Background
Chronic heart failure (CHF) is a complex clinical syndrome in which the heart’s function cannot meet the demand of the body’s healthy blood circulation. In CHF, the useful circulating blood volume is reduced to decrease the rate of renal perfusion, which in turn activates the sympathetic nervous system (SNS), leading to increased blood catecholamine levels, which stimulates the juxtaglomerular apparatus cells to secrete and release more renin to ultimately activate the renin-angiotensin system (RAS) [
1‐
4]. Activation of the SNS and RAS have compensatory effects during the early stage of CHF. However, by the late stage, these activations will promote the development and progression of CHF, with gradual deterioration of cardiac function.
Moreover, SNS activation in CHF will have direct effects on kidney function. Elevated catecholamine acts on the adrenergic receptor (AR) of the kidney tissue, contributing to a series of conditions, including renal interstitial inflammatory cell infiltration, renal fibrosis, and renal tubular necrosis, ultimately resulting in altered renal hemodynamics. When the RAS is activated after heart failure develops, the plasma level of angiotensin II (Ang II) also increases by mediating renal vasomotor and sodium retention, as well as the proliferation, hypertrophy, and profibroblast effect of kidney mesangial cells and the extracellular matrix. There are three kinds of α
1-AR subtypes in the kidney tissue, within which α
1A-AR and α
1D-AR mainly regulate renal vasoconstriction, while α
1B-AR mediates the proliferation and hypertrophy of vascular smooth muscle cells, and together with α
1A-AR promotes sodium and water reabsorption in the proximal tubules [
5‐
7].
Nitrates have beneficial effects on CHF by expanding the vein, coronary artery, and small peripheral arteries to reduce blood reflow and thus the cardiac preload, thereby improving myocardial blood supply and the cardiac afterload, respectively. However, few studies have examined whether long-acting nitrates can inhibit excessive activation of the SNS and RAS by regulating the expression of AR and angiotensin receptor (ATR) in the kidneys under a condition of CHF. Therefore, in this study, we examined the influence of the administration of long-acting nitrates to a rat model of heart failure induced by myocardial infarction on the expression of AR and ATR in the kidney. These results can lay the foundation for a renal protective effect of long-acting nitrate as a treatment or preventive strategy in patients with CHF.
Methods
Experimental animals and the establishment of the CHF model
Clean inbred male Wistar rats (10 weeks old, 250–280 g) were obtained from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). All rats were barrier-housed in the clean animal room with the temperature maintained at 22 ± 3 °C and relative humidity of 50 ± 20% at Capital Medical University affiliated Beijing Anzhen Hospital. The experimental protocol was approved by the institutional ethics committee (permission license: SCXK-2012-0001), and the rats were fed with standardized rat chow and water throughout the experiment.
A total of 90 Wistar rats were randomly divided into three main groups: healthy control group (CTL,
n = 9), sham-operated group (sham,
n = 8), and CHF model rats (
n = 73). The CHF model was induced by ligation of the left anterior descending (LAD) artery. In brief, the rats were anesthetized by intra-abdominal injection of 1% pentobarbital sodium and set on a ventilator for small animals (VT 7–8 ml/kg, respiratory rate 70 per minute, I/E ratio 1:2). Anterior myocardial infarction was created by ligation of the LAD artery near the main pulmonary artery. Four weeks later, echocardiography was performed to evaluate left ventricular (LV) function based on the left ventricular ejection fraction (LVEF); rats with an LVEF ≤45% were considered to have CHF and were included in subsequent experiments [
8].
These CHF rats were then randomly divided into the following five treatment groups: CHF model group (CHF, n = 9), in which the rats received intragastric administration of saline once a day; low-dose nitrate group (n = 9), receiving intragastric administration of 3.6 mg/kg isosorbide-5-mononitrate (IS-5-MN; sustained-release capsules, 50 mg/capsule, UCB Pharma Co. Ltd.) in 2 ml saline once a day; high-dose nitrate group (n = 9), receiving intragastric administration of 7.2 mg/kg IS-5-MN in 2 ml saline once a day; positive drug control group (olmesartan; n = 9), receiving intragastric administration of 3 mg/kg olmesartan (Olmesartan Medoxomil Tablets, 20 mg/pill, Daiichi Sankyo Co. Ltd.) in 2 ml saline once a day; and high-dose nitrate combined with olmesartan group (nitrate + olmesartan, n = 9), receiving intragastric administration with 7.2 mg/kg IS-5-MN and 3 mg/kg olmesartan in 2 ml saline once a day. Sham-operated rats were subject to the same procedure as those in the CHF-induced model but without ligation of the LAD artery. Both the CTL and sham groups received intragastric administration with saline once a day. The treatment lasted for 6 weeks. Rats were anesthetized by intraperitoneal (i.p) injection with sodium pentobarbital (40 mg/kg). And then all blood samples were taken from the abdominal aorta of rats. After blood sampling procedures the animals were euthanized by an injection of sodium pentobarbital (150 mg/kg) administered through the abdominal aorta.
