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) at concentrations of 0.1 μM, 1 μM, and 10 μM for 30 min. The cells were then treated with AGEs 200 μg/mL and incubated for 24 h. For the control group, the cells were treated with the vehicle. In each well, the cells were rinsed three times with PBS (Thermo-Fisher Scientific, MA, USA) for 5 min. Subsequently, 100 μL of 2.5 μM 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) (Millipore, Merck KGaA, Darmstadt, Germany) or 100 μL of 5 μM MitoSox red (Invitrogen, CA, USA) was added to each well, according to the study design, after which the cells were incubated for 30 min in the dark. Cytosolic and mitochondrial oxidative stress levels were next quantified by measuring the fluorescence density at excitation and emission wavelengths of 485 nm and 530 nm, respectively, using the TECAN Infinite M1000 plate reader (Thermo Fisher Scientific, MA, USA).
Protein concentrations were determined using a BCA protein assay kit. Equal amounts (20 μg/lane) of protein were separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) at 70–120 V. The proteins were next transferred to polyvinylidene difluoride (PVDF) membranes through electroblotting for 60 min at 100 V. The transferred membranes were then blocked with 5% non-fat milk [1 g non-fat milk dissolved in 20 mL Tris-buffered saline with 0.05% Tween-20 (TBST)] for 2 h on a shaking table. Subsequently, the membranes were incubated with primary antibodies, including anti-eNOS (1:1000; Abcam, MA, USA), p-eNOS Ser 1177 (1:1000; Abcam, MA, USA), RAGEs (1:1000; Abcam, MA, USA), sirtuin-3 (SIRT3; 1:1000; SantaCruz, Texas, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:2000; GeneTex, CA, USA) at 4 °C overnight on a shaking table. The membranes were then probed with their respective secondary antibodies conjugated with horseradish peroxidase including rabbit IgG antibody and mouse IgG antibody (1:10000; GeneTex, CA, USA) at room temperature for 50 min. After the probing process, each of the membranes was washed three times with TBST for 5 min. The bands were then visualized using an enhanced chemiluminescence detection kit (ECL kit) (Millipore, Merck KGaA, Darmstadt, Germany). The protein bands were quantified using Image J software.
Intact cells were seeded on a 8-well chamber slide at a density of 1.7 × 104 cells/well and cultured overnight. On the second day, different groups of the cultured cells were treated with the vehicle, AGEs 200 μg/mL, AGEs 200 μg/mL + spironolactone 10 μM, and AGEs 200 μg/mL + spironolactone 10 μM + Tenovin-6 1 μM; the treated cells were incubated for 24 h. Next, the cells were fixed with 4% paraformaldehyde and incubated with the diluted primary antibody rabbit anti- nicotinamide adenine dinucleotide phosphate oxidase (NOX-2; 1:100; Thermo-Fisher Scientific, MA, USA), followed by incubation overight at 4 °C. Nuclei were stained with 4′, 6′-diamidino-2-phenylindole (DAPI) (excitation wavelength, 543 nm; emission wavelength, 573 nm). Subsequently, the cells were probed with a fluorochrome-conjugated secondary antibody (Donkey anti-Rabbit IgG, Alexa Fluor 594;1:500; Thermo-Fisher Scientific, MA, USA) and were incubated for 1 h at room temperature. Cells on different chamber slides were examined in a confocal imaging system (Zeiss LSM 510 META, Heidelberg, Germany).
To avoid subjective bias when assessing the results of the animal experiments and cell culture assays, the analyses of the study results were performed by CKC, SMY, and CCT, who were blinded to the experimental conduction.
Continuous variables are expressed as mean ± standard error from at least three experiments. Differences between before and after treatments in the same group were determined with the paired student’s t test. Differences between three or more groups were determined through one-way analysis of variance (ANOVA) with post-hoc analysis with LSD method. A p value < 0.05 was considered as statistically significant. All statistical analyses were performed using SPSS 16.0, IBM, Chicago IL, USA.
