Cardiovascular and Renal Effects of 5-Aminoimidazole-4-Carboxyribonucleoside (AICAR) in the Deoxycorticosterone Acetate (DOCA)-Salt Rat Model of Hypertension

Abstract

Previous studies suggest that exercise training mitigates blood pressure in hypertensive animal models. While exercise training may be an effective therapeutic for hypertension treatment and prevention, many patients diagnosed with hypertension can be noncompliant with lifestyle modifications that involve physical activity or are unable to participate in strenuous activity due to the severity of their hypertension. AMP-activated protein kinase (AMPK) is responsive during metabolic stressors, plays a vasoprotective role in endothelial function, and is stimulated during exercise training. Studies have suggested that signaling pathways activated by AMPK may play a protective role in hypertensive models given AMPK’s involvement in redox balancing. The purpose of this study is to determine the therapeutic potential of AICAR (5-aminoimidazole-4-carboxamide-3-ribonucleoside), a potent AMPK stimulator, for ameliorating hypertension. Uninephrectomized Sprague Dawley rats received DOCA-salt treatment and daily administration of either AICAR (100mg/kg/day) or vehicle (20% DMSO) subcutaneously for 21 days. Cardiovascular and renal function were assessed by radiotelemetry, tail cuff, proteinuria, and GFR. No differences in final MAP were detected in AICAR and vehicle treated DOCA-salt groups, respectively (183.6 ± 26.2 and 179.4 ±26.5 mmHg). Notably, no changes in renal injury or function were detected with AICAR treatment as shown by the final GFR and proteinuria. From these findings we concluded that exogenous administration of AICAR does not attenuate hypertension in the DOCA-salt model despite increased AMPK activation.

INTRODUCTION

Known as the “silent killer”, an estimated 46% of adults with hypertension are unaware of their condition, and only one third of Americans have their hypertension controlled [1]. This multifaceted condition has increasingly adverse outcomes with respect to the morbidity and mortality associated with cardiovascular disease (CVD), metabolic syndromes, and chronic kidney disease (CKD). Hypertension can be preventable with lifestyle modifications, like dietary changes such as lowered sodium intake and increasing physical activity. This is frequently the most effective treatment and the most cost-effective, however, there are many challenges with patient adherence. It is estimated that compliance rates for short term therapy were between 70–80% while compliance with lifestyle modification was 20–30% [2].

Exercise training has been shown to ameliorate a wide variety of cardiovascular disorders and has antihypertensive effects through a myriad of mechanisms [3], as demonstrated in several preclinical murine studies [4–7]. Recent studies have suggested that some of the metabolic changes observed after exercise training can be stimulated pharmacologically [8]. Many of these responses are hypothesized to be mediated through the activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK) which is activated in exercise [9–11]. In the context of hypertension, AMPK is reported to be an important regulator of long-term blood pressure control [12], given its ability to maintain cellular energy homeostasis. Some studies have already addressed this using AMPK activator metformin, which reports antihypertensive effects in hypertensive DOCA-salt rats, as well as improvements in renal and cardiovascular function [13,14]. However, given that metformin has more than one mechanism of action and an indirect activator of AMPK, the variable effects create conflicting results [12,15]. AICAR (5-amino-4-imidazole carboxamide ribonucleoside), is a direct activator of AMPK as an AMP-mimetic and has been studied in metabolic syndrome and ischemic preconditioning. Notably, a 2008 study showed AICAR alone improved endurance by 44% in sedentary mice, mimicking the effects of exercise [16]. Other studies have demonstrated AICAR-induced AMPK activation and its ability to promote vascular relaxation independently of endothelium and nitric oxide, while also reducing smooth muscle contraction, highlighting its therapeutic potential in hypertension models [17,18].

AMPK is a major contributor to the beneficial metabolic adaptations and cardiovascular effects observed in exercise [19]. Therefore, targeting this mechanism pharmacologically may provide an effective treatment for both noncompliant patients and those diagnosed with uncontrolled hypertension. However, the direct effects of AMPK activation in the context of salt-sensitive hypertension are poorly defined in the literature. Therefore, we aimed to address this by targeting AMPK pharmacologically with 5-aminoimidazole-3-carboxyribonucleoside (AICAR) in the deoxycorticosterone acetate (DOCA) salt-sensitive hypertension model and evaluate the therapeutic potential of AICAR in hypertension. We hypothesized that AICAR administration throughout a high sodium intake challenge will mitigate the development of hypertension and improve renal function in the DOCA-salt hypertensive rat.

