Hypertension and metabolic syndrome, both of which increase with age, are multifactorial disorders. Their etiology is complex, making it challenging to isolate involved genes. This study aimed to characterize the hepatic gene expression in spontaneously hypertensive rats (SHR) at different ages. Blood pressure in SHR was determined by tail-cuff method at one and three months of age. Hepatic RNA was isolated and gene expression was compared using microarrays. Comparison between SHR and normotensive rats revealed significant variation in gene expression: 98 genes were upregulated and 122 were downregulated in SHR; while 88 genes were upregulated and 139 genes were downregulated in age-matched normotensive rats. Furthermore, within the SHR group, 110 genes were found to be upregulated and 168 genes downregulated across different ages. Analyses via the Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes pathways revealed that several genes are potentially implicated in both, hypertension and metabolic syndrome. The results suggest that SHR display variations in gene expression due to aging, and when compared to normotensive rats. These variations could contribute to the development of hypertension and metabolic syndrome. Microarray studies involving older rats are necessary to further validate these findings.
Hypertension and metabolic syndrome (MetS) are diseases of multifactorial origin, that significantly contribute to high morbidity and mortality around the world [
In recent years there have been significant advances in genetic mapping of both hypertension and MetS [
Male spontaneously hypertensive rats (SHR, aged 1 month as prehypertensive stage, and 3 months as hypertensive stage, n = 2), and Wistar Kyoto rats (WKY, aged 1 month, and 3 months, both ages as normotensive stage, n = 2), used as a control group, were obtained from the animal facility of the Institute of Cell Physiology at the National Autonomous University of Mexico. All animal housing, care, and procedures were conducted in accordance with Mexican Regulations for Animal Care and Use (NOM-062-ZOO-1999, SAGARPA, Mexico), and the Guide for the Care and Use of Laboratory Animals, as promulgated by the U.S. National Institutes of Health (8th edition, 2011). Animals were maintained in a pathogen-free environment under controlled conditions (22˚C ± 2˚C, 40% - 60% humidity, 12 hours light/12 hours dark cycle), with free access to tap water, and fed a standard rat chow ad libitum throughout the experimental periods, except during the overnight (12 - 16 h) fasting period before being euthanized. All experimental protocols were approved by the Ethics Committee of the Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México (Protocol number 1368).
The indirect measurement of blood pressure was carried out using a tail-cuff device (Automatic Blood Pressure Computer, Model LE 5007; Letica, Panlab, Spain), as described previously [
Rats were sacrificed by cervical dislocation, after which their livers were rapidly removed, rinsed in phosphate buffer solution at 4˚C, and frozen in cryogenic vials at −80˚C until RNA extraction.
Total RNA was extracted from 50 mg of the frozen livers’ samples using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), as per the manufacturer’s protocol. The concentration and purity of the total RNA were determined spectrophotometrically at 260/280 nm using an AgilentTM Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), ensuring all ratios exceeded 1.8. The integrity of the RNA was confirmed through electrophoresis on a 1.5% denaturing agarose gel.
Gene expression in liver samples from SHR and WKY rats was analyzed using rat microarrays. Three independent experiments were conducted: the first microarray compared the gene expression profile of 1-month-old SHR with age-matched WKY; the second microarray compared the gene expression profile of 3-month-old SHR with age-matched WKY; and the third microarray compared the gene expression profile of 1-month-old with 3-month-old SHR.
The Rattus norvegicus 70-mer oligo library from OPERON Oligo Sets (http://omad.operon.com/), containing 5000 gene-specific oligonucleotide probes representing the better-known genes, were suspended in 40 μM of Micro Spotting solution (TeleChem International Inc., Portland, OR, USA). SuperAmine-coated slides (25 × 75 mm, TeleChem) were printed in duplicate and fixed at 80˚C for 4 hours. For pre-hybridization, the slides were re-hydrated with water vapor at 60˚C, then fixed with two cycles of UV light (1200J). After being boiled for 2 min at 92˚C, the slides were washed with 95% ethanol for 1 min and pre-hybridized in 5X SSC, 0.1% SDS, and 1% BSA for 1 hour at 42˚C. The slides were then washed and dried for further hybridization.