Echocardiography
Echocardiography was performed to evaluate LV function before treatment and at the end of the experiment (6 weeks later). The rats were anesthetized, and B-mode measurement in the LV short-axis view (papillary muscle level) was performed with a 12-MHz phased array transducer (Vevo 2100 High Resolution Imaging System, Visual Sonics Inc. Toronto, Canada). LVEF was measured and averaged for three consecutive cardiac cycles.
Plasma renin activity (PRA) and Ang II concentration
At the end of the treatment period, blood samples were collected from the abdominal aorta, and plasma was separated immediately and stored at − 80°C until analysis. For renin activity determination, the plasma was incubated with rabbit angiotensinogen at 37 °C for 60 min and the renin concentration was measured according to the standard protocol of the radioimmunoassay (RIA) kit (IBL, Hamburg, Germany), expressed as nanograms per milliliter per hour.
For Ang II determination, the plasma was incubated with antiserum (anti-rabbit) for 6 h, and then with 125I-labeled Ang II for 18 h at 4 °C. Antibody-bound Ang II was separated from the free Ang II using donkey anti-rabbit-coated cellulose. After incubation for 30 min at room temperature and centrifugation at 5000 rpm for 15 min at 4 °C, the concentration of Ang II in each sample was read with the RIA kit according to a prepared standard curve.
Reverse transcription-polymerase chain reaction (RT-PCR)
The kidneys were removed after blood collection, and specimens of the renal cortex were obtained and frozen in liquid nitrogen at − 80°C until analysis.
Total RNA was extracted from the rat renal cortex by Trizol reagent, and an ultraviolet spectrophotometer was used to detect the concentration of total RNA for each sample. cDNA was synthesized and amplified with a Promega RT-PCR Kit (Madison, WI, USA) according to the manufacturer instructions using sense and antisense oligonucleotide primers synthesized by Bejing SBS Genetech (Bejing, China). To quantify the transcripts obtained by RT-PCR amplification,
Gapdh was used as an internal standard, and the target mRNA (α
1A-AR, α
1B-AR, α
1D-AR, β
1-AR, β
2-AR, β
3-AR, AT
1R, and AT
2R) levels were normalized to that of
Gapdh. The target genes and
Gapdh were amplified according to the parameters shown in Table
1.
Table 1
Primer subsequence and annealing temperature of α1, β adrenergic receptor and Angiotensin II receptor subtypes
β1-AR | sense:5′-GGGCAACGTTGGTGATCG-3′ | 213 bp | 58 °C |
antisense:5′-CTGGCCGTCACACATAGCAC-3′ |
β2-AR | sense:5′-GAGACCCTGTGCGTGATTGC-3′ | 388 bp | 58 °C |
antisense: 5′-CCTGCTCCACCTGGCTGAGG-3′ |
β3-AR | sense:5′-AGTGGGACTCCTCGTAATG-3′ | 444 bp | 59 °C |
antisense: 5′-CGCTTAGCTACGAAC-3’ |
α1A-AR | sense:5′-CAAGGCCTCAAGTCCGGCCT-3’ | 156 bp | 58 °C |
antisense:5′-CTCTCGAGAAAACTTGAGCAG-3’ |
α1B-AR | sense:5′-ATCGTGGCCAAGAGGACCAC-3’ | 287 bp | 62 °C |
antisense: 5′-CTCTCGAGAAAACTTGAGCAG-3’ |
α1D-AR | sense:5′-CGTGTGCTCCTTCTACCTACC-3’ | 304 bp | 58 °C |
antisense:5′-GCACAGGACGAAGACACCCAC-3’ |
AT1R | sense: 5′-CGTCATCCATGACTGTAAAATTTC-3’ | 306 bp | 53 °C |
antisense: 5′-GGGCATTACATTGCCAGTGTG-3’ |
AT2R | sense: 5′-GTGTGGGCCTCCAAACCATTGCTA-3’ | 445 bp | 61 °C |
antisense: 5′-TTGCTGCCACCAGCAGAAAG-3’ |
GAPDH | sense:5′-TGCACCACCAACTGCTTAGC-3’ | 196 bp | 57 °C |
antisense: 5′-GGCATGGACTGTGGTCATGAG-3’ |
RT-PCR products were resolved by electrophoresis on a 1.5% agarose gel (BioRad, USA), and stained with ethidium bromide for visualization on a Bio-rad scanner. Densitometry was used for relative semi-quantitative assessment of expression levels.