METHODS

Animals and Study Design.

All procedures were approved by the University of Arizona Institutional Animal Care and Use Committee (Protocol # 19–554) and were performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animals were housed in a temperature-controlled room (23° C) with a 12:12-h on-off light schedule. These experiments were conducted using the DOCA-salt rat model, a well-established preclinical model that recapitulates salt-sensitive hypertension and renal dysfunction [20]. An overview of the experimental protocol is depicted in Figure 1. Sixteen uninephrectomized male Sprague-Dawley rats (weight 275g; age:10–12 week) were implanted with telemeters, as previously described [21], allowed one week to recover, then underwent a 3-wk DOCA salt treatment (100mg DOCA s.c.; 0.9% saline ad libitum), as previously published [22]. All surgical procedures occurred in vivo under 2% isoflurane anesthesia. Animals were randomly assigned one of the following treatments at D0: 1) DOCA-salt +AICAR (100 mg/kg) (n=8); 2) DOCA-salt+ Vehicle (20% DMSO solution and 0.9% saline solution) (n=8). Treatment was administered daily via subcutaneous injection for 21 days. At the end of the protocol, animals were euthanized, allometric data were recorded, and tissue samples were collected for subsequent analysis.

Figure 1. Protocol Timeline for Experiments.

Animals underwent a five-week study where they underwent two weeks of surgeries, including unilateral nephrectomies, radiotelemetry implants, and DOCA implants prior to receiving daily subcutaneous injections of AICAR (100mg/kg s.i.d.) for three weeks. Animals were euthanized at the end of the study and tissue was collected for analysis. (Created on Biorender).

Hemodynamic Measures:

All rats were implanted with radiotelemeters (Stellar-TSE Systems; Model PTA-M) for continuous measurement of ambulatory arterial pressure and heart rate over 21 days, as previously described [23]. In addition to telemeters, blood pressure was measured by noninvasive tail-cuff plethysmography (Visitech BP1000, USA) at study end. Glomerular filtration rate (GFR) was measured the penultimate day of experiment in unrestrained, conscious rats by transcutaneous measurement of FITC-sinistrin clearance (5mg/100g body weight) using a transdermal GR monitor (Medibeacon, USA), as our laboratory has previously described [24].

Western Blot:

Western blotting was carried out as previously reported [25]. Protein (50 μg) was separated by electrophoresis on 4–20% SDS polyacrylamide separating gels (Bio-Rad) then transferred to nitrocellulose membranes. The membranes were incubated for 1 hour in blocking solution (5% non-fat milk). Membranes were incubated in blocking solution containing commercially available antibodies overnight at 4°C [anti-phospho(Thr172)-AMPKα (40H9), 0.1 μg/ml; Anti-AMPKα (23A3), 0.1 μg/ml; Cell Signaling Technology]. Membranes were washed and incubated for 1 hour with the appropriate horseradish peroxidase-conjugated secondary antibodies (0.01 μg/ml) and incubated in chemiluminescent substrate (West-Femto; Pierce, Grand Island, NY). The immunoreactive band images were quantified using ImageJ (NIH) software.

Urine and Serum Biochemical Analysis:

All animals were placed in metabolic cages for 24 hours and given 0.9% saline ad libitum. Urine samples were collected and processed for urinalysis and proteinuria analysis. Protein was analyzed using BCA assay (ThermoFisher; Cat #23225) and Urine creatinine assays (Arbor Assays; Cat #K002-H1) were used to assess proteinuria. Serum samples collected at baseline and necropsy days were analyzed for creatinine concentration using a commercially available assay per manufacturer’s instructions (Arbor Assays USA; Cat # KB02-H1D). Baseline (Day 0) urine samples missing from Vehicle (4 of 8) and AICAR (3 of 8) due to sample loss.