Comparisons of different groups in terms of renal function before and after treatments
BUN before treatment (mg/dL)
BUN after treatment
Cr before treatment
Cr after treatment
Body weight (g)
15.83 ± 1.6
15.33 ± 3.88
0.73 ± 0.05
0.7 ± 0.18
588.83 ± 44.74
125.50 ± 5.58
73.33 ± 4.18
35.15 ± 4.18#
45.83 ± 4.96*,$
0.82 ± 0.20
1.7 ± 0.43*,$
551.67 ± 25.10
164.83 ± 7.31$
86.33 ± 4.5$
CKD + ALT-711
32.5 ± 7.48#
46 ± 17.32*,$
0.90 ± 0.22
1.82 ± 0.76$
585.67 ± 48.30
161.83 ± 5.95$
83.50 ± 4.72$
CKD + SPL
37.5 ± 8.17#
43.83 ± 18.09$
1.08 ± 0.33
1.32 ± 0.33$
562.50 ± 54.24
165.17 ± 6.18$
85.00 ± 3.58$
The major findings of our study include the following: First, spironolactone ameliorated endothelial dysfunction in a 5/6 nephrectomy renal failure model. Second, spironolactone could inhibit the AGEs/RAGE axis through the downregulation of RAGE protein expression, thereby disrupting the self-promoting positive feedback loop of the AGEs/RAGE axis and its associated inflammatory cascade. [16, 17] Third, spironolactone may reverse the downregulatory effect of SIRT3 caused by the AGEs/RAGE axis, thereby reducing mitochondrial reactive oxygen species (ROS) production, and the NOX-2 expression. The overall oxidative stress reduction may result in eNOS activation.
Spironolactone improves endothelial dysfunction in the setting of CKD
Previous studies have suggested that mineralocorticoid receptor antagonist could ameliorate endothelial dysfunction in the setting of heart failure, diet-induced obesity, and a streptozocin-induced diabetic model. [11, 18, 19] However, whether spironolactone can ameliorate endothelial dysfunction in the setting of non-diabetic nephropathy is unclear. Our study provides evidence that spironolactone could ameliorate endothelial dysfunction, an early indicator of atherosclerosis, in a non-diabetic renal failure model. Unlike previous studies [20, 21], our study failed to demonstrate that treatment with spironolactone could improve renal function. Our study design may explain this discrepancy, we used 5/6 radical surgical nephrectomy to create the renal failure model, and such an advanced stage of renal failure renders treatment with spironolactone ineffective. Though our study failed to demonstrate significant differences of plasma creatinine levels after the treatment between Groups 2,3 and 4. A lower renal function decline rate could be observed in Group 4 in comparison with those of Group 2 and 3. This indicates that spironolactone and ALT-711 may have differential effects and mechanism on renal function. Our study cannot exclude the possibility that by application of other renal failure models, spironolactone and ALT-711 may improve endothelial dysfunction through improving renal function by different pathways. However, based on current evidence, our study suggests that instead of improving renal function, spironolactone ameliorated endothelial dysfunction in the 5/6 nephrectomy model through directly antagonizing the vasculopathy effect caused by uremic toxin. AGEs are well-known uremic toxins, and our study suggest that spironolactone may alleviate the AGEs-induced vasculopathy.
Mitochondrial protection role of SIRT3
The sirtuins belong to a highly conserved family of NAD+- dependent enzymes that regulate lifespan in lower organisms. The role of sirtuins to prevent senescence, insulin resistance, and vascular atherosclerosis has been proposed.20 The SIRT3 is present in mitochondria and has been proposed to reduce mitochondrial ROS production and maintain mitochondrial integrity. SIRT3 can deacetylate global mitochondrial proteins, such as acetyl coenzyme A synthetase 2, and complex 1 of the electron transport chain, and activate manganese superoxide dismutase, thereby reducing mitochondrial ROS production. [22, 23, 24, 25] Furthermore, SIRT3 can deacetylate FOXO3, thereby protecting mitochondria from oxidative damage.  Our finding that the AGEs/RAGE axis activation could downregulate the production of SIRT3 in HAECs is in line with those in the literature.  Our study further suggests that pretreatment with spironolactone in the HAECs can reverse SIRT3 production and reduce mitochondrial oxidative stress.