Western blot
Total protein was extracted after homogenizing the rat renal cortex, and the concentration was determined with a bicinchoninic acid protein assay kit. The proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes, which were incubated with the primary antibodies rabbit anti-β1-AR (1:200), anti-β2-AR (1:200), anti-β3-AR (1:200), anti-α1A-AR (1:200), anti-α1B-AR (1:200), anti-α1D-AR (1:200), anti-AT1R (1:200), anti-AT2R (1:200), and anti-GAPDH (1:200) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight, followed by incubation with goat anti-rabbit fluorescent (IRDye-conjugated) secondary antibodies (1:10,000; Rockland Immunochemicals, Gilbertsville, PA, USA) for 2 h at room temperature. The images were quantified by the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). Levels of proteins were normalized to that of GAPDH.
Histopathology
The kidneys were perfused with formalin, collected, and fixed in paraffin. Fixed specimens were then cut into 4-μm sections and stained with hematoxylin and eosin following standard procedures. Images were captured with a Nikon Labophot 2 microscope equipped with a Sony CCD-Iris/RGB color video camera attached to a computerized imaging system (Nikon, Japan).
Statistical analysis
Data are expressed as mean ± standard error of the mean. Differences between groups were analyzed by Student’s t-test or one-way analysis of variance, followed by the Newman-Keuls test in GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). P < 0.05 was considered statistically significant.
Discussion
Kidneys are vital organs to maintain the body’s blood pressure, as well as the water and electrolyte balance, and the SNS and RAS regulate their physiological functions. After heart failure develops, SNS is activated and the release of catecholamine increases.
In this experiment, the renal tissue showed significant impairment in the CHF group. Inflammatory cell infiltration and tubular necrosis were reduced in all treatment groups to different extents, demonstrating a renal protective effect. However, due to the original design of the study, no quantitative analysis of the histology was performed. We only described the pathological changes under the optical microscope. As the study was completed some time ago and some pathological specimen had been damaged, quantitative analysis of histology could not be performed retrospectively.
In our study, a CHF model was established by ligation of the LV, resulting in a decrease of the LVEF, and an increase of plasma PRA and Ang II levels, indicating excessive activation of the RAS and confirming the successful establishment of the model [
9]. However, after treatment of long-acting nitrate, the plasma PRA and Ang II levels decreased. This is likely attributed to a reduction of the venous return and cardiac preload, thereby improving coronary blood supply, and reducing cardiac afterload through expansion of the vein, coronary arteries, and small arteries, respectively.
Moreover, we found that CHF significantly downregulated α
1A-AR, but did not affect the expression of the other subtypes α
1B-AR and α
1D-AR. The result is consistent with previous findings [
5,
6], suggesting α
1A-AR internalization resulting from persistent activation of the SNS in a condition of heart failure, and further weakens its regulatory effect on renal hemodynamics, while RAS activation inhibits the down-regulation of α
1D-AR and maintains α
1B-AR at a relatively low baseline expression level. Such receptor-mediated regulation has been shown to play an essential role in maintaining renal perfusion and normal renal function [
10,
11]. The up-regulation of α
1A-AR expression induced by long-acting nitrate treatment may indicate that nitrate improved cardiac function and inhibited excessive activation of the local renal SNS, which then normalized AR expression under the pathological condition to restore its renal protective effect.
There are three β-AR subtypes in the kidney tissue: β
1-, β
2-, and β
3-AR. Stimulating β
1-AR and β
2-AR could mediate dilation of the renal arteries and glomerular mesangial cells to regulate blood flow and the glomerular filtration rate (GFR), increase sodium and chloride reabsorption, and increase the secretion of renin and erythropoietin. Besides, β
1-AR plays a dominant role in the regulation of renal function [
3,
12‐
14]. β
3-AR can activate nitric oxide synthase (NOS) and promote NO release, and thus cause dilation of the glomerular afferent arteries to adjust the renal perfusion rate [
10].