Histological Analysis of renal injury:

Kidney samples were collected for histological assessment of injury, as previously described [26]. At nephrectomy and necropsy, kidneys were excised and bisected, fixed in 10% formalin for 24–48 hours, and embedded in paraffin wax. Tissues were sectioned by the University of Arizona TACMASR laboratory, then stained with Masson Trichrome staining per manufacturer’s instructions (Sigma Aldrich USA; Cat # HT15). Assessment of renal injury was scored through glomerular fibrosis and measured through ImageJ software.

Statistical analysis.

All data are presented as mean ± SEM, and statistical significance was accepted when p < 0.05. Comparisons between the groups and over time were made with a two-way repeat measures ANOVA and unpaired t-test. Single/endpoint measurements were compared by an unpaired Student’s t-test. Statistical calculations were made with GraphPad Prism version 10 (GraphPad Software, San Diego, CA).

RESULTS

Cardiovascular Response to AICAR.

Endpoint arterial pressure was assessed in all animals (n=8/group) by tail-cuff plethysmography, where no difference in systolic blood pressure was detected between treatment groups (171 ± 7mmHg DOCA+Vehicle; 169 ± 10mmHg DOCA+AICAR) (Figure 2A). Mean arterial pressure (MAP) and heart rate (HR) were also measured continuously over 3 weeks by implanted radiotelemeters in a subset of animals in each group (n=4–5/group). There was a progressive increase (p<.05) in the 24-hour MAP over time in the DOCA + vehicle (saline) group (183.6±13.1 mmHg), as well as in the DOCA + AICAR group (179.4±15.3 mmHg) (Figure 2B–C). There were no main effects of AICAR treatment detected in the MAP response. Moreover, there was also no detectable effect of AICAR treatment in any 24-hour mean heart rate responses measured (Figure 2D–E). Final heart weights at necropsy yielded no detectable difference between DOCA + vehicle and DOCA + AICAR groups (4.20±0.18 vs. 4.08±0.09 g/kg bodyweight).

Figure 2. Cardiovascular Responses to DOCA-Salt and AICAR Treatment:

(A) Final systolic blood pressure was measured by tail-cuff plethysmography in all DOCA+Vehicle and DOCA+AICAR groups over 21 days (n=8/group). No difference was detected between the Vehicle and AICAR-treated groups (Statistical analysis: unpaired two-tailed Student’s t-test). 24-hour hemodynamic measurements were collected continuously by Stellar radiotelemetry in a subset of both DOCA+Vehicle and DOCA+AICAR groups (n=4–5/group). (B) Mean arterial pressure (MAP) progressively increased in both DOCA+Vehicle and DOCA+AICAR groups (C) as did the change from baseline (protocol day 0) MAP (ΔMAP). There were no differences in the cardiovascular parameters measured as shown in (D) heart rate (HR) and (E) change from baseline (protocol day 0) HR (ΔHR). Statistical analysis: Two-way repeated measures ANOVA with Bonferroni post-hoc test, presented as means ± SEM. DOCA+Vehicle: n=5; DOCA+AICAR: n=4.

Effects of AICAR on Renal Morphometrics and Function:

Final renal weight were similarly Urine samples were collected at necropsy to measure urinary protein-to-creatinine ratio (UPCR) as indirect indicators of glomerular damage. Both DOCA+Vehicle and DOCA+AICAR groups had elevated UPCR at Day 21 (421.8 ± 98.41 DOCA+Vehicle vs. 503.6 ± 97.58 in DOCA+AICAR, p<0.05) compared to their baseline (96.24 ± 7.043 DOCA+Vehicle vs. 147.6 ± 4.551 in DOCA+AICAR, p<0.05), however, there was no effect of treatment (Figure 3). There were no differences in GFR detected between DOCA+Vehicle vs. DOCA+AICAR (0.841±0.240 DOCA+Vehicle vs. 0.758 ±0.131 DOCA+AICAR mL/min/100g bodyweight) (Figure 4). Finally no differences were detected in final renal weights between DOCA + vehicle and DOCA + AICAR groups (6.504±0.221 vs. 6.615±0.411 g/kg bodyweight).