Interaction between NOX and mitochondrial ROS
As illustrated in Fig. 8, NOX2 expression increased when HAECs were pretreated with tenovin-6 (SIRT3 inhibitor). This may be explained by the connection between mitochondrial ROS production and NOX.  A crosstalk between NOX and mitochondrial ROS production has been a well-known example of ROS-induced ROS release (RIRR). ROS generated from mitochondria could stimulate phosphoinositide 3-kinase and translocate the Rac-1, thereby activating NOX. The ROS generated from mitochondria may be transient and their stimulation of NOX may provide a more sustained, self-amplified ROS production.  Our findings indicate that AGEs/RAGE axis activation could downregulate the SIRT3 expression and increase mitochondrial ROS production. Such increased mitochondrial ROS production may further stimulate NOX2 production, further augment cytosolic ROS production, and ultimately lead to endothelial dysfunction. The pharmacologic role of spironolactone in ameliorating endothelial dysfunction may involve the reversal of this pathway.
Our study has several limitations. First, instead of screening all NOX subtypes, we examined only the interaction between mitochondrial ROS and NOX2. The main reason is that NOX2 is the prototype of NOX  and has been proposed to influence endothelial dysfunction in humans.  Second, increased cytosolic oxidative stress may not be generated only from mitochondria and NOX, we cannot exclude the possibility that intracellular oxidative stress originated from other pathways such as xanthine oxidase, lipoxygenase, cyclooxygenase, and uncoupled eNOS. However, a previous study suggests that spironolactone enhances eNOS protein expression through inhibiting NOX, not xanthine oxidase or uncoupled eNOS ; in addition, the interaction between mitochondrial ROS and NOX is a well known model for RIRR.  Therefore, we propose that spironolactone may attenuate the AGEs/RAGE-induced endothelial dysfunction through inhibiting cytosolic ROS generation from mitochondria and NOX. Third, uremic toxins are not limited to AGEs; thus, we cannot exclude the possibility that spironolactone can ameliorate endothelial dysfunction induced by uremic toxins other than AGEs. Fourth, compared with Group 2, we failed to demonstrate significantly lower plasma creatinine level in Group 4. This implied that spironolactone failed to improve renal function in 5/6 radial nephrectomy renal failure model. However, due to limited numbers with inadequate statistical power, the interpretation of the study result should be cautious. In addition, numerically higher BUN/Creatinine ratio after the treatment in Group 4 was noted. This indicated that the renal protection effect of spironolactone may be further confounded by its diuretic effect. [32, 33] Fifth, we only provide evidence that spironolactone could improve endothelial dysfunction in a CKD rat model. We strongly recommend that the beneficial effects of spironolactone on cardiovascular outcomes should be weighed against the potential risk of electrolyte imbalance, particularly in the setting of chronic renal failure.
The contribution of each authors is as follows: (1) WCC, SMY, and CCT conducted the study conception and design. (2) WCC, LAS, and LSH conducted the experiments. (3) WCC, CKC, SMY, and CCT analyzed and interpreted the experimental data. (4) WCC, LAS, and LSH drafted the manuscript. All authors read and approved the final manuscript.
This study was supported by grants from China Medical University Hospital. (DMR-106-160; DMR-107-012). The financing institution did not have any role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
Ethics approval and consent to participate
All animal study protocols were approved by the Institutional Animal Use and Care Committee of China Medical University (protocol number: 104–272).
Consent for publication
The authors report no competing interest.
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