Our results showed that β
1- and β
2-AR expression in the heart failure model group was significantly down-regulated, which was consistent with the findings of Fung et al. [
12]. One reason for this change could be that stimulation of the SNS increases β-AR kinase activity, leading to down-regulation and desensitization of β-AR. In turn, these receptors can affect renal perfusion and GFR to cause renal damage. This decrease of β
1- and β
2-AR expression in the kidney was reversed after nitrate therapy, suggesting that local SNS activity decreases in the kidney after heart function improves so that the receptor expression reverts to the average level under the pathological condition [
12]. By contrast, β
3-AR in the kidney tissue showed a relatively low baseline expression level, and no significant changes were found among the groups.
AT
1R and AT
2R are the two ATR subtypes expressed in the renal vasculature and renal tubules [
15]. Binding of Ang II to AT
1R causes renal vasoconstriction, promotes renal sodium and water reabsorption, and enhances cell proliferation, whereas binding of Ang II to AT
2R can increase NO release, which further contributes to renal vessel dilation, increasing sodium and water excretion, and inhibiting cell proliferation and hypertrophy [
16,
17].
In this study, AT
1R expression was markedly up-regulated in the heart failure group, while AT
2R expression was significantly down-regulated, suggesting changes of ATR expression levels in the kidney during CHF, which could contribute to renal interstitial edema, inflammatory cell infiltration, glomerular fibrosis, and tubular necrosis, ultimately affecting renal perfusion and reserve function [
18,
19]. AT
1R expression was down-regulated while AT
2R was up-regulated after nitrate therapy, which may be related to the fact that nitrates can cause renal vascular relaxation, increase renal perfusion, and inhibit RAS activation. Up-regulation of AT
2R can suppress the expression of AT
1R, thereby the receptor adjustment protected against impaired renal function during CHF [
20,
21].
As a selective AT
1R antagonist, olmesartan can inhibit RAS activation, and reduce plasma PRA and Ang II levels as well as the renal vascular sensitivity to catecholamines [
1,
14]. In contrast to other AT
1R antagonists, olmesartan has an inverse activating effect when binding to AT
1R, and thus inactivate AT
1R and alleviate damage to the kidney induced by Ang II [
22,
23]. Moreover, the combination of Ang II and AT
2R plays a role in physiological renal protection [
24].
Compared with monotherapy, high doses of nitrates combined with olmesartan had a more significant effect on improving cardiac function, reducing PRA and Ang II levels, and normalized the receptor expression level, namely by lowering the expression level of AT
1R and increasing the expression levels of α
1A-, β
1-, β
2-AR, and AT
2R. These results may relate to the fact that olmesartan blocks AT
1R and promotes the rise of the tissue endothelial NOS/inducible NOS level and NO synthesis, and by inducing the up-regulation of AT
2R expression. Thus, olmesartan elevates endothelial NOS levels and increases the bioavailability of NO, which further enhances the vasodilating effect of nitrates [
25,
26]. In this study, regulation of receptor expression by high-dose nitrates was significantly superior to that of the low-dose group, which may indicate that large doses of nitrates are required to expand renal vessels more sufficiently.
During CHF, the plasma Ang II level and catecholamine secretion rise, and the sympathetic nerve is activated so that the RAS and SNS interaction increases, resulting in changes of AR and ATR expression in the kidney. Previous studies have shown that Ang II acts on AT
1R to down-regulate the expression of α
1-AR, and also phosphorylates and desensitizes β
1-AR through the PLC/PKC/c-src/PI3K pathway, eventually deteriorating renal function in heart failure [
6,
14]. After nitrate therapy, the receptors were inversely regulated, among which β
3-AR promotes the synthesis of NO by activating NOS, thus mediating renal vasodilation, resulting in reduced AT
1R expression [
27,
28]. This indicates that nitrate can impact the interaction between AR and ATR subtypes, and can improve renal perfusion, inhibit the abnormal proliferation of mesangial cells, reduce interstitial edema and inflammatory cell infiltration, postpone the progression of glomerular fibrosis and tubular necrosis, and finally protect renal function.
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