Figure 3. Urinary protein to creatinine ratio (UPCR):

UPCR increased at Protocol Day 21 in both DOCA+Vehicle and DOCA+AICAR groups compared to Day 0 values at the start of the experiment. Statistical analysis: Two-way ANOVA with Bonferroni post-hoc test. DOCA+Vehicle: n=8; DOCA+AICAR: n=8. Data presented as mean ± SEM, *p<0.05. Day 0 urine samples missing from Vehicle (4 of 8) and AICAR (3 of 8) due to sample loss.

Figure 4. Glomerular Filtration Rate (GFR):

GFR was measured in a subset of animals on Day 21 at the end of the study. No difference was observed between the DOCA+Vehicle and DOCA+AICAR groups. unpaired two-tailed Student’s t-test. DOCA+Vehicle: n=4; DOCA+AICAR: n=5. Data are presented as mean ± SEM.

Quantification of Renal Injury:

Kidney samples collected at nephrectomy and necropsy were utilized as a paired comparison to quantify changes in glomerular blue to red ratio to measure glomerular fibrosis and injury. Interestingly, an increase (p<.05) in fibrosis compared to baseline prehypertensive control (tissues collected at unilateral nephrectomy) was detected only in the DOCA+AICAR group. No difference in final fibrosis measurements were detected between DOCA+Vehicle and DOCA+AICAR. (DOCA+AICAR, 0.474 ± 0.034 baseline; *0.559, ± 0.020 final; *p<0.05 vs baseline) (DOCA+Vehicle 0.457 ± 0.038 baseline; 0.526 ± 0.034 final) (Figure 5).

Figure 5. Quantification of renal fibrosis:

Renal fibrosis was assessed in a subset of animals’ tissues collected at nephrectomy and necropsy timepoints by quantifying glomerular blue to red ratio (B:R) using ImageJ. Statistical analysis: Two-way repeated measures ANOVA with Bonferroni post-hoc test. DOCA+Vehicle: n=4; DOCA+AICAR: n=5. Data are presented as mean ± SEM (*p<0.05).

AMPK Activation in Renal Tissue:

To confirm drug delivery, we measured phosphorylation level of AMPK by detecting phosphorylated Thr172 (pAMPKα) and total AMPK activity in the kidney by Western Blot. AICAR administration increased (p<.05) AMPKα phosphorylation, indicating enhanced AMPK activation in the DOCA+AICAR group compared to DOCA+Vehicle. Data are presented as the ratio of phosphorylated AMPK to total AMPK (DOCA+Vehicle 1.00±0.118, DOCA+AICAR *1.39 ±0.058 au) (Figure 6).

Figure 6. Western blot analysis of Renal AMPKα phosphorylation:

The activation of AMPK in renal parenchyma was assessed in a subset of animal tissue samples by measuring phospho-AMPKα with respect to total AMPKα by Western blotting with anti-AMPKα p-Thr¹⁷² Ab. Results are shown as the ratio of phosphorylated AMPK (pAMPKα) to total AMPK. DOCA+AICAR groups had higher (p<.05) phosphorylation of AMPKα compared to DOCA+Vehicle. DOCA+Vehicle: n=3; DOCA+AICAR: n=3. Statistical analysis: unpaired two-tailed Student’s t-test. Data are presented as mean ± SEM (*p<0.05).

DISCUSSION

The aim of this study was to investigate the therapeutic potential of AICAR administration to ameliorate the hemodynamic parameters in a DOCA-salt model of hypertensive rats. Contradictory to our initial hypothesis that AICAR treatment would mitigate the hypertensive and renal dysfunction response to DOCA, we failed to detect any improvements in the major disease progression indicators. Foremost, the hypertension observed in our DOCA model was not mitigated with AICAR treatment, nor did we observe any improvement in the renal function or injury parameters. This occurred despite an effective activation of AMPK, which suggests AMPK activation, specifically by AICAR, is an ineffective treatment/target for the salt-sensitive hypertension.