Ten μg of total RNA were used for complementary DNA (cDNA) synthesis, which incorporated dUTP-Alexa555 or dUTP-Alexa647, employing the First-Strand cDNA Labeling Kit (Invitrogen). The incorporation of a fluorophore was analyzed by measuring the absorbance at 555 nm for Alexa555, and 650 nm for Alexa647. Equal quantities of labeled cDNA were hybridized using the UniHyb hybridization solution (TeleChem). The arrays were incubated for 14 h at 42˚C, then washed three times with 1X SCC and 0.05% SDS at room temperature.
The acquisition and quantification of array images were performed on a GenePix 4100A with its accompanying software, GenePix (Molecular Devices, Sunnyvale, CA, USA); all images were captured at a resolution of 10 μm. For each spot, the Alexa555 and Alexa647 density mean values, as well as the background mean values, were calculated using the ArrayPro Analyzer software (Media Cybernetics, Rockville, MD, USA).
Microarray data analysis was carried out using the genArise software, developed in our Informatics Unit (http://www.ifc.unam.mx/genarise/). GenArise performs several operations, including background correction, lowess normalization, intensity filtering, replicates analysis, and selecting differentially expressed genes. The goal of genArise is to identify genes that show substantial evidence of differential expression. The Database for Annotation, Visualization, and Integrated Discovery (DAVID Bioinformatics database) was employed to identify functionally related genes categories. Differentially expressed genes were considered significant and selected based on a change in expression of at least 1.5-fold, and a p-value of ≤0.05. The DAVID software analyzed significant enrichment of differentially expressed genes within Gene Ontology (GO) terms, and involves assessment of advanced pathway analysis [
Statistical analysis was performed using commercially available GraphPad Prism version 4.0 software (La Jolla, CA, USA). The data are expressed as the mean ± SEM. Statistical significance was determined using Student’s t-test, with the significance set at p ≤ 0.05.
The body weights were 83 ± 1.9 g in WKY vs. 64.7 ± 1.3 g in SHR at 1-month-old (p ≤ 0.05), while 310.1 ± 3.3 g in WKY vs. 251.9 ± 4 g in SHR at 3-month-old (p ≤ 0.05).
The systolic blood pressure of SHR was similar to that of WKY rats at 1 month-old (113 ± 2 mmHg in WKY vs. 112 ± 3 mmHg in SHR at pre-hypertensive stage). However, it was higher at 3-month-old (112 ± 3 mmHg in WKY vs. 184 ± 4 mmHg in SHR at the hypertensive stage). Blood pressure increased in SHR from 1- to 3-month-old (p ≤ 0.5), whereas it did not change with age in WKY. The study model (SHR) emulates the phenomenon observed in humans, where blood pressure increases with age.
Three independent microarray analyses were performed. The first examined gene expression in the liver of 1-month-old SHR vs. age-matched WKY. The second assessed the gene expression of 3-month-old SHR vs. age-matched WKY. The third compared the gene expression profile of 1-month-old vs. 3-month-old SHR. The hierarchical clusters show the differential expression of all the transcripts; a color scale is included with the heat map to visualize the consistency of the expression patterns within each group of samples (
Data from the microarrays were analyzed using the DAVID database software to identify genes differentially expressed in SHR, compared with WKY at both ages (p ≤ 0.05). All identified differences are shown in
Pathway analysis was conducted through the DAVID bioinformatics database to identify biological processes. The most enriched databases of pathways from our study were obtained through KEGG, as shown in
Gene Ontology enrichment analysis is based on the knowledge of various biological elements, and KEGG annotations contain information on more than 200 pathways. The identified GO functions were predominantly associated with adrenergic signaling, renin secretion, aldosterone synthesis and secretion, and calcium signaling in SHR rats. The KEGG pathway analysis revealed differentially expressed genes highlighted with red stars, as demonstrated in a representative selected pathway, i.e., aldosterone synthesis and secretion (
| Number of genes | 1 month-old | 3 month-old | Common in both |
|---|---|---|---|
| Up-regulated | 98 | 88 | 12 |
| Down-regulated | 122 | 139 | 10 |
| Total | 220 | 227 | 22 |
p ≤ 0.05, fold change ≤1.5 or >1.5.