The first line of clinical recommendations for preventing and managing hypertension is through lifestyle modifications, whether it is limiting dietary sodium intake, improved physical activity levels, and others [27]. There is a large pool of data supporting the beneficial role of moderate exercise in patient trials [3] and in the preclinical literature in models of hypertension [4,5]. These data demonstrate both the improved cardiovascular effects and favorable renal effects; however, the underlying molecular pathways primarily mediating these effects remains unclear. Of the several pathways changed with exercise that have putative beneficial effects on cardiovascular and renal health, AMPK is often central to these effects. And since exercise, while largely considered as a cost-effective and efficient approach for managing hypertension and other cardiovascular and metabolic conditions, may not be the most inclusive for individuals with sedentary lifestyles, physical disabilities, or those diagnosed with severe uncontrolled hypertension [27]. This unmet need for this patient population requires careful pharmacological interrogation to potential recapitulate the beneficial effects with exercise.

Of the available therapeutic agents for cardiovascular disease, AMPK is a master regulator of energy homeostasis and metabolic pathways [28]. Recent evidence points to a vasoprotective role for AMPK activation that may be mediated through inducing endothelial NO production and regulating vascular redox balance [28–30]. AICAR has been implicated in the modulation of blood pressure in hypertension [31], and utilizing this AMPK activator as a pharmacological intervention is associated with decreased blood pressure in multiple hypertensive rat models [32,33]. Studies involving acute administration of AICAR in healthy humans have demonstrated its modest vasodilatory effects, characterized by increased heart rate and decreased mean arterial blood pressure [34]. Similar benefits including improvement in vascular endothelial function, have been observed in elderly hypertensive patients [35] and hypertensive rats [36].

Although we successfully demonstrated increased activation of phosphorylated AMPK in the kidneys of AICAR treated animals, we failed to detect an effect of AICAR on hypertension development, proteinuria, or renal dysfunction in the DOCA-salt rats. These data are in line with previous reports in the Dahl salt-sensitive rat models studies that have shown similar findings regarding a lack of effect in metformin and AICAR on the development of salt-sensitive hypertension [12]. While the current study does not support a role for systemic administration of AICAR in mitigating salt-sensitive hypertension and its associated pathologies, alternative targets and modes of administration may offer further explanation.

Indeed, the DOCA-salt hypertensive model is marked by increased peripheral inflammatory markers, markedly intrarenal oxidative stress, and inflammatory cytokines, and this has a temporal relationship with the hypertension progression [37]. Interestingly, prior studies from other groups have demonstrated that increased central inflammatory markers are also observed in the DOCA-salt model, specifically in the hypothalamus [38]. Moreover, a recent study investigated the regulatory effects AMPK activation in the hypothalamus on blood pressure periventricular nucleus (PVN) [39]. This study reported that with central AICAR administration in a model of renovascular hypertension, there was an overall and sympathetic nerve activity through repeated central administration of AICAR in the attenuation of central oxidative stress and inflammatory cytokine production, decreased sympathetic nerve activity, and lower arterial pressure [39]. Fu and colleagues followed up on these studies and demonstrated that the central mechanism by which activation of AMPK in the PVN decreases hypertension is by downregulating the ERK1/2-NF-κB pathway [40]. The PVN’s involvement in blood pressure regulation through sympathetic nerve activity and inflammation not only highlights the importance of targeting this region to address hypertension, but also provides insight into how AMPK modulates inflammatory and oxidative stress pathways centrally to regulate hypertension. Notably, AICAR has low blood-brain-barrier permeability [41], which may account for the discrepancy of cardiovascular effects between the current study and others employing a central administration technique. Though it remains unclear if systemic administration of AICAR can activate hypothalamic AMPK, targeted central administration of AICAR should be tested in a future study to compare to the current study.

One paradigm that remains untested in these studies, and in the context of salt-sensitive hypertension, is the effect of combined treatment with AICAR and exercise. Several studies have shown additive effects of AICAR on top of exercise adaptations [8,42–44], yet these effects have largely focuses on exercise capacity, metabolic function, or muscle mass maintenance under various conditions and pathologies. Since aerobic exercise alone has been reported to mitigate DOCA-salt hypertension [45], we can not rule out the possibility of combined aerobic exercise and AICAR treatment to have an additive effect on blood pressure in the DOCA-salt model. Interestingly, a recent study from Abdallah et al. [46] compared the separate and combined effects of exercise and eugenol – a strong antioxidant. While eugenol-alone did not improve the DOCA-salt blood pressure, combined exercise and eugenol obviated the hypertension, even greater than the effects achieved with exercise alone. Addressing the combinatorial effects of exercise and AICAR will be an aim for a future research project.

Study Limitations.

These studies are not without limitations of scope, design, and methodology. Firstly, the study does not include a normotensive group with the treatment of AICAR. This was due to ethical considerations, as there was no improvement detected in any cardiovascular and renal parameters observed in the DOCA-salt model with AICAR treatment. Additionally, the DOCA-salt model was reliably reproduced and measured by multiple repeat measurements to show a pathological change from baseline (normotensive) conditions.

An additional limitation of the current study is that only one dose of AICAR was tested in this model. Previous work has demonstrated an increase of AMPKα and AMPK activity upregulation in the kidney with AICAR treatment [47,48]. The dose utilized in this paper (100mg/kg/day) was previously confirmed to be effective in activating AMPK in various experimental models and tissues [12,32,49,50]. In the present study, we observed a progressive increase in blood pressure over the 21-day DOCA-salt treatment, regardless of experimental drug (AICAR) or vehicle treatment. The lack of AICAR effects could be simply due to an insufficient dose. However, the increased renal AMPKα phosphorylation observed with AICAR treatment in this study does not support this. Importantly, the scope of AMPKα phosphorylation was only measured in renal tissue, and did not include other cardiovascular tissues such as arterial vessels and heart. AMPK is highly expressed in the kidney and reduced renal AMPK activity has often been associated with impaired renal function in multiple metabolic disease associated nephropathies [51]. These considerations and findings suggest that the lack of efficacy observed in the present study may not stem solely from dosage, but may be attributed to the salt-sensitive hypertensive phenotypes in models such as the DOCA-salt and Dahl SS [12] rat – both of which are not sensitive to AICAR treatment. While these salt-sensitive models of hypertension fail to respond to AICAR, other models of hypertension and/or renal injury that are not directly driven by high sodium intake, such as the cisplatin-induced renal injury model [50], the reduced uterine perfusion pressure model of hypertension in pregnancy [32], and insulin resistance syndrome [52]. Indeed, comparative studies across these models may highlight effective mechanisms and hypertensive subtypes to target with AMPK agonists, whereas other phenotypes associated with salt-sensitivity may not be.

Finally, changes in other circulating parameters, such as serum ion content and osmolality were not collected or measured, limiting the overall scope and interpretation of these findings. Important, sodium intake effects AMPK activation and may be specifically mediated by osmolality [53]. Data presented by Fraser and colleagues proposed a role for AMPK in regulation of sodium reabsorption and demonstrated that AMPK activity increased in the kidney in response to sodium loading, and further examination revealed that rats receiving a low-sodium diet had reduced total AMPKα1 expression in their kidneys [53]. The unexpected ability of both high- and low-salt diets to modify AMPK activation was further supported by in vitro studies performed in macula densa cells. These experiments demonstrated that AMPK activation is mediated by changes in osmolality and not salt concentration, given that increasing osmolality in the normal-salt medium to the level of high-salt medium produced an equivalent increase in p-Thr¹⁷² and AMPK activity [53]. It is therefore plausible that any potential effect of AMPK activation from AICAR administration may be quenched under a high-salt diet challenge and therefore functionally ineffective. Further interrogation of AICAR or other AMPK agonists in salt-sensitive hypertension vs. others may reveal a novel targeted approach and differentiating mechanism(s).

Conclusions.

These data indicates that AICAR was not an effective regulator of blood pressure nor renal function within the hypertensive DOCA-salt model, despite activation of renal AMPK. While there is ample evidence on the effects of AMPK activator metformin, there is some variability on the effects of AICAR which may require further consideration regarding the route of administration and dosage. The varying effects appear to be model dependent, which highlights specificity of impact and potential uninterrogated mechanism(s). Indeed, expanding the research in exercise mimetics and AMPK application has the potential to offer valuable insight into alternative therapeutic strategies for hypertensive patients, particularly those who are unable or unwilling to engage in physical activity. However, further research is required to explore the full therapeutic potential of AMPK activators such as AICAR to this pathological condition.